U.S. patent application number 17/540823 was filed with the patent office on 2022-03-24 for bioactive polypeptides for improvements in plant protection, growth and productivity.
The applicant listed for this patent is Spogen Biotech Inc.. Invention is credited to Michelle Leslie, Brian Thompson.
Application Number | 20220087268 17/540823 |
Document ID | / |
Family ID | 1000005999985 |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220087268 |
Kind Code |
A1 |
Thompson; Brian ; et
al. |
March 24, 2022 |
BIOACTIVE POLYPEPTIDES FOR IMPROVEMENTS IN PLANT PROTECTION, GROWTH
AND PRODUCTIVITY
Abstract
Bioactive priming polypeptides are provided that are useful when
applied to plants in agricultural formulations. Methods of using
the formulations containing the bioactive priming polypeptides are
also provided which are applied exogenously to the surface of a
plant or a plant cell membrane or endogenously to the interior of a
plant or to a plant cell. The bioactive priming polypeptides when
applied to a plant, a plant part, or a plant growth medium or a
rhizosphere in an area surrounding the plant or the plant part
increase growth, yield, health, longevity, productivity, and/or
vigor of a plant or a plant part and/or decrease abiotic stress in
the plant or the plant part and/or protect the plant or the plant
part from disease, insects and/or nematodes, and/or increase the
innate immune response of the plant or the plant part and/or change
plant architecture.
Inventors: |
Thompson; Brian; (Creve
Coeur, MO) ; Leslie; Michelle; (Webster Groves,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Spogen Biotech Inc. |
St. Louis |
MO |
US |
|
|
Family ID: |
1000005999985 |
Appl. No.: |
17/540823 |
Filed: |
December 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17350746 |
Jun 17, 2021 |
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17540823 |
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16929422 |
Jul 15, 2020 |
11046735 |
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17350746 |
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16041059 |
Jul 20, 2018 |
10717767 |
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16929422 |
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62534710 |
Jul 20, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/195 20130101;
C12N 1/20 20130101; A01N 63/22 20200101; C12P 21/02 20130101; A01N
63/25 20200101; C07K 14/27 20130101; C07K 14/21 20130101; C12N
2510/02 20130101; A01N 63/10 20200101; C07K 14/245 20130101; A01N
37/46 20130101; A01N 63/50 20200101; C07K 14/32 20130101 |
International
Class: |
A01N 63/50 20060101
A01N063/50; C07K 14/195 20060101 C07K014/195; C07K 14/32 20060101
C07K014/32; C12N 1/20 20060101 C12N001/20; C12P 21/02 20060101
C12P021/02; A01N 63/10 20060101 A01N063/10; A01N 63/22 20060101
A01N063/22; C07K 14/27 20060101 C07K014/27; A01N 63/25 20060101
A01N063/25; C07K 14/21 20060101 C07K014/21; A01N 37/46 20060101
A01N037/46; C07K 14/245 20060101 C07K014/245 |
Claims
1.-206. (canceled)
207. A method for treating a bacterial or fungal infection in a
citrus plant, the method comprising injecting at least one
antimicrobial peptide into the citrus plant.
208. The method of claim 207, wherein the injecting comprises
injecting into a trunk or phloem of the plant.
209. The method of claim 208, wherein the injecting comprises
injecting into the trunk of the plant.
210. The method of claim 207, wherein the bacterial infection
comprises Huanglongbing (HLB), Asian Citrus Greening disease or
Citrus Canker disease.
211. The method of claim 210, wherein the bacterial infection
comprises Huanglongbing (HLB).
212. The method of claim 207, wherein the bacterial infection is
caused by a bacterial species comprising any one of Candidatus
liberibacter spp., Xanthomonas citri, and Xanthomonas axonopodis
pv.
213. The method of claim 212, wherein the bacterial species
comprises Candidatus liberibacter spp.
214. The method of claim 213, wherein the Candidatus liberibacter
spp. comprises liberibacter asiaticus.
215. The method of claim 207, wherein the antimicrobial peptide is
derived from a plant.
216. The method of claim 214, wherein the antimicrobial peptide is
derived from a plant.
217. The method of claim 215, wherein the antimicrobial peptide is
derived from a Citrus spp. plant.
218. The method of claim 216, wherein the antimicrobial peptide is
derived from a Citrus spp. plant.
219. The method of claim 207, wherein the antimicrobial peptide
comprises cysteine bonds.
220. The method of claim 215, wherein the antimicrobial peptide
comprises cysteine bonds.
221. The method of claim 217, wherein the antimicrobial peptide
comprises cysteine bonds.
222. The method of claim 207, wherein the antimicrobial peptide is
produced in a bacterial expression system.
223. The method of claim 215, wherein the antimicrobial peptide is
produced in a bacterial expression system.
224. The method of claim 217, wherein the antimicrobial peptide is
produced in a bacterial expression system.
225. The method of claim 219, wherein the antimicrobial peptide is
produced in a bacterial expression system.
226. The method of claim 207, wherein the antimicrobial peptide
comprises a thionin or thionin-like polypeptide, a defensin, a
flagellin or flagellin-associated polypeptide, or a harpin or
harpin-like polypeptide.
227. The method of claim 226, wherein the antimicrobial peptide is
a thionin, thionin-like polypeptide or defensin.
228. The method of claim 226, wherein the thionin, thionin-like
polypeptide or defensin is injected in combination with a flagellin
or flagellin-associated polypeptide, and/or a harpin or a
harpin-like polypeptide.
229. The method of claim 227, wherein the thionin, thionin-like
polypeptide or defensin comprises any one of SEQ ID NOs:
650-749.
230. The method of claim 229, wherein the thionin-like polypeptide
comprises SEQ ID NO: 651.
231. The method of claim 207, wherein the antimicrobial peptide is
fused to a phloem targeting sequence to form a fusion
polypeptide.
232. The method of claim 231, wherein the phloem targeting sequence
comprises any one of SEQ ID NOs: 641-649.
233. The method of claim 232, wherein the phloem targeting sequence
comprises SEQ ID NO: 641.
234. The method of claim 227, wherein the thionin, thionin-like
polypeptide or defensin is fused to a phloem targeting sequence to
form a fusion polypeptide.
235. The method of claim 234, wherein the phloem targeting sequence
comprises any one of SEQ ID Nos: 641-649.
236. The method of claim 235, wherein the phloem targeting sequence
comprises SEQ ID NO: 641.
237. The method of claim 226, wherein the flagellin or
flagellin-associated peptide comprises any one of SEQ ID NOs: 226,
1-225,227-375, 526, 528, 530, 532, 534, 536, 538, 540, 541, 751,
752, 754-766, 571-579, and 753.
238. The method of claim 237, wherein the flagellin or flagellin
associated peptide comprises SEQ ID NO: 226, SEQ ID NO: 571 or SEQ
ID NO: 752.
239. The method of claim 226, wherein the harpin or harpin-like
polypeptide comprises any one of SEQ ID NOs: 587, 589, 591, 593,
594 and 595.
240. The method of claim 239, wherein the harpin or harpin-like
polypeptide comprises SEQ ID NO: 587.
241. The method of claim 207, wherein treating the bacterial
infection comprises a prophylactic treatment, treatment,
prevention, and decreased disease progression on or in the plant or
plant part.
242. The method of claim 207, wherein the citrus plant comprises
Sweet orange (Citrus sinensis, Citrus maxima x Citrus reticulata),
Bergamot Orange (Citrus bergamia, Citrus limetta x Citrus
aurantium), Bitter Orange, Sour Orange or Seville Orange (Citrus
aurantium, Citrus maxima x Citrus reticulata), Blood Orange (Citrus
sinensis), Orangelo or Chironja (Citrus paradisi x Citrus
sinensis), Mandarin Orange (Citrus reticulate), Trifoliate Orange
(Citrus trifoliata), Tachibana Orange (Citrus tachibana),
Clementine (Citrus clementina), Cherry Orange (Citrus kinokuni),
Lemon (Citrus limon, Citrus maxima x Citrus medica), Indian Wild
Orange (Citrus indica), Imperial Lemon (Citrus limon, Citrus medica
x Citrus paradisi), Lime (Citrus latifoli, Citrus aurantifolia),
Meyer Lemon (Citrus meyen); hybrids of Citrus x meyer with Citrus
maxima, Citrus medica, Citrus paradisi and/or Citrus sinensis,
Rough Lemon (Citrus jambhin), Volkamer Lemon (Citrus volkameriana),
Ponderosa Lemon (Citrus limon x Citrus medica) Kaffir Lime (Citrus
hystrix or Mauritius papeda), Sweet Lemon, Sweet Lime, or Mosambi
(Citrus limetta), Persian Lime or Tahiti Lime (Citrus latifolia),
Palestine Sweet Lime (Citrus limettioides), Winged Lime (Citrus
longispina), Australian Finger Lime (Citrus australasica),
Australian Round Lime (Citrus australis), Australian Desert or
Outback Lime (Citrus glauca), Mount White Lime (Citrus garrawayae),
Kakadu Lime or Humpty Doo Lime (Citrus gracilis), Russel River Lime
(Citrus inodora), New Guinea Wild Lime (Citrus warburgiana), Brown
River Finger Lime (Citrus wintersii), Mandarin Lime (Citrus
limonia; (hybrids with Citrus reticulata x Citrus maxima x Citrus
medica), Carabao Lime (Citrus pennivesiculata), Blood Lime (Citrus
australasica x Citrus limonia) Limeberry (Triphasia brassii,
Triphasia grandifolia, Triphasia trifolia), Grapefruit (Citrus
paradisi; Citrus maxima x Citrus xsinensis), Tangarine (Citrus
tangerina), Tangelo (Citrus tangelo; Citrus reticulata x Citrus
maxima or Citrus paradisi), Minneola Tangelo (Citrus reticulata x
Citrus paradisi), Orangelo (Citrus paradisi x Citrus sinensis),
Tangor (Citrus nobilis; Citrus reticulata x Citrus sinensis),
Pummelo or Pomelo (Citrus maxima), Citron (Citrus medica), Mountain
Citron (Citrus halimii), Kumquat (Citrus japonica or Fortunella
species), Kumquat hybrids (Calamondin, Fortunella japonica;
Citranqequat, Citrus ichangensis; Limequat, Citrofortunella
floridana; Orangequat, hybrid between Satsuma mandarin x Citrus
japonica or Fortunella species; Procimequat, Fortunella hirdsiie;
Sunquat, hybrid between Citrus meyeri and Citrus japonica or
Fortunella species; Yuzuquat, hybrid between Citrus ichangensis and
Fortunella margarita), Papedas (Citrus halimii, Citrus indica,
Citrus macroptera, Citrus micrantha), Ichang Papeda (Citrus
ichangensis), Celebes Papeda (Citrus celebica), Khasi Papeda
(Citrus latipes), Melanesian Papeda (Citrus macroptera), Ichang
Lemon (Citrus ichangensis x Citrus maxima), Yuzu (Citrus
ichangensis x Citrus reticulata), Cam sanh (Citrus reticulata x
Citrus maxima), Kabosu (Citrus sphaerocarpa), Sudachi (Citrus
sudachi), Alemow (Citrus macrophylla), Biasong (Citrus micrantha),
Samuyao (Citrus micrantha), Kalpi (Citrus webber), Mikan (Citrus
unshiu), Hyuganatsu (Citrus tamurana), Manyshanyegan (Citrus
mangshanensis), Lush (Citrus crenatifolia), Amanatsu or Natsumikan
(Citrus natsudaidai), Kinnow (Citrus nobilis x Citrus deliciosa),
Kiyomi (Citrus sinensis x Citrus unshiu), Oroblanco (Citrus maxima
x Citrus paradisi), Ugli (Citrus reticulata x Citrus maxima and/or
Citrus x paradisi), Calamondin (Citrus reticulata x Citrus
japonica), Chinotto (Citrus myrtifolia, Citrus aurantium or Citrus
pumila), Cleopatra Mandarin (Citrus reshni), Daidai (Citrus
aurantium or Citrus daidai), Laraha (Citrus aurantium), Satsuma
(Citrus unshiu), Naartjie (Citrus reticulata x Citrus nobilis),
Rangpur (Citrus limonia; or hybrid with Citrus sinensis x Citrus
maxima x Citrus reticulata), Djeruk Limau (Citrus amblycarpa),
lyokan, anadomikan (Citrus iyo), Odichukuthi (Citrus odichukuthi),
Ougonkan (Citrus flaviculpus), Pompia (Citrus monstruosa), Taiwan
Tangerine (Citrus depressa), Shonan gold (Citrus flaviculpus or
Citrus unshiu), Sunki (Citrus sunki), Mangshanyen (Citrus
mangshanensis, Citrus nobilis), Clymenia (Clymenia platypoda,
Clymenia polyandra), Jabara (Citrus jabara), Mandora (Mandora
cyprus), Melogold (Citrus grandis x Citrus paradisii/Citrus
maxima/Citrus grandis), Shangjuan (Citrus ichangensis x Citrus
maxima), Nanfengmiju (Citrus reticulata), or ShikwAsai (Citrus
depressa).
243. The method of claim 232, wherein the citrus plant comprises
Bergamot Orange (Citrus bergamia), Sour or Bitter Orange (Citrus
aurantium), Sweet Orange (Citrus macrophylla), Key Lime (Citrus
aurantiifolia), Grapefruit (Citrus paradisi), Citron (Citrus
medica), Mandarin Orange (Citrus reticulate), Lemon (Citrus limon,
or hybrids with Citrus medica x Citrus maxima, Citrus limonia,
Citrus medica x Citrus maxima x Citrus medica), Sweet Lime (Citrus
limetta), Kaffir Lime, (Citrus hystrix or Mauritius papeda), Lemon
hybrid or Lumia (Citrus medica x Citrus limon), (Citrus medica x
Citrus maxima x Citrus medica), Omani Lime (Citrus aurantiifolia,
Citrus medica x Citrus micrantha), Jambola (Citrus grandis), Kakadu
Lime or Humpty Doo Lime (Citrus gracilis), Pomelo (Citrus
retkulata), Tangor (Citrus nobilis), or Sour Lime or Nimbuka
(Citrus acida).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 17/350,746, filed on Jun. 17, 2021, which is a divisional of
U.S. application Ser. No. 16/929,422, filed on Jul. 15, 2020, which
is a divisional of U.S. application Ser. No. 16/041,059, filed on
Jul. 20, 2018, issued as U.S. Pat. No. 10,717,767 on Jul. 21, 2020,
which claims the benefit of U.S. Provisional Application No.
62/534,710, filed on Jul. 20, 2017. Each of the above-cited
applications is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] Bioactive priming polypeptides are provided which can be
delivered in agricultural formulations. The polypeptides can be
applied to crops to achieve agronomically desirable outcomes such
as enhanced phenotypes in plants (e.g., those that exhibit
protection against pest, disease agents and abiotic stress),
increased plant growth, productivity and yield.
BACKGROUND OF THE INVENTION
[0003] Conventional methods to achieve desired agronomic phenotypes
such as increased yield, disease prevention, disease resistance,
and improved abiotic stress tolerance have utilized mostly
selective breeding, grafting, transgenic and agrochemical
approaches.
[0004] Bioactive Priming Polypeptides Involved in Plant Defense
Responses
[0005] Plants possess an immune system that detects and protects
against microbes that can cause disease. Antimicrobial peptides
(AMPs) in plants are often the first line of defense against
invading pathogens and are involved in the initiation of defense
responses that can impart innate immunity to a plant. Many AMPs are
generically active against various kinds of infectious agents. They
are generally classified as antibacterial, anti-fungal, anti-viral
and/or anti-parasitic.
[0006] The resistance of given plant species against certain
pathogenic organisms that can contact a plant surface and colonize
it, is based on highly specialized recognition systems for
molecules produced only by certain microbes (for example, specific
bacterial or fungal strains). Plants sense potential microbial
invaders by using pattern-recognition receptors (PRRs) to recognize
the pathogen-associated molecular patterns (PAMPs) associated with
them.
[0007] Flagellin/Flagellin-Associated Polypeptides
[0008] Flagellins and flagellin-associated polypeptides derived
from those flagellins have been reported primarily to have
functional roles in innate immune responses in plants. These
polypeptides are derived from highly conserved domains of
eubacterial flagellin. Flagellin is the main building block of the
bacterial flagellum. The flagellin protein subunit building up the
filament of bacterial flagellum can act as a potent elicitor in
cells to mount defense-related responses in various plant
species.
[0009] "Flagellin" is a globular protein that arranges itself in a
hollow cylinder to form the filament in a bacterial flagellum.
Flagellin is the principal substituent of bacterial flagellum, and
is present in flagellated bacteria. Plants can perceive, combat
infection and mount defense signaling against bacterial microbes
through the recognition of conserved epitopes, such as the stretch
of 22 amino acids (Flg22) located in the N-terminus of a full
length flagellin coding sequence. The elicitor activity of Flg22
polypeptide is attributed to this conserved domain within the
N-terminus of the flagellin protein (Felix et al., 1999). Plants
can perceive bacterial flagellin through a pattern recognition
receptor (PRR) at the plant's cell surface known as flagellin
sensitive receptor, which is a leucine-rich repeat receptor kinase
located in the plasma membrane and available at the plant cell
surface. In plants, the best-characterized PRR is FLAGELLIN SENSING
2 (FLS2), which is highly conserved in both monocot and dicot
plants.
[0010] In Arabidopsis, the innate immune response to Flg22 involves
a host recognition protein complex that contains the FLS2 leucine
rich repeat (LRR) receptor kinase (Gomez-Gomez L. and Boller T.,
"FLS2: An LRR receptor-like kinase involved in the perception of
the bacterial elicitor flagellin in Arabidopsis," Molecular Cell 5:
1003-1011, 2000). In Arabidopsis thaliana, FLS2 is a PRR that
determines flagellin perception and is specific for the binding of
the flagellin-associated polypeptide(s). For example, the binding
of Flg22 to the outer plant FLS2 membrane-bound receptor triggers a
signaling cascade that is involved in the innate immune response
that induces the plant to mount a highly specific
signaling-associated cascade that is involved in the activation of
pattern-triggered immunity (Chinchilla et al., "The Arabidopsis
receptor kinase FLS2 binds Flg22 and determines the specificity of
flagellin perception," Plant Cell 18: 465-476, 2006). Thus, the
binding of Flg22 to the Arabidopsis FLS2 membrane-bound receptor
promotes the first step of activation in which the binding elicits
an activation cascade for defense responses in the plant. The
Flg22-FLS2 interaction can also lead to the production of reactive
oxygen species (ROS) that contribute to the induction of an
oxidative burst, cellular medium alkalinization, downstream
induction of pathogen-responsive genes and defense-related
responses which then can impart disease resistance to a plant
(Felix G. et al., "Plants have a sensitive perception system for
the most conserved domain of bacterial flagellin," The Plant
Journal 18: 265-276, 1999, Gomez-Gomez L. and Boller T., "FLS2: An
LRR receptor-like kinase involved in the perception of the
bacterial elicitor flagellin in Arabidopsis," Molecular Cell 5:
1003-1011, 2000, Meindi et al., "The bacterial elicitor flagellin
activates its receptor in tomato cells according to the
address-message concept," The Plant Cell 12: 1783-1794, 2000). In
tomato, high affinity binding of Flg22 to a FLS receptor was
observed using both intact cells as well as to microsomal membrane
preparations. In this study, the binding of Flg22 to the FLS2
receptor(s) at the plasma membrane surface was nonreversible under
physiological conditions, which reflects an uptake process of the
Flg22 elicitor with import into the tomato cells (Meindi et al.,
"The bacterial elicitor flagellin activates its receptor in tomato
cells according to the address-message concept," The Plant Cell 12:
1783-1794, 2000). Recognition of Flg22 by FLS2 triggers both local
and systemic plant immune responses. The Flg22-bound, activated
FLS2 receptor complex is internalized into plant cells by
endocytosis and moves systemically throughout the plant (Jelenska
et al., "Flagellin peptide flg22 gains access to long-distance
trafficking in Arabidopsis via its receptor, FLS2," Journal of
Experimental Botany 68: 1769-1783, 2017), which may contribute
towards systemic Flg22 immune responses.
[0011] Flagellin receptor perception mediation involving Flg22 is
highly conserved across divergent plant taxa (Taki et al.,
"Analysis of flagellin perception mediated by flg22 receptor OsFLS2
in rice," Molecular Plant Microbe Interactions 21: 1635-1642,
2008). Submicromolar concentrations of synthetic polypeptides
comprising between 15-22 or 28 amino acids from conserved domains
of a flagellin protein, act as elicitors to initiate defense
responses in a variety of plant species.
[0012] Generation of transgenic plants has been used to confirm the
flagellin-specific PAMPs that bind to the flagellin-specific PRRs.
Ectopic expression of FLS2 in Arabidopsis plants showed a direct
correlation between the flagellin responses and FLS2 expression
levels, which indicate that FLS2 is involved in the recognition of
flagellin (a signal of bacterial presence) and leads to the
activation of defense responses in plants (Gomez-Gomez L. and
Boller T., "FLS2: An LRR receptor-like kinase involved in the
perception of the bacterial elicitor flagellin in Arabidopsis,"
Molecular Cell 5: 1003-1011, 2000). Transgenic plants expressing
the flagellin binding receptor have shown efficacy against certain
pathogens. Flagellin binding to FLS2 was involved in the initiation
of expression of specific MAP kinase transcription factors that
function downstream of the flagellin receptor FLS2. Mutant plants
(fls2) lacking in the FLS2 receptor are insensitive to Flg22
(Gomez-Gomez L. and Boller T., "FLS2: An LRR receptor-like kinase
involved in the perception of the bacterial elicitor flagellin in
Arabidopsis," Molecular Cell 5: 1003-1011, 2000), and impaired in
Flg22 binding to the FLS2 receptor. Mutant plants (fls2) also
exhibited enhanced susceptibility to infection and disease when
treated with pathogenic bacteria (Zipfel et al., "Bacterial disease
resistance in Arabidopsis through flagellin perception," Nature
428: 764-767, 2004).
[0013] Traditionally, methods to improve disease resistance have
capitalized on these and other such findings and have taken a
transgenic approach. Transgenic plants and seeds transformed with a
Flagellin-Sensing (FLS) receptor protein (WO2016007606A2
incorporated herein by reference in its entirety) or with
transcription factors involved in downstream signaling of FLS
(WO2002072782A2 incorporated herein by reference in its entirety)
have produced plants that confer disease resistance to certain
pathogenic microorganisms. In another example, transgenic plants
expressing Flagellin-Sensing (FLS3) receptor also have exhibited
enhanced resistance to disease compared to non-transgenic plants
not expressing the FLS3 receptor (WO2016007606A2 incorporated
herein by reference in its entirety).
[0014] Plant Defensins/Thionins
[0015] Plant defensins are also characterized as anti-microbial
peptides (AMPs). Plant defensins contain several conserved
cysteinyl residues that form disulphide bridges and contribute to
their structural stability. Defensins are among the best
characterized cysteine-rich AMPs in plants. Members of the defensin
family have four disulfide bridges that fold into a globular
structure. This highly conserved structure bestows highly
specialized roles in protecting plants against microbial pathogenic
organisms (Nawrot et al., "Plant antimicrobial peptides," Folia
Microbiology 59: 181-196, 2014).
[0016] Thionins are cystine-rich plant AMPs classified in the
defensin family and typically comprise 45-48 amino acid residues,
in which 6-8 of these amino acids are cysteine that form 3-4
disulfide bonds in higher plants. Thionins have been found to be
present in both monocot and dicot plants and their expression can
be induced by infection with various microbes (Tam et. al.,
"Antimicrobial peptides from plants," Pharmaceuticals 8: 711-757,
2015). Particular amino acids of thionins such as Lys1 and Tyr13,
which are highly conserved, have been found to be vital to the
functional toxicity of these AMPs.
[0017] Harpin and Harpin-Like (HpaG-Like)
[0018] Similar to the flagellins or the flagellin-associated
polypeptides, harpins comprise a group of bacterial-derived
elicitors that are derived from larger precursor proteins. Harpins
are critical for the elicitation of a hypersensitive response (HR)
when infiltrated into the intercellular space or apoplast of plant
cells (Kim et al., "Mutational analysis of Xanthomonas harpin HpaG
identifies a key functional region that elicits the hypersensitive
response in nonhost plants," Journal of Bacteriology 186:
6239-6247, 2004). Application of the distant harpin-like
(HpaG-like) bioactive priming polypeptide(s) to a plant provides an
alternative conduit to protect a plant from disease and insect
pressure. Harpins utilize a type III secretion system that enable
the transport of proteins across the lipid bilayers that makeup the
plant plasma cell membrane. The binding of harpins to the surface
of the plasma cell membrane can trigger an innate immune response
that resembles those triggered by pathogen-associated molecular
patterns (PAMPs) and are known to activate PAMP-triggered immunity
(Engelhardt et al., "Separable roles of the Pseudomonas syringae
pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore
formation and activation of plant immunity," The Plant Journal 57:
706-717, 2009). Mutational analysis of a harpin-like HpaG derived
polypeptide showed that the 12 amino acid residues between Leu-39
and Leu50 of the original 133 amino acid harpin elicitor precursor
protein was critical to the elicitation of a hypersensitive (HR)
and subsequent innate immune responses in tobacco (Kim et al.,
"Mutational analysis of Xanthomonas harpin HpaG identifies a key
functional region that elicits the hypersensitive response in
nonhost plants," Journal of Bacteriology 186: 6239-6247, 2004).
This indicates that a specific amino acid region of harpins
(similar to the other AMPs) is responsible for the elicitation
responses. Harpins, such as HpaG-like can be used to enhance
resistance to not only plant pathogens but also to insects (Choi et
al., "Harpins, multifunctional proteins secreted by gram-negative
plant pathogenic bacteria," Molecular Plant Microbe Interactions
26: 1115-1122, 2013). Harpin has been used to induce disease
resistance in plants and protect plants from colonization and
feeding by insect phloem-feeding insects, such as aphids (Zhang et
al., "Harpin-induced expression and transgenic overexpression of
phloem protein gene At.PP2A1 in Arabidopsis repress phloem feeding
of the green peach aphid Myzus persicae," BMC Plant Biology 11:
1-11, 2011).
[0019] Elongation Factor Tu (EF-Tu)
[0020] Elongation factor Tu is an abundant protein found in
bacteria and acts as a pathogen-associated molecular pattern (PAMP)
to initiate signaling cascades that are involved in plant disease
resistance and plant innate immunity to microbial pathogenic
organisms. Interestingly, some EF-Tu polypeptides are also found to
exist in plants. The first 18 amino acid residues of the N-terminus
of EF-Tu from Escherichia coli, termed elf18, is known to be a
potent inducer of PAMP-triggered immune responses in plants (Zipfel
et al., "Perception of the bacterial PAMP EF-Tu by the Receptor EFR
restricts Agrobacterium-mediated transformation," Cell 125:
749-760, 2006). Polypeptides derived from E. coli EF-Tu are
perceived by the plant cell-surface localized receptor EF-Tu
receptor (EFR) (Zipfel et al., 2006). EF-Tu binding and activation
of EFR follow a similar mode of action compared to that of the Flg
peptide-FLS2 receptor complex (Mbengue et al., "Clathrin-dependent
endocytosis is required for immunity mediated by pattern
recognition receptor kinases," Proc Natl Acad Sci U.S.A. 113:
11034-9, 2016).
[0021] Growth Altering Bioactive Priming Polypeptides
[0022] Phytosulfokines (PSK.alpha.)
[0023] Phytosulfokines (PSK) belong to a group of sulfated plant
polypeptides that are encoded by precursor genes that are
ubiquitously present and highly conserved in higher plants (Sauter
M., "Phytosulfokine peptide signaling," Journal of Experimental
Biology 66: 1-9, 2015). PSK genes are encoded by small gene
families that are present in both monocots and dicots and encode a
PSK polypeptide(s) that can be active as either a pentapeptide or a
C-terminally truncated tetrapeptide (Lorbiecke R, Sauter M,
"Comparative analysis of PSK peptide growth factor precursor
homologs," Plant Science 163: 348-357, 2002).
[0024] The phytosulfokine protein is targeted to the secretory
pathway in plants by a conserved signal polypeptide (Lorbiecke R,
Sauter M, "Comparative analysis of PSK peptide growth factor
precursor homologs," Plant Science 163: 348-357, 2002). Processing
of the phytosulfokine precursor protein involves sulfonylation by a
tyrosylprotein sulfotransferase within the plant secretory pathway,
specifically the trans-Golgi followed by secretion and proteolytic
cleavage in the apoplast in order to produce PSK (Sauter M.,
"Phytosulfokine peptide signaling," Journal of Experimental Biology
66: 1-9, 2015). After PSK is processed from the larger precursor
polypeptide, the polypeptide undergoes tyrosine sulphation (Ryan et
al., "Polypeptide hormones," The Plant Cell Supplement, S251-S264,
2002). The secreted polypeptide is then perceived at the cell
surface by a membrane-bound receptor kinase of the leucine-rich
repeat family (Sauter M., "Phytosulfokine peptide signaling,"
Journal of Experimental Biology 66: 1-9, 2015 where PSK can then
bind to the specialized PSK receptor (for example, PSK1 from
Arabidopsis) which has a leucine-rich repeat region located on the
plant plasma membrane surface. Specific binding of PSK was detected
in plasma membrane fractions from cell suspension cultures derived
from rice and maize and the binding to the receptor was shown to
initiate and stimulate cell proliferation (Matsubayashi et al.,
"Phytosulfokine-.alpha., a sulfated pentapeptide, stimulates the
proliferation of rice cells by means of specific high- and
low-affinity binding sites," Proceedings National Academy of
Science USA 94:13357-13362, 1997).
[0025] Phytosulfokines (PSK) serve as sulfated growth factors with
biostimulant activities and are involved in the control of the
development of root and shoot apical meristems, growth regulation
and reproductive processes. PSKs have also been reported to
initiate cell proliferation, differentiation of quiescent tissues
and are involved in the formation and stimulation and
differentiation of tracheary elements (Matsubayashi et al., "The
endogenous sulfated pentapeptide phytosulfokine-.alpha. stimulates
tracheary element differentiation of isolated mesophyll cells of
zinnia, Plant Physiology 120: 1043-1048, 1999). PSK signaling has
also been reported to be involved in the regulation of root and
hypocotyl elongation that occurs in Arabidopsis seedlings
(Kutschmar et al., "PSK-.alpha. promotes root growth in
Arabidopsis," New Phytologist 181: 820-831, 2009).
[0026] Root Hair Promoting Polypeptide (RHPP)
[0027] Root hair promoting polypeptide (RHPP) is a 12 amino acid
fragment derived from soybean Kunitz trypsin inhibitor (KTI)
protein, which was detected from soybean meal that was subjected to
degradation using an alkaline protease from Bacillus circulans
HA.sub.12 (Matsumiya Y. and Kubo M. "Soybean and Nutrition, Chapter
11: Soybean Peptide: Novel plant growth promoting peptide from
soybean," Agricultural and Biological Sciences, Sheny H. E.
(editor), pgs. 215-230, 2011). When applied to soybean roots, RHPP
was shown to accumulate in the roots and promote root growth
through the stimulation of cell division and root hair
differentiation in Brassica.
SUMMARY OF THE INVENTION
[0028] A polypeptide is provided for bioactive priming of a plant
or a plant part to increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part and/or
decrease abiotic stress in the plant or the plant part and/or
protect the plant or the plant part from disease, insects and/or
nematodes, and/or increase the innate immune response of the plant
or the plant part and/or change plant architecture. The polypeptide
comprises either:
[0029] (a) a flagellin or flagellin-associated polypeptide and an
amino acid sequence of the flagellin or flagellin-associated
polypeptide comprises any one of SEQ ID NOs: 226, 1-225, 227-375,
526, 528, 530, 532, 534, 536, 538, 540, 541, 751 and 752; or
[0030] (b) a mutant flagellin or flagellin-associated polypeptide
and an amino acid sequence of the mutant flagellin or
flagellin-associated polypeptide comprises any one of SEQ ID NOs:
571-579 and 753; or
[0031] (c) a mutant flagellin or flagellin-associated polypeptide
and an amino acid sequence of the mutant flagellin or
flagellin-associated polypeptide comprises any one of SEQ ID NOs:
580-585; or
[0032] (d) a retro inverso Flg22 polypeptide and an amino acid
sequence of the retro inverso Flg22 polypeptide comprises any one
of SEQ ID NOs: 376-450, 527, 531, 533, 535, 537 and 539; or
[0033] (e) a retro inverso FlgII-28 polypeptide and an amino acid
sequence of the retro inverso FlgII-28 polypeptide comprises any
one of SEQ ID NOs: 451-525; or
[0034] (f) a retro inverso Flg15 polypeptide and an amino acid
sequence of the retro inverso Flg15 polypeptide comprises SEQ ID
NO: 529; or
[0035] (g) a harpin or harpin-like polypeptide and an amino acid
sequence of the harpin or harpin-like polypeptide comprises any one
of SEQ ID NOs: 587, 589, 591, 593, 594 and 595; or
[0036] (h) a retro inverso harpin or harpin-like polypeptide and an
amino acid sequence of the retro inverso harpin or harpin-like
polypeptide comprises any one of SEQ ID NOs: 588, 590, 592, 596 and
597; or
[0037] (i) a root hair promoting polypeptide (RHPP) and an amino
acid sequence of the RHPP comprises any one of SEQ ID Nos: 600, 603
and 604; or
[0038] (j) a Kunitz Trypsin Inhibitor (KTI) polypeptide and an
amino acid sequence of the KTI polypeptide comprises SEQ ID No:
602; or
[0039] (k) a retro inverso root hair promoting polypeptide (RI
RHPP) and an amino acid sequence of the RI RHPP comprises any one
of SEQ ID NO: 601, 605 and 606; or
[0040] (l) an elongation factor Tu (EF-Tu) polypeptide and an amino
acid sequence of the EF-Tu polypeptide comprises any one of SEQ ID
NOs: 607-623; or
[0041] (m) a retro inverso elongation factor Tu (RI EF-Tu)
polypeptide and an amino acid sequence of the RI EF-Tu polypeptide
comprises any one of SEQ ID NOs: 624-640; or
[0042] (n) a fusion polypeptide comprising SEQ ID NO: 750; or
[0043] (o) a phytosulfokine (PSK) polypeptide and an amino acid
sequence of the PSK polypeptide comprises SEQ ID NO: 598; or
[0044] (p) a retro inverso phytosulfokine (RI PSK) polypeptide and
an amino acid sequence of the RI PSK polypeptide comprises SEQ ID
NO: 599; or
[0045] (q) a thionin or thionin-like polypeptide and an amino acid
sequence of the thionin or thionin-like polypeptide comprises any
one of SEQ ID NOs: 650-749, and
[0046] optionally, wherein the flagellin or flagellin-associated
polypeptide of (a), the mutant flagellin or flagellin-associated
polypeptide of (c), the harpin or harpin-like polypeptide of (g),
the PSK polypeptide of (o), and the thionin or thionin-like
polypeptide of (q) either: contains a chemical modification; is a
variant having an amino acid insertion, deletion, inversion,
repeat, duplication, extension, or substitution within the amino
acid sequence; is part of a fusion protein; or contains a protease
recognition sequence.
[0047] A composition is provided for bioactive priming of a plant
or a plant part to increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part and/or
decrease abiotic stress in the plant or the plant part and/or
protect the plant or the plant part from disease, insects and/or
nematodes, and/or increase the innate immune response of the plant
or the plant part and/or change plant architecture. The composition
comprises either: the polypeptide as described above or any
combination thereof, and an agrochemical or a carrier; or any
combination of the polypeptides.
[0048] A seed coated with the polypeptide or the composition as
described herein is also provided.
[0049] A recombinant microorganism that expresses or overexpresses
a polypeptide is also provided. The polypeptide comprises the
polypeptides as described above for the composition.
[0050] Methods are provided for increasing growth, yield, health,
longevity, productivity, and/or vigor of a plant or a plant part
and/or decreasing abiotic stress in the plant or the plant part
and/or protecting the plant or the plant part from disease, insects
and/or nematodes, and/or increasing the innate immune response of
the plant or the plant part and/or changing plant architecture. The
method can comprise applying the polypeptide or the composition as
described herein to a plant, a plant part, or a plant growth medium
or a rhizosphere in an area surrounding the plant or the plant part
to increase growth, yield, health, longevity, productivity, and/or
vigor of the plant or the plant part and/or decrease abiotic stress
in the plant or the plant part and/or protect the plant or the
plant part from disease, insects and/or nematodes, and/or increase
the innate immune response of the plant or the plant part and/or
change the plant architecture.
[0051] Alternatively, the method can comprise applying the
polypeptide or the composition as described herein to a plant
growth medium to increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part to be grown
in the plant growth medium and/or decrease abiotic stress in the
plant or the plant part to be grown in the plant growth medium
and/or protect the plant or the plant part to be grown in the plant
growth medium from disease, insects and/or nematodes, and/or
increase the innate immune response and/or change plant
architecture of the plant or the plant part to be grown in the
plant growth medium.
[0052] Another method comprises applying the recombinant
microorganism as described herein to a plant, a plant part, or a
plant growth medium or a rhizosphere in an area surrounding the
plant or the plant part to increase growth, yield, health,
longevity, productivity, and/or vigor of the plant or the plant
part and/or decrease abiotic stress in the plant or the plant part
and/or protect the plant or the plant part from disease, insects
and/or nematodes, and/or increase the innate immune response of the
plant or the plant part and/or change the plant architecture. The
recombinant microorganism expresses the polypeptide and expression
of the polypeptide is increased as compared to the expression level
the polypeptide in a wild-type microorganism of the same kind under
the same conditions.
[0053] A method of producing a polypeptide comprising producing a
fusion protein comprising any polypeptide as described herein and
an enterokinase (EK) cleavage site via fermentation, the
enterokinase cleavage site enhancing activity and stability of the
polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 shows the Bt.4Q7Flg22 bioactive priming polypeptide
in its native L configuration (SEQ ID NO: 226) and the
corresponding retro inverso or D configuration form (SEQ ID NO:
375).
[0055] FIG. 2 illustrates total harvestable yield in corn that
received foliar applications with Bt.4Q7Flg22 (SEQ ID NO:226) in 12
locations (panel A) and retro inverso (RI) version of Bt.4Q7Flg22
(SEQ ID NO: 375) bioactive priming polypeptides in 10 locations
(panel B) and reported in Bu/Ac as compared to yield in the
non-treated control.
[0056] FIG. 3 illustrates total harvestable yield in corn that
received foliar applications with Bt.4Q7Flg22 bioactive priming
polypeptide (SEQ ID NO: 226) in 6 locations and reported in Bu/Ac
as compared to yield in the non-treated control.
[0057] FIG. 4 illustrates total harvestable yield in soybean that
received foliar applications with Bt.4Q7Flg22 (SEQ ID NO: 226)
(panel A) and retro inverso (RI) Bt.4Q7Flg22 (SEQ ID NO: 375)
(panel B) bioactive priming polypeptides in 11 locations and
reported in Bu/Ac as compared to yield in the non-treated
control.
[0058] FIG. 5 illustrates total harvestable yield in corn that
received foliar applications with Ec.Flg22 (SEQ ID NO: 526) (panel
A) and retro inverso with Ec.Flg22 (SEQ ID NO: 527) (panel B)
bioactive priming polypeptides in 12 locations and reported in
Bu/Ac as compared to yield in the non-treated control.
[0059] FIG. 6 is directed to a reactive oxygen species (ROS)
activity assay using Bt.4Q7Flg22 in combination with different
concentrations of cellobiose as an additive in corn (panel A) or in
soybeans (panel B).
[0060] FIG. 7 is directed to a reactive oxygen species (ROS)
activity assay using Bt.4Q7Flg22 at different concentrations to
identify the peak activity and timing for the assay.
[0061] FIG. 8 is directed to the application delivery using
thionins to influence (decrease) the growth of Agrobacterium strain
GV3101 in a rate dependent manner.
[0062] FIG. 9 is directed to the application delivery of Bt4Q7
Flg22 polypeptides tagged or untagged with thionins to decrease the
growth of Candidatus liberibacter spp in HLB infected citrus trees.
Data represent quantitative PCR results (Ct values) of C.
liberibacter in leaf samples taken from treated infected trees.
[0063] FIG. 10 is directed to the application delivery to citrus in
trees injected with 1.times. or 10.times.Bt.4Q7Flg22 (SEQ ID NO:
226) to decrease the growth of Candidatus liberibacter spp in HLB
infected citrus trees. Data represent quantitative PCR results (Ct
values) of C. liberibacter in leaf samples taken from treated
infected trees.
[0064] FIG. 11 is directed to `Valencia` orange trees injected with
1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit set per limb.
[0065] FIG. 12 is directed to `Valencia` orange trees injected with
1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit growth as measured in centimeters.
[0066] FIG. 13 is directed to `Valencia` orange trees injected with
1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit set as indicated by estimated fruit volume per limb.
[0067] FIG. 14 is directed to `Ruby Red` grapefruit trees injected
with 1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit set per limb.
[0068] FIG. 15 is directed to `Ruby Red` grapefruit trees injected
with 1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit growth as measured in centimeters.
[0069] FIG. 16 is directed to `Ruby Red` grapefruit trees injected
with 1.times. or 10.times. Bt.4Q7Flg22 (SEQ ID NO: 226) to increase
fruit set as indicated by estimated fruit volume per limb.
DEFINITIONS
[0070] When the articles "a," "an," "one," "the," and "said" are
used herein, they mean "at least one" or "one or more" unless
otherwise indicated.
[0071] The terms "comprising," "including," and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
[0072] "Abiotic stress" as used herein is defined as an
environmental condition that can have a negative impact on a plant.
Abiotic stress can include: temperature (high or low) stress,
radiation stress (visible or UV), drought stress, cold stress, salt
stress, osmotic stress, nutrient-deficient or high metal stress, or
water stress that results in water deficit, flooding or anoxia.
Other abiotic stress factors include dehydration, wounding, ozone,
and high or low humidity.
[0073] "Bioactive priming" refers to an effect of the polypeptides
as described herein to improve a plant or a plant part. Bioactive
priming can increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part and/or
decrease abiotic stress in the plant or the plant part and/or
protect the plant or the plant part from disease, insects and/or
nematodes, and/or increase the innate immune response of the plant
or the plant part and/or change plant architecture.
[0074] A "bioactive priming polypeptide" as used herein may be used
interchangeably with the term "priming agent(s)" and as described
for the classes of polypeptides of the: flagellin and
flagellin-associated polypeptides, harpin and harpin-like
polypeptide (HpaG-like), thionins, elongation factor Tu (EF-Tu) and
its polypeptides, phytosulfokine .alpha. (PSK.alpha.), kunitz
trypsin inhibitor (KTI), and root hair promoting polypeptide
(RHPP), as well as any retro inverso polypeptides thereof.
[0075] A "colorant" as used herein acts as a visual product
identifier for product branding and application. Colorants can
include, but are not limited to, dyes and pigments, inorganic
pigments, organic pigments, polymeric colorants, and formulated
pigment coating dispersions available in a variety of highly
concentrated shades.
[0076] "Endogenously" applied as used herein refers to an
application to the inside of a plant surface. Small bioactive
priming polypeptides are particularly suited for signalling and
communication within a plant. Inside a plant surface refers to a
surface internal to any plant membrane or plant cell. Internal
could be used to mean either extracellular or intracellular to a
plant cell and is inclusive of xylem, phloem, tracheids, etc.
Endogenous can refer to movement systemically or through a plant
such as referring to cell to cell movement in a plant. Endogenous
application can include delivery of bioactive priming polypeptides
using recombinant endophytic bacteria or fungi, wherein the
endophytic microorganism is delivered externally to the plant and
through natural mechanisms moves internally to the plant.
[0077] "Exogenously" applied as used herein refers to an
application to the outside of a plant surface. A plant surface can
be any external plant surface, for example a plasma membrane, a
cuticle, a trichome, a leaf, a root hair, seed coat, etc.
[0078] "-associated" or "-like" polypeptides as used herein refers
to polypeptides derived from or structurally similar to the recited
polypeptide but having an amino acid sequence and/or source
distinct from the recited polypeptide. For example, the
thionin-like protein from Brassica rapa (SEQ ID NO: 694) has a
different sequence than thionin from Brassica napus (SEQ ID NOs
693) but is structurally and functionally similar.
[0079] A "foliar treatment" as used herein refers to a composition
that is applied to the above ground parts or foliage of a plant or
plant part and may have leaves, stems, flowers, branches, or any
aerial plant part, for example, scion.
[0080] "Injection" as described herein can be used interchangeably
with vaccination or immunization and provides a process whereby the
bioactive priming polypeptides are delivered endogenously to a
plant or plant part.
[0081] "Inoculation" means to deliver-bacteria or living
microorganisms that produce the priming polypeptide to a plant or
plant part. Inoculation can also refer to the delivery of the
priming polypeptide for passive entry through the stomata or any
opening in or on a plant or plant part. A "plant" refers to but is
not limited to a monocot plant, a dicot plant, or a gymnosperm
plant. The term "plant" as used herein includes whole plants, plant
organs, progeny of whole plants or plant organs, embryos, somatic
embryos, embryo-like structures, protocorms, protocorm-like bodies,
and suspensions of plant cells. Plant organs comprise, shoot
vegetative organs/structures (e.g., leaves, stems and tubers),
roots, flowers and floral organs/structures (e.g., bracts, sepals,
petals, stamens, carpels, anthers and ovules), seed including
embryo, endosperm, and seed coat and fruit (the mature ovary),
plant tissue (e.g., phloem tissue, vascular tissue, ground tissue,
and the like) and cells (e.g., guard cells, egg cells, trichomes
and the like). The class of plants that can be used in the methods
described herein is generally as broad as the class of higher
plants, specifically angio-sperms monocotyledonous (monocots) and
dicotyledonous (dicots) plants and gymnosperms. It includes plants
of a variety of ploidy levels, including aneuploid, polyploid,
diploid, haploid, homozygous and hemizygous. The plants described
herein can be monocot crops, such as, sorghum, maize, wheat, rice,
barley, oats, rye, millet, and triticale. The plants described
herein can also be dicot crops, such as apple, pear, peach, plum,
orange, lemon, lime, grapefruit, kiwi, pomegranate, olive, peanut,
tobacco, tomato, etc. Also, the plants can be horticultural plants
such as rose, marigold, primrose, dogwood, pansy, geranium,
etc.
[0082] A plant "biostimulant" is any substance or microorganism
applied to a plant or a plant part that is used to enhance
nutrition efficiency, abiotic stress tolerance and/or any other
plant quality trait(s).
[0083] A "plant cell" as used herein refers to any plant cell and
can comprise a cell at the plant surface or internal to the plant
plasma membrane, for example, an epidermal cell, a trichome cell, a
xylem cell, a phloem cell, a sieve tube element, or a companion
cell.
[0084] A "plant part" as described herein refers to a plant cell, a
leaf, a stem, a flower, a floral organ, a fruit, pollen, a
vegetable, a tuber, a corm, a bulb, a pseudobulb, a pod, a root, a
rhizome, a root ball, a root stock, a scion, or a seed.
[0085] A "polypeptide" as described herein refers to any protein,
peptide or polypeptide.
[0086] "Priming" or "peptide priming" as used herein refers to a
technique used to improve plant performance. In particular priming
is a process whereby the bioactive priming polypeptides are applied
either exogenously or endogenously to a plant, plant part, plant
cell or to the intercellular space of a plant that results in
outcomes that provide benefits to a plant, such as enhanced growth,
productivity, abiotic stress tolerance, pest and disease tolerance
or prevention.
[0087] A "retro-inverso" polypeptide as used herein refers to a
polypeptide chain of a natural derived polypeptide from a
normal-all-L chain reconfigured and built using non-naturally
occurring D-amino acids in reverse order of the naturally occurring
L-amino acids. The all-D-amino acid form and the parent chain
containing all L-form are topological mirrorings of the protein
structure.
[0088] A "seed treatment" as used herein refers to a substance or
composition that is used to treat or coat a seed. Sample seed
treatments include an application of biological organisms, chemical
ingredients, inoculants, herbicide safeners, micronutrients, plant
growth regulators, seed coatings, etc. provided to a seed to
suppress, control or repel plant pathogens, insects, or other pests
that attack seeds, seedlings or plants or any useful agent to
promote plant growth and health.
[0089] A "synergistic" effect refers to an effect arising between
the interaction or cooperation of two or more bioactive priming
polypeptides, substances, compounds, or other agents to produce a
combined effect greater than the sum of their separate effects.
[0090] A "synergistic effective concentration" refers to the
concentration(s) of two or more bioactive priming polypeptides,
substances, compounds or other agents that produces an effect
greater than the sum of the individual effects.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0091] There is a growing need for bioactive polypeptides that act
as "priming agents" to provide benefits to agriculture. The use of
bioactive "priming" polypeptides in agricultural practices provides
a paradigm shift for integrated crop management practices for
example, to manage disease, abiotic stress and yield programs.
Bioactive (naturally occurring, recombinant or synthetic) priming
polypeptides are delivered in agricultural formulations.
Compositions and methods of using the bioactive priming
polypeptides are described to supply a multi-tiered treatment
regime to apply to crops to achieve agronomically desirable
outcomes. Such desirable outcomes include enhanced phenotypes in
plants such as those that exhibit protection against pest, disease
agents and abiotic stress, as well as increased plant growth,
productivity and yield. More specifically, the bioactive priming
polypeptides or formulations of the bioactive priming polypeptides
can be applied using various treatment regimes, exogenously and/or
endogenously to a plant or plant part, and have been discovered to
increase growth, yield, health, longevity, productivity, and/or
vigor of a plant or a plant part and/or decrease abiotic stress in
the plant or the plant part and/or protect the plant or the plant
part from disease, insects and/or nematodes, and/or increase the
innate immune response of the plant or the plant part and/or change
plant architecture.
[0092] Specific classes of synthetically derived or naturally
occurring bioactive priming polypeptides including flagellins and
flagellin-associated polypeptides (including those conserved among
the Bacillus genera), thionins, harpin-like polypeptide
(HpaG-like), elongation factor Tu (EF-Tu), phytosulfokine
(PSK.alpha.) and root hair promoting polypeptide (RHPP) were
selected for their distinct modes of action and can be used
individually or in combination with other polypeptides to
accommodate the specific agricultural needs described above. They
can be used in the place of or in addition to commercially
available agrochemicals, biostimulants, supplemental bioactives
and/or pesticidal compounds.
[0093] Combinations of the bioactive priming polypeptides are also
provided that are applied in synergistically effective amounts to
provide control of pests, pathogens and additionally provide
benefits to enhance plant growth and promote plant health.
I. Polypeptides
[0094] The bioactive priming polypeptides are provided as naturally
occurring, recombinant or chemically synthesized forms derived from
bacteria or plants. The bioactive priming polypeptides are provided
in both the normal L and non-natural retro-inverso D amino-acid
forms. In addition, bioactive priming polypeptides are provided
that contain non-natural modifications, including N-terminal and
C-terminal modifications, cyclization, .beta.-amino and D-amino
acid containing, and other chemical modifications that enhance
stability or performance of the polypeptides. For example,
flagellin and the Flg-associated polypeptides comprising 22 amino
acids in length and derived from the full coding region of
flagellin were initially isolated and identified from a proprietary
genome assembled for bacterial strain, Bacillus thuringiensis 4Q7.
These Flg22 derived polypeptides were provided in the standard (L)
and retro-inverso (D) forms. They are described as Bt.4Q7Flg22 and
retro-inverso (RI) Bt.4Q7Flg22. Other bacterial derived bioactive
priming polypeptides are Ec.Flg22 (Escherichia coli), HpaG-like
(Xanthomonas spp.), while the plant derived polypeptides include
thionins (Citrus spp. and other plant species), PSK.alpha.
(Arabidopsis thaliana and other plants), EF-Tu (both bacterial or
plant derived) and RHPP (Glycine max).
[0095] The bioactive priming polypeptides can include full-length
proteins and are provided as naturally occurring, synthetic or
recombinant forms derived from bacteria or plants. For example,
flagellin, EF-Tu, KTI, and HpaG can all be delivered to plants.
[0096] The bioactive priming polypeptides can also be delivered as
fusion partners to other protein sequences, including protease
cleavage sites, binding proteins, and targeting proteins for
specific delivery to plants or plant parts.
[0097] Also provided are signature, signal anchor sorting and
secretion sequences that can be naturally or chemically synthesized
and targeting sequences, such as phloem-targeting sequences that
are produced along with the bioactive priming polypeptide(s) using
recombinant microorganisms and either used as fusion or assistance
polypeptides with the bioactive priming polypeptides as described
herein.
[0098] Non-naturally occurring polypeptides are also described
herein. More specifically, a polypeptide is provided for bioactive
priming of a plant or a plant part to increase growth, yield,
health, longevity, productivity, and/or vigor of a plant or a plant
part and/or decrease abiotic stress in the plant or the plant part
and/or protect the plant or the plant part from disease, insects
and/or nematodes, and/or increase the innate immune response of the
plant or the plant part and/or change plant architecture. The
polypeptide comprises either:
[0099] (a) a flagellin or flagellin-associated polypeptide and an
amino acid sequence of the flagellin or flagellin-associated
polypeptide comprises any one of SEQ ID NOs: 226, 1-225, 227-375,
526, 528, 530, 532, 534, 536, 538, 540, and 541; or
[0100] (b) a mutant flagellin or flagellin-associated polypeptide
and an amino acid sequence of the mutant flagellin or
flagellin-associated polypeptide comprises any one of SEQ ID NOs:
571-579; or
[0101] (c) a mutant flagellin or flagellin-associated polypeptide
and an amino acid sequence of the mutant flagellin or
flagellin-associated polypeptide comprises any one of SEQ ID NOs:
580-585; or
[0102] (d) a retro inverso Flg22 polypeptide and an amino acid
sequence of the retro inverso Flg22 polypeptide comprises any one
of SEQ ID NOs: 376-450, 527, 531, 533, 535, 537 and 539; or
[0103] (e) a retro inverso FlgII-28 polypeptide and an amino acid
sequence of the retro inverso FlgII-28 polypeptide comprises any
one of SEQ ID NOs: 451-525; or
[0104] (f) a retro inverso Flg15 polypeptide and an amino acid
sequence of the retro inverso Flg15 polypeptide comprises SEQ ID
NO: 529; or
[0105] (g) a harpin or harpin-like polypeptide and an amino acid
sequence of the harpin or harpin-like polypeptide comprises any one
of SEQ ID NOs: 587, 589, 591, 593, 594 and 595; or
[0106] (h) a retro inverso harpin or harpin-like polypeptide and an
amino acid sequence of the retro inverso harpin or harpin-like
polypeptide comprises any one of SEQ ID NOs: 588, 590, 592, 596 and
597; or
[0107] (i) a root hair promoting polypeptide (RHPP) and an amino
acid sequence of the RHPP comprises any one of SEQ ID Nos: 600, 603
and 604; or
[0108] (j) a Kunitz Trypsin Inhibitor (KTI) polypeptide and an
amino acid sequence of the KTI polypeptide comprises SEQ ID No:
602; or
[0109] (k) a retro inverso root hair promoting polypeptide (RI
RHPP) and an amino acid sequence of the RI RHPP comprises any one
of SEQ ID NO: 601, 605 and 606; or
[0110] (l) an elongation factor Tu (EF-Tu) polypeptide and an amino
acid sequence of the EF-Tu polypeptide comprises any one of SEQ ID
NOs: 607-623; or
[0111] (m) a retro inverso elongation factor Tu (RI EF-Tu)
polypeptide and an amino acid sequence of the RI EF-Tu polypeptide
comprises any one of SEQ ID NOs: 624-640; or
[0112] (n) a fusion polypeptide comprising SEQ ID NO: 750; or
[0113] (o) a phytosulfokine (PSK) polypeptide and an amino acid
sequence of the PSK polypeptide comprises SEQ ID NO: 598; or
[0114] (p) a retro inverso phytosulfokine (RI PSK) polypeptide and
an amino acid sequence of the RI PSK polypeptide comprises SEQ ID
NO: 599; or
[0115] (q) a thionin or thionin-like polypeptide and an amino acid
sequence of the thionin or thionin-like polypeptide comprises any
one of SEQ ID NOs: 650-749, and
[0116] optionally, wherein the flagellin or flagellin-associated
polypeptide of (a), the mutant flagellin or flagellin-associated
polypeptide of (c), the harpin or harpin-like polypeptide of (g),
the PSK polypeptide of (o), and the thionin or thionin-like
polypeptide of (q) either: contains a chemical modification; is a
variant having an amino acid insertion, deletion, inversion,
repeat, duplication, extension, or substitution within the amino
acid sequence; is part of a fusion protein; or contains a protease
recognition sequence.
[0117] Flagellins and Flagellin-Associated Polypeptides
[0118] The polypeptide can include a flagellin or
flagellin-associated polypeptide.
[0119] The flagellin or flagellin-associated polypeptide can be
derived from a Bacillus, a Lysinibacillus, a Paenibacillus, an
Aneurinibacillus genus bacterium, or any combination thereof.
[0120] One of the main classes of bioactive priming polypeptides as
described herein are the flagellin(s) and the flagellin-associated
priming polypeptide(s). Conserved full and partial length amino
acid flagellin coding sequences were identified from various
species of Bacillus and non-Bacillus bacteria using methods as
described herein.
[0121] Flagellin is a structural protein that forms the main
portion of flagellar filaments from flagellated bacterial species
that can show conservation in the N-terminal and C-terminal regions
of the protein but can be variable in the central or mid part
(Felix G. et al., "Plants have a sensitive perception system for
the most conserved domain of bacterial flagellin," The Plant
Journal 18: 265-276, 1999). The N- and C-terminal conserved regions
from flagellins that form the inner core of the flagellin protein
may have roles in the polymerization of the protein into a
filament, in the motility and transport of the protein and in the
surface attachment of a peptide fragment to the plant cell
membrane/cell surface receptors of a plant.
[0122] Full or partial flagellins (Table 1-2) and the
flagellin-associated polypeptides derived from those Bacillus and
non-Bacillus flagellins (Tables 3 and 5) are provided.
[0123] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 1-768, or any combination thereof.
[0124] Flagellin-associated bioactive priming polypeptides are
produced from flagellin coding polypeptides (such as the precursor
proteins of Flg22). More specifically, a polypeptide or a cleaved
fragment derived from the polypeptide is provided to achieve a
bioactive priming Flg polypeptide that can be used to prime or
treat a plant. The cleavage of the Flg22 fragment from larger
precursors can be accomplished through introduction of proteolytic
cleavage sites near the Flg22 to facilitate processing of the
active biopeptide from the larger polypeptide.
[0125] The flagellin-associated bioactive priming polypeptides can
be derived from full length flagellin proteins (or precursor
proteins from Flg-associated polypeptides from a Bacillus, a
Lysinibacillus, a Paenibacillus, or an Aneurinibacillus or other
non-related genera bacterium). For example, PCR purified DNA from
the flagellin-associated polypeptides such as Flg22 and FlgII-28
(Bacillus genera) and Flg15 and Flg22 (E. coli) are cloned into a
recombinant vector, amplified to achieve adequate amounts of
purified DNA that is then sequenced using conventional methods
known and used by one of ordinary skill in the art. The same
methods can be used with the flagellin coding or the flagellin
partial sequences (Table 1), N- or C-terminal flagellin
polypeptides (Table 2) and any of the Flg-associated polypeptides
(Tables 3-5).
[0126] The flagellin or flagellin-associated polypeptide can be
derived from any member of Eubacteria that contains the conserved
22 amino acid region that is recognized by the plants. Preferred
flagellin or flagellin-associated polypeptides can be derived from
a Bacillus, a Lysinibacillus, a Paenibacillus, an Aneurinibacillus
genus bacterium, or any combination thereof. Additional preferred
flagellin and Flg22 sequences can be obtained from the
gammaproteobacteria, which contain conserved 22 amino acid
sequences of >68% identity.
[0127] Conserved Flagellin Sequences from Bacillus
[0128] The flagellin-associated bioactive priming polypeptides
correspond to the N-terminal conserved domains of Bacillus spp. and
other Eubacterial flagellin and are provided as synthetic,
recombinant or naturally occurring forms. The flagellin bioactive
priming polypeptides of Flg22, Flg15 and FlgII-28 (Table 3) were
identified and act as potent elicitors on a wide range of crops and
vegetables to prevent and treat the spread of select disease(s)
while synergistically stimulating and promoting growth responses in
plants.
[0129] The flagellin and flagellin-associated bioactive priming
polypeptides as described herein are provided for use individually
or in combination with other bioactive priming polypeptides as
described herein, and include conserved full and partial flagellins
from Bacillus (Table 1), conserved N- and C-terminal regions from
flagellin polypeptides (Table 2), Bacillus derived Flg22 and
FlgII-28-derived bioactive priming polypeptides (Table 3) and
retro-inverso sequences that are mirror images derived from the
Bacillus Flg22 and FlgII-28 (Table 4). The underlined portion of
the sequences in Tables 1 and 3 represent identified signal anchor
sorting or secretion sequences, and signal anchoring sequences,
respectively. Other non-Bacillus derived polypeptide and proteins
are also described that are functional equivalents and can be
utilized in similar fashion (Table 5).
TABLE-US-00001 TABLE 1 Conserved flagellin sequences from Bacillus
SEQ ID NO: Full or Partial Flagellin Coding Sequence-Amino Acid
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINSASDDAAGLAIATRMKAR
SEQ ID NO: 1
EGGLNVAGRNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTKGNQASL Bacillus
thuringiensis
QKEFAQLTEQIDYIAKNTQFNDQQLLGTADKKIKIQTLDTGSTNPAQIEITLNSV strain 4Q7
KSADLGLDVQIGDEGDAESTAAADPTSAKQAIDAIDAAITTVAGQRATLGATLNR
FEFNANNLKSQETSMADAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTP QMVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINSASDDAAGLAIATRMKAR
SEQ ID NO: 2
EGGLNVAGRNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTKGNQASL Bacillus
thuringiensis,
QKEFAQLTEQIDYIAKNTQFNDQQLLGTADKKIKIQTLDTGSTNPAQIEITLNSV strain
KSADLGLDVQIGDEGDAESTAAADPTSAKQAIDAIDAAITTVAGQRATLGATLNR HD1002
FEFNANNLKSQETSMADAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTP QMVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINSASDDAAGLAIATRMKAR
SEQ ID NO: 3
EGGLNVAGRNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTKGNQASL Bacillus
thuringiensis,
QKEFAQLTEQIDYIAKNTQFNDQQLLGTADKKIKIQTLDTGSTNPAQIEITLNSV strain
HD-789 KSADLGLDVQIGDEGDAESTAAADPTSAKQAIDAIDAAITTVAGQRATLGATLNR
FEFNANNLKSQETSMADAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTP QMVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINSASDDAAGLAIATRMKAR
SEQ ID NO: 4
EGGLNVAGRNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTKGNQASL Bacillus
cereus QKEFAQLTEQIDYIAKNTQFNDQQLLGTADKKIKIQTLDTGSTNPAQIEITLNSV
strain G9842
KSADLGLDVQIGDEGDAESTAAADPTSAKQAIDAIDAAITTVAGQRATLGATLNR
FEFNANNLKSQETSMADAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTP QMVSKLLQ
Flagellin MRIGTNVLSMNARQSLYENEKHMNVAMEHLATGKKLNNASDNPANIAIVTRMHAR
SEQ ID NO: 5
ASGMRVAIRNNEDAISMLRTAEAALQTVTNILQRMRDLAVQSANGTNSNKNRHSL Bacillus
thuringiensis
NKEFQSLTEKIGYIGETTEFNDLSVFEGQNRPITLDDIGHTINMMKHIPPSPTQH
serovarindiana strain
DIKISTEQEARAAILKIEDALQSVSLHRADLGAMINRLQFNIENLNSQSMALTDA HD521
ASLIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSNSMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
6 ESGLGVAADNTQNGMSLIRTADSAMNSVSNILLRMRDIANQSANGTNTNENKSAL Bacillus
thuringiensis
QKEFAQLQKQITYIAENTQFNDKNLLNEDSEVKIQTLDSSKGEQQITIDLKAVTL strain CTC
EKLNIKDIAIGKADAADKPVTPGATVDQKDLDSVTDKIAALTETSSKADIDAIQS
SLDNFKASMTPEDVKTLEDALKGFKTGQANPADAGVDAIQDALSKVKLPTATAAA
PAADADKSDALAAIAAIDAALTKVADNRATLGATLNRLDFNVNNLKSQSSSMASA
ASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
7 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARENGLGVAANNTQDGMSLIRTAD Bacillus
SAMNSVSNILLRMRDLANQSANGTNTDDNQKALDKEFSALKEQIDYISKNTEFND
thuringiensis
KKLLNGENKTIAIQTLDNADTTKQININLADSSTSALQIDKLTISGKTTDTTKTE
serovaryunnanensis
TITVTDDEIKAAKTDIDEFNDAKKALADLKAETSAGKADGSTDDEIKTAVSNFTK strain
IEBC-T20001 SFEKIQKFMNDSDIKTVQTEIEKFDAAAPALDKAKGMGIAFTSAMDPKAGTITKA
ATRQNASDAIKSIDAALETIASNRATLGATLNRLDFNVNNLKSQSSSMAAAASQI
EDADMAKEMSEMTKFKILNEAGISMLSQANV Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
8 ENGLGVAANNTQDGMSLIRTADSALQSVSNILLRMRDLANQSANGTNTDENKAAM Bacillus
thuringiensis
EKEFGQLKDQIKYITDNTQFNDKNLLDAASGTTKSIAIQTLDSDQASTQIEIKIA serovar
tolworthi GSSLAALGLDKVQIGQETVAQKDLDVLTKAMGRLAAPDADATTRDLDVQVAKDAF
DKVKGFIADPAQAKAVERAFEDYTAAEAGKEEDAAKAIDAAYKKVTGLTAGTTGT
VDAHNAVNKIDAALKTVADNRATLGATLNRLDFNVNNLKSQSASMASAASQIEDA
DMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNSMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
9 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDIANQSANGTNTDKNQVAL Bacillus
cereus strain
QKEFGELQKQIDYIAKNTQFNDKNLLSGKAGAPDQALEINIQTLDSSDPNQQIKI FM1
SLDSVSTAQLGVKDLQIGSSSITQQQLDTLDNAMKRLETASTTAAVRDQDVADAK
AAFENVKGFFSEGNVDSINRAFTDFANETTNKDDKAEAIYALYNNATLITKPTPD
ASNPASVDPANAIKKIDQAIEKIASSRATLGATLNRLDFNVNNLKSQQSSMASAA
SQVEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNVLSMNARQSFYENEKRMNVAIEHLATGKKLNHASDNPANVAIVTRMHAR SEQ ID NO:
10 TSGIHVAIRNNEDAISMLRTAEAALQTVTNILQRMRDVAVQSANGTNSNKNRDSL Bacillus
cereus strain
NKEFQSLTEQIGYIDETTEFNDLSVFDRQNCPVTLDDIGHTVNVTKHIPPSPTQH FM1
DINISTEQEARAAIRKIEETLQNVSLHRADLGAMINQLQFNIENLNSQSTALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVYKLLQS Flagellin
MDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGMSLIRTADSALN SEQ ID NO:
11 SVSNILLRMRDIANQSANGTNTADNQQALQKEFGQLKEQISYIADNTEFNDKTLL Bacillus
thuringiensis
KADNSVKIQTLDSADTNKQISIDLKGVTLNQLGLDTVNIGSEKLSAESLNVAKAT strain MC28
MARLVKADQNADPSTFALDVNTAKESFDKIKGFIANKTNVQNVENAFNDYAVADP
ADKADKADAIQAAFNTAITGLTAGTPNTSNPSSAVDSIDAALKTVASNRATLGAT
LNRLDFNVNNLKSQSASMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQAN QTPQMVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQAKMSNSMDRLSSGKRINNASDDAAGLAIATRMRSR
SEQ ID NO: 12
EGGLNVAARNTEDGMSLIRTADSALNSVSNILLRMRDLANQSASGTNTDKNQAAM Bacillus
QKEFDQLKEQIQYIADNTEFNDKKLLDGSNSTINIQTLDSHDKNKQITISLDSAS
bombysepticus
LKNLDIKDLAIGSATINQTDLDTATNSMKRLATPATDGKVLAQDIADAKAAFNKV strain Wang
QSAYTPAEVDKIQDAFKAYDKLAADPASKATDIADAAKNVNTVFGTLATPTATKF
DPSSAVEKIDKAIETIASSRATLGATLNRLDFNVTNLKSQENSMAASASQIEDAD
MAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
13 MSNSMDRLSSGKRINNASDDAAGLAIATRMRSREGGLNVAARNTEDGMSLIRTAD Bacillus
thuringiensis
SALNSVSNILLRMRDLANQSASGTNTDKNQAAMQKEFDQLKEQIQYIADNTEFND serovar
kenyae KKLLDGSNSTINIQTLDSHDKNKQITISLDSASLKNLDIKDLAIGSATINQTDLD
TATNSMKRLATPATDGKVLAQDIADAKAAFNKVQSAYTPAEVDKIQDAFKAYDKL
AADPASKDTDIADAAKNVNTVFGTLATPTATKFDPSSAVEKIDKAIETIASSRAT
LGATLNRLDFNVTNLKSQENSMAASASQIEDADMAKEMSEMTKFKILNEAGISML
SQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNSMDRLSSGKRINNASDDAAGLAIATRMRSR SEQ ID NO:
14 EGGLNVAARNTEDGMSLIRTADSALNSVSNILLRMRDLANQSASGTNTDKNQAAM Bacillus
thuringiensis
QKEFDQLKEQIQYIADNTEFNDKKLLDGSNSTINIQALDSHDKNKQITISLDSAS serovar
kenyae LKNLDIKDLAIGSATINQTDLDTATNSMKRLATPATDGKVLAQDIADAKAAFNKV
QSAYTPAEVDKIQDAFKAYDKLAADPASKDTDIADAAKNVNTVFGTLATPTATKF
DPSSAVEKIDKAIETIASSRATLGATLNRLDFNVTNLKSQENSMAASASQIEDAD
MAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin (A-type)
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
15 ENGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTGDNQKAL Bacillus
cereus DKEFSALKEQIDYISKNTEFNDKKLLNGDNKTIAIQTLDNADTSKQININLADSS
TSALKIEKLTISGSTAIAGKTEKVTITAEDIKAAEEDIKAFTQAQEGLANLVKEV
KDTDGSVKTPGSTPDDIKKAVTAFTESFEKMKKFMNDEDITKVEEKIKAFDAASP
DLDAAKEMGTAFTAAMKPAAGEITKAAMKPNASDAIKSIDEALETIASNRATLGA
TLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQA
NQTPQMVSKLLQ Flagellin (A-type)
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNNASDNPANIAIVTRMHAR SEQ ID NO:
16 ASGMRLAIRNNEDTISMLRTAEAALQTLTNILQRMRDLAVQSANGTNSNKNRDSL Bacillus
cereus NKEFQSLTEQIGYIGETTEFNDLSVFDGQNRPVTLDDIDHTINMTKHIPPSPTQH
DIKISTEQEARAAILKIEEALQSVSIHRADLGSMINRLQFNIENLNSQSMALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANVAIVTRMHAR SEQ ID NO:
17 ASGMRVAIRNNEDAISMLRTAEAALQTVTNVLQRMRDVAVQSANGTNLNKNRDSL Bacillus
thuringiensis
NNEFQSLTEQIGYIDETTAFNDLSVFDGQNRPVTLDDIGHTVNVTKHISPSPTQH serovar
finitimus DINISTEQEARAAIRKIEEALQNVSLYRADLGAMINRLQFNIENLNSQSTALTDA
strain YBT-020 ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVYKLLQS
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR
SEQ ID NO: 18
ESGLNVAADNTQNGMSLIRTADSAMNSVSNILLRMRDIANQSANGTNTDSNKSAL Bacillus
thuringiensis
QKEFAELQKQITYIADNTQFNDKNLLKEDSEVKIQTLDSSKGEQQIGIDLKAVTL serovar
finitimus EKLGINNISIGKADGTTEGTKADLTALQAAAKKLEKPDTGTMEKDVKDAKEEFDK
strain YBT-020
VKASLSDEDVKKIEAAFGEFDKDKTNTTKASDIFNAIKDVKLADKAAAAPAPADL
TKFKAALDKLQTPNAGTMVDDVKDAKDEFEKIKGSLSDADAQKIQAAFEEFEKAN
TDDSKASAIYNLAKDVKVNATDTTTGTDKDTTTSTDKDAALAAIAAIDAALTKVA
DNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEA
GISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
19 ESGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTAENKAAM Bacillus
cereus QKEFGELKDQIKYISENTQFNDQHLLNAAKGSTNEIAIQTLDSDSSSKQIKITLQ
stain B4264 GASLDSLDIKDLQIGSGSTVSQTDLDVLDATMTRVKTATGATRDVDVQAAKSAFD
KVKGLMTKPAEVKAIERAFEDYNAGKTDALATAIEAAYTANKTGLPAPAAAAGTV
DALGAITKIDAALKTVADNRATLGATLNRLDFNVNNLKSQSASMASAASQIEDAD
MAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
20 ESGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTSDNQKAL Bacillus
thuringiensis
DKEFSALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTTKQININLADSS serovar
nigeriensis TTALNIDKLSIEGTGNKTITLTAADIAKDKANIDAVGTAKTALAGLTGTPAAAAI
NSAVADFKTAFAKADKNLMSDAQIKAVTDAITAFEADATPDLTKAKAIGTAYTAP
AAGDITKASPNASEAIKSIDAALDTIASNRATLGATLNRLDFNVNNLKSQSSSMA
SAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
21 ESGLGVAANNTQDGMSURTADSALNSVSNILLRMRDIANQSANGTNTADNQQALQ Bacillus
thuringiensis
KEFGQLKEQISYIADNTEFNDKTLLKADNSVKIQTLDSADTNKQISIDLKGVTLN
QLGLDTVNIGSETLSAESLNVAKATMARLVKADQNADPSTFALDVNTAKESFDKI
KGFITNKTNVQNVENAFNDYTVADPADKADKADAIQAAFNTAITGLTAGTPNTSN
PSSAVDAIDAALKTVASNRATLGATLNRLDFNVNNLKSQSASMASAASQIEDADM
AKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNVLSMNARQSLYENEKRMNVAMEHFATGKKLNHASDNPANVAIVTRMHAR SEQ ID NO:
22 ASGMRVAIRNNEDAISMLRTAEAALQTVMNILQRMRDLAVQSANGTNSNKNRDSL Bacillus
thuringiensis
NKEFQSLTEQIGYIGETTEFNDLSVFDGQNRPVTLDDIGHTVNVTKHTSPSPTKH serovar
konkukian DIKISTEQEARAAIRKIEEALQNVSLHRADFGAMINRLQFNIENLNSQSMALTDA
strain 97-27 ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR
SEQ ID NO: 23
ESGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTADNQQAL Bacillus
thuringiensis
QKEFGQLKEQISYIADNTEFNDKTLLKADNSVKIQTLDSADTNKQISIDLKGVTL serovar
konkukian NQLGLDTVNIGSETLSAESLNVAKATMARLVKADQNADPSTFALDVNTAKESFDK
strain 97-27
IKGFITNKTNVQNVENAFNDYTVADPADKADKADAIQAAFNTAITGLTAGTPNTS
NPSSAVDAIDAALKTVASNRATLGATLNRLDFNVNNLKSQSASMASAASQIEDAD
MAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin protein FlaA
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANIVIVTRMYAR SEQ ID NO:
24 ASGMRVAIRNNEDAISMLRTAEAALQTVTNILQHMRDFAIQSANGTNSNTNRDSL Bacillus
thuringiensis
NKEFQSLTEPIGYIGETTEFNDLSVFDGQNRPITLDDIGHTINMTKHIPPSPTQH serovar
thuringiensis
DIKISTEQEARAAIRKIEEALQNVSLHRADLGSMINRLQFNIENLNSQSMALIDT strain
IS5056 ASQVEDADMAQEISDFLKFKLLTAVALSVVSQANQIPQIVSKLLQS Flagellin
protein FlaA
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
25 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDISNQSANGTNTDKNQSAL Bacillus
thuringiensis
DKEFAALKDQIDYISKNTEFNDQKLLDGSKKSIAIQTLDNADTNKQIDIQLSNVS serovar
thuringiensis
TKELKLDTLSIEGSSSKTFTITADDMLAVGTANATAKAKAGTLKGLNVTTGDLTA strain
IS5056 AKTDVQDFRAAFDKVKGFMGSTEVTNIEKALTKFDGDQSLANAKAIGDALTSDLA
TTIAKDQTYSKNVSNASSAIASIDAALESIASNRATLGATLNRLDFNVNNLKSQS
SSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin B
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
26 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDISNQSANGTNTDKNQSAL Bacillus
thuringiensis
DKEFAALKDQIDYISKNTEFNDQKLLDGSKKSIAIQTLDNADTNKQIDIQLSNVS strain
Bt407 TKELKLDTLSIEGSSSKTFTITADDMLAVGTANATAKAKAGTLKGLNVTTGDLTA
AKTDVQDFRAAFDKVKGFMGSTEVTNIEKALTKFDGDQSLANAKAIGDALTSDLA
TTIAKDQTYSKNVSNASSAIASIDAALESIASNRATLGATLNRLDFNVNNLKSQS
SSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
27 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDISNQSANGTNTDKNQSAL Bacillus
thuringiensis
DKEFAALKDQIDYISKNTEFNDQKLLDGSKKSIAIQTLDNADTNKQIDIQLSNVS serovar
chinensis CT-
TKELKLDTLSIEGSSSKTFTITADDMLAVGTANATAKAKAGTLKGLNVTTGDLTA 43
AKTDVQDFRAAFDKVKGFMGSTEVTNIEKALTKFDGDQSLANAKAIGDALTSDLA
TTIAKDQTYSKNVSNASSAIASIDAALESIASNRATLGATLNRLDFNVNNLKSQS
SSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
28 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGISLIRTAD Bacillus
thuringiensis
SAMNSVSNILLRMRDLANQSANGTNTNENQAALNKEFDALKEQIDYISTNTEFND serovar
canadensis KKLLDGSNKTIAVQTLDNADTSKQININLSNVSTKELGLDTLSIGTDKVEKTVYD
ATTKAFADLGAKTGADKAAFDADVTAAMKEFDKVKPFMSADDVKKIETKLEDYNK
ANDAGAQTAAQALGKEFATLTKLETTDLKANASGAIASIDTALKNIASNRATLGA
TLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQA
NQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
29 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGISLIRTAD Bacillus
thuringiensis
SAMNSVSNILLRMRDLANQSANGTNTNENQAALNKEFDALKEQIDYISTNTEFND serovar
galleriae KKLLDGSNKTIAVQTLDNADTSKQININLSNVSTKELGLSTLSIGTDKVEKTVYD
ATTKAFADLGAKTGTDKAAFAADVTAAMKEFDKVKPFMSADDVKKIETKLEDYNK
ANDAGAEAAAQALGKEFATLTKLETTDLKANASGAIASIDTALKNIASNRATLGA
TLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQA
NQTPQMVSKLLQ Flagellin N-terminal
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR helical
region ESGLSVAANNTQDGMSLIRTADSAMNSVSNILLRMRDLSNQSANGTNTDENQQAL SEQ
ID NO: 30 NKEFAALKDQIDYISKNTEFNDKKLLDGSNKSIAIQTLDNADTTKQINIDLSNVS
Bacillus TDTLNISGLTINGKKDITVTISDKDIANAATDIGKATSAQQGLADLTDTTPAVPD
weihenstephanensis
TPAVIGTGTAGNPQFPAVKGTPEIPGSSPAEIAKAVDDFKQAFNKVKGLMSDSAV
SAMEQKFATFEKDKSLANAKDIGTAFSAPIAGNITKGEQNASGAIKSIDAALEKI
ASNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNE
AGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
31 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGMSLIRTAD Bacillus
thuringiensis
SALNSVSNILLRMRDIANQSANGTNTGDNQKALDKEFSALKEQIDYISKNTEFND serovar
ostriniae KKLLNGDNKSIAIQTLDNADTAKQININLADSSTKALNIDTLSIAGTTDKTITIT
AKDLTDNKTTLDALKTAKDDLAKLDDKSDQATIDKAVDAFKTAFNNVDKNLLSDK
AIEGITEKMTAFDGTHTAAAAIGAAYTEPTAADIKKSAPNASGAIKSIDAALETI
ASNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNE
AGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANVAIVTRMHAR SEQ ID NO:
32 ASGMRVAIRNNEDALSMLRTAEATLQTVANILQRMRDLAVQSSNDTNSNKNRDSL Bacillus
thuringiensis
NKEFQSLTEQISYIGETTEFNDLSVFDGQNRPVTLDDIGHTVNVTKHISPSPTQH
DIKISTEQEARAAIRKIEEALQNVLLHRADLGAMINRLQFNIENLNSQSMALTDA
ASRIEDADMAQEMSDFLKFKLLSEVALSMVSQANQIPQMVSELLQS Flagellin
MRINTNINSMRTQEYMRQNQTKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
33 ENGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDLANQSANGTNTDDNQKAL Bacillus
thuringiensis
DKEFSALKEQIDYISKNTEFNDKKLLNGENKTIAIQTLDNADTTKQININLADSS
TSALQIDKLTISGKTTDTTKTQTITVTDDEIKAAKTDIDEFNDAKKALADLKAES
APSKGDGSSDDEIKEAVSNFKKSFEKIQKFMNDSDIKTVQTEIEKFDAAAPALDK
AKGMGIAFTSAMDPKAGTITKAATRQNASDAIKSIDAALETIASNRATLGATLNR
LDFNVNNLKSQSSSMAAAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTP QMVSKLLQ
Flagellin MTGITINLEIDFFAYYRFSICRKVNIKKWGFLIMRINTNINSMRTQEYMRQNQTK
SEQ ID NO: 34
MSNAMDRLSSGKRINNASDDAAGLAIATRMRARENGLGVAANNTQDGMSLIRTAD Bacillus
thuringiensis
SAMNSVSNILLRMRDLANQSANGTNTDDNQKALDKEFSALKEQIDYISKNTEFND serovar
KKLLNGENKTIAIQTLDNADTTKQININLADSSTSALQIDKLTISGKTTDTTKTQ
pondicheriensis
TITVTDDEIKAAKTDIDEFNDAKKALADLKAESAPSKGDGSSDDEIKEAVSNFKK
SFEKIQKFMNDSDIKTVQTEIEKFDAAAPALDKAKGMGIAFTSAMDPKAGTITKA
ATRQNASDAIKSIDAALETIASNRATLGATLNRLDFNVNNLKSQSSSMAAAASQI
EDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin B
MSIMRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANIVIVTRM SEQ ID NO:
35 YARASGMRVAIRNNEDAISMLRTAEAALQTVTNILQHMRDFAIQSANGTNSNTNR Bacillus
thuringiensis
DSLNKEFQSLTEPIGYIGETTEFNDLSVFDGQNRPITLDDIGHTINMTKHIPPSP serovar
Berliner TQHDIKISTEQEARAAIRKIEEALQNVSLHRADLGSMINRLQFNIENLNSQSMAL
IDTASQVEDADMAQEISDFLKFKLLTAVALSVVSQANQIPQIVSKLLQ S Flagellin A
MARITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQDYMRQNQAK SEQ ID NO:
36 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGMSLIRTAD Bacillus
thuringiensis
SAMNSVSNILLRMRDISNQSANGTNTDKNQSALDKEFAALKDQIDYISKNTEFND serovar
Berliner QKLLDGSKKSIAIQTLDNADTNKQIDIQLSNVSTKELKLDTLSIEGSSSKTFTIT
ADDMLAVGTANATAKAKAGTLKGLNVTTGDLTAAKTDVQDFRAAFDKVKGFMGST
EVTNIEKALTKFDGDQSLANAKAIGDALTSDLATTIAKDQTYSKNVSNASSAIAS
IDAALESIASNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEM
TKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASNNPANVAIVTRMHAR SEQ ID NO:
37 ASGMRVAIRNNEDAISMLRTAEAALQTVTNVLQRMRDVAVQSANGTNSSKNRDSL Bacillus
cereus strain
NKEFQSLTEQIGYIDETTEFNDLSVFDGQNRTVTLDDIGHTVNVTKHIPPSPTQH Q1
DINISTEQEARAAIRKIEEALQNVSLHRADLGAMINRLQFNIENLNSQSTALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
38 ESGLSVAADNTQNGMSLIRTADSAMNSVSNILLRMRDIANQSANGTNTDKNQVAL Bacillus
cereus strain
QKEFAALKEQITYIADNTQFNDKNLLNGNQTINIQTLDSHDSTKQIGIDLKSATL Q1
EALGIKDLTVGAVGSTEAKNYVDAKEALAKNVAANEFIDAKKALDGNAIAKGYVE
AKTAFDDAKPEVKALVSNYTDALAALAKDDTNDDLKKDVADTKALMDANTVAKTY
FEAKTAHDGADQAIKDIVTTYDSKLGALDDAANKAISDFDKAKAAFDESPAAKEL
VKTMDDAKQAATQNNTANAYLVAKAAAELAPNDADKKAELENATKALEKDDTAKG
LVKTYENAKEALNPANAMPLDAVKQIDAALKTVADNRATLGATLNRLDFNVNNLK
SQSSAMAASASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNFLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANIAIVTRMHAR SEQ ID NO:
39 ANGMRVAIRNNEDAISMLRTAEAALQTVMNILQRMRDLAIQSANSTNSNKNRDSL Bacillus
thuringiensis
NKEFQSLTEQISYIGETTEFNDLSVFDGQNRPVTLDDIGHTVHISKSIPPPSPTQ serovar
morrisoni HDIKISTEQEARAAILKIEEALQSVSLHRADLGAMINRLHFNIENLNSQSMALTD
AASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQTKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
40 ENGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTSDNQKAL Bacillus
thuringiensis
DKEFSALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTTKQININLADSS serovar
neoleonensis
TSALNIDKLSIEGTGNKTITLTAADIAKDKTNIDAVGTAKTALAGLTGTPAAAAI
NSAVADFKTAFAKADKNLMSDAQIKSVTDAITAFEADATPDLTKAKAIGTAYTAP
AAGDITKASPNASEAIKSIDAALDTIASNRATLGATLNRLDFNVNNLKSQSSSMA
SAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLNMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
41 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGMSLIRTAD Bacillus
thuringiensis
SALNSVSNILLRMRDIANQSANGTNTGDNQKALDKEFSALKEQIDYISKNTEFND serovar
morrisoni KKLLNGDNKSIAIQTLDNADTAKQININLADSSTKALNIDTLSIAGTTDKTITIT
AKDLTDNKATLDALKTAKADLAKLDDKSDQATIDKAVDAFKTAFNNVDKNLLSDK
AIEGITDKMTAFDGTHTAAAAIGTAYTEPTAGDITKSAPNASGAIKSIDAALETI
ASNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNE
AGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
42 ESGLGVAANNTQDGMSURTADSALNSVSNILLRMRDIANQSANGTNTGDNQKALD Bacillus
thuringiensis
KEFSALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTAKQININLADSST serovar
morrisoni KALNIDTLSIAGTTDKTITITAKDLTDNKATLDALKTAKADLAKLDDKSDQATID
KAVDAFKTAFNNVDKNLLSDKAIEGITDKMTAFDGTHTAAAAIGTAYTEPTAGDI
TKSAPNASGAIKSIDAALETIASNRATLGATLNRLDFNVNNLKSQSSSMASAASQ
IEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
43 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDIANQSANGTNTNGNQAAL Bacillus
thuringiensis
NKEFDALKQQINYISTNTEFNDKKLLDGSNKTIAIQTLDNADTSKKIDIQLADVS serovar
jegathesan TKSLNIDKLKIGGVSKETTDAVGDTFTKLSTTATTDMGALKIEVEAAMKEFDKVK
GAMSAEDAKAVTDKLDAFNTAAAATNDAATIAAAKALGAAFDKTKVEMADPNASV
AAIDSALENIASNRATLGATLNRLDFNVNNLKSQQSSMASAASQIEDADMAKEMS
EMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
44 ESGLGVAANNTQDGMALIRTADSAMNSVSNILLRMRDIANQSANGTNTDKNQAAL Bacillus
cereus stain
QKEFGELQKQIDYIAGNTQFNDKNLLDGSNPSISIQTLDSADQSKQISIDLKSAT ATCC 10987
LEALGIKDLTVGATENTLAKATITAKDAFDAAKDASDAAKKEIDAAAKDTPSKND
AQLAKEYIEAKATLATLKPTDATYAAKAAELDAATTALNDNAKVLVDGYEKKLTT
TKTKEAEYTAAKEQSTKSTAAADLVTKYETAKSNALGNDIAKEYLEAKTAYEANK
NDISSKSRFEAAETELNKDITANKAAKVLVETYEKAKTAGTTEKSLVAVDKIDEA
LKTIADNRATLGATLNRLDFNVNNLKSQSASMASAASQIEDADMAKEMSEMTKFK
ILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MTGITINLEIDFFAYYRFSICRKVNIKKWGFLIMRINTNINSMRTQEYMRQNQAK SEQ ID NO:
45 MSNAMDRLSSGKRINNASDDAAGLAIATRMRARESGLGVAANNTQDGMSLIRTAD Bacillus
thuringiensis
SAMNSVSNILLRMRDLANQSANGTNTNENQAALNKEFDALKEQINYISTNTEFND serovar
monterrey KKLLDGSNKTIAIQTLDNADTSKKIDIKLADVSTESLKIDKLKIGGVSKETTDAV
SETFTKLSTTKTTDKDALKAEVEAAMKEFDKVKGAMSTEDAKAVTDKLGLFNTAA
AGTDDTAIATAAKNLGAAFDKTKVNMADPNASVAAIDSALENIASNRATLGATLN
RLDFNVNNLKSQQSSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQT PQMVSKLLQ
Flagellin MRIGTNVLSLNARQSLYENEKRMNVAMEHLATGKKLNNASDNPANIAIVTRMHAR
SEQ ID NO: 46
ASSMRVAIRNNEDAISMLRTAEAALQTVTNVLQRMRDLAVQSANDTNSNKNRDSL Bacillus
cereus strain
NKEFQSLTEQIGYIDETTDFNDLSVFDGQNRTVTLDDIGHTVNVTKHIPPSPTQH NC7401
DINISTEQEARAAIRKIEEALQNVSLHRADLGAMINRLQFNIENLNSQSTALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
47 ESGLGVASNNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTNENKAAM Bacillus
cereus strain
QKEFGELKEQIKYIAENTQFNDQHLLNADKGITKEIAIQTLDSDSDSKQIKIKLQ NC7401
GSSLEALDIKDLQIGNTELAQKDLDLLNATMDRLDATVPGTRDVDVQAAKDAFDK
VKGFYTNSDSVKAIERAFEDYATASTAGTAKADAATAIKAAFDLAANKVGKPATG
GAQGSANSLGAITKIDAALKTVADNRATLGATLNRLDFNVNNLKSQASSMAAAAS
QVEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin (A-type)
MRINTNINSLRTQEYMRQNQAKMSNSMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
48 ESGLNVAANNTQDGMSLIRTADSALGSVSNILLRMRDLANQSANGTNTSDNQAAM Bacillus
cereus strain
QKEFAELQKQITYIADNTQFNDKNLLQSNSSINIQTLDSSDGNQQIGIELKSASL AH820
KSLGIEDLAIGASVNPLAKATVEASEAYDKAKADTAAFAKSIADTAATGTGAAKA
DAAAVDAYIKEADPTAKGNLYTGLTADQKKLADEHNTLKAAEDGKKAELTMATTK
STADGTAKGLVDAYDNAKSDAMNDPKAKAYLEAKMAYEKDTSNVANKQKLDSTKE
AMEKDPASKDLVVKLDAAKAAATNGTPLDAVSKIDAALKTVADNRATLGATLNRL
DFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQ MVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR
SEQ ID NO: 49
ESGLGVASNNTQDGMSLIRTADSALNSVSNILLRMRDLANQSANGTNTNENKAAM Bacillus
cereus AFI187
QKEFGELKEQIKYIAENTQFNDQHLLNADKGITKEIAIQTLDSDSDSKQIKIKLQ
GSSLEALDIKDLQIGNTELAQKDLDLLNATMDRLDATVPGTRDVDVQAAKDAFDK
VKGFYTNSDSVKAIERAFEDYATASTAGTAKADAATAIKAAFDLAANKVGKPATG
GAQGSANSLGAITKIDAALKTVADNRATLGATLNRLDFNVNNLKSQASSMAAAAS
QVEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MDFFAYYRFSICRKVNIKKWGFFYMRINTNINSMRTQEYMRQNQAKMSNAMDRLS SEQ ID NO:
50 SGKRINNASDDAAGLAIATRMRARESGLGVASNNTQDGMSLIRTADSALNSVSNI Bacillus
cereus LLRMRDLANQSANGTNTNENKAAMQKEFGELKEQIKYIAENTQFNDQHLLNADKG
ITKEIAIQTLDSDSDSKQIKIKLQGSSLEALDIKDLQIGNTELAQKDLDLLNATM
DRLDATVPGTRDVDVQAAKDAFDKVKGFYTNSDSVKAIERAFEDYATASTAGTAK
ADAATAIKAAFDLAANKVGKPATGGAQGSANSLGAITKIDAALKTVADNRATLGA
TLNRLDFNVNNLKSQASSMAAAASQVEDADMAKEMSEMTKFKILNEAGISMLSQA
NQTPQMVSKLLQ Flagellin protein Fla
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
51 ESGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTGDNQKAL Bacillus
cereus DKEFSALKEQIDYISKNTEFNDKKLLNGENTSIAIQTLDSADTAKQININLADSS
TSALLIDKLSISGAGAGTALAGVATADINAAGTKQAALSGLTGSKTTDELDDAVK
EFKTEFDKVKSGLSAENADKITAAMDKYTNNKTLDNAKAIGDLYKTMAPADSTVV
GTAGTKGQALIDLNATATGDTAQKRQVAVDAFKDDFDKIKGGLNAQDAAKVTAAL
DKFNKADGSGNTLENAQEIGKVFAEVAAGSTKSNASDAIKSIDKALETIASNRAT
LGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAKEMSEMTKFKILNEAGISML
SQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQTKMSNAMDRLSSGKRINNASDDAAGLAIATRMRSR SEQ ID NO:
52 EGGLNVAARNTEDGMSLIRTADSALNSVSNILLRMRDLANQSASETNTSKNQAAM Bacillus
thuringiensis
QKEFDQLKEQIQYIADNTEFNDKKLLDGSNSTINIQTLDSHDKNKQITISLDSAS Strain
HD-771 LKNLDITDLAIGSNTVNKNDLDTLNNSMKRLETAAADAAVQAQDVTDAKNAFNKV [51]
KSGYTPAEVEKMEDAFKAYDKVVADPAKTDALLKAAAEKINTEFKTLTAPTATAF
DPSSSVEKIDKAIETIASSRATLGATLNRLDFNVTNLKSQENSMAASASQIEDAD
MAKEMSEIVITKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQTKMSNAMDRLSSGKRINNASDDAAGLAIATRMRSR SEQ ID NO:
53 EGGLNVAARNTEDGMSLIRTADSALNSVSNILLRMRDLANQSASETNTSKNQAAM Bacillus
thuringiensis
QKEFDQLKEQIQYIADNTEFNDKKLLDGSNSTINIQTLDSHDKNKQITISLDSAS serovar
sotto LKNLDITDLAIGSNTVNKNDLDTLNNSMKRLETAAADAAVQAQDVTDAKNAFNKV [52]
KSGYTPAEVEKMEDAFKAYDKVVADPAKTDALLKAAAEKINTEFKTLTAPTATAF
DPSSSVEKIDKAIETIASSRATLGATLNRLDFNVTNLKSQENSMAASASQIEDAD
MAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MGVLNMRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIAT SEQ ID NO:
54 RMRARENGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTGD Bacillus
thuringiensis
NQKALDKEFSALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTSKQINID serovar
Novosibirsk LANTSTSSLKIDKLSIEGKGNQTIAITAADIAKDTNIAALTSAQGKLAALTGTPA
PAALTTAVDEFKAAFEKVDKNLMSDTQITGIENAIKAYDGATTKTLALAQAVGTA
YTAPTPGDITKELPNASSSIKSIDAALETIASNRATLGATLNRLDFNVNNLKSQA
SSMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MGVLNMRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIAT SEQ ID NO:
55 RMRARESGLGVAANNTQDGISLIRTADSAMNSVSNILLRMRDLANQSANGTNTSE
Bacillus thuringiensis
NQAALDKEFGALKEQINYISTNTEFNDKKLLDGSNETIAIQTLDNADEGKKIDIK serovar
londrina LANVSTDSLKIDKLTIGGAAQKTVDAVADKFNALKTTTTTDKAAIQTEVDAVMKE
FDKVKGSMSAEDAKVITDKLKDYNDAADTDTAKATAAKDLGAAFDKTKVNIANPN
AAVAAIDSALENIASNRATLGATLNRLDFNVNNLKSQSSSMASAASQIEDADMAK
EMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNVLSMNARQSLYENEKRMNVAMEHLATGKKLNHASNNPANIAIVTRMHAR SEQ ID NO:
56 ASGMRVAIRNNEDALSMLRTAEAALQTVTNILQRMRDLAVQSANVTNSNKNRNSL Bacillus
cereus strain
NKEFQSLTEQISYIGETTEFNDLSVFDGQNRPVTLDDIGYTVNVTKHTPPSPTQH E33L
DIKISTEQEARAAIRKIEEALQNVSLHRADLGSMMNRLQFNIENLNSQSMALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSTAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
57 ESGLGVAANNTQDGISLIRTADSAMNSVSNILLRMRDLANQSANGTNTDKNQGAL Bacillus
cereus strain
DKEFAALKEQIDYISKNTEFNDKKLLDGSNKAIAIQTLDSDDKGKQIDISLSDTS E33L
TTALKINNLSIAANGLGIGSGKELVGVADNTIANASAEALKKLDGTTGDTDVKRS
NAVKAFTDQYKDLKVAMNAKDVETIDAAIKKFEGANTLENAQAIGAAFEGAAKAT
LTTDINNATLTSKALSDLDTDSTTETRKAAMKDFVAAFDKVKGSMNSSDVTKISD
AIDRFSKTDDSGNTLEAARAIGDAFKAATTNGKTSTATDANSAIKAIDEALETIA
SNRATLGATLNRLDFNVNNLKNQASSMASAASQVEDADMAKEMSEMTKFKILNEA
GISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSTAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
58 ESGLGVAANNTQDGISLIRTADSAMNSVSNILLRMRDLANQSANGTNTDKNQAAL Bacillus
cereus DKEFNALKEQIDYISKNTEFNDKKLLDGSNKSIAVQTLDNADTSKQININLSNTS
strain FRI-35
TKALEINSLTISGTTPIAGKNETSKITAEQMTAASDALEKFKTAQEGLANLTEPT
KGSDGKPEAGTGSSNEDIVKAVKAFKEAFKNIQPLMSDTDITTVQNKIDLFDEDA
PDLSAAKLIGTTFEESMKPVADKEITKAAVKPNASDAIAAIDAALTKVADNRATL
GATLNRLDFNVNNLKSQASSMASAASQVEDADMAKEMSEMTKFKILNEAGISMLS
QANQTPQMVSKLLQ Flagellin
MRIGTNVLSLNARQSLYENEKRMNVAMEHLATGKKLNNASDNPANIAIVTRMHAR SEQ ID NO:
59 ASGMRVAIRNNEDAISMLRTAEAALQTVTNVLQRMRDLAVQSANGTNSNKNRDSL Bacillus
cereus NKEFQSLTEQIGYIDETTEFNNLSVFDGQNRPVTLDDIGHTVNVTKHIPPFPTQH
strain FRI-35
DINISTEQEARAAIRKIEEALQNVSLHRADLGAMINRLQFNIENLNSQSTALTDA
ASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQVPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAH SEQ ID NO:
60 ESGLSVAARNTSDGISLIRTADSALQSVSNILLRMRDIANQTANGTNKDTDIEAL Bacillus
thuringiensis
GKEFAALKEQITYVSDNTKFNGRELLKGGDDINIQTYDGSDESQQIKIKISELDL
SSLDTGEVTDSDTARGTVSTLDDAITNIASKRAELGATLNRLDYNTQNVNSEAAS
MAASASQIEDADMAKEMSEMTKFKILSEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAH SEQ ID NO:
61 ESGLSVAARNTSDGISLIRTADSALQSVSNILLRMRDIANQTANGTNKDTDIEAL Bacillus
cereus strain
GKEFAALKEQITYVSDNTKFNGRELLKGGDDINIQTYDGSDESQQIKIKISELDL ATCC 4342
SSLDTGEVTDSDTARGTVSTLDDAITNIASKRAELGATLNRLDYNTQNVNSEAAS
MAASASQIEDADMAKEMSEMTKFKILSEAGISMLSQANQTPQMVSKLLQ Flagellin
MRIGTNFLSMNARQSLYENEKRMNVAMEHLATGKKLNHASDNPANIAIVTRMHAR SEQ ID NO:
62 ANGMRVAIRNNEDAISMLRTAEAALQTVMNILQRMRDLAIQSANSTNSNKNRDSL Bacillus
thuringiensis
NKEFQSLTEQISYIGETTEFNDLSVFDGQNRPVTLDDIGHTVHISKSIPPPSPTQ
HDIKISTEQEARAAILKIEEALQSVSLHRADLGAMINRLHFNIENLNSQSMALTD
AASRIEDADMAQEMSDFLKFKLLTEVALSMVSQANQIPQMVSKLLQS Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
63 ESGLGVAANNTQDGMSLIRTADSALNSVSNILLRMRDIANQSANGTNTGDNQKAL Bacillus
thuringiensis
DKEFSALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTAKQININLADSS
TKALNIDTLSIAGTTDKTITITAKDLTDNKATLDALKTAKADLAKLDDKSDQATI
DKAVDAFKTAFNNVDKNLLSDKAIEGITDKMTAFDGTHTAAAAIGTAYTEPTAGD
ITKSAPNASGAIKSIDAALETIASNRATLGATLNRLDFNVNNLKSQSSSMASAAS
QIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINHNITALNTYRQFNNANNAQAKSMEKLSSGQRINSASDDAAGLAISEKMRGQ SEQ ID NO:
64 IRGLDQASRNAQDGVSLIQTAEGALNETHDILQRMRELVVQAGNGTNKTEDLDAI Bacillus
aryabhattai QDEIGSLIEEIGGETDSKGISDRAQFNGRNLLDGSLDITLQVGANAGQQVNLKIG
DMSAGALGADTDSDGAADAFVNSINVKDFATTSFDDQLAIIDGAINQVSEQRSGL
GATQNRLDHTINNLSTSSENLTASESRIRDVDYALAA Flagellin
MRINTNINSMRTQEYMRQNQDKMNTSMNRLSSGKQINSASDDAAGLAIATRMRAK SEQ ID NO:
65 EGGLNVGAKNTQDGMSALRTMDSALNSVSNILLRMRDLATQSATGTNQGNDRESL Bacillus
manliponensis
DLEFQQLTEEITHIAEKTNFNGNALLSGSGSAINVQLSDAAEDKLTIAAIDATAS
TLLKGAVDVKTEDKADAAITKIDQAIQDIADNRATYGSQLNRLDHNLNNVNSQAT
NMAAAASQIEDADMAKEMSEMTKFKILSEAGVSMLSQANQTPQMVSKLLQ Flagellin
MRIGSWTATGMSIVNHMNRNWNAASKSMLRLSSGYRINSAADDAAGLAISEKMRG SEQ ID NO:
66 QIRGLTMASKNIMDGVSLIQTAEGALNETHAIVQRMRELAVQAATDTNTDDDRAK
Lysinibacillus sp.
LDLEFQELKKEIDRISTDTEFNTRTLLNGDYKDNGLKIQVGANSGQAIEVKIGDA strain
GLAGIGLSTESIATREGANAALGKLDEATKNVSMERSRLGAYQNRLEHAYNVAEN BF-4
TAINLQDAESRIRDVDIAKEMMNMVKSQILAQVGQQVLAMHMQQAQGILRLLG Flagellin
MKIGSWTATGMSIVNHMNRNWNAASKSMLRLSSGYRINSAADDAAGLAISEKMRG SEQ ID NO:
67 QIRGLTMASKNIMDGVSLIQTAEGALNETHAIVQRMRELAVQAATDTNTDDDRAK
Lysinibacillus sp.
LDLEFQELKKEIDRISTDTAFNTRTLLNGDYKDNGLKIQVGANSGQAIEVKIGDA strain
GLAGIGLSTESIATREGANAALGKLDEATKNVSMERSRLGAYQNRLEHAYNVAEN 13S34_air
TAINLQDAESRIRDVDIAKEMMHMVKSQILAQVGQQVLAMHIQQAQGILRLLG Flagellin
MIISHNLTALNTMNKLKQKDLAVSKSLGKLSSGLRINGASDDAAGLAISEKMRGQ SEQ ID NO:
68 IRGLNQASRNIQDGISLIQVADGAMQEIHSMLQRMNELAVQASNGTYSGSDRLNI
Paenibacillus sp.
QSEVEQLIEEIDEIAGNTGFNGIKLLNGNNEKTEKTEKTGSVVSVNNPPNNKLIT strain
HW567 ISSPVGTSVSEILNNLLTVFNEAKNGQVGDSDSKRVSSKFTLSINNDELSIVCDT
GDGFLLSGGSPNLFYQGYIGGSYKYKFTEFINENDFINIMDIGGANGGDTLKFNF
SSISKEPEEQKEQKGLTLQIGANSGETLNIKLPNVTTSAIGISSIDVSTIPNAES
SLSSISAAIDKVSAERARMGAYQNRLEHSRNNVVTYAENLTAAESRIRDVDMAKE
MMELMKNQIFTQAGQAMLLQTNTQPQAILQLLK Flagellin
MRINTNINSMRTQEYMRQNQAKMSNAMDRLSSGKRINNASDDAAGLAIATRMRAR SEQ ID NO:
69 ESGLGVAANNTQDGMSLIRTADSAMNSVSNILLRMRDLANQSANGTNTKENQDAL Bacillus
anthracis DKEFGALKEQIDYISKNTEFNDKKLLNGDNKSIAIQTLDNADTAKQININLADSS
TKALNIDSLTISGSKDATITITAEDITAASAEITAAKGARTALANLKDTPADPTK
DPAASTPAEIKAAVDDFKGKFEKIKGLMNDTDVKAVEEKIKEFETTSTLAKAQAI
GTAFTTGMEPKAGNITKNVPAASSSIKAIDSALETIASNRATLGATLNRLDFNVN
NLKSQSSAMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKL LQ
Flagellin MQKSQYKKMGVLKMRINTNINSMRTQEYMRQNQDKMNVSMNRLSSGKRINSAADD
SEQ ID NO: 70
AAGLAIATRMRARQSGLEKASQNTQDGMSLIRTAESAMNSVSNILTRMRDIAVQS Bacillus
anthracis SNGTNTAENQSALQKEFAELQEQIDYIAKNTEFNDKNLLAGTGAVTIGSTSISGA
EISIETLDSSATNQQITIKLANTTAEKLGIDATTSNISISGAASALAAISALNTA
LNTVAGNRATLGATLNRLDRNVENLNNQATNMASAASQIEDADMAKEMSEMTKFK
ILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINTNINSMRTQEYMRQNQDKMNVSMNRLSSGKRINSAADDAAGLAIATRMRAR SEQ ID NO:
71 QSGLEKASQNTQDGMSURTAESAMNSVSNILTRMRDIAVQSSNGTNTAENQSALQ Bacillus
anthracis KEFAELQEQIDYIAKNTEFNDKNLLAGTGAVTIGSTSISGAEISIETLDSSATNQ
QITIKLANTTAEKLGIDATTSNISISGAASALAAISALNTALNTVAGNRATLGAT
LNRLDRNVENLNNQATNMASAASQIKDADKAKEMSEMTKFKILNEAGISMLSQAN QTPQMVSKLLQ
Flagellin MRINTNINSMRTQEYMRQNQDKMNVSMNRLSSGKRINSAADDAAGLAIATRMRAR
SEQ ID NO: 72
QSGLEKASQNTQDGMSLIRTAESAMNSVSNILTRMRDIAVQSSNGTNTAENQSAL Bacillus
anthracis QKEFAELQEQIDYIAKNTEFNDKNLLAGTGAVTIGSTSISGAEISIETLDSSATN
QQITIKLANTTAEKLGIDATTSNISISGAASALAAISALNTALNTVAGNRATLGA
TLNRLDRNVENLNNQATNMASAASQIEDADMAKEMSEMTKFKILNEAGISMLSQA NQTPQIVIV
Flagellin MNVSMNRLSSGKRINSAADDAAGLAIATRMRARQSGLEKASQNTQDGMSLIRTAE
SEQ ID NO: 73
SAMNSVSNILTRMRDIAVQSSNGTNTAENQSALQKEFAELQEQIDYIAKNTEFND Bacillus
anthracis KNLLAGTGAVTIGSTSISGAEISIETLDSSATNQQITIKLANTTAEKLGIDATTS
strain FI9401
NISISGAASALAAISALNTALNTVAGNRATLGATLNRLDRNVENLNNQATNMASA
ASQIEDADMAKEMSEMTKFKILNEAGISMLSQANQTPQMVSKLLQ Flagellin
MRINHNITALNTYRQFNNANNAQAKSMEKLSSGQRINSASDDAAGLAISEKMRGQ SEQ ID NO:
74 IRGLDQASRNAQDGVSLIQTAEGALNETHDILQRMRELVVQAGNGTNKTEDLDAI Bacillus
megaterium QDEIGSLIEEIGGEADSKGISDRAQFNGRNLLDGSLDITLQVGANAGQQVNLKIG
strain WSH-002
DMSAGALGADTNSDGAADAFVNSINVKDFTATSFDDQLAIIDGAINQVSEQRSGL
GATQNRLDHT1NNLSTSSENLTASESR1RDVDYALAA Flagellin
MRINHNLPALNAYRNLAQNQIGTSKILERLSSGYRINRASDDAAGLAISEKMRGQ SEQ ID NO:
75 IRGLEQGQRNTMDGVSLIQTAEGALQEIHEMLQRMRELAVQAANGTYSDKDKKAI
Aneurinibacillus sp.
EDEINQLTAQIDQIAKTTEFNGIQLIGDSDSTSLQDVKIQYGPKKEDSLTLELTT XH2
QPEADPPFAAGCKADKASLKIDNVDVISDPEGAIETFKAAIDQVSRIRSYFGAIQ
NRLEHVVNNLSNYTENLTGAESRIRDADMAKEMTEFTRFNIINQSATAMLAQANQ
LPQGVLQLLKG
[0130] N- and C-Terminal Conserved Regions of Flagellin
[0131] The flagellin or flagellin-associated polypeptide can
comprise a truncated N-terminal polypeptide and an amino acid
sequence of the truncated N-terminal polypeptide can comprise SEQ
ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
104, 106, 108, 109, 110, 112, 114, 116, 118, 120, 122, 124, 126,
128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,
180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,
206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 752, or any
combination thereof.
[0132] The flagellin or flagellin-associated polypeptide can
comprise a truncated C-terminal polypeptide and an amino acid
sequence of the truncated C-terminal polypeptide can comprise SEQ
ID NO: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,
131, 133, 135, 137, 141, 143, 145, 147, 149, 151, 153, 155, 157,
159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,
185, 187, 189, 191, 201, 203, 205, 207, 209, 211, 213, 215, 217,
219, 221, 223, 225, or any combination thereof.
[0133] N-terminal and C-terminal conserved regions were identified
from full length flagellin sequences from diverse strains of
Bacillus spp. and other Eubacteria (Table 2). Conserved N- and
C-terminal domains were identified using BLAST multiple alignment
software and assigned functional annotations based on individual
hits searching against Bacillus and other Eubacterial bacterial
databases. The start site for the N-terminal region of the coding
sequences is bolded methionine (M). The conserved domains are
provided as amino acid sequences N-terminus (left column) and
C-terminus (right column).
TABLE-US-00002 TABLE 2 N- and C-terminal conserved regions of
flagellins SEQ ID NO: Conserved N-terminus Conserved C-terminus
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM IDAAITTVAGQRATLGATLNRFE N-SEQ
ID NO: 76 SNAMDRLSSGKRINSASDDAAGLAIATRM FNANNLKSQETSMADAASQIE C-SEQ
ID NO: 77 KAREGGLNVAGRNTQDGMSLIRTADSALN DADMAKEMSEMTKFKILNEAG
Bacillus thuringensis SVSNILLRMRDLANQSANGTNTKGNQASL
ISMLSQANQTPQMVSKLLQ strain 4Q7 QKEFAQLTEQIDYIAKNTQFNDQQLLGTAD [CDS
of SEQ ID NO: 1] KKIKIQTL Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM
IDAAITTVAGQRATLGATLNRFE N-SEQ ID NO: 78
SNAMDRLSSGKRINSASDDAAGLAIATRM FNANNLKSQETSMADAASQIE C-SEQ ID NO: 79
KAREGGLNVAGRNTQDGMSLIRTADSALN DADMAKEMSEMTKFKILNEAG Bacillus
thuringiensis, SVSNILLRMRDLANQSANGTNTKGNQASL ISMLSQANQTPQMVSKLLQ
strain HD1002 QKEFAQLTEQIDYIAKNTQFNDQQLLGTAD [CDS of SEQ ID NO: 2]
KKIKIQTL Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM
IDAAITTVAGQRATLGATLNRFE N-SEQ ID NO: 80
SNAMDRLSSGKRINSASDDAAGLAIATRM FNANNLKSQETSMADAASQIE C-SEQ ID NO: 81
KAREGGLNVAGRNTQDGMSLIRTADSALN DADMAKEMSEMTKFKILNEAG Bacillus
thuringiensis, SVSNILLRMRDLANQSANGTNTKGNQASL ISMLSQANQTPQMVSKLLQ
strain HD-789 QKEFAQLTEQIDYIAKNTQFNDQQLLGTAD [CDS of SEQ ID NO: 3]
KKIKIQTL Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM
IDAAITTVAGQRATLGATLNRFE N-SEQ ID NO: 82
SNAMDRLSSGKRINSASDDAAGLAIATRM FNANNLKSQETSMADAASQIE C-SEQ ID NO: 83
KAREGGLNVAGRNTQDGMSLIRTADSALN DADMAKEMSEMTKFKILNEAG Bacillus cereus
SVSNILLRMRDLANQSANGTNTKGNQASL ISMLSQANQTPQMVSKLLQ strain G9842
QKEFAQLTEQIDYIAKNTQFNDQQLLGTAD [CDS of SEQ ID NO: 4] KKIKIQTL
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM QLDAALTKVADNRATLGATLNR N-SEQ
ID NO: 84 SNSMDRLSSGKRINSAADDAAGLAIATRM LDFNVNNLKSQENSMAASASQ C-SEQ
ID NO: 85 KAREGGLNVAARNTQDGMSLIRTADSALN IEDADMAKEMSEMTKFKILNEA
Bacillus thuringiensis SVSNILLRMRDLANQSATGTNTTKNQVAL
GISMLSQANQTPQMVSKLLQ serovar indiana strain
NKEFAALKEQITYIADNTQFNDKNLLKSTQ HD521 EIKIQTL [CDS of SEQ ID NO: 5]
Flagellin WGFLIMRINTNINSMRTQEYMRQNQAK AIAAIDAALTKVADNRATLGATL N-SEQ
ID NO: 86 MSNSMDRLSSGKRINNASDDAAGLAIATR NRLDFNVNNLKSQSSSMASAA C-SEQ
ID NO: 87 MRARESGLGVAADNTQNGMSLIRTADSA SQIEDADMAKEMSEMTKFKILN
Bacillus thuringiensis MNSVSNILLRMRDIANQSANGTNTNENKS
EAGISMLSQANQTPQMVSKLL strain CTC ALQKEFAQLQKQITYIAENTQFNDKNLLNE Q
[CDS of SEQ ID NO: 6] DSEVKIQTLDS Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ ID NO: 88
SNAMDRLSSGKRINNASDDAAGLAIATRM SSMAAAASQIEDADMAKEMSE C-SEQ ID NO: 89
RARENGLGVAANNTQDGMSLIRTADSAM MTKFKILNEAGISMLSQAN Bacillus
NSVSNILLRMRDLANQSANGTNTDDNQK thuringiensis
ALDKEFSALKEQIDYISKNTEFNDKKLL serovar yunnanensis strain IEBC-T20001
[CDS of SEQ ID NO: 7] Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM
IDAALKTVADNRATLGATLNRL N-SEQ ID NO: 90
SNAMDRLSSGKRINNASDDAAGLAIATRM DFNVNNLKSQSASMASAASQIE C-SEQ ID NO:
91 RARENGLGVAANNTQDGMSLIRTADSAL DADMAKEMSEMTKFKILNEAG Bacillus
thuringiensis QSVSNILLRMRDLANQSANGTNTDENKAA ISMLSQANQTPQMVSKLLQ
serovar tolworthi MEKEFGQLKDQIKYITDNTQFNDKNLLDA [CDS of SEQ ID NO:
8] Flagellin MGVLNMRINTNINSMRTQEYMRQNQAK RATLGATLNRLDFNVNNLKSQQ
N-SEQ ID NO: 92 MSNSMDRLSSGKRINNASDDAAGLAIATR SSMASAASQVEDADMAKEMS
C-SEQ ID NO: 93 MRARESGLGVAANNTQDGMSLIRTADSA EMTKFKILNEAGISMLSQANQT
Bacillus cereus strain FM1 MNSVSNILLRMRDIANQSANGTNTDKNQ PQMVSKLLQ
[CDS of SEQ ID NO: 9] VALQKEFGELQKQIDYIAKNTQFND Flagellin
MGVLNMRIGTNVLSMNARQSFYENEKR RADLGAMINQLQFNIENLNSQS N-SEQ ID NO: 94
MNVAIEHLATGKKLNHASDNPANVAIVTR TALTDAASRIEDADMAQEMSD C-SEQ ID NO: 95
MHARTSGIHVAIRNNEDAISMLRTAEAAL FLKFKLLTEVALSMVSQANQIP Bacillus
cereus strain FM1 QTVTNILQRMRDVAVQSANGTNSNKNRD QMVYKLLQ [CDS of SEQ
ID NO: 10] SLNKEFQSLTEQIGYIDETTEFND Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM AVDSIDAALKTVASNRATLGATL N-SEQ ID NO: 96
SNAMDRLSSGKRINNASDDAAGLAIATRM NRLDFNVNNLKSQSASMASAA C-SEQ ID NO: 97
RARESGLGVAANNTQDGMSLIRTADSALN SQIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis SVSNILLRMRDIANQSANGTNTADNQQAL EAGISMLSQANQTPQMVSKLL
strain MC28 QKEFGQLKEQISYIADNTEFNDKTLL Q [CDS of SEQ ID NO: 11]
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM LGATLNRLDFNVTNLKSQENS N-SEQ
ID NO: 98 SNSMDRLSSGKRINNASDDAAGLAIATRM MAASASQIEDADMAKEMSEM C-SEQ
ID NO: 99 RSREGGLNVAARNTEDGMSLIRTADSALN TKFKILNEAGISMLSQANQTPQ
Bacillus bombysepticus SVSNILLRMRDLANQSASGTNTDKNQAA MVSKLLQ strain
Wang MQKEFDQLKEQIQYI [CDS of SEQ ID NO: 12] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVTNLKSQE N-SEQ ID NO: 100
SNSMDRLSSGKRINNASDDAAGLAIATRM NSMAASASQIEDADMAKEMSE C-SEQ ID NO:
101 RSREGGLNVAARNTEDGMSLIRTADSALN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDLANQSASGTNTDKNQAA QMVSKLLQ serovar kenyae
MQKEFDQLKEQIQYI [CDS of SEQ ID NO: 13] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVTNLKSQE N-SEQ ID NO: 102
SNSMDRLSSGKRINNASDDAAGLAIATRM NSMAASASQIEDADMAKEMSE C-SEQ ID NO:
103 RSREGGLNVAARNTEDGMSLIRTADSALN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDLANQSASGTNTDKNQAA QMVSKLLQ serovar kenyae
MQKEFDQLKEQIQYI [CDS of SEQ ID NO: 14] Flagellin (A-type)
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ ID NO: 104
SNAMDRLSSGKRINNASDDAAGLAIATRM SSMASAASQIEDADMAKEMSE C-SEQ ID NO:
105 RARENGLGVAANNTQDGMSLIRTADSAL MTKFKILNEAGISMLSQANQTP Bacillus
cereus NSVSNILLRMRDLANQSANGTNTGDNQK QMVSKLLQ [CDS of SEQ ID NO: 15]
ALDKEFSALKEQIDYISKNTEFNDKKLL Flagellin (A-type)
GFLNMRIGTNVLSMNARQSLYENEKRMN RADLGSMINRLQFNIENLNSQS N-SEQ ID NO:
106 VAMEHLATGKKLNNASDNPANIAIVTRMH MALTDAASRIEDADMAQEMS C-SEQ ID NO:
107 ARASGMRLAIRNNEDTISMLRTAEAALQTL DFLKFKLLTEVALSMVSQANQIP Bacillus
cereus TNILQRMRDLAVQSANGTNSNKNRDSLNK QMVSKLLQ [CDS of SEQ ID NO:
16] EFQSLTEQIGYIGETTEFND Flagellin GVLNMRINTNINSMRTQEYMRQNQAKM
AIDAALTKVADNRATLGATLNR N-SEQ ID NO: 108
SNAMDRLSSGKRINNASDDAAGLAIATRM LDFNVNNLKSQSSSMASAASQI C-SEQ ID NO:
109 RARESGLNVAADNTQNGMSLIRTADSAM EDADMAKEMSEMTKFKILNEA Bacillus
thuringiensis NSVSNILLRMRDIANQSANGTNTDSNKSA GISMLSQANQTPQMVSKLLQ
serovar finitimus LQKEFAELQKQITYIADNTQFNDKNLLKEDS strain YBT-020
EVKIQTLDS [CDS of SEQ ID NO: 17] Flagellin
GVLNMRINTNINSMRTQEYMRQNQAKM AAIDAALTKVADNRATLGATLN N-SEQ ID NO: 110
SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO:
111 RARESGLNVAADNTQNGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis NSVSNILLRMRDIANQSANGTNTDSNKSA EAGISMLSQANQTPQMVSKLL
serovar finitimus LQKEFAELQKQITYIADNTQFNDKNLLKEDS Q strain YBT-020
EVKIQTLDS [CDS of SEQ ID NO: 18] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM TVADNRATLGATLNRLDFNVN N-SEQ ID NO: 112
SNAMDRLSSGKRINNASDDAAGLAIATRM NLKSQSASMASAASQIEDADM C-SEQ ID NO:
113 RARESGLGVAANNTQDGMSLIRTADSALN AKEMSEMTKFKILNEAGISMLS Bacillus
cereus SVSNILLRMRDLANQSANGTNTAENKAA QANQTPQMVSKLLQ stain B4264
MQKEFGELKDQIKYISENTQFNDQHLL [CDS of SEQ ID NO: 19] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM AIKSIDAALDTIASNRATLGATLN N-SEQ ID NO:
114 SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID
NO: 115 RARESGLGVAANNTQDGMSLIRTADSALN QIEDADMAKEMSEMTKFKILN
Bacillus thuringiensis SVSNILLRMRDIANQSANGTNTSDNQKAL
EAGISMLSQANQTPQMVSKLL serovar nigeriensis
DKEFSALKEQIDYISKNTEENDKKLL Q [CDS of SEQ ID NO: 20] Flagellin
WGFLIMRINTNINSMRTQEYMRQNQAK AVDAIDAALKTVASNRATLGAT N-SEQ ID NO: 116
MSNAMDRLSSGKRINNASDDAAGLAIATR LNRLDFNVNNLKSQSASMASA C-SEQ ID NO:
117 MRARESGLGVAANNTQDGMSLIRTADSA ASQIEDADMAKEMSEMTKFKIL Bacillus
thuringiensis LNSVSNILLRMRDIANQSANGTNTADNQQ NEAGISMLSQANQTPQMVSKL
[CDS of SEQ ID NO: 21] ALQKEFGQLKEQISYIADNTEFND LQ Flagellin
WGFLIMRINTNINSMRTQEYMRQNQAK AVDAIDAALKTVASNRATLGAT N-SEQ ID NO: 118
MSNAMDRLSSGKRINNASDDAAGLAIATR LNRLDFNVNNLKSQSASMASA C-SEQ ID NO:
119 MRARESGLGVAANNTQDGMSLIRTADSA ASQIEDADMAKEMSEMTKFKIL Bacillus
thuringiensis LNSVSNILLRMetRDIANQSANGTNTADN NEAGISMLSQANQTPQMVSKL
serovar konkukian QQALQKEFGQLKEQISYIADNTEENDKTLL LQ strain 97-27
[CDS of SEQ ID NO: 22] Flagellin WGFLIMRINTNINSMRTQEYMRQNQAK
AIASIDAALESIASNRATLGATLN N-SEQ ID NO: 120
MSNAMDRLSSGKRINNASDDAAGLAIATR RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO:
121 MRARESGLGVAANNTQDGMSLIRTADSA QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis MNSVSNILLRMRDISNQSANGTNTDKNQS EAGISMLSQANQTPQMVSKLL
serovar konkukian ALDKEFAALKDQIDYISKNTEENDQKLL Q strain 97-27 [CDS
of SEQ ID NO: 23] Flagellin protein FlaA
GFLNMRINTNINSMRTQEYMRQNQAKM AIASIDAALESIASNRATLGATLN N-SEQ ID NO:
122 SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID
NO: 123 RARESGLGVAANNTQDGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis NSVSNILLRMRDISNQSANGTNTDKNQSA EAGISMLSQANQTPQMVSKLL
serovar thuringiensis LDKEFAALKDQIDYISKNTEFNDQKLL Q strain IS5056
[CDS of SEQ ID NO: 24] Flagellin protein FlaA
GFLNMRINTNINSMRTQEYMRQNQAKM AIASIDAALESIASNRATLGATLN N-SEQ ID NO:
124 SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID
NO: 125 RARESGLGVAANNTQDGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis NSVSNILLRMRDISNQSANGTNTDKNQSA EAGISMLSQANQTPQMVSKLL
serovar thuringiensis LDKEFAALKDQIDYISKNTEFNDQKLL Q strain IS5056
[CDS of SEQ ID NO: 25] Flagellin B GFLNMRINTNINSMRTQEYMRQNQAKM
AIASIDAALESIASNRATLGATLN N-SEQ ID NO: 126
SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO:
127 RARESGLGVAANNTQDGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis NSVSNILLRMRDISNQSANGTNTDKNQSA EAGISMLSQANQTPQMVSKLL
strain Bt407 LDKEFAALKDQIDYISKNTEFNDQKLL Q [CDS of SEQ ID NO: 26]
Flagellin GFLNMINTNINSMRTQEYMRQNQAKM AIASIDAALESIASNRATLGATLN N-SEQ
ID NO: 128 SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS
C-SEQ ID NO: 129 RARESGLGVAANNTQDGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN
Bacillus thuringiensis NSVSNILLRMRDISNQSANGTNTDKNQSA
EAGISMLSQANQTPQMVSKLL serovar chinensis CT-43
LDKEFAALKDQIDYISKNTEFNDQKLL Q [CDS of SEQ ID NO: 27] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ ID NO: 130
SNAMDRLSSGKRINNASDDAAGLAIATRM SSMASAASQIEDADMAKEMSE C-SEQ ID NO:
131 RARESGLGVAANNTQDGISLIRTADSAMN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDLANQSANGTNTNENQAAL QMVSKLLQ serovar
Canadensis NKEFDALKEQIDYISTNTEENDKKLL [CDS of SEQ ID NO: 28]
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ
ID NO: 132 SNAMDRLSSGKRINNASDDAAGLAIATRM SSMASAASQIEDADMAKEMSE
C-SEQ ID NO: 133 RARESGLGVAANNTQDGISLIRTADSAMN
MTKFKILNEAGISMLSQANQTP Bacillus thuringiensis
SVSNILLRMRDLANQSANGTNTNENQAAL QMVSKLLQ serovar galleriae
NKEFDALKEQIDYISTNTEENDKKLL [CDS of SEQ ID NO: 29] Flagellin
N-terminal helical GVLNMRINTNINSMRTQEYMRQNQAKM
RATLGATLNRLDFNVNNLKSQS region SNAMDRLSSGKRINNASDDAAGLAIATRM
SSMASAASQIEDADMAKEMSE N-SEQ ID NO: 134 RARESGLSVAANNTQDGMSLIRTADSAM
MTKFKILNEAGISMLSQANQTP C-SEQ ID NO: 135
NSVSNILLRMRDLSNQSANGTNTDENQQ QMVSKLLQ Bacillus
ALNKEFAALKDQIDYISKNTEFNDKKLL weihenstephanensis [CDS of SEQ ID NO:
30] Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM IDAALETIASNRATLGATLNRLD
N-SEQ ID NO: 136 SNAMDRLSSGKRINNASDDAAGLAIATRM
FNVNNLKSQSSSMASAASQIED C-SEQ ID NO: 137
RARESGLGVAANNTQDGMSLIRTADSALN ADMAKEMSEMTKFKILNEAGIS Bacillus
thuringiensis SVSNILLRMRDIANQSANGTNTGDNQKAL MLSQANQTPQMVSKLLQS
serovar ostriniae DKEFSALKEQIDYISKNTEENDKKLL [CDS of SEQ ID NO: 31]
Flagellin WGFLIMRINTNINSMRTQEYMRQNQTK LGATLNRLDFNVNNLKSQSSSM N-SEQ
ID NO: 138 MSNAMDRLSSGKRINNASDDAAGLAIATR AAAASQIEDADMAKEMSEMT C-SEQ
ID NO: 139 MRARENGLGVAANNTQDGMSLIRTADSA KFKILNEAGISMLSQANQTPQM
Bacillus thuringiensis MNSVSNILLRMRDLANQSANGTNTDDNQ VSKLLQ [CDS of
SEQ ID NO: 32] KALDKEFSALKEQIDYISKNTEFNDKKLL Flagellin
WGFLIMRINTNINSMRTQEYMRQNQTK LGATLNRLDFNVNNLKSQSSSM N-SEQ ID NO: 140
MSNAMDRLSSGKRINNASDDAAGLAIATR AAAASQIEDADMAKEMSEMT C-SEQ ID NO: 141
MRARENGLGVAANNTQDGMSLIRTADSA KFKILNEAGISMLSQANQTPQM
Bacillus thuringiensis MNSVSNILLRMRDLANQSANGTNTDDNQ VSKLLQ [CDS of
SEQ ID NO: 33] KALDKEFSALKEQIDYISKNTEFNDKKLL Flagellin
WGFLIMRINTNINSMRTQEYMRQNQTK RATLGATLNRLDFNVNNLKSQS N-SEQ ID NO: 142
MSNAMDRLSSGKRINNASDDAAGLAIATR SSMAAAASQIEDADMAKEMSE C-SEQ ID NO:
143 MRARENGLGVAANNTQDGMSLIRTADSA MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis MNSVSNILLRMRDLANQSANGTNTDDNQ QMVSKLLQ serovar
pondicheriensis KALDKEFSALKEQIDYISKNTEENDKKLL [CDS of SEQ ID NO:
34] Flagellin B GFLNMRINTNINSMRTQDYMRQNQAKM
AIASIDAALESIASNRATLGATLN N-SEQ ID NO: 144
SNAMDRLSSGKRINNASDDAAGLAIATRM RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO:
145 RARESGLGVAANNTQDGMSLIRTADSAM QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis NSVSNILLRMRDISNQSANGTNTDKNQSA EAGISMLSQANQTPQMVSKLL
serovar Berliner LDKEFAALKDQIDYISKNTEFNDQKLL Q [CDS of SEQ ID NO:
35] Flagellin A GFLNMARITINLEIDFFAYYRFSICRKVNIKK
AIASIDAALESIASNRATLGATLN N-SEQ ID NO: 146
WGFLNMRINTNINSMRTQDYMRQNQAK RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO: 147
MSNAMDRLSSGKRINNASDDAAGLAIATR QIEDADMAKEMSEMTKFKILN Bacillus
thuringiensis MRARESGLGVAANNTQDGMSLIRTADSA EAGISMLSQANQTPQMVSKLL
serovar Berliner MNSVSNILLRMRDISNQSANGTNTDKNQS Q [CDS of SEQ ID NO:
36] ALDKEFAALKDQIDYISKNTEFNDQKLL Flagellin
GVLYMRINTNINSMRTQEYMRQNQAKM TVADNRATLGATLNRLDFNVN N-SEQ ID NO: 148
SNAMDRLSSGKRINNASDDAAGLAIATRM NLKSQSSAMAASASQIEDADM C-SEQ ID NO:
149 RARESGLSVAADNTQNGMSLIRTADSAM AKEMSEMTKFKILNEAGISMLS Bacillus
cereus strain Q1 NSVSNILLRMRDIANQSANGTNTDKNQVA QANQTPQMVSKLLQ [CDS
of SEQ ID NO: 37] LQKEFAALKEQITYIADNTQFNDKNLLNGN QTINIQTLDSHDST
Flagellin GVLYMRINTNINSMRTQEYMRQNQAKM TVADNRATLGATLNRLDFNVN N-SEQ
ID NO: 150 SNAMDRLSSGKRINNASDDAAGLAIATRM NLKSQSSAMAASASQIEDADM
C-SEQ ID NO: 151 RARESGLSVAADNTQNGMSLIRTADSAM
AKEMSEMTKFKILNEAGISMLS Bacillus cereus strain Q1
NSVSNILLRMRDIANQSANGTNTDKNQVA QANQTPQMVSKLLQ [CDS of SEQ ID NO: 38]
LQKEFAALKEQITYIADNTQFNDKNLLNGN QTINIQTLDSHDST Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM LGATLNRLDFNVNNLKSQSSSM N-SEQ ID NO: 152
SNAMDRLSSGKRINNASDDAAGLAIATRM ASAASQIEDADMAKEMSEMTK C-SEQ ID NO:
153 RARESGLGVAANNTQDGMSLIRTADSALN FKILNEAGISMLSQANQTPQM Bacillus
thuringiensis SVSNILLRMRDIANQSANGTNTGDNQKAL VSKLLQ serovar
morrisoni DKEFSALKEQIDYISKNTEENDKKLL [CDS of SEQ ID NO: 39]
Flagellin GFLNMRINTNINSMRTQEYMRQNQTKM AIKSIDAALDTIASNRATLGATLN
N-SEQ ID NO: 154 SNAMDRLSSGKRINNASDDAAGLAIATRM
RLDFNVNNLKSQSSSMASAAS C-SEQ ID NO: 155 RARENGLGVAANNTQDGMSLIRTADSAL
QIEDADMAKEMSEMTKFKILN Bacillus thuringiensis
NSVSNILLRMRDIANQSANGTNTSDNQKA EAGISMLSQANQTPQMVSKLL serovar
neoleonensis LDKEFSALKEQIDYISKNTEFNDKKLL Q [CDS of SEQ ID NO: 40]
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ
ID NO: 156 SNAMDRLSSGKRINNASDDAAGLAIATRM SSMASAASQIEDADMAKEMSE
C-SEQ ID NO: 157 RARESGLGVAANNTQDGMSLIRTADSALN
MTKFKILNEAGISMLSQANQTP Bacillus thuringiensis
SVSNILLRMRDIANQSANGTNTGDNQKAL QMVSKLLQ serovar morrisoni
DKEFSALKEQIDYISKNTEENDKKLL [CDS of SEQ ID NO: 41] Flagellin
GFLNMRINTNINSMRTQEYMRQNQAKM RATLGATLNRLDFNVNNLKSQS N-SEQ ID NO: 158
SNAMDRLSSGKRINNASDDAAGLAIATRM SSMASAASQIEDADMAKEMSE C-SEQ ID NO:
159 RARESGLGVAANNTQDGMSLIRTADSALN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDIANQSANGTNTGDNQKAL QMVSKLLQ serovar
morrisoni DKEFSALKEQIDYISKNTEENDKKLL [CDS of SEQ ID NO: 42]
Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM LGATLNRLDFNVNNLKSQQSS N-SEQ
ID NO: 160 SNAMDRLSSGKRINNASDDAAGLAIATRM MASAASQIEDADMAKEMSEM C-SEQ
ID NO: 161 RARESGLGVAANNTQDGMSLIRTADSAM TKFKILNEAGISMLSQANQTPQ
Bacillus thuringiensis NSVSNILLRMRDIANQSANGTNTNGNQA MVSKLLQ serovar
jegathesan ALNKEFDALKQQINYISTNTEFNDKKLLDGS [CDS of SEQ ID NO: 43]
NKTIAIQTLD Flagellin GVLNMRINTNINSMRTQEYMRQNQAKM
DKIDEALKTIADNRATLGATLNR N-SEQ ID NO: 162
SNAMDRLSSGKRINNASDDAAGLAIATRM LDFNVNNLKSQSASMASAASQI C-SEQ ID NO:
163 RARESGLGVAANNTQDGMALIRTADSAM EDADMAKEMSEMTKFKILNEA Bacillus
cereus stain NSVSNILLRRDIANQSANGTNTDKNQAAL GISMLSQANQTPQMVSKLLQ
ATCC 10987 QKEFGELQKQIDYIAGNTQFNDK [CDS of SEQ ID NO: 44] Flagellin
WGFLIMRINTNINSMRTQEYMRQNQAK RATLGATLNRLDFNVNNLKSQQ N-SEQ ID NO: 164
MSNAMDRLSSGKRINNASDDAAGLAIATR SSMASAASQIEDADMAKEMSE C-SEQ ID NO:
165 MRARESGLGVAANNTQDGMSLIRTADSA MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis MNSVSNILLRMRDLANQSANGTNTNENQ QMVSKLLQ serovar
monterrey AALNKEFDALKEQINYISTNTEFNDKKLL [CDS of SEQ ID NO: 45]
Flagellin WGFFYMRINTNINSMRTQEYMRQNQAK TVADNRATLGATLNRLDFNVN N-SEQ
ID NO: 166 MSNAMDRLSSGKRINNASDDAAGLAIATR NLKSQASSMAAAASQVEDAD C-SEQ
ID NO: 167 MRARESGLGVASNNTQDGMSLIRTADSAL MAKEMSEMTKFKILNEAGISM
Bacillus cereus strain NSVSNILLRMRDLANQSANGTNTNENKAA
LSQANQTPQMVSKLLQ NC7401 MQKEFGELKEQIKYIAENTQFNDQHLL [CDS of SEQ ID
NO: 46] Flagellin WGFFYMRINTNINSMRTQEYMRQNQAK TVADNRATLGATLNRLDFNVN
N-SEQ ID NO: 168 MSNAMDRLSSGKRINNASDDAAGLAIATR NLKSQASSMAAAASQVEDAD
C-SEQ ID NO: 169 MRARESGLGVASNNTQDGMSLIRTADSAL
MAKEMSEMTKFKILNEAGISM Bacillus cereus strain
NSVSNILLRMRDLANQSANGTNTNENKAA LSQANQTPQMVSKLLQ NC7401
MQKEFGELKEQIKYIAENTQFNDQHLL [CDS of SEQ ID NO: 47] Flagellin
(A-type) GVLNMRINTNINSLRTQEYMRQNQAKMS IDAALKTVADNRATLGATLNRL N-SEQ
ID NO: 170 NSMDRLSSGKRINNASDDAAGLAIATRMR DFNVNNLKSQSSSMASAASQIE
C-SEQ ID NO: 171 ARESGLNVAANNTQDGMSLIRTADSALGS
DADMAKEMSEMTKFKILNEAG Bacillus cereus strain
VSNILLRMRDLANQSANGTNTSDNQAAM ISMLSQANQTPQMVSKLLQ AH820
QKEFAELQKQITYIADNTQFNDKNLL [CDS of SEQ ID NO: 48] Flagellin
WGFFYMRINTNINSMRTQEYMRQNQAK TVADNRATLGATLNRLDFNVN N-SEQ ID NO: 172
MSNAMDRLSSGKRINNASDDAAGLAIATR NLKSQASSMAAAASQVEDAD C-SEQ ID NO: 173
MRARESGLGVASNNTQDGMSLIRTADSAL MAKEMSEMTKFKILNEAGISM Bacillus cereus
AH187 NSVSNILLRMRDLANQSANGTNTNENKAA LSQANQTPQMVSKLLQ [CDS of SEQ ID
NO: 49] MQKEFGELKEQIKYIAENTQFNDQHLL Flagellin
WGFFYMRINTNINSMRTQEYMRQNQAK TVADNRATLGATLNRLDFNVN N-SEQ ID NO: 174
MSNAMDRLSSGKRINNASDDAAGLAIATR NLKSQASSAAAASQVEDADMA C-SEQ ID NO:
175 MRARESGLGVASNNTQDGMSLIRTADSAL KEMSEMTKFKILNEAGISMLSQ Bacillus
cereus NSVSNILLRMRDLANQSANGTNTNENKAA ANQTPQMVSKLLQ [CDS of SEQ ID
NO: 50] MQKEFGELKEQIKYIAENTQFNDQHLL Flagellin protein Fla
GFLNMRINTNINSMRTQEYMRQNQAKM LGATLNRLDFNVNNLKSQSSSM N-SEQ ID NO: 176
SNAMDRLSSGKRINNASDDAAGLAIATRM ASAASQIEDADMAKEMSEMTK C-SEQ ID NO:
177 RARESGLGVAANNTQDGMSLIRTADSALN FKILNEAGISMLSQANQTPQM Bacillus
cereus SVSNILLRMRDIANQSANGTNTGDNQKAL VSKLLQ [CDS of SEQ ID NO: 51]
DKEFSALKEQIDYISKNTEFNDKKLL Flagellin GFLNMRINTNINSMRTQEYMRQNQTKM
RATLGATLNRLDFNVTNLKSQE N-SEQ ID NO: 178
SNAMDRLSSGKRINNASDDAAGLAIATRM NSMAASASQIEDADMAKEMSE C-SEQ ID NO:
179 RSREGGLNVAARNTEDGMSLIRTADSALN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDLANQSASETNTSKNQAAM QMVSKLLQ Strain HD-771
QKEFDQLKEQIQYI [CDS of SEQ ID NO: 52] Flagellin
GFLNMRINTNINSMRTQEYMRQNQTKM RATLGATLNRLDFNVTNLKSQE N-SEQ ID NO: 180
SNAMDRLSSGKRINNASDDAAGLAIATRM NSMAASASQIEDADMAKEMSE C-SEQ ID NO:
181 RSREGGLNVAARNTEDGMSLIRTADSALN MTKFKILNEAGISMLSQANQTP Bacillus
thuringiensis SVSNILLRMRDLANQSASETNTSKNQAAM QMVSKLLQ serovar sotto
QKEFDQLKEQIQYI [CDS of SEQ ID NO: 53] Flagellin
MGVLNMRINTNINSMRTQEYMRQNQAK AIKAIDEALETIASNRATLGATLN N-SEQ ID NO:
182 MSTAMDRLSSGKRINNASDDAAGLAIATR RLDFNVNNLKNQASSMASAAS C-SEQ ID
NO: 183 MRARESGLGVAANNTQDGISLIRTADSAM QVEDADMAKEMSEMTKFKILN
Bacillus thuringiensis NSVSNILLRMRDLANQSANGTNTDKNQG
EAGISMLSQANQTPQMVSKLL serovar Novosibirsk
ALDKEFAALKEQIDYISKNTEFNDKKLL Q [CSD of SEQ ID NO: 54] Flagellin
MGVLNMRINTNINSMRTQEYMRQNQAK AIDSALENIASNRATLGATLNRL N-SEQ ID NO:
184 MSNAMDRLSSGKRINNASDDAAGLAIATR DFNVNNLKSQSSSMASAASQIE C-SEQ ID
NO: 185 MRARESGLGVAANNTQDGISLIRTADSAM DADMAKEMSEMTKFKILNEAG
Bacillus thuringiensis NSVSNILLRMRDLANQSANGTNTSENQAA
ISMLSQANQTPQMVSKLLQ serovar Londrina LDKEFGALKEQINYISTNTEFNDKKLL
[CDS of SEQ ID NO: 55] Flagellin MGVLNMRINTNINSMRTQEYMRQNQAK
LGATLNRLDFNVNNLKNQASS N-SEQ ID NO: 186
MSTAMDRLSSGKRINNASDDAAGLAIATR MASAASQVEDADMAKEMSE C-SEQ ID NO: 187
MRARESGLGVAANNTQDGISLIRTADSAM MTKFKILNEAGISMLSQANQTP Bacillus
cereus strain E33L NSVSNILLRMRDLANQSANGTNTDKNQG QMVSKLLQ [CDS of
SEQ ID NO: 56] ALDKEFAALKEQIDYISKNTEFNDKKLL Flagellin
MGVLNMRINTNINSMRTQEYMRQNQAK ATLNRLDFNVNNLKNQASSMA N-SEQ ID NO: 188
MSTAMDRLSSGKRINNASDDAAGLAIATR SAASQVEDADMAKEMSEMTK C-SEQ ID NO: 189
MRARESGLGVAANNTQDGISLIRTADSAM FKILNEAGISMLSQANQTPQM Bacillus cereus
strain E33L NSVSNILLRMRDLANQSANGTNTDKNQG [CDS of SEQ ID NO: 57]
ALDKEFAALKEQIDYISKNTEFNDKKLL Flagellin WGFFYMRINTNINSMRTQEYMRQNQAK
AIAAIDAALTKVADNRATLGATL N-SEQ ID NO: 190
MSTAMDRLSSGKRINNASDDAAGLAIATR NRLDFNVNNLKSQASSMASAA C-SEQ ID NO:
191 MRARESGLGVAANNTQDGISLIRTADSAM SQVEDADMAKEMSEMTKFKIL Bacillus
cereus NSVSNILLRMRDLANQSANGTNTDKNQA NEAGISMLSQANQTPQMVSKL strain
FRI-35 ALDKEFNALKEQIDYISKNTEFNDKKL LQ [CDS of SEQ ID NO: 58]
Flagellin WGFFYMRIGTNVLSLNARQSLYENEKRM AIRKIEEALQNVSLHRADLGAMI
N-SEQ ID NO: 192 NVAMEHLATGKKLNNASDNPANIAIVTR
NRLQFNIENLNSQSTALTDAAS C-SEQ ID NO: 193
MHARASGMRVAIRNNEDAISMLRTAEAA RIEDADMAQEMSDFLKFKLLTE Bacillus cereus
LQTVTNVLQRMRDLAVQSANGTNSNKNR VALSMVSQANQVPQMVSKLL strain FRI-35
DSLNKEFQSLTEQIGYIDETTEFNN Q [CDS of SEQ ID NO: 59] Flagellin
LVPFAVWLAMSRIRRRILDTDCKAESAVRIK MAASASQIEDADMAKEMSEM N-SEQ ID NO:
194 EIPSDVLRAATERPLSCARIRVAIARPAASSE TKFKILSEAGISMLSQANQTPQ C-SEQ
ID NO: 195 ALLIRLPLDKRSIALLILAWFWRMYSCVRML MVSKLLQ Bacillus
thuringiensis LMFVLILMLRTP [CDS of SEQ ID NO: 60] Flagellin
AVWLAMSRIRRRILDTDCKAESAVRIKEIPS SMAASASQIEDADMAKEMSE N-SEQ ID NO:
196 DVLRAATERPLSCARIRVAIARPAASSEALLI MTKFKILSEAGISMLSQANQTP C-SEQ
ID NO: 197 RLPLDKRSIALLILAWFWRMYSCVRMLLMF QMVSKLLQ Bacillus cereus
strain VLILMLRTP ATCC 4342 [CDS of SEQ ID NO: 61] Flagellin
GFLNMRIGTNFLSMNARQSLYENEKRMN LGAMINRLHFNIENLNSQSMAL N-SEQ ID NO:
198 VAMEHLATGKKLNHASDNPANIAIVTRMH TDAASRIEDADMAQEMSDFLK C-SEQ ID
NO: 199 ARANGMRVAIRNNEDAISMLRTAEAALQT FKLLTEVALSMVSQANQIPQM
Bacillus thuringiensis VMNILQRMRDLAIQSANSTNSNKNRDSLN VSKLLQ [CDS of
SEQ ID NO: 62] KEFQSLTEQISYI Flagellin GFLNMRINTNINSMRTQEYMRQNQAKM
LGATLNRLDFNVNNLKSQSSSM N-SEQ ID NO: 200
SNAMDRLSSGKRINNASDDAAGLAIATRM ASAASQIEDADMAKEMSEMTK C-SEQ ID NO:
201 RARESGLGVAANNTQDGMSLIRTADSALN FKILNEAGISMLSQANQTPQM Bacillus
thuringiensis SVSNILLRMRDIANQSANGTNTGDNQKAL VSKLLQ [CDS of SEQ ID
NO: 63] DKEFSALKEQIDYI Flagellin MRINHNITALNTYRQFNNANNAQAKSME
IDGAINQVSEQRSGLGATQNRL N-SEQ ID NO: 202
KLSSGQRINSASDDAAGLAISEKMRGQIRG DHTINNLSTSSENLTASESRIRD C-SEQ ID NO:
203 LDQASRNAQDGVSLIQTAEGALNETHDILQ VDYALAA Bacillus aryabhattai
RMRELVVQAGNGTNKTEDLDAIQDEIGSLI [CDS of SEQ ID NO: 64]
EEIGGETDSKGISDRAQFNGRNLLDGSLDIT LQVGA Flagellin
MRINTNINSMRTQEYMRQNQDKMNTSM IDQAIQDIADNRATYGSQLNRL N-SEQ ID NO: 204
NRLSSGKQINSASDDAAGLAIATRMRAKE DHNLNNVNSQATNMAAAASQ C-SEQ ID NO: 205
GGLNVGAKNTQDGMSALRTMDSALNSVS IEDADMAKEMSEMTKFKILSEA Bacillus
manliponensis NILLRMRDLATQSATGTNQGNDRESLDLE GVSMLSQANQTPQMVSKLLQ
[CDS of SEQ ID NO: 65] FQQLTEEITHIAEKTNFNGNALLSGSGSAIN VQLS
Flagellin MRIGSWTATGMSIVNHMNRNWNAASKS LDEATKNVSMERSRLGAYQNRL N-SEQ
ID NO: 206 MLRLSSGYRINSAADDAAGLAISEKMRGQI EHAYNVAENTAINLQDAESRIR
C-SEQ ID NO: 207 RGLTMASKNIMDGVSLIQTAEGALNETHAI
DVDIAKEMMNMVKSQILAQV Lysinibacillus sp. strain
VQRMRELAVQAATDTNTDDDRAKLDLEF GQQVLAMHMQQAQGILRLLG BF-4
QELKKEIDRISTDTEFNTRTLLNGDYKDNGL [CDS of SEQ ID NO: 66] KIQVG
Flagellin MKIGSWTATGMSIVNHMNRNWNAASKS LDEATKNVSMERSRLGAYQNRL N-SEQ
ID NO: 208 MLRLSSGYRINSAADDAAGLAISEKMRGQI EHAYNVAENTAINLQDAESRIR
C-SEQ ID NO: 209 RGLTMASKNIMDGVSLIQTAEGALNETHAI
DVDIAKEMMHMVKSQILAQV Lysinibacillus sp. strain
VQRMRELAVQAATDTNTDDDRAKLDLEF GQQVLAMHIQQAQGILRLLG 13S34_air
QELKKEIDRISTDTAFNTRTLLNGDYKDNGL [CDS of SEQ ID NO: 67] KIQVG
Flagellin MIISHNLTALNTMNKLKQKDLAVSKSLGKL ISAAIDKVSAERARMGAYQNRL
N-SEQ ID NO: 210 SSGLRINGASDDAAGLAISEKMRGQIRGLN
EHSRNNVVTYAENLTAAESRIR C-SEQ ID NO: 211
QASRNIQDGISLIQVADGAMQEIHSMLQR DVDMAKEMMELMKNQIFTQA Paenibacillus
sp. strain MNELAVQASNGTYSGSDRLNIQSEVEQLIE GQAMLLQTNTQPQAILQLLK
HW567 EIDEIAGNTGFNGIKLLNGNNEKTEKTEK [CDS of SEQ ID NO: 68]
Flagellin MRINTNINSMRTQEYMRQNQAKMSNA IDSALETIASNRATLGATLNRLDF N-SEQ
ID NO: 212 MDRLSSGKRINNASDDAAGLAIATRMRAR NVNNLKSQSSAMASAASQIED
C-SEQ ID NO: 213 ESGLGVAANNTQDGMSLIRTADSAMNSV
ADMAKEMSEMTKFKILNEAGIS Bacillus anthracis
SNILLRMRDLANQSANGTNTKENQDALDK MLSQANQTPQMVSKLLQ [CDS of SEQ ID NO:
69] EFGALKEQIDYISKNTEFNDKKLLNGDNKSI AIQTL
Flagellin MQKSQYKKMGVLKMRINTNINSMRTQEY ALNTVAGNRATLGATLNRLDR N-SEQ
ID NO: 214 MRQNQDKMNVSMNRLSSGKRINSAADD NVENLNNQATNMASAASQIED C-SEQ
ID NO: 215 AAGLAIATRMRARQSGLEKASQNTQDGM ADMAKEMSEMTKFKILNEAGIS
Bacillus anthracis SLIRTAESAMNSVSNILTRMRDIAVQSSNG MLSQANQTPQMVSKLLQ
[CDS of SEQ ID NO: 70] TNTAENQSALQKEFAELQEQIDYIAKNTEF
NDKNLLAGTGAVTIGSTSISGAEISIETL Flagellin MRINTNINSMRTQEYMRQNQDKMNVS
ALNTVAGNRATLGATLNRLDR N-SEQ ID NO: 216
MNRLSSGKRINSAADDAAGLAIATRMRAR NVENLNNQATNMASAASQIKD C-SEQ ID NO:
217 QSGLEKASQNTQDGMSLIRTAESAMNSVS ADKAKEMSEMTKFKILNEAGIS Bacillus
anthracis NILTRMRDIAVQSSNGTNTAENQSALQKE MLSQANQTPQMVSKLLQ [CDS of
SEQ ID NO: 71] FAELQEQIDYIAKNTEFNDKNLLAGTGAVTI GSTSISGAEISIETL
Flagellin MRINTNINSMRTQEYMRQNQDKMNVS ALNTVAGNRATLGATLNRLDR N-SEQ ID
NO: 218 MNRLSSGKRINSAADDAAGLAIATRMRAR NVENLNNQATNMASAASQIED C-SEQ
ID NO: 219 QSGLEKASQNTQDGMSLIRTAESAMNSVS ADMAKEMSEMTKFKILNEAGIS
Bacillus anthracis NILTRMRDIAVQSSNGTNTAENQSALQKE MLSQANQTPQMV [CDS
of SEQ ID NO: 72] FAELQEQIDYIAKNTEFNDKNLLAGTGAVTI GSTSISGAEISIETL
Flagellin MNVSMNRLSSGKRINSAADDAAGLAIATR LNTALNTVAGNRATLGATLNRL
N-SEQ ID NO: 220 MRARQSGLEKASQNTQDGMSLIRTAESA DRNVENLNNQATNMASAASQI
C-SEQ ID NO: 221 MNSVSNILTRMRDIAVQSSNGTNTAENQS
EDADMAKEMSEMTKFKILNEA Bacillus anthracis strain
ALQKEFAELQEQIDYIAKNTEFNDKNLLAG GISMLSQANQTPQMVSKLLQ H9401
TGAVTIGSTSISGAEISIETL [CDS of SEQ ID NO: 73] Flagellin
MRINHNITALNTYRQFNNANNAQAKSME IIDGAINQVSEQRSGLGATQNR N-SEQ ID NO:
222 KLSSGQRINSASDDAAGLAISEKMRGQIRG LDHTINNLSTSSENLTASESRIRD C-SEQ
ID NO: 223 LDQASRNAQDGVSLIQTAEGALNETHDILQ VDYALAA Bacillus
megaterium strain RMRELVVQAGNGTNKTEDLDAIQDEIGSLI WSH-002
EEIGGEADSKGISDRAQFNGRNLLDGSLDIT [CDS of SEQ ID NO: 74] LQVGA
Flagellin MRINHNLPALNAYRNLAQNQIGTSKILERL FKAAIDQVSRIRSYFGAIQNRLE
N-SEQ ID NO: 224 SSGYRINRASDDAAGLAISEKMRGQIRGLE
HVVNNLSNYTENLTGAESRIRD C-SEQ ID NO: 225
QGQRNTMDGVSLIQTAEGALQEIHEMLQ ADMAKEMTEFTRFNIINQSATA
Aneurinibacillus sp. XH2 RMRELAVQAANGTYSDKDKKAIEDEINQL
MLAQANQLPQGVLQLLKG [CDS of SEQ ID NO: 75]
TAQIDQIAKTTEFNGIQLIGDSDSTSLQDVK
[0134] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 226-300, or any combination thereof.
[0135] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise SEQ ID NO: 226.
[0136] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 301-375, or any combination thereof.
[0137] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise SEQ ID NO: 301.
[0138] The flagellin-derived polypeptide sequence for Bt4Q7Flg22
(SEQ ID NO: 226) was identified from a proprietary "In house"
library from Bacillus thuringiensis (Bt.) strain 4Q7. Conserved
primers to full length flagellin from E. coli were used to screen
the Bt.4Q7 strain library and identify a functional
flagellin-associated bioactive priming Flg22 polypeptide.
TABLE-US-00003 TABLE 3 Flagellin polypeptides Flg22 and FlgII-28
identified from Bacillus spp. SEQ ID NO: Peptide Flg22 Flg22-8L4Q7
DRLSSGKRINSASDDAAGLAIA SEQ ID NO: 226 Bacillus thuringiensis strain
4Q7 Flg22 DRLSSGKRINSASDDAAGLAIA SEQ ID NO: 227 Bacillus
thuringiensis, strain HD1002 Flg22 DRLSSGKRINSASDDAAGLAIA SEQ ID
NO: 228 Bacillus thuringiensis, strain HD-789 Flg22
DRLSSGKRINSASDDAAGLAIA SEQ ID NO: 229 Bacillus cereus strain G9842
Flg22 EHLATGKKLNNASDNPANIAIV SEQ ID NO: 230 Bacillus thuringiensis
serovar indiana strain HD521 Flg22 DRLSSGKRINNASDDAAGLAIAT SEQ ID
NO: 231 Bacillus thuringiensis strain CTC Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 232 Bacillus thuringiensis
serovar yunnanensis strain IEBC-T20001 Flg22 DRLSSGKRINNASDDAAGLAIA
SEQ ID NO: 233 Bacillus thuringiensis serovar tolworthi Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 234 Bacillus cereus strain FM1
Flg22 EHLATGKKLNHASDNPANVAIV SEQ ID NO: 235 Bacillus cereus strain
FM1 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 236 Bacillus
thuringiensis strain MC28 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO:
237 Bacillus bombysepticus strain Wang Flg22 DRLSSGKRINNASDDAAGLAIA
SEQ ID NO: 238 Bacillus thuringiensis serovar kenyae Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 239 Bacillus thuringiensis
serovar kenyae Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 240 Bacillus
cereus Flg22 EHLATGKKLNNASDNPANIAIV SEQ ID NO: 241 Bacillus cereus
Flg22 EHLATGKKLNHASDNPANVAIV SEQ ID NO: 242 Bacillus thuringiensis
serovar finitimus strain YBT-020 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ
ID NO: 243 Bacillus thuringiensis serovar finitimus strain YBT-020
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 244 Bacillus cereus stain
B4264 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 245 Bacillus
thuringiensis serovar nigeriensis Flg22 DRLSSGKRINNASDDAAGLAIA SEQ
ID NO: 246 Bacillus thuringiensis Flg22 EHFATGKKLNHASDNPANVAIV SEQ
ID NO: 247 Bacillus thuringiensis serovar konkukian strain 97-27
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 248 Bacillus thuringiensis
serovar konkukian strain 97-27 Flg22 EHLATGKKLNHASDNPANIVIV SEQ ID
NO: 249 Bacillus thuringiensis serovar thuringiensis strain IS5056
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 250 Bacillus thuringiensis
serovar thuringiensis strain IS5056 Flg22 DRLSSGKRINNASDDAAGLAIA
SEQ ID NO: 251 Bacillus thuringiensis strain Bt407 Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 252 Bacillus thuringiensis
serovar chinensis CT-43 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 253
Bacillus thuringiensis serovar canadensis Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 254 Bacillus thuringiensis
serovar galleriae Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 255
Bacillus weihenstephanensis Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO:
256 Bacillus thuringiensis serovar ostriniae Flg22
EHLATGKKLNHASDNPANVAIV SEQ ID NO: 257 Bacillus thuringiensis Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 258 Bacillus thuringiensis Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 259 Bacillus thuringiensis
serovar pondicheriensis Flg22 EHLATGKKLNHASDNPANIVIV SEQ ID NO: 260
Bacillus thuringiensis serovar Berliner Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 261 Bacillus thuringiensis
serovar Berliner Flg22 EHLATGKKLNHASNNPANVAIV SEQ ID NO: 262
Bacillus cereus strain Q1 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO:
263 Bacillus cereus strain Q1 Flg22 EHLATGKKLNHASDNPANIAIV SEQ ID
NO: 264 Bacillus thuringiensis serovar morrisoni Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 265 Bacillus thuringiensis
serovar neoleonensis Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 266
Bacillus thuringiensis serovar morrisoni Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 267 Bacillus thuringiensis
serovar morrisoni Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 268
Bacillus thuringiensis serovar jegathesan Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 269 Bacillus cereus stain ATCC
10987 Flg22 from Flagellin A DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 270
Bacillus thuringiensis serovar monterrey Flg22
EHLATGKKLNNASDNPANIAIV SEQ ID NO: 271 Bacillus cereus strain NC7401
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 272 Bacillus cereus strain
NC7401 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 273 Bacillus cereus
strain AH820 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 274 Bacillus
cereus AH187 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 275 Bacillus
cereus Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 276 Bacillus cereus
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 277 Bacillus thuringiensis
Strain HD-771 [51] Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 278
Bacillus thuringiensis serovar sotto [52] Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 279 Bacillus thuringiensis
serovar Novosibirsk Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 280
Bacillus thuringiensis serovar londrina Flg22
EHLATGKKLNHASNNPANIAIV SEQ ID NO: 281 Bacillus cereus strain E33L
Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 282
Bacillus cereus strain E33L Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO:
283 Bacillus cereus strain FRI-35 Flg22 EHLATGKKLNNASDNPANIAIV SEQ
ID NO: 284 Bacillus cereus strain FRI-35 Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 285 Bacillus thuringiensis Flg22
DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 286 Bacillus cereus strain ATCC
4342 Flg22 EHLATGKKLNHASDNPANIAIV SEQ ID NO: 287 Bacillus
thuringiensis Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 288 Bacillus
thuringiensis Flg22 EKLSSGQRINSASDDAAGLAIS SEQ ID NO: 289 Bacillus
aryabhattai Flg22 NRLSSGKQINSASDDAAGLAIA SEQ ID NO: 290 Bacillus
manliponensis Flg22 LRLSSGYRINSAADDAAGLAIS SEQ ID NO: 291
Lysinibacillus sp. strain BF-4 Flg22 LRLSSGYRINSAADDAAGLAIS SEQ ID
NO: 292 Lysinibacillus sp. strain 13S34_air Flg22
GKLSSGLRINGASDDAAGLAIS SEQ ID NO: 293 Paenibacillus sp. strain
HW567 Flg22 DRLSSGKRINNASDDAAGLAIA SEQ ID NO: 294 Bacillus
anthracis Flg22 NRLSSGKRINSAADDAAGLAIA SEQ ID NO: 295 Bacillus
anthracis Flg22 NRLSSGKRINSAADDAAGLAIA SEQ ID NO: 296 Bacillus
anthracis Flg22 NRLSSGKRINSAADDAAGLAIA SEQ ID NO: 297 Bacillus
anthracis Flg22 NRLSSGKRINSAADDAAGLAIA SEQ ID NO: 298 Bacillus
anthracis strain H9401 Flg22 EKLSSGQRINSASDDAAGLAIS SEQ ID NO: 299
Bacillus megaterium strain WSH-002 Flg22 ERLSSGYRINRASDDAAGLAIS SEQ
ID NO: 300 Aneurinibacillus sp. XH2 SEQ ID NO: Peptide Flg15
Flg15-Bt4Q7 RINSAKDDAAGLAIA SEQ ID NO: 752 Modified FLG15-Bt4Q7;
Syn01 Bacillus thuringiensis strain 4Q7 SEQ ID NO: Peptide FgII-28
FlgII-28-Bt.4Q7 SVSNILLRMRDLANQSANGTNTKGNQAS SEQ ID NO: 301
Bacillus thuringiensis strain 4Q7 FlgII-28
SVSNILLRMRDLANQSANGTNTKGNQAS SEQ ID NO: 302 Bacillus thuringiensis,
strain HD1002 FlgII-28 SVSNILLRMRDLANQSANGTNTKGNQAS SEQ ID NO: 303
Bacillus thuringiensis, strain HD-789 FlgII-28
SVSNILLRMRDLANQSANGTNTKGNQAS SEQ ID NO: 304 Bacillus cereus strain
G9842 FlgII-28 TVTNILQRMRDLAVQSANGTNSNKNRHS SEQ ID NO: 305 Bacillus
thuringiensis serovar indiana strain HD521 FlgII-28
SVSNILLRMRDIANQSANITNTNENKSA SEQ ID NO: 306 Bacillus thuringiensis
strain CTC FlgII-28 SVSNILLRMRDLANQSANGTNTDDNQKA SEQ ID NO: 307
Bacillus thuringiensis serovar yunnanensis strain IEBC-T20001
FlgII-28 SVSNILLRMRDLANQSANGTNTDENKAA SEQ ID NO: 308 Bacillus
thuringiensis serovar tolworthi FlgII-28
SVSNILLRMRDIANQSANGTNTDKNQVA SEQ ID NO: 309 Bacillus cereus strain
FM1 FlgII-28 TVTNILQRMRDVAVQSANGTNSNKNRDS SEQ ID NO: 310 Bacillus
cereus strain FM1 FlgII-28 SVSNILLRMRDIANQSANGTNTADNQQA SEQ ID NO:
311 Bacillus thuringiensis strain MC28 FlgII-28
SVSNILLRMRDLANQSASGTNTDKNQAA SEQ ID NO: 312 Bacillus bombysepticus
strain Wang FlgII-28 SVSNILLRMRDLANQSASGTNTDKNQAA SEQ ID NO: 313
Bacillus thuringiensis serovar kenyae FlgII-28
SVSNILLRMRDLANQSASGTNTDKNQAA SEQ ID NO: 314 Bacillus thuringiensis
serovar kenyae FlgII-28 SVSNILLRMRDLANQSANGTNTGDNQKA SEQ ID NO: 315
Bacillus cereus FlgII-28 TNILQRMRDLAVQSANGTNSNKNRDSLN SEQ ID NO:
316 Bacillus cereus FlgII-28 TNVLQRMRDVAVQSANGTNLNKNRDSLN SEQ ID
NO: 317 Bacillus thuringiensis serovar finitimus strain YBT-020
FlgII-28 SVSNILLRMRDIANQSANGTNTDSNKSA SEQ ID NO: 318 Bacillus
thuringiensis serovar finitimus strain YBT-020 FlgII-28
SVSNILLRMRDLANQSANGTNTAENKAA SEQ ID NO: 319 Bacillus cereus stain
B4264 FlgII-28 SVSNILLRMRDIANQSANGTNTSDNQKA SEQ ID NO: 320 Bacillus
thuringiensis serovar nigeriensis FlgII-28
SVSNILLRMRDIANQSANGTNTADNQQA SEQ ID NO: 321 Bacillus thuringiensis
FlgII-28 TVMNILQRMRDLAVQSANGTNSNKNRDS SEQ ID NO: 322 Bacillus
thuringiensis serovar konkukian strain 97-27 FlgII-28
SVSNILLRMRDIANQSANGTNTADNQQA SEQ ID NO: 323 Bacillus thuringiensis
serovar konkukian strain 97-27 FlgII-28
TVTNILQHMRDFAIQSANGTNSNTNRDS SEQ ID NO: 324 Bacillus thuringiensis
serovar thuringiensis strain IS5056 FlgII-28
SVSNILLRMRDISNQSANGTNTDKNQSA SEQ ID NO: 325 Bacillus thuringiensis
serovar thuringiensis strain IS5056 FlgII-28
SVSNILLRMRDISNQSANGTNTDKNQSA SEQ ID NO: 326 Bacillus thuringiensis
strain Bt407 FlgII-28 SVSNILLRMRDISNQSANGTNTDKNQSA SEQ ID NO: 327
Bacillus thuringiensis serovar chinensis CT-43 FlgII-28
SVSNILLRMRDLANQSANGTNTNENQAA SEQ ID NO: 328 Bacillus thuringiensis
serovar canadensis FlgII-28 SVSNILLRMRDLANQSANGTNTNENQAA SEQ ID NO:
329 Bacillus thuringiensis serovar galleriae FlgII-28
SVSNILLRMRDLSNQSANGTNTDENQQA SEQ ID NO: 330 Bacillus
weihenstephanensis FlgII-28 SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID NO:
331 Bacillus thuringiensis serovar ostriniae FlgII-28
TVANILQRMRDLAVQSSNDTNSNKNRDS SEQ ID NO: 332 Bacillus thuringiensis
FlgII-28 SVSNILLRMRDLANQSANGTNTDDNQKA SEQ ID NO: 333 Bacillus
thuringiensis FlgII-28 SVSNILLRMRDLANQSANGTNTDDNQKA SEQ ID NO: 334
Bacillus thuringiensis serovar pondicheriensis FlgII-28
TVTNILQHMRDFAIQSANGTNSNTNRDS SEQ ID NO: 335 Bacillus thuringiensis
serovar Berliner FlgII-28 SVSNILLRMRDISNQSANGTNTDKNQSA SEQ ID NO:
336 Bacillus thuringiensis serovar Berliner FlgII-28
TVTNVLQRMRDVAVQSANGTNSSKNRDS SEQ ID NO: 337 Bacillus cereus strain
Q1 FlgII-28 SVSNILLRMRDIANQSANGTNTDKNQVA SEQ ID NO: 338 Bacillus
cereus strain Q1
FlgII-28 TVMNILQRMRDLAIQSANSTNSNKNRDS SEQ ID NO: 339 Bacillus
thuringiensis serovar morrisoni FlgII-28
SVSNILLRMRDIANQSANGTNTSDNQKA SEQ ID NO: 340 Bacillus thuringiensis
serovar neoleonensis FlgII-28 SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID
NO: 341 Bacillus thuringiensis serovar morrisoni FlgII-28
SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID NO: 342 Bacillus thuringiensis
serovar morrisoni FlgII-28 SVSNILLRMRDIANQSANGTNTNGNQAA SEQ ID NO:
343 Bacillus thuringiensis serovar jegathesan FlgII-28
SVSNILLRMRDIANQSANGTNTDKNQAA SEQ ID NO: 344 Bacillus cereus stain
ATCC 10987 FlgII-28 from Flagellin A SVSNILLRMRDLANQSANGTNTNENQAA
SEQ ID NO: 345 Bacillus thuringiensis serovar monterrey FlgII-28
TVTNVLQRMRDLAVQSANDTNSNKNRDS SEQ ID NO: 346 Bacillus cereus strain
NC7401 FlgII-28 SVSNILLRMRDLANQSANGTNTNENKAA SEQ ID NO: 347
Bacillus cereus strain NC7401 FlgII-28 SVSNILLRMRDLANQSANGTNTSDNQAA
SEQ ID NO: 348 Bacillus cereus strain AH820 FlgII-28
SVSNILLRMRDLANQSANGTNTNENKAA SEQ ID NO: 349 Bacillus cereus AH187
FlgII-28 SVSNILLRMRDLANQSANGTNTNENKAA SEQ ID NO: 350 Bacillus
cereus FlgII-28 SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID NO: 351
Bacillus cereus FlgII-28 SVSNILLRMRDLANQSASETNTSKNQAA SEQ ID NO:
352 Bacillus thuringiensis Strain HD-771 [51] FlgII-28
SVSNILLRMRDLANQSASETNTSKNQAA SEQ ID NO: 353 Bacillus thuringiensis
serovar sotto [52] FlgII-28 SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID NO:
354 Bacillus thuringiensis serovar Novosibirsk FlgII-28
SVSNILLRMRDLANQSANGTNTSENQAA SEQ ID NO: 355 Bacillus thuringiensis
serovar londrina FlgII-28 TVTNILQRMRDLAVQSANVTNSNKNRNS SEQ ID NO:
356 Bacillus cereus strain E33L FlgII-28
SVSNILLRMRDLANQSANGTNTDKNQGA SEQ ID NO: 357 Bacillus cereus strain
E33L FlgII-28 SVSNILLRMRDLANQSANGTNTDKNQAA SEQ ID NO: 358 Bacillus
cereus strain FRI-35 FlgII-28 TVTNVLQRMRDLAVQSANGTNSNKNRDS SEQ ID
NO: 359 Bacillus cereus strain FRI-35 FlgII-28
SVSNILLRMRDIANQTANGTNKDTDIEA SEQ ID NO: 360 Bacillus thuringiensis
FlgII-28 SVSNILLRMRDIANQTANGTNKDTDIEA SEQ ID NO: 361 Bacillus
cereus strain ATCC 4342 FlgII-28 TVMNILQRMRDLAIQSANSTNSNKNRDS SEQ
ID NO: 362 Bacillus thuringiensis FlgII-28
SVSNILLRMRDIANQSANGTNTGDNQKA SEQ ID NO: 363 Bacillus thuringiensis
FlgII-28 ETHDILQRMRELVVQAGNGTNKTEDLDA SEQ ID NO: 364 Bacillus
aryabhattai FlgII-28 SVSNILLRMRDLATQSATGTNQGNDRES SEQ ID NO: 365
Bacillus manliponensis FlgII-28 ETHAIVQRMRELAVQAATDTNTDDDRAK SEQ ID
NO: 366 Lysinibacillus sp. strain BF-4 FlgII-28
ETHAIVQRMRELAVQAATDTNTDDDRAK SEQ ID NO: 367 Lysinibacillus sp.
strain 13S34_air FlgII-28 EIHSMLQRMNELAVQASNGTYSGSDRLN SEQ ID NO:
368 Paenibacillus sp. strain HW567 FlgII-28
SVSNILLRMRDLANQSANGTNTKENQDA SEQ ID NO: 369 Bacillus anthracis
FlgII-28 SVSNILTRMRDIAVQSSNGTNTAENQSA SEQ ID NO: 370 Bacillus
anthracis FlgII-28 SVSNILTRMRDIAVQSSNGTNTAENQSA SEQ ID NO: 371
Bacillus anthracis FlgII-28 SVSNILTRMRDIAVQSSNGTNTAENQSA SEQ ID NO:
372 Bacillus anthracis FlgII-28 SVSNILTRMRDIAVQSSNGTNTAENQSA SEQ ID
NO: 373 Bacillus anthracis strain H9401 FlgII-28
ETHDILQRMRELVVQAGNGTNKTEDLDA SEQ ID NO: 374 Bacillus megaterium
strain WSH-002 FlgII-28 EIHEMLQRMRELAVQAANGTYSDKDKKA SEQ ID NO: 375
Aneurinibacillus sp. XH2
[0139] Retro-Inverso Flagellin-Associated Polypeptides
[0140] Bioactive Flg polypeptide(s) useful for priming can be
created in a non-natural isomeric or retro-inverso (RI) form.
[0141] The retro-inverso Flg polypeptides can exhibit enhanced
binding affinity for the FLS receptor protein(s). Plant flagellin
receptors, like FLS2, can recognize a retro inverso Flg polypeptide
fragment such as either Flg22 or FlgII-28 located within the
N-terminal conserved domain of flagellin. The retro-inverso forms
of these Flg polypeptides are provided as biologically active
forms, which can recognize and interact with the Flg-associated or
FLS receptor protein on the surface of the plant cell membrane.
[0142] Retro-inverso Flg polypeptides can possess an increased
activity and stability to proteolytic degradation at the plant
membrane surface. For example, retro inverso forms of Bacillus
Flg22 or FlgII-28 polypeptides can increase activity and stability
of the Flg polypeptide(s) and increase protection against
proteolytic degradation at the plant surface or root surface. The
retro inverso forms also exhibit enhanced stability when applied in
a field, or on or in a soil.
[0143] Retro-inverso polypeptides are topological mirror images of
the native structures of the parent polypeptide. Retro inverso
synthetic forms of the polypeptide sequences are created by
reversing the polypeptide sequences and using retro-all-D or
retro-enantio-peptides. The all D-chain amino acid Flg
polypeptide(s) adopts a "mirror image" of the three-dimensional
structure of its related L-peptide or L-chain amino.
[0144] This is further accomplished by creating a retro-inverso
alteration of any of the parent Flg polypeptide derived from
Bacillus or other Eubacteria in Table 3. Retro-inverso polypeptides
that were designed to the Flg22 (RI Flg22: SEQ ID NOs: 376-450),
and FlgII-28 (RI-FlgII-28: SEQ ID NOs: 451-525) are provided in
Table 4. Retro inverso forms of Ec.Flg22 (SEQ ID NO: 526) and
EcFlg15 (SEQ ID NO: 529) as provided in Table 5 were also created
from E. coli derived sequences.
[0145] The polypeptide can include a retro inverso Flg22
polypeptide.
[0146] The polypeptide can comprise a retro inverso FlgII-28
polypeptide.
[0147] Any of the flagellin-associated bioactive priming
polypeptides comprising Bacillus or from other Eubacteria Flg22 or
FlgII-28 polypeptides in Table 3 can be used in their
retro-inversed forms (referenced in Table 4).
[0148] Retro inverso forms of the Flg bioactive priming
polypeptides as referenced herein can be provided in any of three
forms where the inversion of amino acid chirality contains the
normal-all-D (inverso), all-L (retro) and/or retro-all-D
(retro-inverso) or a combination of these forms to achieve the
desired phenotypes in a plant.
[0149] The Bacillus-derived L-Flg22 and L-FlgII-28 polypeptides in
Table 3 and the E.c. native L-Flg22 and L-Flg15 polypeptides in
Table 5 were synthetically generated via retro-inverso engineering
to form retro-inverso D-Flg22 polypeptide (SEQ ID NO: 376-450),
D-FlgII-28 (SEQ ID NO: 451-525), and E.c. D-Flg22 polypeptide (SEQ
ID NO: 527, 529).
[0150] The inversion of amino acid chirality (all-L to all-D) for
Bt.4Q7 Flg22 (SEQ ID NO: 376), which is provided as a small linear
polypeptide fragment and is referred to as a retro inverso
modification was achieved by a reversal of the direction of the
polypeptide backbone and described below.
TABLE-US-00004
(.sup.DA.sup.DIA.sup.DL.sup.DG.sup.DA.sup.DA.sup.DD.sup.DD.sup.DS.sup.DA.-
sup.DS.sup.DN.sup.DI.sup.DR.sup.DK.sup.DG.sup.DS.sup.DS.sup.DL.sup.DR.sup.-
DD)
[0151] The retro inverso all D-chain amino acid Flg22 polypeptide
adopts a "mirror image" of the three-dimensional structure of its
related native L-Bt.4Q7Flg 22 polypeptide and this all L-chain has
an equivalent mirror image to the all D Bt.4Q7Flg22 polypeptide.
All L-amino acid residues are replaced by their D-enantiomers
leading to all D-peptides or retro all D-isomer-peptides containing
amide linkages. The native L-amino acid chain form of Bt.4Q7 Flg22
polypeptide chain reversed to generate the retro-inverso synthetic
all-D confirmation that is prepared by replacing all the L-amino
acid residues with their corresponding D-enantiomers.
[0152] FIG. 1 provides a diagrammatic representation of a natural
(all L) Bt.4Q7 Flg22 and its retro inverso or mirror image to form
an all D Bt.4Q7 Flg22 enantiomeric polypeptide. The retro-inverso
Flg polypeptide that corresponds to Bt.4Q7 Flg22 (SEQ ID NO: 226)
is described as SEQ ID NO: 376.
[0153] In the case of short polypeptides, such as Flg22, Flg15 and
FlgII-28, the mirroring of the side chain positions in a
conformational change from L-to-D conversion states results in a
mirroring of symmetry transformations of the side chains as
well.
[0154] Retro-all-D analogues have been found to possess biological
activity (Guptasarma, "Reversal of peptide backbone direction may
result in mirroring of protein structure, FEBS Letters 310:
205-210, 1992). The retro-inverso D-Flg polypeptide(s) can assume a
side chain topology in its extended conformation that is similar to
a corresponding native L-Flg polypeptide sequence, thus emulating
biological activities of the native L-parent molecule while fully
resistant to proteolytic degradation thus increasing stability when
the polypeptide contacts the plant or the surrounding
environment.
[0155] Retro-inverso Flg bioactive priming polypeptides are
described in Table 4 or Table 5. Retro inverso Flg-associated
bioactive priming polypeptides provided in Table 4 were selected
for their enhanced activity and stability and their ability to
survive under varying conditions and environments. Based on their D
enantiomer nature, they are more resistant to proteolytic
degradation and can survive and exist in harsher environmental
conditions.
TABLE-US-00005 TABLE 4 Retro-inverso flagellin polypeptides from
Flg22 and FlgII-28 from Bacillus SEQ ID NO: Peptide Flg22 RI
Bt.4Q7Flg22 AIALGAADDSASNIRKGSSLRD SEQ ID NO: 376 Bacillus
thuringiensis strain 4Q7 RI Flg22 AIALGAADDSASNIRKGSSLRD SEQ ID NO:
377 Bacillus thuringiensis, strain HD1002 RI Flg22
AIALGAADDASNIRKGSSLRD SEQ ID NO: 378 Bacillus thuringiensis, strain
HD-789 RI Flg22 AIALGAADDSASNIRKGSSLRD SEQ ID NO: 379 Bacillus
cereus strain G9842 RI Flg22 VIANAPNDSANNLKKGTALHE SEQ ID NO: 380
Bacillus thuringiensis serovar indiana strain HD521 RI Flg22
TAIAGAADDSANNIRKGSSLRD SEQ ID NO: 381 Bacillus thuringiensis strain
CTC RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 382 Bacillus
thuringiensis serovaryunnanensis strain IEBC-T20001 RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 383 Bacillus thuringiensis
serovar tolworthi RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 384
Bacillus cereus strain FM1 RI Flg22 VIAVNAPNDSAHNLKKGTALHE SEQ ID
NO: 385 Bacillus cereus strain FM1 RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 386 Bacillus thuringiensis strain MC28 RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 387 Bacillus bombysepticus strain
Wang RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 388 Bacillus
thuringiensis serovar kenyae RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID
NO: 389 Bacillus thuringiensis serovar kenyae RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 390 Bacillus cereus RI Flg22
VIAINAPNDASNNLKKGTALHE SEQ ID NO: 391 Bacillus cereus RI Flg22
VIANAPNDSAHNLKKGTALHE SEQ ID NO: 392 Bacillus thuringiensis serovar
finitimus strain YBT-020 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO:
393 Bacillus thuringiensis serovar finitimus strain YBT-020 RI
Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 394 Bacillus cereus stain
B4264 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 395 Bacillus
thuringiensis serovar nigeriensis RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 396 Bacillus thuringiensis RI Flg22
VIANAPNDSAHNLKKGTAFHE SEQ ID NO: 397 Bacillus thuringiensis serovar
konkukian strain 97-27 RI Flg22 AIALGAADDSANNRKGSSLRD SEQ ID NO:
398 Bacillus thuringiensis serovar konkukian strain 97-27 RI Flg22
VIVINAPNDSAHNLKKGTALHE SEQ ID NO: 399 Bacillus thuringiensis
serovar thuringiensis strain IS5056 RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 400 Bacillus thuringiensis serovar thuringiensis strain
IS5056 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 401 Bacillus
thuringiensis strain Bt407 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID
NO: 402 Bacillus thuringiensis serovar chinensis CT-43 RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 403 Bacillus thuringiensis
serovar canadensis RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 404
Bacillus thuringiensis serovar galleriae RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 405 Bacillus weihenstephanensis
RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 406 Bacillus
thuringiensis serovar ostriniae RI Flg22 VIANAPNDSAHNLKKGTALHE SEQ
ID NO: 407 Bacillus thuringiensis RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 408 Bacillus thuringiensis RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 409 Bacillus thuringiensis
serovar pondicheriensis RI Flg22 VIVINAPNDASHNLKKGTALHE SEQ ID NO:
410 Bacillus thuringiensis serovar Berliner RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 411 Bacillus thuringiensis
serovar Berliner RI Flg22 VIAVANPNNSAHNLKKGTALHE SEQ ID NO: 412
Bacillus cereus strain Q1 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID
NO: 413 Bacillus cereus strain Q1 RI Flg22 VIANAPNDSAHNLKKGTALHE
SEQ ID NO: 414 Bacillus thuringiensis serovar morrisoni RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 415 Bacillus thuringiensis
serovar neoleonensis RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 416
Bacillus thuringiensis serovar morrisoni RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 417 Bacillus thuringiensis
serovar morrisoni RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 418
Bacillus thuringiensis serovar jegathesan RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 419 Bacillus cereus stain ATCC
10987 RI Flg22 from Flagellin A AIALGAADDASNNIRKGSSLRD SEQ ID NO:
420 Bacillus thuringiensis serovar monterrey RI Flg22
VIANAPNDSANNLKKGTALHE SEQ ID NO: 421 Bacillus cereus strain NC7401
RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 422 Bacillus cereus
strain NC7401 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 423
Bacillus cereus strain AH820 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID
NO: 424 Bacillus cereus AH187 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ
ID NO: 425 Bacillus cereus RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID
NO: 426 Bacillus cereus RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO:
427 Bacillus thuringiensis Strain HD-771 [51] RI Flg22
AIALGAADDANNIRKGSSLRD SEQ ID NO: 428 Bacillus thuringiensis serovar
sotto [52] RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 429 Bacillus
thuringiensis serovar Novosibirsk RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 430 Bacillus thuringiensis serovar londrina RI Flg22
VIAINAPNNSAHNLKKGTALHE SEQ ID NO: 431 Bacillus cereus strain E33L
RI Flg22 AIALGAADDSANNIRKGSSLRD
SEQ ID NO: 432 Bacillus cereus strain E33L RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 433 Bacillus cereus strain FRI-35
RI Flg22 VIAINAPNDSANNLKKGTALHE SEQ ID NO: 434 Bacillus cereus
strain FRI-35 RI Flg22 AIALGAADDSANNIRKGSSLRD SEQ ID NO: 435
Bacillus thuringiensis RI Flg22 AIALGAADDANNIRKGSSLRD SEQ ID NO:
436 Bacillus cereus strain ATCC 4342 RI Flg22 VIANAPNDSAHNLKKGTALHE
SEQ ID NO: 437 Bacillus thuringiensis RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 438 Bacillus thuringiensis RI
Flg22 SIALGAADDSASNIRQGSSLKE SEQ ID NO: 439 Bacillus aryabhattai RI
Flg22 AIALGAADDSASNIQKGSSLRN SEQ ID NO: 440 Bacillus manliponensis
RI Flg22 SIALGAADDAASNIRYGSSLRL SEQ ID NO: 441 Lysinibacillus sp.
strain BF-4 RI Flg22 SIALGAADDAASNIRYGSSLRL SEQ ID NO: 442
Lysinibacillus sp. strain 13S34_air RI Flg22 SIAGLAADDSAGNIRLGSSLKG
SEQ ID NO: 443 Paenibacillus sp. strain HW567 RI Flg22
AIALGAADDSANNIRKGSSLRD SEQ ID NO: 444 Bacillus anthracis RI Flg22
AIALGAADDAASNIRKGSSLRN SEQ ID NO: 445 Bacillus anthracis RI Flg22
AIALGAADDAASNIRKGSSLRN SEQ ID NO: 446 Bacillus anthracis RI Flg22
AIALGAADDAASNIRKGSSLRN SEQ ID NO: 447 Bacillus anthracis RI Flg22
AIALGAADDAASNIRKGSSLRN SEQ ID NO: 448 Bacillus anthracis strain
H9401 RI Flg22 SIALGAADDSASNIRQGSSLKE SEQ ID NO: 449 Bacillus
megaterium strain WSH-002 RI Flg22 SIALGAADDSARNIRYGSSLRE SEQ ID
NO: 450 Aneurinibacillus sp. XH2 SEQ ID NO: Peptide Flg15 RI
Flg15-8t4Q7 AIALGAADDKASNIR SEQ ID NO: 767 Modified FLG15-Bt4Q7;
Syn01 Bacillus thuringiensis strain 4Q7 SEQ ID NO: Peptide FlgII-28
RI FlgII-28-8L4Q7 SAQNGKTNTGNASQNALDRMRLLINSVS SEQ ID NO: 451
Bacillus thuringiensis strain 4Q7 RI FlgII-28
SAQNGKTNTGNASQNALDRMRLLINSVS SEQ ID NO: 452 Bacillus thuringiensis,
strain HD1002 RI FlgII-28 SAQNGKTNTGNASQNALDRMRLLINSVS SEQ ID NO:
453 Bacillus thuringiensis, strain HD-789 RI FlgII-28
SAQNGKTNTGNASQNALDRMRLLINSVS SEQ ID NO: 454 Bacillus cereus strain
G9842 RI FlgII-28 SHRNKNSNTGNASQVALDRMRQLINTVT SEQ ID NO: 455
Bacillus thuringiensis serovar indiana strain HD521 RI FlgII-28
ASKNENTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 456 Bacillus thuringiensis
strain CTC RI FlgII-28 AKQNDDTNTGNASQNALDRMRLLINSVS SEQ ID NO: 457
Bacillus thuringiensis serovaryunnanensis strain IEBC-T20001 RI
FlgII-28 AAKNEDTNTGNASQNALDRMRLLINSVS SEQ ID NO: 458 Bacillus
thuringiensis serovar tolworthi RI FlgII-28
LAVQNKDTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 459 Bacillus cereus strain
FM1 RI FlgII-28 SDRNKNSNTGNASQVAVDRMRQLINTVT SEQ ID NO: 460
Bacillus cereus strain FM1 RI FlgII-28 AQQNDATNTGNASQNAIDRMRLLINSVS
SEQ ID NO: 461 Bacillus thuringiensis strain MC28 RI FlgII-28
AAQNKDTNTGSASQNALDRMRLLINSVS SEQ ID NO: 462 Bacillus bombysepticus
strain Wang RI FlgII-28 AAQNKDTNTGSASQNALDRMRLLINSVS SEQ ID NO: 463
Bacillus thuringiensis serovar kenyae RI FlgII-28
AAQNKDTNTGSASQNALDRMRLLINSVS SEQ ID NO: 464 Bacillus thuringiensis
serovar kenyae RI FlgII-28 AKQNDGTNTGNASQNALDRMRLLINSVS SEQ ID NO:
465 Bacillus cereus RI FlgII-28 NLSDRNKNSNTGNASQVALDRMRQLINT SEQ ID
NO: 466 Bacillus cereus RI FlgII-28 NLSDRNKNLNTGNASQVAVDRMRQLVNT
SEQ ID NO: 467 Bacillus thuringiensis serovar finitimus strain
YBT-020 RI FlgII-28 ASKNSDTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 468
Bacillus thuringiensis serovar finitimus strain YBT-020 RI FlgII-28
AAKNEATNTGNASQNALDRMRLLINSVS SEQ ID NO: 469 Bacillus cereus stain
B4264 RI FlgII-28 AKQNDSTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 470
Bacillus thuringiensis serovar nigeriensis RI FlgII-28
AQQNDATNTGNASQNAIDRMRLLINSVS SEQ ID NO: 471 Bacillus thuringiensis
RI FlgII-28 SDRNKNSNTGNASQVALDRMRQLINMVT SEQ ID NO: 472 Bacillus
thuringiensis serovar konkukian strain 97-27 RI FlgII-28
AQQNDATNTGNASQNAIDRMRLLINSVS SEQ ID NO: 473 Bacillus thuringiensis
serovar konkukian strain 97-27 RI FlgII-28
SDRNTNSNTGNASQIAFDRMHQLINTVT SEQ ID NO: 474 Bacillus thuringiensis
serovar thuringiensis strain IS5056 RI FlgII-28
ASQNKDTNTGNASQNSIDRMRLLINSVS SEQ ID NO: 475 Bacillus thuringiensis
serovar thuringiensis strain IS5056 RI FlgII-28
ASQNKDTNTGNASQNSIDRMRLLINSVS SEQ ID NO: 476 Bacillus thuringiensis
strain Bt407 RI FlgII-28 ASQNKDTNTGNASQNSISRMRLLINSVS SEQ ID NO:
477 Bacillus thuringiensis serovar chinensis CT-43 RI FlgII-28
AAQNENTNTGNASQNALDRMRLLINSVS SEQ ID NO: 478 Bacillus thuringiensis
serovar canadensis RI FlgII-28 AQQNEDTNTGNASQNSLDRMRLLINSVS SEQ ID
NO: 479 Bacillus thuringiensis serovar galleriae RI FlgII-28
AQQNEDTNTGNASQNSLDRMRLLINSVS SEQ ID NO: 480 Bacillus
weihenstephanensis RI FlgII-28 AKQNDGTNTGNASQNAIDRMRLLINSVS SEQ ID
NO: 481 Bacillus thuringiensis serovar ostriniae RI FlgII-28
SDRNKNSNTDNSSQVALDRMRQLINAVT SEQ ID NO: 482 Bacillus thuringiensis
RI FlgII-28 AKQNDDTNTGNASQNALDRMRLLINSVS SEQ ID NO: 483 Bacillus
thuringiensis RI FlgII-28 AKQNDDTNTGNASQNALDRMRLLINSVS SEQ ID NO:
484 Bacillus thuringiensis serovar pondicheriensis RI FlgII-28
SDRNTNSNTGNASQIAFDRMHQLINTVT SEQ ID NO: 485 Bacillus thuringiensis
serovar Berliner RI FlgII-28 ASQNKDTNTGNASQNSIDRMRLLINSVS SEQ ID
NO: 486 Bacillus thuringiensis serovar Berliner RI FlgII-28
SDRNKSSNTGNASQVAVDRMRQLVNTVT SEQ ID NO: 487 Bacillus cereus strain
Q1 RI FlgII-28 AVQKDTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 488 Bacillus
cereus strain Q1
RI FlgII-28 SDRNKNSNTSNASQIALDRMRQLINMVT SEQ ID NO: 489 Bacillus
thuringiensis serovar morrisoni RI FlgII-28
AKQNDSTNIGNASQNAIDRMRLLINSVS SEQ ID NO: 490 Bacillus thuringiensis
serovar neoleonensis RI FlgII-28 AKQNDGTNTFNASQNAIDRMRLLINSVS SEQ
ID NO: 491 Bacillus thuringiensis serovar morrisoni RI FlgII-28
AKQNDGTNTFNASQNAIDRMRLLINSVS SEQ ID NO: 492 Bacillus thuringiensis
serovar morrisoni RI FlgII-28 AAQNGNTNTFNASQNAIDRMRLLINSVS SEQ ID
NO: 493 Bacillus thuringiensis serovar jegathesan RI FlgII-28
AAQNKDTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 494 Bacillus cereus stain
ATCC 10987 RI FlgII-28 from Flagellin A
AAQNENTNTGNASQNALDRMRLLINSVS SEQ ID NO: 495 Bacillus thuringiensis
serovar monterrey RI FlgII-28 SDRNKNSNTDNASQVALDRMRQLVNTVT SEQ ID
NO: 496 Bacillus cereus strain NC7401 RI FlgII-28
AAKNENTNTGNASQNALDRMRLLINSVS SEQ ID NO: 497 Bacillus cereus strain
NC7401 RI FlgII-28 AAQNDSTNTGNASQNALDRMRLLINSVS SEQ ID NO: 498
Bacillus cereus strain AH820 RI FlgII-28
AAKNENTNTGNASQNALDRMRLLINSVS SEQ ID NO: 499 Bacillus cereus AH187
RI FlgII-28 AAKNENTNTGNASQNALDRMRLLINSVS SEQ ID NO: 500 Bacillus
cereus RI FlgII-28 AKQNDGTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 501
Bacillus cereus RI FlgII-28 AAQNKSTNTESASQNALDRMRLLINSVS SEQ ID NO:
502 Bacillus thuringiensis Strain HD-771 [51] RI FlgII-28
AAQNKSTNTESASQNALDRMRLLINSVS SEQ ID NO: 503 Bacillus thuringiensis
serovar sotto [52] RI FlgII-28 AKQNDGTNTGNASQNAIDRMRLLINSVS SEQ ID
NO: 504 Bacillus thuringiensis serovar Novosibirsk RI FlgII-28
AAQNESTNTGNAQNALDRMRLLINSVS SEQ ID NO: 505 Bacillus thuringiensis
serovar londrina RI FlgII-28 SNRNKNSNTVNASQVALDRMRQLINTVT SEQ ID
NO: 506 Bacillus cereus strain E33L RI FlgII-28
AGQNKDTNTNASQNALDRMRLLINSVS SEQ ID NO: 507 Bacillus cereus strain
E33L RI FlgII-28 AAQNKDTNTGNASQNALDRMRLLINSVS SEQ ID NO: 508
Bacillus cereus strain FRI-35 RI FlgII-28
SDRNKNSNTGNASQVALDRMRQLVNTVT SEQ ID NO: 509 Bacillus cereus strain
FRI-35 RI FlgII-28 AEIDTDKNTGNATQNAIDRMRLLINSVS SEQ ID NO: 510
Bacillus thuringiensis RI FlgII-28 AEIDTDKNTGNATQNAIDRMRLLINSVS SEQ
ID NO: 511 Bacillus cereus strain ATCC 4342 RI FlgII-28
SDRNKNSNTSNASQIALDRMRQLINMT SEQ ID NO: 512 Bacillus thuringiensis
RI FlgII-28 AKQNDGTNTGNASQNAIDRMRLLINSVS SEQ ID NO: 513 Bacillus
thuringiensis RI FlgII-28 ADLDETKNTGNGAQVVLERMRQLIDHTE SEQ ID NO:
514 Bacillus aryabhattai RI FlgII-28 SERDNGQNTGTAQTALDRMRLLINSVS
SEQ ID NO: 515 Bacillus manliponensis RI FlgII-28
KARDDDTNTDTAAQVALERMRQVIAHTE SEQ ID NO: 516 Lysinibacillus sp.
strain BF-4 RI FlgII-28 KARDDDTNTDTAAQVALERMRQVIAHTE SEQ ID NO: 517
Lysinibacillus sp. strain 13S34_air RI FlgII-28
NLRDSGSYTGNSAQVALENMRQLMSHIE SEQ ID NO: 518 Paenibacillus sp.
strain HW567 RI FlgII-28 ADQNEKTNTGNASQNALDRMRLLINSVS SEQ ID NO:
519 Bacillus anthracis RI FlgII-28 ASQNEATNTGNSSQVAIDRMRTLINSVS SEQ
ID NO: 520 Bacillus anthracis RI FlgII-28
ASQNEATNTGNSSQVAIDRMRTLINSVS SEQ ID NO: 521 Bacillus anthracis RI
FlgII-28 ASQNEATNTGNSSQVAIDRMRTLINSVS SEQ ID NO: 522 Bacillus
anthracis RI FlgII-28 ASQNEATNTGNSSQVIADRMRTLINSVS SEQ ID NO: 523
Bacillus anthracis strain H9401 RI FlgII-28
ADLDETKNTGNGAQVVLERMRQLIDHTE SEQ ID NO: 524 Bacillus megaterium
strain WSH-002 RI FlgII-28 AKKDKSYTGNAAQVALERMRQLMEHIE SEQ ID NO:
525 Aneurinibacillus sp. XH2
[0156] Flg Sequences from Various Organisms
TABLE-US-00006 TABLE 5 Flagellin-associated Flg22 and Flg15
polypeptides from other organisms Peptide-Amino SEQ ID NO: Acid
Flagellin (Flg22) ERLSSGLRIN SEQ ID NO: 526 SAKDDAAGQA Escherichia
coli IA Flagellin (Retro-Inverso AIAQGAADDK Flg22) ASNIRLGSSL SEQ
ID NO: 527 RE Escherichia coli Flagellin (Flg15) RINSAKDDAA SEQ ID
NO: 528 GQAIA Escherichia coli Flagellin (Retro-Inverso AIAQGAADDK
Flg15) ASNIR SEQ ID NO: 529 Escherichia coli Flagellin (Flg22)
QRLSTGSRIN SEQ ID NO: 530 SAKDDAAGLQ Pseudomonas aeruginosa IA
Flagellin (Retro Inverso AIQLGAADDK Flg22) ASNIRSGTSL SEQ ID NO:
531 RQ Pseudomonas aeruginosa Flagellin (Flg22) QRLSSGLRIN SEQ ID
NO: 532 SAKDDAAGLA Xanthomonas spp. IS X. campestris & X. citri
Flagellin (Retro Inverso SIALGAADDK Flg22) ASNIRLGSSL SEQ ID NO:
533 RQ Xanthomonas spp. X. campestris & X. citri Flagellin
(Flg22) QRLSSGLRIN SEQ ID NO: 534 SAKDDAAGQA Erwinia amylovora IS
Flagellin (Retro Inverso SIAQGAADDK Flg22) ASNIRLGSSL SEQ ID NO:
535 RQ Erwinia amylovora Flagellin (Flg22) TRLSSGKRIN SEQ ID NO:
536 SAADDAAGLA Burkholderia phytofirmans IS Flagellin (Retro
Inverso SIALGAADDA Flg22) ASNIRKGSSL SEQ ID NO: 537 RT Burkholderia
phytofirmans Flagellin (Flg22) NRLSSGKRIN SEQ ID NO: 538 TAADDAAGLA
Burkholderia ubonensis IS Flagellin (Retro Inverso SIALGAADDA
Flg22) ATNIRKGSSL SEQ ID NO: 539 RN Burkholderia ubonensis
Flagellin (Flg22) TRLSSGLKIN SEQ ID NO: 540 SAKDDAAGLQ Pseudomonas
syringae IA Flagellin (Retro Inverso AIQLGAADDK Flg22) ASNIKLGSSL
SEQ ID NO: 541 RT Pseudomonas syringae Flagellin (Flgll-28)
ESTNILQRMR (SEQ ID NO: 751) ELAVQSRNDS Pseudomonas syringae
NSATDREA Flagellin (Retro Inverso AERDTASNSD Flgll-28) NRSQVALERM
(SEQ ID NO: 768) RQLINTSE Pseudomonas syringae
Sequences that Assist in Directing Flagellins or
Flagellin-Associated Polypeptides to the Plant
[0157] The signature, signal anchor sorting and secretion sequences
can be used separately or together in combination with any of the
flagellin or flagellin-associated polypeptides as described herein.
These assistance sequences are useful for the efficient delivery of
the flagellin polypeptides to the plant cell membrane surface.
Other assistance sequences can also assist with the translocation
of the Flg polypeptide fragment across the plasma membrane.
Delivery of flagellins and flagellin-associated polypeptides to the
plasma membrane surface of a plant (or plant part) can contribute
to downstream signalling processes and result in beneficial
outcomes to a plant or a plant part, such as enhanced plant health
and productivity.
[0158] The polypeptide can further comprise an assistance
polypeptide.
[0159] The assistance polypeptide can comprise a signature
polypeptide, and an amino acid sequence of the signature
polypeptide can comprise any one of SEQ ID NOs: 542-548, listed in
Table 6, or any combination thereof. For example, the amino acid
sequence of the signature polypeptide can comprise SEQ ID NO:
542.
[0160] The assistance polypeptide can comprise a signal anchor
sorting polypeptide, and an amino acid sequence of the signal
anchor sorting polypeptide can comprise any one of SEQ ID NOs:
549-562, listed in Table 7, or any combination thereof. For
example, the amino acid sequence of the signal anchor sorting
polypeptide can comprise SEQ ID NO: 549.
[0161] The flagellin or flagellin-associated polypeptide can be
produced recombinantly by a microorganism. For example, the
microorganism can comprise a Bacillus, a Pseudomonas, a
Paenibacillus, Aneurinibacillus or a Lysinibacillus.
[0162] The assistance polypeptide can comprise a secretion
polypeptide, and an amino acid sequence of the secretion
polypeptide can comprise any one of SEQ ID NOs: 563-570, or any
combination thereof. For example, the amino acid sequence of the
secretion polypeptide can comprise SEQ ID NO: 563.
[0163] These three types of assistance sequences are further
described in Table 6 (N-terminal signature sequences), Table 7
(signal anchor sorting sequences) and Table 8 (secretion
sequences).
[0164] Also provided are "assistance" sequences having conserved
signature (Table 6; SEQ ID NOs: 542-548), signal anchor sorting
(Table 7; SEQ ID NOs: 549-562) and secretion (Table 8; SEQ ID NOs:
563-570) sequences in combination with any of the
flagellin-associated polypeptides as described herein. Particularly
useful are combinations of the signature, signal anchor sorting and
secretion assistance sequences with the native L-Flg polypeptides
(Table 3. SEQ ID NOs: 226-375) or any of the retro inverso Flg22
polypeptides (Table 4. SEQ ID NOs: 376-525) for providing efficient
delivery of the Flg polypeptides to the extracellular plant
membrane surface, such as the surface of a plant or plant part.
[0165] N-Terminal Signature Sequences
[0166] Amino acid "signature" sequences conserved within Bacillus,
Lysinibacillus, Paenibacillus or Aneurinibacillus bacteria (genera)
and other Eubacterial generas can function in targeting flagellin
polypeptides to the appropriate Flg-associated receptor protein(s),
such as FLS receptors that have an exposed binding site at the
plant cell membrane surface and can be used to enhance Flg
polypeptide-receptor binding leading to an increased activation
potential of the Flg-associated receptor(s). Flagellin signature
sequences as identified in Table 6 are useful for targeting and
stably delivering the Flg polypeptides for binding to the FLS or
FLS-like receptor(s) therefore increasing the contact and binding
between the membrane receptor and the Flg polypeptide.
[0167] Conserved N-terminal signature sequences (SEQ ID NO:
542-548) can be used in combination with any of the
flagellin-associated polypeptides as described herein. Of
particular utility are the signature sequences used in combination
with the native L-Flg polypeptides (L-Flg22 SEQ ID NOs: 226-300;
L-FlgII-28 SEQ ID NOs: 301-375) or any of the retro inverso D-Flg
polypeptides (D-Flg22 SEQ ID NOs: 376-450; FlgII-28 SEQ ID NO:
451-525) or any of the other Flg-associated sequences provided in
Table 5 (SEQ ID NOs: 526-541) to provide efficient delivery of the
Flg-associated polypeptides to the plant membrane surface.
[0168] Signature sequences assist with Flg22 and FlgII-28 bioactive
priming polypeptide sequences in binding to the appropriate
Flg-associated receptor(s) in order to activate the receptor(s)
making it functionally active.
TABLE-US-00007 TABLE 6 Flagellin-associated N-terminal signature
sequences Flagellin Signature SEQ ID NO: Sequences SEQ ID NO: 542
GFLN SEQ ID NO: 543 WGFLI SEQ ID NO: 544 MGVLN SEQ ID NO: 545 GVLN
SEQ ID NO: 546 WGFFY SEQ ID NO: 547 LVPFAVWLA SEQ ID NO: 548
AVWLA
[0169] N-Terminal Signal Anchor Sorting Sequences
[0170] Amino acid "signal anchor sorting" sequences conserved
within Bacillus, Lysinibacillus, Aneurinibacillus and Paenibacillus
genera and other Eubacterial generas' bacteria can function in
anchoring and localizing the flagellin-associate polypeptides to
the plant cell membrane surface and assist in high affinity binding
to the appropriate Flg-associated receptor(s) thereby increasing
the activation potential of the bound receptor(s).
[0171] Conserved signal anchor sequences (SEQ ID NO: 549-562; Table
7) are located downstream of the pre-cleaved or full-length coding
or partial coding flagellin sequences, for example, as described
herein (SEQ ID NOs: 1-75; Table 1).
[0172] The signal anchor sorting domains as described herein are
useful in membrane attachment. They can be used to aid in the
localization and binding of Flg-associated polypeptides to a
surface membrane receptor and have some functional similarity at
the amino acid level to proteins that are endosomal (vesicular)
trafficked or destined for targeting to the secretory pathway. Such
signal anchor sorting sequences as described herein that are useful
for anchoring the Flg bioactive priming polypeptides to the plant
cell membrane are also used to enhance the membrane integration of
the bioactive priming Flg polypeptides into the plant cell.
[0173] Such sequences as described in Table 7 may further be
functionally annotated as import receptor signal anchor sequences,
which can be used to improve targeting or delivery and efficient
membrane anchoring of Flg-associated polypeptides to a plant and
assist with membrane integration into the cytosol of the plant
cell.
[0174] Combining the signal anchor sequences (SEQ ID NOs: 549-562;
Table 7) with any of the flagellins or flagellin-associated
bioactive priming polypeptides as described herein is useful to
facilitate the attachment and import of these flagellin-associated
polypeptide(s) into the plant.
[0175] Such signal anchor sorting sequences can be used in
combination with the Flg-associated polypeptides, and are useful
for targeting, efficient membrane anchoring, membrane integration
and Golgi-to-lysosomal/vacuolar trafficking. The signal anchor
sorting sequences are used to stably deliver the Flg polypeptides
to the plant membrane surface and integrally incorporate them into
the plant.
[0176] Such sequences as described herein contain di-leucine amino
acids that are referenced to confer endocytosis functionalities in
plant systems (Pond et al. 1995, "A role for acidic residues in
di-leucine motif-based targeting to the endocytic pathway", Journal
of Biological Chemistry 270: 19989-19997, 1995).
[0177] Such signal anchor sorting sequences as described can also
be used to efficiently deliver systemic signals to infection sites
and stimulate a plant's innate immunity in plant cells.
TABLE-US-00008 TABLE 7 Flagellin-associated signal anchor sorting
sequences SEQ ID NO: Signal Anchor Sequence SEQ ID NO: 549
LLGTADKKIKIQ SEQ ID NO: 550 LLKSTQEIKIQ SEQ ID NO: 551 LLNEDSEVKIQ
SEQ ID NO: 552 LGVAANNTQ SEQ ID NO: 553 LLRMRDLANQ SEQ ID NO: 554
LQRMRDVAVQ SEQ ID NO: 555 LLRMRDISNQ SEQ ID NO: 556 LLRMRDIANQ SEQ
ID NO: 557 LQKQIDYIAGNTQ SEQ ID NO: 558 LLIRLPLD SEQ ID NO: 559
QRMRELAVQ SEQ ID NO: 560 TRMRDIAVQ SEQ ID NO: 561 TRMRDIAVQ SEQ ID
NO: 562 QRMRELVVQ
[0178] C-Terminal Secretion Sequences
[0179] Conserved sequences located in the C-terminus of
flagellin(s) are further described as secretion sequences (SEQ ID
NO: 563-570; Table 8).
[0180] Conserved sequences were identified in the C-terminus of the
Bacillus, Lysinibacillus, and Paenibacillus bacteria (genera) and
other Eubacterial genera derived flagellin proteins and comprise 6
amino acids, for example LGATLN, LGSMIN, or LGAMIN. These sequences
were functionally annotated using BLAST against the bacterial
databases as motifs that have highest homology to secretion
polypeptides. The 6 amino acid conserved polypeptides identified
were found most similar to those found in type III secretion
systems in E. coli. Type III export systems have been cited to be
involved in the translocation of polypeptides across the plant cell
membrane. The filament assembly of flagellin is dependent on the
availability of flagellins to be secreted and may require
chaperones that assist in the secretory process.
[0181] These secretion polypeptides as described herein may be used
in combination with any of the flagellin-associated polypeptides as
described herein to deliver these polypeptides/peptides into the
cytosol of the host plant thus providing beneficial outcomes to a
plant.
TABLE-US-00009 TABLE 8 C-terminal flagellin-associated secretion
sequences Flagellin Secretion SEQ ID NO: polypeptides SEQ ID NO:
563 LGATLN SEQ ID NO: 564 LGATQN SEQ ID NO: 565 LAQANQ SEQ ID NO:
566 LGAMIN SEQ ID NO: 567 LGSMIN SEQ ID NO: 568 MGAYQN SEQ ID NO:
569 LGAYQN SEQ ID NO: 570 YGSQLN
[0182] The signature (SEQ ID NO: 542-548; Table 6), signal anchor
sorting (SEQ ID NO: 549-562; Table 7) and secretion (SEQ ID NO:
563-570; Table 8) sequences as provided herein can be used with any
of the flagellin polypeptides or the flagellin-associated
polypeptides to promote growth and provide health and protective
benefits to a plant or a plant part.
Modification of Flg Polypeptide Sequences Function
[0183] Any of the L or D Flg-associated sequences provided in
Tables 3, 4 or 5 can be similarly modified as fused to any of the
assistance sequences as described in Table 6-8. For one example,
fusion of any of these assistance sequences will present a
modification to the Bt.4Q7Flg22 bioactive priming polypeptide
sequence identified as SEQ ID NO: 226.
Mutations to Flg-Associated Polypeptides to Increase Responsiveness
to Reactive Oxygen Species or Polypeptide Stability
[0184] The polypeptide can comprise a mutant flagellin or
flagellin-associated polypeptide.
[0185] The mutant flagellin or flagellin-associated polypeptide can
be derived from a Bacillus, a Lysinibacillus, a Paenibacillus, or
an Aneurinibacillus genus bacterium. Other polypeptides from other
Eubacterial classes, including Enterobacteraciae, can also be used
in the same fashion. Other generas of interest include Pseudomonas,
Escherichia, Xanthomonas, Burkholderia, Erwinia, and others.
[0186] The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 226, 289, 290, 291, 293, 294, 295, 300, 437, 532, 534, 536,
538, 540, 571-586 and 751-768. For example, the amino acid sequence
of the flagellin or flagellin-associated polypeptide can comprise
any one of SEQ ID NOs: 226, 293, 295, 300, 540, 571, 574 and 752,
or any combination thereof.
[0187] Any bioactive priming polypeptide, whether naturally
occurring or non-natural, can be further modified via chemical
modification to increase performance as well as stability of the
polypeptides. Such bioactive priming polypeptides include flagellin
polypeptides, retro inverso polypeptides, harpin derived
polypeptides, harpin-like derived polypeptides, EF-Tu polypeptides,
thionin polypeptides, RHPP polypeptides, and PSK polypeptides.
Specific sequences that can be chemically modified include SEQ ID
NOs: 226-592, 594-601, 603-749, and 751-766.
[0188] These bioactive priming polypeptides can also be conjugated
to other moieties, including a plant binding domain and a
polypeptide, a plant part binding domain and a polypeptide, and
other carriers such as oils, plastics, beads, ceramic, soil,
fertilizers, pellets, and most structural materials.
[0189] The flagellin or flagellin-associated polypeptide can be
modified chemically on its N or C terminus. Common modification of
the N and C-termini include: acetylation, lipid addition, urea
addition, pyroglutamyl addition, carbamate addition, sulfonamide
addition, alkylamide addition, biotinylation, phosphorylation,
glycosylation, PEGylation, methylation, biotinylation, acid
addition, amide addition, ester addition, aldehyde addition,
hydrazide addition, hydroxyamic acid addition, chloromethyl ketone
addition, or addition of purification tags. These tags can increase
activity of the polypeptides, increase stability, add protease
inhibitor abilities to the polypeptides, block proteases directly,
allow for tracking, and help in binding to plant tissues.
[0190] The flagellin or flagellin-associated polypeptide can be
modified via crosslinking or cyclization. Crosslinking can bind
polypeptides either to each other or to a secondary surface or
moiety to help in delivery or stability of the polypeptides.
Cyclization can be performed, for example, to both increase
activity of the polypeptide as well as prevent protease interaction
with the polypeptide.
[0191] Sequence modifications or mutations can be made to any amino
acid sequence(s) as described in Tables 4 and 5 and replaced with
any of the 20 standard amino acid sequences known in nature or
replaced with a nonstandard or non-canonical amino acid sequence,
such as selenocysteine, pyrrolysine, N-formylmethione, etc. For
example, modifications or mutations can be made to the internal
sequences as shown in SEQ ID NO: 571, to the C-terminis as shown in
SEQ ID NO: 572 or SEQ ID NO: 753, or to the N terminus as shown in
SEQ ID NO: 573 to produce Flg polypeptides with enhanced ROS
activates and increased functionality in a plant or plant part.
Modified polypeptides also can be truncated at the N or C terminus
as shown in SEQ ID NO: 752 (N-terminus truncation) to further
increase functionality in a plant or plant part. Table 9A
summarizes flagellin polypeptides identified that provide modified
ROS activity.
TABLE-US-00010 TABLE 9A Flagellin polypeptides Flg22 identified
from Bacillus or other bacteria with mutations that provide
modified ROS activity SEQ ID NO: Peptide Fig22 Flg22-Bt4Q7
DRLSSGKRINSA KDDAAGLAIA SEQ ID NO:- 571 Bacillus thuringiensis
strain 4Q7 Modified FLG22-Bt4Q7 (S13K); Syn01 Flg22-Bt4Q7
DRLSSGKRINSASDDAAGLQIA SEQ ID NO: 572 Bacillus thuringiensis strain
4Q7 Modified FLG22-Bt4Q7 (A20Q); Syn02 Flg22-Bt4Q7
RLSSGKRINSASDDAAGLAIA SEQ ID NO: 573 Bacillus thuringiensis strain
4Q7 Modified FLG22-Bt4Q7 (D1Q); Syn03 Flg22-Bt4Q7
RLSSGKRINSASDDAAGLAIA SEQ ID NO: 574 Bacillus thuringiensis strain
4Q7 Modified FLG22-Bt4Q7 (D1N); Syn06 Caballeronia
megalochromosomata RLSSGKRINSASDDAAGLAIA SEQ ID NO: 575 Flg22-Bt4Q7
DRLSSG RINSASDDAAGLAIA SEQ ID NO: 576 Bacillus thuringiensis strain
4Q7 Modified FLG22-Bt4Q7 (K7Y); Syn07 Flg22-Bt4Q7 DRLSSG
RINSASDDAAGLAIA SEQ ID NO: 577 Bacillus thuringiensis strain 4Q7
Modified FLG22-Bt4Q7 (K7F); Syn08 Flg22-Br4Q7 DRLSSGKRINSASDD
AGLAIA SEQ ID NO: 578 Bacillus thuringiensis Modified FLG22-Bt4Q7
(A16P); Syn05 Flg22-Bt4Q7 DRLSSG RINSASDDAAGLAIA SEQ ID NO: 579
Bacillus thuringiensis strain 4Q7 Modified FLG22-Bt4Q7 (K7Q); Syn09
Flg22-Br4Q7 DRLSSGKRINSASD AAGLAIA SEQ ID NO: 753 Bacillus
thuringiensis strain 4Q7 Modified FLG22-Bt4Q7 (D15P); Syn04
Flg15-Br4Q7 RINSA DDAAGLAIA SEQ ID NO: 752 Bacillus thuringiensis
N-term Truncated Syn01 Bm.Flg22-B1 NRLSSGKQINSASDDAAGLAIA Bacillus
manliponensis SEQ ID NO: 290 Ba.Flg22-B2 NRLSSGKRINSAADDAAGLAIA
Bacillus anthracis SEQ ID NO: 295 Bc.Flg22-B3
DRLSSGKRINNASDDAAGLAIA Bacillus cereus SEQ ID NO: 294 A.
spp.Flg22-B4 ERLSSGYRINRASDDAAGLAIS Aneurinibacillus spp. XH2 SEQ
ID NO: 300 Ba.Flg22-B5 EKLSSGQRINSASDDAAGLAIS Bacillus aryabhattai
SEQ ID NO: 289 P spp.Flg22-B6 GKLSSGLRINGASDDAAGLAIS Paenibacillus
spp. strain HW567 SEQ ID NO: 293 L spp.Flg22-L1
LRLSSGYRINSAADDAAGLAIS Lysinibacillus spp. SEQ ID NO: 291 L
spp.Flg22-L2 EKLSSGLRINRAGDDAAGLAIS Lysinibacillus spp. SEQ ID NO:
580 L spp.Flg22-L3 EKLSSGYKINRASDDAAGLAIS Lysinibacillus spp. SEQ
ID NO: - 581 L spp.Flg22-L4 LRISSGYRINSAADDPAGLAIS Lysinibacillus
spp. 5G9 SEQ ID NO: 582 Lf.Flg22-L5 LRISTGYRINSAADDPAGLAIS
Lysinibacillus fusiformis SEQ ID NO: 583 Lm.Flg22-L6
EKLSSGFRINRAGDDAAGLAIS Lysinibacillus macroides SEQ ID NO: 584
Lx.Flg22-L6 EKLSSGYKINRAGDDAAGLAIS Lysinibacillus xylanilyticus SEQ
ID NO: 585 Pa.Flg22 QRLSTGSRINSAKDDAAGLQIA Pseudomonas aeruginosa
SEQ ID NO: 530 Ec.Flg22 ERLSSGLRINSAKDDAAGQAIA Escherichia coli SEQ
ID NO: 586 Xcc.Flg22 QRLSSGLRINSAKDDAAGLAIS Xanthomonas campestris
pv campestris strain 305 or (Xanthomonas citri pv. citri) SEQ ID
NO: 532 Ea. Flg22 QRLSSGLRINSAKDDAAGQAIS Erwinia amylovora SEQ ID
NO: 534 Bp. Flg22 TRLSSGKRINSAADDAAGLAIS Burkholderia phytofirmans
strain Ps1A1 SEQ ID NO: 536 Bu.Flg22 NRLSSGKRINTAADDAAGLAIS
Burkholderia ubonensis SEQ ID NO: 538 Ps.Flg22
TRLSSGLKINSAKDDAAGLQIA Pseudomonas syringae pv. actinidiae ICMP
19096 SEQ ID NO: 540
Core Active Domain of Flg22
[0192] The underlined portions of the sequences in Table 9A
represent the core active domain of Flg22. This core domain
comprises, for example, SEQ ID NO: 754 with up to one, two or three
amino acid substitutions (represented by SEQ ID NOs 755-765) that
can promote growth, disease reduction and/or prevention in crops
and ornamental plants. For ease of reference, this core domain is
represented as the consensus sequence having the SEQ ID NO: 766.
The various native and mutant Flg22 polypeptides comprising SEQ ID
NOs 754-765 are described along with the consensus sequence in
Table 9B, below. Therefore, the polypeptides can further comprise a
core sequence. The core sequence can comprise any one of SEQ ID NOs
754-766.
[0193] The polypeptide can also comprise any polypeptide comprising
any one of SEQ ID NOs 1-753 or 767 to 768 wherein the polypeptide
further comprises the core sequence comprising any one of SEQ ID
NOs: 754-766. The inclusion of the core sequence in the polypeptide
or full-length protein of dissimilar function can increase the
bioactive priming activity of the polypeptide.
TABLE-US-00011 TABLE 9B Flg22 core sequence with variants.
Polypeptides comprising SEQ ID NO: FLG22 core sequence core
sequence SEQ ID NO: 754 RINSASDD SEQ ID NO: 226-229 SEQ ID NO: 289
SEQ ID NO: 299 SEQ ID NO: 536 SEQ ID NO: 572-579 SEQ ID NO: 755
RINNASDD SEQ ID NO: 231-234 SEQ ID NO: 236-240 SEQ ID NO: 243-246
SEQ ID NO: 248 SEQ ID NO: 250-256 SEQ ID NO: 258-259 SEQ ID NO: 261
SEQ ID NO: 263 SEQ ID NO: 265-270 SEQ ID NO: 272-280 SEQ ID NO:
282-283 SEQ ID NO: 285-286 SEQ ID NO: 288 SEQ ID NO: 294 SEQ ID NO:
756 QINSASDD SEQ ID NO: 290 SEQ ID NO: 757 RINSAADD SEQ ID NO:
291-292 SEQ ID NO: 295-298 SEQ ID NO: 582-583 SEQ ID NO: 536 SEQ ID
NO: 582-583 SEQ ID NO: 758 RINGASDD SEQ ID NO: 293 SEQ ID NO: 759
RINRASDD SEQ ID NO: 300 SEQ ID NO: 760 RINSAKDD SEQ ID NO: 526 SEQ
ID NO: 528 SEQ ID NO: 530 SEQ ID NO: 532 SEQ ID NO: 534 SEQ ID NO:
571 SEQ ID NO: 586 SEQ ID NO: 761 RINTAADD SEQ ID NO: 538 SEQ ID
NO: 762 KINSAKDD SEQ ID NO: 540 SEQ ID NO: 763 RINRAGDD SEQ ID NO:
580 SEQ ID NO: 584 SEQ ID NO: 764 KINRASDD SEQ ID NO: 581 SEQ ID
NO: 765 KINRAGDD SEQ ID NO: 585 SEQ ID NO: 766 (R/Q/K)IN(S/N/G/R/T)
Consensus of SEQ ID NO: A(S/A/K/G)DD 755-765 (sequences identified
in this table)
Harpin or Harpin-Like Polypeptides
[0194] The polypeptide can include a harpin or harpin-like
polypeptide.
[0195] The amino acid sequence of the harpin or harpin-like
polypeptide can comprise SEQ ID NOs: 587-592 and 594-597 (Tables 10
and 11),
[0196] The harpin or harpin-like polypeptides can be derived from
Xanthomonas species or diverse bacteria genera including Pantoea
sesami, Erwinia genidensis, Pantoea sesami, or Erwinia
genidensis
[0197] Additional Harpin-like bioactive priming polypeptides can be
derived from the full length HpaG-like protein from Xanthamonas
citri comprising SEQ ID NO: 593.
[0198] Application of HpaG-like polypeptides using the native
L-harpin-like sequence (SEQ ID NO: 587) or retro inverso
D-harpin-like sequence (SEQ ID NO: 588) bioactive priming
polypeptides forms as represented in Tables 10 or 11 are useful to
increase growth and immune responses in plants when applied either
exogenously or endogenously to a plant or plant part. The
retro-inverso HpaG-like (e.g. SEQ ID NO: 588) bioactive priming
polypeptide is particularly useful to enhance the activity and
stability of the HpaG-like polypeptide when applied to plants grown
under or exposed to conditions of abiotic stress. The retro-inverso
HpaG-like form can be used to enhance growth and protection
responses in plants grown under such environments.
TABLE-US-00012 TABLE 10 Harpin-like (HpaG-like) SEQ ID NO: Peptide
Sequence Amino Acid Harpin-like (HpaG-like) NQGISEKQLDQLLTQLIMALLQQ
SEQ ID NO: 587 Xanthomonas species MW 2626.35 Da Harpin-like
(Retro-Inverso HpaG-like) QQLLAMILQTLLQDLQKESIGQN SEQ ID NO: 588
Xanthomonas species MW 2626.35 Da Harpin-like (HpaG-like)
LDQLLTQLIMAL SEQ ID NO: 589 Xanthomonas species MW 2626.35 Da
Harpin-like (Retro-Inverso HpaG-like) LAMILQTLLQDL SEQ ID NO: 590
Xanthomonas species MW 2626.35 Da Harpin-like (HpaG-like)
SEKQLDQLLTQLIMALLQQ SEQ ID NO: 591 Xanthomonas species MW 2626.35
Da Harpin-like (Retro-Inverso HpaG-like) QQLLAMILQTLLQDLQKES SEQ ID
NO: 592 Xanthomonas species MW 2626.35 Da HpaG-Like Protein
MMNSLNTQLGANSSFFQVDPSQNTQSGSNQGNQGISEK SEQ ID NO: 593
QLDQLLTQLIMALLQQSNNAEQGQGQGQGGDSGGQGG Xanthamonas citri
NRQQAGQSNGSPSQYTQMLMNIVGDILQAQNGGGFGG
GFGGGFGGGLGTSLGTSLGTSLASDTGSMQ
TABLE-US-00013 TABLE 11 HpaG-like Homologs from diverse bacterial
genera SEQ ID NO: Peptide amino acid HpaG Homolog
QLEQLMTQLRARLCRLMAM Active Fraction SEQ ID NO: 594 Pantoea sesami
HpaG Homolog QLEQLMTQLRARLKRLMAM Active Fraction SEQ ID NO: 595
Erwinia gerudensis Retro Inverso MAMLRCLRARLQTMLQELQ HpaG Homolog
Active Fraction SEQ ID NO: 596 Pantoea sesami Retro Inverso
MAMLRKLRARLQTMLQELQ HpaG Homolog Active Fraction SEQ ID NO: 597
Erwinia gerudensis
Phytosulfokine (PSK.alpha.) Polypeptides
[0199] The polypeptide can comprise the PSK polypeptide.
[0200] The amino acid sequence of the PSK polypeptide can comprise
SEQ ID NOs: 598-599.
[0201] Phytosulfokine alpha (PSK.alpha.) was originally derived
from Arabidopsis thaliana and is a sulfonated bioactive priming
polypeptide. The PSK.alpha. bioactive priming polypeptide(s) are in
Table 11.
[0202] PSK.alpha. is provided either as a synthetic polypeptide or
a natural polypeptide that is expressed in a recombinant
microorganism, purified and used in agricultural formulations for
applications to plants or plant parts.
TABLE-US-00014 TABLE 12 Phytosulfokine alpha (PSK.alpha.),
sulfonated bioactive priming polypeptides provided as natural and
retro-inverso amino acid sequences SEQ ID NO: Peptide Sequence
Amino Acid Phytosulfokine (PSK.alpha.)
Tyr(SO.sub.3H)-I-Tyr(SO.sub.3H)-TQ SEQ ID NO: 598 Arabidopsis
thaliana MW 845 Da Phytosulfokine
QT-Tyr(SO.sub.3H)-I-Tyr(SO.sub.3H) (Retro Inverso PSK.alpha.) SEQ
ID NO: 599 Arabidopsis thaliana MW 845 Da
Root Hair Promoting polypeptide (RHPP)
[0203] The polypeptide can comprise a RHPP
[0204] The amino acid sequence of the RHPP can comprise SEQ ID NO:
600-601 and 603-606. For example, the amino acid sequence of the
RHPP can comprise SEQ ID NO: 600.
[0205] A combination of the polypeptide comprising an RHPP and a
polypeptide comprising a flagellin or flagellin associated
polypeptide is also provided. The flagellin or flagellin associated
polypeptide can comprise any one of SEQ ID NO: 226, 752, and 571.
In some instances, the polypeptide comprises an RHPP comprising SEQ
ID NO: 600 and a flagellin comprising SEQ ID NO: 226.
[0206] The polypeptide can comprise the PSK polypeptide, the RHPP,
the harpin or harpin-like polypeptide, or a combination
thereof.
[0207] Additional RHPP bioactive priming polypeptides can be
derived from the full length Kunitz Trypsin Inhibitor protein from
Glycine max comprising SEQ ID NO: 602. The RHPP polypeptide can be
modified via C-terminal amidation, N-terminal acetylation or other
modification. The RHPP bioactive priming polypeptide can be
obtained through addition of crude protease digest of kunitz
trypsin inhibitor and/or soybean meal.
[0208] RHPP originally derived for soybean (Glycine max) can be
provided, for example, as a foliar application to produce
beneficial phenotypes in corn, soybean and other vegetables.
TABLE-US-00015 TABLE 13 Amino acid sequence for RHPP forward and
retro-inverso sequences SEQ ID NO: Peptide Sequence Amino Acid Root
Hair Promoting Peptide GGIRAAPTGNER (RHPP) SEQ ID NO: 600 Glycine
max MW 1198.20 Da Root Hair Promoting Peptide RENGTPAARIGG (Retro
Inyerso RHPP) SEQ ID NO: 601 Glycine max MW 1198.20 Da Kunltz
Trypsin Inhibitor
MKSTIFFALFLFCAFTTSYLPSAIADFVLDNEGNPLENGGTYYILSDITAF SEQ ID NO: 602
GGIRAAPTGNERCPLTVVQSRNELDKGIETIISSPYRIRFIAEGHPLSLKF Glycine Max
DSFAVIMLCVGIPTEWSVVEDLPEGPAVKIGENKDAMDGWFRLERVS
DDEFNNYKLVFCPQQAEDDKCGDIGISIDHDDGTRRLVVSKNKPLVV
QFQKLDKESLAKKNHGLSRSE
TABLE-US-00016 TABLE 14 Homologs of RHPP from Glycine spp. Peptide
Sequence SEQ ID NO: Amino Acid Homolog RHPP GGIRATPTENER SEQ ID NO:
603 Glycine max Homolog RHPP GGIRVAATGKER SEQ ID NO: 604 Glycine
max/Glycine sofa
[0209] The polypeptide can include a retro inverso (RI) RHPP.
[0210] The retro inverso RHPP can comprise SEQ ID NO& 601, 605
or 606.
[0211] The retro inverso (RI) RHPP can be modified via C-terminal
amidation or N-terminal acetylation.
TABLE-US-00017 TABLE 15 Retro inverso amino acid sequences for
homologs of RHPP from Glycine spp. Peptide Sequence SEQ ID NO:
Amino Acid Homolog RHPP RENETPTARIGG SEQ ID NO: 605 Glycine max
Homolog RHPP REKGTAAVRIGG SEQ ID NO: 606 Glycine max/Glycine
sofa
Elongation Factor Tu (EF-Tu) Polypeptides
[0212] The polypeptide can comprise an EF-Tu polypeptide.
[0213] Peptides derived from elongation factor Tu (EF-Tu) can be
used separately or in combination with the other bioactive priming
polypeptides as described herein such as in combination with Flg22
polypeptides to provide multiple modes of defense against
pathogenic organisms, generally bacterial and fungal microorganisms
but also including other infection agents, such as viruses.
[0214] Table 16 provides preferred N-terminal polypeptides derived
from various EF-Tu bioactive priming polypeptides selected from
both plants and bacteria. The EF-Tu derived polypeptides can be any
length from 18 to 26 amino acids or less than 26 amino acids in
length. Table 17 further provides retro-inverse (all-D) versions of
EF-Tu polypeptides derived from bacteria and algae.
[0215] The amino acid sequence of the EF-Tu polypeptide can
comprise and one of SEQ ID NOs: 607-640.
[0216] The amino acid sequence of the EF-Tu polypeptide can
comprise SEQ ID NO: 616 or 617.
[0217] The EF-Tu polypeptide can be modified via N-terminal
acetylation. For example, the EF-Tu polypeptide can be modified via
N-terminal acetylation and comprise any of SEQ ID NOs: 607, 608,
610, 611, 613, 614, 616, 617, 619, or 622.
TABLE-US-00018 TABLE 16 N-terminal acetylated and central
polypeptides derived from elongation factors (EF-Tu) existing in
plant, bacterial and algae species Length amino SEQ ID NO: acids
Peptide amino acid Chloroplastic EF-Tu 18 Ac-ARGKFERKKPHVNIGTIG SEQ
ID NO: 607 (acetylated) Arabidopsis lyrata Chloroplastic EF-Tu 26
Ac-ARGKFERKKPHVNIGTIGHVDHGKTT SEQ ID NO: 608 (acetylated)
Arabidopsis lyrata Chloroplastic EF-Tu 50
EKPNVKRGENKWVDKIYELMDSVDSYIPIPTRQTELPFLLAVEDVFS SEQ ID NO: 609 ITG
Arabidopsis lyrata N-terminus of EF-Tu 18 Ac-ARQKFERTKPHINIGTIG SEQ
ID NO: 610 (acetylated) Euglena gracilis N-terminus of EF-Tu 26
Ac-ARQKFERTKPHINIGTIGHVDHGKTT SEQ ID NO: 611 (acetylated) Euglena
gracilis EF-Tu fragment 50
KNPKITKGENKWVDKILNLMDQVDSYIPTPTRDTEKDFLMAIEDVL SEQ ID NO: 612 SITG
Euglena gracilis N-terminus of EF-Tu 18 Ac-AKGKFERTKPHVNVGTIG SEQ
ID NO: 613 (acetylated) Acidovorax avenae N-terminus of EF-Tu 26
Ac-AKGKFERTKPHVNVGTIGHVDHGKTT SEQ ID NO: 614 (acetylated)
Acidovorax avenae EF-Tu fragment 50
KLALEGDKGPLGEQAIDKLAEALDTYIPTPERAVDGAFLMPVEDVF SEQ ID NO: 615 SISG
Acidovorax spp. N-terminus of EF-Tu 18 Ac-AKAKFERSKPHVNIGTIG SEQ ID
NO: 616 (acetylated) Bacillus cereus N-terminus of EF-Tu 26
Ac-AKAKFERSKPHVNIGTIGHVDHGKTT SEQ ID NO: 617 (acetylated) Bacillus
cereus EF-Tu fragment 50
SALKALQGEAEWEEKIIELMAEVDAYIPTPERETDKPFLMPIEDVFS SEQ ID NO:618 ITG
Bacillus cereus N-terminus of EF-Tu 26
Ac-AKGKFERTKPHVNVGTIGHVDHGKTT SEQ ID NO: 619 (acetylated)
Burkholderia spp. EF-Tu fragment 50
KLALEGDTGELGEVAIMNLADALDTYIPTPERAVDGAFLMPVEDV SEQ ID NO: 620 FSISG
Burkholderia spp. EF-Tu fragment 50
RLALDGDQSEIGVPAILKLVDALDTFIPEPTRDVDRPFLMPVEDVFS SEQ ID NO: 621 ISG
Xanthomonas campestris N-terminus of EF-Tu 26
Ac-AKEKFERSKPHVNVGTIGHVDHGKTT SEQ ID NO: 622 (acetylated)
Pseudomonas spp. EF-Tu 50
MALEGKDDNEMGTTAVKKLVETLDSYIPEPERAIDKPFLMPIEDVF SEQ ID NO: 623 SISG
Pseudomonas spp.
TABLE-US-00019 TABLE 17 Retro Inverso polypeptides derived from
elongation factors (EF-Tu) existing in bacterial and algaespecies
Length amino SEQ ID NO: acids Peptide amino acid RI Chloroplastic
EF-Tu 18 GITGINVHPKKREFKGRA SEQ ID NO: 624 Arabidopsis lyrata RI
Chloroplastic EF-Tu 26 TTKGHDVHGITGINVHPKKREFKGRA SEQ ID NO: 625
Arabidopsis lyrata RI Chloroplastic EF-Tu 50
GTISFVDEVALLFPLETQRTPIPIYSDVSDMLEYIKDVWKNEGRKVN SEQ ID NO: 626 PKE
Arabidopsis lyrata RI N-terminus of EF-Tu 18 GITGINIHPKTREFKQRA SEQ
ID NO: 627 Euglena gracilis RI N-terminus of EF-Tu 26
TTKGHDVHGITGINIHPKTREFKQRA SEQ ID NO: 628 Euglena gracilis RI EF-Tu
fragment 50 GTISLVDEIAMLFDKETDRTPTPIYSDVQDMLNLIKDVWKNEGKTI SEQ ID
NO: 629 KPNK Euglena gracilis RI N-terminus of EF-Tu 18
GITGVNVHPKTREFKGKA SEQ ID NO: 630 Acidovorax avenae RI N-terminus
of EF-Tu 26 TTKGHDVHGITGVNVHPKTREFKGKA SEQ ID NO: 631 Acidovorax
avenae RI EF-Tu fragment 50
GSISFVDEVPMLFAGDVAREPTPIYTDLAEALKDIAQEGLPGKDGE SEQ ID NO: 632 LALK
Acidovorax spp. RI N-terminus of EF-Tu 18 GITGINVHPKSREFKAKA SEQ ID
NO: 633 Bacillus cereus RI N-terminus of EF-Tu 26
TTKGHDVHGITGINVHPKSREFKAKA SEQ ID NO: 634 Bacillus cereus RI EF-Tu
fragment 50 GITSFVDEIPMLFPKDTEREPTPIYADVEAMLEIIKEEWEAEGQLAK SEQ ID
NO: 635 LAS Bacillus cereus RI N-terminus of EF-Tu 26
TTKGHDVHGITGVNVHPKTREFKGKA SEQ ID NO: 636 Burkholderia spp. RI
EF-Tu fragment 50 GSISFVDEVPMLFAGDVAREPTPIYTDLADALNMIAVEGLEGTDGE
SEQ ID NO: 637 LALK Burkholderia spp. RI EF-Tu fragment 50
GSISFVDEVPMLFPRDVDRTPEPIFTDLADVLKLIAPVGIESQDGDL SEQ ID NO: 638 ALR
Xanthomonas campestris RI N-terminus of EF-Tu 26
TTKGHDVHGITGVNVHPKSREFKEKA SEQ ID NO: 639 Pseudomonas spp. RI EF-Tu
50 GSISFVDEIPMLFPKDIAREPEPIYSDLTEVLKKVATTGMENDDKGE SEQ ID NO: 640
LAM Pseudomonas spp.
Thionins and Thionin-Targeting Polypeptides
[0218] The polypeptide can comprise the thionin or thionin-like
polypeptide.
[0219] The thionin or thionin-like polypeptide can be fused to a
phloem targeting sequence to form a fused polypeptide, the amino
acid sequence of the phloem targeting sequence comprising any one
of SEQ ID NOs: 641-649, or any combination thereof, for delivering
the fused polypeptide to vascular tissue or cells and/or phloem or
phloem-associated tissue or cells in the plant or plant part.
[0220] The amino acid sequence of the phloem targeting sequence can
comprise SEQ ID NO: 641.
[0221] More specifically, targeting sequences useful for targeting
AMP polypeptides, such as thionins or Flg polypeptides to the
vascular tissues (xylem and phloem) can be extremely useful for
treating diseases that colonize restricted tissues involved in the
transport of fluids and nutrients (e.g., water soluble nutrients,
sugars, amino acids, hormones, etc.). Vascular tissues such as the
xylem transport and store water and water-soluble nutrients and the
phloem cells transport sugars, proteins, amino acids, hormones and
other organic molecules in plants.
[0222] Preferred vascular/phloem targeting polypeptides useful for
targeting the thionins and flagellin-associated polypeptides as
described herein are provided in Table 18.
TABLE-US-00020 TABLE 18 Phloem targeting polypeptides SEQ ID NO:
Vascular/Phloem targeting polypeptides Phloem targeting peptide
MSTATFVDIIIAILLPPLGVFLRFGCGVEFWICLVL Synthetic TLLGYIPGIIYAIYVLTK
SEQ ID NO: 641 Salt stress induced targeting peptide
MGSETFLEVILAILLPPVGVFLRYGCGVEFWICLL Citrus clementine
LTVLGYIPGIIYAIYVLVG SEQ ID NO: 642 Hypothetical protein CICLE
MGTATCVDIILAVILPPLGVFLKFGCKAEFWICLL Citrus trifoliate
LTILGYIPGIIYAVYVITK SEQ ID NO: 643 Hypothetical protein CICLE
MADEGTATCIDIILAIILPPLGVFLKFGCKVEFWIC Citrus sinensis
LLLTIFGYIPGIIYAVYAITKN SEQ ID NO: 644 Low temperature and salt
responsive protein MADGSTATCVDILLAVILPPLGVFLKFGCKAEFW Citrus
sinensis ICLLLTILGYIPGIIYAVYAITKK SEQ ID NO: 645 Hypothetical
protein CICLE FYKQKYQVQITKAVTQNPKHFFNQSSCFLTLNFI Citrus
LFHFTLFKNQSKMADGSTATCVDILLAVILPPLG clementine
VFLKFGCKAEFWICLLLTILGYIPGIIYAVYAITKK SEQ ID NO: 646 Low temperature
and salt responsive protein MSTATFVDIIIAILLPPLGVFLRFGCGVEFWICLVL
Arabidopsis thaliana TLLGYIPGIIYAIYVLTK SEQ ID NO: 647
Cold-inducible protein MSTATFVDIIIAVLLPPLGVFLRFGCGVEFWICLV Cameline
sativa LTLLGYIPGIIYAIYVLTK SEQ ID NO: 648 Low temperature and salt
responsive protein MGTATCVDIIIAILLPPLGVFLRFGCGVEFWICLV Arabidopsis
lyrata LTLLGYIPGILYALYVLTK SEQ ID NO: 649
[0223] A synthetic version of a phloem targeting polypeptide (SEQ
ID NO: 641) is particularly useful in targeting anti-microbial
polypeptides to the phloem sieve tube and companion cells.
[0224] Anti-microbial thionin polypeptides are also provided (Table
19) and are utilized with the phloem targeting sequences provided
in Table 18 for targeting the thionin sequences into the phloem
tissues of citrus as well as other plants.
[0225] The amino acid sequence of the thionin or thionin-like
polypeptide can comprise an one of SEQ ID NOs: 650-749 such as SEQ
ID NO: 651.
TABLE-US-00021 TABLE 19 Thionin and thionin-like sequences SEQ ID
NO: Sequences-Amino Acid Thionin-like protein RTCESQSHRFKGPCS
Synthetic RDSNCATVCLTEGFS SEQ ID NO: 650 GGDCRGFRRRCRCTR PCVFDEK
Thionin-like protein RVCQSQSHHFHGACF Citrus sinensis
SHHNCAFVCRNEGFS SEQ ID NO: 651 GGKCRGVRRRCFCSK LC Thionin-like
protein KSCCKDIMARNCYNV Avena sativa CRIPGTPRPVCATTC SEQ ID NO: 652
RCKIISGNKCPKDYP K Thionin-like protein RTCESQSHRFKGPCS Synthetic
RDSNCATVCLTEGFS SEQ ID NO: 653 GGDCRGFRRRCRCTR PCVFDEK Thionin-like
protein MDSRSFGLLPLLLLI Citrus sinensis LLTSQMTVLQTEARL SEQ ID NO:
654 CESQSHRFHGTCVRS HNCDLVCRTEGFTGG RCRGFRRRCFCTRIC Proteinase
inhibitor MKSFFGIFLLLLILF se60-like protein ASQEIMVPAEGRVCQ Citrus
paradise SQSHHFHGACFSHHN SEQ ID NO: 655 CAFVCRNEGFSGGKC
RGVRRRCFCSKLC Defensin precursor MKSFFGIFLLLLILF Citrus Clementina
ASQMMVPAEGRVCQS SEQ ID NO: 656 QSHHFHGACFSHHNC AFVCRNEGFSGGKCR
GARRRCFCSKLC defensin precursor MKSFFGIFLLLLILF Citrus Clementina
ASQEMMVPAEGRVCQ SEQ ID NO: 657 SQSHHFHGACFSHHN CAFVCRNEGFSGGKC
RGARRRCFCSKLC Thionin-like protein MKSFFGIFLLLLILF Citrus
Clementina ASQMMVPAEGRVCQS SEQ ID NO: 658 QSHHFHG ACFSHHNCAFVCRNE
GFSGGKCRGARRRCF CSKLC Thionin-like peptide MANSMRFFATVLLLA
Nicotiana benthamiana LLVMATEMGPMTIAE SEQ ID NO: 659
ARTCESQSHRFKGPC SRDSNCATVCLTEGF SGGDCRGFRRRCFCT RPC Thionin-like
protein MAKSMRFFATVLLLA Nicotiana sylvestris LLVMATEMGPTTIAE SEQ ID
NO: 660 ARTCESQSHRFKGPC SRDSNCATVCLTEGF SGGDCRGFRRRCFCT RPC
Thionin-like protein MANSMRFFATVLLLT Nicotiana tabaccum
LLVMATEMGPMTIAE SEQ ID NO: 661 ARTCESQSHRFKGPC SRDSNCATVCLTEGF
SGGDCRGFRRRCFCT RPC Thionin-like protein MANSMRFFATVLLIA Nicotiana
LLVMATEMGPMTIAE tomentosiformis ARTCESQSHRFKGPC SEQ ID NO: 662
SRDSNCATVCLTEGF SGGDCRGFRRRCFCT RPC Thionin-like protein
MANSMRFFATVLLIA Nicotiana tabaccum LLVTATEMGPMTIAE SEQ ID NO: 663
ARTCESQSHRFKGPC SRDSNCATVCLTEGF SGGDCRGFRRRCFCT RPC Defensin class
1 MANSMRFFATVLLLT Nicotiana alata LLFMATEMGPMTIAE SEQ ID NO: 664
ARTCESQSHRFKGPC ARDSNCATVCLTEGF SGGDCRGFRRRCFCT RPC Leaf thionin
MGSIKGLKSWICVLV Avena sativa LGIVLEQVQVEGKSC SEQ ID NO: 665
CKDIMARNCYNVCRI PGTPRPVCATTCRCK IISGNKCPKDYPKLH GDPD Leaf thionin
MGSIKGLKSVVICVL Avena sativa VLGIVLEHVQVEGKS SEQ ID NO: 666
CCKDTTARNCYNVCR IPGTPRPVCATTCRC KIISGNKCPKDYPKL HGDLD Thionin Class
1 LGLVVAQTQVDAKSC Tulipa gesneriana CPSTAARNCYNVCRF SEQ ID NO: 667
PGTPRPVCAATCGCK IITGTKCPPDYPKLG WSTFQNSDVADKALD VVDEALHVAKEVMKE
AVERCNNACSEVCTK GSYAVTA Thionin-like MERKSLGFFFFLLLI protein Class
1 LLASQEMVVPSEARV Vitis vinifera CESQSHKFEGACMGD SEQ ID NO: 668
HNCALVCRNEGFSGG KCKGLRRRCFCTKLC Thionin-like MERKSLGFFFFLLLI
protein Class 1 LLASQMVVPSEARVC Vitis vinifera ESQSHKFEGACMGDH SEQ
ID NO: 669 NCALVCRNEGFSGGK CKGLRRRCFCTKLC defensin Ec-AMP-D1
MERSVRLFSTVLLVL Citrus sinensis LLLASEMGLRAAEAR SEQ ID NO: 670
ICESQSHRFKGPCVS KSNCAAVCQTEGFHG GHCRGFRRRCFCTKR C Antimicrobial
LCNERPSQTWSGNCG Protein 1 NTAHCDKQCQDWEKA (Ah-Amp1) SHGACHKRENHWKCF
Aesculus hippocastanum CYFNC SEQ ID NO: 671 hypothetical protein
MAKNSTSPVSLFAIS DCAR LIFFLLANSGSITEV Dacus carota DGKVCEKPSLTWSGK
SEQ ID NO: 672 CGNTQHCDKQCQDWE GAKHGACHSRGGW KCFCYFEC Cysteine-rich
NLCERASLTWTGNCG antimicrobial protein NTGHCDTQCRNWESA Clitoria
ternatea KHGACHKRGNWKCFC SEQ ID NO: 673 YFNC hypothetical
MAKKSSSFCLSAIFL protein DCAR VLLLVANTGMVREVD Dacus carota
GALCEKPSLTWSGNC SEQ ID NO: 674 RNTQHCDKQCQSWEG AKHGACHKRGNWKCF
CYHAC Thionin-like MAKKLNAVTVSAIFL Bupleurum kaoi WFLIASYSVGAAKEA
SEQ ID NO: 675 GAEGEVVFPEQLCER ASQTWSGDCKNTKNC DNQCIQWEKARHGAC
HKRGGKWMCFCYFDK C defensin Dm-AMP1 = ELCEKASKTWSGNCG cysteine-rich
NTGHCDNQCKSWEGA antimicrobial protein AHGACHVRNGKHMCF Dahlia
merckii CYFNC SEQ ID NO: 676 Thionin-like MAKISVAFNAFLLLL
Helianthus annuus FVLAISEIGSVKGEL SEQ ID NO: 677 CEKASQTWSGTCGKT
KHCDDQCKSWEGAAH GACHVRDGKHMCFCY FNCSKAQKLAQDKLR AEELAKEKIEPEKAT AKP
Thionin MAKNSVAFFALLLLI Cynara cardunculus CILTISEFAVVKGEL var.
scolymus CEKASKTWSGNCGNT SEQ ID NO: 678 RHCDDQCKAWEGAAH
GACHTRNKKHMCFCY FNCPKAEKLAQDKLK AEELARDKVEAKEVP HFKHPIEPIHHP
Thionin MAKQWVSFFALAFIV Cynara cardunculus FVLAISETQTVKGEL var.
scolymus CEKASKTWSGNCGNT SEQ ID NO: 679 KHCDDQCKSWEGAAH
GACHVRNGKHMCFCY FNSCAEADKLSEDQI EAGKLAFEKAEKLDR DVKKAVPNVDHP
defensin-like protein MAQKVNSALIFSAIF 1-DCAR-like VLFLVASYSVTVAEG
Daucus carota subsp. ARAGAEGEVVYPEAL Sativus CERASQTWTGKCQHT SEQ ID
NO: 680 DHCDNQCIQWENARH GACHKRGGNWKCFCY FDHC low-molecular-weight
MASSYTLMLFLCLSI cysteine-rich FLIASTEMMAVEARI defensin
CERRSKTWTGFCGNT Arabidopsis lyrata RGCDSQCKSWERASH SEQ ID NO: 681
GACHAQFPGFACFCY FNC Thionin-like protein MAKSSTSYLVFLLLV Parthenium
hysterophorus LVVAISEIASVNGKV SEQ ID NO: 682 CEKPSKTWFGNCKDT
EKCDKRCMEWEGAKH GACHQRESKYMCFCY FDCDP putative defensin
MASSYTLMLFLCLSI AMP1 protein FLIASTEMMAVEGRI Arabidopsis thaliana
CERRSKTWTGFCGNT SEQ ID NO: 683 RGCDSQCKRWERASH GACHAQFPGFACFCY FNC
Thionin-like MASSYTLLLFVCLSI Eutrema salsugineum FFIASTEMMMVEGRV
SEQ ID NO: 684 CERRSKTWTGFCGNT RGCDSQCKRWERASH GACHAQFPGFACFCY FNC
defensin-like MAKLLGYLLSYALSF Vitis vinifera LTLFALLVSTEMVML SEQ ID
NO: 685 EAKVCQRPSKTWSGF CGSSKNCDRQCKNWE GAKFIGACHAKFPGV ACFCYFNC
Knottin MAKSLSSFATFLALL Corchorus olitorius CLFFLLSTPNEMKMA
SEQ ID NO: 686 EAKICEKRSQTWSGW CGNSSHCDRQCKNWE NARHGSCHADGLGWA
CFCYFNC Knottin MEMKMAEGKICEKRS Corchorus olitorius QTWSGWCGNSSHCDR
SEQ ID NO: 687 QCKNWENARHGSCHA DGLGWACFCYFNC Thionin-like protein
MASSLKLMLFLCLSI Camelina sativa FLIASTEMMTVEGRT SEQ ID NO: 688
CERRSKTWTGFCGNT RGCDSQCRSWEGASH GACHAQFPGFACFCY FNC Thionin-like
protein MAKVVGNSAKMIVAL Cucumis sativus LFLLALMLSMNEKQG SEQ ID NO:
689 WEAKVCERRSKTWSG WCGNTKHCDRQCKNW EGATHGACHAQFPGR ACFCYFNC
Thionin-like protein MIDAFNYKQFSTVKG Cynara cardunculus
KICEKPSKTWFGKCQ var. scolymus DTTKCDKQCIEWEDA SEQ ID NO: 690
KHGACHERESKLMCF CYYNCGPPKNTPPGT PPSPP Thionin-like MASSYKLILFLCLSI
Capsella rubella FLIASFEMMAVEGRI SEQ ID NO: 691 CQRRSKTWTGFCGNT
RGCDSQCKRWERASH GACHAQFPGFACFCY FNC Thionin MMAVEGRICERRSKT
Arabidopsis thaliana WTGFCGNTRGCDSQC SEQ ID NO: 692 KRWERASHGACHAQF
PGFACFCYFNC Thionin MASSYTRLLLLCLSI Brassica napus FLIASTEVMMVEGRV
SEQ ID NO: 693 CQRRSKTWTGFCGNT RGCDSQCKRWERASH GACHAQFPGFACFCY FNC
Thionin-like protein MASSYARLLLLCLSI Brassica rapa FLIASTEVMMVEGRV
SEQ ID NO: 694 CQRRSKTWTGFCGNT RGCDSQCKRWERASH GACHAQFPGFACFCY FNC
Thionin-like protein MASSLKLMLFLCLSI Camelina sativa
FLIASTEMMTVEGRT SEQ ID NO: 695 CERRSKTWTGFCGNT RGCDSQCRRWEHASH
GACHAQFPGFACFCY FNC defensin-like protein MASYTRLLLLCLSIF Brassica
napus LIASTEVMMVEGRVC SEQ ID NO: 696 QRRSKTWTGFCGNTR
GCDSQCKRWERASHG ACHAQFPGFACFCYF NC Thionin-like protein
MVMLEAKVCQRPSKT Vitis vinifera WSGFCGSSKNCDRQC SEQ ID NO: 697
KNWEGAKHGACHAKF PGVACFCYFNC Thionin-like protein MTKSFILVALLCICF
Brassica napus ILLSPTEMRLTLNAC SEQ ID NO: 698 LKLAEAKICEKYSQT
WSGRCTKTSHCDRQC INWEDARHGACHQDK HGRACFCYFNCKK Thionin-like protein
MASSYTVFLLLCLSI Raphanus sativus FLIASTEVMMVEGRV SEQ ID NO: 699
CQRRSKTWTGFCGNT RGCDSQCKRWEHASH GACHAQFPGFACFC YFNC Thionin-like
MASSYTLLLFLCLSI Arabis alpine FLIVSTEMMMVEGRI SEQ ID NO: 700
CERRSKTWTGFCANT RGCDSQCKRWERASH GACHAQFPGVACFCY FNC Thionin-like
protein MAKVVGNSAKMIVAF Cucumis melo LFLLALTLSMNEKQG SEQ ID NO: 701
VVEAKVCERRSKTWS GWCGDTKHCDRQCKN WEGAKHGACHAQFPG RACFCYFNC
Thionin-like protein MAASLVYRLSSVILI Erythranthe guttate
VLLLFIMLNNEVMVV SEQ ID NO: 702 ESRLCERRSKTWTGF CGSSNNCNNQCRNWE
RASHGACHAQFPGFA CFCYFNC Thionin-like protein MAKFQVSSTIFFALF
Sesamum indicum FCFLLLASNEAKICQ SEQ ID NO: 703 RMSKTWSGVCLNSGN
CDRQCRNWERAQHGA CHRRGLGFACLCYFK C Thionin-like protein
MAKNSVAFFAFLLIL Eclipta prostrata FVLAISEIGSVKGEL SEQ ID NO: 704
CEKASQTWSGTCRIT SHCDNQCKSWEGAAH GACHVRGGKHMCFCY FSHCAKAEKLTQDKL
KAGHLVNEKSEADQK VPVTP Gamma thionin Cynara MAKNTKVSAFLFVFL
cardunculus var. FVFFLVVHSVTAFAI scolymus RFKCFDTDMLLKVIA SEQ ID
NO: 705 DMVVGMKGIEKVCRR RSKTWSGYCGDSKHC DQQCREWEGAEHGAC
HHEGLGRACFCYFNC Art v 1 precursor MAAGLLVFVLAISEI Ambrosia
artemisiifolia ASVKGKLCEKPSVTW SEQ ID NO: 706 SGKCKVKQTDKCDKR
CIEWEGAKHGACHKR DSKASCFCYFDCDPT KNPGPPPGAPKGKAP APSPPSGGGGEGGGE
GGGER Art v 1 precursor MAAGLLVFVLAISEI Ambrosia ASVKGKLCEKPSLTW
artemi679siifolia SGKCKVKQTDKCDKR SEQ ID NO: 707 CIEWEGAKHGACHKR
DSKATCFCYFDCDPT KNPGPPPGAPKGKAP APSPPSGGGAPPPSG GEGGER Thionin-like
protein MAKLHSSALCFLIIF Jatropha curcas LFLLVSKEMAVTEAK SEQ ID NO:
708 LCQRRSKTWSGFCGD PGKCNRQCRNWEGAS HGACHAQFPGFACFC YFKC
Thionin-like protein MAKAPKSVSYFAFFF Nelumbo nucifera
ILFLLASSEIQKTKK SEQ ID NO: 709 LCERRSKTWSGRCTK TQNCDKQCKDWEYAK
HGACFIGSWFNKKCY CYFDC Thionin-like protein MAKLLSRLSIPLIVF Pyrus x
VFLLILLASTEVAMV bretschneideri EARICQRRSKTWSGF SEQ ID NO: 710
CANTGNCNRQCTNWE GALHGACHAQFPGVA CFCYFRC Low-molecular-weight
MAKLHFPTLLCLFIF cysteine-rich LFLLVSTEMQVTQAK protein LCR78
precursor VCQRRSKTWSGFCGS Ricinus communi TKNCDRQCKNWEGAL SEQ ID
NO: 711 HGACHAQFPGVACFC YFKCGGER homologue of Art v 1
KLCEKPSVTWSGKCK precursor VKQTDKCDKRCIEWE Ambrosia artemisiifolia
GAKFIGACHKRDSKA SEQ ID NO: 712 SCFCYFDCDPTKNPG PPPGAPKGKAPAPSP
PSGGGAPPPSGGEGG GD homologue of Art v 1 KLCEKPSVTWSGNKV precursor
KQTDKCDKRCIEWEG Ambrosia artemisiifolia AKHGACHKRDSKASC SEQ ID NO:
713 FCYFDCDPTKNPGPP PGAPKGKAPAPSPPS GGGAPPPSGGEGGGD GGGGRR
Thionin-like protein MAKLLSHLLFYPILF Prunus mume LFLFIFLASTEVAILE
SEQ ID NO: 714 ARICQRRSKT WSGFCGNTRNCNRQC RNWEGALRGACHAQF
PGFACFCYFRC Knottin MAKTLQLFALFFIVI Corchorus olitorius
LLANQEIPVAEAKLC SEQ ID NO: 715 QKRSKTWTGICIKTK NCDNQCKKWEKAEHG
ACHRQGIGFACFCYF NQKKC Knottin MAKFVSTVALLFALF Corchorus olitorius
ILLASFDEGMMPMAE SEQ ID NO: 716 AKVCSKRSKTWSGFC NSSANCNKQCREWED
AKHGACHFEFPGFAC FCYFNC Thionin-like protein MNSKVILALLVCFLL Solanum
pennellii IASNEMQGGEAKVCG SEQ ID NO: 717 RRSSTWSGLCLNTGN
CNTQCIKWEHASSGA CHRDGFGFACFCYFN C Thionin-like protein
MAKLLGYHLVYPILF Frogaria vesca LFIFLLLASTEMGML subsp. Vesco
EARICQRRSKTWTGL SEQ ID NO: 718 CANTGNCHRQCRNWE GAQRGACHAQFPGFA
CFCYFNC Knottin MAKFVSVALLLALFI Corchorus capsularis
LVASFDEGMVPMAEA SEQ ID NO: 719 KLCSKRSKTWSGFCN SSANCNRQCREWEDA
KHGACHFEFPGFACF CYFDC Thionin-like protein MQGGEARVCERRSST Solanum
tuberosum WSGPCFDTGNCNRQC SEQ ID NO: 720 INWEHASSGACHREG
IGSACFCYFNC Defensin 1.2-like MAKTLKSVQFFALFF protein PDF1.2-1
LVILLAGSEMTAVEA Dimocarpus longan LCSKRSKTWSGPCFI SEQ ID NO: 721
TSRCDRQCKRWENAK HGACHRSGWGFACFC YFNKC Thionin-like protein
MAKAATIVTLLFAAL
Camelina sativa VFFAALETPTMVEAQ SEQ ID NO: 722 KLCERPSGTWSGVCG
NSNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like MAKFASIIAFLFAAL
Arabis alpine VLFASFEAPTMVEAQ SEQ ID NO: 723 KYCEKPSGTWSGVCG
NSNACNNQCINLEGA RHGSCNYVFPYYRCI CYFQC Thionin-like MAMSLKSVHFFALFF
Theobroma cacao IVVLLANQEMPVAEA SEQ ID NO: 724 KLCQKRSKTWTGPCI
KTKNCDHQCRKWEKA QHGACHWQWPGFACF CYVNC Thionin-like MAKLVSPKAFFVFLF
Amborella trichopoda VFLLISASEFSGSEA SEQ ID NO: 725 KLCQKRSRTWSGFCA
NSNNCSRQCKNLEGA RFGACHRQRIGLACF CYFNC low-molecular-weight
MAKSATIVTLFFAAL cysteine-rich 67 VFFAALEAPMVVEAQ Arabidopsis
thaliana KLCERPSGTWSGVCG SEQ ID NO: 726 NSNACKNQCINLEKA
RHGSCNYVFPAHKCI CYFPC Thionin-like MAKFASIITLLFAAL Arabis alpine
VLFASLEAPTMVEAQ SEQ ID NO: 727 KLCQRPSGTWSGVCG NNGACKNQCINLEKA
RHGSCNYVFPYHRCI CYFPC Thionin-like MAKVASIIALLFAAL Brassica juncea
VLFAAFEAPTMVEAQ SEQ ID NO: 728 KLCERPSGTWSGVCG NNNACKNQCINLEKA
RHGSCNYVFPAHKCI CYFPC Thionin-like MAKFASIIALLFAAL Brassica
oleracea VLFAALEAPTMVEAQ var. oleracea KLCERPSGTWSGVCG SEQ ID NO:
729 NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MAKPATIVTLLFAAL Camelina sativa VFFAALETPTMVEAQ SEQ ID NO: 730
KLCERPSGTWSGVCG NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MAKSATIVTLLFAAL Camelina sativa VFFAALETPTMVEAQ SEQ ID NO: 731
KLCERPSGTWSGVCG NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MAKFASIIAPLFAVL Brassica napus VLFAAFEAPTMVEAQ SEQ ID NO:732
KLCERPSGTWSGVCG NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MAKFASIITLLFAAL Eutrema salsugineum VLFAVFEGPTMVEAQ SEQ ID NO: 733
KLCERPSGTWSGVCG NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Cysteine-rich
MAKFASIIALLFAAL antifungal protein VLFAAFEAPTMVEAQ Raphanus sativus
KLCERPSGTWSGVCG SEQ ID NO: 734 NNNACKNQCINLEKA RHGSCNYVFPAHKCI
CYFPC Thionin-like protein 1 MAKFASIVSLLFAAL Raphanus sativus
VLFTAFEAPAMVEAQ SEQ ID NO: 735 KLCERPSGTWSGVCG NNNACKNQCINLEKA
RHGSCNYVFPAHKCI CYFPC Thionin-like protein 1 MNTKVILALLFCFLL
Raphanus sativus VASNEMQVGEAKVCQ SEQ ID NO: 736 RRSKTWSGPCINTGN
CSRQCKQQEDARFGA CHRSGFGFACFCYFK C Thionin-like MAKFASIIAPLFAAL
Brassica rapa VLFAAFEAPTMVEAQ SEQ ID NO: 737 KLCERPSGTWSGVCG
NNNACKNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like MNTKLILALMFCFLL
Solanum pennellii IASNEMQVGEAKVCQ SEQ ID NO: 738 RRSKTWSGPCINTGN
CSRQCKQQEDARFGA CHRSGFGFACFCYFK C Thionin-like MAKFTTTFALLFAFF
Citrus Clementina ILFAAFDVPMAEAKV SEQ ID NO: 739 CQRRSKTWSGLCLNT
GNCSRQCKQQEDARF GACHRQGIGFACFCY FKC Thionin-like MAKFTSIIVLLFAAL
Brassica rapa VLFAGFEAPTMVEAQ SEQ ID NO: 740 KLCERPSGTWSGVCG
NNNACKNQCIRLEKA RHGSCNYVFPARKCI CYFPC Thionin-like MAKFASIITLLFAAL
Eutrema salsugineum VLFATFAPTMVEAKL SEQ ID NO: 741 CERPSGTWSGVCGNN
NACKSQCQRLEGARH GSCNYVFPAHKCICY FPC Thionin-like MAKFASIITLLFAAL
Eutrema salsugineum VLFATFEAPTMVEAK SEQ ID NO: 742 LCERPSGTWSGVCGN
NNACKSQCQRLEGAR HGSCNYVFPAHKCIC YFPC Thionin-like MAKFASIIAFFFAAL
Heliophila VLFAAFEAPTIVEAQ coronopifolia KLCERPSGTWSGVCG SEQ ID NO:
743 NNNACRNQCINLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MAKVASIVALLFPAL Brassica oleracea VIFAAFEAPTMVEAQ SEQ ID NO: 744
KLCERPSGTWSGVCG NNNACKNQCIRLEKA RHGSCNYVFPAHKCI CYFPC Thionin-like
MSKFYTVFMFLCLAL Cicer arietinum LLISSWEVEAKLCQR SEQ ID NO: 745
RSKTWSGPCIITGNC KNQCKNVEHATFGAC HRQGFGFACFCYFNC H Thionin-like
MAKSVASITTAFAUF Citrus Clementina AFFILFASFGVPMAE SEQ ID NO: 746
AKVCQRRSKTWSGPC LNTGKCSRQCKQQEY ARYGACYRQGAGYAC YCYFNC Thionin-like
MAKSVASITTAFALI Citrus sinensis FAFFILFASFEVPMA SEQ ID NO: 747
EAKVCQRRSKTWSGP CLNTGKCSRHCKQQE DARYGACYRQGTGYA CFCYFEC
Thionin-like MAKFTTTFALLFAFF Citrus sinensis ILFAAFDVPMAEAKV SEQ ID
NO: 748 CQLRSKTWSGLCLNT GNCSRQCKQQEDARF GACHRQGIGFACFCY FKC
Ec-AMP-D1 MERSVRLFSTVLLVL Citrus sinensis LLLASEMGLRAAEAR SEQ ID
NO: 749 ICESQSHRFKGPCVS KSNCAAVCQTEGFHG GHCRGFRRRCFCTKR C
[0226] The polypeptide can comprise a fusion protein.
[0227] Table 20 (SEQ ID NO: 750) describes the sequences used to
make a translational fusion using the nucleotide sequence that
encodes the synthetic phloem targeting polypeptide (SEQ ID NO: 641)
with a synthetic thionin polypeptide (SEQ ID NO: 650). The upper
case (not bald) font sequence identifies the phloem targeting
sequence, the upper case bald font identifies the fusion of these
two peptide sequences (Table 20) that codes for the phloem targeted
bioactive priming polypeptide.
TABLE-US-00022 TABLE 20 Translational fusion of a phloem targeting
sequence with a thionin derived polypeptide Translational fusion
phloem targeting sequence with thionin polypeptide (synthetic): SEQ
ID NO: 750 MSTATFVDIIIAILLPPLGVFLRFGCGVEFWICLVLTLLGYIPGIIYAIY
VLTKRTCESQSHRFKGPCSRDSNCATVCLTEGFSGGDCRGFRRRCRCTRP CVFDEK
Additional Modifications
[0228] In addition, polypeptides can be chemically synthesized with
D-amino acids, .beta.2-amino acids, .beta.3-amino acids, homo amino
acids, gamma amino acids, peptoids, N-methyl amino acids, and other
non-natural amino acid mimics and derivatives.
[0229] The polypeptides may be modified by either natural
processes, such as posttranslational processing, or by chemical
modification techniques that are well known in the art.
Modifications can occur anywhere in a polypeptide, including the
polypeptide backbone, the amino acid side-chains and the amino or
carboxyl termini. The same type of modification may be present in
the same or varying degrees at several sites in a polypeptide.
Also, a polypeptide may contain many types of modifications.
[0230] Peptides may be branched, for example, as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched, and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods.
[0231] Modifications include acetylation, acid addition, acylation,
ADP-ribosylation, aldehyde addition, alkylamide addition,
amidation, amination, biotinylation, carbamate addition,
chloromethyl ketone addition, covalent attachment of a nucleotide
or nucleotide derivative, cross-linking, cyclization, disulfide
bond formation, demethylation, ester addition, formation of
covalent cross-links, formation of cysteine-cysteine disulfide
bonds, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation, hydrazide
addition, hydroxyamic acid addition, hydroxylation, iodination,
lipid addition, methylation, myristoylation, oxidation, PEGylation,
proteolytic processing, phosphorylation, prenylation,
palmitoylation, addition of a purification tag, pyroglutamyl
addition, racemization, selenoylation, sulfonamide addition,
sulfation, transfer-RNA mediated addition of amino acids to
proteins such as arginylation, ubiquitination, and urea addition.
(see, e.g., Creighton et al. (1993) Proteins--Structure and
Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York; Johnson, ed. (1983) Posttranslational Covalent
Modification Of Proteins, Academic Press, New York; Seifter et al.
(1990) Meth. Enzymol., 182: 626-646; Rattan et al. (1992) Ann. N.Y.
Acad. Sci., 663: 48-62; and the like).
[0232] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides described herein. Such variants
include deletions, insertions, inversions, repeats, duplications,
extensions, and substitutions (e.g., conservative substitutions)
selected according to general rules well known in the art so as
have little effect on activity.
[0233] The polypeptide can comprise an amino acid sequence having
at least 70% identity to any one of SEQ ID NOs. 1-768 wherein the
polypeptide has bioactive priming activity.
[0234] The polypeptide can comprise an amino acid sequence having
at least 75% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0235] The polypeptide can comprise an amino acid sequence having
at least 80% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0236] The polypeptide can comprise an amino acid sequence having
at least 85% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0237] The polypeptide can comprise an amino acid sequence having
at least 90% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0238] The polypeptide can comprise an amino acid sequence having
at least 95% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0239] The polypeptide can comprise an amino acid sequence having
at least 98% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
[0240] The polypeptide can comprise an amino acid sequence having
at least 99% identity to any one of SEQ ID NOs. 1-768, wherein the
polypeptide has bioactive priming activity.
II. Preparation of Bioactive Priming Polypeptides
[0241] Methods and approaches are provided for cloning, genetically
modifying and expressing the bioactive priming polypeptides (for
example, flagellins) and the bioactive priming polypeptides (for
example, Bt.4Q7Flg22) using those methods well understood and
commonly used by one of ordinary skill in the art. The methods
described herein can be used with any of the bioactive priming
polypeptides as described herein and therefore include any of the
flagellins, flagellin-associated polypeptides, thionins,
harpin-like (HpaG-like), EF-Tu, PSK.alpha. or RHPP and/or any
combinations thereof.
[0242] Bioactive priming polypeptides can be provided as a free
polypeptide, immobilized on the surface of a particle, or
impregnated on or into a matrix. Several expression systems can be
used for the production of free polypeptide.
[0243] The flagellin-derived full-coding, partial coding (flagellin
polypeptides) and flagellin-associated polypeptides can be
overexpressed in Bacillus strain, for example, Bacillus
thuringiensis strain BT013A, in Bacillus cereus or in Bacillus
subtilis. The flagellins and flagellin-derived polypeptides are
cloned using an appropriate expression vector to allow for the
abundant production of the polypeptide.
[0244] For example, in order to facilitate cloning of the target
nucleotides that encode the bioactive priming polypeptide(s) as
described herein, an E. coli compatible shuttle vector pSUPER was
constructed by fusing the pBC plasmid backbone described above with
the E. coli pUC57 cloning vector at compatible BamHI restriction
endonuclease sites. The resulting, pSUPER vector carries dual
selection markers (ampicillin selection in E. coli and tetracycline
selection in Bacillus spp). Cloning was performed by PCR
amplification of target nucleotides with specific primers
synthesized with 15 bp overlapping the pSUPER insertion site.
Specific gene encoding polypeptides were fused to the pSUPER vector
with In-Fusion HD Cloning Kit (Clontech). Sequence verified pSUPER
constructs were amplified using the pBC suitable backbone Reverse
and Forward primers. The resulting PCR products were self-ligated
to generate the pBC plasmid that was used to transform the B30
donor Bacillus spp. strain. The final construct was verified to be
completely intragenic by Sanger sequencing.
[0245] The bioactive priming polypeptides/peptides as described
herein are produced in large amounts for field and grower
applications by using a free expression system that can utilize a
Bacillus subtilis and/or Bacillus thuringiensis strain as the
designated heterologous expression strain. The base expression
plasmid designated pFEe4B consists of an E. coli section (=e) and a
Bacillus section (=pFE). The e section was derived from pUC19 and
enables selection and amplification of the vector in E. coli for
cloning purposes. It comprises the beta-lactamase gene (bla)
conferring resistance to beta-lactam antibiotics such as ampicillin
and other penicillin derivatives, as well as an E. coli origin of
replication allowing vector multiplication. The pFE section
provides selection and plasmid amplification in Bacillus spp. and
drives expression of the heterologous polypeptide/peptide of
interest. As such it contains a gene conferring resistance to
tetracycline (tetL), as well as the gene for a replication protein
(repU) responsible for amplifying the plasmid in Bacillus spp.,
both of which were derived from the native Bacillus cereus plasmid
pBC16. The expression cassette of pFEe4B contains a secretion
signal (amyQ), a cloning site and a terminator (rspD), the former
resulting in secretion of the expressed protein/peptide from the
host strain cells into the surrounding medium, and the latter
preventing transcription beyond the open reading frame of interest.
Expression in pFEe4B is driven by a modified autoinducible
promoter, which initiates expression once the culture reaches a
sufficient optical density. In the pFEe4b expression system,
expression is controlled by an IPTG-inducible promoter sequence
from Bacillus subtilis. This promoter consists of a modified
constitutive promoter combined with the E. coli lac repressor
(lacl) and a ribosome binding site. Thus, expression from
pFEe4B-encoded polypeptides/peptides depends on the presence of
suitable induction agents such as isopropyl
beta-D-1-thiogalactopyranoside (IPTG). However other pFe systems
useful for expression of the polypeptides as described herein do
not rely on such induction systems for their expression. The pFEe4
plasmid further harbors the E. coli lacl gene under control of the
Bacillus licheniformis penicillase promoter to prevent expression
of polypeptide/peptide as described herein in absence of any
induction agent.
[0246] Other commercially available expression vectors, for
example, any of those derived from Bacillus subtilis, can also be
useful. Other expression vectors were selected for producing the
recombinant bioactive priming polypeptides due to the following
desired criteria: the recombinant microorganism is non-pathogenic
and is considered as generally regarded as safe (GRAS) organisms,
it has no significant bias in codon usage and it is capable of
secreting extracellular proteins directly into the culture medium
providing for a cell free version(s) of the bioactive priming
polypeptides.
[0247] Other expression systems common in the art can be utilized
to express bioactive priming polypeptides in a similar manner.
[0248] The bioactive priming polypeptides as described herein can
be produced and purified either by the use of a protein tag(s)
using affinity purification or by using column protease cleavage
methods which release the un-tagged polypeptide(s). Methods of
using this approach to make free versions of the bioactive priming
polypeptides are commonly known and understood by one of ordinary
skill in the art.
[0249] Protein tags usually comprise a relatively small sequence of
amino acids incorporated into a translated polypeptide, basically
providing a molecular tether for the bioactive priming polypeptide
of interest. They are commonly used to aid in the expression and
purification of recombinant polypeptides. The polyhistidine (His)
tag was selected for the purposes of affinity purification of the
bioactive priming polypeptides as described. A His tag can be fused
to either the N- or C-terminus of a polypeptide. His tags are
frequently combined with other tags for dual-labeling. Tags for the
bioactive priming polypeptides can be useful to affinity purify
them. The tags can also be cleaved off of the bioactive priming
polypeptides using specific proteases and column-specific protease
cleavage methods to release the purified un-tagged bioactive
priming polypeptide or full-length precursor protein of interest.
These methods are also common and well known to one of ordinary
skill in the art. Other tags that can be utilized are known in the
art, and include FLAG tags, antibody epitopes, streptavidin/biotin,
among other purification tools. Another useful tag is a glutathione
S-transferase (GST) tag.
[0250] Protein tags can be provided within the plasmid to produce
the polypeptide. Ideally, the plasmid comprises, alongside the
sequence encoding the polypeptide of interest, a secretion signal
(e.g., the amyE or amyQ secretion signal) to promote secretion, and
a protein tag (e.g., glutathione S transferase) to enhance the
stability of the polypeptide, thereby enhancing production and
stability. In preferred cases, the protein tag (e.g., GST) is
linked to the polypeptide using a linker sequence comprising a
consensus cleavage sequence. This can allow the addition of a
targeted kinase that can cleave the tag and release the purified,
isolated polypeptide. A suitable consensus cleavage sequence can
comprise an enterokinase cleavage sequence (SEQ ID NO: 772), which
can be cleaved by simple application of a bovine enterokinase, for
example.
[0251] Therefore, a method is provided for producing a polypeptide
comprising producing a fusion protein comprising any polypeptide
described herein and an Enterokinase (EK) cleavage site via
fermentation, the EK cleavage site serving to enhance activity and
stability of the polypeptide. The fusion protein encoded by the
plasmid can further comprise a protein tag (e.g., a poly-histidine
(His) tag, a FLAG tag, an antibody epitope, streptavidin/biotin,
glutathione S-transferase (GST), or any combination thereof),
wherein the enterokinase cleavage site comprises a linking region
connecting the polypeptide and the protein tag. The fusion protein
can also comprise a secretion signal. The secretion signal can
comprise an amyE or amyQ secretion signal (e.g., SEQ ID NO: 769),
or it can comprise any one of SEQ ID NOs 563-570 as described
above. The polypeptide comprising the enterokinase (EK) cleavage
site can be more stable and produced in higher yields using
fermentation than a polypeptide lacking the enterokinase (EK)
cleavage site. When desired, an enterokinase (e.g., a bovine
enterokinase) can be applied to the fusion protein to activate
(e.g., isolate) the polypeptide of interest. The enterokinase can
be applied on-site to enable maximum stability of the bioactive
priming polypeptide prior to administration.
[0252] The bioactive priming polypeptides can be provided in a
synthetic form using commercially available peptide synthesis
technologies to produce high purity polypeptides. Synthetic
production of the bioactive priming polypeptides utilizes general
solid-phase peptide synthesis methodologies that are well known to
one of ordinary skill in the art. Chemical synthesis methodologies
include: a stepwise assembly of peptides from amino acid
precursors, whereby peptide elongation proceeds via a coupling
reaction between amino acids, followed by the removal of a
reversible protecting group. Solid phase peptide synthesis is used
to add a covalent attachment step that links the nascent peptide
chain to an insoluble polymeric support whereby the anchored
peptide can be extended by a series of cycles. These extension
reactions are driven to completion and then the synthesized
polypeptide is removed from the solid support by filtration and
washing steps. MS and HPLC analyses are performed after the
completion of synthesis and purification.
[0253] Any of the bioactive priming polypeptides as described
herein for flagellin-associated polypeptides (Tables 1-5),
harpin-like (HpaG-like) polypeptides (Table 10 and 11),
phytosulfokine (PSK.alpha.) polypeptides (Table 12), RHPP (Table
13-15), elongation factor Tu (EF-Tu polypeptides) (Tables 16 and
17), thionin and thionin-like polypeptides (Table 19) can be
provided in synthetic forms.
[0254] Additionally, such methods can be used for making and using
conserved assistance sequences preferably named signature (SEQ ID
NOs: 542-548), signal anchor sorting (SEQ ID NOs: 549-562) and
secretion (SEQ ID NOs: 563-570) sequences.
[0255] Retro inverso can also be made synthetically or chemically
manufactured. Synthetic polypeptides produced in the all-D
confirmation are prepared by replacing all the L-amino acid
residues with their D-enantiomers resulting in a reversed or
retro-all-D-isomer Flg polypeptide. Solid phase synthesis is used
to prepare the retro-inverso versions of the Flg polypeptide(s).
After synthesis and purification of the retro-inverso
polypeptide(s), the amino acid composition is confirmed using mass
spectrometry of the Flg polypeptide(s). The purity of the
retro-inverso polypeptide(s) is then confirmed at a level greater
or equal to 95% using HPLC analysis. The retro-inverso versions of
the Flg polypeptide(s) are further characterized using HPLC
retention time, relative molecular mass and amino acid composition
values (IC50 .mu.M). Retro inverso production using recombinant DNA
technology generally involves the use of non-ribosomal protein
synthesis mechanisms.
[0256] Retro-inverso synthetic Flg bioactive priming polypeptides
prepared by solid phase synthesis are tested for their capacity to
bind to the FLS2 or alternative FLS receptors, for example, FLS3
also found in plants. Competitive ELISA experiments are used to
confirm the binding affinities of retro inverso Flg-associated
polypeptides to plant FLS receptors.
Recombinant Bacteria that Express Bioactive Priming
Polypeptides
[0257] A recombinant microorganism that expresses or overexpresses
a polypeptide is also provided. The polypeptide comprises the
polypeptides as described above for the composition. For example,
the polypeptide can comprise: the flagellin or flagellin-associated
polypeptide of (a); or the mutant flagellin or flagellin-associated
polypeptide of (b); or the mutant flagellin or flagellin-associated
polypeptide of (c); or the harpin or harpin-like polypeptide of
(g); or the RHPP of (i); or the KTI polypeptide of (j); or the
EF-Tu polypeptide of (l); or the fusion polypeptide of (n); or the
PSK polypeptide of (o); or the thionin or thionin-like polypeptide
of (q).
[0258] The polypeptide can be overexpressed by the microorganism.
The recombinant microorganism can comprise a microorganism that is
capable of making recombinant bioactive priming polypeptides or
their precursors in an effective manner. The preferred
microorganism would be from the genus Bacillus, a bacterium of the
genus Paenibacillus, a fungus of the genus Penicillium, a bacterium
of the genus Glomus, a bacterium of the genus Pseudomonas, a
bacterium of the genus Arthrobacter, a bacterium of the genus
Paracoccus, a bacterium of the genus Rhizobium, a bacterium of the
genus Bradyrhizobium, a bacterium of the genus Azospirillum, a
bacterium of the genus Enterobacter, a bacterium of the genus
Escherichia, or any combination thereof.
[0259] The recombinant microorganism can comprise a bacterium of
the genus Bacillus, a bacterium of the genus Paenibacillus, or any
combination thereof.
[0260] For example, the microorganism can comprise Bacillus
mycoides, Bacillus pseudomycoides, Bacillus cereus, Bacillus
thuringiensis, Bacillus megaterium, Bacillus subtilis, Bacillus
firmus, Bacillus aryabhattai, Bacillus amyloliquefaciens, Bacillus
licheniformis, Bacillus circulans, Bacillus flexus, Bacillus
nealsonii, Bacillus pumulis, Paenibacillus genus bacterium or a
combination thereof.
[0261] Methods and approaches are commonly used by one of ordinary
skill in the art to determine and verify the genus and species of
the bacteria. A common method provides chromosomal DNA isolated
from the bacteria with PCR amplification of the 16s rRNA region
using universal primers (ACTCCTACGGGAGGCAGCAGT) and
(GGGTTGCGCTCGTTG/AC). The PCR amplicons are then purified and
sequenced for correct identification of the appropriate bacterial
strain, for example a specific strain in the genera of
Bacillus.
[0262] Sample protocols are generally known to one in the art for
the preparation of chromosomal DNA, transformation of the DNA of
genes encoding the polypeptides using a plasmid, producing the
polypeptides in a host bacterium, for example, a Bacillus
strain.
[0263] The Bacillus strains provided can produce any bioactive
priming polypeptide as described herein or a combination thereof.
For example, the strain can comprise:
(a) Bacillus aryabhattai CAP53 (NRRL No. B-50819), (b) Bacillus
aryabhattai CAP56 (NRRL No. B-50817), (c) Bacillus flexus BT054
(NRRL No. B-50816), (d) Paracoccus kondratievae NC35 (NRRL No.
B-50820), (e) Bacillus mycoides BT155 (NRRL No. B-50921), (f)
Enterobacter cloacae CAP12 (NRRL No. B-50822), (g) Bacillus
nealsonii BOBA57 (NRRL No. NRRL B-50821), (h) Bacillus mycoides
EE118 (NRRL No. B-50918), (i) Bacillus subtilis EE148 (NRRL No.
B-50927), (j) Alcaligenes faecalis EE107 (NRRL No. B-50920), (k)
Bacillus mycoides EE141 (NRRL NO. B-50916), (l) Bacillus mycoides
BT46-3 (NRRL No. B-50922), (m) Bacillus cereus family member EE128
(NRRL No. B-50917), (n) Paenibacillus massiliensis BT23 (NRRL No.
B-50923), (o) Bacillus cereus family member EE349 (NRRL No.
B-50928), (p) Bacillus subtilis EE218 (NRRL No. B-50926), (q)
Bacillus megaterium EE281 (NRRL No. B-50925), (r) Bacillus cereus
family member EE-B00377 (NRRL B-67119); (s) Bacillus pseudomycoides
EE-B00366 (NRRL B-67120), (t) Bacillus mycoides EE-B00363 (NRRL
B-67121), (u) Bacillus pumilus EE-B00143 (NRRL B-67123), (v)
Bacillus thuringiensis EE-B00184 (NRRL B-67122), (w) Bacillus
mycoides EE116 (NRRL No. B-50919), (x) Bacillus cereus family
member EE417 (NRRL No. B-50974), (y) Bacillus subtilis EE442 (NRRL
No. B-50975), (z) Bacillus subtilis EE443 (NRRL No. B-50976), (aa)
Bacillus cereus family member EE444 (NRRL No. B-50977), (bb)
Bacillus subtilis EE405 (NRRL No. B-50978), (cc) Bacillus cereus
family member EE439 (NRRL No. B-50979), (dd) Bacillus megaterium
EE385 (NRRL No. B-50980), (ee) Bacillus cereus family member EE387
(NRRL No. B-50981), (ff) Bacillus circulans EE388 (NRRL No.
B-50982), (gg) Bacillus thuringiensis EE319 (NRRL No. B-50983),
(hh) Bacillus cereus family member EE377 (NRRL No. B-67119), (ii)
Bacillus mycoides EE363 (NRRL No. B-67121), (jj) Bacillus
pseudomycoides EE366 (NRRL No. B-67120); (kk) Bacillus
thuringiensis BT013A (NRRL No. B-50924);
[0264] or any combination thereof. Each of these strains has been
deposited with the United States Department of Agriculture (USDA)
Agricultural Research Service (ARS), having the address 1815 North
University Street, Peoria, Ill. 61604 U.S.A., and are identified by
the NRRL deposit numbers provided in parentheses. Strains (a)-(d),
(f), and (g) were deposited on Mar. 11, 2013. Strains (e), (hHq),
(w), and (kk) were deposited on Mar. 10, 2014. Strains (xHff) were
deposited on Sep. 10, 2014. Strain (gg) was deposited on Sep. 17,
2014. Strains (rHv), (hh), (ii), and (j) were deposited on Aug. 19,
2015. Bacillus thuringiensis BT013A is also known as Bacillus
thuringiensis 4Q7.
[0265] The isolation and characterization of these strains are
described in the Examples found within International Publication
No: WO/2017/161091, incorporated herein by reference in its
entirety. For ease of identification of the organism, International
Publication No: WO/2017/161091 A1 also provides the partial 16S
ribosomal RNA sequences for each of these strains in a sequence
list and in Table 17.
[0266] Any of the recombinant microorganisms can be used to
overexpress a bioactive priming polypeptide as described herein for
a flagellin-associated polypeptide (Tables 1-5), a harpin or
harpin-like (HpaG-like) polypeptide (Table 10 or 11), a
phytosulfokine (PSK.alpha.) polypeptide (Table 12), RHPP (Table
13-15), an EF-Tu polypeptide (Table 16-17, and a thionin or
thionin-like polypeptide (Table 19).
[0267] The recombinant microorganism can comprise a mixture of two
or more of any of the recombinant microorganisms described
herein.
[0268] The recombinant microorganism can be inactivated.
Inactivation results in microorganisms that are unable to
reproduce. Inactivation of microorganisms can be advantageous, for
example because it allows for delivery of the microorganism to a
plant or a plant growth medium while reducing or eliminating any
detrimental effects that the live microorganism may have on a plant
or on the environment. The recombinant microorganism can be
inactivated by any physical or chemical means, e.g., by heat
treatment, gamma irradiation, x-ray irradiation, UV-A irradiation,
UV-B irradiation, or treatment with a solvent such as
glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid,
bleach, chloroform, or phenol, or any combination thereof.
III. Compositions
[0269] A composition is provided for bioactive priming of a plant
or a plant part to increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part and/or
decrease abiotic stress in the plant or the plant part and/or
protect the plant or the plant part from disease, insects and/or
nematodes, and/or increase the innate immune response of the plant
or the plant part and/or change plant architecture. The composition
comprises either the polypeptide as described herein or any
combination thereof, and an agrochemical or a carrier; or any
combination of the polypeptides as described herein.
[0270] The composition can consist essentially of the bioactive
priming polypeptides or polypeptides as described herein.
[0271] The composition can comprise a majority of the bioactive
priming polypeptides with the remainder of the composition being
agrochemicals or carriers. More specifically, the composition can
comprise from about 0.00001% to about 95% of the polypeptides, from
about 0.1 to about 80 wt. % of the agrochemicals, and from about 5
to about 50 wt. % carrier based on the total weight of the
composition. Alternatively, the composition can comprise from about
0.01 to about 5 wt. % of the polypeptides, from about 0.2 to about
70 wt. % of the agrochemicals, and from about 10 to about 30 wt. %
carrier based on the total weight of the composition, or the
composition can comprise from about 0.05 wt. % to about 1 wt. % of
the polypeptides, from about 30 to about 60 wt. % of the
agrochemicals, and from about 40 to about 69 wt. % carrier based on
the total weight of the composition. Alternatively, the composition
can comprise any detectable amount of the polypeptides, and from
about 0.1 to about 80 wt. % of the agrochemicals and from about 5
to about 50 wt. % of the carrier, based on the total weight of the
composition.
[0272] The composition can include either an agrochemical or a
carrier which is associated with the polypeptide in nature.
[0273] The agrochemical can be non-naturally occurring in
combination with the polypeptide.
[0274] The agrochemical can include, but is not limited to, a
preservative, a buffering agent, a wetting agent, a surfactant, a
coating agent, a monosaccharide, a polysaccharide, an abrading
agent, a pesticide, an insecticide, an herbicide, a nematicide, a
bacteriocide, a fungicide, a miticide, a fertilizer, a
biostimulant, a colorant, a humectant, an osmoprotectant, an
antibiotic, an amino acid, a biological control agent, or a
combination thereof.
[0275] When the composition includes an amino acid, the amino acid
can be provided separately from the amino acids that comprise the
polypeptide. For example, an isolated amino acid can be used.
Suitable amino acids include any natural or unnatural amino acids.
For example, the composition can comprise cysteine.
[0276] The agrochemical can comprise an acid such as an acid that
is present from chemical synthesis of any polypeptide described
herein. For example, hydrochloric acid, acetic acid, or
trifluoroacetic acid can be present if the polypeptide is
synthesized such as by fermentation.
[0277] When the agrochemical is an acid, it can comprise from about
0.001 to about 30 wt. %, from about 0.01 to about 20 wt. %, or from
about 0.1 to about 5 wt. % of the total weight of the
composition.
[0278] Unless otherwise specified, each agrochemical can comprise
from about 0.1 to about 60 wt. %, from about 0.5 to about 50 wt. %,
or from about 10 to about 30 wt. % of the total weight of the
composition.
[0279] When the composition includes a preservative, the
preservative can comprise those based on dichlorophene and
benzylalcohol hemi formal (PROXEL from ICI or ACTICIDE RS from Thor
Chemie and KATHON MK from Dow Chemical) and isothiazolinone
derivatives such as alkylisothiazolinones and benzisothiazolinones
(ACTICIDE MBS from Thor Chemie). As further examples, suitable
preservatives include MIT (2-methyl-4-isothiazolin-3-one), BIT
(1,2-benzisothiazolin-3-one, which can be obtained from Avecia,
Inc. as PROXEL GXL as a solution in sodium hydroxide and
dipropylene glycol),
5-chloro-2-(4-chlorobenzyl)-3(2H)-isothiazolone,
5-chloro-2-methyl-2H-isothiazol-3-one,
5-chloro-2-methyl-2H-isothiazol-3-one,
5-chloro-2-methyl-2H-isothiazol-3-one-hydrochloride,
4,5-dichloro-2-cyclohexyl-4-isothiazolin-3-one,
4,5-dichloro-2-octyl-2H-isothiazol-3-one,
2-methyl-2H-isothiazol-3-one, 2-methyl-2H-isothiazol-3-one-calcium
chloride complex, 2-octyl-2H-isothiazol-3-one, benzyl alcohol
hemiformal, or any combination thereof.
[0280] When the composition includes a buffering agent, the
buffering agent can comprise potassium, phosphoric acid, a
phosphate salt, citric acid, a citrate salt, a sulfate salt, MOPS,
or HEPES. The buffering agent can stabilize the polypeptide in the
composition.
[0281] When the composition includes a wetting agent, the wetting
agent can comprise organosilicones, polyoxyethoxylates,
polysorbates, polyethyleneglycol and derivatives thereof,
ethoxylates, crop oils, and polysaccharides.
[0282] When the composition includes a surfactant, the surfactant
can comprise a heavy petroleum oil, a heavy petroleum distillate, a
polyol fatty acid ester, a polyethoxylated fatty acid ester, an
aryl alkyl polyoxyethylene glycol, a
polyoxyethylenepolyoxypropylene monobutyl ether, an alkyl amine
acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl
phosphate, an alcohol ethoxylate, an alkylphenol ethoxylate, an
alkyphenol ethoxylate, an alkoxylated polyol, an alky polyethoxy
ether, an alkylpolyoxethylene glycerol, ethoxylated and soybean oil
derivatives, an organosilicone-based surfactant or any combination
thereof. Surfactants can be included in a range of compositions
including those for foliar use.
[0283] When the composition includes a coating agent, the coating
agent can comprise a tackifier, polymers, filling agents, or
bulking agents.
[0284] The tackifier can include, but is not limited to,
carboxymethylcellulose and natural and synthetic polymers in the
form of powders, granules, or latexes, such as gum Arabic, chitin,
polyvinyl alcohol and polyvinyl acetate, as well as natural
phospholipids, such as cephalins and lecithins, and synthetic
phospholipids. Tackifiers include those composed preferably of an
adhesive polymer that can be natural or synthetic without
phytotoxic effect on the seed to be coated. Additional tackifiers
that can be included, either alone or in combination, include, for
example, polyesters, polyether esters, polyanhydrides, polyester
urethanes, polyester amides; polyvinyl acetates; polyvinyl acetate
copolymers; polyvinyl alcohols and tylose; polyvinyl alcohol
copolymers; polyvinylpyrolidones; polysaccharides, including
starches, modified starches and starch derivatives, dextrins,
maltodextrins, alginates, chitosanes and celluloses, cellulose
esters, cellulose ethers and cellulose ether esters including
ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,
hydroxypropylcelluloses and carboxymethylcellulose; fats; oils;
proteins, including casein, gelatin and zeins; gum arabics;
shellacs; vinylidene chloride and vinylidene chloride copolymers;
lignosulfonates, in particular calcium lignosulfonates;
polyacrylates, polymethacrylates and acrylic copolymers;
polyvinylacrylates; polyethylene oxide; polybutenes,
polyisobutenes, polystyrene, polybutadiene, polyethyleneamines,
polyethylenamides; acrylamide polymers and copolymers;
polyhydroxyethyl acrylate, methylacrylamide monomers; and
polychloroprene, or any combination thereof. Tackifiers can be used
in a range of compositions including those for seed treatment.
[0285] When the composition includes an abrading agent, the
abrading agent can comprise talc, graphite, or a combination of
both.
[0286] A humectant is a hygroscopic substance that assists with the
retention of moisture. When the composition includes a humectant,
the humectant can comprise: glycerol, glycerin, a glycerol
derivative (e.g. glycerol monosterate, glycerol triacetate,
triacetin, propylene glycol, hexylene glycol, or butylene glycol),
triethylene glycol, tripolypropylene glycol, glyceryl triacetate,
sucrose, tagatose, a sugar alcohol or a sugar polyol (e.g glycerol,
sorbitol, xylitol, mannitol, or mantitol), a polymeric polyol (e.g.
polydextrose, a collagen, an aloe or an aloe vera gel), or an alpha
hydroxy acid (e.g. lactic acid, honey, molasses, quillaia, sodium
hexametaphosphate, lithium chloride or urea). Synthetic humectants
can also comprise: butylene glycol, and tremella extract.
[0287] When the composition includes a pesticide, the pesticide can
comprise an insecticide, a herbicide, a fungicide, a bacteriocide,
a nematicide, a miticide, or any combination thereof.
[0288] When the composition includes an insecticide, the
insecticide can comprise clothianidin, imidacloprid, an
organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl
phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic
substituted urea, a hydrocarbon ester, a biologically-based
insecticide, or any combination thereof. For example, the
insecticide can comprise clothianidin or imidacloprid.
[0289] The agrochemical can comprise an herbicide. The herbicide
can comprise 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor,
ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulfuron
methyl bensulide, bentazon, bispyribac sodium, bromacil,
bromoxynil, butylate, carfentrazone, chlorimuron, 2-chlorophenoxy
acetic acid, chlorsulfuron, chlorimuron ethyl, clethodim,
clomazone, clopyralid, cloransulam, CMPP-P-DMA, cycloate, DCPA,
desmedipham, dicamba, dichlobenil, diclofop, 2,4-dichlorophenol,
dichlorophenoxyacetic acid, dichlorprop, dichlorprop-P, diclosulam,
diflufenzopyr, dimethenamid, dimethyl amine salt of
2,4-dichlorophenoxyacetic acid, diquat, diuron, DSMA, endothall,
EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P,
flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin,
fluometuron, fluroxypyr, fluorxypyr 1-methyleptylester, fomesafen,
fomesafen sodium salt, foramsulfuron, glufosinate,
glufosinate-ammonium, glyphosate, halosulfuron,
halosulfuron-methyl, hexazinone, 2-hydroxyphenoxy acetic acid,
4-hydroxyphenoxy acetic acid, imazamethabenz, imazamox, imazapic,
imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron,
mazapyr, MCPA, MCPB, mecoprop, mecoprop-P, mesotrione,
metolachlor-s, metribuzin, metsulfuron, metsulfuron-methyl,
molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon,
oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid,
pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine,
prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithiobac,
pyroxasulfone, quinclorac, quizalofop, rimsulfuron, sethoxydim,
siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron,
tebuthiuron, terbacil, thiazopyr, thifensulfuron,
thifensulfuron-methyl, thiobencarb, tralkoxydim, triallate,
triasulfuron, tribenuron, tribemuron-methyl, triclopyr,
trifluralin, triflusulfuron, or any combination thereof.
[0290] When the composition includes a nematicide, the nematicide
can comprise Bacillus firmus, fluopyram, antibiotic nematicides
such as abamectin; carbamate nematicides such as acetoprole,
Bacillus chitonosporus, chloropicrin, benclothiaz, benomyl,
Burholderia cepacia, carbofuran, carbosulfan, and cleothocard;
dazomet, DBCP, DCIP, alanycarb, aldicarb, aldoxycarb, oxamyl,
diamidafos, fenamiphos, fosthietan, phosphamidon, cadusafos,
chlorpyrifos, diclofenthion, dimethoate, ethoprophos,
fensulfothion, fostiazate, harpins, heterophos, imicyafos,
isamidofos, isazofos, methomyl, mecarphon, Myrothecium verrucaria,
Paecilomyces lilacinus, Pasteuria nishizawae (including spores
thereof), phorate, phosphocarb, terbufos, thionazin, triazophos,
tioxazafen, dazomet, 1,2-dichloropropane, 1,3-dichloropropene,
furfural, iodomethane, metam, methyl bromide, methyl
isothiocyanate, xylenol, or any combination thereof. For example,
the nematicide can comprise Bacillus firmus strain i-2580,
Pasteuria nishizawae (including spores thereof), or fluopyram.
[0291] When the composition includes a bacteriocide, the
bacteriocide can comprise streptomycin, penicillins, tetracyclines,
oxytetracycline, kasugamycin, ampicillin, oxolinic acid,
chlorotetracycline, copper oxide, or any combination thereof. For
example, the bacteriocide can comprise oxytetracycline.
[0292] Biological control agents are broadly defined as
microorganisms that can be used instead of synthetic pesticides or
fertilizers. When the composition includes a biological control
agent, the biological control agent can comprise Bacillus
thuringiensis, Bacillus megaterium, Bacillus mycoides isolate J,
Bacillus methylotrophicus, Bacillus vallismortis, Chromobacterium
subtsugae, Deiftia acidovorans, Streptomyces lydicus, Streptomyces
colombiensis, Streptomyces galbus K61, Penicillium bilaii, a
lipopeptide-producing Bacillus subtilis strain, a
lipopeptide-producing Bacillus amyloliquefaciens strain, a Bacillus
firmus strain or a Bacillus pumilus strain.
[0293] The agrochemical can include a fungicide. The fungicide can
comprise aldimorph, ampropylfos, ampropylfos potassium, andoprim,
anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil,
benomyl, benzamacril, benzamacryl-isobutyl, benzovindflupyr,
bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S,
boscalid, bromuconazole, bupirimate, buthiobate, calcium
polysulphide, capsimycin, captafol, captan, carbendazim, carvon,
quinomethionate, chlobenthiazone, chlorfenazole, chloroneb,
chloropicrin, chlorothalonil, chlozolinate, clozylacon, cufraneb,
cymoxanil, cyproconazole, cyprodinil, cyprofuram, debacarb,
dichlorophen, diclobutrazole, diclofluanid, diclomezine, dicloran,
diethofencarb, dimethirimol, dimethomorph, dimoxystrobin,
diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione,
ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos,
epoxiconazole, etaconazole, ethirimol, etridiazole, famoxadon,
fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan,
fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentin
hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumetover,
fluoromide, fluoxastrobin fluquinconazole, flurprimidol,
flusilazole, flusulfamide, flutolanil, flutriafol, folpet,
fosetyl-aluminium, fosetyl-sodium, fthalide, fuberidazole,
furalaxyl, furametpyr, furcarbonil, furconazole, furconazole-cis,
furmecyclox, guazatine, hexachlorobenzene, hexaconazole,
hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine
albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP),
iprodione, irumamycin, isoprothiolane, isovaledione, kasugamycin,
kresoxim-methyl, copper preparations, such as: copper hydroxide,
copper naphthenate, copper oxychloride, copper sulphate, copper
oxide, oxine-copper and Bordeaux mixture, mancopper, mancozeb,
maneb, meferimzone, mepanipyrim, mepronil, metconazole, metalzxyl,
methasulfocarb, methfuroxam, metiram, metomeclam, metsulfovax,
mildiomycin, myclobutanil, myclozolin, nickel
dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofurace,
oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, oxyfenthiin,
paclobutrazole, pefurazoate, penconazole, pencycuron, phosdiphen,
picoxystrobin, pimaricin, piperalin, polyoxin, polyoxorim,
probenazole, prochloraz, procymidone, propamocarb,
propanosine-sodium, propiconazole, propineb, prothiocinazole,
pyrazophos, pyrifenox, pyrimethanil, pyroquilon, pyroxyfur,
quinconazole, quintozene (PCNB), a strobilurin, sulphur and sulphur
preparations, tebuconazole, tecloftalam, tecnazene, tetcyclasis,
tetraconazole, thiabendazole, thicyofen, thifluzamide,
thiophanate-methyl, tioxymid, tolclofos-methyl, tolylfluanid,
triadimefon, triadimenol, triazbutil, a triazole, triazoxide,
trichlamide, tricyclazole, triclopyr, tridemorph, trifloxystrobin,
triflumizole, triforine, uniconazole, validamycin A, vinclozolin,
viniconazole, zarilamide, zineb, ziram and also Dagger G, OK-8705,
OK-8801,
a-(1,1-dimethylethyl)-(3-(2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol,
a-(2,4-dichlorophenyl)-[3-fluoro-3-propyl-1H-1,2,4-triazole-1-ethanol,
a-(2,4-dichlorophenyl)-[3-methoxy-a-methyl-1H-1,2,4-triazole-1-ethanol,
a-(5-methyl-1,3-dioxan-5-yl)-[3-[[4-(trifluoromethyl)-phenyl]-methylene]--
1H-1,2,4-triazole-1-ethanol,
(5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1H-1,2,4-triazol-1-yl)-3-octan-
one, (E)-a-(methoxyimino)-N-methyl-2-phenoxy-phenylacetamide,
1-isopropyl{2-methyl-1-[[[1-(4-methylphenyl)-ethyl]-amino]-carbonyl]-prop-
yl}carbamate,
1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-ethanone-O-(phenyl
methyl)-oxime, 1-(2-methyl-1-naphthalenyl)-1H-pyrrole-2,5-dione,
1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidindione,
1-[(diiodomethyl)-sulphonyl]-4-methyl-benzene,
1-[2-(2,4-dichlorophenyl)-1, 3-dioxolan-2-yl]-methyl]-1H-imidazole,
1-[[2-(4-chlorophenyl)-3-phenyloxiranyl]-methyl]-1H-1,2,4-triazole,
1-[1-[2-[(2,4-dichlorophenyl)-methoxy]-phenyl]-ethenyl]-1H-imidazole,
1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinole,
2',6'-dibromo-2-methyl-4'-trifluoromethoxy-4'-trifluoro-methyl-1,
3-thiazole-carboxanilide,
2,2-dichloro-N-[1-(4-chlorophenyl)-ethyl]-1-ethyl-3-methyl-cyclopropaneca-
rboxamide, 2,6-dichloro-5-(methylthio)-4-pyrimidinyl-thiocyanate,
2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide,
2,6-dichloro-N-[[4-(trifluoromethyl)-phenyl]-methyl]-benzamide,
2-(2,3,3-triiodo-2-propenyl)-2H-tetrazole,
2-[(1-methylethyl)-sulphonyl]-5-(trichloromethyl)-1,3,4-thiadiazole,
2-[[6-deoxy-4-O-(4-O-methyl-(3-D-glycopyranosyl)-a-D-glucopyranos
yl]-amino]-4-methoxy-1H-pyrrolo [2,3-d]pyrimidine-5-carbonitrile,
2-aminobutane, 2-bromo-2-(bromomethyl)-pentanedinitrile,
2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxam-
ide,
2-chloro-N-(2,6-dimethylphenyl)-N-(isothiocyanatomethyl)-acetamide,
2-phenylphenol (OPP),
3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-pyrrole-2,5-dione,
3,5-dichloro-N-[cyano[(1-methyl-2-propynyl)-oxy]-methyl]-benzamide,
3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile,
3-[2-(4-chlorophenyl)-5-ethoxy-3-isoxazolidinyl]-pyridine,
4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-I-sulphonam-
ide, 4-methyl-tetrazolo[1,5-a]quinazolin-5(4H)-one,
8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4,
5]decane-2-methanamine, 8-hydroxyquinoline sulphate,
9H-xanthene-2-[(phenylamino)-carbonyl]-9-carboxylic hydrazide,
bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophenedicar-
boxylate,
cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol,
cis-4-[3-[4-(1,1-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl-morp-
holine hydrochloride, ethyl [(4-chlorophenyl)-azo]-cyanoacetate,
potassium bicarbonate, methanetetrathiol-sodium salt, methyl
1-(2,3-dihydro-2,2-dimethyl-inden-1-yl)-1H-imidazole-5-carboxylate,
methyl
N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate,
methyl N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate,
N-(2,3-dichloro-4-hydroxyphenyl)-1-methyl-cyclohexanecarboxamide,
N-(2,6-dimethyl phenyl)-2-methoxy-N-(tetra
hydro-2-oxo-3-furanyl)-acetamide, N-(2,6-dimethyl
phenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)-acetamide,
N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitro-benzenesulphonamide,
N-(4-cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine,
N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine,
N-(5-chloro-2-methylphenyl)-2-methoxy-N-(2-oxo-3-oxazolidinyl)-acetamide,
N-(6-methoxy)-3-pyridinyl)-cyclopropanecarboxamide,
N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide,
N-[3-chloro-4,5-bis(2-propinyloxy)-phenyl]-N'-methoxy-methanimidamide,
N-formyl-N-hydroxy-DL-alanine-sodium salt, 0,0-diethyl
[2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate, O-methyl
S-phenyl phenylpropylphosphoramidothioate, S-methyl
1,2,3-benzothiadiazole-7-carbothioate, and
spiro[2H]-1-benzopyrane-2,1'(3'H)-isobenzofuran]-3'-one,
N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide,
tetramethylthioperoxydicarbonic diamide, methyl
N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate,
4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1-H-pyrrol-3-carbonitril, or
any combination thereof.
[0294] When the polypeptides are formulated or applied in
combination with commercially available fungicides, the
compositions can provide an extra layer of protection for enhancing
disease prevention or spread in a plant. The combination of the
polypeptides with a fungicide can protect a plant against a primary
or secondary fungal infection which may occur if the plant has
become compromised or weakened due to exposure to abiotic stress or
disease.
[0295] The strobilurin fungicide can comprise a Strobilurin A, a
Strobilurin B, a Strobilurin C, a Strobilurin D, a Strobilurin E, a
Strobilurin F, a Strobilurin G, a Strobilurin H, an Azoxystrobin, a
Trifloxystrobin, a Kresoxim methyl, a Fluoxastrobin, Picoxystrobin,
or any combination thereof.
[0296] The strobilurin fungicide can comprise a non-naturally
occurring strobilurin fungicide such as an Azoxystrobin, a
Trifloxystrobin, a Kresoxim methyl, a Fluoxastrobin, or any
combination thereof. For example, the strobilurin fungicide can
comprise a Trifloxystrobin, Fluoxastrobin or Picoxystrobin.
Strobilurin fungicides are used to control a range of fungal
diseases, including water molds, downy mildews, powdery mildews,
leaf spotting and blighting fungi, fruit rotters, and rusts. They
are useful for treating a variety of crops, including cereals,
field crops, fruits, tree nuts, vegetables, turfgrasses, and
ornamentals.
[0297] The triazole fungicide can comprise prothioconazole,
imidazole, imidazil, prochloraz, propiconazole, triflumizole,
diniconazole, flusilazole, penconazole, hexaconazole,
cyproconazole, myclobutanil, tebuconazole, difenoconazole,
tetraconazole, fenbuconazole, epoxiconazole, metconazole,
fluquinconazole, triticonazole, or any combination thereof.
[0298] The bioactive priming polypeptides can be delivered in
combination with strobilurins and triazole fungicides, especially
fluoxastrobin or trifloxystrobin in combination with
prothioconazole.
[0299] In addition, the fungicide can comprise azoxystrobin,
carboxin, difenoconazole, fludioxonil, fluxapyroxad, ipconazole,
mefenoxam, pyraclostrobin, silthiofam, sedaxane, thiram,
triticonazole or any combination thereof.
[0300] In addition to foliar applied fungicides as described
herein, the bioactive priming polypeptides can be provided in
combination with a fungicide, an insecticide, a nematicide, a
bacteriocide, and a miticide or any agrochemical which is a
biological agent.
[0301] The agrochemical can include a fertilizer. The fertilizer
can comprise ammonium sulfate, ammonium nitrate, ammonium sulfate
nitrate, ammonium chloride, ammonium bisulfate, ammonium
polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous
ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate,
calcium ammonium nitrate, calcium sulfate, calcined magnesite,
calcitic limestone, calcium oxide, calcium nitrate, dolomitic
limestone, hydrated lime, calcium carbonate, diammonium phosphate,
monoammonium phosphate, magnesium nitrate, magnesium sulfate,
potassium nitrate, potassium chloride, potassium magnesium sulfate,
potassium sulfate, sodium nitrates, magnesian limestone, magnesia,
urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated
urea, polymer-coated urea, isobutylidene diurea,
K.sub.2SO4-Mg.sub.2SO.sub.4, kainite, sylvinite, kieserite, Epsom
salts, elemental sulfur, marl, ground oyster shells, fish meal, oil
cakes, fish manure, blood meal, rock phosphate, super phosphates,
slag, bone meal, wood ash, manure, bat guano, peat moss, compost,
green sand, cottonseed meal, feather meal, crab meal, fish
emulsion, humic acid, or any combination thereof.
[0302] The fertilizer can comprise a liquid fertilizer or a dry
fertilizer.
[0303] The agrochemical can comprise a micronutrient fertilizer
material, the micronutrient fertilizer material comprising boric
acid, a borate, a boron frit, copper sulfate, a copper frit, a
copper chelate, a sodium tetraborate decahydrate, an iron sulfate,
an iron oxide, iron ammonium sulfate, an iron frit, an iron
chelate, a manganese sulfate, a manganese oxide, a manganese
chelate, a manganese chloride, a manganese frit, a sodium
molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc
carbonate, a zinc frit, zinc phosphate, a zinc chelate, or any
combination thereof.
[0304] The agrochemical can comprise an insecticide, the
insecticide comprising an organophosphate, a carbamate, a
pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate,
a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon
ester, a biologically-based insecticide, or any combination
thereof.
[0305] When the composition includes a biostimulant, the
biostimulant can comprise a seaweed extract, an elicitor, a
polysaccharide, a monosaccharide, a protein extract, a soybean
extract, a humic acid, a plant hormone, a plant growth regulator,
or any combination thereof.
[0306] A variety of colorants may be employed, including organic
chromophores classified as nitroso, nitro, azo, including monoazo,
bisazo, and polyazo, diphenylmethane, triarylmethane, xanthene,
methane, acridine, thiazole, thiazine, indamine, indophenol, azine,
oxazine, anthraquinone, phthalocyanine, or any combination
thereof.
[0307] The composition can further comprise a carrier.
[0308] The carrier of the composition can include, but is not
limited to, water, peat, wheat, bran, vermiculite, clay,
pasteurized soil, calcium carbonate, calcium bicarbonate, dolomite,
gypsum, bentonite, a clay, a rock phosphate, a phosphorous
compound, titanium dioxide, humus, talc, alginate, activated
charcoal, or a combination thereof.
[0309] The composition can be in the form of an aqueous solution, a
slurry or dispersion, an emulsion, a solid such as a powder or
granule, or any other desirable form for applying the composition
to a plant or plant part.
[0310] Bioactive priming polypeptides such as the flagellin and
flagellin-associated polypeptides, thionin (defensin family),
harpin-like HpaG, EF-Tu or other growth promoting or altering
bioactive priming polypeptides such as PSK.alpha. and RHPP can be
provided as compositions that can either be exogenously and/or
endogenously applied to a plant or a plant part and provide
enhanced plant growth, productivity and enhanced health of that
plant or plant part as described in more detail below.
[0311] The bioactive priming polypeptides can be added separately
or in combination as a composition that are useful as applications
to provide a benefit to plants and/or plant parts.
[0312] In combination, the polypeptides may be formulated and
delivered in a purified polypeptide form either as a genetic fusion
on the same recombinant vector, or separately using different
recombinant vectors.
[0313] The bioactive priming polypeptides can also be created and
delivered to a plant or plant part as polypeptides from multiple
actives in a fusion protein. Examples of this include delivery of
multiple flagellin associated polypeptides produced in series with
protease cleavage sites between each polypeptide as is within the
skill of one of ordinary skill in the art. Such fusion proteins can
include any combination of the bioactive priming polypeptides as
described herein, including bioactive priming polypeptides from
different classes, such as combinations of flagellin associated
polypeptides with RHPP. Bioactive priming polypeptides can also be
utilized as protein fusions to plant binding domains, which can
direct the polypeptides to distinct locations within the plant
where they are most desired or needed for their activities to be
beneficial.
[0314] Additionally, the polypeptides may be added to formulations
provided in a synthetic compound form.
[0315] The flagellin and flagellin-associated bioactive priming
polypeptides as described herein can be provided individually or in
combination containing at least two to multiple bioactive priming
polypeptides to provide a composition that meets the specific needs
of a plant over a wide range of desired host responses and cropping
systems.
[0316] When a composition includes the retro-inverso form of a Flg
bioactive priming polypeptide (for example, RI Bt.4Q7 Flg 22 (SEQ
ID NO: 376), the polypeptide exhibits enhanced stability and less
degradation over time providing for more activity at the plant cell
membrane surface, which enhances the ability of the polypeptide to
bind to the receptor and be taken into the plant. Retro inverso
forms of such Flg-associated bioactive priming polypeptides are
used to provide enhanced stability of the agriculturally applied
formulation whereby the Flg polypeptide(s) exhibits enhanced
protection from proteolytic cleavage, which contributes to an
overall greater activity and shelf life of the composition.
[0317] When the polypeptide comprises an RHPP polypeptide, the
composition can further comprise a flagellin or flagellin
associated polypeptide. The RHPP polypeptide can comprise SEQ ID
NO: 600. The amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 1-525, 532, 534, 536, 538, 540, 571-586, and 751-752, or any
combination thereof. For example, the flagellin or flagellin
associated polypeptide can comprise any one of SEQ ID NO: 226, 571,
and 752. In some instances, the RHPP polypeptide can comprise SEQ
ID NO: 600 and the flagellin or flagellin associated polypeptide
can comprise SEQ ID NO: 226.
[0318] The polypeptides can be formulated in combination with an
assistance polypeptide. The signature (SEQ ID NOs: 542-548), signal
anchor sorting (SEQ ID NOs: 549-562) and secretion (SEQ ID NOs:
563-570) polypeptides can be combined with the bioactive priming
polypeptides as described for targeting the polypeptides/peptides
(Tables 1-5) to the plant cell membrane surface for improved
binding and activation of the Flg-associated receptors. This means
for efficient delivery and binding of the polypeptide to a plant
provides growth promoting benefits, as well as enhanced protection
to the plant or plant part.
[0319] For example, the harpin or HpaG-like bioactive priming
polypeptides as described herein can be used in combination with
the assistance polypeptides as described in Tables 6-8), signature
polypeptides (SEQ ID NO: 542-548), signal anchor sorting (SEQ ID
NO: 549-562) and/or secretion (SEQ ID NO: 563-570) polypeptides.
These assistance polypeptides used in combination with the
HpaG-like bioactive priming polypeptides are useful to target and
deliver the harpin-like bioactive priming polypeptides to the plant
cell membrane surface enhancing the contact with the plant cell
membrane and provide a conduit facilitating efficient contact and
entry of harpin-like (HpaG-like) into the plant or to the plant
cell milieu (apoplast).
[0320] One or more of the EF-Tu polypeptides can be combined,
optionally, with the flagellin or flagellin-associated polypeptide.
The amino acid sequence of the EF-Tu polypeptide or polypeptides
can comprise SEQ ID NOs: 616 and/or 617. The amino acid sequence of
the flagellin or flagellin-associated polypeptide can comprise any
one of SEQ ID NOs: 1-525, 532, 534, 536, 538, 540, 571-586, and
751-753 or any combination thereof. For example, the amino acid
sequence of the flagellin or flagellin-associated polypeptide can
comprise SEQ ID NO: 571. As another example, the composition can
comprise an EF-Tu polypeptides comprising SEQ ID NOs: 616 and 617,
and a flagellin or flagellin associated polypeptide comprising SEQ
ID NO: 226, 571, 572, or combinations thereof. As another example,
the EF-Tu polypeptide or polypeptides having SEQ ID NOs 616 and/or
617 can be combined with a flagellin or flagellin associated
polypeptide having SEQ ID NO: 226. Alternatively, the composition
can comprise one or more EF-Tu polypeptides alone (e.g., comprising
SEQ ID NOs 616 and/or 617). The EF-Tu polypeptides (e.g., SEQ ID
Nos 616 and 617) can be further modified via N-terminal
acetylation.
[0321] Additionally, the EF-Tu polypeptide or the EF-Tu polypeptide
and the flagellin or flagellin-associated polypeptide can be
combined with the harpin or harpin-like polypeptide. For example,
the amino acid sequence of the harpin or harpin-like polypeptide
can comprise SEQ ID NO: 587.
[0322] The composition can comprise any one of the following
combinations: (a) the flagellin or flagellin-associated
polypeptides and the amino acid sequences of the flagellin or
flagellin-associated polypeptides comprise SEQ ID NOs: 571, 295,
300, 293, and 580; or 295, 300, 293, and 580; or 571, 295, 293, and
580; or 571, 300, 293, and 580; or 571, 293 and 580; or 571, 295,
293; or (b) the flagellin or flagellin-associated polypeptide and
the amino acid sequence of the flagellin or flagellin-associated
polypeptide comprises SEQ ID NO: 226 and cellobiose, cellulose,
chitin, chitosan or any combination thereof; or (c) the flagellin
or flagellin-associated polypeptide and the amino acid sequence of
the flagellin or flagellin-associated polypeptide comprises SEQ ID
NO: 226 and the harpin or harpin-like polypeptide and the amino
acid sequence of the harpin or harpin-like polypeptide comprises
SEQ ID NO: 591; or (d) the harpin or harpin-like polypeptide and
the amino acid sequence of the harpin or harpin-like polypeptide
comprises SEQ ID NO: 587 and the PSK polypeptide and the amino acid
sequence of the PSK polypeptide comprises SEQ ID NO: 598; or (e)
the flagellin or flagellin-associated polypeptide and the amino
acid sequence of the flagellin or flagellin-associated polypeptide
comprises SEQ ID NO: 226, 752, or 571 or any combination thereof
and the EF-Tu polypeptides and the amino acid sequences of the
EF-Tu polypeptides comprise SEQ ID NOs: 616 and 617; or (f) the
flagellin or flagellin-associated polypeptide and the amino acid
sequence of the flagellin or flagellin-associated polypeptide
comprises SEQ ID NO: 226, 540, 752, or 571 or any combination
thereof; or (g) the RHPP polypeptide and the amino acid sequence of
the RHPP polypeptide comprises SEQ ID NO: 600; or (h) the flagellin
or flagellin-associated polypeptide and the amino acid sequences of
the flagellin or flagellin-associated polypeptide comprises SEQ ID
NO: 226, 540, 226, 752, or 571 or any combination thereof and the
RHPP polypeptide and the amino acid sequence of the RHPP
polypeptide comprises SEQ ID NO: 600; or (i) the flagellin or
flagellin-associated polypeptide and the amino acid sequence of the
flagellin or flagellin-associated polypeptide comprises SEQ ID NO:
226 and the RHPP polypeptide and the amino acid sequence of the
RHPP polypeptide comprises SEQ ID NO: 600.
IV. Applications
[0323] The agricultural composition and methods described herein
can be used with any species of plant and/or the seeds thereof. The
compositions and methods are typically used with seeds that are
agronomically important.
[0324] The seed can be a transgenic seed from which a transgenic
plant can grow that incorporates a transgenic event that confers,
for example, tolerance to a particular herbicide or combination of
herbicides, increased disease resistance, enhanced tolerance to
insects, drought, stress and/or enhanced yield.
[0325] The seed can comprise a breeding trait, including for
example, a disease tolerant breeding trait.
[0326] In some instances, the seed includes at least one transgenic
trait and at least one breeding trait.
[0327] The bioactive priming polypeptide compositions and methods
for applying the polypeptides can be used for the treatment of any
suitable seed type, including, but not limited to, row crops and
vegetables. For example, one or more plants or plant parts or the
seeds of one or more plants can comprise abaca (manila hemp) (Musa
textilis), alfalfa for fodder (Medicago sativa), alfalfa for seed
(Medicago sativa), almond (Prunus dulcis), anise seeds (Pimpinella
anisum), apple (Malus sylvestris), apricot (Prunus armeniaca),
areca (betel nut) (Areca catechu), arracha (Arracacia
xanthorrhiza), arrowroot (Maranta arundinacea), artichoke (Cynara
scolymus), asparagus (Asparagus officinalis), avocado (Persea
americana), bajra (pearl millet) (Pennisetum americanum), bambara
groundnut (Vigna subterranea), banana (Musa paradisiaca), barley
(Hordeum vulgare), beans, dry, edible, for grains (Phaseolus
vulgaris), beans, harvested green (Phaseolus and Vigna spp.), beet,
fodder (mangel) (Beta vulgaris), beet, red (Beta vulgaris), beet,
sugar (Beta vulgaris), beet, sugar for fodder (Beta vulgaris),
beet, sugar for seeds (Beta vulgaris), bergamot (Citrus bergamia),
betel nut (Areca catechu), black pepper (Piper nigrum), black
wattle (Acacia mearnsii), blackberries of various species (Rubus
spp.), blueberry (Vaccinium spp.), Brazil nut (Bertholletia
excelsa), breadfruit (Artocarpus altilis), broad bean, dry (Vicia
faba), broad bean, harvested green (Vicia faba), broccoli (Brassica
oleracea var. botrytis), broom millet (Sorghum bicolor), broom
sorghum (Sorghum bicolor), Brussels sprouts (Brassica oleracea var.
gemmifera), buckwheat (Fagopyrum esculentum), cabbage, red, white,
Savoy (Brassica oleracea var. capitata), cabbage, Chinese (Brassica
chinensis), cabbage, for fodder (Brassica spp.), cacao (cocoa)
(Theobroma cacao), cantaloupe (Cucumis melo), caraway seeds (Carum
carvi), cardamom (Elettaria cardamomum), cardoon (Cynara
cardunculus), carob (Ceratonia siliqua), carrot, edible (Daucus
carota spp. sativa), carrot, for fodder (Daucus carota sativa),
cashew nuts (Anacardium occidentale), cassava (manioc) (Manihot
esculenta), castor bean (Ricinus communis), cauliflower (Brassica
oleracea var. botrytis), celeriac (Apium graveolens var. rapaceum),
celery (Apium graveolens), chayote (Sechium edule), cherry, all
varieties (Prunus spp.), chestnut (Castanea sativa), chickpea (gram
pea) (Cicer arietinum), chicory (Cichorium intybus), chicory for
greens (Cichoium intybus), chili, dry (all varieties) (Capsicum
spp. (annuum)), chili, fresh (all varieties) (Capsicum spp.
(annuum)), cinnamon (Cinnamomum verum), citron (Citrus medica),
citronella (Cymbopogon citrates; Cymbopogon nardus), clementine
(Citrus reticulata), clove (Eugenia aromatica; Syzygium
aromaticum), clover for fodder (all varieties) (Trifolium spp.),
clover for seed (all varieties) (Trifolium spp.), cocoa (cacao)
(Theobroma cacao), coconut (Cocos nucifera), cocoyam (Colocasia
esculenta), coffee (Coffea spp.), cola nut, all varieties (Cola
acuminata), colza (rapeseed) (Brassica napus), corn (maize), for
cereals (Zea mays), corn (maize), for silage (Zea mays), corn
(maize), for vegetable (Zea mays), corn for salad (Valerianella
locusta), cotton, all varieties (Gossypium spp.), cottonseed, all
varieties (Gossypium spp.), cowpea, for grain (Vigna unguiculata),
cowpea, harvested green (Vigna unguiculata), cranberry (Vaccinium
spp.), cress (Lepidium sativum), cucumber (Cucumis sativus),
currants, all varieties (Ribes spp.), custard apple (Annona
reticulate), dasheen (Colocasia esculenta), dates (Phoenix
dactylifera), drumstick tree (Moringa oleifera), durra (sorghum)
(Sorghum bicolour), durum wheat (Triticum durum), earth pea (Vigna
subterranea), edo (eddoe) (Xanthosoma spp.; Colocasia spp.),
eggplant (Solanum melongena), endive (Cichorium endivia), fennel
(Foeniculum vulgare), fenugreek (Trigonella foenum-graecum), fig
(Ficus carica), filbert (hazelnut) (Corylus avellana), fique
(Furcraea macrophylla), flax for fiber (Linum usitatissimum), flax
for oil seed (linseed) (Linum usitatissimum), formio (New Zealand
flax) (Phormium tenax), garlic, dry (Allium sativum), garlic, green
(Allium sativum), geranium (Pelargonium spp.; Geranium spp.),
ginger (Zingiber officinale), gooseberry, all varieties (Ribes
spp.), gourd (Lagenaria spp; Cucurbita spp.), gram pea (chickpea)
(Cicer arietinum), grape (Vitis vinifera), grapefruit (Citrus
paradisi), grapes for raisins (Vitis vinifera), grapes for table
use (Vitis vinifera), grapes for wine (Vitis vinifera), grass
esparto (Lygeum spartum), grass, orchard (Dactylis glomerata),
grass, Sudan (Sorghum bicolor var. sudanense), groundnut (peanut)
(Arachis hypogaea), guava (Psidium guajava), guinea corn (sorghum)
(Sorghum bicolor), hazelnut (filbert) (Corylus avellana), hemp
fiber (Cannabis sativa spp. indica), hemp, manila (abaca) (Musa
textilis), hemp, sun (Crotalaria juncea), hempseed (marijuana)
(Cannabis sativa), henequen (Agave fourcroydes), henna (Lawsonia
inermis), hop (Humulus lupulus), horse bean (Vicia faba),
horseradish (Armoracia rusticana), hybrid maize (Zea mays), indigo
(Indigofera tinctoria), jasmine (Jasminum spp.), Jerusalem
artichoke (Helianthus tuberosus), jowar (sorghum) (Sorghum
bicolor), jute (Corchorus spp.), kale (Brassica oleracea var.
acephala), kapok (Ceiba pentandra), kenaf (Hibiscus cannabinus),
kohlrabi (Brassica oleracea var. gongylodes), lavender (Lavandula
spp.), leek (Allium ampeloprasum; Allium porrum), lemon (Citrus
limon), lemongrass (Cymbopogon citratus), lentil (Lens culinaris),
lespedeza, all varieties (Lespedeza spp.), lettuce (Lactuca sativa
var. capitata), lime, sour (Citrus aurantifolia), lime, sweet
(Citrus limetta), linseed (flax for oil seed) (Linum
usitatissimum), licorice (Glycyrrhiza glabra), litchi (Litchi
chinensis), loquat (Erobotrya japonica), lupine, all varieties
(Lupinus spp.), Macadamia (Queensland nut) (Macadamia spp.
ternifolia), mace (Myristica fragrans), maguey (Agave atrovirens),
maize (corn) (Zea mays), maize (corn) for silage (Zea mays), maize
(hybrid) (Zea mays), maize, ordinary (Zea mays), mandarin (Citrus
reticulata), mangel (fodder beet) (Beta vulgaris), mango (Mangifera
indica), manioc (cassava) (Manihot esculenta), maslin (mixed
cereals) (mixture of Triticum spp. and Secale cereale), medlar
(Mespilus germanica), melon, except watermelon (Cucumis melo),
millet broom (Sorghum bicolor), millet, bajra (Pennisetum
americanum), millet, bulrush (Pennisetum americanum), millet,
finger (Eleusine coracana), millet, foxtail (Setaria italica),
millet, Japanese (Echinochloa esculenta), millet, pearl (bajra,
bulrush) (Pennisetum americanum), millet, proso (Panicum
miliaceum), mint, all varieties (Mentha spp.), mulberry for fruit,
all varieties (Morus spp.), mulberry for silkworms (Morus alba),
mushrooms (Agaricus spp.; Pleurotus spp.; Volvariella), mustard
(Brassica nigra; Sinapis alba), nectarine (Prunus persica var.
nectarina), New Zealand flax (formio) (Phormium tenax), Niger seed
(Guizotia abyssinica), nutmeg (Myristica fragrans), oats, for
fodder (Avena spp.), oil palm (Elaeis guineensis), okra
(Abelmoschus esculentus), olive (Olea europaea), onion seed (Allium
cepa), onion, dry (Allium cepa), onion, green (Allium cepa), opium
(Papaver somniferum), orange (Citrus sinensis), orange, bitter
(Citrus aurantium), ornamental plants (various), palm palmyra
(Borassus flabellifer), palm, kernel oil (Elaeis guineensis), palm,
oil (Elaeis guineensis), palm, sago (Metroxylon sagu), papaya
(pawpaw) (Carica papaya), parsnip (Pastinaca sativa), pea, edible
dry, for grain (Pisum sativum), pea, harvested green (Pisum
sativum), peach (Prunus persica), peanut (groundnut) (Arachis
hypogaea), pear (Pyrus communis), pecan nut (Carya illinoensis),
pepper, black (Piper nigrum), pepper, dry (Capsicum spp.),
persimmon (Diospyros kaki; Diospyros virginiana), pigeon pea
(Cajanus cajan), pineapple (Ananas comosus), pistachio nut
(Pistacia vera), plantain (Musa sapientum), plum (Prunus
domestica), pomegranate (Punica granatum), pomelo (Citrus grandis),
poppy seed (Papaver somniferum), potato (Solamum tuberosum), palm,
kernel oil (Elaeis guineensis), potato, sweet (Ipomoea batatas),
prune (Prunus domestica), pumpkin, edible (Cucurbita spp.),
pumpkin, for fodder (Cucurbita spp.), pyrethum (Chrysanthemum
cineraraefolium), quebracho (Aspidosperma spp.), Queensland nut
(Macadamia spp. temifolia), quince (Cydonia oblonga), quinine
(Cinchona spp.), quinoa (Chenopodium quinoa), ramie (Boehmeria
nivea), rapeseed (colza) (Brassica napus), raspberry, all varieties
(Rubus spp.), red beet (Beta vulgaris), redtop (Agrostis spp.),
rhea (Boehmeria nivea), rhubarb (Rheum spp.), rice (Oryza sativa;
Oryza glaberima), rose (Rose spp.), rubber (Hevea brasiliensis),
rutabaga (swede) (Brassica napus var. napobrassica), rye (Secale
cereale), ryegrass seed (Lolium spp.), safflower (Carthamus
tinctorius), sainfoin (Onobrychis viciifolia), salsify (Tragopogon
porrifolius), sapodilla (Achras sapota), satsuma
(mandarin/tangerne) (Citrus reticulata), scorzonera (black salsify)
(Scorzonera hispanica), sesame (Sesamum indicum), shea butter (nut)
(Vitellaria paradoxa), sisal (Agave sisalana), sorghum (Sorghum
bicolor), sorghum, broom (Sorghum bicolor), sorghum, durra (Sorghum
bicolor), sorghum, guinea corn (Sorghum bicolor), sorghum, jowar
(Sorghum bicolor), sorghum, sweet (Sorghum bicolor), soybean
(Glycine max), soybean hay (Glycine max), spelt wheat (Triticum
spelta), spinach (Spinacia oleracea), squash (Cucurbita spp.),
strawberry (Fragaria spp.), sugar beet (Beta vulgaris), sugar beet
for fodder (Beta vulgaris), sugar beet for seed (Beta vulgaris),
sugarcane for fodder (Saccharum officinarum), sugarcane for sugar
or alcohol (Saccharum officinarum), sugarcane for thatching
(Saccharum officinarum), sunflower for fodder (Helianthus annuus),
sunflower for oil seed (Helianthus annuus), sunhemp (Crotalaria
juncea), swede (Brassica napus var. napobrassica), swede for fodder
(Brassica napus var. napobrassica), sweet corn (Zea mays), sweet
lime (Citrus limetta), sweet pepper (Capsicum annuum), sweet potato
(Lopmoea batatas), sweet sorghum (Sorghum bicolor), tangerine
(Citrus reticulata), tannia (Xanthosoma sagittifolium), tapioca
(cassava) (Manihot esculenta), taro (Colocasia esculenta), tea
(Camellia sinensis), teff (Eragrostis abyssinica), timothy (Phleum
pratense), tobacco (Nicotiana tabacum), tomato (Lycopersicon
esculentum), trefoil (Lotus spp.), triticale, for fodder (hybrid of
Triticum aestivum and Secale cereale), tung tree (Aleurites spp.;
Fordii), turnip, edible (Brassica rapa), turnip, for fodder
(Brassica rapa), urena (Congo jute) (Urena lobata), vanilla
(Vanilla planifolia), vetch, for grain (Vicia sativa), walnut
(Juglans spp., especially Juglans regia), watermelon (Citrullus
lanatus), wheat (Triticum aestivum), yam (Dioscorea spp.), or yerba
mate (Ilex paraguarensis).
[0328] The compositions and methods disclosed herein can also be
applied to turf grass, ornamental grass, flowers, ornamentals,
trees, and shrubs.
[0329] The compositions comprising the bioactive priming
polypeptides are also suitable for use in the nursery, lawn and
garden, floriculture or the cut flower industry and provide
benefits for enhanced plant productivity, protection health, vigor
and longevity. For example, they can be applied to perennials,
annuals, forced bulbs, or pseudo bulbs, herbs, groundcovers, trees,
shrubs, ornamentals (e.g., orchids, etc.), tropicals, and nursery
stock.
[0330] The compositions comprising the bioactive priming
polypeptides are suitable for treating plants, plant parts and
plant propagation material(s), for example, any plant or plant
part, such as seeds, roots, stems, floral organs, root stocks,
scions, bulb, pseudobulbs, rhizomes, tubers, etc.
[0331] The bioactive priming polypeptides can be applied as seed
treatments to treat for a number of pests, diseases, nutrient
deficiencies while enhancing plant growth and productivity.
[0332] Seed coating or dressing compositions can be, for example, a
liquid carrier composition, a slurry composition, or a powder
composition applied with conventional additives that are provided
to make the seed treatment have sticky qualities to stick to and
coat the seeds. Suitable additives for a seed composition comprise:
talcs, graphites, gums, stabilizing polymers, coating polymers,
finishing polymers, slip agents for seed flow and plantability,
cosmetic agents and cellulosic materials such as carboxymethyl
cellulose and the like. The bioactive priming polypeptide seed
treatments can further comprise colorant agents and other such
additives.
[0333] The bioactive priming polypeptides can be applied
individually as seed treatments or in combination with other
additives such as fungicides, insecticides, inoculants, plant
growth regulators, plant growth promoting microbes, fertilizers and
fertilizer enhancers, seed nutrients, biological control agents,
herbicidal antidotes and seedling disease treatments and with other
conventional seed treatments.
[0334] The seed treatment composition as described herein can be
applied to seeds in a suitable carrier such as water or a powder
that is not harmful to the seeds or the environment. The seeds are
then planted in conventional fashion.
[0335] Preferred seed treatments such as Bt.4Q7Flg22 (SEQ ID NO:
226 or SEQ ID NO: 571), Ec.Flg22 (SEQ ID NO: 526) and Gm.RHPP (SEQ
ID NO: 600) are useful to enhance seedling development, decrease
the time for germination, increase the number of seeds that
germinate, and enhance seedling survivability. In addition, the
seed treatment compositions enhance seed protection from
microbial-based diseases which are known to contact the seed or the
soil surrounding the seed and spread during early seedling
establishment.
[0336] The seed treatment composition can comprise a polypeptide as
described herein and a fungicide, an insecticide, a nematacide, a
biological control agent, a biostimulant, a microbe, or any
combination thereof.
[0337] The seed treatment composition can comprise a polypeptide as
described herein and clothianidin, Bacillus firmus, metalaxyl, or
any combination thereof.
[0338] The seed treatment composition can comprise a polypeptide as
described herein, clothianidin and fluopyram.
[0339] The seed treatment can comprise a polypeptide as described
herein, metalaxyl and fluopyram.
[0340] The bioactive priming polypeptides can be applied directly
to the seed as a solution or in combination with other commercially
available additives. Solutions containing the water-soluble
polypeptide can be sprayed or otherwise applied to the seed as a
seed slurry or a seed soak. Solids or dry materials containing
soluble bioactive priming polypeptides are also useful to promote
effective seedling germination, growth and protection during early
seedling establishment.
[0341] The bioactive priming polypeptides can be formulated with a
solubilizing carrier such as water, buffer (e.g., citrate or
phosphate buffer) and other treating agents (i.e., alcohol, other
solvents) or any solubilizing agent. In addition, small amounts of
drying agent enhancers, such as lower alcohols, etc. can be
utilized in the composition. Surfactants, emulsifiers and
preservatives can also be added at small (0.5% v/v or less) levels
in order to enhance the stability of the seed coating product.
[0342] Seed treatments containing the bioactive priming
polypeptides can be applied using any commercially available seed
treatment machinery or can also be applied using any acceptable
non-commercial method(s) such as the use of syringes or any other
seed treatment device. General seed treatments coating procedures
using bioactive priming polypeptides can be performed using a
Wintersteiger HEGE 11 (Wintersteiger AG, Austria, Germany) and
applied to the seed of major crops, namely corn, soybean, wheat,
rice and various vegetables. The capacity of this seed treatment
machinery can accommodate a large number of different seed types,
sizes and amounts of seed (20-3000 grams). The seed is loaded into
bowls of the seed treater machinery. The bowl selection depends on
the treatment seed amount required and the size of the bowl
selected: large 14.5 L bowl (500-3000 g seed per coating); medium 7
L bowl (80-800 g seed per coating); and small 1 L bowl (20-100 g
seed per coating). Other larger seed treatment systems are also
available.
[0343] The seed is distributed toward the radial peripheries of the
rotatable bowls via an application of centrifugal force with the
centrifugal coating device. The spinning disc located at the bottom
of the bowl distributes the seed treatment evenly over the seed. At
this point, the spin cycle is started which causes the seeds to
revolve around the bowl center in a circle to evenly coat the
seeds. The process of seed treatment coating is initiated after the
seed is evenly dispersed around the spreader. Seed treatment sample
material (such as a powdered, semi-liquid, liquid or a slurry) can
be applied onto the rotatable disk as the disks are spinning within
the rotatable bowls used to distribute the seed treatment evenly to
provide a uniform coat and dress the surface of the seed.
[0344] A constant air flow delivered using compressed air (2-6
bars) can be provided during seed coating to assist with uniformly
coating the seeds in the bowl. The amount of time for the coating
of the seed depends on the amount of the seed, the viscosity of the
seed treatment and the type of the seed used in the treatment. A
seed treatment calculator is used to adjust for all volumes, for
most major and commercially grown crops and the type of seed
treatment being applied.
[0345] The seeds can be coated using a variety of methods
including, but not limited to, pouring or pumping, drizzling or
spraying an aqueous solution containing the bioactive priming
polypeptides on or over a seed, spraying or applying onto a layer
of seeds either with the use or without the use of a conveyor
system. Suitable mixing devices include tumblers, mixing basins or
drums, or other fluid applicating devices that include basins or
drums used to contain the seed while coating.
[0346] After the seed has been treated and dried, the seeds are
distributed into a larger storage container(s). Seeds are either
air dried or dried with a continuous air stream that passes over
the seeds. Seeds are then transferred into a separate container or
bag for shipment, transfer or storage.
[0347] The bioactive priming polypeptides can further be provided
for delivery to a plant surface or plant plasma membrane as a
foliar spray or a seed treatment to an area surrounding a plant or
a plant part.
[0348] The bioactive priming polypeptide formulation(s) can also be
provided as a seed treatment application or on a matrix such as
immobilized or impregnated on a particle, or a granule such as used
in a broadcast treatment.
[0349] The bioactive priming polypeptides as described herein can
be applied to plants and plant parts using an exogenous application
as a spray, soil treatment, in furrow, seed treatment, dip or wash
or as an endogenous application as an injection, inoculation,
irrigation, infiltration, etc.
[0350] The polypeptides can be applied directly to a plant or to
the area surrounding a plant or plant part.
[0351] They can also be provided on a matrix material which is then
provided to a plant or plant part.
[0352] The compositions containing the flagellin-associated
bioactive priming polypeptides can also be provided for direct
delivery into a plant, plant tissues or a plant cell by various
delivery methods, for example, injection, inoculation or
infiltration (for example, infiltration into the stomata on the
leaf). These polypeptides can also be provided in a manner where
they can move systemically through a plant and influence signaling
cascades in the plant that subsequently produce beneficial and
productive outcomes to the plant or plant part.
[0353] Retro-inverso Flg bioactive priming polypeptides as
described in Table 4 or Table 5 can be applied individually or in
combination with any other flagellin, flagellin-associated or other
bioactive priming polypeptide sequences as described herein.
Combinations of such RI flagellin and flagellin-associated
bioactive priming polypeptides are useful as plant protectants as
well as plant growth promoting enhancers.
[0354] The signature (SEQ ID NO: 542-548; Table 6), signal anchor
sorting (SEQ ID NO: 549-562, Table 7) and secretion assistance
polypeptides (SEQ ID NOs 563-570; Table 8) can be used in
combination with any of the flagellin coding (Table 1), N and/or
C-terminal conserved sequences from Bacillus-derived flagellins
(Table 2), flagellin-associated polypeptides: Flg22 and FlgII-28
(Table 3), the retro inverso forms of Flg22 and FlgII-28 (Table 4)
or any of the other Flgs (Table 5) as described herein.
[0355] For example, any of the Flg-associated bioactive priming
polypeptides or combinations thereof can be provided in individual
formulations and applied either simultaneously, sequentially in
separate formulations or provided as fusion protein(s) that contain
the assistance sequences as described in Tables 6-8 and applied
directly or separately to a plant or plant part.
[0356] Harpin-like polypeptides or RHPP polypeptides can provide
functional benefits when applied both exogenously, for example as a
foliar spray to the plant surface, or provided apoplastically (to
the space outside of the plant cell membrane) or endogenously
(inside a plant cell/plant cell membrane). RHPP polypeptides can
also provide functional benefits when applied as a seed
treatment.
[0357] Foliar or in furrow applications of harpin-like, HpaG-like
polypeptides are useful to enhance growth, increase biomass, and
greenness or chlorophyll production of a plant.
[0358] The PSK.alpha. bioactive priming polypeptide(s) can be
provided for delivery to a plant surface/plant plasma membrane as a
foliar spray or, a seed treatment to an area surrounding a plant,
plant part or a plant cell.
[0359] The compositions containing the PSK.alpha. bioactive priming
polypeptides can also be provided for delivery into a plant, plant
tissues or a plant cell by various delivery methods, for example,
injection, inoculation or infiltration (for example, added directly
or prerequisitely to cell culture).
V. Methods of Use
[0360] Methods are provided for increasing growth, yield, health,
longevity, productivity, and/or vigor of a plant or a plant part
and/or decreasing abiotic stress in the plant or the plant part
and/or protecting the plant or the plant part from disease, insects
and/or nematodes, and/or increasing the innate immune response of
the plant or the plant part and/or changing plant architecture. The
method can comprise applying the polypeptide or the composition as
described herein to a plant, a plant part, or a plant growth medium
or a rhizosphere in an area surrounding the plant or the plant part
to increase growth, yield, health, longevity, productivity, and/or
vigor of the plant or the plant part and/or decrease abiotic stress
in the plant or the plant part and/or protect the plant or the
plant part from disease, insects and/or nematodes, and/or increase
the innate immune response of the plant or the plant part and/or
change the plant architecture.
[0361] Alternatively, the method can comprise applying the
polypeptide or the composition as described herein to a plant
growth medium to increase growth, yield, health, longevity,
productivity, and/or vigor of a plant or a plant part to be grown
in the plant growth medium and/or decrease abiotic stress in the
plant or the plant part to be grown in the plant growth medium
and/or protect the plant or the plant part to be grown in the plant
growth medium from disease, insects and/or nematodes, and/or
increase the innate immune response and/or change plant
architecture of the plant or the plant part to be grown in the
plant growth medium.
[0362] Another method comprises applying the recombinant
microorganism as described herein to a plant, a plant part, or a
plant growth medium or a rhizosphere in an area surrounding the
plant or the plant part to increase growth, yield, health,
longevity, productivity, and/or vigor of the plant or the plant
part and/or decrease abiotic stress in the plant or the plant part
and/or protect the plant or the plant part from disease, insects
and/or nematodes, and/or increase the innate immune response of the
plant or the plant part and/or change the plant architecture. The
recombinant microorganism expresses the polypeptide and expression
of the polypeptide is increased as compared to the expression level
the polypeptide in a wild-type microorganism of the same kind under
the same conditions.
[0363] Methods using the bioactive priming polypeptides are also
provided to increase the overall plant productivity in a field,
orchard, planting bed, nursery, timberland, farm, lawn, garden,
garden center or acreage. Applications and methods using the
bioactive priming polypeptides are also useful for increasing plant
growth, health and productivity in diverse crops (monocots and
dicots), for example, corn, wheat, rice, sugarcane, soybean,
sorghum, potatoes and a variety of vegetables.
[0364] A "bioactive polypeptide priming" approach is also provided
by direct application of the polypeptides, which can be applied
either exogenously to a plant cell surface or endogenously to the
interior of a plant and/or a plant cell. The polypeptides are
provided for delivery to the plant surface or plasma cell membrane
or to the interior of a plant, plant tissue or cell and are useful
for regulating developmental processes that result in enhanced
growth phenotypes such as increases in overall biomass, vegetative
growth, seed fill, seed size, and number of seed that contribute to
increases in the total yield of crop plants.
[0365] Application of the retro-inverso Flg polypeptides provided
in agricultural formulations can result in enhanced plant
protection from diseases and abiotic stresses while synergistically
enhancing growth, productivity and yield while maintaining
increased plant health with enhanced plant performance for longer
periods of time.
[0366] Selection of the native L (Table 3) or the retro-inverso D
(Table 4) forms of the Flg-associated polypeptides can depend on
the environment, the plant/crop, or the combination of plant/crop
and environment. In addition, the timing of the treatment
application (for example, a foliar spray application) during the
growing season are all relevant considerations. The retro inverso
Flg bioactive priming polypeptides have enhanced binding affinity
to cell surface membranes. Due to these features, the RI forms of
the Flg bioactive priming polypeptides can be used to improve
abiotic stress tolerance in a plant or plant part.
[0367] Additionally, the retro inverso forms of RI Ec.Flg22 and RI
Bt.4Q7Flg22 can be useful to stimulate the closure of stomata under
conditions of drought and heat stress and improve yields under
those conditions. Control of stomatal closure using Flg-associated
bioactive priming polypeptide applied to a plant during periods of
environmental stress can assist in the regulation of water loss and
stabilize turgor pressure in a plant when environmental conditions
are unfavorable.
[0368] In the methods, the polypeptide or the composition can
comprise: the Flg22 polypeptide and an amino acid sequence of the
Flg22 polypeptide comprising any one of SEQ ID NOs: 226-300 and
571-573; the retro inverso Flg22 polypeptide and an amino acid
sequence of the retro inverso Flg22 polypeptide comprising any one
of SEQ ID NO: 376-450; or any combination thereof to protect the
plant or the plant part from disease and/or increase the innate
immune response of the plant or the plant part.
[0369] In the methods, the polypeptide or the composition can
comprise: the FlgII-28 polypeptide and an amino acid sequence of
the FlgII-28 polypeptide comprising any one of SEQ ID NOs: 301-375;
the retro inverso FlgII-28 polypeptide and an amino acid sequence
of the retro inverso FlgII-28 polypeptide comprising any one of SEQ
ID NO: 451-525; or any combination thereof to protect the plant or
the plant part from disease and/or increase the innate immune
response of the plant or the plant part.
[0370] In the methods, the polypeptide or the composition can
comprise the FlgII-28 polypeptide and an amino acid sequence of the
Flg22 polypeptide can comprise any one of SEQ ID NO: 226, 571, or
752 and/or EF-Tu polypeptides, the amino acid sequence of the EF-Tu
polypeptides comprising SEQ ID NOs: 616 and 617, to protect the
plant or the plant part from disease and/or increase the innate
immunity of the plant or plant part. In the methods, the amino acid
sequence of the flagellin or flagellin-associated polypeptide can
comprise any one of SEQ ID NOs: 226, 289, 290, 291, 293, 294, 295,
300, 437, 532, 534, 536, 538, 540, 571-586, and 751-766 or any
combination thereof to protect the plant or the plant part from
disease, insects or nematodes. These are polypeptides with mutant
sequences exhibiting increased activity to reactive oxygen species.
For example, the amino acid sequence of the flagellin or
flagellin-associated polypeptide can comprise any one of SEQ ID
NOs: 226, 293, 295, 300, 540, 571 574, 751 and 752 or any
combination thereof.
[0371] The disease can comprise Asian citrus greening, Huanglonging
(HLB) disease, Asian soybean rust, Sclerotinia stem rot (or white
mold), Pseudomonas leaf spot, or Cercospora leaf blight.
[0372] In the methods, the polypeptide or the composition can
comprise the Flg22 polypeptide and an amino acid sequence of the
Flg22 polypeptide comprising any one of SEQ ID NOs: 226-300 and
571-573 or any combination thereof.
[0373] In the methods, the polypeptide or the composition can
comprise the FlgII-28 polypeptide and an amino acid sequence of the
FlgII-28 polypeptide comprising any one of SEQ ID NOs: 301-375 or
751 or any combination thereof.
[0374] In the methods, the polypeptide or the composition can
comprise the Flg22 polypeptide and the FlgII-28 polypeptide, an
amino acid sequence of the Flg22 polypeptide comprising any one of
SEQ ID NOs: 226-300 and 571-573 or any combination thereof and an
amino acid sequence of the FlgII-28 polypeptide comprising any one
of SEQ ID NOs: 301-375 or 751 or any combination thereof. The
polypeptide or the composition can further comprise the retro
inverso Flg22 polypeptide, the retro inverso FlgII-28 polypeptide
or a combination thereof, an amino acid sequence of the retro
inverso Flg22 polypeptide comprising any one of SEQ ID NO: 376-450
or any combination thereof and an amino acid sequence of the retro
inverso FlgII-28 polypeptide comprising any one of SEQ ID NO:
451-525 or any combination thereof.
[0375] In the methods, the polypeptide or the composition can
comprise the RHPP polypeptide and/or the RI RHPP polypeptide to
increase the yield, the growth and/or the productivity of the plant
or plant part and/or change the plant architecture.
[0376] When the method includes a polypeptide or composition
comprising the RHPP polypeptide and/or the RI RHPP polypeptide, the
growth can comprise root growth, root length, root biomass,
nodulation, total biomass, above ground biomass, or any combination
thereof. When the polypeptide or composition comprises the RHPP
polypeptide, the amino acid sequence of the RHPP polypeptide can
comprise SEQ ID NO: 600.
[0377] When the method includes a polypeptide or composition
comprising the RHPP polypeptide and/or the RI RHPP polypeptide, the
plant can comprise soybean, the growth can comprise overall root
length, root biomass, nodulation, nodules per plant, total biomass,
above ground biomass, or any combination thereof, and the
productivity can comprise number of total pods or pods per
node.
[0378] The plant architecture can comprise beneficial outcomes to
the plant or plant part. For example, the beneficial outcomes can
include increased planting density capability for a field of the
plants.
[0379] In the methods, the polypeptide or the composition can
comprise the harpin-like polypeptide or the RHPP polypeptide to
protect the plant or the plant part from disease, insects and/or
nematodes, and/or increase the innate immune response of the plant
or the plant part.
[0380] In the methods, the polypeptide or the composition can
comprise the PSK polypeptide to increase yield of the plant or the
plant part in environments prone to heat and drought.
[0381] The polypeptide, the composition, or the recombinant
microorganism can be applied just prior to floral formation or at
the pre-flowering stage.
[0382] In the methods, the polypeptide or the composition can
comprise the PSK polypeptide, the RHPP, the harpin or harpin-like
polypeptide, or a combination thereof to increase growth of the
plant or the plant part.
[0383] The growth can comprise root and floral apical meristems,
floral organ production, fruit development, fruit production,
number of floral organs, size of floral organs, or a combination
thereof.
[0384] In the methods, the polypeptide or the composition can
comprise the PSK polypeptide and the harpin or harpin-like
polypeptide to increase growth and productivity of the plant or the
plant part in an environment prone to both stress and non-stress
conditions for plant growth.
[0385] In the methods, the polypeptide or the composition can
comprise the thionin or thionin-like polypeptide.
[0386] The thionin or thionin-like polypeptide can be fused to a
phloem targeting sequence to form a fused polypeptide, the amino
acid sequence of the phloem targeting sequence comprising any one
of SEQ ID NOs: 641-649, or any combination thereof, for delivering
the fused polypeptide to vascular tissue or cells and/or phloem or
phloem-associated tissue or cells in the plant or plant part.
[0387] In the methods, protecting the plant or the plant part from
disease can comprise prophylactic treatment, treatment, prevention
and decreased disease progression on or in the plant or plant
part.
[0388] The disease can comprise Asian citrus greening disease
(HLB), Citrus canker disease, Cercospora leaf blight or a bacteria
causing disease.
[0389] The bacteria causing disease can comprise bacterial leaf
blight, bacterial leaf streak, bacterial stalk rot, bacterial leaf
spot, bacterial leaf scorch, bacterial top rot, bacterial stripe,
chocolate spot, Goss's bacterial wilt and blight, Holcus spot,
purple leaf sheath, seed rot, seedling blight, Stewart's disease
(bacterial wilt), corn stunt, Fire Blight, Pierce's disease, citrus
variegated chlorosis, citrus canker, Pseudomonas syringae serovars,
or a combination thereof.
[0390] In the methods, the polypeptide or the composition further
can comprise the flagellin or flagellin-like polypeptide, and an
amino acid sequence of the flagellin or flagellin-like polypeptide
comprising any one of SEQ ID NOs: 226-525 and 571-573 or any
combination thereof.
[0391] In the methods, the polypeptide, the composition, or the
recombinant microorganism can be applied exogenously to the plant,
the plant part, or the plant growth medium.
[0392] In the methods, the polypeptide, the composition, or the
recombinant microorganism can be applied endogenously to the plant
or the plant part.
[0393] The plant part can include a plant cell, a leaf, a branch, a
stem, a flower, a foliage, a floral organ, a fruit, pollen, a
vegetable, a tuber, a rhizome, a corm, a bulb, a pseudobulb, a pod,
a root, a root ball, a root stock, a scion, or a seed.
[0394] In the methods, the polypeptide, the composition, or the
recombinant microorganism can be applied to a surface of the plant,
a foliage of the plant or a surface of a seed of the plant.
[0395] In the methods, the polypeptide, the composition, or the
recombinant microorganism can be applied to the surface of the seed
and the plant or the plant part is grown from the seed.
[0396] In the methods, the polypeptide, the composition, or the
recombinant microorganism can be applied as a foliar
application.
[0397] The plant can be a fruit plant or a vegetable plant, and the
method provides increased yield of fruits or vegetables.
[0398] In methods where the bioactive priming polypeptides are
applied two or more times during a growing season, the first
application can occur at or before the V2 stage of development, and
subsequent applications can occur before the plant flowers. For
example, the first application can occur as a seed treatments,
at/or before the VE stage of development, at or before the V1 stage
of development, at or before the V2 stage of development, at or
before the V3 stage of development, at or before the V4 stage of
development, at or before the V5 stage of development, at or before
the V6 stage of development, at or before the V7 stage of
development, at or before the V8 stage of development, at or before
the V9 stage of development, at or before the V10 stage of
development, at or before the V11 stage of development, at or
before the V12 stage of development, at or before the V13 stage of
development, at or before the V14 stage of development, at or
before the V15 stage of development, at or before the VT stage of
development, at or before the R1 stage of development, at or before
the R2 stage of development, at or before the R3 stage of
development, at or before the R4 stage of development, at or before
the R5 stage of development, at or before the R6 stage of
development, at or before the R7 stage of development, or at or
before the R8 stage of development. By way of example, the first
application can occur at or before the germination stage, at or
before the seedling stage, at or before the tillering stage, at or
before the stem elongation stage, at or before the booting stage,
or at or before the heading stage. For example, where the Feekes
scale is used to identify the stage of growth of a cereal crop, the
first application can occur at or before stage 1, at or before
stage 2, at or before stage 3, at or before stage 4, at or before
stage 5, at or before stage 6, at or before stage 7, at or before
stage 8, at or before stage 9, at or before stage 10, at or before
stage 10.1, at or before stage 10.2, at or before stage 10.3, at or
before stage 10.4, or at or before stage 10.5.
[0399] Abiotic Stress
[0400] Abiotic stress causes significant crop loss and can result
in major reductions in crop production and yield potential. The
bioactive priming polypeptides and compositions as described herein
can be used as chemical priming agents to increase tolerance of a
plant to one or more abiotic stresses. Thus, the flagellin
polypeptides, flagellin-associated polypeptides of Flg22 or
FlgII-28 derived from Bacillus species, Flg15 and Flg22 derived
from E. coli and other organisms (Table 5) and the RHPP
polypeptides derived from Glycine max (Tables 13 to 15) are useful
for increasing the tolerance of a plant, group of plants, field of
plants and/or the parts of plants to abiotic stress. The
polypeptides and compositions as described herein impart abiotic
stress tolerance to a plant or plant part. The abiotic stress
tolerance imparted to a plant or plant part are to abiotic stresses
that include, but are not limited to: temperature stress, radiation
stress, drought stress, cold stress, salt stress, osmotic stress,
nutrient-deficient or high metal stress, and water stress that
results from water deficit, flooding or anoxia. Chemical priming
using the bioactive priming polypeptides and compositions as
described herein are applied to a plant or plant part offering a
versatile approach to protect the plant or plant part against
individual, multiple or combined abiotic stresses.
[0401] The polypeptides and compositions as described herein are
effective to protect a plant against abiotic stressors when applied
as an above ground foliar application to a plant, a plant part, a
plant root, a plant seed, a plant growth medium, or the area
surrounding a plant or the area surrounding a plant seed. For
example, for trees, one or more applications can be applied at
different growth timings of trees, including timings before, during
or after flushes; before, during, or after fruit set; or before or
after fruit harvest.
[0402] The methods described herein chemically prime the plant for
protection against abiotic stress(es) in such a way that the plant
has already prepared and initiated defense mechanisms that can be
activated faster and increase tolerance to an abiotic stress or
multiple stressors occurring simultaneously or at different times
during the growing season.
[0403] The retro inverso forms of the Flg22 polypeptides as
described herein can be applied externally as a foliar spray
application (or using other application methods as well, for
example as a root drench) during times of excessive heat, water,
and drought stress and be used to protect a plant against drought,
heat stress and/or other abiotic stresses that can affect stomatal
aperture and oscillation that commonly occur with transpiration
loss through a plant.
[0404] In the methods, the polypeptide or the composition can
comprise: the Flg22 polypeptide and an amino acid sequence of the
Flg22 polypeptide comprising any one of SEQ ID NOs: 226-300 and
571-573 or any combination thereof; the retro inverso Flg22
polypeptide and an amino acid sequence of the retro inverso Flg22
polypeptide comprising any one of SEQ ID NO: 376-450 or any
combination thereof; or any combination thereof to decrease abiotic
stress in the plant or the plant part and/or protect the plant or
the plant part from disease and/or increase the innate immune
response of the plant or the plant part.
[0405] In the methods, the polypeptide or the composition can
comprise: the FlgII-28 polypeptide and an amino acid sequence of
the FlgII-28 polypeptide comprising any one of SEQ ID NOs: 301-375
or any combination thereof; the retro inverso FlgII-28 polypeptide
and an amino acid sequence of the retro inverso FlgII-28
polypeptide comprising any one of SEQ ID NO: 451-525 or any
combination thereof; or any combination thereof to decrease abiotic
stress in the plant or the plant part and/or protect the plant or
the plant part from disease and/or increase the innate immune
response of the plant or the plant part.
[0406] In the methods, the polypeptide or the composition can
comprise: the retro inverso Flg22 polypeptide and an amino acid
sequence of the retro inverso Flg22 polypeptide comprising any one
of SEQ ID NO: 376-450 or any combination thereof; the retro inverso
FlgII-28 polypeptide and an amino acid sequence of the retro
inverso FlgII-28 polypeptide comprising any one of SEQ ID NO:
451-525 or any combination thereof; or any combination thereof to
decrease abiotic stress in the plant or the plant part and/or
protect the plant or the plant part from disease and/or increase
the innate immune response of the plant or the plant part.
[0407] In the methods, the polypeptide or the composition can
comprise the RHPP polypeptide and an amino acid sequence of the
RHPP polypeptide comprises SEQ ID NO: 600, 603, 604 or any
combination thereof; the Kunitz Trypsin Inhibitor (KTI) polypeptide
and an amino acid sequence of the KTI polypeptide comprises SEQ ID
NO: 602; the retro-inverso RHPP polypeptide and an amino acid
sequence of the RI RHPP comprises SEQ ID NO 601, 605, 606 or any
combination thereof; or any combination thereof to decrease abiotic
stress in the plant or the plant part and/or protect the plant or
the plant part from disease and/or increase the innate immune
response of the plant or the plant part.
[0408] The abiotic stress can comprise heat stress, temperature
stress, radiation stress, drought stress, cold stress, salt stress,
nutrient-deficient stress, high metal stress, water stress, osmotic
stress, or any combination thereof.
Balancing Immune Response with Plant Growth and Development
[0409] Although immune responses can provide protection of plants
from pathogen attack, excessive immune responses may have negative
impacts on plant growth. Therefore, balancing enhanced immunity or
disease prevention and protection in a plant with an increased
growth promoting response is a desired combination to optimize
plant health.
[0410] Bioactive priming polypeptides that are useful for enhancing
immune responses as described herein can be combined with
polypeptides that provide positive impacts on plant growth and
productivity. The polypeptide combinations are specifically
selected for their distinct modes of action/regulation when applied
to a plant or plant part. However, some of the bioactive priming
polypeptides (Flgs, HpGa-like, PSK.alpha., thionins) are perceived
by receptor-like proteins, followed by a process that initiates
their entry and transport in the plant which results in functional
outcomes while others are taken into the plant by active absorption
(e.g., RHPP). For example, PSK.alpha. and the Flg-associated
polypeptides such as Flg22, Flg25 and FlgII-28 are perceived by a
leucine-rich receptor kinase located on the surface of the plasma
membrane and involve a complex signaling pathway involved in the
pathogen-triggered responses leading to immunity, disease
resistance or disease prevention (Kutschmar et al. "PSK.alpha.
promotes root growth in Arabidopsis," New Phytologist 181: 820-831,
2009).
[0411] The bioactive priming polypeptides as described herein such
as Flg22 HpaG-like polypeptides and thionins can act as elicitors
and exhibit antimicrobial activity (e.g., anti-pesticide;
bacterial, fungal, or viral activity). Specific combinations of
polypeptides are provided, for example, the combination of
flagellin- and harpin-associated bioactive priming polypeptides are
useful for preventing and protecting plants from pathogenic
diseases and serve a dual utility when they are applied together
with those other polypeptides, for example, PSK.alpha. and RHPP,
that enhance plant growth and productivity in a plant, plant part,
and/or field of plants.
[0412] The combinations of bioactive priming polypeptides as
described herein can be applied exogenously as a foliar spray, in
furrow treatment, seed treatment, drench or wash or endogenously to
a plant to stimulate both the immune responsiveness and growth
characteristics of the plant that collectively result in improved
yield performance. They can also provide protection and growth
benefits to the different parts of the plant (for example, leaves,
roots, tubers, corms, rhizomes, bulbs, pseudobulbs, flowers, pods,
fruits, and growing meristems).
[0413] The combined foliar application or sequential applications
of PSK.alpha. with HpaG-like bioactive priming polypeptides can be
useful for enhancing growth of plants under standard (non-stress or
optimal growth) environments or of plants exposed to abiotic stress
(for example, heat, and water deficit stress).
[0414] Foliar application treatments using the X.spp HpaG-like and
the At.PSK.alpha. bioactive priming polypeptides have different
modes of action when applied on plants in optimal (non-stress) and
in stress environments. The two classes of bioactive priming
polypeptides are useful either provided sequentially or in
combination in a foliar application and can improve plant growth in
an environment that is with or without abiotic stress(es).
[0415] X.spp.HpGa-like provides a plant growth benefit to corn in a
non-stress environment where temperature, water, nutrients and
other environmental parameters were conducive to optimal plant
growth. On the other hand, At.PSK.alpha. applied as a foliar spray
provides a benefit to plant growth under environmental conditions
of heat and drought or water deficit stress. Thus, when used in
combination in formulation together as foliar applications they can
span both non-stress and stress environments and provide additive
benefits to the growth of corn plants grown in a variety of
environmental conditions.
[0416] Increases in plant productivity and growth for At.PSK.alpha.
is also seen in soybean plants grown in environments with and
without abiotic stress. Soybean plants that receive a foliar
application with a formulation containing the bioactive priming
polypeptide At.PSK.alpha. and are grown under conditions of heat
and drought stress have increased yield over control soybean plants
that received water and surfactant with no bioactive priming
polypeptide.
[0417] When X. spp. HpaG-like and At.PSK.alpha. are applied as a
foliar spray together, they are useful to provide synergistic
effects for plant production under normal and stressed
environments. At.PSK.alpha. exhibits increased overall growth in
corn when applied as a spray application, whereas X. spp. HpaG-like
polypeptide results in the opposite trend. Thus, applying the two
bioactive priming polypeptides together can act to balance plant
growth in "heat stressed" environments such that the changes in
plant growth compared to control plants are greater than the sum of
the effects of the bioactive priming polypeptides applied
individually.
[0418] The synergistic interaction of these two classes of
bioactive priming polypeptides enhance plant growth under heat
stressed environments (e.g., greater growth rates with increased
plant biomass).
[0419] Any of the bioactive priming polypeptides as described
herein can be applied one or more times to a plant either in
combination or individually to enhance growth and productivity of a
plant. Multiple applications can be applied to promote yield
benefits over the growing season with applications tailored to the
conditions in the environment, for example if a period of hot and
dry weather is expected during the growth season, an additional
spray of bioactive priming polypeptides that promote growth under
abiotic stress can alleviate negative impacts to the plant.
[0420] Foliar Application of Phytosulfokine Alpha (PSK.alpha.) to
Increase Yield
[0421] A method is provided for applying At.PSK.alpha. as a foliar
application to actively growing soybean plants to provide a yield
advantage in environments with heat and drought stress. For
example, a means of applying a composition containing bioactive
priming At.PSK.alpha. polypeptide is provided as a foliar spray to
soybean at V1-V4 stage using application methods as described
herein. Soybean plants treated with foliar applications of
At.PSK.alpha. can be grown in field environments under conditions
that produced a non-stress and stress (heat and water deficit)
environments. Treatment with At.PSK.alpha. can result in growth and
yield benefits in plants grown in a variety of environmental
conditions including abiotic stressors.
[0422] Any of the RHPP bioactive priming polypeptides provided in
Tables 12-14 can be applied as a foliar, in furrow, seed treatment
or root drench application to a plant surface.
[0423] Foliar application of RHPP results in the alteration of
plant architecture.
[0424] A method is provided where the RHPP polypeptide is applied
as a foliar application to plants and results in a distinct leaf
architecture (corn) and an enhanced root system (soybean). The
increase in leaf angle and root biomass using a foliar treatment
with RHPP has impactful advantages for use in agriculture in two
major agriculture crops (corn and soybean).
[0425] Application of RHPP to Alter Plant Architecture
[0426] Applying the bioactive priming polypeptide, RHPP, as a
foliar application to V5-V8 corn results in a distinct leaf
architecture phenotype with an upright leaf orientation and more
erect leaves. This is particularly relevant with higher planting
densities used to maximize yield in a field environment. Foliar
applications of the RHPP polypeptide in maize (corn) is useful for
changing the leaf angle thus contributing to a smaller leaf angle
which results in an upright leaf orientation. This phenotype can be
beneficial for increasing the leaf area index, reducing maize shade
syndrome, and improving photosynthetic efficiency. In addition,
providing RHPP as a foliar formulation to maximize canopy
development and total light penetrance is key to increasing
vegetative growth of the plants prior to the initiation of the
grain filling stage.
[0427] Maize plants exhibit leaf curl or changes in their leaf
architecture to a more upright leaf orientation to conserve water
and enhance plant tolerance to drought and heat. The upright
changes to the leaf phenotype for corn after application with the
RHPP bioactive priming polypeptide(s) compositions are useful and
provide an alternative non-breeding approach for shaping leaf
architecture and enhancing tolerance to drought and heat.
[0428] An upright leaflet orientation phenotype in corn plants
functions in the reduction of leaf temperatures, whole plant
transpiration and in the improvement of water use efficiency, as
well as provide architectural changes to the plant canopy which can
allow for higher density plantings that result in substantial
increases in yield.
[0429] Application of the bioactive priming polypeptide, RHPP, to
soybeans can also provide benefits. For example, foliar application
of RHPP to flowering soy can increase pod set. Pod set is a stage
in soybean development occurring from the middle of R4 to the
middle of R5 that contributes directly to yield. Initial pod set is
marked by the emergence of a 3/4 inch pod at one of the four
uppermost nodes on the main stem. It then progesses to the full pod
stage where pod growth is rapid and seed development begins. An
increase in pod set is quantified by an increase in yield (i.e the
pod number per node on a plant or the overall number of pods per
plant).
[0430] RHPP to Increase Root Biomass and Yield
[0431] Soybean plants treated with foliar applied RHPP (SEQ ID NO:
600) bioactive priming polypeptide(s) can exhibit increased pod
filling and a more-complete pod filing compared to non-treated
plants which can be the result of increases in nitrogen
fixation.
[0432] Root architecture, particularly a root system with a rapid
exploitation of deep soil can optimize nitrogen capture and water
uptake which is especially important in drying and nitrogen
depleted soils. An RHPP polypeptide(s) as described herein when
applied as a foliar treatment to soybean plants results in a root
phenotype that is useful for water and mineral (nitrogen)
acquisition, especially in nitrogen-deficient soils. Increasing
nutrient uptake efficiency by enhancing root architecture is a key
factor for improving plant productivity when used with soybean
cultivation practices in a wide range of soil types.
[0433] Enhanced root biomass that results from a foliar application
of RHPP provided at the early vegetative stages for soybean VE-V5
or V2-V3 stage of development results in a root system with rapid
exploitation of deep soil (deep roots), and greater overall
increases in root biomass. For example, a root hair promoting
bioactive priming polypeptide such as RHPP (SEQ ID NO: 600) can be
applied as a foliar treatment to soybean plants at the V2 to V3
stage of development to result in an overall increase in root
biomass. Other notable enhancements in addition to root biomass are
the production of longer lateral roots, increases in root
branching, root hairs and increases in the root absorptive surface
area.
[0434] RHPP can be applied as a foliar treatment at key
developmental stages (VE-V8 or V2-V8) or in environments where a
rapid increase in root production is desired, such as dry or
nutrient poor soil types. Soil types in particular may affect root
development and expansion. For example, if plants have a hard time
emerging in a clay soil, it may affect root formation and root
proliferation. Increasing root mass may not only beneficially
effect plant emergence but also contribute to plant establishment.
In addition, nodule formation and number are important because the
bacteria that inhabit the nodules pull nitrogen from the air
allowing soybeans to convert it into the nitrogen that they need to
grow and produce seeds.
[0435] The RHPP polypeptides (Tables 13-15) can be used to increase
nodule formation and nodule production of soybean roots when
applied using any of these treatment application methods which can
be applied directly to the soil, as a soil drench, as an in furrow
treatment, or as a foliar application to the above ground plant
parts.
[0436] Increase in nodules can result in increased nitrogen
fixation by nitrogen fixing bacteria that inhabit the root nodules,
such as Rhizobium leguminosarum or japonicum. Nodule formation can
be seen shortly after VE and can increase nitrogen fixation.
Effective nodulation of soybean roots results in higher yields and
higher quality seed production, protein and oil per seed or acre
basis. Soybean plants have fully formed first trifoliate leaves at
the V1-V2 stage of development which is estimated to be the peak
time for nitrogen fixation.
[0437] The combination application of Gm.RHPP bioactive priming
polypeptide with various fertilizer treatment(s) can provide a
yield boost and is recommended especially for crop management
applications in nitrogen depleted soils.
[0438] Bacterial Disease
[0439] Methods of using the bioactive priming polypeptides such as
the flagellin-associated polypeptides or the thionin-like
polypeptides as described herein are useful for the prevention,
treatment and control of bacterial diseases in corn and
particularly useful for the treatment of bacterial leaf streak
disease in corn caused by Xanthomonas vasicola pv. vasculorum, also
recognized as Xanthomnas campestris pv. vasculorum.
[0440] Surveys indicate that bacterial leaf streak disease has
spread and may be widely distributed throughout the U.S. Corn Belt
(Western Indiana, Illinois, Iowa, Missouri, Eastern Nebraska and
Eastern Kansas). Disease spread is most prevalent where corn is
planted on corn in crop rotation practices. The bacterial leaf
streak disease can cause infection on dent corn (field) seed corn,
popcorn and sweet corn. The symptoms on corn include narrow to
brown yellow streaks and brown yellow strips between the leaf
veins. Lesions usually develop on lower or older plant leaves and
initially spread to the higher or younger leaves on the plant.
Yellow discoloration also may be present around lesions.
[0441] The bacterial leaf streak disease of corn presumably
survives in previously infected host debris. Bacterial exudates
found on surfaces of infected leaf tissues can serve as secondary
inocula. The bacterium is spread by wind, splashing rain, and
possibly by irrigation water. The pathogen penetrates corn leaves
through natural openings such as stomata, which can result in a
banded pattern of lesions occurring across leaves. Colonization of
leaf tissues apparently is restricted by main veins.
[0442] Because the disease is caused by a bacterial pathogen, the
current use of bactericides is problematic to control it. For
example, most bactericides act as contact products and are not
systemic and thus they will not be absorbed or taken into the plant
via other mechanisms. Bactericide treatments may require repeated
applications as the bactericide may be washed off with rain or
wind, thus rendering them uneconomical or impractical for use in
some corn crops.
[0443] Current disease management practices to date recommend crop
rotation practices (such as corn, soybean and then back to corn)
and the implementation of sanitation practices, such as cleaning
equipment between field usage to slow disease progression.
[0444] Foliar applications of the Flg (Tables 4-5) and thionin
polypeptides (Table 19) or combinations of the two classes provide
an alternative approach for treating the disease. Foliar
applications with these bioactive priming polypeptides provided as
a spray to the leaf surface of either asymptomatic or symptomatic
plants provides a means to prevent, treat, and control the
bacterial leaf streak disease in corn.
[0445] Alternatively, the flagellin- and thionin bioactive priming
polypeptides or combinations thereof can be useful for the
prevention, treatment and control of other bacterial diseases that
infect corn (Table 21).
TABLE-US-00023 TABLE 21 Bacteria causing diseases in corn Corn
Disease Bacteria Causing Diseases Bacterial leaf blight and stalk
rot Pseudomonas avenae subsp. avenae Bacterial leaf spot
Xanthomonas campestris pv. holcicola Bacterial leaf streak
Xanthomonas vasicola Bacterial stalk rot Enterobacter dissolvens;
Erwinia dissolvens Bacterial stalk and top rot Erwinia carotovora
subsp. carotovora Erwinia chrysanthemi pv. zeae Bacterial stripe
Pseudomonas andropogonis Chocolate spot Pseudomonas syringae pv.
coronafaciens Goss's bacterial wilt and blight Clavibacter
michiganensis subsp. (leaf freckles and wilt) nebraskensis;
Corynebacterium michiganense pv. nebraskense Holcus spot
Pseudomonas syringae pv. syringae van Hall Purple leaf sheath
Hemiparasitic bacteria Seed rot-seedling blight Bacillus subtilis
Stewart's disease (bacterial wilt) Erwinia stewartii Corn stunt
(achapparramiento, Spiroplasma kunkelii maize stunt, Mesa Central
or Rio Grande maize stunt)
Cercospora Leaf Blight Disease of Soybean
[0446] Cercospora is a fungal pathogen that causes the disease
Cercospora leaf blight of soybean. Cercospora leaf blight also
referred to as the purple seed stain disease infects both the
leaves and seeds of soybeans. Cercospora infection of soybean seeds
diminishes seed appearance and quality. The causal organism of
Cercospora leaf blight is Cercospora kikuchii, which overwinters in
soybean residue and in the seed coats. Spread of the disease occurs
when the spores from the fungus are spread to soybean plants from
infected residue, weeds or other infected soybean plants. Disease
spread and symptom development are accelerated during periods of
warm and wet weather. Symptom development usually begins after
flowering and appears as circular lesions on soybean leaves as
reddish brown to purple spots that can merge to form lesions.
Symptoms are apparent in the upper canopy, usually in the uppermost
three or four trifoliate leaves. Infected soybean plants exhibit
worsening symptoms as the crop matures, and premature defoliation
of affected leaves may occur during pod-fill. Cercospora symptom
development may also appear as lesions on stems, leaf petioles and
pods. Seeds are infected through the attachment to the pod.
Cercospora infected seeds show a purple discoloration, which can
appear as specks or blotches covering the entire seed coat.
[0447] Foliar applications of flagellin or flagellin-associated
polypeptides (Tables 4-5) provide an alternative approach for
treating the disease. Foliar applications with these bioactive
priming polypeptides provided as a spray to the leaf surface of
either asymptomatic or symptomatic plants provides a means to
prevent, treat, and control Cercospora Leaf Blight in soybeans.
Foliar applications of Flg22 derived from Bacillus thuringiensis,
particularly at high use rates (e.g. 4.0 Fl. oz/Ac), can provide a
means of managing early symptom development and provide healthier
more vigorous soybean plants grown in field locations that have
been impacted by Cercospora.
[0448] Specific combinations of bioactive priming polypeptides that
can be useful for treating or reducing the symptoms of Cercospora
include: a flagellin or flagellin-associated polypeptide having an
amino acid sequence comprising SEQ ID NO 226, 751 or 752; an RHPP
polypeptide having a sequence comprising SEQ ID NO: 600; or a
combination of a flagellin associated polypeptide having an amino
acid sequence comprising any one of SEQ ID NOs 226, 751 and 572 and
an RHPP polypeptide having the amino acid sequence comprising SEQ
ID NO: 600.
[0449] For example, a useful combination of bioactive priming
polypeptides for treating, or reducing the symptoms of Cercospora
on a plant or plant part is a flagellin polypeptide having an amino
acid sequence comprising SEQ ID NO: 226 alone or in combination
with an RHPP polypeptide having an amino acid sequence comprising
SEQ ID NO: 600. Additional treatments can further comprise a
fungicide in combination with these bioactive priming
polypeptides.
Asian Soybean Rust Disease
[0450] Asian soybean rust is a fungal disease caused by Phakopsora
pachyrhizi. Its etiology and symptoms are similar to Cercospora and
the bioactive priming polypeptide combinations useful for treating
it are similar as well. Specifically, combinations of bioactive
priming polypeptides that can be useful for treating or reducing
the symptoms of Asian soybean rust include: a flagellin or
flagellin-associated polypeptide having an amino acid sequence
comprising SEQ ID NO 226, 751 or 752; an RHPP polypeptide having a
sequence comprising SEQ ID NO: 600; or a combination of a flagellin
associated polypeptide having an amino acid sequence comprising any
one of SEQ ID NOs 226, 751 and 572 and an RHPP polypeptide having
the amino acid sequence comprising SEQ ID NO: 600.
[0451] For example, a useful combination of bioactive priming
polypeptides for treating, or reducing the symptoms of Asian
soybean rust on a plant or plant part is a flagellin polypeptide
having an amino acid sequence comprising SEQ ID NO: 226 alone or in
combination with an RHPP polypeptide having an amino acid sequence
comprising SEQ ID NO: 600. Additional treatments can further
comprise a fungicide in combination with these bioactive priming
polypeptides.
Holcus Spot
[0452] Holcus spot is a bacterial disease caused by Pseudomanas
syringae pv. actinidae. Methods are described herein for using
flagellin or flagellin associated polypeptides to restrict growth
of P. syringae and thus prevent or treat the disease of Holcus spot
in a plant or a plant part. Flagellin or flagellin associated
polypeptides useful for the treatment of P. syringae include any
polypeptides having amino acid sequences comprising any one of SEQ
ID NOs: 226, 540, 751, and 572 or any combination thereof.
Sclerotinia Stem Rot (White Mold) Disease
[0453] Sclerotinia sclerotiorum is a plant pathogenic fungus that
causes a disease caused white mold. It is also known as cottony
rot, water soft rot, stem rot, drop, crown rot, and blossom blight.
Diagnostic symptoms of the white rot include black resting
structures known as sclerotia and white fuzzy growths of mycelium
on the infected plant. The sclerotia, in turn, produce a fruiting
body that produces spores in a sac. Sclerotinia can affect
herbaceous, succulent plants, particularly fruits and vegetables,
or juvenile tissue on woody ornamentals. It can also affect legumes
or tuberous plants like potatoes. White mold can affect a host at
any stage of growth, including seedlings, mature plants, and
harvested products. It is usually found on tissues with high water
content and close proximity to soil. Left untreated, pale to dark
brown lesions on the stem at the soil line are covered by a white,
fluffy mycelial growth. This affects the xylem which leads to
chlorosis, wilting, leaf drop, and death. White mold can also occur
on fruit in the field or in storage and is characterized by white
fungal mycelium covering the fruit and its subsequent decay.
Flagellin or flagellin associated polypeptides useful for the
treatment of S. sclerotiorum include any polypeptides having amino
acid sequences comprising any one of SEQ ID NOs: 226, 540, 571,
751, and 752.
Pseudomonas Leaf Spot
[0454] Pseudomonas syringae pv. actinidiae (PSA) is a devastating
plant pathogen causing bacterial canker of both green- (Actinidiae
deliciosa) and yellow-flesh (Actinidiae chinesis) kiwi plants
throughout zones of kiwi production, causing severe harvest loss in
New Zealand, China, and Italy. In New Zealand alone, cumulative
revenue losses to the most devastating biovar PSA-V are predicted
to approach $740 million New Zealand leaves Dollars (NZD) by 2025
(Agribusiness and Economics Research Institute of Lincoln
University "The Costs of Psa-V to the New Zealand Kiwifruit
Industry and the Wider Community"; May 2012). PSA-V colonizes the
outer and inner surfaces of the kiwi plant and can spread through
the xylem and phloem tissues. Disease symptoms of PSA-V on kiwi
include bacterial leaf spot, bacterial canker of the trunk, red
exudates, blossom rot, discoloration of twigs, and ultimately
dieback of kiwi vines. The standard method of control for PSA-V
currently employs frequent foliar applications of metallic copper
to kiwi vines which is predicted to lead to the selection of
copper-resistant form of the pathogen and loss of disease control.
Novel methods of control are urgently needed.
[0455] Flagellin or flagellin associated peptides useful for the
treatment of Pseudomanas syringase, particularly in kiwis, include
any polypeptides having amino acid sequences comprising SEQ ID NO:
226, 540, 752, and/or 571.
Asian Citrus Greening (Huanglonging) Disease
[0456] The methods described herein incorporate a different
approach to combating disease and additionally providing benefits
of increasing the overall productivity of a plant. This approach is
specifically directed to providing either exogenous or endogenous
applications of the bioactive priming polypeptides that include
thionins to combat disease in plants.
[0457] The thionin and thionin-like polypeptides (Table 19) and
compositions thereof are useful for the prevention, treatment and
control of Asian citrus greening also referred to as Huanglonging
(HLB) disease, a devastating disease for citrus. HLB disease is
widely distributed and has been found in most commercial and
residential sites in all counties that have commercial citrus
orchards.
[0458] Methods are described herein for using the thionin
polypeptides (SEQ ID NOs: 650-749) to prevent the spread of and in
the treatment of HLB disease.
[0459] Asian citrus greening disease is transmitted by the Asian
citrus psyllid, Diaphorina citri or the two-spotted citrus psyllid,
Trioza erytreae Del Guercio, which are both characterized as
sap-sucking, hemipteran bug(s) in the family Psyllidae and have
been implicated in the spread of citrus greening, a disease caused
by a highly fastidious phloem-inhabiting bacteria, Candidatus
Liberibacter asiaticus (Halbert, S. E. and Manjunath, K. L, "Asian
citrus psyllids Stemorrhyncha: Psyllidae and greening disease of
citrus: A literature review and assessment of risk in Florida,"
Florida Entomologist 87: 330-353, 2004). Asian citrus greening or
Huanglongbing disease is considered fatal for a citrus tree once
the tree becomes infected.
[0460] The early symptoms of the disease on leaves are vein
yellowing and an asymmetrical chlorosis referred to as blotchy
mottle, which is the most diagnostic symptom of the disease.
Infected trees are stunted and sparsely foliated with a blotchy
mottling appearing on the foliage. Early symptoms of yellowing may
appear on a single shoot or branch and with disease progression,
the yellowing can spread over the entire tree. Afflicted trees may
show twig dieback, and fruit drop. Fruit are often few in number,
small, deformed or lopsided and fail to color properly, remaining
green at the end and display a yellow stain just beneath the
peduncle (stem) on a cut fruit.
[0461] The Asian citrus greening disease may also be graft
transmitted when citrus rootstocks are selected for and grafted to
scion varieties.
[0462] Management of citrus greening disease has proven difficult
and therefore current methods for control of HLB have taken a
multi-tiered integrated disease and pest management approach using
1) the implementation of disease-free nursery stock and rootstock
used in grafting, 2) the use of pesticides and systemic
insecticides to control the psyllid vector, 3) the use of
biological control agents such as antibiotics., 4) the use of
beneficial insects, such as parasitic wasps that attack the
psyllid, and 5) breeding for new citrus germplasm with increased
resistance to the citrus greening causing bacteria (Candidatus
Liberibacter spp.). The use of cultural and regulatory measures to
prevent the spread of the disease is also part of the integrated
management approach. Many aspects involved in the management of
citrus greening are costly both monetarily and in respect to losses
in citrus production.
[0463] Interveinal application of a thionin polypeptide or mixture
of thionin polypeptides can be delivered directly into the phloem
(e.g., phloem cells including phloem sap, phloem companion cells
and phloem sieve tube elements) where Candidatus Liberibacter can
reside.
[0464] The thionins can be produced using an expression system
where they can be fused to a phloem targeting sequence(s) (Table
18) and then uniquely delivered to the same vicinity where the
bacteria can reside in the citrus plant.
[0465] The phloem targeted thionin bioactive priming polypeptides
are useful for treating citrus plants to prevent, reduce or
eliminate the spread of the Asian citrus greening disease or
Huanglonging (HLB) by directly targeting the bacterium, Candidatus
Liberibacter asiaticus
[0466] These phloem targeted thionins can be delivered by injection
into the phloem of a shrub or tree. Additionally, they can be
delivered by spraying, washing, or adding as a soak or a drench to
the soil or area surrounding a plant.
[0467] Any of the phloem targeting sequences (Table 18; SEQ ID NOs:
641-649) can be used in combinations with the thionin and
thionin-like polypeptides (Table 19; SEQ ID NOs: 650-749).
[0468] The bacteria that cause HLB, Candidatus Liberibacter
asiaticus is difficult to isolate and culture. In order to test
individual thionins and thionins with the phloem targeting
sequences to determine if they are useful for the treatment of HLB
disease, Agrobacterium tumefaciens can be used as a model organism
to test the effectiveness on reducing the cell titer or growth of
Agrobacterium prior to using the thionin or thionin combinations in
an orchard setting.
[0469] The "peptide priming" methods provided herein with the
thionins and/or thionin-like polypeptides (Table 19) can also be
used in combination flagellin and flagellin-associated polypeptides
(Tables 1-5). Combinations of the thionin- and flagellin-associated
bioactive priming polypeptides can be used to prophylactically
pre-treat a citrus plant by applying the bioactive priming
polypeptide or a composition containing the polypeptide prior to
the onset or appearance of any infection-related symptoms on the
citrus shrubs or trees. This pretreatment increases resistance to
the disease pathogen that causes citrus greening (Candidatus
Liberibacter spp.).
[0470] The thionins provided in combination with the flagellin
associated bioactive priming polypeptides provide a more
comprehensive approach to disease prevention and management. The
thionin and flagellin associated bioactive priming polypeptides use
two distinct modes of action to prevent disease and the spread of
disease.
[0471] The thionin-flagellin bioactive priming polypeptide
combinations can also be used with any other integrated management
approach for disease control prescribed for HLB including, but are
not limited to, (1) the use of disease-free nursery stock and/or
rootstocks for grafting, (2) the use of pesticides and/or systemic
insecticides to control the disease-causing psyllid, (3) the use of
biological control agents such as injections of antibiotics or
parasitic insects that controls the psyllid, (4) breeding new
varieties of citrus germplasm with increased resistance to the
bacteria responsible for Asian citrus greening disease, (5)
controlling parasitic plants (for example, dodder) that may spread
the disease, or (6) any combination thereof.
[0472] A synthetic version of a phloem targeting polypeptide (SEQ
ID NO: 641) is particularly useful in targeting anti-microbial
polypeptides to the phloem sieve tube and companion cells and can
be useful for treating various bacterial diseases of plants, such
as bacterial leaf streak, Asian citrus greening or Huanglonging and
citrus canker.
[0473] In addition, flagellin or flagellin associated polypeptides
are useful for treating Asian citrus greening, especially when used
in combination with a bacteriocide. For instance, flagellin or
flagellin associated polypeptides having amino acid sequences
comprising any one of SEQ ID NOs: 226, 571, and 752 can be used.
Preferably, the bacteriocide comprises oxytetracycline.
Citrus Canker
[0474] "Peptide priming" methods were developed for use with the
bioactive priming thionin and flagellin-associated polypeptides as
described in Table 19 (thionins) and Tables 1-5 (flagellin and
flagellin-associated polypeptides) to prophylactically treat citrus
plants prior to any visible symptoms of the citrus canker disease
or as a treatment once the onset of disease symptoms become
apparent.
[0475] Citrus canker occurs primarily in tropical and sub-tropical
climates and has been reported to occur in over thirty countries
including spread of infection reported in Asia, Africa, the Pacific
and Indian Oceans Islands, South America, Australia, Argentina,
Uruguay, Paraguay, Brazil and the United States. Citrus canker is a
disease caused by the bacterium, Xanthomonas axonopodis pv. citri
or pv. aurantifolii (also referred as Xanthomonas citri subsp.
citn) that infect foliage, fruit and young stems. Symptoms of
citrus canker infection on leaves, and fruit of the citrus
shrubs/trees can result in leaf-spotting, leaf lesions,
defoliation, die back, deformation of fruit, fruit rind-blemishing,
pre-mature fruit drop, and canker formation on leaves and fruits.
Diagnostic symptoms of citrus canker include a characteristic
yellow halo that surrounds the leaf lesions and a water-soaked
margin that develops around the necrotic tissue on the leaves of
the citrus plant. The citrus canker pathogen can spread through the
transport of infected fruit, plants, and equipment. Dispersal can
also be facilitated by the wind and rain. Overhead irrigation
systems may also facilitate movement of the citrus canker causing
pathogen. Infected stems can harbor the citrus canker causing
bacteria (Xanthomonas axonopodis pv. citn) in the stem lesions for
transmission to other citrus plants. Insects, such as the Asian
leafminer (Phyllocnistis citrella) also disemminate the
disease.
[0476] In general, citrus plants susceptible to the citrus canker
disease include orange, sweet orange, grapefruit, pummelo, mandarin
tangerine, lemon, lime, swingle acid lime, palestine sweet lime,
tangerine, tangelo, sour orange, rough lemon, citron, calamondin,
trifoliate orange and kumquat. World-wide, millions of dollars are
spent annually on prevention, sanitation, exclusion, quarantine and
eradication programs to control citrus canker (Gottwald T. R.
"Citrus Canker," The American Phytopathological Society, The Plant
Health Instructor 2000/updated in 2005). Treatment for the disease
has included application of antibiotics or disinfectants, the use
of copper-based bactericidal sprays, and pesticide applications for
Asian leafminer control.
[0477] The bioactive priming polypeptide combination comprising the
thionin and the flagellin-associated polypeptides can be applied to
a citrus plant or citrus plant part (e.g., rootstock, scion,
leaves, roots, stems, fruit, and foliage) using application methods
that can comprise: spraying, inoculating, injecting, soaking,
infiltrating, washing, dipping and/or provided to the surrounding
soil as an in furrow treatment.
[0478] The methods are provided using the bioactive priming
polypeptides comprising the thionin and/or flagellin-associated
polypeptides to pre-treat citrus plants or citrus plant parts
(e.g., root stock, scion, leaves, roots, stems, fruit, and foliage)
prior to any visible occurrence of symptoms. They are also useful
for providing an increase in resistance to the citrus canker
pathogen resulting in a reduction in disease symptoms.
[0479] Additionally, the methods of using the bioactive priming
polypeptides such as the flagellin and flagellin-associated
polypeptides are useful to treat citrus plants or citrus plant
parts (e.g., root stock, scion, leaves, roots, stems, fruit, and
foliage) once the early onset of citrus canker disease symptoms or
when the symptoms of the disease become apparent.
[0480] Application of the Flg polypeptides for treating citrus
plants to prevent, reduce or eliminate the spread of the citrus
canker disease can be delivered by injecting into the phloem of a
shrub or tree, spraying, washing, adding as a soak or a drench to
the soil or soil area surrounding a plant or provided in
furrow.
[0481] Thionin bioactive priming polypeptides as described herein
(Table 17) can be applied individually or in combination with any
of the flagellin-associated Flg polypeptides (Tables 1-5) as a
foliar treatment or spray or as an injection and are useful for the
prevention of infestation of citrus plants from insects such as the
Asian leafminer (Phyllocnistis citrella) that have been identified
in the dissemination of the bacteria (Xanthomonas axonopodis pv.
citri) that cause the citrus canker disease.
Citrus Plants
[0482] Any of the methods described herein to provide improved
plant health, disease tolerance or disease treatment applications
to treat or prevent Asian citrus greening (HLB) or citrus canker
are suitable for use with any citrus plants and shrubs/trees.
[0483] The thionin or flagellin-associated polypeptides or
compositions comprising the thionin or flagellin-associated
polypeptides as described herein can be applied to any citrus shrub
and/or tree and to any agronomically-important citrus hybrid or
citrus non-hybrid plant, and are useful for prophylactically
treating the citrus to prevent the onset of an infection or
providing treatment after an infection has occurred.
[0484] Citrus plant species for use of the methods described herein
include, but are not limited to: Sweet orange (Citrus sinensis,
Citrus maxima x Citrus reticulata), Bergamot Orange (Citrus
bergamia, Citrus limetta x Citrus aurantium), Bitter Orange, Sour
Orange or Seville Orange (Citrus aurantium, Citrus maxima x Citrus
reticulata), Blood Orange (Citrus sinensis), Orangelo or Chironja
(Citrus paradisi x Citrus sinensis), Mandarin Orange (Citrus
reticulate), Trifoliate Orange (Citrus trifoliata), Tachibana
Orange (Citrus tachibana), Clementine (Citrus clementina), Cherry
Orange (Citrus kinokuni), Lemon (Citrus limon, Citrus maxima x
Citrus medica), Indian Wild Orange (Citrus indica), Imperial Lemon
(Citrus limon, Citrus medica x Citrus paradisi), Lime (Citrus
latifoli, Citrus aurantifolia), Meyer Lemon (Citrus meyeri);
hybrids of Citrus x meyeri with Citrus maxima, Citrus medica,
Citrus paradisi and/or Citrus sinensis), Rough Lemon (Citrus
jambhin), Volkamer Lemon (Citrus volkameriana), Ponderosa Lemon
(Citrus limon x Citrus medica) Kaffir Lime (Citrus hystrix or
Mauritius papeda), Sweet Lemon, Sweet Lime, or Mosambi (Citrus
limetta), Persian Lime or Tahiti Lime (Citrus latifolia), Palestine
Sweet Lime (Citrus limettioides), Winged Lime (Citrus longispina),
Australian Finger Lime (Citrus australasica), Australian Round Lime
(Citrus australis), Australian Desert or Outback Lime (Citrus
glauca), Mount White Lime (Citrus garrawayae), Kakadu Lime or
Humpty Doo Lime (Citrus gracilis), Russel River Lime (Citrus
inodora), New Guinea Wild Lime (Citrus warburgiana), Brown River
Finger Lime (Citrus wintersii), Mandarin Lime (Citrus limonia;
(hybrids with Citrus reticulata x Citrus maxima x Citrus medica),
Carabao Lime (Citrus pennivesiculata), Blood Lime (Citrus
australasica x Citrus limonia) Limeberry (Triphasia brassii,
Triphasia grandifolia, Triphasia trifolia), Grapefruit (Citrus
paradisi; Citrus maxima x Citrus x sinensis), Tangarine (Citrus
tangerina), Tangelo (Citrus tangelo; Citrus reticulata x Citrus
maxima or Citrus paradisi), Minneola Tangelo (Citrus reticulata x
Citrus paradisi), Orangelo (Citrus paradisi x Citrus sinensis),
Tangor (Citrus nobilis; Citrus reticulata x Citrus sinensis),
Pummelo or Pomelo (Citrus maxima), Citron (Citrus medica), Mountain
Citron (Citrus halimii), Kumquat (Citrus japonica or Fortunella
species), Kumquat hybrids (Calamondin, Fortunella japonica;
Citranqequat, Citrus ichangensis; Limequat, Citrofortunella
floidana; Orangequat, hybrid between Satsuma mandarin x Citrus
japonica or Fortunella species; Procimequat, Fortunella hirdsiie;
Sunquat, hybrid between Citrus meyeri and Citrus japonica or
Fortunella species; Yuzuquat, hybrid between Citrus ichangensis and
Fortunella margarita), Papedas (Citrus halimii, Citrus indica,
Citrus macroptera, Citrus micrantha), Ichang Papeda (Citrus
ichangensis), Celebes Papeda (Citrus celebica), Khasi Papeda
(Citrus latipes), Melanesian Papeda (Citrus macroptera), Ichang
Lemon (Citrus ichangensis x Citrus maxima), Yuzu (Citrus
ichangensis x Citrus reticulata), Cam sanh (Citrus reticulata x
Citrus maxima), Kabosu (Citrus sphaerocarpa), Sudachi (Citrus
sudachi), Alemow (Citrus macrophylla), Biasong (Citrus micrantha),
Samuyao (Citrus micrantha), Kalpi (Citrus webberi), Mikan (Citrus
unshiu), Hyuganatsu (Citrus tamurana), Manyshanyegan (Citrus
mangshanensis), Lush (Citrus crenatifolia), Amanatsu or Natsumikan
(Citrus natsudaidai), Kinnow (Citrus nobilis x Citrus deliciosa),
Kiyomi (Citrus sinensis x Citrus unshiu), Oroblanco (Citrus maxima
x Citrus paradisi), Ugli (Citrus reticulata x Citrus maxima and/or
Citrus x paradisi), Calamondin (Citrus reticulata x Citrus
japonica), Chinotto (Citrus myrtifolia, Citrus aurantium or Citrus
pumila), Cleopatra Mandarin (Citrus reshni), Daidai (Citrus
aurantium or Citrus daidai), Laraha (Citrus aurantium), Satsuma
(Citrus unshiu), Naartjie (Citrus reticulata x Citrus nobilis),
Rangpur (Citrus limonia; or hybrid with Citrus sinensis x Citrus
maxima x Citrus reticulata), Djeruk Limau (Citrus amblycarpa),
lyokan, anadomikan (Citrus iyo), Odichukuthi (Citrus odichukuthi),
Ougonkan (Citrus flaviculpus), Pompia (Citrus monstruosa), Taiwan
Tangerine (Citrus depressa), Shonan gold (Citrus flaviculpus or
Citrus unshiu), Sunki (Citrus sunki), Mangshanyen (Citrus
mangshanensis, Citrus nobilis), Clymenia (Clymenia platypoda,
Clymenia polyandra), Jabara (Citrus jabara), Mandora (Mandora
cyprus), Melogold (Citrus grandis x Citrus paradisiil Citrus
maxima/Citrus grandis), Shangjuan (Citrus ichangensis x Citrus
maxima), Nanfengmiju (Citrus reticulata), and ShikwAsai (Citrus
depressa).
[0485] The thionin and/or flagellin-associated priming polypeptides
can be applied to any citrus plant, shrub/tree used for medicinal
or cosmetic/health and beauty purposes, such as Bergamot Orange
(Citrus bergamia), Sour or Bitter Orange (Citrus aurantium), Sweet
Orange (Citrus macrophylla), Key Lime (Citrus aurantiifolia),
Grapefruit (Citrus paradisi), Citron (Citrus medica), Mandarin
Orange (Citrus reticulate), Lemon (Citrus limon, or hybrids with
Citrus medica x Citrus maxima, Citrus limonia, Citrus medica x
Citrus maxima x Citrus medica), Sweet Lime (Citrus limetta), Kaffir
Lime, (Citrus hystrix or Mauritius papeda), Lemon hybrid or Lumia
(Citrus medica x Citrus limon), (Citrus medica x Citrus maxima x
Citrus medica), Omani Lime (Citrus aurantiifolia, Citrus medica x
Citrus micrantha), Jambola (Citrus grandis), Kakadu Lime or Humpty
Doo Lime (Citrus gracilis), Pomelo (Citrus retkulata), Tangor
(Citrus nobilis), and Sour Lime or Nimbuka (Citrus acida).
[0486] Exemplary important citrus hybrids for fruit production are:
Sweet Orange (Citrus sinensis), Bitter Orange (Citrus aurantium),
Grapefruit (Citrus paradisi), Lemon (Citrus limon), Persian Lime
(Citrus latifolia), Key Lime (Citrus aurantiifolia), Tangerine
(Citrus tangerine) and Rangpur (Citrus limonia).
[0487] Additionally, any of the bioactive priming polypeptides,
compositions, and methods as described herein can be applied to any
citrus plant, shrub/tree used as a rootstock and/or a scion
germplasm. The methods are particularly useful for rootstocks
commonly used in grafting of citrus to enhance the merits of the
scion varieties, which can include tolerance to drought, frost,
disease or soil organisms (for example, nematodes). Such citrus
plants that provide useful rootstocks include: Sour or Bitter
Orange (Citrus aurantium), Sweet Orange (Citrus macrophylla),
Trifoliate Orange (Poncirus trifoliata), Rough Lemon (Citrus
jambhin), Volkamer Lemon (Citrus volkameriana), Alemow (Citrus
macrophylla), Cleopatra Mandarin (Citrus reshini), Citrumelo
(hybrids with x Citroncirus species), Grapefruit (Citrus paradisi),
Rangpure Lime (Citrus limonia), Palestine Sweet Lime (Citrus
limettioides) and Troyer Citrange (Citrus sinensis x Poncirus
trifoliata or Citrus sinensis x Citrus trifoliata) and Citrange
(Citrus sinensis x Poncirus trifoliata or C. sinensis x C.
trifoliata).
Use of Retro-Inverso Flg Bioactive Priming Polypeptides to Treat
and Reduce Citrus Greening
[0488] Combinations of flagellin-associated polypeptides paired
with their retro-inverso counterparts can be used to treat and
reduce the greening effect on citrus that results in Asian citrus
greening or Huanglongbing disease.
[0489] An early symptom of HLB in citrus is the yellowing of leaves
on an individual limb or in one sector of a tree's canopy. Leaves
that turn yellow from HLB will show an asymmetrical pattern of
blotchy yellowing or mottling of the leaf, with patches of green on
one side of the leaf and yellow on the other side. As the HLB
disease progresses, the fruit size becomes smaller, and the juice
turns bitter. The fruit can remain partially green and tends to
drop prematurely.
[0490] Treatment combinations of Flg polypeptides with their
retro-inverso (RI) forms can be used to minimize the effect on
citrus fruit greening. Such treatment combinations can be applied
on HLB-infected trees. The retro-inverso forms will compete with
the native forms of Flg polypeptides for binding to the
FLS-associated receptor(s) at the plant surface and thus
inhibit/delay the symptom formation of greening associated with HLB
disease. The native Flg22 and RI Flg22 combinations assist with a
fine tuned immune response to reduce and even eliminate the
disease-causing bacteria, Candidatus Liberibacter asiaticus, while
preventing acute symptom development, such as leaf yellowing and
citrus fruit greening.
EXAMPLES
[0491] The following non-limiting examples are provided to further
illustrate the present invention.
Example 1: Application of Bt.4Q7Flg22 and Retro-Inverso Bt4Q7Flg22,
and Ec.Flg22 and Ec. RI Flg22 to Corn
[0492] The effect of Bt.4Q7Flg22 (SEQ ID NO: 226) and retro-inverso
Bt.4Q7Flg22 (SEQ ID NO: 376), as well as Ec. Flg22 (SEQ ID NO 526)
and Ec. RI Flg22 (SEQ ID 527) bioactive priming polypeptides on
corn (BECK'S 5828 YH, 6175YE) yield was determined in 10 separate
locations in the US Midwest (FIG. 2 and FIG. 3).
[0493] Field seed beds at each location were prepared using
conventional or conservation tillage methods for corn plantings.
Fertilizer was applied as recommended by conventional farming
practices and remained consistent between the US Midwest locations.
Herbicides were applied for weed control and supplemented with
cultivation when necessary. Four-row plots, 17.5 feet (5.3 meters)
long were planted at all locations. Corn seed was planted 1.5 to 2
inches (3.8 to 5.1 cm) deep, to ensure normal root development, at
28,000 to 36,000 plants per acre with row widths of 30 inch (76.2
cm) rows with seed spacing of approximately 1.6 to 1.8 seeds per
foot. Each hybrid was grown in at least three separate plots
(replicates) at each location to account for field variability.
[0494] Native Bt.4Q7Flg22 bioactive priming polypeptide (SEQ ID NO:
226) and its retro-inverso polypeptide (SEQ ID NO: 376) were
chemically synthesized via solid phase peptide synthesis and
formulated at 0.33 Fl. oz/Ac (24.1 mL/hectare, Ha) use rate. The
final concentration in the spray tank was 25 nM after dilution in
carrier rate of 10 gallons water/Ac (37.85 L/Ha). Native
Bt.4Q7Flg22 bioactive priming polypeptides were applied during
first and second year field trials to measure effects across a
multi-year growing season. Retro-inverso polypeptides were applied
during the first year field trials to compare with native
Bt.4Q7Flg22. Bioactive priming polypeptides were applied as foliar
spray applications at 0.33 Fl. oz/Ac (24.1 mL/Ha) use rate during
the V5-V8 development stage. Each polypeptide was applied with a
non-ionic surfactant at 0.5%. The effect of bioactive priming
polypeptides was measured as the absolute changes in yield in
bushels per acre (Bu/Ac). Additionally, the win rate was
calculated: the percentage of testing locations at which one
treatment has a yield advantage over other treatments (in this
case, as compared to the non-treated control plants).
[0495] FIG. 2, panel A shows that during the first year field
trials, foliar spray application of Bt.4Q7Flg22 (SEQ ID NO: 226)
resulted in an average yield increase of 11.60 Bu/Ac (728.1 kg/Ha)
and a win rate of 90% across the 10 locations compared to the
non-treated control corn plants. FIG. 2, panel B shows that foliar
spray applications of retro inverso Bt.4Q7Flg22 bioactive priming
polypeptide (SEQ ID NO: 376) resulted in an average yield increase
of +11.90 Bu/Ac (746.9 kg/Ha) and a win rate of 70% across the 10
locations in the US Midwest compared to the non-treated control
corn plants. In both figures, locations (1-12) are reported on the
x-axis and absolute change in yield Bu/Ac is reported on the y-axis
and above or below the bar graphs at each location. Ec. Flg22
polypeptide delivered to corn yielded 8.2 bu/Ac (514.7 kg/Ha)
advantage with a 80% win rate across the 10 sites. The retro
inverso version of Ec. RI Flg22 did not yield as well, giving 1.9
bu/c across the 10 sites with a 50% win rate.
[0496] The second year field trials were conducted using large acre
field trials at 10-11 locations in the US MidWest (IL, IN, IA) and
employed foliar spray application of the Bt.4Q7Flg22 bioactive
priming polypeptide (SEQ ID NO: 226) provided to V8 corn plants
(Dekalb 5064). Foliar spray application of Bt.4Q7Flg22 was applied
at a use rate of 0.33 fluid ounces per acre (Fl. oz/Ac). As shown
in FIG. 3, foliar application using the Bt.4Q7Flg22 bioactive
priming polypeptide resulted in an average yield increase of +4.8
Bu/Ac over the control across the 11 locations with a win rate of
83%. Locations 1-6 are reported on the x-axis and absolute change
in yield Bu/Ac is reported on the y-axis and above or below the bar
graphs at each location.
[0497] First year field trials using foliar treatments using
Bt.4Q7Flg22 bioactive priming polypeptide (SEQ ID NO: 226) applied
to corn hybrid (BECK's 5828 YH) shown in FIG. 2 (panel A) resulted
in over a +11 Bu/Ac (690.4 kg/Ha) increase in yield over the
non-treated control plants. Second year field trials applied to V8
corn plants (Dekalb 5064) shown in FIG. 3 resulted in an almost+5
Bu/Ac (313.8 kg/Ha) increase compared to the yield of the
non-treated control plants. The combined average for the two corn
hybrids resulted in a 2-year average yield increase of +8.0 Bu/Ac
(50.2 kg/Ha) across locations with a win rate of 86% represented
for the multiple year growing season.
[0498] A third study was performed with Bt.4Q7 Flg22 bioactive
priming polypeptide tested as a V5-V8 application to corn at 3
rates and applied with a non-ionic surfactant. The final use rates
were 0.33 Fl. oz/Ac, 4 Fl. oz/Ac, 8 Fl. oz/Ac (24.1 mL/Ha, 292.3
mL/Ha, 584.6 mL/Ha), resulting in approximate final concentrations
of 25 nM, 300 nM, 600 nM respectively. Each study was performed at
between 10 and 11 sites. The end results of the study at V5-V8 at
0.33 Fl. oz/Ac (24.1 mL/Ha) was 5.75 Bu/Ac or 360.9 kg/Ha
advantage, at 4 Fl. oz/Ac a 3.77 bu/Ac or 236.6 kg/Ha advantage,
and at 8 Fl. oz/Ac a 5.05 Bu/Ac or 317 kg/Ha.
Example 2: Application of Bt.4Q7Flg22 to V8 Corn with Fungicide
[0499] Foliar treatments with Bt.4Q7Flg22, with and without a
commercially available fungicide, STRATEGO YLD, were conducted to
determine if synergistic effects resulted from the combinations of
the Bt.4Q7Flg22 bioactive priming polypeptide with the fungicide.
Foliar spray application of Bt.4Q7Flg22 (SEQ ID NO: 226) alone or
in combination with STRATEGO YLD was assessed on corn plants
(hybrid Dekalb 5064) at the V8 stage of development.
[0500] Replicated trials were conducted at 6-8 locations throughout
the US Midwest (IA, IL, IN) using replicated trials. Corn plants
were grown as described in Example 1. Plots were maintained using
the individual grower's production practices and each plot was
replicated 3-4 times. When used, STRATEGO YLD fungicide (a
combination of prothioconazole and trifloxystrobin) was applied
using the recommended label rates (4.0 Fl. oz/Ac or 292.3 mL/Ha))
at each location. Foliar treatment applications consisted of the
following treatments: (a) non-treated control, (b) STRATEGO YLD
fungicide alone, and Bt.4Q7Flg22 (SEQ ID NO: 226) delivered in a
free peptide form provided with (c) and without (d) the fungicide.
Bt.4Q7Flg22 was applied at a use rate of 0.33 or 4.0 fluid ounces
per acre (Fl. oz/Ac) or (24.1 or 292.3 mL/Ha).
[0501] Corn yield in bushels per acre (Bu/Ac) was reported at all
locations as an average yield for the replicated trials at each
location. The change in yield in Bu/Ac for corn plants receiving
foliar applications with the STRATEGOYLD fungicide were normalized
to the average yield for the control corn plants for the 6
locations (Table 22).
[0502] Foliar treatments with Bt.4Q7Flg22 provided at 0.33 Fl.
oz/Ac (24.1 mL/Ha) provided yield benefits over the non-treated
control corn plants with a +4.84 Bu/Ac (303.8 kg/Ha) increase
observed across the 6 locations. Foliar treatment using only the
fungicide application of STRATEGO YLD also provided a yield benefit
in corn of +4.88 Bu/Ac (306.3 kg/Ha) over the control plants.
Application of the free peptide, Bt.4Q7Flg22, at 0.33 Fl. oz/Ac
(24.1 mL/Ha) combined with STRATEGO YLD fungicide at 4.0 Fl. oz/Ac
demonstrated a synergistic effect, resulting in an average of
+10.72 Bu/Ac (672.9 kg/Ha) over the non-treated control plants.
Therefore, the Bt.4Q7Flg22 polypeptide and fungicide treatment
combination resulted in a synergistic effect at the 0.33 Fl. oz/Ac
(24.1 mL/Ha) use rate for the polypeptides and 4.0 Fl. oz/Ac (292.3
mL/Ha) use rate for the fungicide.
TABLE-US-00024 TABLE 22 Foliar treatment of corn with Bt.4Q7Flg22
bioactive priming polypeptide applied with a fungicide to increase
yield in corn Average Total Average Bu/Ac Application Yield
Increase Use Rate Bu/Ac compared to Treatment - Corn Fl. oz/Ac (6
locations) Control Control -- 187.37 -- Bt.4Q7Flg22 0.33 192.21
+4.84 STRATEGO YLD 4.0 193.80 +4.88 Bt.4Q7Flg22 + 0.33 207.86
+10.72 STRATEGO YLD 4.0
[0503] A second study looking at the combination of Ec. Flg22 with
STRATEGO was also performed at 8 sites as replicated trials in the
same fashion as above. Ec. Flg22 at 4 Fl. oz/Ac (292.3 mL/Ha) added
1.3 (Bu/Ac) (81.6 kg/Ha) on top of STRATEGO YLD with a 63% win
percentage over 8 sites. This demonstrates that both Flg22
polypeptides were able to add benefit over a commercial fungicide,
STRATEGO YLD.
Example 3: Application of Bt.4Q7 Flg22, Retro-Inverso Bt4Q7Flg22,
Ec.Flg22, Retro Inverso Ec.Flg22 or RHPP to R2 Soybean--Increased
Yield
[0504] Foliar application using, Bt.4Q7 Flg22 bioactive priming
polypeptide (SEQ ID NO: 226; FIG. 4, panel A), the retro-inverso
(RI) Bt.4Q7Flg22 (SEQ ID NO: 376; FIG. 4, panel B) from Bacillus
thuringiensis strain 4Q7 and root hair promoting polypeptide (RHPP,
SEQ ID NO: 600) derived from Glycine max were applied individually
to soybean plants (commercial hybrid Beck's 294 NR) at the R2 stage
of development using a use rate of 0.33 Fl. oz/Ac or 24.1 mL/Ha
(Flg22 polypeptides) or 4.0 Fl. oz/Ac or 292.3 mL/Ha (RHPP).
Cultivation methods employed in Example 1 were followed in growing
soybean seeds. Soybean seed (commercial hybrid Beck's 294 NR) was
planted 1.5 to 2 inches (3.8 to 5.1 cm) deep to assure normal root
development. Soybean seed was planted at approximately on average
150,000 plants per acre with row widths of 30 inch (76.2 cm) rows
with seed spacing of approximately 7 to 8 seeds per foot (0.3
meter).
[0505] Yield results in bushels per acre (Bu/Ac) are reported for
soybean grown in 11 separate US Midwest locations harvested in
October (FIG. 4). Soybean yield (Bu/Ac) is also reported as
averaged across all of the locations as the change in yield (Bu/Ac)
normalized to the control soybean plants. Soybean yield following
foliar application with Bt.4Q7Flg22 (SEQ ID NO: 226) and the RI
Bt.4Q7Flg22 (SEQ ID NO: 376) was compared to yield of non-treated
soybean plants and plotted in FIG. 4. Locations 1-11 are reported
on the x-axis. Absolute change in yield (Bu/Ac) as compared to the
non-treated control soybean plants is reported on the y-axis and
above or below the bar graphs at each location. Average yield
across all 11 locations are reported and highlighted with the black
bar. Soybean yield for the Bt4Q7Flg22 and RI Bt.4Q7Flg22 foliar
treated plants showed similar trends at the 11 different locations.
Spray application using RI Bt.4Q7Flg22 on soybean resulted in an
average yield increase of 0.90 Bu/Ac (60.5 kg/Ha) for the 2 soybean
hybrids across 11 locations compared to the soybean non-treated
control plants. Yield results for the natural (all L) Bt.4Q7Flg22
in soybean was neutral (-0.1 Bu/Ac or -6.7 kg/Ha)) when compared
across the locations. Yield data represented across 11 individual
US Midwestern locations resulted in a win rate of 64%, for both the
RI Bt.4Q7Flg22 and Bt.4Q7Flg22 spray application treatments as
compared to the control or non-treated soybean plants. The addition
of RHPP polypeptide at 4 Fl. oz/Ac (292.3 mL/Ha) in the same study
increased yield by 1.2 Bu/Ac (80.7 kg/Ha) compared to control.
[0506] A second study was performed to test Ec. Flg22 and Ec. RI
Flg22 polypeptides as R2 foliar treatments on soybeans with a
carrier rate of 10 gallons/Ac (93.5 L/Ha) water and NIS surfactant.
A concentration of 100 nM was obtained in the tank for each
treatment. The application of the Ec. Flg22 lead to a 0.9 Bu/Ac
(60.5 kg/Ha) increase with a 82% win rate for the 11 sites, and the
Ec. RI Flg22 lead to a 0.6 Bu/Ac (40.3 kg/Ha) increase with a 80%
win rate over 10 sites. Also included was the RHPP polypeptide as a
seed treatment, with 1.2 bu/Ac (80.7 kg/Ha) over 11 sites at 73%
win rate.
[0507] A third study at the same 11 sites was performed adding a
foliar fertilizer alone or with RHPP at 8 fl oz/Ac (584.6 mL/Ha).
The addition of RHPP on top of the foliar fertilizer gave 1 Bu/AC
(67.2 kg/Ha) advantage across the 11 sites.
Example 4: Foliar Spray Application of Flg22 Polypeptides to
Soybeans
[0508] Foliar treatments with Bt.4Q7Flg22, Ec.Flg22, and RHPP at
4.0 and 8.0 Fl. oz/Ac (292.3 and 584.6 mL/Ha) were tested at the R2
timing on soybean varieties over 11 sites with 10 gallons/Ac (93.5
L/Ha) water with 0.5% NIS surfactant. At the higher dose of 8 Fl.
oz/Ac (584.6 mL/Ha), the Ec. Flg22 polypeptide gave a 0.74 Bu/Ac
(49.8 kg/Ha) advantage and the Bt.4Q7 Flg22 gave a 0.88 Bu/Ac (59.2
kg/Ha) advantage. The lower rate of 4 Fl. oz/Ac (292.3 mL/Ha) for
RHPP gave 0.31 Bu/Ac (20.9 kg/Ha) yield advantage.
Example 5: Application of Escherichia coli Flagellin Polypeptides
to Increase Yield--Corn
[0509] The effect of flagellin polypeptides derived from
Escherichia coli on corn yield was then tested. Corn plants (Beck's
5828 YH) received an initial spray application at the V5-V8 stage
of development with formulations containing the Ec. Flg22 bioactive
priming polypeptide (SEQ ID NO: 526) and the retro-inverso RI
Ec.Flg22 (SEQ ID NO: 527) from Escherichia coli applied at a use
rate of 0.33 Fl. oz/Ac (24.1 mL/Ha). Yield results in bushels per
acre (Bu/Ac) were determined for corn grown in the 12 separate
locations harvested in October.
[0510] FIG. 5 depicts yield across these 12 locations, normalized
to the non-treated control plants and shown as an increase or a
decrease in Bu/Ac compared to the control. Yield data represented
for 12 individual locations in Illinois resulted in a win rate of
50%. Corn plants that received a foliar spray application of
Ec.Flg22 bioactive priming polypeptide (FIG. 5, panel A) resulted
in an average yield increase of +8.2 Bu/Ac (514.7 kg/Ha) across the
12 locations over non-treated plants. Corn plants that received the
foliar spray applications of the retro inverso RI Ec.Flg22
bioactive priming polypeptide (FIG. 5, panel B) resulted in an
average yield increase of +1.9 Bu/Ac 119.3 kg/Ha) across the 12
locations as compared to the non-treated control corn plants.
Therefore, application of foliar sprays containing the Ec.Flg22
(SEQ ID NO: 526) bioactive priming polypeptides provided a
beneficial growth response and yield benefit to corn plants when
applied at the V5-V8 stage of development.
Example 6: Foliar Application of Escherichia coli Flagellin
polypeptides to V2-V3
[0511] Soybean to Increase Plant Height
[0512] Foliar application of the Ec.Flg22 (SEQ ID NO: 526) and
retro inverso RI Ec.Flg22 (SEQ ID NO: 527) was applied to soybean
(Beck's 297NR). Plants were grown in an environmentally controlled
growth room. Seed was planted directly into 39.7 cm.sup.3 pots
containing Timberline top soil at a depth of 2.54 cm, with 2 seeds
per pot. After planting, 50 mL of room temperature water was added
to each pot to allow for germination. The pots were kept in an
artificial lighted growth room receiving approximately 300 .mu.mol
m.sup.-2 s.sup.-1 (light photons) for a 13/11 light/day cycle and a
21.degree. C. day/15.degree. C. night temperature range. Plants
received the same watering and fertilizer regimes.
[0513] Foliar treatments using both the native and retro inverso
forms of Ec.Flg22 were applied to 3-week-old soybean plants at the
V2 to V3 stage of development using a use rate of 0.33 Fl. oz/Ac
(24.1 mL/Ha). Plant height (cm) was measured just prior to the
foliar application delivered at 3 weeks and then again 2 weeks
later when the plants were 5-weeks-old. Two replicate trials were
conducted using 18 plants per trial.
[0514] As described in Table 23, foliar application of the Ec.Flg22
polypeptide to soybean at the V2-V3 stage of development increased
plant height, compared to the control (water only treatment) plants
(Table 23). Foliar application using the Ec.Flg22 (SEQ ID NO: 526)
and the retro-inverso Ec.Flg22 (SEQ ID NO: 527) bioactive priming
polypeptides resulted in +13% and +16% increases in plant height
when normalized to the control non-treated soybean plants
(normalized to 100%).
TABLE-US-00025 TABLE 23 Foliar application of flagellin polypeptide
increases plant height for soybean Foliar Height (cm) Height (cm)
at Percentage Treatment at 3 weeks 5 weeks height of control
Soybean Ec.Flg22 (1 .mu.M) 40.17 (5.83) 64.79 (8.40) 113.2%
Ec.Flg22-Retro 36.57 (6.00) 66.46 (5.77) 116.1% Inverso (1
.mu.M)
Example 7: Application of Flg22 and Retro Inverso Flg22 in
Corn--Plant Height
[0515] Corn (Beck's hybrid 5828 YH) plants were grown in an
environmentally controlled growth room as described in Example 6.
Plants were measured three weeks after emergence and then treated
with foliar applications of natural (L) and retro-inverso (D) forms
of Flg22 polypeptides from Bacillus thuringiensis (Bt.4Q7Flg22, SEQ
ID Nos 226 and 376) and Escherichia coli (Ec.Flg22, SEQ ID NOs:
526-527). Bioactive priming polypeptides were applied as free
polypeptides at a concentration of 1 .mu.M. Control plants were
treated with water alone. After an additional 2 weeks of growth,
plant height was measured (at 5 weeks).
[0516] The change in plant height (.DELTA. height cm) between the 2
week and 5 week interval time points was measured and normalized to
the growth of water-treated control plants. Three replicate trials
were conducted using 9 plants per trial equaling a total of 27
measurements per treatment (Table 24). There were no differences in
the plant height measured between the EcFlg22, the Bt.4Q7Flg22 or
the water treated control plants at the 3-week measurement time
point. The greatest change in plant height from 3 to 5 weeks was
reported for corn plants that received the Ec.Flg22 foliar
application (.DELTA.=17.60 cm). These plants also achieved a +8.3%
increase in height compared to control plants at the 5 week
measurement mark. The two retro inverso polypeptides (RI Ec.Flg22
and RI Bt.4Q7Flg2) and the natural Bt.4Q7Flg22 similarly increased
plant height when compared to the control treatment with increases
reported from approximately +2% to +4%.
TABLE-US-00026 TABLE 24 Foliar application of Ec.Flg22 and Bt.Flg22
polypeptides on corn resulted in increases in plant height Height
Height .DELTA. Height Foliar (cm) at 3 (cm) at 5 .DELTA. Normalized
as Treatment weeks weeks Height a percentage Corn (STDEV) (STDEV)
(cm) of control height Ec. Flg22 1 .mu.M 47.00 (8.30) 64.60 (6.93)
17.60 +8.3% Ec. Flg22 Retro 48.62 (6.62) 62.00 (4.07) 13.38 +3.9%
inverso 1 .mu.M Bt 4Q7Flg 22 50.10 (6.79) 61.89 (7.03) 5.40 +3.7% 1
.mu.M Bt 4Q7Flg22 49.05 (4.28) 61.03 (7.13) 11.98 +2.3% Retro
inverso 1 .mu.M
Example 8: Application of Retro Inverso Flg22 Bioactive Priming
Polypeptides to Promote Growth Under Stress--Corn
[0517] Abiotic stress causes significant crop loss and can result
in major reductions in crop production and yield potential. The
flagellin compositions and flagellin-associated bioactive priming
polypeptides can be used as chemical priming agents to increase
tolerance of a plant to one or more abiotic stresses. Foliar
treatments using the Ec.Flg22 and Bt.4Q7Flg22 and the retro inverso
(RI) forms of both of these bioactive priming polypeptides were
conducted to determine if these foliar applied polypeptides could
provide a protective advantage against heat and drought stress.
[0518] Corn (Beck's hybrid 5828 YH) seed was planted and grown as
described in Example 6 with the difference that a 16 hour day/8
hour night light-cycle was followed. Temperature was cycled from
21.degree. C./day to 15.degree. C./night with 75% humidity. The
light cycle still provided a uniform approximately 300 .mu.mol
m.sup.-2 s.sup.-1, adequate light for plant growth. Plants were
measured at 3 weeks after emergence and were then treated with
foliar applications of natural or the retro-inverso (RI) forms of
Ec.Flg22 (SEQ ID NOs 526-527) or Bt.4Q7Flg22 (SEQ ID NOs: 226 and
376) at 1 .mu.M concentrations. Control plants were treated only
with water. A week after the spray treatments were applied, the
plants were subdivided into 2 groupings where one group remained in
the same standard growth environment as described and the other
group was transferred to an environment that provided heat and
water deficit stress. In the heat stress environment, the
temperature was elevated using heat maps from 21.degree. C. to
27.degree. C. for 18 hours per day for a period of 5 days. Plants
were left un-watered for the heat stress duration to further
simulate a water deficit stress. Change in plant height (cm) was
measured 2 weeks later at 5 weeks and reported as a percentage of
the height of the control (water) plants. Measurements are reported
as the combined average of two trials with 9 replicate plants per
trial (Table 25).
[0519] As shown in Table 25, both natural forms of the bioactive
priming polypeptides (Ec.Flg22 and Bt.4Q7Flg22) increased plant
growth, as measured by control plant height, when applied under
non-stressed conditions. The two treatments resulted in plants that
reached heights 103% and 108% of their respective controls.
However, only corn plants treated with the retro inverso Flg22
polypeptides (both retro inverso Ec.Flg22 and Bt.4Q7Flg22) showed
enhanced plant growth compared to control plants when grown in both
normal and heat/water stressed environments. Plants treated with
Ec. Flg22 Retro inverso reached 103% of their control heights in
both conditions. Plants treated with Bt.4Q7Flg22 reached 102% and
almost 108% of their counterpart control's heights in non-stressed
and stressed conditions, respectively.
[0520] Therefore, corn plants that were treated with the retro
inverso Flg22 polypeptides (RI-Ec.Flg22 and RI-Bt.4Q7Flg22)
exhibited increased growth as indicated by increased percentage in
plant height over the control plants. This result suggests that the
retro-inverso forms are more stable in form and able to survive
without proteolytic breakdown in harsher environments or situations
conducive to abiotic stress. Thus, they may offer a protective
advantage to plants that are subjected to abiotic stress
environments.
TABLE-US-00027 TABLE 25 Foliar application of Ec.Flg22 and Bt.Flg22
to corn grown in non-stress and stress environments Foliar Stressed
.DELTA. Height Treatment in Non-Stressed .DELTA. Height (cm)
Normalized as a Corn (Non-heat Normalized as a percentage
percentage of stressed) of control height control height Ec. Flg22
1 .mu.M 108.3% 95.8% Ec. Flg22 Retro 103.9% 103.5% inverso 1 .mu.M
Bt. 4Q7Flg 22 103.7% 100.1% 1 .mu.M Bt.4Q7Flg22 Retro 102.3% 107.8%
inverso 1 .mu.M
Example 9: Heat and Water Deficit Stress after Application of
Foliar Flg22 Polypeptide to V2-V3 Corn
[0521] In a separate experiment, corn plants, grown as described in
Example 8, were treated with Bt.4Q7Flg22 along with a surfactant
before exposure to heat and water deficit stress. Three replicate
trials of 18 corn plant replicates per trial were grown in an
environmentally controlled growth room until the V2-V3 stage of
development. Each plant was treated with foliar sprays containing
0.1% surfactant with or without Bt.4Q7Flg22 (1 .mu.M final
concentration). A week after the spray treatments were applied, the
plants were transferred to an environment that provided a heat
stress and water deficit stress. Heat stress was applied using heat
mats to raise the temperature in the environment from 21.degree. C.
to 27.degree. C. During the period of heat stress, the plants were
left unwatered. The corn plants remained in the simulated abiotic
stress environment for one week and then plant height (cm) was
re-measured (Table 26).
[0522] As shown in Table 26, in two out of the three trials,
application of Bt.4Q7Flg22 polypeptide applied as a foliar spray
(Trials 1 and 3) resulted in significant increased growth (height
measured in cm) in corn plants as compared to the control plants
treated with the surfactant alone. Foliar treatment with the
Bt.4Q7Flg22 bioactive priming polypeptide resulted in an almost 13%
increase in plant height in Trial 1 and more than a 33% increase in
Trial 3 compared to the control (surfactant alone treated)
plants.
TABLE-US-00028 TABLE 26 Change in plant height in corn with
application of Bt.4Q7Flg22 .DELTA. Height Height (cm) Height (cm)
Normalized as before stress after stress a percentage Treatments 2
weeks 4 weeks .DELTA. Height of control Corn (STDEV) (STDEV) (cm)
height Trial 1 Surfactant (0.1%) 17.62 (2.32) 27.96 (3.02) +10.34
100.0% Bt.4Q7Flg22 17.72 (2.08) 29.39 (3.04) +11.68 112.9% (1
.mu.M) Trial 2 Surfactant (0.1%) 15.93 (1.22) 25.26 (1.99) +9.32
100.0% Bt.4Q7Flg22 16.03 (1.97) 25.31 (6.29) +9.28 99.5% (1 .mu.M)
Trial 3 Surfactant (0.1%) 13.16 (2.28) 21.43 (2.89) +8.28 100.0%
Bt.4Q7Flg22 14.99 (1.97) 26.02 (3.21) +11.03 133.2% (1 .mu.M)
Example 10: Seed Treatment Using the Flg22 Polypeptides--Corn and
Soy
[0523] Corn seed from two separate hybrids (hybrid BECK's 5828 AM
and 4606 P2) was treated with Bt.4Q7Flg22 (SEQ ID NO: 226)
bioactive priming polypeptides with final slurry concentrations of
0.25 .mu.M or 1.0 .mu.M (Table 27) applied to the surface of each
seed. The seed applications were provided using a 40 .mu.M
polypeptide stock diluted to the appropriate concentration in a
slurry containing a fungicide, insecticide, beneficial bacteria,
colorant and seed finisher (EverGol Energy (0.031 mg ai/seed),
PONCHO/VOTiVO (0.6 mg ai/seed), Peridium 1006 (5 fl oz/cwt or 147.9
mL/cwt) and Pro-Ized Red Colorant (normal) (0.5 fl oz/cwt). Seed
treatment was applied using a Wintersteiger HEGE II (Wintersteiger
AG, Austria, Germany).
[0524] Seed was planted in 12 locations in the U.S. Midwest (IA,
IL, IN). Sixteen randomized replicate blocks were harvested per
each of the Flg22 polypeptide treatments consisting of Bt.4Q7Flg22
applied at 0.25 .mu.M and 1.0 .mu.M slurry concentration.
[0525] Table 27 shows that seed treatment with the Bt.4Q7Flg22
bioactive priming polypeptide applied at what would be the
equivalent of a 40 .mu.M polypeptide solution at a rate of 0.035 or
0.14 Fl. oz (2.6 or 10.2 mL/Ha) of polypeptide solution per unit of
corn seed resulted in enhanced yield with averages of +2.1 Bu/Ac
(131.8 kg/Ha) increases for the low rate and +5.3 Bu/Ac increases
for the high rate application as compared to non-treated control
seed (no seed treatment).
TABLE-US-00029 TABLE 27 Seed treatment on corn using Flg22
polypeptides Peptide Equivalent Average Average Average Change in
concentration Application Total Total Total Yield Yield Bu/Ac in
seed Rate Yield Yield Bu/Ac compared to Treatment coating Fl. oz
/unit Bu/Ac Bu/Ac Hybrid 1 the Control Corn slurry corn seed Hybrid
1 Hybrid 2 and 2 Seed Control -- -- 206.65 184.27 197.32 --
Bt.4Q7Flg22 0.25 .mu.M 0.035 fl oz 218.36 182.88 200.62 +2.1 of 40
.mu.M peptide solution/ unit Bt.4Q7Flg22 1.0 .mu.M 0.14 fl oz of
213.44 187.48 202.62 +5.3 40 .mu.M peptide solution/ unit
[0526] A second study was set up to test the ability of Ec.Flg22,
Ec.RI Flg22, Bt.4Q7Flg22, and Bt.4Q7R1 Flg22 to promote yield in
corn. Replicated trials with 12 locations were set up as above. The
Bt.4Q7 Flg22 gave 2.8 bushels or 71.1 kg at 50% win rate, the
Bt.4Q7 RI Flg22 polypeptide gave 0.5 Bu/Ac. The Ec. Flg22
polypeptide gave 2.8 Bu/Ac (175.8 kg/Ha) advantage at 70% win rate,
and the Ec. RI Flg22 gave no benefit.
[0527] A third study was set up to look at soybean seed treatment
benefits of Bt.4Q7 Flg22, RI Bt.4Q7Flg22, Ec.RI Flg22, and RHPP as
a seed treatment on soybean. Over a 12 location study, the RHPP
polypeptide gave 0.4 Bu/AC (26.9 kg/Ha) at 64% win rate, the Bt.4Q7
Flg22 polypeptide gave 1.3 Bu/Ac (87.4 kg/Ha) at 64%, the Bt.4Q7 RI
Bt.4Q7Flg22 polypeptide gave 0.3 Bu/Ac (20.2 kg/Ha) at 55%, and the
Ec. RI Flg22 gave 1.8 Bu/Ac (121.1 kg/Ha) at 73% win rate.
Example 11: Application of Flagellin Bioactive Priming Polypeptides
to Tomatoes--Increased Yield
[0528] Foliar application treatments of Bt.4Q7 Flg22 (SEQ ID NO:
226) and Ec. Flg22 (SEQ ID NO: 526) were applied as an exogenous
spray at the pre-bloom stage and used to increase yield in
tomatoes.
[0529] Small scale plots were designed to simulate commercial
growing conditions for tomatoes. Two hybrids of tomatoes, JetSetter
(Trial 1) and Better Big Boy (Trial 2) were started as transplants
in the greenhouse 42 to 56 days prior to planting in the raised
field beds. Tomatoes were transplanted once soil temperatures three
inches (7.6 cm) beneath the soil surface reach 60.degree. F.
(15.5.degree. C.). Tomatoes were grown on raised beds covered with
black plastic mulch. Plants were grown using drip irrigation and
fertilizer applied following grower guidelines throughout the
growing season to ensure optimum plant growth and yields. Small
raised bed plots were designed to simulate the planting densities
used by commercial growers that generally plant 2,600 to 5,800
plants per acre in single rows with 18 to 30 inches (46 to 76 cm)
between plants in the row on 5- to 6.5-ft (1.5 to 2 m) centers.
[0530] Foliar treatments of Bt.4Q7 Flg22 and Ec. Flg22 at low and
high use rates of 1 Fl. oz/Ac (73.1 mL/Ha) and 20 Fl. oz/Ac (1461.5
mL/Ha), respectively, were applied on the two hybrids at early
bloom (first flower) stage. Replicated trials were conducted at the
University of Missouri (Columbia, Mo.) in July. Control plants were
treated with equal volumes (use rates) of water. Effects of the
foliar treatments on increasing yield in tomatoes were determined
and reported as normalized to the water control treatment. The
average percentage change in yield over the average control yield
is reported in the Table 28.
[0531] Foliar application of both the Bt.4Q7Flg22 and Ec.Flg22
bioactive priming polypeptides increased tomato fruit yield for
each hybrid at both the low and high use rate. When results for the
two hybrids were averaged, low and high application use rates for
Bt.4Q7 Flg22 increased tomato yield+25% and +17%, respectively,
over the control plants. Similarly, low and high application use
rates for the Ec. Flg22 treatments resulted in an average increase
in tomato yield of +43% and +46% over the control plants for the
two hybrids.
TABLE-US-00030 TABLE 28 Foliar treatment to increase yield in
different hybrids of tomato Trial 1: Percent Trial 2: Change in
Percent Change Average Trials Yield over in Yield over 1 & 2
Avg. Control; Avg. Control; Percent Change Hybrid: Hybrid: Better
Yield over Avg. Foliar Treatment Jetsetter Big Boy Control Bt.4Q7
Flg22 +49% +1% +25% (1 Fl. oz/Ac) Bt.4Q7 Flg22 +22% +12% +17% (20
Fl. oz/Ac) Ec. Flg22 +61% +25% +43% (1 Fl. oz/Ac) Ec. Flg22 +72%
+21% +46% (20 Fl. oz/Ac)
Example 12: Foliar Treatment of Tomato Plants with a Formulation of
Bt.4Q7 Flg22
[0532] In another experiment, tomato plants (hybrid: Better Boy),
cultivated as described in the previous example, were treated with
a formulation of Bt.4Q7 Flg22 at the first bloom stage. The
formulation used consisted of the retro inverso D RI Bt.4Q7 Flg22
applied with 0.01% (v/v) non-ionic surfactant. The formulation was
applied to tomato foliage using application use rates of 1 Fl.
oz/Ac (73.1 mL/Ha) in two replicate winter tomato trials conducted
in Florida. At harvest, the yield was measured as the number of
fruits per plant, the weight (grams) per fruit and the total yield
(lbs/Ac). Table 29 reports the yield as a percent comparison or
change to the non-treated control (water only) plants.
[0533] Foliar treatment using the Bt.4Q7 Flg22 formulation applied
at 1 Fl. oz/Ac (73.1 mL/Ha) increased yield of Better Boy tomatoes
an average of 21% compared to the non-treated (water alone) control
plants. This increase for Better Boy tomatoes corresponded to both
an increase in number of fruits per plant and an increase in the
fruit weight (Table 29).
TABLE-US-00031 TABLE 29 Foliar treatment with a Flg22 bioactive
priming polypeptide to increase yield in tomato Percent Change in
Number of Percent Change Percent Change Fruits per Plant in
Weight/Fruit in Yield (lbs/Ac) Compared to Compared to Compared to
Treatment Control Control Control Bt.4Q7 Flg22 +12% +9% +21% 1 Fl.
oz/Ac
Example 13: Application of Flagellin Bioactive Priming Polypeptides
to Peppers-Increased Yield
[0534] Foliar treatments of Bt.4Q7 Flg22 (SEQ ID NO: 226) and
Ec.Flg22 (SEQ ID NO: 526) were applied as an exogenous spray at the
first-bloom stage and used to increase yield in two pepper
varieties.
[0535] Foliar treatments of Bt.4Q7 Flg22 and Ec.Flg22 bioactive
priming polypeptides were applied using small scale plots designed
to simulate commercial growing conditions for peppers (Capsicum).
Two varieties of pepper: Red Knight (RK) and Hungarian Hot Wax
(HHW) were grown from 6-week old transplants in raised beds covered
with black plastic mulch that had good water-holding
characteristics and a pH of 5.8-6.6. Plants were grown using drip
irrigation and fertilizer applied following grower guidelines
throughout the growing season to ensure optimum plant growth and
yields. Small raised bed plots were designed to simulate the
planting densities used by commercial growers that generally plant
approximately 10,000-14,000 plants per acre in double rows 14-18
inches (35.6 to 46 cm) apart on plastic mulched beds with 16-24
inches (40.6 to 61 cm) between plants in the row and with the beds
spaced 5.0-6.5 feet (40.6 to 70 cm) apart from their centers. A
single row of peppers also can be planted on each bed (5,000-6,500
plants per acre or 12,355-16,062 plants per hectare).
[0536] Foliar applications with compositions containing Bt.4Q7
Flg22 and Ec.Flg22 were applied at the first flower stage at an
application use rate of 1 Fl. oz/Ac (low rate) or 73.1 mL/Ha and 20
Fl. oz/Ac (high rate) or 1461.5 mL/Ha on both pepper plants and
compared to the control (water applied at same use rate). Effects
of the foliar applications on pepper yield were determined for two
separate harvests using a once over harvest approach and normalized
to the yield of the control plants. The average percentage change
in yield for each treatment over the yield for the control plants
is reported as pounds/acre (lbs/Ac) in Table 29.
[0537] Foliar treatment of peppers using either the Bt.4Q7 Flg22 or
Ec.Flg22 bioactive priming polypeptides resulted in overall average
increases in pepper yield (lbs/Ac) with both the low and high
application use rates and for both the RK and HHW pepper varieties.
The combined yield averages for the RK and HHW varieties were +53%
higher (low rate: 1 Fl. oz/Ac or 73.1 mL/Ha) and +25% higher (high
rate: 20 Fl. oz/Ac) for Bt.4Q7 Flg22 foliar treated peppers
compared to the control pepper plants. Alternatively, the combined
yield average increases for the RK and HHW varieties were +30%
higher (low rate: 1 Fl. oz/Ac) and +47% higher (high rate: 20 Fl.
oz/Ac or 1461.5 mL/Ha) for Ec. Flg22 foliar treated peppers
compared to the control pepper plants.
[0538] Differences existed in how the two pepper varieties
responded to the foliar treatments and in the resultant yield
advantages provided to both pepper varieties (Table 30).
Substantial yield increases were seen in the HHW variety as
compared to the RK variety of peppers and the control or
non-treated plants with yield increases of +77% (low: 1 Fl. oz/Ac
or 73.1 mL/Ha) and +42% (high: 20 Fl. oz/Ac or 1461.5 mL/Ha) over
the control or non-treated pepper plants for the Bt.4Q7Flg22.
Additionally, low use rates of the Bt.4Q7Flg22 (1 Fl. oz/Ac) and
high use rates of the Ec. Flg22 (20 Fl oz/Ac) polypeptides were the
most effective at increasing yield in the HHW variety (both yielded
a +72% increase over the control plants).
TABLE-US-00032 TABLE 30 Foliar treatment of Flg22 to increase yield
in different varieties of pepper Avg. Percent Avg. Percent Combined
Change Yield Change Yield Total Avg. Percent Total Weight Weight
(lbs/Ac) Change Yield (lbs/Ac) (Hungarian Hot Total Number Red
Knight Wax) (lbs/Ac) Foliar Treatment 2 Replicate Trials 2
Replicate Trials RK and HHW Bt.4Q7Flg22: +29% +77% +53% 1 Fl. oz/Ac
Ec.Flg22: +30% +29% +30% 1 Fl. oz/Ac Bt.4Q7Flg22: +8% +42% +25% 20
Fl. oz/Ac Ec.Flg22: +22% +72% +47% 20 Fl. oz/Ac
Example 14: Application of Flagellin Bioactive Priming Polypeptides
to Squash-Increased Yield
[0539] Foliar treatment of Bt.4Q7Flg22 were applied exogenously on
Ambassador squash at the first bloom stage using two separate
formulations (formulation 1=F1 and formulation 2=F2). Formulation 1
(F1) consists of the native L Bt.4Q7 Flg22 bioactive priming
polypeptide applied with 0.01% (v/v) non-ionic surfactant.
Formulation 2 (F2) consists of the D RI Bt.4Q7 Flg22 applied with
0.01% (wv) non-ionic surfactant. Both formulations F1 and F2 were
applied to squash foliage using application use rate of 1 Fl. oz/Ac
(73.1 mL/Ha). Yield comparisons were made between the plants
treated with the foliar Bt.4Q7Flg22 F1 and F2 spray applications
compared to the control (water) or non-treated squash plants.
Squash plants were cultivated in sandy loam soil as follows. 2.5 cm
holes were cut in 2.5 ft. (0.76 m) wide plastic covered mounds, two
rows per mound, holes spaced 1.5 ft (0.46 m) apart within each row.
Rows were staggered within the mound. Mounds were spaced 4 ft (1.2
m) apart. Three squash seeds were planted per hole and thinned to a
single plant per hole 14 days after planting. Drip irrigation
tubing was laid in the center of each mound, and plants were
watered as necessary.
[0540] Squash plants were grown from seed in raised beds until
bloom, and foliar treated in the same Florida (FL) location using
two replicated trials or two separate harvests. Yield for the
foliar Bt.4Q7Flg22 applied F1 and F2 treated plants is reported as
the number of squash per plant, the weight (grams) per squash and
the total squash yield (lbs/Ac) and represented as a percentage
change as compared to non-treated control plants (Table 31).
[0541] Foliar treatments of Bt.4Q7Flg22 using the two formulations
F1 and F2 resulted in an increased yield advantage when foliar
applied on squash (Ambassador) at the pre-bloom stage compared to
the non-treated control plants. The number of squash per plant,
weight per squash and overall average percent change in yield
(lbs/Ac) all were increased in the Bt.4Q7Flg22 F1 and F2 treated
plants compared to the control or non-treated plants. The squash
plants treated with both the Bt.4Q7Flg22 F1 and F2 formulations had
similar trend increases in the number of squash per plant, weight
per squash and overall average percent change in yield (lbs/Ac),
however squash plants that received the F1 foliar application
showed increases in the number of squash per plant and in the total
yield of squash over the plants that received the F2
formulation.
TABLE-US-00033 TABLE 31 Foliar treatment with a composition of
Flg22 polypeptides to increase yield in squash Percent Change in
Percent Number of Squash Change in Percent Change per Plant
Weight/Squash in Yield (lbs/Ac) Compared to Compared to Compared to
Treatment Control Control Control Bt.4Q7Flg22 +7% +2% +9% 1 Fl.
oz/Ac Formulation 1 Bt.4Q7Flg22 +4% +2% +6% 1 Fl. oz/Ac Formulation
2
Example 15. Screening Flg Polypeptides for Reactive Oxygen Species
(ROS) Production in Corn and Soybean
[0542] Codon usage was performed to generate mutations in the
Bt.4Q7Flg22 to better match the host organism and the binding of
the Flg22 polypeptide to the FLS receptor at the plant cell
surface. A probabilistic approach was used to generate three
variants of the native Bt.4Q7Flg22 that were designed to have
preferred amino acid signatures for corn and soybean and to perform
equal to or better than the native Bt.4Q7Flg22 (SEQ ID NO: 226) in
ROS activity assays. These variants possessed mutations to the
internal segment (SEQ ID NO: 571), or the C-terminus (SEQ ID NO:
572) or the N terminus (SEQ ID NO: 573) and were designated as
Bt.4Q7Flg22-Syn01, Bt.4Q7Flg22-Syn02 and Bt.4Q7Flg22-Syn03,
respectively. Bt.4Q7Flg22-Syn01 and Bt.4Q7Flg22-Syn03 were then
measured in relation to their native forms at a variety of
concentrations.
[0543] Fresh plant tissues from corn (hybrid 5828 YX) and soybean
(hybrid 297 R4) leaves were cut into uniform samples and floated on
150 .mu.L of sterile water in a 96-well white, low luminescence
plate. The plate was placed under growth lights that had a 16-hour
light/8-hour dark cycles at a consistent temperature of 22.degree.
C.
[0544] For corn samples, aerial tissue from V1 to V4 stage corn
plants was cut away from the plant above the soil line using a
clean razor blade. The cotyledon and sheath were removed. 1-mm
slices were cut through the stalk from the base of the plant until
approximately 1.3 cm below the first leaf node. Each corn section
was placed in an individual well of the 96-well plate.
[0545] For soybean samples, fully expanded trifoliate leaves were
removed from V1-V3 stage plants. Leaf discs (12.6 mm.sup.2) were
cut from the leaf blades using a 4-mm diameter clean, sharpened
cork borer. Discs were cut in half using a clean razor blade, and
each disc half was placed in an individual well of the 96-well
plate.
[0546] Native Flg22 polypeptide (SEQ ID NO: 226) or Flg22
polypeptides containing the described mutations (SEQ ID NOs 571 or
573) stocks were prepared in either sterile, deionized water or 100
mM sodium phosphate (pH 7.8-8.0) buffer with 0.1% Tween-20. After
18-24 hours, the water was removed from each well of the 96-well
plate. Plant tissue samples were treated with a 100 .mu.L
elicitation solution containing 1:100 dilution of Flg22 polypeptide
stock (concentration range from 250 picomolar (pM) to 10 micromolar
(.mu.M)), 34 .mu.g/mL luminol, and 20 .mu.g/mL horseradish
peroxidase. Recognition of the Flg22 polypeptide by the plant
tissue resulted in activation of immune signaling and the
production of apoplastic reactive oxygen species (ROS). In the
presence of ROS (H.sub.2O.sub.2), horseradish peroxidase catalyzed
the oxidation of luminol and production of visible light. Relative
light units (RLUs) were recorded with a GLOMAX 96 microplate
luminometer (Promega Corporation) using a 0.5 s integration; 2.6
min intervals over a time course of 40 minutes.
[0547] For data analysis, total RLUs produced were calculated for
each sample over the entire 40 min time course. Significant
outliers beyond the interquartile range were excluded from
analysis. Total RLUs in each condition (n=6-16) were normalized to
the average RLU for Bt.4Q7Flg22 at 25 nM and reported as a
percentage (%) of the Bt.4Q7Flg22 control (Table 32).
[0548] The synthetic mutagenized Bt.4Q7Flg22-Syn01 version had
increased ROS activities at a range of concentrations (0.25-100 nM)
while Bt.4Q7Flg22-Syn03 was more varied and showed increased ROS
activities at 0.25 nM, 1 nM, 10 nM, 25 nM, and 100 nM
concentrations as compared to the native version of Flg22 or
Bt.4Q7Flg22. The synthetic version of Bt.4Q7Flg22-Syn01 treatment
using 5 nM resulted in the largest change in ROS activity over the
native version or Bt.4Q7Flg22. ROS activities for Bt.4Q7Flg22-Syn03
showed a more varied response over the range of concentrations
added.
TABLE-US-00034 TABLE 32 Flg generated synthetic mutants (Syn-01 and
Syn-03) have more activity in the ROS assay than the native
Bt.4Q7Flg22 over a wide range of concentrations. Bt.4Q7Flg22
Concentration (SEQ ID Bt.4Q7Flg22-Syn01 Bt.4Q7Flg22-Syn03 (nM) NO:
226) (SEQ ID NO: 571) (SEQ ID NO: 573) 0.25 8.12 23.56 14.86 0.5
14.86 55.00 14.86 1 23.85 57.04 41.47 5 57.00 113.00 57.00 10 76.85
116.34 85.70 25 100.00 118.05 111.40 100 113.74 120.83 162.29 1000
127.76 121.20 97.67
Example 16: ROS Screening Assays to Identify Functionally Active
Flg Polypeptides for Corn and Soybean
[0549] Based on the results from preliminary studies in Example 15,
the following concentrations were chosen to screen ROS activities
of a wide range of Flg22 polypeptides in corn and soybean: 5 nM in
corn (hybrid 5828 YX) and 100 nM in soybean (hybrid 297 R4). ROS
activity assays were then used to identify the best Flg22 bioactive
priming polypeptide candidates for individual treatment use of corn
and soybean and to identify those candidates that were active for
both corn and soybean.
[0550] Corn and soybean leaf tissues were harvested from plants and
ROS assays were performed as previously described in Example 15 for
the mutant polypeptides listed in Table 33. Total RLUs produced
were calculated for each sample over the entire 40 min time course.
Significant outliers beyond the interquartile range were excluded
from analysis. Comparisons of ROS activity on corn (hybrid 5828 YX)
and soybean (hybrid 297 R4) were made and reported as the
percentage (%) of relative light units (RLU) compared to the
average RLU values at the 25 nM Bt.4Q7Flg22 treatment concentration
(Table 33).
[0551] Table 33 summarizes the relative activity for a variety of
mutant Flg22 polypeptides compared to native Bt.4Q7Flg22 alongside
the standard deviation in for each condition (STDEV).
TABLE-US-00035 TABLE 33 ROS activity comparisons for various Flg22
polypeptides in corn and soybean Corn (5828 YX) Soybean (297 R4 5
nM polypeptide 100 nM polypeptide Amino Acid Avg. Avg. SEQ ID NO:
Sequence Activity (%) STDEV Activity (%) STDEV Bt.4Q7Flg22
DRLSSGKRINSA 100 -- 100 -- Bacillus SDDAAGLAIA thuringiensis SEQ ID
NO: 226 Bt.Flg22-Syn01 DRLSSGKRINSA 142.9 39.3 112 3.0 Mutant S13K
KDDAAGLAIA Bacillus thuringiensis SEQ ID NO: 571 Bt.Flg22-Syn02
DRLSSGKRINSA 78.3 26 68.7 14.0 Mutant A20Q SDDAAGLQIA Bacillus
thuringiensis SEQ ID NO: 572 Bt.Flg22-Syn03 QRLSSGKRINSA 122.1 29.5
113.5 42.6 Mutant D1Q SDDAAGLAIA Bacillus thuringiensis SEQ ID NO:
573 Bm.Flg22-B1 NRLSSGKQINSA 106.0 25.2 74.6 4.9 Bacillus
SDDAAGLAIA manliponensis SEQ ID NO: 290 Ba.Flg22-B2 NRLSSGKRINSA
134.7 56.8 83.0 26.6 Bacillus anthracis ADDAAGLAIA SEQ ID NO: 295
Bc.Flg22-B3 DRLSSGKRINNA 80.3 18.4 90.0 35.5 Bacillus cereus
SDDAAGLAIA SEQ ID NO: 294 A spp.Flg22-B4 ERLSSGYRINRA 78.1 20.1
133.1 23.9 Aneurini-bacillus SDDAAGLAIS spp. XH2 SEQ ID NO: 300
Ba.Flg22-B5 EKLSSGQRINSA 27.1 2.3 42.2 7.4 Bacillus SDDAAGLAIS
aryabhattai SEQ ID NO: 289 P spp.Flg22-B6 GKLSSGLRINGA 135.3 31.6
112.5 22.8 Paenibacillus spp. SDDAAGLAIS strain HW567 SEQ ID NO:
293 L spp.Flg22-L1 LRLSSGYRINSA 26.6 3.6 64.1 14.1 Lysinibacillus
spp. ADDAAGLAIS SEQ ID NO: 291 L spp.Flg22-L2 EKLSSGLRINRA 104.5
1.2 128.6 29.5 Lysinibacillus spp. GDDAAGLAIS SEQ ID NO: 580 L
spp.Flg22-L3 EKLSSGYKINRA 36.4 7.9 96.9 20.6 Lysinibacillus spp.
SDDAAGLAIS SEQ ID NO: 581 L spp.Flg22-L4 LRISSGYRINSAA 60.1 5.9
117.9 25.7 Lysinibacillus spp. DDPAGLAIS SG9 SEQ ID NO: 582
Lf.Flg22-L5 LRISTGYRINSAA 59.3 5.8 111.6 27.5 Lysinibacillus
DDPAGLAIS fusiformis SEQ ID NO: 583 Lm.Flg22-L6 EKLSSGFRINRA 58.7
19.4 112.3 42.3 Lysinibacillus GDDAAGLAIS macroides SEQ ID NO: 584
Lm.Flg22-L6 EKLSSGYKINRA 33.7 1.4 77.0 19.2 Lysinibacillus
GDDAAGLAIS xylanilyticus SEQ ID NO: 585 Pa.Flg22 QRLSTGSRINSA 116.0
32.5 88.6 22.2 Pseudomonas KDDAAGLQIA aeruginosa SEQ ID NO: 530
Ec.Flg22 ERLSSGLRINSA 95.0 46.7 116.8 13.3 Escherichia coli
KDDAAGQAIA SEQ ID NO: 586 Xcc.Flg22 QRLSSGLRINSA 143.3 5.2 96.4
17.6 Xanthomonas KDDAAGLAIS campestris pv campestris strain 305 or
(Xanthomonas citri pv. citri) SEQ ID NO: 532 Ea.Flg22 QRLSSGLRINSA
125.2 9.2 91.9 10.1 Erwinia amylovora KDDAAGQAIS SEQ ID NO: 534
Bp.Flg22 TRLSSGKRINSA 111.2 14.0 67.2 3.0 Burkholderia ADDAAGLAIS
phytofirmans strain PsJN SEQ ID NO: 536 Bu.Flg22 NRLSSGKRINTA 92.9
12.7 91.1 12.9 Burkholderia ADDAAGLAIS ubonensis SEQ ID NO: 538
Ps.Flg22 TRLSSGLKINSA 154.4 20.7 113.1 19.6 Pseudomonas KDDAAGLQIA
syringae pv. actinidiae ICMP 19096 SEQ ID NO: 540
[0552] Based on the results from T able 33, a number of predictions
could be made based on the effect of different mutations on Flg22
polypeptides on ROS activity in corn and soybean. Table 34
describes ROS activity observed or predicted for a variety of
targeted mutations. Briefly, replacements at the first amino acid
(D1N, D1Q or D1T) have or likely will result in strong recognition
and/or activation of the Flg22 receptor in corn. Mutations in the
inner segment, K7Y, K7F and A16P, will likely have similar positive
results in soybean. Of the tested polypeptides, Bt.4Q7Flg22-Syn01
(S13K) and Bt.4Q7Flg22-Syn03 (D1Q) had the strongest ROS-inducing
activity in corn and soybean.
TABLE-US-00036 TABLE 34 Result summary of mutant versions of native
Bt.4Q7Flg22 SEQ ID NO Amino Acid Sequence Description of ROS
activity results SEQ ID NO: 226 DRLSSGKRINSASDDA Bt.4Q7Flg22 AGLAIA
Bacillus thuringiensis (native version and used as the standard
comparison) SEQ ID NO: 571 DRLSSGKRINSAKDDA S13K mutation: Strong
ROS activation in AGLAIA both corn and soybean SEQ ID NO: 572
DRLSSGKRINSASDDA A20Q mutation: Negative ROS activation AGLQIA in
both corn and soybean SEQ ID NO: 573 QRLSSGKRINSASDDA D1Q mutation:
Strong ROS activation in AGLAIA both corn and soybean SEQ ID NO:
574 NRLSSGKRINSASDDA D1N mutation: Strong ROS activation in AGLAIA
both corn and soybean*(predicted) SEQ ID NO: 575 TRLSSGKRINSASDDA
D1T mutation: Strong ROS activation in AGLAIA both corn and
soybean*(predicted) SEQ ID NO: 576 DRLSSGYRINSASDDA K7Y mutation:
Strong ROS activation in AGLAIA only soybean*(predicted) SEQ ID NO:
577 DRLSSGFRINSASDDA K7F mutation: Strong ROS activation in AGLAIA
only soybean*(predicted) SEQ ID NO: 578 DRLSSGKRINSASDDP A16P
mutation: Strong ROS activation in AGLAIA only soybean *
(predicted) SEQ ID NO: 579 DRLSSGKRINSASDDA K7Q mutation: Strong
reduction in ROS AGLAIA activation in both corn and
soybean*(predicted)
Example 17: ROS Activity Assays to Identify Combinations of Flg
Polypeptides for Corn and Soybean
[0553] Corn and soybean leaf tissues were harvested from plants and
ROS assays were performed as previously described in Example 15.
The relative ROS activity of different Flg22 variants, alone or in
combination, were assessed to identify the preferred combinations
of Flg22 polypeptides that when applied together provided the
highest ROS activity response for bath corn and soybean. Results
are summarized in Table 35.
TABLE-US-00037 TABLE 35 Flg22 combinations with increased ROS
activities in corn and soybean Corn (5828 YX) Soybean (297 R4) 5 nM
polypeptide 100 nM polypeptide Flagellin Amino Acid Avg. Avg.
Composition Sequence Activity (%) STDEV Activity (%) STDEV
Bt.4Q7Flg22 DRLSSGKRINS 100 -- 100 -- Bacillus thuringiensis
ASDDAAGLAIA SEQ ID NO: 226 Bt.Flg22-Syn01 DRLSSGKRINS 122.48 31.69
83.54 36.21 Bacillus thuringiensis AKDDAAGLAIA SEQ ID NO: 571
Ba.Flg22-B2 NRLSSGKRINS 142.53 7.45 97.59 68.59 Bacillus antrhacis
AADDAAGLAIA SEQ ID NO: 295 A spp.Flg22-B4 ERLSSGYRINR 53.64 1.45
106.37 16.48 Aneurinbacillus spp. ASDDAAGLAIS XH2 SEQ ID NO: 300 P
spp.Flg22-B6 GKLSSGLRING 103.61 37.59 132.95 54.72 Paenibacillus
spp. ASDDAAGLAIS strain HW567 SEQ ID NO: 293 L spp.Flg22-L2
EKLSSGLRINR 113.04 28.89 138.86 53.66 Lysinibacillus spp.
AGDDAAGLAIS SEQ ID NO: 574 FLG22-Syn01 polypeptide 148.52 6.30
132.35 53.99 +B2+B4 combinations as +B6+L2 described above FLG22B2
polypeptide 128.31 0.65 139.74 55.00 +B4 combinations as +B6+L2
described above FLG22-Syn01 polypeptide 122.81 29.81 124.51 67.31
+B2 combinations as +B6+L2 described above FLG22-Syn01 polypeptide
119.17 8.02 100.97 25.95 +B4 combinations as +B6+L2 described above
FLG22-Syn01 polypeptide 124.67 8.69 103.45 34.03 +B6+L2
combinations as described above FLG22-Syn01 polypeptide 143.02 7.08
120.67 24.76 +B2+B4 combinations as described above
Example 18: ROS Activity Assay with Cellobiose Additive--Corn and
Soybean
[0554] Cellobiose is a glucose disaccharide and a building block
for cellulose polymer. Chemically, it is glucose-beta-1-4-glucose,
a reducing sugar that consists of two .beta.-glucose molecules
linked by a .beta. (1-4) bond. Cellobiose is obtained by the
breakdown of cellulose or lichenin and yields glucose upon
hydrolysis. Treatments using Bt.4Q7Flg22 were compared with and
without cellobiose in ROS activity assays to determine if
cellobiose can act an elicitor to increase ROS production in
reactions containing Flg22 polypeptide. The specific treatments
conducted using ROS assays with corn (FIG. 6, panel A) and soybean
(FIG. 6, panel B) leaf assays were: Bt.4Q7Flg22 at 25 nM;
Bt.4Q7Flg22 at 25 nM+cellobiose at 100 .mu.M; Bt.4Q7Flg22 at 25
nM+cellobiose at 1 mM; 100 mM sodium phosphate buffer control; and
cellobiose alone (100 .mu.M).
[0555] Corn and soybean leaf tissues were harvested from plants as
previously described in Example 15. Flg22 bioactive priming
polypeptide stocks were prepared in either sterile, deionized water
or 100 mM sodium phosphate (pH 7.8-8.0) buffer with 0.1% Tween-20.
After 18-24 hours, the water was removed from each well of the
96-well plate. Samples were treated with a 100 .mu.L solution
containing Bt.4Q7Flg22 (SEQ ID NO: 226, 25 nM), cellobiose (100
.mu.M or 1 mM), 34 .mu.g/mL luminol, and 20 .mu.g/mL horseradish
peroxidase. Recognition of the Flg22 polypeptide by the plant
tissue resulted in activation of immune signaling and the
production of apoplastic reactive oxygen species (ROS). In the
presence of ROS (H.sub.2O.sub.2), horseradish peroxidase catalyzed
the oxidation of luminol and production of visible light. Relative
Light Units (RLUs) were recorded with a GLOMAX 96 microplate
luminometer (Promega Corporation) using 0.5 s integration; 2.6 min
intervals over a time course of 40 minutes.
[0556] For data analysis, the average RLU per treatment (n=6-16
samples, +/-standard error of the means) was graphed over the time
course (FIG. 6). Significant outliers beyond the interquartile
range were excluded from analysis.
[0557] The average RLU across the experiment for each treatment is
graphed in FIG. 6, panel A (corn) and panel B (soybeans). While ROS
production was observed in both plant tissue in treatments only
containing the Flg22 polypeptide (white circles), the addition of
cellobiose at 1 mM resulted in significant ROS activity in both
plant tissues (black circles). Addition of cellobiose at lower
concentrations (100 uM) did not alter ROS activity compared to
Bt.4Q7Flg22 alone (comparison of white and grey circles in FIG. 6,
panel A) and did not lead to any ROS production in soybean (grey
circles in FIG. 6, panel B). Notably the combination of Bt.4Q7Flg22
at 25 nM and cellobiose at 1 mM resulted in more ROS production in
soybean (ROS peak at approximately 25,000 RLU) as compared to corn
leaves (ROS peak at approximately 75,000 RLU).
Example 20: Application of Phytosulfokine (PSK.alpha.) to Increase
Yield--Corn and Soybean
[0558] The effect of Phytosulfokine alpha (PSK.alpha.), a
sulfonated bioactive priming polypeptide derived from Arabidopsis
thaliana, on corn and soybean yield was tested. Corn and soybeans
were cultivated in the field as described in Example 1 and 3.
Arabidopsis thaliana PSK.alpha. (SEQ ID NO: 598) was applied at a
final concentration of 1 .mu.M in foliar spray with a surfactant
and provided using a uniform application to the above ground plant
parts of corn (hybrid 5140RR) and soybean (hybrid 375 NR).
At.PSK.alpha. formulations were applied at the V5-V8 stage of
development in corn and the V1-V4 stage of development in soybean.
Corn and soybean plants treated with At.PSK.alpha. were compared to
non-treated control plants (water). Treated plants were randomized
at one location in four replicate blocks for comparisons to the
controls. Yield was reported in Bushels per acre (Bu/Ac).
[0559] Table 36 depicts how foliar application of At.PSK.alpha.
resulted in yield increases in both corn and soybean yield trials.
Both corn and soybean had positive yield increases in the field
with foliar formulations containing At.PSK.alpha. applied at the
V5-V8 stage of development in corn and the V1-V4 stage of
development in soybean. On average, corn had a +3 Bu/Ac (188.3
kg/Ha) increase in overall yield in the field and soybean had a
+0.8 Bu/Ac (53.8 kg/Ha) yield increase.
TABLE-US-00038 TABLE 36 Foliar application of At.PSK.alpha. to corn
and soybean result in yield increases (Bu/Ac) Bu/Ac Foliar Corn
Bu/Ac Foliar Soybean 3.0 0.8
Example 21: Foliar Application of Phytosulfokine Alpha (PSK.alpha.)
to Increase Yield--Soybean
[0560] A method is provided wherein applying At.PSK.alpha. as a
foliar application to actively growing soybean plants provides a
yield advantage in environments with heat and drought stress.
[0561] Soybean plants were grown as described in Example 6. The
At.PSK.alpha. polypeptide (SEQ ID NO: 598) was applied as a foliar
spray to the plants at the V1-V4 stage. Soybean plants treated with
foliar applications of At.PSK.alpha. and control plants treated
with water and surfactant alone were then grown in conditions
described in Examples 7-9 that produced a non-stress and stress
(heat and water deficit) environments. Table 37 describes the
percentage change or increase in height reported for soybean plants
treated with At.PSK.alpha. as a foliar spray at the V1-V4 growth
stage. At.PSK.alpha. application resulted in a +3.5% increase in
height in the non-stress environment and a +4.3% increase in height
in stress environments reported in Bushels per acre (Bu/Ac) as
compared to the non-treated control soybean plants.
TABLE-US-00039 TABLE 37 Yield increases in soybean treated with a
foliar application of At.PSK.alpha. and grown in non-stress and
stress environments Percentage (%) change in Height in Percentage
(%) change in Height in Non-Stress Environment Stress Environment
Soybean with At.PSK.alpha. over control Soybean with At.PSK.alpha.
over control 3.5% 4.3%
Example 22: Application of RHPP to Alter Plant
Architecture--Corn
[0562] Root hair promoting polypeptide (RHPP, SEQ ID NO: 600)
originally derived for soybean (Glycine max) is provided as a
foliar application to produce beneficial phenotypes in corn.
[0563] Native and retro inverso RHPP (SEQ ID NOs 600-601) will be
applied to corn plants at the V5-V8 stages. Retro inverso RHPP may
be modified with C-terminal amidation prior to application.
Treatment with RHPP in this way is expected to result in a distinct
leaf architecture phenotype with an upright leaf orientation and
more erect leaves. The increase in leaf angle has impactful
advantages for use in agriculture in this area. This is
particularly relevant with higher planting densities used to
maximize yield in a field environment. Foliar applications of the
RHPP polypeptide in maize (corn) is useful for changing the leaf
angle thus contributing to a smaller leaf angle which results in an
upright leaf orientation. This phenotype can be beneficial for
increasing the leaf area index, reducing maize shade syndrome, and
improving photosynthetic efficiency. In addition, providing RHPP as
a foliar formulation to maximize canopy development and total light
penetrance is key to increasing vegetative growth of the plants
prior to the initiation of the grain filling stage.
Example 23: Application of RHPP to Increase Root Biomass and Yield
Parameter-Soybean
[0564] Effective nodulation of soybean roots result in higher
yields and higher quality seed production, protein and oil per seed
or acre basis. This could be due to increased nitrogen fixation
since nodulale formation increases nitrogen fixation. To determine
whether root hair promoting bioactive priming polypeptide, RHPP
(SEQ ID NO: 600) could modulate root biomass and nodulation and
thereby improve nitrogen fixation, soybean plants (hybrid Morsoy
38X52 and Beck's hybrid 297R4) were treated with foliar application
of RHPP (300 nM) at the R1-R2 stage of development.
Increased Plant Biomass and Nodulation
[0565] RHPP bioactive priming polypeptide (SEQ ID NO: 600,
originally derived from Glycine max) was applied as foliar
treatment to 4-week-old hybrid soybean (Morsoy variety) with 0.1%
(v/v) non-ionic surfactant (ALLIGARE SURFACE.TM.) using a spray
bottle and delivering approximately 1.25 ml/plant. The experiment
was conducted using a total of 8 plants per trial per treatment
group. The pots were kept in an artificial lighted growth room
receiving a light level of approximately 300 .mu.mol m.sup.-2
s.sup.-1 for a 16/8 light/day cycle and a 21.degree. C.
day/15.degree. C. night temperature range. Growth parameters of
nodule counts, root biomass and total biomass per plant were
measured at 15 days post the foliar application and compared
between the foliar treatments consisting of ALLIGARE SURFACE
surfactant (0.1% v/v) as a control and the RHPP polypeptide (300
nM) containing the ALLIGARE SURFACE surfactant (0.1% v/v). Average
growth parameters as described were normalized to the control
plants that received the surfactant alone treatment (Table 38).
[0566] Nodulation counts on the roots of each plant treated with a
foliar application of RHPP were compared to the number of nodules
on the control plants treated with 0.1% (v/v) surfactant alone.
RHPP treatment resulted in approximately two times the number of
nodules on the roots of each soybean plant compared to control
(surfactant) treatment. Soybean plants receiving the foliar
application of the RHPP polypeptide also exhibited an increase in
root biomass and total overall plant biomass which when normalized
to the control resulted in an increase of more than 20% in root
biomass and 8% in total biomass.
TABLE-US-00040 TABLE 38 Increases in plant biomass and nodulation
in soybean (Morsoy variety) after foliar application with RHPP
bioactive priming polypeptide (n = 8 replicate plants) RHPP Control
(300 nM + RHPP treatment (surfactant 0.1% 0.1% v/v normalized as a
Growth v/v ALLIGARE ALLIGARE percentage of the Parameters SURFACE)
SURFACE) surfactant control Average nodule 8.88 15.13 170.42% count
per plant Root biomass (g) 1.76 2.13 120.57% Total biomass (g)
42.64 46.24 108.44%
Increased Plant Growth
[0567] RHPP bioactive priming polypeptide (SEQ ID NO: 600) was also
applied as foliar treatment to R1 stage hybrid soybean (Beck's
297R4) with 0.1% (v/v) non-ionic surfactant (ALLIGARE SURFACE)
using a spray bottle delivering approximately 1.2 ml/plant. This
experiment was performed to look at the effects of RHPP on plant
growth and was conducted using a total of 18 plants per treatment
group. The pots were kept in an artificial lighted growth room
receiving a light level of approximately 300 .mu.mol m.sup.-2
s.sup.-1 for a 1816 light/day cycle and a 21.degree. C.
day/15.degree. C. night temperature range. R1 stage soybean plants
were treated with nothing (non-treated control), ALLIGARE SURFACE
surfactant applied at a concentration of 0.1% (v/v) or the RHPP
polypeptide (300 nM) applied in combination with ALLIGARE SURFACE
surfactant (0.1% v/v). Height for each plant was recorded at the
time of spray and again at 16 days post foliar application and
average growth parameters were compared between foliar treatments
(Table 39).
[0568] Soybean plants that received the foliar application of RHPP
polypeptide (300 nM+0.1% ALLIGARE SURFACE) had increased plant
growth (plant height) and an increased change in plant height as
compared to the plants that received the surfactant alone and
non-treated control (Table 39).
TABLE-US-00041 TABLE 39 Increases in plant growth in soybean
(Beck's 297R4) with foliar application with RHPP bioactive priming
polypeptide (n = 18 replicate plants) Control (surfactant RHPP Non-
0.1% (300 nM + 0.1% treated ALLIGARE ALLIGARE Growth Parameters
Control SURFACE) SURFACE) Height (cm) 19.3 19.4 19.9 Change in
height (cm) 4.0 3.7 4.6
Example 24: Application of RHPP in Combination with a
Fertilizer--Soybean
[0569] The Gm.RHPP bioactive priming polypeptide (SEQ ID NO: 600)
was applied as a foliar application with a liquid foliar
fertilizer, N-RAGE MAX (21-1-3 N-P-K), to two soybean varieties
(AG3536 and AG3832). Foliar application of RHPP was applied at 1
Fl. oz/Ac or 73.1 mL/Ha (300 nM concentration) with the recommended
use rate of the fertilizer for soybeans (1 to 2 gal/Ac (9.4 to 18.8
L/Ha), or equal to Nitrogen 2.16 lbs/gal (0.29 kg/L); Phosphate
P.sub.2O.sub.5 0.10 lbs/gal and soluble potash (K.sub.2O 0.31
lbs/gal or 0.4 kg/L). Foliar application of the combination RHPP,
fertilizer treatment was provided to two soybean varieties (AG3536
and AG3832) at the R2 stage (recommended stages R1 to R6) of
development in 5 locations across the US Midwest (IA, IL, IN).
Foliar application of Gm.RHPP with the N-RAGE MAX provided a yield
advantage of 1.9 Bu/Ac (127.8 kg/Ha) compared to the control
treatment and on average a 1.4 Bu/Ac (94.2 kg/Ha) increase compared
to those plants that received the fertilizer alone treatment for
variety 1 (AG3536) (Table 40).
TABLE-US-00042 TABLE 40 Application of RHPP plus a fertilizer
Average Bu/Ac Average Change Average Average Total Yield compared
Application Total Yield Total Yield Bu/Ac to Control Treatment Use
Rate Bu/Ac Bu/Ac Variety 1 Variety 1 Soybean Fl. oz/Ac Variety 1
Variety 2 and 2 and 2 Control -- 62.50 62.16 62.33 -- N-rage 128
63.04 60.29 61.66 -0.67 Max RHPP 4.0 65.42 61.04 63.23 +0.9 RHPP +
4.0 64.40 60.96 62.68 +0.35 N-RAGE 128 MAX
Example 25: Application of RHPP Bioactive Priming Polypeptides to
Tomatoes-Increased Yield
[0570] Foliar application treatments of Gm.RHPP (SEQ ID NO: 600)
was applied as an exogenous spray at the pre-bloom stage and used
to increase yield in tomatoes. Two tomato hybrids (JetSetter and
Better Big Boy) were planted in small scale plots as described in
Example 12. Foliar treatment of Gm.RHPP was applied at an
application use rate of 1 Fl. oz/Ac (73.1 mL/Ha) and 20 Fl. oz/Ac
(1461.5 mL/Ha) to the two hybrids, JetSetter (Trial 1) and Better
Big Boy (Trial 2), at early bloom (first flower) stage. Replicated
trials were conducted at the US Midwest (Missouri) in July. The
foliar treatment of Gm.RHPP on tomato plants was compared to the
control (water applied at same use rate). Effects of the foliar
treatments on increasing yield in tomatoes were determined and
reported as normalized to the water control treatment and reported
as the average percentage change in yield over the average control
yield in Table 41.
[0571] The average yield represented as a percent change over the
control plants was reported separately for the two trials and as
the average for the two tomato hybrids. Foliar application using
Gm.RHPP resulted in an increase in tomato fruits for each of the
two trials when applied at a use rate of 1 Fl. oz/Ac (73.1 mL/Ha).
Application of Gm.RHPP resulted in an average increase in tomato
yield of +52% over the control plants for the two hybrids with
individual average increases of +93% for the Jetsetter hybrid and
+10% for the Better Big Boy compared to the control plants.
TABLE-US-00043 TABLE 41 Foliar treatment of RHPP to increase yield
in different hybrids of tomato Trial 1: Percent Trial 2: Change in
Percent Change Average Trials Yield over in Yield over 1 & 2
Avg. Control; Avg. Control; Percent Change Hybrid: Hybrid: Better
Yield over Avg. Foliar Treatment Jetsetter Big Boy Control Gm.RHPP
+93% +10% +52% (1 Fl. oz/Ac)
Example 26: Application of RHPP to Peppers--Increased Yield
[0572] Foliar treatment of Gm.RHPP (SEQ ID NO: 600) was applied as
an exogenous spray at the first-bloom stage to increase yield in
two pepper varieties. Foliar treatment of Gm.RHPP was applied using
small scale plots designed to simulate commercial growing
conditions for peppers (Capsicum) as described in Example 13.
Foliar applications with the Gm.RHPP bioactive priming polypeptide
were applied at the first flower stage, on two varieties of pepper,
Red Knight (RK) and Hungarian Hot Wax (HHW). The foliar Gm.RHPP
treatments were applied using an application use rate of 1 Fl.
oz/Ac (73.1 mL/Ha) on the RK and HHW pepper plants and compared to
the control (water applied at same use rate). Effects of the foliar
applications on pepper yield were determined for two separate
harvests using a once over harvest approach and normalized to the
yield of the control plants. The average percentage change in yield
over the yield for the control plants is reported in Table 42, as
the percent change per total weight (lbs/Ac) of peppers harvested.
Average percent change in yield is reported for the 2 replicate
harvests (trials) for the RK and HHW pepper varieties and then as a
combined average for both varieties.
TABLE-US-00044 TABLE 42 Foliar treatment of RHPP to increase yield
in different varieties of pepper Combined Avg. Percent Avg. Percent
Change Yield Avg. Percent Change Yield Total Weight Change Yield
Total Total Number (lbs/Ac) Weight (lbs/Ac) (lbs/Ac) Foliar
Treatment Red Knight Hungarian Hot Wax RK and HHW Gm.RHPP +87% +46%
+67% 1 Fl. oz/Ac
[0573] Percent average yield for RK and HHW peppers that received
the Gm.RHPP applied at the use rate of 1 Fl. oz/Ac (73.1 mL/Ha) was
increased by 87% for RK and 46% for HHW peppers compared to the
control plants. The combined average for both pepper varieties was
reported as an average 67% increase for the percent change in yield
in the foliar Gm.RHPP treated peppers over the non-treated (water)
control pepper plants (Table 42).
Example 27: Application of Harpin-Like and ALPSK.alpha.
Polypeptides to Corn
[0574] Harpins can provide functional benefits when applied both
exogenously, for example as a foliar spray to the plant surface, or
provided apoplastically (the space outside of the plant cell
membrane) or endogenously (inside a plant cell/plant cell
membrane). Synthetic harpin bioactive priming polypeptide,
HpaG-like (Xanthomonas spp., SEQ ID NO: 587) was applied
exogenously to the surface of corn plants at the V2-V3 stage of
development. Additionally, the effect of exogenous application of
Phytosulfokine alpha (PSK.alpha.), a sulfonated bioactive priming
polypeptide derived from Arabidopsis thaliana, on corn growth was
tested.
[0575] Corn (Beck's hybrid 5828 YH) plants were grown in an
environmentally controlled growth room. Corn seed was planted
directly into 39.7 cm.sup.3 pots containing Timberline top soil at
a depth of 2.54 cm, with 2 seeds per pot. After planting, 50 mL of
room temperature water was added to each pot to allow for
germination. The pots were kept in an artificial lighted growth
room receiving approximately 300 .mu.mol m.sup.-2 s.sup.-1 (light
photons) for a 16/8 light/day cycle and a 21.degree. C.
day/15.degree. C. night temperature range. Plants received the same
watering and fertilizer regimes.
[0576] Plant height (cm) was measured at 3 weeks after emergence.
Bioactive priming polypeptides for HpaG-like (SEQ ID NO: 587),
provided as a synthetic 23 amino acid polypeptide, and
At.PSK.alpha. (SEQ ID NO: 598) were then applied to the corn plants
as a foliar spray at final concentrations of 1 .mu.M for HpaG-like
and 100 mM for PSK.alpha. bioactive priming polypeptides. Control
plants were treated with surfactant (0.01% v/v) alone. A week after
the spray treatments were applied, the plants were subdivided into
2 groupings where one group remained in the same standard growth
environment described above and the other group was transferred to
an environment that provided heat and water deficit stress. For the
heat and water deficit treatments, the growth room environment
(with the exception of temperature and watering/fertilizer cycles)
remained similar to the standard growth environment). Heat stress
was applied using heat mats to raise the temperature in the
environment from 21.degree. C. to 27.degree. C. During the period
of heat stress, the plants were left unwatered to simulate a water
deficit stress. Change in plant height (cm) was measured at 5 weeks
and reported as normalized to or as a percentage of the height of
the control (water) plants. Measurements are reported as the
combined average of two trials with 9 replicate plants per trial
(Table 43) and are presented as a percentage of growth over control
corn plants that received water plus surfactant (0.01% v/v)
standardized to measure 100% (Table 43).
TABLE-US-00045 TABLE 43 Changes in Plant height of corn plants
treated with X. spp. HpaG-like and At. PSK.alpha. Plant Height
Plant Normal- Height ized as Normal- a per- ized as centage a per-
Height Height of centage Height (cm) (cm) control of (cm) and after
non- after height control Treatment (STDEV) stress stress Non-
height Corn 3 weeks 5 weeks 5weeks stress Stress X, spp. 47.23
(6.11) 64.10 (5.53) 45.50 (4.37) 107.4% 89.4% HpaG-like (1 .mu.M)
At.PSK.alpha. 49.36 (8.00) 58.62 (4.84) 54.88 (2.79) 98.2% 107.8%
(100 nM)
[0577] Foliar application using the HpaG-like polypeptide showed an
improved growth phenotype in normal environments, but not stressed
environments, when compared to the control plants, while foliar
application of PSK.alpha. exhibited an improved growth phenotype
when grown under conditions of heat and water deficit stress but
not in the non-stressed environment.
[0578] In a separate set of replicated trials, similar changes in
growth rates resulted from the foliar applications of HpaG-like
(SEQ ID NO: 587) and PSK.alpha. (SEQ ID NO: 598). Table 44 shows
the percentage change in plant growth for corn receiving X. spp.
HpaG-like polypeptide (1 .mu.M final concentration) and
At.PSK.alpha. (100 nM final concentration) applied as foliar
treatments and measured by changes in plant height compared to
control (water plus 0.01% v/v surfactant) plants grown in optimal
(non-stress) and in stress environments. This suggests that the
combined foliar application or sequential applications of
PSK.alpha. with HpaG-like bioactive priming polypeptides may be
useful for enhancing growth of plants growth under standard
(non-stress or optimal growth) environments or of plants exposed to
abiotic stress (for example, heat, and water deficit stress).
TABLE-US-00046 TABLE 44 Foliar application treatments using the
Xspp HpaG-like and the At.PSK.alpha. polypeptides on corn grown
under non-stress and stress conditions Plant Height (cm) Plant
Height (cm) Percentage Change Percentage Change Compared to Control
Compared to Control (0.01% surfactant) (0.01% surfactant) Treatment
Non-Stress Stress Xspp. HpaG-like (1 .mu.M) +5.0% -6.1% At.
PSK.alpha. (100 nM) -11.8% +6.1%
Example: 28 Combination of Bt.4Q7Flg22 or Ec.Flg22 with RHPP
[0579] The bioactive priming polypeptides, Bt.4Q7Flg22 and
Ec.Flg22, were combined with RHPP and accessed for yield benefits
in soybean. The combination of either Bt.4Q7Flg22 (SEQ ID NO: 226)
or Ec.Flg22 (SEQ ID NO: 526) and RHPP (SEQ ID NO: 600) were foliar
applied to two varieties of soybean (AG2836, Variety 1; AG3536,
Variety 2) in 7 locations across the US Midwest (IA, IL and
IA).
[0580] Foliar application using Bt.4Q7 Flg22 bioactive priming
polypeptide (SEQ ID NO: 226; FIG. 4, panel A) and Ec.Flg22 (SEQ ID
NO: 526; FIG. 4, panel B) and RHPP (SEQ ID NO: 600) were applied
individually to soybean plants (commercial hybrid Beck'S 294 NR) at
the R2 stage of development using varying use rates of 0.33, 4.0,
8.0, and 16.0 Fl. oz/Ac or (24.1 mL/Ha, 292.3 mL/Ha, 584.6 mL/Ha,
1169.2 mL/Ha). Average yield (harvested in September) in bushels
per acre (Bu/Ac) is reported for soybean grown in 7 separate
locations and reported individually for both soybean varieties and
as a combined average yield (Table 45). Soybean yield (Bu/Ac) is
also reported as the change in yield (Bu/Ac) normalized to the
control soybean plants for both varieties.
TABLE-US-00047 TABLE 45 Flg polypeptides and RHPP polypeptides
increase yield in soybean Average Average Average Average Total
Bu/Ac Total Total Yield Increase Yield Yield Bu/Ac compared
Application Bu/Ac Bu/Ac Variety to Control Treatment Use Rate
Variety Variety 1 Variety 1 Soybean Fl. oz/Ac 1 2 and 2 and 2
Control -- 59.53 61.61 60.57 -- Bt.4Q7Flg22 0.33 60.33 61.61 61.02
+0.45 Bt.4Q7Flg22 4.0 57.61 64.19 60.90 +0.33 Bt.4Q7Flg22 8.0 59.05
63.86 61.45 +0.88 Ec.Flg22 0.33 58.62 63.58 61.10 +0.53 Ec.Flg22
4.0 58.02 63.91 60.74 +0.17 Ec.Flg22 8.0 58.27 64.35 61.31 +0.74
Gm.RHPP 0.33 59.15 62.44 60.92 +0.35 Gm RHPP 4.0 58.61 66.35 61.83
+1.26 Gm RHPP 8.0 59.47 62.46 61.08 +0.51 Bt.4Q7Flg22 + 4.0 61.14
64.88 63.18 +2.61 Gm.RHPP 4.0 Ec.Flg22 + 4.0 59.56 62.46 61.08
+0.51 Gm.RHPP 16
[0581] Soybean variety AG3536 (Variety 2) consistently outperformed
AG3536 (Variety 1) for yield Bu/Ac in all 7 locations across the US
Midwest. Foliar applications with the Bt.4Q7Flg22, Ec.Flg22 and
RHPP applied individually at the 3 different use rates (0.33, 4.0
and 8.0 Fl. oz/Ac) or (24.1 mL/Ha, 292.3 mL/Ha, 584.6 mL/Ha) all
resulted in a yield advantage over the non-treated control plants.
The RHPP applied foliarly using a 4.0 Fl. oz/Ac (292.3 mL/Ha) use
rate resulted in the largest yield increase of +1.26 Bu/Ac (84.7
kg/Ha) over the control plants compared to the other bioactive
priming polypeptides applied separately. However, the combination
of Bt.4Q7Flg22 with RHPP provided an additional yield advantage
resulting in a +2.61 Bu/Ac (175.5 kg/Ha) over the non-treated
soybean control plants. This increase in yield seen from soybean
plants treated with foliar applications of Bt.4Q7Flg22 combined
with RHPP illustrates a synergistic effect achieved by combining
the bioactive priming polypeptides where the increase in yield of
the combination was greater than the sum of the two polypeptides
applied separately.
Example 29: Use of Agrobacterium tumefaciens to Test Effectiveness
of Thionins in Treating HLB Disease
[0582] Agrobacterium tumefaciens strain GV3101 was inoculated into
Luria broth medium (LB) and grown for 20 hours. Initially the
optical density (OD) of the culture was measured at a wavelength of
600 nm using a spectrophotometer and normalized to a low starting
density. The cultures were then divided equally and treated with
similar proportions of thionins that are representative of mixtures
used to treat citrus trees. The ratios of Cs.thionin (SEQ ID NO:
651), As.thionin (SEQ ID NO: 652) and Mt.thionin (SEQ ID NO: 653)
used were 10.0%, 2.0%, 0.40%, 0.08%, and 0.02% and were prepared to
match the 20 mL total volume of filtrate of each of the thionin
mixtures that is used as a treatment per tree. Each thionin mixture
was also compared to control mixtures containing only: filtrate,
minimal media (LB), or a tetracycline (Tet) antibiotic (10 .mu.g/mL
per culture). Each bar represents a combined OD measure of 3
replicates. After incubation with the thionin and antibiotic
mixtures, the optical density (OD 600) was measured again to
determine if growth of the Agrobacterum cultures was reduced or
inhibited.
[0583] As is shown in FIG. 8, the Cs.thionin, As.thionin and
Mt.thionin treatments all showed a dose dependent response and
decreased growth of the Agrobacterium cultures compared to the
filtrate, minimal media (LB) or antibiotic (Tet) controls.
Example 30: Treatment of Candidatus Liberibacter asiaticus
Infection with Thionins
[0584] Use of thionins to treat Candidatus Liberibacter asiaticus
infection will be tested in citrus trees from an orchard located in
central Florida (Okeechobee county). Treatment of a total of 26
trees will use formulation mixtures of thionin (SEQ ID NO: 620; 621
and 622) either with or without a phloem localization sequence (SEQ
ID NO: 611) to target the thionins specifically to the phloem where
Candidatus Liberibacter asiaticus reside. Inoculation of Valencia
orange (Citrus sinensis) trees with these formulations of thionins
and mixtures thereof will be conducted using a low-pressure
injection device, BRANDT ENTREE. Four total thionin treatments
including water as a negative control and oxytetracycline as a
positive control will be applied to 5 year-old trees. The citrus
trees will be randomized into treatment blocks for control
(non-treated), thionin treated and positive control antibiotic
(oxytetracycline) treated tree plots. Thionins fused to a phloem
targeting sequence will be expressed in a pBC vector, and thionin
containing filtrate will be collected from the expressed cells. A
total volume of 20 mL containing a mixture of thionins: Cs.thionin
(SEQ ID NO: 651), As.thionin (SEQ ID NO: 652) and Ms.thionin (SEQ
ID NO: 653) will be provided as 20 mL total volume of filtrate. The
thionin treated citrus trees will be compared to the non-treated
(control) trees and trees that received a separate positive control
of an antibiotic, oxytetracycline, applied with a concentration of
2 grams/tree. Levels of infection of trees with Candidatus
Liberibacter asiaticus will be confirmed by qPCR detection or
amplification using 16S rRNA gene specific primers and nested
primers to detect the HLB disease [Sequence 5'>>3':(forward)
HLB as TCGAGCGCGTATGCAATACG; (reverse) HLBr
GCGTTATCCCGTAGAAAAAGGTAG; HLBpc (probe) AGACGGFTGAGTAACGCG labeled
with fluorescein reporter dye].
[0585] Plants will be treated in March and leaf samples will be
collected one month later in April. Average bacterial counts for
Candidatus Liberibacter asiaticus will be assessed along with
visual symptomology ranking scores for leaf blotch mottling or
signs of yellowing of leaves and stems.
[0586] Fruit size, shape and level of fruit development or maturity
will be collected for 20 representative fruits per tree.
Longitudinal length (major diameter, cm) and width (minor diameter,
cm, the average of the largest and smallest widths if the fruit is
not symmetrical). Fruit shape will be measured by the ratio of
width to length. Total fruit weight will be obtained and divided by
the total number of fruits (20) to provide an average fruit weight
(grams). Total fruit weight will be collected and represented in
kg/tree.
[0587] Acid-corrected .sup.oBrix (.sup.oBrix.sub.c) values of juice
obtained from the juiced (squeezed) grapefruit and orange fruit
will be obtained per tree following the USDA minimum standards for
.sup.oBrix.sub.c laboratory analytical methods. Percent acid (%,
w/v) will also be measured. The .sup.oBrix reading on a
refractometer for a juice to be reconstituted equals the value of
the desired acid-corrected .sup.oBrix subtracted of the acid
contribution and temperature effect. The total titratable acidity
(% acid) of the reconstituted juice will also be calculated based
on the reconstituted .sup.oBrix and Brix/Acid ratio and adjusted
using an acid correction and temperature correction factors (JBT
FoodTech Laboratory Manual, "Procedures for Analysis of Citrus
Products, Sixth Edition).
[0588] Bacterial cell counts will be calculated using real time
fluorescent PCR, quantitative polymerase chain reaction (qPCR)
techniques to detect only live bacterial and subtract out
background DNA including naked DNA or DNA from dead cells (Davis
and Brlansky, "Quantification of live Candidatus Liberibacter
asiaticus" populations using real-time PCR and propidium
monoazide", Plant Disease 97: 1158-1167, 2013). Colony counts
specific for Candidatus Liberibacter asiaticus (CLas) cells will be
measured in the leaves collected from the thionin treated,
non-treated (control) and positive control (antibiotic) treated
trees. Calculations for live bacterial titers will be obtained from
the DNA yield obtained by qPCR, fit into a regression equation to
correlate target copy number to total bacterial counts and
represented on a log scale of live cells per gram tissue.
Comparisons of titers from treated and non-treated trees will be
matched with the degree of disease severity or disease symptoms,
such as the classic blotchy mottling on the leaves, deformed or
lopsided fruit and greening fruit, etc. for both the red grapefruit
and Valencia orange trees.
Example 31: Use of Retro-Inverso Flg Bioactive Priming Polypeptides
to Treat and Reduce Citrus Greening
[0589] Combinations of flagellin-associated polypeptides paired
with their retro-inverso counterparts can be used to treat and
reduce the greening effect on citrus that results in Asian citrus
greening or Huanglongbing disease (HLB).
[0590] An early symptom of HLB in citrus is the yellowing of leaves
on an individual limb or in one sector of a tree's canopy. Leaves
that turn yellow from HLB will show an asymmetrical pattern of
blotchy yellowing or mottling of the leaf, with patches of green on
one side of the leaf and yellow on the other side. As the HLB
disease progresses, the fruit size becomes smaller, and the juice
turns bitter. The fruit can remain partially green and tends to
drop prematurely.
[0591] The retro-inverso forms of Flg22 can compete with native
forms of Flg22 for binding to the FLS-associated receptor(s) at the
plant surface and thus inhibit/delay the symptom formation of
greening associated with HLB disease. Using native Flg22 and RI
combinations will assist with a fine tuned immune response to
reduce and even eliminate the disease-causing bacteria, Candidatus
Liberibacter asiaticus and thus prevent acute symptom development,
such as leaf yellowing and citrus fruit greening.
[0592] Treatment combinations of Flg polypeptides with their
retro-inverso (RI) forms will be used to minimize the effect of HLB
infection on citrus fruit greening. Thirty-four commercial
grapefruit, Citrus paradise Macfad., and six sweet orange, Citrus
sinensis (L.) trees, with or without symptoms of HLB disease, will
be treated using flagellin bioactive priming polypeptide
combinations described in Table 46, below, using a low pressure
injection device called BRANDT enTREE to distribute the Flg
polypeptides into the interior of the tree.
TABLE-US-00048 TABLE 46 Combinations of Flg22 native and
retro-inverso Flg22 bioactive priming polypeptides Treatments SEQ
ID NO: Concentration nM Bt.4Q7Flg22 226 50 nM Bt.4Q7Flg22 226 100
mM RI Bt.4Q7Flg22 376 50 nM RI Bt.4Q7Flg22 376 100 mM Bt.4Q7Flg22 +
RI 226 & 376 50 nM Bt.4Q7Flg22 Bt.4Q7Flg22 + RI 226 & 376
100 mM Bt.4Q7Flg22 Ec.Flg22 526 50 nM Ec.Flg22 526 100 mM RI
Ec.Flg22 527 50 nM RI Ec.Flg22 527 100 mM Ec.Flg22 + RI Ec.Flg22
526 & 527 50 nM Ec.Flg22 + RI Ec.Flg22 526 & 527 100 mM
[0593] Leaf tissue samples from these treated grapefruit and sweet
orange trees will be analyzed using the ROS assay as described in
Example 15. Sampling will be conducted in orchard groves from March
to August in central Florida. The sample of citrus orchards will be
assumed to be representative of the state. The orchard sampled will
have a minimum acreage of 2 hectares (range of 2-24 Ha and an
average of 5.2 Ha). Selected orchard citrus trees will be randomly
selected with the non-treated control trees nested in each
randomized plot. Leaf tissues from the grapefruit and orange trees
will be collected from trees of approximately the same age. Leaves
will be sampled at similar locations on the trees and only from
trees that had a new flush of growth at the time of sampling. In
the orchards selected for sampling, similar cultural practices will
be maintained and include flood irrigation and weed management with
herbicides. However, the selected orchards will not receive any
pesticide application for a minimum of 30 days before leaf sampling
for the ROS assays. Two replicate trials of 10 grapefruit trees
exhibiting symptomology of HLB disease will be randomly sampled per
orchard and compared to 14 grapefruit trees (non-infected control)
sampled that do not exhibit any symptoms. Similar leaf sampling
will be performed in sweet orange (four infected samples compared
to 2 uninfected controls). Trees will be selected to be
representative of the whole orchard. A nested analysis of variance
(ANOVA) will be performed to determine the statistical significance
of any differences in ROS activities observed from treatment of the
control and infected HLB citrus leaf samples.
Example 32: Foliar Application of the Flg22 Polypeptide Reduces
Cercospora Leaf Blight Disease of Soybean
[0594] Foliar application of the Bt.4Q7Flg22 bioactive priming
polypeptide (SEQ ID NO: 226) derived from Bacillus thuringiensis
and Bacillus pseudomycoides expressing Bt.4Q7Flg22 (H1) were
applied to soybean plants (commercial hybrid Beck's 294 NR) at the
V3 stage of development that were grown at 3 separate US Midwestern
locations that were known to previously have Cercospora infection
in the fields.
[0595] A Cercospora leaf blight rating scale (percentage of leaf
area affected) was used to rate disease severity in all field
experiments. The percentage of leaf area affected was calculated
using a visual key based on the ASSESS image analysis for plant
disease quantification (Chagas Ferreira da Silva, LSU Master's
Theses, 2014). Symptom ranking as a percentage was done for the
uppermost trifoliate leaves
[0596] The results are described in Table 47. Visually, soybean
plants that received the foliar treatments of the Bt.4Q7Flg22
bioactive priming polypeptide and Bacillus pseudomycoides
expressing Bt.4Q7Flg22 (H1) had increased vigor as compared to the
non-treated control plants. The control plants showed an increase
in early symptom development at the 4 week observation time point,
30% as compared to 20% with the Bt.4Q7Flg22 treatment (0.33 Fl.
oz/Ac or 24.1 mL/Ha) and approximately 5% with the Bt.4Q7Flg22
treatment (4.0 Fl. oz/Ac or 292.3 mL/Ha). Soybean plants receiving
the Bacillus pseudomycoides expressing the Bt.4Q7Flg22 (H1)
treatment also showed less early symptom development as a result of
Cercospora infection than the non-treated control plants, 20% at 4
weeks (0.33 Fl. oz/Ac or 24.1 mL/Ha) and 10% (4.0 Fl. oz/Ac or
292.3 mL/Ha). At 8 weeks post application, the non-treated control
plants showed 50% visual symptom damage on the upper foliage of the
plant (top 3-4 trifoliate leaves). The symptom ranking for plants
that received the foliar treatments of the Bt.4Q7Flg22 polypeptide
(0.33 Fl. oz/Ac or 24.1 mL/Ha) was comparable to the non-treated
control plants at 8 weeks post foliar treatment. However, the
soybean plants that received the foliar treatments of Bt.4Q7Flg22
polypeptide (4.0 Fl. oz/Ac) and Bacillus pseudomycoides expressing
Bt.4Q7Flg22 (H1) (0.33 Fl. oz/Ac or 24.1 mL/Ha) and 4.0 Fl. oz/Ac
or 292.3 mL/Ha) showed considerably less apparent symptoms and
damage. Overall the treatment of the Bt.4Q7Flg22 polypeptide (4.0
Fl. oz/Ac or 292.3 mL/Ha) was effective at the prevention of early
symptom development from Cercospora infection as compared to the
non-treated plants that showed blight and purple coloration
symptoms as well as defoliation. Therefore, foliar application of
Bt.4Q7Flg22 polypeptide applied at a higher application use rate
(eg. 4.0 Fl. oz/Ac or 292.3 mL/Ha)) can provide a means of managing
early symptom development and provide healthier more vigorous
soybean plants grown in field locations that have been impacted by
Cercospora.
TABLE-US-00049 TABLE 47 Foliar treatment of soybean plant with
Bt.4Q7Flg22 and Bp. expressing Bt.4Q7Flg22 resulted in disease
reduction and symptom development of Cercospora on soybean Percent
of Disease Application Use Area covering Disease Area, Treatment-
Rate plant, 4 weeks 8 weeks Post Soybean Fl. oz/Ac Post Application
Application Control -- 30% 50% Bt.4Q7Flg22 0.33 Fl. oz/Ac 20% 50%
Bt.4Q7Flg22 4.0 Fl. oz/Ac 5% 35% H1 Bt.4Q7Flg22 0.33 Fl. oz/Ac 20%
40% H1 Bt.4Q7Flg22 4.0 Fl. oz/Ac 10% 30%
Example 33: Application to Corn--Enhanced Normalized Difference
Vegetation Index (ENDV) Analysis
[0597] Enhanced Normalized Difference Vegetation Index (ENDVI) is
an indicator of live, photosynthetically-active green vegetation
and was used to compare the effectiveness of treatments in field
trials using remote sensing technology. In the ENDVI index, values
ranging from -1.0 to 0.1, are indicative of unhealthy plants with
decreased photosynthesis, whereas values approaching 1 are
indicative of lush greenness, high photosynthetic capacity, and
increased biomass. Healthy plants strongly absorb visible light
from the 400-700 nm spectral wavelength range and reflect the
wavelengths in the near-infrared light from 700-1100 nm. ENDVI
measurements can correspond to certain vegetative properties, such
as plant biomass or greenness, absorption of light by plant
canopies, photosynthetic capacity (e.g., leaf area index, biomass,
and chlorophyll concentration). ENDVI images were collected using a
BGNIR camera (Zenmuse X3) attached to a drone (DJI MATRICE 100)
specifically created to capture images and filter different
wavelengths of light during the capture. The camera uses sensors to
capture visible and near-infrared bands of the electromagnetic
spectrum. Healthy plants with large amounts of vegetation or
biomass reflect green (G) and near-infrared (NIR) light, while
absorbing both blue (B) and red light. Plants that are less healthy
or that have less above-ground biomass reflect more visible and
less NIR light. ENDVI uses both NIR and G as the reflective
channels while using B as the absorption channel. The ENDVI formula
below adds the NIR and green channels together for the reflective
channel. The blue channel is multiplied by two to compensate for
the NIR and G channels being added together. The ENDVI equation
uses the following calculation for the NIR, G, and B channels to
provide a ratio value as a single output.
E .times. .times. N .times. .times. D .times. .times. V .times.
.times. I .times. = ( N .times. .times. I .times. .times. R + Green
) - ( 2 * Blue ) ( N .times. .times. I .times. .times. R + Green )
+ ( 2 * Blue ) ##EQU00001##
[0598] Corn seed (DEKALB hybrid DKC 58-89) treated with a seed
treatment comprising EVERGOL fungicide (7.18% propiconazole, 3.59%
penflufen and combined with 5.74% metalaxyl) and PONCHO/VOTiVO 500
(a mixture of 40.3% clothianidin insecticide and 51.6% Bacillus
firmus 1-1582, a microbial agent) was planted in the US Midwest
(IL). Various foliar treatments containing Bt.4Q7Flg22 and a
synthetic version of Bt.4Q7Flg22 (Syn01Flg22 as described in Table
48) were applied to corn plants at the V5-V7 stage of development.
BGNIR images were collected by drone flight, 50 m above the trial
plot, three weeks after each foliar treatment and after the corn
canopy had fully closed. Individual BGNIR images were processed
using drone display image analysis software to create a single
orthomosaic image of the trial plot that was further analyzed with
Fiji imaging software. Within the orthomosaic image, plot regions
to identify individual foliar treatments in a field and the
replicates per each treatment were clearly established using GPS
coordinates in each field trial. The treatment replicates
identified for imaging were consistent in size. For each foliar
treatment, three replicates were collected with two rows imaged per
each replicated plot. Within each replicate, the average intensity
of light was measured for each of the image channels [blue, green,
and near infrared, (visualized as red)] on a scale of 0-255, with
Intensity 0=0% reflection (black pixel) and Intensity 255=100%
reflection (white pixel). These average B, G and NIR light
intensities were used to calculate an ENDVI value using the ENDVI
algorithm for plant health (greenness) for each replicated plot.
The ENDVI values were then averaged for the three plot replicates
as reported in Tables 49 and 50. ENDVI values for the treatment
applications were compared to the control treatments in each plot.
Control treatments consisted of corn plants grown from seed that
was treated with a base seed treatment only and received no foliar
treatments. Foliar treatment compositions were as described using
the application use rates as specified in Table 48.
TABLE-US-00050 TABLE 48 Compositions of foliar Flg22 treatments for
testing on corn and soybean Application Use Rate Fluid ounce/ acre
(Fl. oz/Ac) Milliliters/ Composition Foliar Formulation hectare
(mL/Ha) Composition 1 Bt.4Q7Flg22 (SEQ ID NO: 226) 4 Fl. oz/Ac or
16.7 .mu.M PROXEL BC 292.3 mL/Ha preservative: 330.7 .mu.M (BIT);
53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) Composition 2 Bt.4Q7Flg22 (SEQ
ID NO: 226) 4 Fl. oz/Ac or 16.7 .mu.M 11.6 mM 292.3 mL/Ha Sodium
Phosphate Dibasic combined with 4.2 mM Citric Acid Monohydrate pH
5.6 PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) Composition 3 Bt.4Q7Flg22 (SEQ ID NO: 226) 4 Fl. oz/Ac
or 16.7 .mu.M 1.67 mM Sodium 292.3 mL/Ha Phosphate Buffer, pH 5.7
PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M
(MIT) Composition 4 Bt.4Q7Flg22 (SEQ ID NO: 226) 4 Fl. oz/Ac
(Flg22) 16.7 .mu.M + Cellobiose: 320 mM 292.3 mL/Ha 1.67 mM Sodium
Phosphate 8 Fl. oz/Ac Buffer, pH 5.7 PROXEL BC (Cellobiose)
preservative: 330.7 .mu.M; 584.6 mL/Ha 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) Composition 5 Bt.4Q7Flg22 (SEQ ID NO: 226) 48 Fl. oz/Ac
or 16.7 .mu.M 1.67 mM 3,507.6 mL/Ha Sodium Phosphate Buffer, pH 5.7
PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M
(MIT) Composition 6 Syn01Flg22 (SEQ ID NO: 571) 4 Fl. oz/Ac or 16.7
.mu.M 1.67 mM Sodium 292.3 mL/Ha Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
Composition 7 Syn01Flg22 (SEQ ID NO: 571) 0.4 Fl. oz/Ac or 16.7
.mu.M 1.67 mM Sodium 29.23 mL/Ha Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
Composition 8 Syn01Flg22 (SEQ ID NO: 571) 0.4 Fl. oz/Ac (Flg22)
16.7 .mu.M + Cellobiose: 320 mM 29.23 mL/Ha 1.67 mM Sodium
Phosphate 8 Fl. oz/Ac Buffer, pH 5.7 PROXEL BC (Cellobiose)
preservative: 330.7 .mu.M; 50.1 584.6 mL/Ha .mu.M (CMIT); 21.71
.mu.M (MIT) Composition 9 At.Flg22-B4 (SEQ ID NO: 300) 4 Fl. oz/Ac
or 1.67 mM Sodium Phosphate 292.3 mL/Ha Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
[0599] Foliar compositions contained 0.1% (v/v) PROXEL BC
preservative, an aqueous dispersion of a blend of 330.7 mM
1,2-benzisothiazolin (BIT), 53.5 mM
5-chloro-2-methyl-4-isolthiazolin-3-one (CMIT), and 26.1 mM
2-methyl-4-isothiazolin-3-one (MIT). Foliar compositions were
applied at the indicated rates (Fl. oz/Ac or mL/Ha) in a carrier
volume of 20 gallons/acre water with 0.1% (v/v) Alligare
Surface.TM. non-ionic surfactant
[0600] As shown in Table 49, foliar applications with compositions
containing Bt.4Q7Flg22 and Syn01Flg 22 applied to corn at the V5-V7
stage of development resulted in increased ENDVI measurement ratio
values as compared or normalized to plants that received no foliar
treatment (seed treatment control). The Bt.4Q7Flg22 compositions
provided as a foliar treatment in buffered formulations
(compositions 2, 3, 4 and 5; sodium phosphate pH 5.6-5.7) resulted
in plants with higher ENDVI ratio values compared to the plants
that received the Bt.4Q7Flg22 provided as a non-buffered
composition (composition 1) applied at 4 Fl. oz/Ac or 292.3 mL/Ha.
However, ENDVI ration values of Bt.4Q7Flg22 treated plants
(compositions 1-5) were all increased relative to the non-treated
control plants. Corn plants that received the foliar treatment
application of composition 4 consisting of the Bt4Q7Flg22
polypeptide combined with cellobiose in a phosphate buffered
formulation provided at 8 Fl. oz/Ac or 584.6 mL/Ha use rate
resulted in a +9% increase in the average ENDVI ratio value over
the control (seed treatment only) plants. Plants that received
foliar applications of composition 1 and 5 which differed in
composition only in the respective application use rates (4 and 48
Fl. oz/Ac or 292.3 mL/Ha and 3,507.6 mL/Ha) resulted in plants with
similar ENDVI ratio values (+3% and +4%) as compared to the control
plants. Importantly, the higher rate of 48 Fl. oz/Ac or 3,507.6
mL/Ha resulted in no detectable phytotoxicity, which would have
been observed as a reduced ENDVI value compared to the control
(seed treatment only). A synthetic derived variant version of
Bt.4Q7Flg22 (Syn01Flg22) compositions 6, 7, and 8 were also
provided as a foliar spray to V5-V7 corn plants. The Syn01Flg22
polypeptide provided in a phosphate buffered formulation
(composition 6 and composition 7) were compared according to the
application use rates. The Syn01Flg22 polypeptide (composition 6)
that was provided to corn plants using a higher application use
rate (4 Fl. oz/Ac or 292.3 mL/Ha) resulted in a decreased ENDVI
ratio value or had a lesser percentage increase in ENDVI as
compared to the Syn01Flg22 polypeptide (composition 7) applied to
plants using a 0.4 Fl. oz/Ac or 29.23 mL/Ha application use rate, a
change of 8% between the composition 6 and 7 treatments. The
Syn01Flg22 (composition 8) had the addition of cellobiose and
similar to composition 7 was provided at an application use rate of
0.4 Fl. oz/Ac or 29.23 mL/Ha. Foliar application of Syn01Flg22
(composition 7) was compared to Syn01Flg22 combined with cellobiose
(320 mM) (composition 8). The Syn01Flg22 composition 7 and
composition 8 had similar increases in ENDVI measurement ratios
resulting in a +10% increase as compared to control plants or a +8%
increase compared to plants that received the Syn01Flg22
(composition 6) provided at the higher 4 Fl. oz/Ac or 292.3 mL per
hectare (Ha) use rate.
TABLE-US-00051 TABLE 49 ENDVI outputs provided for foliar Flg22
treatments on corn hybrid DKC 52-61 Percentage AVG Change in ENDVI
Normalized to Treatment ENDVI (STDEV) Control Plants* Seed
Treatment Control 0.253 (0.027) -- Bt.4Q7Flg22 Composition 1 0.259
(0.019) +3% Bt.4Q7Flg22 Composition 2 0.269 (0.021) +6% Bt.4Q7Flg22
Composition 3 0.272 (0.003) +7% Bt.4Q7Flg22 + Cellobiose 0.276
(0.009) +9% Composition 4 Bt.4Q7Flg22 Composition 5 0.263 (0.010)
+4% Syn01Flg22 Composition 6 0.258 (0.016) +2% Syn01Flg22
Composition 7 0.277 (0.018) +10% Syn01Flg22 + Cellobiose 0.279
(0.015) +10% Composition 8 *Normalized to seed treatment control:
EVERGOL and PONCHO/VOTiVO 500
[0601] Corn seed (DEKALB hybrid DKC 52-61) was also treated with
Roundup POWERMAX (active ingredient glyphosate, 48.7% in the form
of potassium salt) in combination with the Bt.4Q7Flg22 composition
3. Roundup POWERMAX was applied using the recommended use rate on
the specimen label of 24 Fl oz/Ac. The Bt.4Q7Flg22 (composition 3)
was applied at a rate of 4.0 Fl. oz/Ac or 292.3 mL/Ha. Results are
shown in Table 50.
TABLE-US-00052 TABLE 50 ENDVI with foliar applications of Flg22
polypeptides combined with an herbicide on corn (hybrid DKC 52-61)
Percentage AVG Change Treatment in ENDVI Normalized Formulation
Code ENDVI to Roundup Application Use Rate (STDEV)
POWERMAXTreatment Roundup POWERMAX 0.271 (0.006) -- Roundup
POWERMAX + 0.293 (0.005) +8% Bt.4Q7Flg22 (Composition 3)
*Normalized to RoundUp POWERMAX foliar treatment
[0602] As shown in Table 50, Roundup POWERMAX applied to corn at
the V5-V7 stage of development as a foliar herbicide combined with
the Bt.4Q7Flg22 (composition 3) resulted in an increased ENDVI
measurement ratio, an increase of +8% compared to the treatment
with the Roundup POWERMAX applied without the Bt.4Q7Flg22
polypeptide.
Example 34: Application of Bioactive Priming Polypeptides to V4-V7
Corn--Increased Yield
[0603] Large acre corn trials were planted from corn seed (DEKALB
hybrids: DKC 52-61, DKC 58-89, and DKC 65-81) coated with a seed
treatment comprising EVERGOL fungicide (7.18% propiconazole, 3.59%
penflufen, and 5.74% metalaxyl) with PONCHO/VOTiVO 500 (a mixture
of clothianidin insecticide and a microbial agent, Bacillus firmus
1-1582). Corn field trials were planted in 8 locations throughout
the US Midwest (IN, IL, & IA). Field seed beds at each location
were prepared using conventional or conservation tillage methods
for corn plantings. Fertilizer was applied as recommended by
conventional farming practices which remained consistent between
the US Midwest locations. Herbicides were applied for weed control
and supplemented with cultivation when necessary. Four-row plots,
5.3 meters were planted at all locations. Corn seed was planted 3.8
to 5.1 cm deep to ensure normal root development. Corn was planted
at approximately on average of 42,000 plants per acre or 103,782
plants per hectare with an average row width of 0.8 meters with
seed spacing of 1.6 to 1.8 seeds per every 30 cm.
[0604] Corn plants at approximately the V5 stage of development
received foliar applications using a foliar composition comprising
a Bt.4Q7Flg22 (SEQ ID NO: 226) polypeptide and a synthetic version
of Bt.4Q7Flg22 which is described as Syn01Fflg22 (SEQ ID NO: 571)
polypeptide. The foliar compositions comprising the Bt.4Q7Flg22
polypeptide and a synthetic version of Bt.4Q7Flg22 polypeptide were
applied to 3 corn hybrids (DEKALB hybrids: hybrid 1: DKC 52-61;
hybrid 2: DKC 58-89; hybrid 3: DKC 65-81) planted in 8 locations
throughout the US Midwest (IN, IL, & IA). Corn plants received
foliar treatments using the concentrations and application use
rates as described in Table 51. Corn yield (Bu/Ac) was collected
and reported as the average yield (Bu/Ac) across the locations (8
locations for hybrid 1, 7 locations for hybrid 2 and 6 locations
for hybrid 3) and as the average change in Bu/Ac compared to the
base seed treatment (ST) control treated with surfactant alone in
Table 51.
TABLE-US-00053 TABLE 51 Foliar treatment using Bt.4Q7Flg22 and a
Syn01Flg22 synthetic mutant-increase yield in corn Average Change
Application in Yield Bu/Ac Use Rate compared to Foliar Treatment
Fl. oz/Ac Average Yield Surfactant (Concentration) (mL/hectare)
Bu/Ac Control Bt.4Q7Flg22 4.0 Fl. oz/Ac 201.43 +1.14 (16.7 .mu.M)
(292.3 mL/hectare) (Composition 1) Bt.4Q7Flg22 4.0 Fl. oz/Ac 205.43
+2.55 (16.7 .mu.M) + (292.3 mL/hectare) Cellobiose 8.0 Fl. oz/Ac
(320 mM) (584.6 mL/hectare) (Composition 4) Syn01Flg22 4.0 Fl.
oz/Ac 203.79 +0.90 (16.7 .mu.M) (292.3 mL/hectare) (Composition 6)
Syn01Flg22 0.4 Fl. oz/Ac 204.36 +1.48 (16.7 .mu.M) (29.2
mL/hectare) (Composition 7) (16.7 .mu.M) + 0.4 Fl. oz/Ac 204.47
+1.59 Cellobiose (29.2 mL/hectare) (320 mM) 8.0 Fl. oz/Ac
(Composition 8) (584.6 mL/hectare)
[0605] Corn plants at approximately the V5 stage of development
received foliar applications using a foliar composition comprising
a Bt.4Q7Flg22 and a synthetic version Syn01Flg22 of Bt.4Q7Flg22
polypeptides. The Bt.4Q7Flg22 and a synthetic version of
Bt.4Q7Flg22 polypeptides were also combined with cellobiose (320
mM), a reducing sugar, consists of two .beta.-glucose molecules
linked by a .beta.-(1.fwdarw.4) bond and provided as an elicitor
treatment to enhance the effect of the Flg22 polypeptide. Both the
Bt.4Q7Flg22 and the Syn01Flg22 provided in combination with
cellobiose to corn plants resulted in an enhanced yield boast over
the Bt.4Q7Flg22 and the Syn01Flg22 foliar applied polypeptides. A
positive increase in yield of +2.55 Bu/Ac or 160 kg/Ha resulted in
the corn plants that received the Bt.4Q7Flg22 foliar treatment with
cellobiose as compared to the +1.14 BuAc or 71.6 kg/Ha increase in
yield for the Bt. 4Q7Flg22 foliar treatment provided alone. There
was also a positive increase in yield of +1.59 Bu/Ac or 99.8 kg/Ha
resulted in the corn plants that received the Syn01Flg22 (0.2 Fl.
oz/Ac or 14.6 mL/Ha) foliar treatment provided in combination with
cellobiose as compared to the +1.48 BuAc or 92.9 kg/Ha increase in
yield for the Syn01Flg22 foliar treatment provided at the same
application use rate. Whereas, the Bt.4Q7Flg22 and the Syn01Flg22
provided as foliar treatments to corn plants at the V5 stage of
development using a 4.0 Fl. oz use rate or 292.3 mL/Ha provided a
slightly lower increase in yield +1.14 Bu/Ac (71.6 kg/Ha) and +0.90
Bu/Ac (56.5 kg/Ha) as compared to the combinations of the two Flg22
polypeptides with cellobiose.
Example 35: Combination of a Synthetic-Derived Flg22 (Syn01Flg22)
and a Fungicide
[0606] In a further study, large acre yield trials were conducted
using a foliar application comprising a compositions of the
Bt.4Q7Flg22 polypeptide and a synthetic derived polypeptide from
Bt.4Q7Flg22 (Syn01Flg22) provided with a broad-spectrum fungicide,
STRATEGO YLD (10.8% prothioconazole and 32.3% thiofloxystrobin).
STRATEGO YLD is a commercially available fungicide suitable for use
as an early season foliar application for corn was applied as a
foliar spray following the recommendations on the specimen label at
a use rate of 4.0 fluid ounces per acre (Fl. oz/Ac) (292.3
mL/hectare). Corn plants at approximately the V5 stage of
development received foliar applications using a foliar composition
comprising the Bt.4Q7Flg22 polypeptide and Syn0Flg22, the synthetic
version of Syn01Flg22 polypeptide combined with the STRATEGO YLD
fungicide. Foliar treatments were applied to 2 corn hybrids (DEKALB
hybrids: hybrid 1: DKC 52-61; hybrid 2: DKC 58-89) planted in 2
locations Iowa. Corn yield (Bu/Ac) was collected and reported as
the average yield (Bu/Ac) across the 2 locations for both hybrids
and as the average change in Bu/Ac compared to the corn plants
grown from seed that received the base seed treatment (ST) and only
the foliar application with the STRATEGOYLD fungicide (Table
52).
TABLE-US-00054 TABLE 52 Corn yield foliar applications of a
synthetic mutant of Bt.4Q7Flg22 combined with a fungicide
Application Use Average Change Rate Average in Yield Bu/Ac Foliar
Treatment Fl. oz/Ac Yield Compared to (Concentration) (mL/hectare)
Bu/Ac Fungicide Control STRATEGO YLD 4.0 Fl. oz/Ac 223.13 --
Fungicide (292.3 mL/ hectare) STRATEGO YLD 4.0 Fl. oz/Ac 228.62
+5.49 Fungicide + (292.3 mL/ Bt.4Q7Flg22 hectare) (SEQ ID NO: 226)
4.0 Fl. oz/Ac (16.7 .mu.M) (292.3 mL/ (Composition 3) hectare)
STRATEGO YLD 4.0 Fl. oz/Ac 228.96 +5.83 Fungicide + (292.3 mL/
Syn01Flg22 hectare) (SEQ ID NO: 571) 4.0 Fl. oz/Ac (16.7 .mu.M)
(292.3 mL/ (Composition 6) hectare) The base seed treatment (ST)
consisted of EVERGOL fungicide + PONCHO/VOTIVO 500. The STRATEGO
YLD fungicide was applied at the concentration and application use
rate as recommended on the specimen label.
[0607] Foliar application to V5 corn plants with the Bt.4Q7Flg22
and the Syn01Flg22 polypeptides that were provided in combination
with a fungicide, STRATEGO YLD at the concentrations and
application use rates as specified in Table 5 above resulted in a
more than a +5 Bu/Ac. The Syn01Flg22 polypeptide foliar treatment
resulted in slightly higher corn yields of +5.84 Bu/Ac (366.6
kg/Ha) than the corn plants that received the Bt.4Q7Flg22
polypeptide treatment which resulted in average yields of +5.50
Bu/Ac (345.2 kg/Ha) as compared to the plants that received the
foliar treatment with only the STRATEGO YLD fungicide.
Example 36: Seed Treatment with Flg22 Polypeptides to Increase
Yield in Corn
[0608] In other studies, large acre yield trials were conducted
using a base seed treatment consisting of .RTM.EVERGOL fungicide
(7.18% propiconazole, 3.59% penflufen and combined with 5.74%
metalaxyl) and PONCHO/VOTiVO 500 (a mixture of 40.3% clothianidin
insecticide and 51.6% Bacillus firmus 1-1582, a microbial agent)
provided in combination with various Flg22 polypeptides. Seed
treatments were applied to 3 corn hybrids (BECK's 4919V2, 5140HR
and 5828YX) planted in 8 locations throughout the US Midwest (IN,
IL, & IA). Seed treatment compositions of the Flg22
polypeptides were applied as described in Table 53 as Fl. oz per
unit of corn or soy seeds in a total slurry volume containing the
base seed treatment Bt.4Q7Flg22 from Bacillus thuringiensis
(Composition 10) and Pa.Flg22 from Paenibacillus alvei (Composition
11). Final concentration of polypeptide in the slurry for
Compositions 10 and 11 was 1 uM.
TABLE-US-00055 TABLE 53 Compositions of Flg22 seed treatments for
testing on corn and soybean Application Use Rate Fluid ounce/unit
corn or soy (Fl. oz/unit) Milliliters/unit Composition Seed
Treatment Formulation (mL/unit) Composition 10 Bt.4Q7Flg22 (SEQ ID
NO: 226) 0.14 Fl. oz/unit or 40.0 .mu.M 11.6 mM Sodium 4.14 mL/unit
Phosphate Dibasic combined with 4.2 mM Citric Acid Monohydrate pH
5.6 Composition 11 Pa.Flg22 (SEQ ID NO: 293) 0.14 Fl. oz/unit or
40.0 .mu.M 11.6 mM Sodium 4.14 mL/unit Phosphate Dibasic combined
with 4.2 mM Citric Acid Monohydrate pH 5.6
[0609] Corn yield (Bu/Ac) was collected and reported as the yield
(Bu/Ac) across the 8 locations averaged for all 3 hybrids. The
average change in Bu/Ac was as compared to the corn plants grown
from seed that received the only the base seed treatment (ST) and
is reported in Table 54.
TABLE-US-00056 TABLE 54 Corn seed treatment with Flg22 Polypeptide
increases yield Average Change in Yield (Bu/Ac) Application Average
compared Use Yield to ST Foliar Treatment Rate (Bu/Ac) control
Bt.4Q7Flg22 0.14 Fl. oz/unit 179.72 +4.73 Bacillus thuringiensis
4.14 mL/unit (SEQ ID NO: 226) (Composition 10) Pa.Flg22 0.14 Fl.
oz/unit 182.24 +3.57 Paenibacillus alvei or 4.14 mL/unit (SEQ ID
NO: 293) (Composition 11)
[0610] Treatment of corn seed with Bt.4Q7Flg22 (SEQ ID NO: 226) and
Pa.Flg22 (SEQ ID NO: 293) polypeptides increased yield as
represented as an average over the 3 corn hybrids and the 8 US
Midwest locations. The Bt.4Q7Flg22 polypeptide provided as a seed
treatment resulted in an even greater yield advantage or a +4.73
Bu/Ac (296.9 kg/Ha) compared to the control plants. The Pa.Flg22
applied as a seed treatment also resulted in a yield gain with a
+3.57 Bu/Ac (224 kg/Ha) over corn plants grown from seed that
received only the base seed treatment. Thus, Flg22 polypeptides
obtained from different species of bacteria (Bacillus and
Paenibacillus) both resulted in substantial yield increases when
applied as a seed treatment on corn seed.
Example 37: Application of Flg22 Polypeptides with Cellobiose to
Increase Yield in Corn
[0611] Large acre corn trials were planted from corn seed (DEKALB
hybrids: DKC 52-61, DKC 58-89, and DKC 65-81) containing a seed
treatment comprising EVERGOL fungicide (7.18% propiconazole, 3.59%
penflufen and combined with 5.74% metalaxyl) combined with
PONCHO/VOTiVO 500 (a mixture of clothianidin insecticide and a
microbial agent, Bacillus firmus 1582). Corn plants at
approximately the V5 stage of development received foliar
applications using an agricultural composition comprising a
Bt.4Q7Flg22 and the synthetic Syn01Flg22 polypeptides were provided
with and without cellobiose (320 mM). The foliar treatments were
applied to 2 corn hybrids (DEKALB hybrids: hybrid 1: DKC 58-89;
hybrid 2: DKC 65-81) planted in 2 locations in the US Midwest (IL)
that experienced drought-like conditions after foliar application,
during the pollination stage of corn development. Corn plants
received the Bt.4Q7Flg22 and Syn01Flg22 foliar treatments using the
concentrations and application use rates as described in Table 48
with a non-ionic surfactant (Alligare Surface.TM. applied at a
final concentration of 0.1% v/v of spray tank volume). Corn yield
(Bu/Ac) was collected and reported as the average yield (Bu/Ac)
across the 2 locations for the 2 hybrids and as the average change
in Bu/Ac compared to yield from corn plants that received only base
seed treatment (ST) and a non-ionic surfactant (Alligare
Surface.TM. applied at a final concentration of 0.1% v/v of spray
tank volume) (Table 55).
TABLE-US-00057 TABLE 55 Combinations of Flg22 polypeptides with
cellobiose-corn Average Change Application in Yield Use Rate
(Bu/Ac) Foliar Fl. oz/Ac Average compared to Treatment (mL/hectare
Yield Surfactant (Concentration) (Ha) (Bu/Ac) control Bt. 4Q7Flg22
4.0 96.46 +3.70 (SEQ ID NO: 226) (292.3 mL/Ha) (16.7 .mu.M ) Bt.
4Q7Flg22 4.0 100.98 +8.22 (SEQ ID NO: 226) (292.3 mL/Ha) (16.7
.mu.M) + 8.0 Cellobiose (584.6 mL/Ha) (320 mM) Bt.4Q7Flg22 48.0
119.37 +26.61 (SEQ ID NO: 226) (3507.6 mL/Ha) (16.7 .mu.M )
Syn01Flg22 4.0 98.48 +5.72 (SEQ ID NO: 571) (292.3 mL/Ha) (16.7
.mu.M ) Syn01Flg22 0.4 102.36 +9.60 (SEQ ID NO: 571) (29.2 mL/Ha)
(16.7 .mu.M ) Syn01Flg22 0.4 108.24 +15.48 (SEQ ID NO: 571) (29.2
mL/Ha) (16.7 .mu.M) + 8.0 Cellobiose (584.6 mL/Ha) (320 mM)
[0612] Foliar treatment applications of Bt.4Q7Flg22 (SEQ ID NO:
226) and a synthetic version of Syn01Flg22 (SEQ ID NO: 571)
resulted in substantial yield gains in corn plants when combined in
a foliar treatment application with cellobiose, a disaccharide that
is used as a secondary stabilization agent for the Flg polypeptide
and vehicle for delivery to the plant membrane surface. The
Bt.4Q7Flg22 polypeptide (16.7 .mu.M) provided with cellobiose (320
mM) as a combination foliar spray applied using 4.0 Fl. oz/Ac
application use rate (Flg22) resulted in a more than doubled yield
gain, a +8.22 Bu/Ac increase or approximately 516 kg/ha over the
control plants in comparison to 4.0 Fl. oz/Ac Bt.4Q7Flg22
polypeptide alone. The Bt.4Q7Flg22 polypeptide applied without
cellobiose resulted in a +3.70 Bu/Ac or 232 kg/Ha increase over the
control plants grown from the surfactant control. Similar increased
yield resulted in corn plants treated with the Syn01Flg22 and the
combination of Syn01Flg22 (16.7 .mu.M) provided in combination with
cellobiose (320 nM) using a 0.2 Fl. oz/Ac application use rate, a
respective increase of +9.60 (602.6 kg/Ha) and +15.48 (971.6 kg/Ha)
compared to the yield obtained from the surfactant control plants.
Additionally, the Bt.4Q7Flg22 (16.7 .mu.M) polypeptide was provided
as a foliar spray application using three different application use
rates of 0.2, 2.0 and 24.0 Fl. oz/Ac (14.6 mL/Ha, 146.2 mL/Ha and
1753.8 mL/Ha) to corn plants at the V5-V7 stage of development. The
Bt.4Q7Flg22 polypeptide delivered using the highest use rate
resulted in a substantially higher yield advantage, an almost +27
Bu/Ac (1694.6 kg/Ha) yield increase over the yield obtained from
the control plants. Overall, Bt.4Q7Flg22 and a synthetic version of
Syn01Flg22 provided protection from drought-like growth conditions
during a critical stage of plant development (i.e. pollination),
resulting in increased yield for all combinations of Bt.4Q7Flg22,
Syn01Flg22 and cellobiose used as foliar applications.
[0613] In another study, seed treatments using Flg22 polypeptides
and combinations of Flg22 polypeptides with cellobiose resulted in
overall yield increases in field trials reported as an average for
four replicated trials (Table 56). Seed treatments were applied to
corn hybrid (BECK's 5828YX) planted in 1 locations in the US
Midwest (Columbia). Seed treatment compositions of Flg22 were
applied as described in Table 56 as 0.14 Fl. oz per unit of corn
seeds in a total slurry volume containing the base seed treatment.
Final concentration of the Flg22 polypeptides in the slurry for
were standardized to 1 uM per seed. The same final concentration of
cellobiose that was applied in combination treatments with the
Flg22 polypeptides was at 1.0 mM per seed. The average yield in
Bu/Ac and the average increase in Bu/Ac as compared to the
untreated control (column 1) and to the Bt.4Q7Flg22 (SEQ ID NO:226)
(column 2) is reported for corn grown from seed that received the
Flg22 polypeptide combination treatments as described below in
Table 56.
TABLE-US-00058 TABLE 56 Seed treatment combinations of Flg22
polypeptides and variants of Flg22 polypeptides with
cellobiose-corn Average Yield Bu/Ac Average (Average Average Change
Change Change in Bu/Ac in Bu/Ac in Bu/Ac compared to Foliar
compared to compared to Bt.4Q7Flg22; Treatment Untreated Untreated
SEQ ID (Concentration) Control) Control NO:226 Base Seed 28.00 --
-1.60 Treatment Control Bt.4Q7Flg22 29.60 +1.60 -- at 1.0 .mu.M
(SEQ ID NO: 226) Syn01Flg22 39.58 +11.58 +9.98 at 1.0 .mu.M (SEQ ID
NO: 571) Syn03Flg22 35.17 +7.17 +5.57 at 1.0 .mu.M (SEQ ID NO: 300)
Pa.Flg22 39.04 +11.04 +9.44 Paenibacillus alvei at 1.0 .mu.M (SEQ
ID NO: 293) La. Flg22 37.13 +9.13 +7.53 Lysinibacillus at 1.0 .mu.M
(SEQ ID NO: 574) Flg22-B2 36.79 +8.79 +7.19 Bacillus at 1.0 .mu.M
(SEQ ID NO: 295) Flg22 Combination 45.39 +17.39 +15.79 Syn01Flg22
(SEQ ID NO: 571) + Flg22-B2 (SEQ ID NO: 295) + At.Flg22-B4 (SEQ ID
NO: 300) at 0.33 .mu.M each Cellobiose 1 mM 39.66 +11.67 +10.07
Bt.4Q7Flg22 36.22 +8.22 +6.62 (SEQ ID NO: 226) at 1.0 .mu.M +
Cellobiose 1 mM Bt.4Q7Flg22 47.06 +19.07 +17.47 (SEQ ID NO: 226) at
0.25 .mu.M Bt.4Q7Flg22 40.62 +12.63 +11.02 (SEQ ID NO: 226) at 0.25
.mu.M + Cellobiose 1 mM Syn01Flg22 32.65 +4.66 +3.06 (SEQ ID NO:
571) at 0.25 .mu.M Bt.4Q7Flg22 (SEQ ID NO: 571) 31.03 +3.03 +1.43
at 0.25 .mu.M + Cellobiose 1 mM
Example 38: Application of Flg22 with Cellobiose Additive to V4-V6
Soybean Increased Yield--Large Acre Yield Trials
[0614] Large acre soybean trials were planted from uncoated soybean
seed. Soybean seed was planted 1.5 to 2 inches deep (approximately
5 cm) to ensure normal root development. Soybean was planted in
12.5' (3.8 meter) plots with an average of 150,500 plants per acre,
row widths of 30 inch rows (0.8 meter) and seed spacing of 7 to 8
seeds per foot (30 cm).
[0615] Agricultural compositions comprising agriculturally
effective amounts of compositions of Bt.4Q7Flg22 (SEQ ID NO: 226),
Syn01Flg22 (SEQ ID NO: 571) and a Flg22 from Aneurinbacillus
thermoaerophilus, At.Flg22-B4 (SEQ ID NO: 300) were applied to
soybean. The Flg22 polypeptide treatments were applied as a foliar
spray at application use rates (Fl. oz/Ac or mL/Ha) as specified in
Table 57 to soybean grown at five US Midwest locations
(participating sites: IA and IL). The soybean plants received
foliar treatments containing Bt.4Q7Flg22 (SEQ ID NO: 226);
Syn01Flg22 (SEQ ID NO: 571) and At.Flg22-B4 (SEQ ID NO: 300) at
approximately the V4-V6 stage of development with a non-ionic
surfactant to facilitate spreading and uptake of treatments
(Alligare Surface.TM. applied at a final concentration of 0.1% v/v
of spray tank volume). Soybean yield was collected for the 3
soybean varieties (Asgrow: AG2733, AG3536 and AG4034) for plants
receiving the Flg22 compositions. Soybean yield was also reported
as the change in yield Bu/Ac compared to the control soybean plants
that received a non-ionic surfactant (Alligare Surface.TM. applied
at a final concentration of 0.1% (v/v) only treatment (Table
57).
[0616] Foliar application of the Bt.4Q7Flg22 and Syn01Flg22
polypeptides were also combined with cellobiose as an additive and
examined for the effect of Flg22 polypeptides combined with the
cellobiose additive on yield increase. Cellobiose is a glucose
disaccharide and a building block for cellulose polymer.
Chemically, it is glucose-beta-1-4-glucose, a reducing sugar that
consists of two .beta.-glucose molecules linked by a .beta. (1-4)
bond. Cellobiose is obtained by the breakdown of cellulose or
lichenin and yields glucose upon hydrolysis. The cellobiose
additive combined with Bt.4Q7Flg22 resulted in an increase in
reactive oxygen species (ROS) activity in soybean. Soybean yield
was collected for the 3 soybean varieties (Asgrow: AG2733, AG3536
and AG4034) for plants receiving the Flg22 compositions with and
without the cellobiose additive and reported as the average yield
(Bu/Ac) for all 3 varieties across locations. Soybean yield was
also reported as the change in yield Bu/Ac compared to the control
soybean plants that received a non-ionic surfactant (Alligare
Surface.TM. applied at a final concentration of 0.1% v/v only
treatment) (Table 57).
TABLE-US-00059 TABLE 57 Soybean yield with foliar treatments using
varying Flg22 polypeptides Application Change Use Rate Fl. in Bu/Ac
oz/Ac Average Over Treatment (mL/hectare Bu/Ac Surfactant
Concentration (Ha) (5 locations) Control Non-ionic Surfactant alone
0.1% v/v spray 61.36 -- Bt.4Q7Flg22 4.0 Fl. oz/Ac 62.85 +1.49 (SEQ
ID NO: 226) (292.3 mL/Ha) 16.7 .mu.M Composition 1 Bt.4Q7Flg22 4.0
Fl. oz/Ac 64.72 +1.56 (SEQ ID NO: 226) (292.3 mL/Ha) 16.7 .mu.M
Composition 2 Bt.4Q7Flg22 4.0 Fl. oz/Ac 63.87 +2.51 (SEQ ID NO:
226) (292.3 mL/Ha) 16.7 .mu.M Composition 3 Bt.4Q7Flg22 4.0 Fl.
oz/Ac 63.15 +1.79 (SEQ ID NO: 226): (292.3 mL/Ha) 16.7 .mu.M + 8.0
Fl. oz/Ac Cellobiose: 320 mM (584.6 mL/Ha) Composition 4
Bt.4Q7Flg22 48.0 Fl. oz/Ac 62.64 +1.28 (SEQ ID NO: 226) (3507.6
16.7 .mu.M mL/Ha) Composition 5 Syn01Flg22 4.0 Fl. oz/Ac 63.12
+1.76 (SEQ ID NO: 571) (292.3 mL/Ha) 16.7 .mu.M Composition 6
Syn01Flg22 0.4 Fl. oz/Ac 62.88 +1.52 (SEQ ID NO: 571) (29.2 mL/Ha)
16.7 .mu.M Composition 7 Syn01Flg22 0.4 Fl. oz/Ac 63.92 +2.56 (SEQ
ID NO: 571) (29.2 mL/Ha) 16.7 .mu.M + 8.0 Cellobiose (320 mM)
(584.6 mL/Ha) Composition 8 At.Flg22-B4 4.0 Fl. oz/Ac 63.66 +2.30
(SEQ ID NO: 300) (292.3 mL/Ha) 16.7 .mu.M Composition 9
[0617] Foliar treatment of the various Flg22 polypeptides,
Bt.4Q7Flg22; Syn01Flg22 and At.Flg22-B4 (Compositions 1-9) all
resulted in yield benefits when applied on soybean at the V4-V6
stage of development compared to the control soybean plants that
were treated with a foliar application of surfactant alone. Foliar
treatment with Bt.4Q7Flg22 (Composition 3) applied at 4.0 Fl. oz/Ac
resulted in a +2.51 Bu/Ac (168.8 kg/Ha) increase over control
plants (surfactant only). The Syn01Flg22 (Composition 6)
polypeptide applied as a foliar treatment using 4.0 Fl. oz/Ac to
soybean plants resulted in a yield gain of +1.76 Bu/Ac (118.4
kg/Ha) compared to the surfactant only control plants. Syn01Flg22
(Composition 7) and Syn01Flg22 with the cellobiose (320 nM)
(Composition 8) applied to soybean plants using a lower application
use rate of 0.2 Fl. oz/Ac resulted in an increase of +1 Bu/Ac with
the addition of the cellobiose additive or an overall +2.56 Bu/Ac
(172.2 kg/Ha) increase in yield over the control plants. The
At.Flg22-B4 (Composition 9) polypeptide applied to soybean (V4-V6)
also resulted in a yield benefit of +2.3 Bu/Ac (154.7 kg/Ha)
compared to the control plants or over 3.5 Bu/Ac (235.4 kg/Ha) as
compared to plants that received treatment with the non-ionic
surfactant only.
[0618] In still another study, seed treatments using Flg22
polypeptides and combinations of Flg22 polypeptides with cellobiose
were used as seed treatments on soybean and resulted in overall
yield increases in field trials reported as an average for four
replicated trials (Table 58). Seed treatments were applied to 1
soybean hybrid (variety) planted in 1 locations in the US Midwest
(Columbia, Mo.). Seed treatment compositions of Flg22 were applied
as described in Table 58 as 0.14 Fl. oz per unit of soybean seeds
in a total slurry and provided to soybean seed that had a base seed
treatment consisting of Poncho VOTiVO 600 FS and Evergol Energy.
The application use rates per each seed treatment were held
constant at 0.14 Fl. oz/Ac or 4.14 mL/unit. Final concentration of
the Flg22 polypeptides in the slurry for were standardized to 1 uM
per seed. The same final concentration of cellobiose that was
applied in combination treatments with the Flg22 polypeptides was
at 1.0 mM per seed. Four replicate plots per each seed treatment
were randomized over the location. The average yield in Bu/Ac and
the average change in Bu/Ac as compared to the control plants that
received only the base seed treatment are reported in Table 58. The
most substantial yield increases were seen with Bt.4Q7Flg22 (SEQ ID
NO: 226) and Syn01Flg22 (SEQ ID NO: 571) when applied as a seed
treatment on soybean delivered at a final concentration of 1.0
.mu.M of the Flg22 polypeptides and resulting in respective average
yield increases of +5.11 (343.7 kg/Ha) and +9.92 (667.1 kg/Ha) over
yield from soybean that received the base seed treatment.
TABLE-US-00060 TABLE 58 Seed treatment combinations of Flg22
polypeptides and variants of Flg22 polypeptides with
cellobiose-soybean Average Yield Bu/Ac (Average Average Change
Change in Bu/Ac in Bu/Ac Foliar compared to compared to Treatment
Untreated Untreated (Concentration) Control) Control Base Seed
41.11 -- Treatment Control Bt.4Q7Flg22 46.22 +5.11 at 1.0 .mu.M
(SEQ ID NO:226) Syn01Flg22 51.09 +9.92 at 1.0 .mu.M (SEQ ID NO:
571) Syn03Flg22 43.61 +2.50 at 1.0 .mu.M (SEQ ID NO: 573) Pa.Flg22
41.39 +0.28 Paenibacillus alvei at 1.0 .mu.M (SEQ ID NO: 293)
An,.Flg22 46.01 +4.90 Aneurillusbacillus at 1.0 .mu.M (SEQ ID NO:
300) Flg22 44.43 +3.32 Bacillus species (Combination of Flg22
sequences) Syn01Flg22 (SEQ ID NO: 571, Flg22- B2 (SEQ ID NO:295)
& Flg22-B4 (SEQ ID NO: 300) at 0.33 .mu.M each Cellobiose 1 mM
43.98 +2.87 Bt.4Q7Flg22 43.40 +2.29 (SEQ ID NO: 226) at 1.0 .mu.M +
Cellobiose 1 mM Bt.4Q7Flg22 43.80 +2.69 (SEQ ID NO: 226) at 0.25
.mu.M Bt.4Q7Flg22 43.52 +2.41 (SEQ ID NO: 226) at 0.25 .mu.M +
Cellobiose 1 mM
Example 39: Application of RHPP to V5 Corn Increased Yield
[0619] Large acre corn trials were planted from corn seed (DEKALB
hybrids: DKC 52-61, DKC 58-89, and DKC 65-81) containing a seed
treatment comprising EVERGOL fungicide (7.18% propiconazole, 3.59%
penflufen and combined with 5.74% metalaxyl) combined with
PONCHO/VOTiVO 500 (a mixture of clothianidin insecticide and a
microbial agent, Bacillus firmus 1582). Corn plants at
approximately the V5 stage of development received a foliar
application using an agricultural composition comprising an Gm.RHPP
polypeptide (SEQ ID NO: 600). The formulated Gm.RHPP polypeptide
(Table 59) was applied to the corn hybrids using an application use
rate of 8.0 Fl. oz/Ac (584.6 mL/Ha) with 0.1% v/v (of spray tank)
non-ionic surfactant (Alligare Surface.TM.). In total, the trial
was conducted at 6 locations in the US Midwest (IL, IN, IA), with
1-2 hybrids per location and 3 replicated plots per hybrid. Corn
yield (Bu/Ac) was collected and reported as the average yield
(Bu/Ac). The average change in Bu/Ac was compared to the yield of
plants grown from the surfactant control and reported as the
combined average yield (Bu/Ac) for the 6 locations (11 replicated
plots in total) and as overall change in Bu/Ac as compared to
control plants. Results are shown in Table 59.
TABLE-US-00061 TABLE 59 Foliar treatment using RHPP-increase yield
in corn Average Yield Bu/Ac (Average Change in Bu/Ac Foliar
compared to Treatment Application surfactant Concentration Rate
only control) Surfactant control -- 206.15 (Control) Gm.RHPP 8.0
Fl. oz/Ac 209.67 (SEQ ID: 600) (584.6 mL/Ha) (+3.52; 100 .mu.M 64%
PROXEL BC win rate) preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT);
21.71 .mu.M (MIT)
[0620] Foliar treatment of plants with the Gm.RHPP polypeptide
resulted in increased yields in corn compared to plants that
received surfactant alone. The average yield per the 6 locations
combined for corn plants that received the Gm.RHPP polypeptide
foliar treatment was slightly more than 209 Bu/Ac as compared to
206 Bu/Ac or for the control plants. The yield for the Gm.RHPP
treated plants was increased by 3.52 Bu/Ac or 220.9 kg/Ha as
compared to the yield from the corn control plants (Table 59).
Example 40: RHPP Polypeptide Increases Pod Number In Soybean
[0621] Soybean (variety MorSoy) plants were grown from seed with 2
seeds planted per pot in a controlled environmental growth room
under conditions of approximately 300 .mu.mol.sup.-2 s.sup.-1
(light photons) for a 13/11 light/day cycle and a 21.degree. C.
day/15.degree. C. night temperature range until the V4 stage of
development. Plants were then placed under a long day conditions
consisting of 16/8 light/day cycle and temperature 21-26.degree. C.
to promote early flowering and speed up progression to the
reproductive (R) growth stage. When the soybean plants had reached
the R1 stage of development a foliar application containing the
Gm.RHPP (SEQ ID NO: 600) polypeptide at a final concentration of
300 nM and a non-ionic surfactant of 0.10% (NIS90:10; Precision
Laboratories, LLC) was applied to soybean. Soybean plants were
provided with the Gm.RHPP formulation and a non-ionic surfactant
only control. Both the Gm.RHPP and the non-ionic surfactant control
treatment were applied to 18 plants per treatment. Six equidistant
sprays were provided approximately 15 cm above per each plant for
complete coverage of foliage. After treatment application, the R1
soybean plants were returned to the control environmental growth
room. After seventeen days, the plants received another foliar
treatment application with the formulation containing the Gm.RHPP
polypeptide and the non-ionic surfactant as well as the non-ionic
surfactant only treatment. Soybean pods of more than 1 mm in length
were counted on the plants after 31 days from the first foliar
spray treatment applications. The average number of pods per plant
and the standard deviation from the overall average are reported
(Table 60). A p value (p<0.05 for significance) was calculated
from a paired T-test comparison between pod number from plants that
received the Gm.RHPP and the non-ionic surfactant control treatment
applications.
TABLE-US-00062 TABLE 60 Number of pods in greenhouse grown soybean
at 31 days after foliar treatment with RHPP Treatment Pod Count
Concentration (STDEV) p-value Non-Ionic Surfactant 1.07 0.0116
(NIS90:10 Control) (+0.44) 0.01% (v/v) Gm.RHPP 2.00 (SEQ ID: 591) +
(+1.22) Surfactant 300 nM *p value <0.05 is statistically
significant
[0622] Foliar application of the Gm.RHPP polypeptide at early
reproductive stage (R1) of soybean plants resulted in an
approximately doubled pod count as compared to plants that received
the non-ionic surfactant control treatment.
Example 41: Flg22 and RHPP Polypeptides Increase Yield in Tomato
and Pepper
[0623] Foliar application treatments of Bt.4Q7Flg22 (SEQ ID NO:
226) and Gm.RHPP (SEQ ID NO: 600) were applied as an exogenous
spray at the pre-bloom stage and used to increase yield in tomatoes
and jalapeno peppers.
[0624] Small scale plots were designed to simulate commercial
growing conditions for tomatoes. Tomato plants, variety Roma were
started from transplants that were grown in a greenhouse for 45
days prior to planting into 2 raised field row beds with 2 feet
(0.6 meters) between each transplant with an average of 30 plants
per row bed. Tomatoes were transplanted three inches beneath the
soil surface once the soil temperature reached 15.6.degree. C.
Tomatoes were grown on raised beds covered with black plastic
mulch. Plants were grown using drip irrigation and fertilizer (80
lbs. or 36.3 kg) nitrogen; 100 lbs. (45.4 kg) phosphate, and 100
lbs. (45.4 kg) potash or potassium) applied following grower
guidelines throughout the growing season to provide for optimum
plant growth and yields. Small raised bed plots were designed to
simulate the planting densities used by commercial growers that
generally plant 2,600 to 5,800 plants per acre in single rows with
45.7 to 76.2 cm between plants in the row on 1.5- to 2-meter
centers. [Orzolek et al., "Agricultural Alternatives: Tomato
Production." University Park: Penn State Extension, 2016].
[0625] Foliar treatments using Bt.4Q7Flg22 and Gm.RHPP were applied
on the tomato plants directly at early bloom (first flower) stage.
The Bt.4Q7Flg22 polypeptide foliar composition was applied using an
application use rate of 4.0 Fl. oz/Ac (292.3 mL/hectare) and the
Gm.RHPP polypeptide foliar composition was applied using an
application use rate of 3.2 Fl. oz/Ac (234 mL/hectare) on tomato
plants in 10 gallons of water per acre with 0.1% v/v non-ionic
surfactant (Alligare.TM. Surface). The Bt.4Q7Flg22 and Gm.RHPP
treated plants were compared to the control plants that received no
foliar treatment application. Plants were treated in replicates of
6 plants, with three replicates per treatment. Effect of the foliar
treatments on the yield obtained from tomatoes was determined and
reported as normalized to no spray control treatment. The average
fruit weight per tomato plant is reported as the combined average
for 2 separate harvests and the average percentage change in fruit
weight as compared to the no-spray control in Table 61.
TABLE-US-00063 TABLE 61 Foliar treatment on Spring-planted tomato
Percentage Change in Fruit Average Weight Fruit Compared Foliar
Weight to No- Treatment (grams) Spray Concentration per Plant
Control No Spray Control 1369.9 -- Bt.4Q7Flg22 1487.8 +8.61% (SEQ
ID NO: 226) 16.7 .mu.M 1.67 mM Sodium Phosphate Buffer, pH 5.7
PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M
(MIT) (Composition 3) Gm.RHPP 1397.1 +1.99% (SEQ ID NO: 591) 100
.mu.M PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT)
[0626] Foliar treatment application with the Bt.4Q7Flg22
polypeptide provided at a concentration of 16.7 .mu.M and an
application use rate of 4.0 Fl. oz/Ac (292.3 mL/hectare) resulted
in an overall increase in the average fruit weight per plant as
reported in total grams and an +8.61% change in fruit weight as
compared to the no spray control. The Gm.RHPP polypeptide provided
at a concentration of 100 .mu.M and a 3.2 Fl. oz/Ac (234
mL/hectare) application use rate also resulted in yield overall
increase in average fruit weight (grams) per plants and an almost
+2% change in fruit weight as compared to the no spray control.
[0627] In another study, foliar treatments with the Bt.4Q7Flg22
(SEQ ID NO: 226) and Gm.RHPP (SEQ ID NO: 600) polypeptides were
applied on jalapeno peppers (Capsicum) plants at early bloom (first
flower) stage. Small-scale plots were designed to simulate
commercial growing conditions for jalapeno peppers. Peppers were
grown for 12-weeks in a controlled growth room and then
transplanted outside in 2 raised beds covered with black plastic
mulch that had good water-holding characteristics and in soil
having a pH of 5.8-6.6. Jalapeno pepper plants were spaced 14-16
inches (38 cm) apart with 16-24 inches (50 cm) between plants
containing approximately 25 plants per row bed. Plants were grown
using drip irrigation and fertilizer applied following grower
guidelines throughout the growing season to provide optimum
conditions for plant growth. The raised bed plots were designed to
simulate the planting densities used by commercial growers that
generally plant approximately plants per acre (5,000-6,500 plants
per acre or 12,355-16,062 plants per hectare) in double rows
35.6-45.7 cm apart with the beds spaced 5.0-6.5 feet (1.52-1.98
meters) apart from their centers (Orzolek et al., "Agricultural
Alternatives: Pepper Production." University Park: Penn State
Extension, 2010).
[0628] Foliar treatments using the Bt.4Q7Flg22 and Gm.RHPP
polypeptides were applied on jalapeno pepper using application use
rates of 2.0 Fl. oz/Ac (146.2 mL/hectare) and 4.0 Fl. oz/Ac (292.3
mL/hectare) for Bt.Flg22 and 3.2 Fl. oz/Ac (234 mL/hectare) for the
Gm.RHPP polypeptide in a spray volume of 10 gallons of water per
acre with 0.1% v/v non-ionic surfactant (Alligare.TM. Surface).
Plants were treated in replicates of 6 plants, with three
replicates per treatment. Replicates with average yield per plant
50% above or 50% below the median yield for the trial were excluded
as outliers. The Bt.4Q7Flg22 and Gm.RHPP polypeptide foliar
treatments applied on jalapeno pepper plants were compared to
plants sprayed with 10 gallons of water per acre with 0.1% v/v
non-ionic surfactant (Alligare.TM. Surface) alone.
[0629] Effects of the Bt.4Q7Flg22 and Gm.RHPP polypeptides used as
foliar spray applications on pepper yield were determined for two
separate harvests using a once over harvest approach. The number of
peppers and the above ground biomass per plant were normalized to
the yield and to the biomass of the pepper control plants that were
treated with surfactant alone (Table 62).
TABLE-US-00064 TABLE 62 Foliar treatment on Spring-planted Jalapeno
pepper Percentage Change Average in Fruit Fruit Weight Foliar
Weight Compared to Treatment (grams) Surfactant and Rate per Plant
Control Surfactant control 123.7 -- (Alligare .TM. Surface; 0.1%
v/v of spray volume) Bt.4Q7Flg22 184.1 +49% (SEQ ID NO: 226) 16.7
.mu.M 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
(Composition 3) 2 fl oz/Ac Bt.4Q7Flg22 173.7 +40% (SEQ ID NO: 226)
16.7 .mu.M 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
(Composition 3) 4 fl oz/Ac Gm.RHPP 156.6 +27% (SEQ ID NO: 591) 100
.mu.M PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) 3.2 fl oz/Ac.
[0630] The Bt.4Q7Flg22 polypeptide applied as a foliar spray
application to Jalapeno pepper at the pre-bloom stage resulted in
substantial increases in average fruit weight per plant, a +49%
increase for 2 Fl. oz/Ac (146.2 mL/hectare) and +40% increase for 4
Fl. oz/Ac (292.3 mL/Ha) as compared to the surfactant only control
plants. The Gm.RHPP polypeptide treatment also applied as a foliar
spray at the pre-bloom stage also resulted in an increased average
fruit weight in Jalapeno peppers per plant with a +27% increase in
the weight of peppers as measured on a per plant basis as compared
to the peppers harvested from the surfactant only control
plants.
Example 42: Application to Squash-Increased Yield
[0631] Foliar treatments containing the Bt.4Q7Flg22 or the Gm.RHPP
polypeptide was applied exogenously as a foliar treatment to
Crookneck squash at the first bloom stage. Foliar treatments with
the Bt.4Q7Flg22 and the Gm.RHPP polypeptide were applied to squash
plants using an application use rate of 2.0 Fl. oz/Ac (146.2
mL/hectare) or 3.2 Fl. oz/Ac (234 mL/hectare), respectively, in a
spray volume of 10 gallons of water per acre with 0.1% v/v
non-ionic surfactant (Alligare.TM. 90). Yield comparisons were made
between the plants treated with the polypeptides compared to
surfactant only control plants, with three replicates per
treatment. Yield for the foliar treated plants that received the
Bt.4Q7Flg22 or Gm.RHPP polypeptide treatment are reported in Table
63 as the average weight (grams) of squash per plant over two
harvests per replicate and represented as a percentage change as
compared to control plants. Replicates with average yield per plant
50% above or 50% below the median yield for the trial were excluded
as outliers.
[0632] Squash plants were cultivated in sandy loam soil as follows.
2.5 cm holes were cut in 0.76 meters wide plastic covered mounds,
two rows per mound, holes spaced 0.46 meters apart within each row.
Rows were staggered within the mound. Mounds were spaced 1.2 meters
apart. Three squash seeds were planted per hole and thinned to a
single plant per hole 14 days after planting. Drip irrigation
tubing was laid in the center of each mound, and plants were
watered as necessary.
TABLE-US-00065 TABLE 63 Foliar treatment with a composition of
Gm.RHPP polypeptide to increase yield in squash Percentage Change
Average in Fruit squash Weight fruit Compared to Foliar weight
Surfactant Treatment (grams) only and Rate per plant control
Surfactant control 716.7 -- (Alligare .TM. 90; 0.1% v/v of spray
volume) Bt.4Q7Flg22 748.4 +4.4% (SEQ ID NO: 226) 16.7 .mu.M 1.67 mM
Sodium Phosphate Buffer, pH 5.7 PROXEL BC preservative: 330.7
.mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 3) 2 fl
oz/Ac Gm.RHPP 748.4 +4.4% (SEQ ID NO: 591) 100.mu.M PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) 3.2
fl oz/Ac.
[0633] Foliar treatment with either Bt.4Q7Flg22 or Gm.RHPP
polypeptide on squash plants at the pre-bloom stage both resulted
in an increased weight of harvested squash fruit by an average by
31.7 grams per plant or +4.4% change in fruit weight as compared to
the surfactant only control plants (Table 63).
Example 43. Flg22 Polypeptide Reduces Severity of White Leaf Spot
on Kale
[0634] In a replicated Fall season kale trial in the Midwest
(Columbia, Mo.), very wet and warm growing conditions led to the
development of white leaf spot on the kale leaves, which is
typically caused by Cercospora brassicicola. The infected kale
plants had received no previous foliar treatments for fungal
disease prevention. To assess the severity of disease, a scoring
rubric (1-5 scale) was established where 1=a healthy plant with
three or fewer white fungal spots, 2=a plant with more four or more
spots and a portion of the foliage is affected by disease,
3=majority of the foliage shows symptoms and up to one leaf has
fallen off due to disease, 4=majority of the foliage shows symptoms
and 2-3 leaves have fallen off due to disease, and 5=majority of
the foliage shows symptoms and four or more leaves have fallen off
due to disease. A single person scored all the plants within the
trial area, and then evenly distributed the plants by disease score
between the treatments in Table 64, with 6 replicated blocks of 6
plants per treatment (total=36 plants per treatment). To test
Bt.4Q7Flg22 (SEQ ID NO: 226) for improvement of disease symptoms on
kale, treatments were applied as a foliar spray at the indicated
rates in Table 64 in a carrier volume of 10 gallons of water per
acre with 0.1% v/v non-ionic surfactant (Alligare.TM. Surface).
Three weeks after foliar treatments, the plants were scored used
the same disease severity rubric. The change in disease score was
calculated for each plant, and the average change in disease score
was determined per treatment. Plants were harvested four days after
assessing disease severity, and yield was measured as plant weight
(grams). Outlying values with weights that were either 50% below or
50% above the median weight for the trial were excluded from the
dataset.
TABLE-US-00066 TABLE 64 Foliar kale treatments for amelioration of
white leaf spot. Yield (Average Application plant weight Average
Use Rate in grams) Change in Foliar Treatment Fl. oz/Ac Relative to
Disease (Concentration) (mL/hectare) control (%) Score Surfactant
n/a 14.6 0.6 point only control (100%) improvement Bt.4Q7Flg22 12.0
Fl. oz/Ac 15.1 1.1 point (SEQ ID NO: 226) (876.9 mL/ (103%)
improvment (100 .mu.M ) hectare) 1.67 mM Sodium Phosphate Buffer,
pH 5.7 PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT);
21.71 .mu.M (MIT) Liquid Copper Label rate 11.5 1.1 point Fungicide
(54.45 Fl oz/Ac) (79%) improvment Liquid Copper Label Rate 12.4 0.8
point Fungicide + (54.45 Fl oz/Ac) + (85%) improvment Bt.4Q7Flg22
12.0 Fl. oz/Ac (SEQ ID NO: 226) (876.9 mL/ (100 .mu.M ) hectare)
1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC preservative:
330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
[0635] Foliar treatment of infected kale plants with formulated
Bt.4Q7Flg22 (SEQ ID NO: 226) led to an improvement in yield and
disease symptoms over the control (Table 64). While untreated
controls had an average plant weight of 14.6 g, plants receiving
foliar Bt.4Q7Flg22 had an average plant weight of 15.1 g and an
average improvement in disease scoring of 0.5 points over control.
The application of copper fungicide improved plant disease scores
to the same extent as Bt.4Q7Flg22, yet decreased yield by 21% (11.5
g) compared to the control. The combination treatment of copper
fungicide with Bt.4Q7Flg22 increased yield to 12.4 g per plant, but
overall Bt.4Q7Flg22 alone gave the greatest yield and plant health
benefit in the trial. In conclusion, Flg22 polypeptides can used to
slow the progression of fungal infections in vegetables and
increase yield under stressful growing conditions.
Example 44: ROS Screening Assays to Determine Compatibility of
Flg22 Polypeptide with Seed Treatments
[0636] Seed treatments were examined for compatibility with the
production of apoplastic reactive oxygen species (ROS) in corn
petiole tissues. Various commercially available seed treatments
were examined for compatibility with the Flg22 polypeptide
(Bt.4Q7Flg22; SEQ ID NO: 226) shown to increase yield when applied
alone as a seed treatment on corn. ROS activity assays were
conducted using corn petiole samples from corn hybrid 5828 YX as
described in Example 15 with the exception that Relative light
units (RLUs) were recorded with a SpectraMax L luminometer (0.5 s
integration; 2.0 min intervals) over a time course of 40 minutes.
Varying concentrations of Bt.4Q7Flg22 (0 and 1000 .mu.M) were
combined with three commercial seed treatments consisting of PPST
2030 (a combination of bacteria, Bacillus subtilis 5.times.108
cfu/mL and Bacillus pumilus 5.times.108 cfu/mL), ILEVO (48.4%
fluopyram) and PONCHO/VOTiVO (a mixture of 40.3% clothianidin and a
microbial agent, Bacillus firmus 1-1582) and tested for the
presence of a ROS response in corn petioles. All three seed
treatments as described were applied using the application use
rates per seed as recommended on the individual specimen label for
each seed treatment. A standard curve was generated using varying
concentrations of the Bt.4Q7Flg22 polypeptide and resulted in a
logarithmic correlation between the RLU and concentration of Flg22
with an R2 of 0.90. The RLU values are the average of 4 separate
measurements (4 treatment wells on each plate) and the increase in
overall ROS (RLU) (times increase over the background) are shown in
parentheses (Table 65).
TABLE-US-00067 TABLE 65 Seed treatment compatibility with Flg22
polypeptide using ROS assay 1 .mu.M 1 .mu.M BL4Q7Flg22 Bt.4Q7Flg22
(SEQ ID NO: 226) (SEQ ID NO: 226) (Full-strength ST) (1:10 dilution
of ST) Seed (Fold increase (X) (Fold increase (X) Treatment
Background over background) over background) PPST 2030 15952.2
109313.7 96129.9 (6.9X) (6.0X) ILeVO 84716.9 548686.2 382365.1
(6.5X) (4.5X) PONCHO/ 17379.7 120788.9 267720.2 VOTiVO (6.9X)
(15.4X)
[0637] ROS production as measured by RLUs were increased with the
addition of 1 .mu.M Bt.4Q7Flg22 when combined with each of the seed
treatments as described in Table 65. The ROS production (RLU
values) with the 1:10 dilution of the seed treatments with the
addition of 1 .mu.M Bt.4Q7Flg22 was also increased as compared to
the back ground RLU level for the seed treatment only or no
Bt.4Q7Flg22 polypeptide. The diluted PONCHO/VOTiVO seed treatment
combined with 1 .mu.M Bt.4Q7Flg22 was increased more than 15.times.
compared to the background or 2.2.times. compared to the
non-diluted PONCHO/VOTiVO treatment applied per seed following the
recommendation on the specimen label. Therefore, the Flg22
polypeptide is detectable by ROS assay when combined with standard
seed treatment base at label rates. When combining such Flg22
polypeptides with a particular seed treatment, adjustment of either
the polypeptide concentration or the seed treatment concentration
can be taken into consideration to ensure an optimal ROS response
in the plant. These demonstrate the activity of the Flg22
polypeptides on plants in the presence of other seed treatment
packages on the market today.
Example 45: Combinations of Flg22 and FlgII-28 Peptides to Increase
ROS Activity in Tomato
[0638] In a separate study, the Flg22 and FlgII-28 polypeptides
derived from distinct regions of flagellin protein were tested
separately and in combination for compatibility of response in
tomato leaves. While Flg22 and FlgII-28 are both microbe-associated
molecular patterns (MAMPs) they may be recognized distinctly by the
Flagellin-sensing 2 (FLS2) and Flagellin-sensing 3 (FLS3)
receptors, respectively (Hind et al., 2016; Nature Plants 2:16128),
and the interactions may differ across plant species. Several Flg22
polypeptides (Bt.4Q7Flg22, SEQ ID NO: 226; Bt.4Q7Flg22-Syn01, SEQ
ID NO: 571 and Ec.Flg22, SEQ ID NO: 526) were compared using ROS
activity assays in tomato to several FlgII-28 polypeptides
(Ps.tomatoFlgII-28, SEQ ID NO: 751; A.sp.FlgII-28, SEQ ID NO:
375).
[0639] Tomato leaves were excised from 4-week-old plants using a
cork borer to generate 4 mm disks. Each disc was cut in half using
the edge of a razor blade, and then each disc half was floated on
150 .mu.L of water in a 96-well plate to rest overnight. The next
day, the water was removed from each well just prior to polypeptide
treatment. The Flg polypeptides as described in Table 66 were added
to water to bring them to a final concentration of 5 nM (Table 67)
and 100 nM (Table 68) in solution with luminol and HRP before
adding to each treatment well. To maintain activity, the
polypeptides were stored in small aliquots to avoid multiple
freezing and thawing. All dilutions to obtain working
concentrations were done in ultrapure water. Polypeptide solutions
were stored at -20.degree. C. for short term usage or -80.degree.
C. for long term storage. RLU values and relative ROS activity
(Tables 67, 68) is reported as the average of 4 measurements. ROS
activity assays were conducted using the methods as previously
reported in Example 15 with the exception that Relative light units
(RLUs) were recorded with a SpectraMax L luminometer (0.5 s
integration; 2.0 min intervals) over a time course of 40
minutes.
TABLE-US-00068 TABLE 66 Flg22 and FlgII-28 Polypeptides from
various sources Flg Polypeptide Amino Acid Description Length
Sequence Bt.4Q7FIg22 22 DRLSSGKRINSASDDAAGLAIA Bacillus
thuringiensis (SEQ ID NO: 226) Syn01Flg22 22 DRLSSGKRINSAKDDAAGLAIA
Synthetic (SEQ ID NO: 571) Ps.tomato FlgII-28 28
ESTNILQRMRELAVQSRNDSNSATDREA Pseudomonas syringae pv. Tomato DC3000
(SEQ ID NO: 751) Ec.Flg22 22 ERLSSGLRINSAKDDAAGQAIA Escherichia
coli (J26) (SEQ ID NO: 526) A.sp.FlgII-28 28
EIHEMLQRMRELAVQAANGTYSDKDKKA Aneurinibacillus sp. XH2 (SEQ ID NO:
300)
TABLE-US-00069 TABLE 67 Comparison of ROS activity of Flg22 and
Flgll-28 polypeptides in tomato leaf tissue Average RLU value (5 nM
Flg polypeptide) (Fold increase (X) over Polypeptide Bt.4Q7Flg22
Treatment treatment) Negative 24423 control (water) (0.7X)
Bt.4Q7Flg22 33118 Bacillus thuringiensis (-) (SEQ ID NO: 226)
Bt.4Q7Flg22-Syn01 116751 Synthetic (3.5 X) (SEQ ID NO: 571)
Ps.tomatoFlgll-28 1019995 Pseudomonas (30.8 X) syringae pv. Tomato
(SEQ ID NO: 751) Ec.Flg22 426307 Escherichia coli (12.9 X) (SEQ ID
NO: 526) Aneurinibacillus.sp. 32980 Flgll-28 (SEQ ID NO: 375) (1.0
X)
TABLE-US-00070 TABLE 68 Flgll-28 polypeptides from gram-negative
Pseudomonas syringae pv. Tomato DC3000 and gram-positive
Aneurinibacillus sp. XH2 trigger ROS production in tomato leaf
tissue Average RLU value with 100 nM Flg polypeptide Polypeptide
Treatment (Fold increase (X) over Concentration Bt.4Q7Flg22
treatment) Negative control (water) 15,824 (0.007X) Bt.4Q7Flg22
2,118,932 (--) Bacillus thuringiensis (SEQ ID NO: 226) 100 nM
Ps.tomatoFlgll-28 3,657,810 (1.7X) Pseudomonas syringae pv. Tomato
DC3000 (SEQ ID NO: 751) 100 nM Bt.4Q7Flg22 4,222,426 (2.0X) (SEQ ID
NO: 226; 100 nM) + Ps.tomatoFlgll-28 (SEQ ID NO: 751; 100 nM)
Aneurinibacillus. sp. Flgll-28 2,844,947 (1.3X) (SEQ ID NO: 375)
100 nM
[0640] It was determined from the results in Table 67 and Table 68
that a second epitope of flagellin, termed FlgII-28 derived from
either Gram-negative Pseudomonas syringae pv.tomato DC3000 or
Gram-positive Aneurinibacillus sp. XH2 (SEQ ID NO: 375) are
sufficient to trigger an immune response (e.g. ROS production) in
tomato (SEQ ID NO: 751) at both 5 nM and 100 nM concentrations. At
the 5 nM concentration, Ps.tomato FlgII-28 had the highest activity
as compared to the other Flg22 and FlgII-28 polypeptides and
resulted in an almost 31 times increase in RLUs as compared to
Bt.4Q7Flg22 at the same concentration, whereas 5 nM A.spp.FlgII28
gave an equally low ROS response to 5 nM Bt.4Q7Flg22. The Flg22
polypeptide (Ec.Flg22; SEQ ID NO: 526) from Gram-negative
Escherichia coli also resulted in increased ROS activity when
applied to tomato leaves, with RLU values 12.9 X over the
Bt.4Q7Flg22 treatment alone. The Bt.4Q7Flg22 (SEQ ID NO: 226)
polypeptide triggered a very low ROS response in tomato leaves at
the 5 nM concentration, but provided a high response at the 100 nM
concentration. Ps.tomato FlgII-28, on the other hand, provided a
strong ROS response in comparison to the negative control (water)
at both tested concentrations. Thus, tomato leaves display
increased sensitivity to Flg polypeptides derived from
gram-negative bacteria Flagellin such as Ps.tomato FlgII-28 and
Ec.Flg22. In addition, a synthetic variant of Bt.4Q7Flg22 termed
Syn01Flg22 (SEQ ID NO: 571) had substantially increased activity
(3.5.times.) as compared to Bt.4Q7Flg22 treatment when tested at
the 5 nM concentration.
[0641] As indicated in Table 68, combinations of Gram-positive
(Bt.4Q7Flg22; SEQ ID NO: 226) and Gram-negative Ps.tomato FlgII-28
(Pseudomonas syringae pv. tomato DC3000; SEQ ID NO: 751) can be
used as a combined foliar application to increase ROS production
over either treatment alone, and enhance plant immunity against
certain pathogenic organisms.
Example 46: Synthetic Flg22Syn01 and Flg-15Syn01 Polypeptides to
Increase ROS Activity in Corn and Soybean
[0642] A truncated version of Syn01Flg22 derived from
Bt.4Q&Flg22 lacking seven N-terminal amino acids was generated,
resulting in the 15 amino acid polypeptide with the sequence
nh2-RINSAKDDAAGLAIA-cooh. This polypeptide, termed Bt.4Q7Syn01Flg15
(SEQ ID NO: 752) is a naturally occurring polypeptide among the
Gram-negative proteobacteria but is absent from Gram-positive
protein sequences. The core sequence required for receptor
interaction, RINSAKDD, is retained in the shortened polypeptide,
and thus the 15-amino acid variant was predicted to be active for
triggering ROS production in plants. To test this, Syn01Flg15 was
compared to Bt.4Q7Flg22 and Syn01Flg22 in ROS assays with both corn
(Table 69) and soybean (Table 70). ROS activity assays were
conducted using the methods as previously reported in Example 15
with the exception that Relative light units (RLUs) were recorded
with a SpectraMax L luminometer (0.5 s integration; 2.0 min
intervals) over a time course of 40 minutes.
TABLE-US-00071 TABLE 69 Flg22Syn01 and Flg15Syn01 variants have
greater activity than Bt.4Q7Flg22 in a ROS activity assay with corn
stalk tissue. Flg Polypeptide Concentration Bt.4Q7Flg22 Syn01Flg22
Syn01Flg15 (nM) (SEQ ID NO: 226) (SEQ ID NO: 571) (SEQ ID NO: 752)
100 33037 54888 n.d. (1X*) (1.6X) 10 6032 17660 14079 (0.2X) (0.5X)
(0.4X) *Relative ROS activity was normalized to the average RLU
values of Bt.4Q7Flg22 (SEQ ID NO: 226). n.d. indicates that a value
was not tested and therefore a relative value was not
determined.
[0643] In the ROS activity assay with corn (Table 69), the
Flg22.quadrature.Syn01 (SEQ ID NO: 571) had the greatest ROS
response in corn stalk tissue at both the 100 nM and 10 nM
concentrations as indicated by the relative respective activities
of 1.6.times. (100 nM) and 0.5.times. (10 nM) as compared to
treatment using Bt.4Q7Flg22 (SEQ ID NO: 226) that has an attenuated
ROS response of 0.2.times. at 10 nM. The shortened version of
Syn01Flg22 (SEQ ID NO: 571) or Syn01Flg15 (SEQ ID NO: 752) also
exhibited a greater ROS response of 0.4.times. at 10 nM, which was
twice the relative ROS activity of Bt.4Q7Flg22 (SEQ ID NO: 226) at
the same concentration.
TABLE-US-00072 TABLE 70 Flg22Syn01 and Flg15Syn01 variants have
greater activity than Bt.4Q7Flg22 in a ROS assay with soybean leaf
tissue Flg Relative ROS Relative ROS Relative ROS Polypeptide
Activity Activity Activity Concentration BL4Q7Flg22 Flg22Syn01
Syn01Flg15 (nM) (SEQ ID NO: 226) (SEQ ID NO: 571) (SEQ ID NO: 752)
100 250,432 315,961 n.d. (1X)* (1.25X) 10 10,754 62,020 42,983
(0.04X) (0.25X) (0.17X) *Relative ROS activity was normalized to
the average RLU values of Bt.4Q7Flg22 (SEQ ID NO: 226). n.d.
indicates that a value was not tested and therefore a relative
value was not determined.
[0644] Likewise, in the ROS activity assay with soybean (Table 70),
the synthetic derived mutant of Bt.4Q7Flg22 described as
Bt.4Q7Flg22.quadrature.Syn01 (SEQ ID NO: 571) also had the greatest
ROS response in soy leaf tissue at both the 100 nM and 10 nM
concentrations as indicated by the relative respective activities
of 1.25.times. (100 nM) and 0.25.times. (10 nM) as compared to
treatment using Bt.4Q7Flg22 (SEQ ID NO: 226) that has a highly
attenuated ROS response of 0.04.times. at 10 nM. The shortened
version of Syn01Flg22 or Syn01Flg15 also exhibited a greater ROS
response of 0.17.times. at 10 nM, which was four times the relative
ROS activity of Bt.4Q7Flg22 at the same concentration.
[0645] Overall, the Syn01Flg22 had higher ROS activity at both
concentrations tested in both corn and soy tissues in comparison to
Bt.4Q7Flg22 (SEQ ID NO: 226). The shortened 15-amino acid
polypeptide Syn01Flg15 was 2-4.times. more active than Bt.4Q7Flg22
and only slightly less active than the 22-amino acid Syn01Flg22 at
10 nM, indicating that key amino acids for eliciting a plant immune
response are retained within the sequence.
Example 47: Chemical Modification to Increase ROS Activity for
Flg22 Polypeptides
[0646] Chemical modifications can be made to Flg22 polypeptides to
increase protein stability against proteolysis and/or promote a
longer duration of activity that can result in greater availability
to the FLS2 receptor. In general, polypeptide modifications can be
utilized to 1) stabilize a polypeptide under adverse conditions or
in the presence of proteases, or 2) provide additional function or
molecular characteristics to the peptide. Modifications for
improved stability include polypeptide cyclization and alternations
at the N- and C-termini. Head-to-tail cyclization (i.e. amide bond
formation between N-terminal amino and C-terminal carboxyl ends)
results in a rigid polypeptide backbone that resists conformational
changes, often stabilizing peptide-receptor binding and protecting
the polypeptide termini from exoproteases. Alternatively,
modification of the polypeptide termini can stabilize polypeptides
through neutralization (C-terminal amidation) and prevention of
N-terminal degradation (N-terminal acetylation). Increased
polypeptide solubility and stability can also be conferred through
the conjugation of a hydrophilic molecule such as polyethylene
glycol (PEG).
[0647] Such modifications used to stabilize Flg22 polypeptides
include PEGylation, cyclization and amidation/acetylation, all of
which are described in Table 71. Stabilization of polypeptides
using PEGylation is carried out by linking the polypeptide to
polyethylene glycol (PEG). Once linked to the polypeptide, each PEG
subunit becomes tightly associated with 2 to 3 water molecules,
which then function in increasing the solubility of the polypeptide
as well as increasing its overall structure to make it less
susceptible to proteolytic degradation and more accessible to the
membrane FLS2 receptor at the plant surface. Cyclization can also
be used to increase the stability of the Flg polypeptide.
Stabilization of a polypeptide can also be obtained using
N-terminal acetylation and C-terminus through amidation where these
modifications generate a closer mimic of the native protein and
therefore may increase the biological activity of the
polypeptide.
TABLE-US-00073 TABLE 71 Modified Flg22 polypeptides Peptide
Description (Reference Code) Modification MW Sequence Bt.4Q7Flg22
Native derived 2229.42 nh2 DRLSSGKRINSASDDAAGLAIA (modified SEQ ID
sequence conh2 NO: 226) from Bacillus thuringiensis Bt.4Q7Flg22
N-terminal 229.3 Ac DRLSSGKRINSASDDAAGLAIA nh2 Mod-1 acetylation
(modified SEQ ID C-terminal NO: 226) amidation Syn05Flg22 Amino
acid 2255.46 Ac DRLSSGKRINSASDDPAGLAIA nh2 (modified SEQ ID
substitution NO: 578) (A16P) N-terminal acetylation C-terminal
amidation Syn05Flg22- PEGylation 2461 peg4 (where x =4) PEG4 before
amide DRLSSGKRINSASDDPAGLAIA conh2 (modified SEQ ID bond NO: 578)
conjugated to Flg22 Syn05Flg22-Cyc Cyclization 2196
Cyc(DRLSSGKRINSASDDPAGLAIA) (modified SEQ ID Head-to-Tail NO:
578)
[0648] The specialized, modified polypeptides as described in Table
71 including Syn05Flg22-Syn05 (J36), Syn05Flg22-PEG (J37) and
Syn05Flg22-Cyc were synthesized by the University of Missouri
Molecular Interactions Core (Columbia, Mo. USA), lyophilized to a
dry powder, and determined to be of the correct MW and desired
purity (>70%) by liquid chromatography-mass spectrometry (LC-MS)
and high-performance liquid chromatography (HPLC), respectively.
Standard synthesis polypeptides including Bt.4Q7Flg22 (SEQ ID NO:
226) and Bt.4Q7Flg22 Mod-1 (SEQ ID NO: 226; J41) were obtained from
Genscript (Piscataway, N.J. USA). All lyophilized polypeptides were
resuspended in ultrapure water to a 10 mM concentration and
serially diluted in ultrapure water to the desired concentration
for testing in soybean and corn ROS assays as described previously
in Example 15.
[0649] For soybean samples, fully expanded trifoliate leaves were
removed from V1 to V3 stage plants (variety Morsoy). Leaf discs (4
mm) were removed using a cork borer and then floated on 150 .mu.L
of water, abaxial side down, overnight before performing the ROS
assay previously described.
[0650] For corn samples, aerial tissue from V1 to V4 stage corn
plants (Beck's hybrid 5828 YX) were prepared as previously
described. The 1-mm excised leaf slices were then floated on 150 uL
of water overnight.
[0651] ROS activity assays were conducted using the methods as
previously reported in Example 15 with the exception that Relative
light units (RLUs) were recorded with a SpectraMax L luminometer
(0.5 s integration; 2.0 min intervals) over a time course of 40
minutes. Relative light units (RLUs) were first plotted over time
using a kinetic time course for each concentration tested, followed
by integration under the curve to calculate total RLU values
produced. Average total RLUs (n=4 samples per treatment) were then
graphed versus polypeptide concentration for each polypeptide for
soybean (Tables 72-73) and corn (Table 74).
[0652] A best fit logarithmic or linear regression (R>0.80) was
fit to the data for each treatment. Using the best-fit regression,
the polypeptide concentration required to reach a total RLU
production of 15,000 total RLU (corn) or a 50,000 total RLU
(soybean) was calculated for each polypeptide and % activity was
compared within each data set to the control treatment (Tables
72-74).
TABLE-US-00074 TABLE 72 Flg22-Bt modified at the N- and C-termini
polypeptides trigger reactive oxygen species production in soybean
Polypeptide % Activity Concentration (compared to (nM) unmodified
Treatment for 5 .times. 10.sup.4 total Bt.4Q7Flg22 (Code) RLU
production (SEQ ID NO: 226) Bt.4Q7Flg22 31.4 100.0% (SEQ ID NO:
226) Bt.4Q7Flg22 Mod-1 29.6 106.14% (SEQ ID NO: 226)
[0653] The Flg22 polypeptide concentration required to result in an
RLU output of 50,000 RLU for the Bt.4Q7Flg22S Mod-1 (SEQ ID NO:
226) was less than the current Bt.4Q7Flg22 (SEQ ID NO: 226) that
has been shown to produce yield gains and impart plant protective
qualities to soybean plants. This indicates that the modification
of Flg22 by N-terminal acetylation and/or C-terminal amidation does
not interfere with polypeptide binding to the FLS2 receptor, and
modifications may be used to produce a more active and/or stable
version of Flg22 as indicated by the +6% increase in activity of
Bt.4Q7Flg22S Mod-1 over Bt.4Q7Flg22 (Table 72).
[0654] Novel polypeptides were generated at the University of
Missouri Molecular Interactions Core (Columbia, Mo.) with a single
amino acid substitution (A16P) in comparison to the Bt.4Q7Flg22
(SEQ ID NO: 226) unmodified polypeptide, resulting in the
Syn05Flg22 (SEQ ID NO: 578) polypeptide which was amenable to
further modification by N-terminal PEGylation Syn05Flg22-PEG (SEQ
ID NO: 578) and Head-to-Tail cyclization Syn05Flg22-Cyc (SEQ ID NO:
578). A soy ROS assay was performed to assess the effect of these
two additional modifications, namely N-terminal PEGylation and
Head-to-Tail cyclization to a Flg22 polypeptide, with results shown
in Table 73.
TABLE-US-00075 TABLE 73 Modified, synthetic Flg22-Bt polypeptides
trigger reactive oxygen species production in soybean Polypeptide %
Activity Concentration (compared (nM) to Syn0Flg22; for 5 .times.
10.sup.4 total SEQ ID Treatment RLU production NO: 578) Syn05Flg22
89.8 100.0% (SEQ ID NO: 578) Syn05Flg22-PEG 64.8 138.6% (SEQ ID NO:
578) Syn05Flg22-Cyc 146.9 61.1% (SEQ ID NO: 578)
[0655] In a soy ROS assay to compare the relative activities of
Syn05Flg2 (SEQ ID NO: 578) to two modified versions of the
polypeptide, the PEGylated polypeptide Syn05Flg22-PEG (SEQ ID NO:
578) required substantially less amount of the polypeptide to
achieve a total of 50,000 RLU, which resulted in an increased
activity of +38% as compared to the non-PEGylated version or
Syn05Flg22 (SEQ ID NO: 578). PEGylation of the N-terminus of the
peptide increases the hydrophilicity of the polypeptide and may
increase affinity for the peptide-binding pocket of the FLS2
receptor. The cyclized version of Syn05Flg22-Cyc (SEQ ID NO: 578),
however, required more polypeptide provided in the ROS activity
assay (+57.1 nM more) compared to the non-cyclized version of
Syn05Flg22 to reach a total RLU production of 50,000 RLU in the
soybean ROS assay. This suggests that the cyclization of the Flg22
polypeptide (Syn05Bt.4Q7Flg22-Cyc) may result in a more rigid
polypeptide backbone with altered binding to the FLS2 receptor,
such that more cyclized peptide is required to reach an equivalent
ROS response. However, increased stability of a cyclized
polypeptide in the environment may compensate for the slight loss
in activity.
TABLE-US-00076 TABLE 74 Modified, synthetic Flg22-Bt polypeptides
trigger reactive oxygen species production in corn Polypeptide %
Activity Concentration (compared to (nM) for unmodified 5 .times.
10.sup.4 total Syn05Flg22 Treatment RLU production (SEQ ID NO: 578)
Syn05Flg22 19.5 100.0% (SEQ ID NO: 578) Syn05Flg22-PEG 15.0 130.3%
(SEQ ID NO: 578)
[0656] In a corn ROS assay, the PEGylated version of Syn05Flg22-PEG
required substantially less amount (almost 5 nM less) of the
polypeptide to achieve a total of 15,000 RLU, which resulted in an
increased activity of +30% as compared to the non-PEGylated version
or Syn05Flg22 (Table 74).
[0657] Modification of Flg22 (Bt) or Syn05Flg22 (Bt) polypeptides
by N-terminal acetylation, N-terminal PEGylation, C-terminal
amidation, and/or head-to-tail cyclization produces a peptide that
retains activity, as measured through ROS assays with corn and soy
tissues. These polypeptides could be used to deliver a further
stabilized Syn05Flg22 (SEQ ID NO: 578) derived polypeptide variant
for agricultural uses (either by foliar application, seed
treatment, in furrow application, application at transplant, or
trunk injection). Cyclization of Syn05Flg22-Cyc may be used to
increase the stability of the polypeptide yet compromised the ROS
activity, likely by affecting the affinity of the synthetic
polypeptide to the membrane FLS2 receptor.
Example 48. Adjuvant Compatibility with Flg22 Polypeptides
[0658] Product formulations using Flg22 polypeptides can generally
include antimicrobial biostatic preservatives such as Proxel and
surfactants. Therefore, the compatibility of these types of
adjuvants were tested using ROS activity assays to determine the
effect in solution on Flg22 responsiveness when used in combination
with such adjuvants. Proxels in general are broad spectrum biocides
for the preservation of many agricultural based products that
protect them against spoilage from bacteria, yeast and fungi.
Surfactants in general are also commonly used in agricultural
formulations to improve the penetration of many agrochemical
products into the plant for improved performance. In this study,
five different Proxels and two different non-ionic surfactants were
tested in formulations combined with Bt.4Q7Flg22 (SEQ ID NO: 226)
for effectiveness in producing a ROS response using a ROS activity
assay in soybean leaves. The different Proxel formulations (Lonza)
are described below in Table 75. Theses Proxel formulations were
mixed with 40 .mu.M Flg22 polypeptide at a range of recommended
label rates by the manufacturer (Lonza), and then diluted into the
ROS assay to a final polypeptide concentration of 100 nM and Proxel
concentrations indicated in Table 75. The tested non-ionic
surfactants were provided in the ROS assay at a range of
recommended label rates by the individual distributer or
manufacturer. The average four sample measurements RLU values
obtained after performing a ROS assay were collected using soybean
leaf disks as previously described in Example 15 with the exception
that Relative light units (RLUs) were recorded with a SpectraMax L
luminometer (0.5 s integration; 2.0 min intervals) over a time
course of 40 minutes. The average of these 4 RLU values is reported
in Table 76.
TABLE-US-00077 TABLE 75 Different PROXEL additives used as
adjuvants in formulations with polypeptides PROXEL Formulations
Chemical Description PROXEL BD20 A 20% aqueous dispersion of
1,2-benzisothiazoline-3-one PROXELBC An aqueous dispersion of a
blend of 1,2-benzisothiazoline-3-one (BIT),
5-chloro-2-methyl-4-isothhiazoline-3-one (CIMT) and 2-methyl-
4-isothiazoline-3-one (MIT) PROXELGXL A 20% aqueous dipropylene
glycol solution of 1,2-benzisothiazoline-3-one PROXELBN An aqueous
dispersion of 1,2-benzisothiazoline-3-one and 2-bromo-2-
nitropropen-1,3-diol PROXEL AQ A solution of
1,2-benzisothiazoline-3-one in water
TABLE-US-00078 TABLE 76 RLU output values from ROS activity assays
in soybean leaves using Flg22 polypeptide formulated using
different Proxel preservatives Treatment Comparison with and
Average RLU values without PROXEL preservative (Fold increase over
Concentration negative control) Mock (water) Negative Control 4823
Bt.4Q7Flg22 81887 (SEQ ID NO: 226 at 100 nM) (17.0X) (No PROXEL
Preservative Added) Bt.4Q7Flg22 89188 (SEQ ID NO: 226 at 100 nM) +
(18.5X) PROXEL BD20 (0.0005988%) Bt.4Q7Flg22 105527 (SEQ ID NO: 226
at 100 nM) + (21.9X) PROXELBD20 (0.00011976%) Bt.4Q7Flg22 136575
(SEQ ID NO: 226 at 100 nM) + (28.3X) PROXELBC (0.0005988%)
Bt.4Q7Flg22 92808 (SEQ ID NO: 226 at 100 nM) + (19.2X) PROXELBC
(0.00011976%) Bt.4Q7Flg22 128410 (SEQ ID NO: 226 at 100 nM) +
(26.6X) PROXELGXL (0.0002994%) Bt.4Q7Flg22 101847 (SEQ ID NO: 226
at 100 nM) + (21.1X) PROXELGXL (0.0008982%) Bt.4Q7Flg22 91554 (SEQ
ID NO: 226 at 100 nM) + (19.0X) PROXELBN (0.0002994%) Bt.4Q7Flg22
105164 (SEQ ID NO: 226 at 100 nM) + (21.8X) PROXELBN (0.00017964%)
Bt.4Q7Flg22 116634 (SEQ ID NO: 226 at 100 nM) + (24.2X) PROXELAQ
(0.0005988%) Bt.4Q7Flg22 98394 (SEQ ID NO: 226 at 100 nM) + (20.4X)
PROXELAQ (0.0035928%)
[0659] All Proxel preservative treatments as described in Table 76
were compatible when used in formulations with the Flg22
polypeptide (Bt.4Q7Flg22; SEQ ID NO: 226) as indicated by the high
RLU values (19.0-28.3.times. fold increase over mock treatment) as
comparable to the Bt.4Q7Flg2 polypeptide control without a Proxel
preservative (17.0.times. fold increase over mock treatment).
TABLE-US-00079 TABLE 77 RLU output values from ROS activity assays
in soybean leaf tissues using Flg22 polypeptide formulated using
different non-ionic surfactants Treatment Comparison with Average
RLU values and without Surfactant (Fold increase over Concentration
negative control) Mock (water) Negative Control 51288 Bt.4Q7Flg22
350503 (SEQ ID NO: 226) (6.8X) 52.2 nM Equivalent: 4.0 Fl. oz/Ac in
10 gallons water/Ac Bt.4Q7Flg22 142478 (SEQ ID NO: 226) (2.8X) 52.2
nM + Silwet-L77 (0.025%) Bt.4Q7Flg22 163517 (SEQ ID NO: 226) (3.2X)
52.2 nM + Silwet-L77 (0.10%) Bt.4Q7Flg22 329295 (SEQ ID NO: 226)
(6.4X) 52.2 nM + NIS90:10 (0.25%) Bt.4Q7Flg22 295726 (SEQ ID NO:
226) (5.8X) 52.2 nM + NIS90:10 (0.5%)
[0660] All surfactant (non-ionic) treatments as described in Table
77 were compatible when mixed at the indicated concentrations with
52.2 nM Flg22 polypeptide (Bt.4Q7Flg22; SEQ ID NO: 226), a
polypeptide concentration equivalent to 4.0 Fl oz/Ac usage rate of
Composition 1 (Bt.4Q7Flg22; SEQ ID NO: 226; 16.7 .mu.M) applied in
water at a spray rate of 10 gallons per acre. Fold-increase in ROS
production over the mock-treated control were comparable between
the Bt.4Q7Flg2 polypeptide control without a surfactant (6.8.times.
over control) versus Bt.4Q7Flg2 polypeptide with non-ionic
surfactant NIS90:10 applied at 0.25% v/v or 0.5% v/v of treatment
solution (5.8-6.4.times.), or slightly lower for Bt.4Q7Flg2
polypeptide with Silwet-L77 applied at 0.025% v/v or 0.1% v/v of
treatment solution (2.8-3.2.times.). Silwet-L77 (Helena), a
non-ionic organosilicone surfactant is formulated as a co-polymer
that has enhanced wetting and spreading characteristics when used
in aqueous sprays. NIS90:10 (Precision Laboratories) is a
low-foaming, non-ionic surfactant that enhances crop protection and
performance by improving spray solution coverage and penetration of
target leaf surfaces. Both the non-ionic surfactants combined with
Bt.4Q7Flg22 permitted ROS production in response to the Flg22
polypeptide in target leaf tissues (Table 77), and as such, are
compatible with Flg22 polypeptide foliar application in the
field.
Example 49: Production of BL4Q7Flg22 Using Fermentation Methods and
Activation by Enterokinase Cleavage for Disease Prevention Trials
in Potato, Lentils and Citrus Trees
[0661] The Bt.4Q7Flg22 (SEQ ID NO: 226) was provided in a
confirmation to stabilize the polypeptide and enhance activity for
an alternative production method, namely bacterial fermentation.
The Bt.4Q7Flg22 polypeptide was combined with an amyQ secretion
signal from Bacillus amyloliquefaciens alpha-amylase) fused to
glutathione S-transferase (GST) and an enterokinase cleavage tag
sequence as described: amyQ secretion signal (Bacillus
amyloliquefaciens alpha-amylase) GST (Schistosoma
japonicum)_linker_Enterokinase cleavage site_Bt.4Q7Flg22_stop codon
(Table 78).
TABLE-US-00080 TABLE 78 Cloning of Bt.4Q7Flg22 with sequences to
increase polypeptide stability and activity Description Amino Acid
Sequences amyQ secretion signal MIQKRKRTVSFRLVLMCTLLFVSLPITKTSA
(Bacillus amyloliquefaciens) SEQ ID NO: 769 GST
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRN (Schistosoma japonicum)
KKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPK SEQ ID NO: 770
ERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLK
MFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAF
PKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHP PK linker GGGGGGS SEQ
ID NO: 771 Enterokinase cleavage tag DDDDK (Consensus cleavage
target for bovine Enterokinase, light chain protease) SEQ ID NO:
772 Bt.4Q7Flg22 DRLSSGKRINSASDDAAGLAIA (SEQ ID NO: 226) (Bacillus
thuringiensis strain 4Q7) *DNA used for cloning from the amy E
secretion signal, GST and Bt.4Q7Flg22 (SEQ ID NO: 226) sequences
came from internal proprietary libraries; production stain code =
H101 (Chloramphenicol resistant)
TABLE-US-00081 TABLE 79 Cloning of Syn01Flg22 (SEQ ID NO: 571) with
sequences to increase polypeptide stability and activity
Description Amino Acid Sequence amyQ secretion MIQKRKRTVSFRLVL
signal MCTLLFVSLPITKTS (Bacillus A amyloliquefaciens) SEQ ID NO:
769 GST MSPILGYWKIKGLVQ (Schistosoma japonicum) PTRLLLEYLEEKYEE SEQ
ID NO: 770 HLYERDEGDKWRNKK FELGLEFPNLPYYID GDVKLTQSMAIIRYI
ADKHNMLGGCPKERA EISMLEGAVLDIRYG VSRIAYSKDFETLKV DFLSKLPEMLKMFED
RLCHKTYLNGDHVTH PDFMLYDALDWLYMD PMCLDAFPKLVCFKK RIEAIPQIDKYLKSS
KYIAWPLQGWQATFG GGDHPPK linker GGGGGGS SEQ ID NO: 771 Enterokinase
DDDDK cleavage tag (Consensus cleavage target for bovine
Enterokinase, light chain protease) SEQ ID NO: 772
Bt.4Q7Flg22-Syn01 DRLSSGKRINSAKDDAA (SEQ ID NO: 226) GLAIA
(Bacillus thuringiensis strain 4Q7) *DNA used for cloning from the
amy E secretion signal, GST and SynFlg22 (SEQ ID NO: 571) sequences
came from internal proprietary libraries. Production strain code =
H114 (Tetracycline resistant)
TABLE-US-00082 TABLE 80 Cloning of thionin-like protein with
sequence for secretion into fermentation growth media Description
Amino Acid Sequence amyQ secretion signal
MIQKRKRTVSFRLVLMCTLLFVSLPITKTSA SEQ ID NO: 769 (Bacillus
amyloliquefaciens) Thionin-like
RTCESQSHRFKGPCSRDSNCATVCLTEGFSGGDCRGFRRR protein CRCTRPCVFDEK (SEQ
ID NO: 650) (Synthetic)
[0662] The sequences in Tables 78, 79 and 80 were cloned into a
standard cloning vector containing an ampicillin selection marker
and either a chloramphenicol (Cm) or Tetracycline (Tet) selection
marker that can replicate in E. coli and then be transferred to
Bacillus subtilis strain K08 for production purposes (Production
strain codes: H101=amyQ-GST-EK-BtFlg22,
H114=amyQ-GST-EK-BtFlg22-Syn01, and H117=amyQ-Thionin-like). The
fermentation production was carried out by starting an overnight
culture in sterile 2XYT media (16 g Bacto tryptone, 10 g yeast
extract, and 5 g NaCl per liter; pH adjusted to 7.0) with 10
.mu.g/mL Cm or Tet, and then diluted into fresh 2XYT media with 10
.mu.g/mL Cm or Tet the following day. Productions were performed
using 50 mL (shake flask) or 3 L (glass bioreactor vessel) media
volumes with a constant temperature of 30.degree. C. Larger scale
up volumes can include 5 L to 1000 L+, including up to 30,000 L
volumes). Bacterial growth was monitored until the culture reached
an optical density of 0.6-1.0, after which Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) was added to a final
concentration of 0.1-1.0 mM to induce production of the
GST-Bt.4Q7Flg22 fusion protein. The induced production continued in
culture conditions for an additional 12-24 hours to produce the
fusion protein which is secreted into the growth media. Upon
secretion, the amyQ secretion tag is cleaved from the fusion
protein. The cultures were then centrifuged at 5000.times.g for 20
min and filtered through a 0.22 .mu.m bottle-top vacuum filter to
remove the bacterial cells. The sterile filtrate was then collected
and used as a foliar treatment on lentil and potato plants in
Sclerotinia disease prevention trials (Example 50) or as a trunk
injection of citrus trees for eradication and prevention of HLB
disease symptoms (Example 51).
[0663] After fermentation, two versions of the Bt.4Q7Flg22 (SEQ ID
NO: 226) polypeptide product were used in potato and lentil disease
prevention trials, one without enterokinase treatment (H101
filtrate, non-activated) and another with activation using a
enterokinase to cleave off the GST tag fused to the Flg22
polypeptide (H101 filtrate EK-activated). To activate Bt.4Q7Flg22,
the addition of 32 U (units) of enterokinase (EK: Enterokinase
light chain; New England BioLabs, Inc, Product No. P8070) was added
per 1 mL of H101 filtrate with an incubation period for 2-3 hours
at 30.degree. C. for the enzymatic release of Bt.4Q7Flg22 from the
GST-EK cleavage site resulting in an activated and released product
comprising the 22 amino acid Bt.4Q7Flg22 polypeptides. For the
citrus tree injection trial, H101 filtrate and H114 filtrate were
EK-activated with the addition of 0.8 U Enterokinse, light chain
(New England Biolabs, Inc, Product No. P8070) per mL of filtrate
with an incubation of 3 hours at 30.degree. C. for the enzymatic
release of Flg22 polypeptide. No activation treatment was required
for release of the thionin-like peptide which was produced without
a GST tag.
Example 50: Foliar Pre-Treatment with Bt.4Q7Flg22 Polypeptides
Protect Lentil and Potato Plants from Sclerotinia Stem Rot (White
Mold) Disease
[0664] Treatment applications of Bt.4Q7Flg22 (SEQ ID NO: 226) were
examined for protection of lentil and potato plants against disease
infection and progression with Sclerotinia sclerotiorum strain MT07
(white mold). Three different versions of the Bt.4Q7Flg22
polypeptides were examined in the disease assessment studies. A
formulated Bt.4Q7Flg22 (100 .mu.M) in sodium phosphate buffer, pH
5.7 and two different versions of Bt.4Q7Flg22 produced using
fermentation methods as described (Example 49) and provided with
and without activation of Flg22 with an enterokinase (EK) and
referred to as H101 filtrate.
[0665] Prior to using the three versions of the polypeptides in
disease protection assays with lentil and potato plants, a ROS
activity assay was performed using corn petiole tissues using
methods as described previously in Example 15 to ensure that the
Bt.4Q7Flg22H101 filtrate, particularly with the EK was active. The
H101 Bt.4Q7Flg22 filtrates without and with enterokinase (EK=8 U/mL
filtrate) activation were compared to the synthetic Bt.4Q7Flg22
(SEQ ID NO: 226) which was used to generate a series of
concentration comparisons to predict the Flg22 concentrations in
the H101 filtrates generated using fermentation procedures.
TABLE-US-00083 TABLE 81 ROS activity assay using Flg22 produced by
fermentation with and without enterokinase activation in corn
Average RLU Change (X) in RLU of Flg22 of Flg22 Flg22 Polypeptide
Polypeptide Polypeptide Compared Concentration Treatment to
Negative Control Bt.4Q7Flg22 6851 0.8X (SEQ ID NO: 226) (1.0 nM)
Bt.4Q7Flg22 9389 1.1X (SEQ ID NO: 226) (5 nM) Bt.4Q7Flg22 12157
1.4X (SEQ ID NO: 226) (25 nM) Bt.4Q7Flg22 25212 3.0X (fermentation
H101 filtrate) With (+) Enterokinase 8 U/mL (0.1 % v/v) Bt.4Q7Flg22
16891 2.0X (fermentation H101 filtrate) Without (-) Enterokinase
(0.1% v/v) *RLU values are reported as an average of 4 separate
measurements after the background RLU levels were subtracted.
[0666] The fermentation produced H101 filtrates of Bt.4Q7Flg22
provided with and without EK activation bath resulted in ROS
activities (RLU values) that were higher than the control (0 nM
Bt.4Q7Flg22). The H101 Bt.4Q7Flg22 filtrates (0.1% v/v) with EK
treatment provided to corn stem in the ROS assay resulted in a
3.0.times. increase in RLU values as compared to the control
treatment without any Flg22 polypeptide and the ROS response was
greater than Bt.4Q7Flg22 (SEQ ID NO: 226) provided at a
concentration of 25 nM; therefore, the estimated Bt.4Q7Flg22
activity in the undiluted EK-activated filtrate was Z 25 .mu.M The
fermentation produced H101 filtrates of Bt.4Q7Flg22 treatments that
were provided without EK still had ROS activity (2.0.times.RLU)
over the negative control treatment but with a lower increase seen
in RLU values as compared to the H101 filtrates of Bt.4Q7Flg22 with
EK. Once it was confirmed that the H101 filtrates had activity in
ROS assays (Table 81) they were assessed in disease protection
studies with potato and lentil.
[0667] Various formulations of Bt.4Q7Flg22 polypeptides were
provided to lentil and potato plants as a foliar pre-treatment to
plants 48 hours prior to inoculation with the Sclerotinia
sclerotiorum fungus and provided in combination with and without a
fungicide (Endura, active ingredient 70% boscalid), which is
effective in the treatment and protection of plants from infection
with the Sclerotinia fungus (white mold). The Bt.4Q7Flg22
formulations were tested using a crop-disease model (Montana State
University, Extension Services Crop Protection) to examine the
effects of each of the foliar pre-treatments on the prevention and
protection against disease and the development of symptoms. All
treatments including the water control were applied to the lentil
and potato plants using an air brush connected to a regulated air
compressor set with an output pressure to the brush at 50 psi.
After the pre-treatment, plants were inoculated with Sclerotinia
sclerotiorum using mycelial plug (agar plug covered with mycelium)
placed with the mycelia side touching the plant stem and placed in
humidity (100%) chambers for a set amount of time.
[0668] Lentils
[0669] Lentil (variety Pennel) plants were grown in soilless media
consisting of a mixture of 1:1 peat moss to perlite in 4'4' pots
with one plant per pot for 24 days in a controlled growth chamber
under growth conditions: 300-400 .mu.mol m.sup.-2 s.sup.-1 (light
photons) for a 13/11 light/day cycle and a 21.degree. C.
day/15.degree. C. night temperature range. The disease studies
included five lentil plants per each of six different foliar
treatments with 6 replicate plants per treatment, a total of 30
plants per foliar treatment as described in Table 35. All of the
foliar treatments used for pre-treatments were applied with the
addition of a non-ionic surfactant (ALLIGARE SURFACE; Alligare,
LLC) to a final concentration of 0.1% (v/v) or a concentration of
alkylpolyoxethylene, glycol derivatives. Each of the Bt.4Q7Flg22
treatments from the formulated and fermentation-derived productions
were provided at an application use rate of 0.1% (v/v) or 300 .mu.L
of product to 300 mL water and provided to each plant in an
equivalent number of sprays completely covering the foliage, using
8 mL of each treatment application for all 30 plants per treatment.
The Endura fungicide pre-treatment was applied at an equivalent
application use rate of 11 Fl. oz/Ac (803.8 mL/Ha) following the
application instructions on the specimen label. The treatments were
randomized using a complete random block design. Approximately 48
hours after the pre-treatment, plants were inoculated with a
Sclerotinia sclerotiorum strain isolated locally in Montana using
mycelial plug (agar plug covered with mycelium) placed with the
mycelia side touching the plant stem. The lentil plants were then
placed in humidity (100%) chamber for a period of 72 hours. At 11
days after inoculation disease symptoms were assessed and scored
and average fresh weight (total weight of each replicate--grams)
were collected (Table 82). Plants were allowed to dry for
approximately 3 weeks, and then dry weight was collected (total
weight per replicate--grams) (Table 82)
[0670] Disease scoring (disease scoring scale 0-7) and fresh weight
and dry weight (grams) were collected for each replicate of five
plants and then averaged for the total number of plants (n=30). The
disease scoring was ranked on a scale of 0-7, with a score of 0
equivalent to no disease and a score of 10 ranked as all plants did
not survive (Table 82).
TABLE-US-00084 TABLE 82 Disease assessment in lentils 10-days post
infection with Sclerotinia sclerotiorum Average Average Total Total
Disease Fresh Dry Scoring Weight per Weight Scale Plant per Plant
0-7 (grams) (grams) Treatment (STDEV) (STDEV) (STDEV) Water control
2.83 4.52 0.95 (.+-.1.84) (.+-.1.18) (.+-.0.21) Endura Fungicide
0.50 2.89 0.68 (.+-.0.84) (.+-.0.30) (.+-.0.05) Formulated 1.33
5.44 0.99 Bt.4Q7Flg22 (.+-.0.82) (.+-.0.48) (.+-.0.07) Endura
Fungicide + 1.0 4.95 0.96 Formulated (.+-.0.89) (.+-.0.59)
(.+-.0.05) Bt.4Q7Flg22 H101 filtrate non- 2.17 4.51 0.85 activated
(.+-.1.47) (.+-.0.93) (.+-.0.06) H101 filtrate EK 2.17 6.11 1.09
activated (.+-.1.33) (.+-.0.69) (.+-.0.11) *p value of .ltoreq.0.1
means there is a statistically significant difference between
treatments and the water control.
[0671] Foliar application of formulated Bt.4Q7Flg22 was compared to
the Endura fungicide, the Endura fungicide combined with formulated
Bt.4Q7Flg22 and the two Bt.4Q7Flg22 treatments provided with the
Flg22 polypeptides produced from the fermentation reactions with
and without EK activation as previously described in Table 82. All
of the foliar treatments in the crop-disease model were compared to
the each other and to water control treated plants and assessed 11
days post inoculation for the appearance of disease symptoms. Each
plant was assigned a disease score from 0-7. The total fresh and
dry weights (grams) were also determined per plant. The Endura
fungicide, a commercially available treatment for Sclerotinia
sclerotium resulted in the least disease symptom development on
lentil compared across all of the foliar treatments with a disease
score of 0.50 whereas, the water treatment (control) resulted in a
disease ranking score of 2.83. Foliar application of the formulated
Bt.4Q7Flg22 treatment to lentil plants resulted in an increased
resistance to Sclerotinia with a disease score of 1.33 (p
value=0.0972) compared to plants that received the water control
treatment. Unlike the Endura fungicide treatment which resulted in
slowed growth compared to the plants treated with the water
control, the formulated Bt.4Q7Flg22 treatment resulted in continued
vigorous growth during early symptom development. The lentil plants
that received the pre-treatment with the formulated Bt.4Q7Flg22 had
an average fresh weight of 5.44 grams per plant compared to plants
treated with the Endura fungicide alone (2.89 g) or the water
control (4.52 g). The combination treatment of the Endura fungicide
with the formulated Bt.4Q7Flg22 polypeptide further increased
protection of the lentil plants from symptom development with a
disease score of 1.0 (p value=0.0524) compared to the plants
treated with the water control. Plant weight (fresh and dry) for
plants that received the pre-treatment with the formulated
Bt.4Q7Flg22 polypeptide was greater than the fresh or dry weights
from plants that received the water control or the Endura fungicide
alone. The Bt.4Q7Flg22 polypeptides provided from the fermentation
derived products (non-EK activated and EK activated) were
equivalent in the disease symptom ranking with a disease score of
2.17, which was less than the disease score of plants treated with
the water only control application. However, the fresh weight per
plant treated with the EK-activated version of the Bt.4Q7Flg22
polypeptide had a significantly increased fresh weight of 6.11
grams (p value=0.0266) as compared to the water treated plants. The
EK-activated version of the Bt.4Q7Flg22 polypeptide also had the
overall highest fresh and dry weights compared to all of the other
treatments in Table 82. Other significant findings of this study
were that the formulated Bt.4Q7Flg22 polypeptide pre-treatment of
lentil plants protected the lentils from fungicide-induced damage.
The average fresh weight of the plants that received the Endura
fungicide was 2.89 g while the formulated Bt.4Q7Flg22 treatment was
5.44 g (p value=2.645.times.10-05). The fermentation produced
Bt.4Q7Flg22 containing the enterokinase (EK) enzyme was used to
cleave the Bt.4Q7Flg22 polypeptide from the GST-EK-Bt.4Q7Flg22 as
previously described. This Bt.4Q7Flg22 filtrate treatment provided
to lentil increased activity of the Flg22 polypeptide thus
resulting in significantly enhanced plant growth during the
infection period compared to the water treated control plants (p
value=1.180.times.10-05). The non-activated EK or
GST-EK-Bt.4Q7Flg22 or non-cleaved Bt.4Q7Flg22 filtrate did not
increase plant growth compared to the plants that received the
water control only treatment (p value=0.9852).
[0672] Potatoes
[0673] Seed potatoes (variety: Russet Burbank) were planted from 2
cm potato sections from which eye buds protrude (1 section per pot)
with the cut side down and planted approximately 7-8 cm deep in
soilless media consisting of a mixture of 1:1 peat moss to perlite
in 10.times.10 cm pots. Potatoes were grown with one plant per pot
for 19 days in a controlled growth chamber under standard
conditions of receiving approximately 300-400 .mu.mol m.sup.-2
s.sup.-1 (light photons) for a 13/11 light/day cycle and a
21.degree. C. day/15.degree. C. night temperature range. 19 days
after planting, the potato plants were pre-treated with the foliar
applications as described in Table 36. The disease studies included
five potato plants per each of six different foliar treatments with
6 replicate plants per treatment, a total of 30 plants per foliar
treatment as described in Table 83. All of the foliar treatments
used for pre-treatments were foliar applied with the addition of a
non-ionic surfactant (ALLIGARE SURFACE; Alligare LLC) to a final
concentration of 0.1% (v/v) or a concentration of
alkylpolyoxethylene, glycol derivatives. Each of the Bt.4Q7Flg22
treatments from the formulated and fermentation derived productions
were provided at an application use rate of 0.1% (v/v) or 300 .mu.L
of product to 300 mL water and provided to each plant in an
equivalent number of sprays completely covering the foliage using
15 mL of each treatment application for all 30 plants per
treatment. The Endura fungicide pre-treatment was applied at an
equivalent application use rate of 11 Fl. oz/Ac (803.8 mL/Ha)
following the application instructions on the specimen label. The
treatments were randomized using a complete random block design.
Approximately 48 hours after the pre-treatment, plants were
inoculated with Sclerotinia sclerotiorum using mycelial plug (agar
plug covered with mycelium) placed with the mycelia side touching
the plant stem and placed in a humid chamber (100%) for 192 hours.
At 16 days after inoculation disease symptoms were assessed and
scored and average stem fresh weight (total stem weight--grams)
were collected (Table 83). Plants were allowed to dry for 12 days,
and then dry weights were recorded (total stem weight--grams)
(Table 83).
[0674] After 48 hours, the potato plants were inoculated with
mycelia plugs placed on the soil near each plant and placed in a
humid misting chamber. The treatments were randomized using a
random block design. Disease scoring (scoring scale 0-6). Stem
fresh and dry weight (grams) were also collected from each plant
and then averaged for the total number of plants (n=30). Stem dry
weight was taken after the plants were fully desiccated at
approximately 12 days after harvest. Disease scores were assessed
16 days after the initial inoculation. The disease scoring was
ranked on a scale of 0-6, with a score of 0 equivalent to no
disease and a score of 6 ranked as all plants did not survive.
TABLE-US-00085 TABLE 83 Disease assessment in potatoes 15-days post
infection with Sclerotinia sclerotiorum Average Average Disease
Fresh Dry Stem Scoring Stem Weight Weight Scale per Plant per Plant
0-6 (grams; "g") (grams; "g") Treatment (STDEV) (STDEV) (STDEV)
Water control 2.83 96.01 12.08 (.+-.1.33) (.+-.17.05) (.+-.2.75)
Endura Fungicide 0.50 110.98 16.86 (.+-.0.84) (.+-.18.32)
(.+-.8.69) Formulated 1.83 113.37 15.01 Bt.4Q7Flg22 (.+-.0.98)
(.+-.14.58) (.+-.3.72) (SEQ ID NO: 226) H101 filtrate non- 2.33
98.08 12.96 activated (.+-.1.03) (.+-.15.34) (.+-.2.77) H101
filtrate EK 1.83 117.76 17.66 activated (.+-.0.75) (.+-.15.83)
(.+-.8.70) *p value of .ltoreq.0.1 means there is a statistically
significant difference between treatments and the water
control.
[0675] Foliar pre-treatment applications using the formulated
Bt.4Q7Flg22 and Bt.4Q7Flg22 polypeptides derived from the
fermentation products (H101 filtrates) were compared for disease
symptom development on potato plants that received the Endura
fungicide and the water control treatment. Foliar application of
formulated Bt.4Q7Flg22 (SEQ ID NO: 226) provided as a pre-treatment
to potato plants resulted in a disease score of 1.83 as compared to
plants that received the water control (disease score=2.83). Plants
that received pre-treatment with the Endura fungicide had the least
disease symptoms with a disease score of 0.50 (p value=0.0045)
compared to plants treated with the water control. The formulated
Bt.4Q7Flg22 polypeptide pre-treatment resulted in plants with an
average disease score similar to the enterokinase activated
Bt.4Q7Flg22 (H101 EK-activated) provided in a filtrate
(fermentation product)--both had disease scores of 1.83. The
non-activated EK or GST-EK-Bt.4Q7Flg22 or non-cleaved Flg22
filtrate (H101 non-activated) provided to plants had a score of
2.33 and was not significantly different from the disease score of
plants that were treated with the water control (p value=0.4835).
However, potato plants that received the pre-treatment with the
EK-activated Bt.4Q7Flg22 filtrate resulted in an increased average
stem fresh and dry weight per plant compared across all treatments
with approximately a 20 g increase in stem fresh weight and an
almost 6 g increase in stem dry weight per plant compared to plants
that received the water control pre-treatment. Plants that received
the formulated Bt.4Q7Flg22 polypeptide all had increased stem fresh
and dry weight as measured on a per plant basis compared to plants
that received the water only control application.
Example 51: Treatment of Candidatus Liberibacter asiaticus
infection with Flg22 and Anti-Microbial Polypeptides
[0676] Bt.4Q7Flg22 formulations were applied by trunk injection
treatments to both Valencia orange (Citrus sinensis) and Ruby Red
grapefruit (Citrus x paradisi) trees. The study was conducted at a
commercial grove orchard located in central Florida (Okeechobee
county). Injection treatment using the Bt.4Q7Flg22 polypeptide (SEQ
ID NO: 226) provided using a 1.times. Low Rate (0.55 micromoles
peptide; 0.138 .mu.M estimated concentration in phloem) and a
10.times. High Rate (5.5 micromoles peptide; 1.38 .mu.M estimated
concentration in phloem) was compared to the non-treated control
trees. The injection treatments were set up using a randomized
complete block design with 10 grapefruit trees (4 years old) per
treatment. The injections were provided in April (2017) at first
flush, a stage in growth from the emergence of leaves until they
expand to full size. Injection of grapefruit trees were conducted
using a low-pressure injection device, BRANDTENTREE (BRANDT).
Leaves from each of the grapefruit trees were sampled at the time
of injection (Day 0), 21 and 56 days post injection. A total of six
leaf samples per tree were selected to represent the population of
leaves on the tree in terms of leaf age, location, and presence of
visual symptoms. Each midrib was separated from the leaf blade and
immediately chopped into very small pieces with a new sterile razor
blade. Leaf samples from each tree were then placed in an
individual tube that was subsequently stored in a freezer at
-80.degree. C. until further processing. DNA extraction and
real-time polymerase chain reaction or quantitative PCR (qPCR)
analysis on these leaves was performed at Southern Gardens Citrus
(Clewiston, Fla.).
[0677] The presences of the CLas bacterial titers in the HLB
infected citrus trees can be determined with quantitative real-time
polymerase chain reaction (qPCR) methods using specific primers to
confirm the presence of the disease (Li, W. B., Hartung, J. S. and
Levy, L. 2008 "Optimized quantification of unculturable `Candidatus
Liberibacter spp.` In host plants using real-time PCR", Plant
Disease 92: 854-861). DNA extraction and quantitative PCR (qPCR)
analysis on these leaves was performed at Southern Gardens Citrus
(Clewiston, Fla.) using HLB primer set targeting the 16S DNA of C.
liberibacter bacteria 5'>>3' (forward): HLB as
TCGAGCGCGTATGCAATACG (SEQ ID NO: 773); (reverse) HLBr:
GCGTTATCCCGTAGAAAAAGGTAG (SEQ ID NO: 774); HLBpc (probe):
AGACGGFTGAGTAACGCG (SEQ ID NO: 775) labeled with an intercalating
fluorescent reporter dye]. Forty cycles of qPCR were conducted and
the fluorescent signal which is proportional to the amount of dsDNA
in solution was measured. The qPCR analysis allows for the
detection of the CLas bacteria in citrus tissue. The cycle
threshold (Ct) values from the qPCR analysis were obtained per each
treatment. The Ct measurement is equivalent to the number of PCR
cycles required to produce a relative threshold level. As in common
practice within the field of molecular biology, the change in Ct
value is reported to indicate the relative quantity of CLas DNA
either in treated vs untreated samples or in treated samples at one
time point vs another time. The higher the Ct value, the greater or
more effective the treatment effect, which is indicated by the
reduction/elimination of CLas bacteria from the tree. A percentage
reduction in bacterial load can be computed as:
% .times. .times. reduction .times. .times. in .times. .times.
sample .times. .times. over .times. .times. time = ( 1 - 2 [ C
.times. t .function. ( inital .times. .times. time ) - Ct
.function. ( later .times. .times. time ) ] ) * 100 .times. %
##EQU00002## or ##EQU00002.2## % .times. .times. reduction .times.
.times. in .times. .times. treated .times. .times. vs . .times.
control .times. .times. sample = ( 1 - 2 [ C .times. t .function. (
control .times. .times. sample ) - Ct .function. ( treated .times.
.times. sample ) ] ) * 1 .times. 0 .times. 0 .times. %
##EQU00002.3##
[0678] The results from the grapefruit trial are shown in FIG. 9.
The average values from the Ct comparisons (n=10 trees per
treatment) obtained from the qPCR analysis from the T0 timepoint
(day of injection), the T21 and the T56 timepoints (21 and 56 days
post injection) are reported with the standard error from the mean
Ct values in FIG. 9 (T0=dark grey bars; T21=white bars; T56=light
gray bars; average Ct values marked with an "x"). Any outlier
values are indicated by the small circles located outside the
standard error bars for each treatment. The control or grapefruit
trees that were not injected had the lowest Ct values in a range of
Ct near 25 for all treatment timepoints. Leaves sampled from
grapefruit trees that received injection treatments with the
1.times. and 10.times.Bt.4Q7Flg22 polypeptide formulations resulted
in slightly higher Ct counts as compared to leaves from the control
trees (FIG. 9). The higher the Ct. value, the greater the treatment
effect for controlling or reducing the infection of the CLas
bacteria from spreading. The average Ct value in leaves taken from
the T21 sampling was greater than the Ct value from the T56
sampling but both were significantly increased over the
non-injected control leaves or leaves from trees that received
injections with the Bt.4Q7Flg22 polypeptide formulations (FIG. 9,
average Ct values marked with "x").
[0679] In another study using Valencia orange (Citrus sinensis)
also conducted at the commercial grove orchard located in central
Florida (Okeechobee county). Injection treatments using
formulations of Bt.4QFlg22 (SEQ ID NO: 226) were compared to
antimicrobial polypeptides known as thionins. Thionin injection was
provided as a mixture of thionin polypeptides (SEQ ID NOs: 651, 652
and 653) which are characterized as "un-tagged" or without a phloem
localization sequence. In addition to the un-tagged thionin
mixture, a "tagged" thionin polypeptide that comprised a phloem
localization sequence (SEQ ID NO: 650) was used as a comparative
injection treatment. The phloem targeted or "tagged" version was
used to target the thionin specifically to the phloem where CLas
bacteria reside and multiply. The injection treatments were applied
to orange trees using a randomized complete block design with a
total of 8 orange trees (8 years old) per treatment for the
untreated control and Bt.4QFlg22 treatments, and a total of 5
orange trees per treatment for the thionin treatments. The
injections were provided in April (2017) at first flush, a stage in
growth from the emergence of leaves until they expand to full size.
Injection of the orange trees were conducted using a low-pressure
injection device, BRANDTENTREE (BRANDT). The Bt.4Q7Flg22
polypeptide 1.times. (0.138 .mu.M) and a 10.times. (1.37 .mu.M)
concentrations, the "untagged" and the "tagged" thionin
polypeptides were all compared to trees that received no injection
treatment (control). Leaves from the orange trees were sampled per
each treatment at the time of injection (Day 0) and at T56, or 56
days post injection.
[0680] A total of six leaf samples per tree, were selected to
represent the population of leaves on the tree in terms of leaf
age, location, and presence of visual symptoms. Each midrib was
separated from the leaf blade and immediately chopped into very
small pieces with a new sterile razor blade. Leaf samples from each
tree were then placed in an individual tube that was subsequently
stored in a freezer at -80.degree. C. until further processing. DNA
extraction and real-time polymerase chain reaction or quantitative
PCR (qPCR) analysis on these leaves was performed at Southern
Gardens Citrus (Clewiston, Fla.) using the methods as described
above for performing Ct analysis.
[0681] Results from the Valencia orange trial are shown in FIG. 10
(T0=dark grey bars; T56=white bars). Leaf tissues from the control
orange trees had the lowest Ct values in a range of Ct near 25-30
for treatment timepoints T0 and T56 indicating that titer levels of
the CLas bacteria did not change in these trees. Both of the
thionin treatments "untagged" and "tagged" had higher average Ct
values in leaves taken from the T56 sampling as compared to the
average Ct values from T56 leaves sampled from the water-injected
controls (FIG. 10; average Ct values marked with "x"). Any outlier
values are indicated by the small circles located outside the
standard error bars for each treatment. Leaves sampled from trees
that received the phloem targeted thionin "tagged" treatment had a
higher average Ct value at T56 compared to leaves from trees that
received non-targeted or "un-tagged" thionin treatment. Leaves
sampled from orange trees that received injection treatments with
the 1.times. and 10.times.Bt.4Q7Flg22 polypeptide formulations
resulted in significantly higher Ct counts from the T0 to T56
timepoints shown by the average increase in Ct at T56 compared to
T0 (FIG. 10; average Ct values marked with "x"). Leaves from trees
injected with both Bt.4Q7Flg22 polypeptide formulations (1.times.
and 10.times.) also had significantly higher Ct values compared to
leaves samples from the control trees. The Bt.4Q7Flg22 are
effective treatments for controlling or reducing the titer levels
of the CLas bacteria in the infected orange trees (FIG. 9).
[0682] The Bt.4Q7Flg22 polypeptides provided as injection
treatments using final concentrations at the 1.times. (0.138 .mu.M)
and 10.times. (1.38 .mu.M) were both effective in reducing CLas
titer levels in the leaf tissue sampled 8 weeks post injection. The
higher concentration of the Bt.4Q7Flg22 polypeptide 10.times. (1.38
.mu.M) however was even more effective resulting in a 37% reduction
(Trial 1) and a 43% reduction (Trial 2) in CLas titer levels.
TABLE-US-00086 TABLE 84 Treatment effectiveness of Bt.4Q7Flg22 on
reducing CLas bacterial titer levels 8 weeks post injection
treatment on citrus (Valencia orange and Ruby Red Grapefruit)
Percentage Reduction in CLas titer Injection Treatment Normalized
to the Control Concentration Trial 1 Trial 2 1X Bt.4Q7Flg22 33% 21%
(SEQ ID NO: 226) 0.138 .mu.M estimated concentration in tree
vasculature 10X Bt.4Q7Flg22 37% 43% (SEQ ID NO: 226) 1.38 .mu.M
estimated concentration in tree vasculature
[0683] Previous results indicate that Bt.4Q7Flg22 (SEQ ID NO: 226)
promotes plant growth throughout periods of disease (Example 50).
To assess for a potential plant growth benefit to injecting
HLB-infected `Valencia` Orange and `Ruby Red` Grapefruit trees with
Bt.4Q7Flg22 (SEQ ID NO: 226), current year growth was measured in
May 2018 for the same trees that were injected with Bt.4Q7Flg22 in
April 2017 and assessed for CLas bacterial titer at the commercial
grove orchard located in central Florida (Okeechobee county). Each
tree was visually assessed for regions of current season growth
with green color to the branches, as compared to old growth
branches that are more woody in appearance with a dark
greenish-brown to brown hue. Three representative branches with new
growth were selected per tree, and the distance in inches from the
start of green growth (oldest node) to the tip of the youngest node
was measured with a flexible measuring tape. Data was collected for
trees injected with 1.times. and 10.times.Bt.4Q7Flg22 (SEQ ID NO:
226) as well as the untreated control, with 8 trees per treatment
for the `Valencia` orange trial and 9-10 trees per treatment for
the `Ruby Red` Grapefruit trial (n=24-30 measurements per
treatment). Only one tree in the `Ruby Red` Grapefruit trial was
lost from the original trial (1.times. Bt.4Q7Flg22 treatment
group), presumably due to hurricane-strength wind damage in
September 2017. For each trial, the average new growth length
(inches) was calculated and normalized to the untreated control
(Table 85).
TABLE-US-00087 TABLE 85 Bt.4Q7Flg22 trunk injection increases new
branch growth in `Valencia` orange and `Ruby Red` grapefruit
Average Flush Flush length Length (% of Trial Treatment (inches)
control) Valencia Orange- Control 7.23 100% Injected April 2017, 1X
Bt.4Q7Flg22 (SEQ 13.33 184% Measured May 2018 ID NO: 226) 0.138
.mu.M estimated concentration in tree vasculature 10X Bt.4Q7Flg22
12.29 170% (SEQ ID NO: 226) 1.38 .mu.M estimated concentration in
tree vasculature Red Grapefruit- Control 8.05 100% Injected April
2017, 1X Bt.4Q7Flg22 (SEQ 10.83 135% Measured May 2018 ID NO: 226)
0.138 .mu.M estimated concentration in tree vasculature 10X
Bt.4Q7Flg22 (SEQ 9.13 113% ID NO: 226) 1.38 .mu.M estimated
concentration in tree vasculature
[0684] These results demonstrate the ability of Flg22 compositions,
which displayed reduced CLas bacterial titer compared to untreated
plants (FIG. 9 and FIG. 10), to also enhance the growth of sweet
orange and grapefruit trees (Table 85). Enhanced branch growth
serves as an indicator of enhanced fruit yield as more leaves are
produced to sustain fruit growth throughout the season. In
comparison to the untreated control, orange and grapefruit trees
receiving the 1.times.Bt.4Q7Flg22 injection in April 2017 had on
average 6.1 more inches (+85%) or 2.8 more inches (+35%) of new
branch growth, respectively. The 10.times. injection dose of
Bt.4Q7Flg22 was also effective at increasing growth, with 5.1 more
inches (70%) and +1.1 more inches (+13%) of new branch growth in
orange and grapefruit trees, respectively. As the
10.times.Bt.4Q7Flg22 injection did not perform better than the
1.times. injection for enhancing growth in 2018, and bacterial
titer reductions were similar in 2017. The 1.times.Bt.4Q7Flg22
injection provides a sufficient response in the plant. Importantly,
growth measurement indicated that no phytotoxicity occurred after
Flg22 trunk injection at either the 1.times. or 10.times. rate.
[0685] As these plants were not 100% cleared of disease-causing
bacteria, these results also demonstrate the ability of the plants
injected with Bt.4Q7Flg22 to continue to grow despite the presence
of HLB-causing bacteria. Provided that CLas strains with antibiotic
resistance are predicted to emerge and become an additional hurdle
for HLB-control, Flg22 injection represents a desirable alternative
to antibiotic treatments for ameliorating plant growth and reducing
bacterial titer. The trees receiving the Flg22 injections in this
example were maintained with a standard commercial citrus treatment
program, which further demonstrates the ability to add Flg22 citrus
injections to standard grower practices.
Example 52: Foliar and Trunk Injection of Flg22 Applied Alone or in
Combination with Antimicrobial or Plant-Health Promoting Compounds
Increase New Shoot Growth in Orange Trees
[0686] In subsequent trials in April (2018), Flg22 formulations
were applied by trunk injection treatments or foliar spray at two
independent trial sites. Trials were designed to 1) test Flg22
polypeptide variants produced synthetically and by fermentation, 2)
compare the efficacy of the Flg22 variant previously used for
citrus injection trials in 2017, Bt.4Q7Flg22 (SEQ ID NO: 226),
versus Syn01Bt.4Q7Flg22 (SEQ ID NO: 571) which was effective as
both a foliar and seed treatment for increasing yield in row crops,
3) compare Flg22 application methods, namely trunk injection versus
foliar spray to the canopy, and 4) test combinatorial treatments
between Flg22 peptides and oxytetracycline injection, L-cysteine,
and Benzo (1,2,3) thiadiazole-7-carbothioic acid-S-methyl ester
(also known as BTH) as the commercially available formulation
ACTIGARD WG. L-cysteine is an essential, proteinogenic amino acid;
and BTH is a salicylic acid analog with increased stability that is
used agriculturally as an activator of plant immune responses and
is approved for application to citrus trees as root drench or
irrigation treatment to prevent citrus canker caused by Xanthomonas
axonopodis pv citri.
[0687] In March 2018, trees were treated at two separate sites.
Three-year old Hamlin orange trees (Citrus sinensis) were treated
at a commercial grove orchard located in central Florida
(Okeechobee County). A similar trial was conducted in a commercial
grove of 6-year old Vemia orange trees on Swingle rootstock at Lake
Wales, Fla. (Polk County). Treatments were applied as listed in
Table 85 below using a low-pressure injection device, BRANDT ENTREE
(BRANDT) for trunk injection or a CO2-pressurized backpack sprayer
that produced a fine mist for foliar spray. Trunk injections were
as described in Example 51. Foliar compositions of Bt.4Q7Flg22 were
diluted in water with a non-ionic surfactant (Precision Labs
NIS90:10; 0.1% v/v of spray tank volume) and evenly applied to the
canopy of the tree at a spray rate of 3 Liters per tree. Blocks of
trees receiving a foliar treatment were spaced in the trial area
with a gap (skipped tree) in between treatment blocks to avoid
drift of treatment into neighboring treatment blocks. Treatments
were applied during the early morning or late evening during a
period of low wind (<5 mph), and conditions were such all spray
treatments dried on leaves within a period of 4 hours. Combination
treatments described in Table 86 were either co-injected in the
same BRANDT ENTREE battle (Citrus Composition 7, Citrus Composition
8) or applied separately as an oxytetracycline injection followed
by a Bt.4Q7Flg22-Syn01 foliar treatment on the same day (Citrus
Composition 11, Citrus Composition 12). For all treatments, 10
trees were used per treatment, separated into two replicated blocks
of five trees each. Citrus compositions 1-8 were applied at bath
the Okeechobee and Polk County groves, while Citrus Compositions
9-12 were applied at Okeechobee grove alone.
TABLE-US-00088 TABLE 86 Treatment compositions tested for
ameliorating the effects of HLB in orange trees Treatment
Composition Formulation Method Application Use Rate Citrus
Bt.4Q7Flg22 Trunk 2.75 mL/tree Composition 1 (SEQ ID NO: 226) 100
.mu.M Injection (estimated 0.138 .mu.M in plant 10 mM Sodium
vasculature) Phosphate Buffer, pH 5.7 Citrus Bt.4Q7Flg22-Syn01 (SEQ
Trunk 2.75 mL/tree Composition 2 ID NO: 571) 100 .mu.M Injection
(estimated 0.138 .mu.M in plant 10 mM Sodium vasculature) Phosphate
Buffer, pH 5.7 Citrus Bt.4Q7Flg22 (SEQ Trunk 80 mL/tree Composition
3 ID NO: 226) Injection (fermentation brothfiltrate) With (+)
Enterokinase 0.8 U/mL Citrus Bt.4Q7Flg22-Syn01 Trunk 80 mL/tree
Composition 4 (SEQ ID NO: 571) Injection (fermentation broth
filtrate) With (+) Enterokinase 0.8 U/mL Citrus Bt.4Q7Flg22-Syn01
Foliar 3.0 mL/tree in a spray carrier Composition 5 (SEQ ID NO:
571) 100 .mu.M Spray volume of 3 L water + 0.1% 10 mM Sodium v/v
Precision Labs NIS90:10 Phosphate Buffer, pH 5.7 Citrus
Bt.4Q7Flg22-Syn01 (SEQ Foliar 12.0 mL/tree in a spray Composition 6
ID NO: 571) 100 .mu.M Spray carrier volume of 3 L water + 10 mM
Sodium 0.1% v/v Precision Labs Phosphate Buffer, pH 5.7 NIS90:10
Citrus Part A Trunk 2.75 mL/tree Composition 7 Bt.4Q7Flg22-Syn01
(SEQ Injection (estimated 0.138 .mu.M in plant ID NO: 571) 100
.mu.M vasculature) 10 mM Sodium Phosphate Buffer, pH 5.7 Part B
Trunk 20 mL/tree ACTIGARDWG Injection (1 g per tree) (Active
Ingredient: 50% Acibenzolar-S-methyl: Benzo (1,2,3) thiadiazole-7-
carbothioic acid-S-methyl ester; BTH) (50 mg/mL solution in water)
Citrus Part A Trunk 2.75 mL/tree Composition 8 Bt.4Q7Flg22-Syn01
(SEQ Injection (estimated 0.138 .mu.M in plant ID NO: 571) 100
.mu.M vasculature) 10 mM Sodium Phosphate Buffer, pH 5.7 Part B
Trunk 20 mL/tree L-Cysteine Injection (60 mg per tree) (3 mg/mL
solution in water) Citrus Part A Foliar 3.0 mL/tree in a spray
carrier Composition Bt.4Q7Flg22-Syn01 (SEQ Spray volume of 3 L
water + 0.1% 10 ID NO: 571) 100.mu. M v/v Precision Labs NIS90:10
10 mM Sodium Phosphate Buffer, pH 5.7 Part B Trunk 20 m L/tree
Oxytetracycline-HCl Injection (0.45 g per tree) (22.5 mg/mL
solution in water) Citrus Part A Foliar 12.0 mL/tree in a spray
Composition Bt.4Q7Flg22-Syn01 (SEQ Spray carrier volume of 3 L
water + 11 ID NO: 571) 100 .mu.M 0.1% v/v Precision Labs 10 mM
Sodium NIS90:10 Phosphate Buffer, pH 5.7 Part B Trunk 20 m L/tree
Oxytetracycline-HCl Injection (0.45 g per tree) (22.5 mg/mL
solution in water)
[0688] To assess for a potential plant growth benefit to injecting
or spraying HLB-infected orange trees with different formulations
of Flg22 polypeptides alone or in combination with antimicrobial or
plant-health promoting compounds, new flush length was measured in
May 2018 for trees that were treated in March 2018 at commercial
groves in Okeechobee county, FL and Polk county, FL. At the time of
treating plants at both locations (March 2018), trees exhibited
darker green leaves with 2018 season fruit beginning to develop. In
the two-month interval between treatment (March 2018) and the time
of tree measurement (May 2018), trees entered a period of spring
flush with new growth visible as very light green, flexible
branches with similarly light green leaves. Each tree was assessed
for new flush, and three representative branches with new growth
were selected per tree. The distance in inches from the start of
light green growth (oldest node) to the tip of the youngest node
was measured with a flexible measuring tape. Data was collected for
10 trees per treatment including the untreated control, for a total
of 30 measurements per treatment. Represented in Table 86 is the
average flush length (inches) for each treatment across the two
grove sites in Okeechobee and Polk counties, with growth normalized
to the untreated control.
TABLE-US-00089 TABLE 87 Flg22 variants applied as either a trunk
injection or foliar spray increase new branch growth in `Hamlin`
and `Vernia` orange trees Average Flush Flush length Treatment
Length (inches) (% of control) Untreated Control 3.08 100% Citrus
Composition 1 3.84 125% Bt.4Q7Flg22 (SEQ ID NO: 226) 2.75 mL/tree
injection Citrus Composition 2 4.48 146% Bt.4Q7Flg22-Syn01 (SEQ ID
NO: 571) 2.75 mL/tree injection Citrus Composition 3 5.18 169%
Bt.4Q7Flg22 (SEQ ID NO: 226) Enterokinase (EK) -activated filtrate
80 mL/tree injection Citrus Composition 4 3.28 107%
Bt.4Q7Flg22-Syn01 (SEQ ID NO: 571) Enterokinase (EK)-activated
filtrate 80 mL/tree injection Citrus Composition 5 3.66 119%
Bt.4Q7Flg22-Syn01 (SEQ ID NO: 571) 1X foliar spray Citrus
Composition 6 3.76 122% Bt.4Q7Flg22-Syn01 (SEQ ID NO: 571) 4X
foliar spray
[0689] Growth measurements of `Hamlin` and `Vemia` new shoots,
taken two months after either trunk injection or foliar spray
application of Flg22 variants, indicated that Bt.4Q7Flg22 (SEQ ID
NO: 226) and Syn01Flg22 (SEQ ID NO: 571) are both effective at
promoting greater growth than the untreated control. On average,
untreated control shoots were 3.08 inches in length, while
Bt.4Q7Flg22-injected trees had 25% longer shoots (3.84 inches) and
Syn01Flg22 injected trees 146% longer shoots (4.48 inches).
Bt.4Q7Flg22 and Syn01Flg22 produced through fermentation methods
described in Example 49 were also effective at increasing shoot
growth when injected into the trunk at a rate of 80 mL/tree. Citrus
composition 3 containing Bt.4Q7Flg22 produced by fermentation of
strain H101 and treated with 0.8 U/mL Enterokinase (New England
Biolabs; Product Code P8070) was the most effective, with shoots
measuring on average 169% (5.18 inches) longer than the untreated
control.
[0690] Foliar application of Flg22 variants, which is effective for
promoting growth of kiwi, soy, lentils, and potatoes under disease
pressure were also tested for the ability to promote growth of
HLB-infected orange trees. Table 88 shows that Citrus Compositions
5 and 6 comprised of a 1.times. or 4.times. dose of
Syn01Bt.4Q7Flg22 (SEQ ID NO: 571), respectively, are also effective
at promoting new shoot growth in orange trees. The 1.times. and
4.times. doses were similarly effective, with the 1.times. foliar
rate measuring 119% longer shoots than the control, and the
4.times. rate measuring 122% longer shoots than the control. These
results show that a foliar application of Flg22 polypeptide can be
used as part of a standard program of care of citrus grove
trees.
TABLE-US-00090 TABLE 88 Injection of Bt.4Q7Flg22-Syn01 in
combination with plant-health promoting compounds increases new
branch growth in `Hamlin' and Vernia` orange trees Average Flush
Flush length Treatment Length (inches) (% of control) Untreated
Control 3.08 100% Citrus Composition 2 4.48 146% Bt.4Q7Flg22-Syn01
(SEQ ID NO: 571) 2.75 mL/tree injection Citrus Composition 7 3.44
112% Bt.4Q7Flg22-Syn01 (SEQ ID NO: 571) 2.75 mL/tree injection +
BTH (ACTIGARD WG; 1 g/tree injection) Citrus Composition 8 5.87
191% Bt.4Q7Flg22-Syn01 (SEQ ID NO: 571) 2.75 mL/tree injection +
L-Cysteine (60 mg/tree injection)
[0691] Next, the combination of Syn01Bt.4QFlg22 (SEQ ID NO: 571)
with BTH (ACTIGARD WG) or L-cysteine was investigated at both the
Melvin locations and Lake Wales groves. Both combination treatments
in Table 88 showed greater new flush length in comparison to the
untreated control, showing that Flg22 polypeptides can be used in
combination with amino acids, plant hormones, or plant
hormone-mimics to improve citrus tree health.
TABLE-US-00091 TABLE 89 Foliar spray application of
Bt.4Q7Flg22-Syn01 in combination with oxytetracycline injection
increases new branch growth in 3-year old `Hamlin` orange trees
Average Flush Flush Length Treatment (see table 85) Length (inches)
(% of control) Untreated Control 1.67 100% Citrus Composition 9
3.80 228% Oxytetracycline-HCl (0.45 g/tree) + Syn01Bt.4Q7Flg22 (SEQ
ID NO: 571) 1X foliar spray Citrus Composition 10 3.32 199%
Oxytetracycline-HCl (0.45 g/tree) + Syn01Bt.4Q7Flg22 (SEQ ID NO:
571) 4X foliar spray
[0692] In a separate trial, the combination of Syn01Flg22 (SEQ ID
NO: 571) and oxytetracycline treatments were observed. On the same
day that trees were injected with oxytetracycline, groups of 10
trees were also sprayed with a foliar application of Syn01Flg22 at
a 1.times. rate or 10.times. rate. These results show that the
antibiotic and polypeptide treatments are compatible and that no
phytoxicity was observed due to the dual treatment. A standard
program could be envisioned where grower alternated tree injections
with foliar treatments for enhanced control of HLB symptoms and for
reducing CLas titer.
Example 53: Disease Protection Using Bt.4Q7Flg22 and Gm.RHPP Foliar
Applications on Soybean Plants to Protect from Diseases Caused by
Phakopsora Pachyrhizi and Cercospora Kikuchii
[0693] Table 90 describes the compositions and corresponding use
rates tested in the following example.
TABLE-US-00092 TABLE 90 BL4Q7Flg22 and Gm.RHPP foliar applications
on soy protect plants from Phakopsora pachyrhizi and Cercospora
kikuchii Application Use Rate Fluid ounce/acre (Fl. oz/Ac)
Composition Foliar Formulation Milliliters/hectare (mL/Ha)
Composition 12 FOX Fungicide 5.48 Fl. oz/Ac or 400 mL/Ha
Composition 13 Bt.4Q7Flg22 (SEQ ID NO: 226) 16.7 .mu.M 2.05 Fl.
oz/Ac or 1.67 mM Sodium Phosphate Buffer, pH 5.7 150 mL/Ha PROXEL
BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) Composition 14 Bt.4Q7Flg22 (SEQ ID NO: 226) 16.7 .mu.M 4.11
Fl. oz/Ac or 1.67 mM Sodium Phosphate Buffer, pH 5.7 300 mL/Ha
PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1
.mu.M (MIT) Composition 15 Gm.RHPP (SEQ ID NO: 600) 100 .mu.M 2.05
Fl. oz/Ac or PROXEL BC preservative: 330.7 .mu.M; 150 mL/Ha 50.1
.mu.M (CMIT); 21.71 .mu.M (MIT) Composition 16 Gm.RHPP (SEQ ID NO:
600) 100 .mu.M 4.11 Fl. oz/Ac or PROXEL BC preservative: 330.7
.mu.M; 300 mL/Ha 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) Composition
17 FOX Fungicide + 5.48 Fl. oz/Ac or Bt.4Q7Flg22 (SEQ ID NO: 226)
16.7 .mu.M 400 mL/Ha + 1.67 mM Sodium Phosphate Buffer, pH 5.7 2.05
Fl. oz/Ac or PROXEL BC preservative: 330.7 .mu.M (BIT); 150 mL/Ha
53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) Composition 18 FOX Fungicide +
5.48 Fl. oz/Ac or Bt.4Q7Flg22(SEQ ID NO: 226) 16.7 .mu.M 400 mL/Ha
+ 1.67 mM Sodium Phosphate Buffer, pH 5.7 4.11 Fl. oz/Ac or PROXEL
BC preservative: 330.7 .mu.M (BIT); 300 mL/Ha 53.5 .mu.M (CMIT);
26.1 .mu.M (MIT) Composition 19 FOX Fungicide + 5.48 Fl. oz/Ac or
Gm.RHPP (SEQ ID NO: 600) 100 .mu.M 400 mL/Ha + PROXEL BC
preservative: 330.7 .mu.M; 2.05 Fl. oz/Ac or 50.1 .mu.M (CMIT);
21.71 .mu.M (MIT) 150 mL/Ha Composition 20 FOX Fungicide + 5.48 Fl.
oz/Ac or Gm.RHPP (SEQ ID NO: 600) 100 .mu.M 400 mL/Ha + PROXEL BC
preservative: 330.7 .mu.M; 300 mL/Ha 50.1 .mu.M (CMIT); 21.71 .mu.M
(MIT) * Foliar compositions contained 0.1% (v/v) PROXEL BC
preservative, an aqueous dispersion of a blend of 330.7 mM
1,2-benzisothiazolin (BIT), 53.5 mM
5-chloro-2-methyl-4-isolthiazolin-3-one (CMIT), and 26.1 mM
2-methyl-4-isothiazolin-3-one (MIT). Foliar compositions were
applied at the indicated rates (Fl. oz/Ac or mL/Ha) in a carrier
volume of 150 L/Ha or 16 gallons/acre water with 0.5% (v/v) AUREO
methylated bean oil surfactant (Composition 13) or with 0.33% (v/v)
Agris Parrafinic mineral oil (stock concentration of 795 g/L or
79.5% (p/v) (Compositions 13-20).
[0694] Replicated field trials were conducted across three
locations in Paraguay (Yatytay, Obligado, and Capitcn Miranda)
using a foliar application comprising a compositions of the
Bt.4Q7Flg22 polypeptide and RHPP polypeptide provided with a
broad-spectrum fungicide, Fox (16.0% prothioconazole and 13.7%
thiofloxystrobin). FOX is a commercially available foliar fungicide
in South America with limited efficacy for preventative and
curative treatment of Asian soybean rust caused by Phakopsora
pachyrhizi and Cercospora leaf blight of soybean caused by
Cercospora kikuchii applied as a foliar spray following the
recommendations on the specimen label at a use rate of 5.48 fluid
ounces per acre (Fl. oz/Ac) (400 mL/hectare). Beginning at the R1
stage of development, soybean plants received two foliar
applications of the compositions described in Table 90 with an
interval of 13-14 days between spray applications. Foliar
treatments were applied to a single soy variety (which one? Same at
all 3 sites) at the three sites, with 4 replicated plots
(3.times.10 meters, 30 m2; with minimum of 6 rows per treatment).
Disease assessments for trials that were naturally infected were
scored for the severity of infection (0-100% of foliage affected)
were scored for 10 plants within each plot for both Asian soybean
rust caused by Phakopsora pachyrhizi and Cercospora leaf blight of
soybean caused by Cercospora kikuchii at the R4-R5 stage of soy
development (4-15 days after second foliar application) with
guidance from Godoy et al (1997; Journal of plant diseases and
protection 104:336-345). Percent phytotoxicity (0-100% of foliage
affected) was also scored at the R4-R5 stage of soy development.
Severity of infection and phytotoxicity were averaged across all
four replicates per site (Total=12 replicates, 3 sites with 4
replicates each). Standard deviation for each treatment between the
three sites was calculated. Untreated control plants at the Yatytay
site displayed 99% defoliation at 11 days post-application of the
second foliar treatment and were scored for defoliation (0-100%
defoliated) at this time. Disease severity, phytotoxicity, and
defoliation results are provided in Table 91 as percentages, with
standard deviation in parentheses.
TABLE-US-00093 TABLE 91 Incidence of Asian Soybean Rust disease
symptoms after foliar application of fungicide and polypeptide
compositions in Paraguay Incidence of Asian Soybean Rust Change in
Asian Defoliation symptoms Soybean (% of after 2 foliar Rust
foliage) Application Use applications symptoms, after 2 foliar Rate
(% of foliage relative to applications Fluid ounce/acre affected);
control (Yatytay (Fl. oz/Ac) N = 12 reps (%);N = 12 only; N = 4
Foliar Milliliters/hectare per reps per reps per Formulation
(mL/Ha) treatment treatment treatment) Untreated Control n/a 35.1%
-- 99% (.+-.13.8%) FOX Fungicide 5.48 Fl. oz/Ac or 19.4% -15.7% 45%
(Composition 12) 400 mL/Ha (.+-.10.3%) (-15.7%) Bt.4Q7FIg22 (SEQ
2.05 Fl. oz/Ac or 22.7% -12.4% 70% ID NO: 226) 16.7 150 mL/Ha
(.+-.14.4%) .mu.M 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 13) Bt.4Q7FIg22 (SEQ 4.11 Fl. oz/Ac or 22.1%
-13.0% 60% ID NO: 226) 16.7 300 mL/Ha (.+-.14.6%) .mu.M 1.67 mM
Sodium Phosphate Buffer, pH 5.7 PROXEL BC preservative: 330.7 .mu.M
(BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 14) Gm.
RHPP (SEQ 2.05 Fl. oz/Ac or 24.6% -10.5% 96% ID NO: 600) 100 150
mL/Ha (.+-.13.4%) .mu.M PROXEL BC preservative: 330.7 .mu.M; 50.1
.mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 15) Gm. RHPP (SEQ 4.11
Fl. oz/Ac or 23.8% -11.3% 70% ID NO: 600) 100 300 mL/Ha (.+-.11.5%)
.mu.M PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) (Composition 16) FOX Fungicide + 5.48 Fl. oz/Ac or 9.3%
-25.8% 25% Bt.4Q7FIg22 (SEQ 400 mL/Ha + (.+-.3.9%) ID NO: 226) 16.7
2.05 Fl. oz/Ac or .mu.M 150 mL/Ha 1.67 mM Sodium Phosphate Buffer,
pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M
(CMIT); 26.1 .mu.M (MIT) (Composition 17) FOX Fungicide + 5.48 Fl.
oz/Ac or 8.0% -27.1% 25% Bt.4Q7FIg22 (SEQ 400 mL/Ha + (+5.1%) ID
NO: 226) 16.7 4.11 Fl. oz/Ac or .mu.M 300 mL/Ha 1.67 mM Sodium
Phosphate Buffer, pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT);
53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 18) FOX Fungicide
+ 5.48 Fl. oz/Ac or 8.3% -26.8% 25% Gm. RHPP (SEQ 400 mL/Ha +
(.+-.5.8%) ID NO: 600) 100 2.05 Fl. oz/Ac or .mu.M 150 mL/Ha PROXEL
BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
(Composition 19) FOX Fungicide + 5.48 Fl. oz/Ac or 11.0% -24.1% 25%
Gm. RHPP (SEQ 400 mL/Ha + (.+-.3.3%) ID NO: 600) 100 300 mL/Ha
.mu.M PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) (Composition 20)
TABLE-US-00094 TABLE 92 Incidence of Cercospora leaf blight
symptoms after foliar application of fungicide and polypeptide
compositions in Paraguay Incidence of Change in Application Use
Cercospora Cercospora Rate symptoms after 2 foliar symptoms, Fluid
ounce/acre applications, (% relative to (Fl. oz/Ac) of foliar
affected); control Milliliters/hectare N = 12 reps per (%);N = 12
reps Foliar Formulation (mL/Ha) treatment per treatment Untreated
Control n/a 19.3% (.+-.5.1%) -- FOX Fungicide 5.48 Fl. oz/Ac or
15.0% (.+-.9.6%) -4.3% (Composition 12) 400 mL/Ha Bt.4Q7FIg22 (SEQ
ID NO: 2.05 Fl. oz/Ac or 16.4% (.+-.7.7%) -2.8% 226) 16.7 .mu.M 150
mL/Ha 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXELBC
preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 13) Bt.4Q7FIg22 (SEQ ID NO: 4.11 Fl. oz/Ac or
15.8% (.+-.6.3%) -3.5% 226) 300 mL/Ha 16.7 .mu.M 1.67 mM Sodium
Phosphate Buffer, pH 5.7 PROXELBC preservative: 330.7 .mu.M (BIT);
53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 14) Gm. RHPP (SEQ
ID NO: 600) 2.05 Fl. oz/Ac or 15.9% (.+-.6.9%) -3.3% 100 .mu.M 150
mL/Ha PROXELBC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) (Composition 15) Gm. RHPP (SEQ ID NO: 600) 4.11 Fl.
oz/Ac or 14.8% (.+-.5.3%) -4.5% 100 .mu.M 300 mL/Ha PROXELBC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
(Composition 16) FOX Fungicide + 5.48 Fl. oz/Ac or 10.6% (.+-.3.9%)
-8.7% Bt.4Q7FIg22 (SEQ ID NO: 400 mL/Ha + 226) 16.7 .mu.M 2.05 Fl.
oz/Ac or 1.67 mM Sodium Phosphate 150 mL/Ha Buffer, pH 5.7 PROXELBC
preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 17) FOX Fungicide + 5.48 Fl. oz/Ac or 10.6%
(.+-.4.4%) -9.2% Bt.4Q7FIg22 (SEQ ID NO: 400 mL/Ha + 226) 16.7
.mu.M 4.11 Fl. oz/Ac or 1.67 mM Sodium Phosphate 300 mL/Ha Buffer,
pH 5.7 PROXELBC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT);
26.1 .mu.M (MIT) (Composition 18) FOX Fungicide + 5.48 Fl. oz/Ac or
11.3% (.+-.4.2%) -8.0% Gm. RHPP (SEQ ID NO: 600) 400 mL/Ha + 100
.mu.M 2.05 Fl oz/Ac or PROXEL BC preservative: 150 mL/Ha 330.7
.mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 19) FOX
Fungicide + 5.48 Fl. oz/Ac or 11.3% (.+-.4.2%) -8.0% Gm. RHPP (SEQ
ID NO: 600) 400 mL/Ha + 100 .mu.M 300 mL/Ha PROXEL BC preservative:
330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition
20)
TABLE-US-00095 TABLE 93 Phytotoxicity after foliar application of
fungicide and polypeptide compositions in Paraguay Application Use
Rate Phytotoxicity (% of Fluid ounce/acre (Fl. foliage affected)
after 2 oz/Ac) foliar applications; Milliliters/hectare N = 12 reps
per Foliar Formulation (mL/Ha) treatment Untreated Control n/a
0.00% (.+-.0.00%) FOX Fungicide 5.48 Fl. oz/Ac or 2.25% (.+-.32.9%)
(Composition 12) 400 mL/Ha Bt.4Q7FIg22 (SEQ ID NO: 226) 16.7 2.05
Fl. oz/Ac or 0.00% (.+-.0.00%) .mu.M 150 mL/Ha 1.67 mM Sodium
Phosphate Buffer, pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT);
53.5 .mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 13) Bt.4Q7FIg22
(SEQ ID NO: 226) 16.7 4.11 Fl. oz/Ac or 0.00% (.+-.0.00%) .mu.M 300
mL/Ha 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 14) Gm.RHPP (SEQ ID NO: 600) 100 .mu.M 2.05 Fl.
oz/Ac or 0.00% (.+-.0.00%) PROXEL BC preservative: 330.7 .mu.M; 150
mL/Ha 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 15) Gm.RHPP
(SEQ ID NO: 600) 100 .mu.M 4.11 Fl. oz/Ac or 0.00% (.+-.0.00%)
PROXEL BC preservative: 330.7 .mu.M; 300 mL/Ha 50.1 .mu.M (CMIT);
21.71 .mu.M (MIT) (Composition 16) FOX Fungicide + 5.48 Fl. oz/Ac
or 2.33% (.+-.0.14%) Bt.4Q7FIg22 (SEQ ID NO: 226) 16.7 400 mL/Ha +
.mu.M 2.05 Fl. oz/Ac or 1.67 mM Sodium Phosphate Buffer, 150 mL/Ha
pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M
(CMIT); 26.1 .mu.M (MIT) (Composition 17) FOX Fungicide + 5.48 Fl.
oz/Ac or 2.25% (.+-.0.25%) Bt.4Q7FIg22 (SEQ ID NO: 226) 16.7 400
mL/Ha + .mu.M 4.11 Fl. oz/Ac or 1.67 mM Sodium Phosphate Buffer,
300 mL/Ha pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5
.mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 18) FOX Fungicide +
5.48 Fl. oz/Ac or 2.42% (.+-.0.29%) Gm.RHPP (SEQ ID NO: 600) 100
.mu.M 400 mL/Ha + PROXEL BC preservative: 330.7 .mu.M; 2.05 Fl.
oz/Ac or 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) 150 mL/Ha
(Composition 19) FOX Fungicide + 5.48 Fl. oz/Ac or 2.17%
(.+-.0.29%) Gm.RHPP (SEQ ID NO: 600) 400 mL/Ha + 100 .mu.M 300
mL/Ha PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71
.mu.M (MIT) (Composition 20)
[0695] Foliar application of Bt.4Q7Flg22 and Gm.RHPP during
reproductive phases of soy development provided increased
protection against Asian soybean rust and Cercospora leaf blight as
compared to the untreated control. Foliar applications of
Bt.4Q7Flg22-treated plants at 150 and 300 mL/Ha displayed
12.4-13.0% less Asian soybean rust leaf area damage and 2.8-3.5%
less Cercospora leaf area damage compared to the untreated control;
and foliar applications of Gm.RHPP-treated plants at 150 and 300
mL/Ha displayed 10.5-11.3% less Asian soybean rust leaf area damage
and 3.3-4.5% less Cercospora leaf area damage compared to the
untreated control. Combination treatments including either
Bt.4Q7Flg22 or RHPP with FOX fungicide increased protection against
Asian Soybean Rust and Cercospora relative to the Fox Fungicide
treatment alone. At the Yatytay site, less defoliation was observed
at the R7 stage of development due to severe disease symptoms upon
Bt.4Q7Flg22 or Gm.RHPP treatments+/-FOX fungicide. While the
untreated control was 99% defoliated at this stage, Bt.4Q7Flg22
treatment at 150 or 300 mL/Ha decreased defoliation to 70 or 60%
with green leaves still visible, respectively. The Gm.RHPP
treatment at 150 or 300 mL/Ha decreased defoliation to 96% or 70%,
respectively. Combination treatment with Bt.4Q7Flg22 or Gm.RHPP
treatments with FOX fungicide decreased defoliation to 25% with
green leaves visible, while Fox fungicide alone decreased
defoliation to only 45% without green leaves visible. Overall,
polypeptide treatments provided increased protection over FOX
Fungicide alone for control of Asian soybean rust and Cercospora
leaf blight. No phytotoxicity was observed for any polypeptide
application alone, and combination of either polypeptide with FOX
fungicide neither significantly increased or decreased
phytotoxicity relative to the FOX Fungicide alone (Table 93).
Example 54. Flg22-PSA Foliar Application on Kiwi Protects Plants
from Pseudomonas syringae pv. actinidiae (PSA-V)
[0696] Pseudomonas syringae pv. actinidiae (PSA) is a devastating
plant pathogen causing bacterial canker of both green- (Actinidiae
deliciosa) and yellow-flesh (Actinidiae chinesis) kiwi plants
throughout zones of kiwi production, causing severe harvest loss in
New Zealand, China, and Italy. In New Zealand alone, cumulative
revenue losses to the most devastating biovar PSA-V are predicted
to approach $740 million New Zealand leaves Dollars (NZD) by 2025
(Agribusiness and Economics Research Institute of Lincoln
University "The Costs of Psa-V to the New Zealand Kiwifruit
Industry and the Wider Community"; May 2012). PSA-V colonizes the
outer and inner surfaces of the kiwi plant and can spread through
the xylem and phloem tissues. Disease symptoms of PSA-V on kiwi
include bacterial leaf spot, bacterial canker of the trunk, red
exudates, blossom rot, discoloration of twigs, and ultimately
dieback of kiwi vines. The standard method of control for PSA-V
currently employs frequent foliar applications of metallic copper
to kiwi vines which is predicted to lead to the selection of
copper-resistant form of the pathogen and loss of disease control.
Novel methods of control are urgently needed.
[0697] To test the sensitivity of kiwi leaves to 22-amino acid
fragments of flagellin, 1 mm slices were cut through Actinidiae
deliciosa Kiwi `Hayward` leaf petioles and floated in 150 .mu.L of
water in a 96-well plate, with one slice per well. Flg22
polypeptides in Table 94 were prepared for the assay by
re-suspending lyophilized polypeptide in deionized water to a
concentration of 10 mM; peptides were then serially diluted to 10
.mu.M in 100 mM sodium phosphate (pH 7.8-8.0) buffer with 0.1%
Tween-20. Water was removed from kiwi leaf petiole samples after 20
hours and replaced with 100 .mu.L of an elicitation solution
containing 100 nM peptide (diluted from 10 .mu.M stock), 34
.mu.g/mL luminol, and 20 .mu.g/mL horseradish peroxidase in
deionized water. Recognition of the Flg22 polypeptide by the plant
tissue resulted in activation of immune signaling and the
production of apoplastic reactive oxygen species (ROS). In the
presence of ROS (H.sub.2O.sub.2), horseradish peroxidase catalyzed
the oxidation of luminol and production of visible light. Relative
light units (RLUs) were recorded with a SpectraMax L luminometer
(0.5 s integration; 2.0 min intervals) over a time course of 40
minutes. In two independent experiments, a total of 6 kiwi leaf
petiole samples were treated with each Flg22 polypeptide in Table
94. The average total RLU and standard error of the means (SEM) was
calculated for each treatment. A two-tailed T-test was used to
determine significance at the 90% confidence level (P<0.1)
between treatments. Relative ROS production was determined for each
polypeptide in comparison to total RLUs for the 100 nM Bt.4Q7Flg22
control.
TABLE-US-00096 TABLE 94 Kiwi leaf petioles are most sensitive to
FIg22-PSA Average Total Relative Light P-value Units (RLUs);
compared to ROS production SEM in 100 nM relative to Treatment
parentheses Bt.4Q7FIg22 Bt4Q7FIg22 (%) 100 nM Bt.4Q7FIg22- 47,457
n/a 100% (SEQ ID NO: 226) (.+-.12,900) 100 nM Syn01FIg22 81,848 p =
0.286 172% (SEQ ID: 571) (.+-.27,631) 100 nM FIg22-PSA 124,550 p =
0.058* 262% (SEQ ID: 540) (.+-.33,555) *Significant difference at
the 90% confidence level
[0698] Across two independent experiments Kiwi `Hayward` leaf
petioles were significantly more sensitive to Flg22 derived from
Pseudomonas syringae pv. actinidiae (Flg22-PSA; SEQ ID NO:540) in
comparison to Flg22 derived from Bacillus thuringiensis strain 4Q7
(Bt.4Q7Flg22; SEQ ID NO: 226). While ROS production was increased
in kiwi leaf petioles in response to the synthetic Syn01Flg22 (SEQ
ID NO: 571) in comparison to Bt.4Q7 Flg22 (SEQ ID NO: 226) the
difference was not significant. Based on these results, Flg22-PSA
(SEQ ID NO: 540) was formulated as indicated at 100 nM final
concentration (Table 94) for disease prevention trials in potted
Kiwi `Hayward` plants in New Zealand.
TABLE-US-00097 TABLE 95 Treatments applied to potted kiwi trial
Product dilution for spray application Milliliters product/Liter
water (mL/L) or Grams product/Liter Composition Foliar Formulation
water (g/L) Composition 21 ChampION++ .TM. (46.1% Copper 0.9 g
Hydroxide; 30% metallic copper ChampION++ .TM./L equivalent) water
Composition 22 FIg22-PSA (SEQ ID NO: 540) 100 4 mL/L water .mu.M 10
mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC preservative: 330.7
.mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M (MIT)
[0699] Foliar compositions contained 0.1% (v/v) PROXEL BC
preservative, an aqueous dispersion of a blend of 330.7 mM
1,2-benzisothiazolin (BIT), 53.5 mM
5-chloro-2-methyl-4-isolthiazolin-3-one (CMIT), and 26.1 mM
2-methyl-4-isothiazolin-3-one (MIT). Foliar compositions were
diluted to the indicated concentrations in water (g/L water or mL/L
water) with 0.05% (v/v) Contact Xcel.TM. non-ionic surfactant. The
diluted products were applied in fine droplets with a pressurized
backpack sprayer to the entire canopy of each plant, until
thoroughly covered.
[0700] To assess the efficacy of Flg22-PSA (SEQ ID NO: 540) for
control of Pseudomonas syringae pv. actinidiae (PSA-V), a potted
kiwi disease trial was conducted in the Bay of Plenty area of New
Zealand by HortEvaluation Ltd in collaboration with NuFarm Limited.
PSA-V symptom-free potted kiwi Actinidiae deliciosa `Hayward`
plants were evenly distributed between the 6 treatment groups, with
12 potted plants per group. One day prior to inoculation with
PSA-V, potted plants were treated with ChampION++.TM., the industry
standard for PSA-V control, or formulated Flg22-PSA according to
the application rates in Table 96 (Treatment groups 3,4) at a plant
nursery in Te Puka, New Zealand. After 24 hours, all plants except
for the uninfected controls were sprayed with 1.times.108 cfu/mL
PSA-V inoculum using a 5 L hand-held pressurized sprayer aimed at
the underside of leaves until thoroughly covered. The uninfected
control was sprayed with water alone. Potted plants were then
transported to Pukehina and placed in an area with overhead misting
for 48 hours to mimic environmental conditions for PSA-V infection,
with uninfected control plants separated from infected plants.
After 48 hours, a subset of plants was then removed from the
misting area and allowed to briefly dry. After the final
treatments, all plants were moved to their final outdoor trial
site, randomized positions in Pukehina. Average daily temperature
at the trial site was 20.75.degree. C. with a total rainfall of 277
mm over 34 days. Additionally, each plant was watered twice a day
for two hours at a time by drip irrigation. Environmental
conditions were favorable for progression of PSA-V disease
symptoms. Plants were visually monitored throughout the trial
period for PSA-V disease assessments, with the same assessor
recording the % of leaf area covered in spots at 6 days after
inoculation (6 DAI), 16 DAI, 23 DAI and 29 DAI. Additionally, each
plant was assessed for treatment phytotoxicity effects at 29 DAI on
a scale of 0-10, with 0=no leaf phytotoxicity and 10=very severe
leaf phytotoxicity symptoms. The average disease scores at 6, 16,
23, and 29 DAI and phytotoxicity score at 29 DAI are reported in
Table 96 for each treatment (n=12 plants per treatment). P-values
were calculated for each treatment vs. the untreated control.
TABLE-US-00098 TABLE 96 FIg22-PSA foliar application reduces PSA-V
disease symptoms in kiwi plants Treatment Application Foliage
Affected (% leaf surface area); group #/Foliar Rate and p-values
vs. untreated control Formulation Timing 6 DAI 16 DAI 23 DAI 29 DAI
Treatment group 1 n/a 0.00% 1.66% 7.89% 18.14% Uninfected plants
Treatment group 2 n/a 15.12% 40.36% 54.64% 67.82% Untreated Control
Treatment group 3 0.9 g/L; 3.23% 12.48% 16.57% 25.20% ChampION++
.TM. One day (p < 0.001) (p < 0.001) (p < 0.001) (p <
0.001) (Composition 21) pre- inoculation Treatment group 4 4 mL/L;
7.31% 29.41% 45.97% 61.91% FIg22-PSA One day (p < 0.001) (p =
0.013) (p = 0.085) (p = 0.190) (Composition 22) pre-
inoculation
[0701] Application of Flg22-PSA significantly reduced PSA-V leaf
spot symptoms (P<0.1; 90% confidence interval) at 6, 16 and 23
DAI in comparison to the untreated control. Combination of
Flg22-PSA pre-treatment further decreased the severity of leaf spot
compared to Flg22-PSA treatment alone at all assessment timepoints
and prolongs the period of significant protection to 29 DAI (14.3%
less leaf spot compared to untreated control; P=0.002). In
conclusion, Flg22-PSA can be used both as a stand-alone treatment
and in combination with other treatments aimed at restricting
pathogen growth. While the industry standard ChampION++.TM. which
is the currently used copper containing treatment to treat PSA
causes mild leaf phytotoxicity (AVE score=1.6), no significant
phytotoxicity was observed for Treatments 3-4 (Table 97). Flg22-PSA
can be used as an alternative to other phytotoxic treatments.
TABLE-US-00099 TABLE 97 FLG22-PSA foliar application does not cause
leaf phytotoxicity of kiwi plants Average Treatment group #/Foliar
Application Rate Phytotoxicity Score Formulation and Timing (0-10);
29 DAI Treatment group 1 n/a 0.0 (.+-.0.0) Uninfected plants
Treatment group 2 n/a 0.0 (.+-.0.0) Untreated Control Treatment
group 3 0.9 g/L; 1.6 (.+-.0.9) ChampION++ .TM. One day pre-
(Composition 21) inoculation Treatment group 4 4 mL/L; 0.1
(.+-.0.3) FIg22-PSA One day pre- (Composition 22) inoculation
Example 55: Polypeptides Derived from Elongation Factor Tu
[0702] Elf18 and Elf26 polypeptides derived from the consensus
Bacillus cereus Elongation Factor-TU (EF-Tu) protein were tested
for ability to produce a ROS response in corn (hybrid 5828 YX), soy
(variety Morsoy), and Arabidopsis thaliana. Polypeptides were
synthesized by Genscript USA (Piscataway, N.J.) using standard
solid-phase synthesis methods and provided as a lyophilized powder
with greater than or equal to 70% purity. Dry powder was
re-suspended to a concentration of 10 mM in ultrapure water, and
then serially diluted in ultrapure water to the concentrations
tested in the ROS assay in Table 98.
[0703] For the ROS assay, Arabidopsis leaves were excised from
4-week-old plants, and using a cork borer 4 mm disks were removed
from the leaves. Each disc was cut in half using the edge of a
razor blade, and then each disc half was floated on 150 .mu.L of
water abaxial side touching the water in a 96-well plate to rest
overnight. The next day, the water was removed from each well just
prior to polypeptide treatment. RLU values and relative ROS
activity was reported as the average of 4 measurements. ROS
activity assays were conducted using the methods as previously
reported in Example 15). ROS activity results are reported in Table
97 below.
TABLE-US-00100 TABLE 98 Elf18 and Elf26 Polypeptides from Bacillus
cereus Amino EF-Tu Polypeptide Acid Description Length Sequence N
terminus of EF Tu 18 Ac-AKAKFERSKPHVNIGTIG-conh2 (modified)
Bacillus cereus (SEQ ID NO: 616) N terminus of EF Tu 26
Ac-AKAKFERSKPHVNIGTIGHVDHGKTT-conh2 (modified) Bacillus cereus (SEQ
ID NO: 617)
TABLE-US-00101 TABLE 99 Comparison of ROS activity of elf18 and
elf26 polypeptides in Arabidopsis leaf tissue Average RLU value
(Fold increase (X) over mock Polypeptide Treatment treatment)
Negative control (water) 82896 (1 X) N terminus of EF Tu (100 nM)
(SEQ ID NO: 616) 264194 (3.2 X) N terminus of EF Tu (100 nM) (SEQ
ID NO: 617) 211383 (2.5 X) Bt.4Q7FIg22 (100 nM) (SEQ ID NO: 226)
258073 (3.1 X) N terminus of EF Tu (100 nM) (SEQ ID NO: 254344 (3.1
X) 616) + Bt.4Q7FIg22 (100 nM) (SEQ ID NO: 226) N terminus of EF Tu
(100 nM) (SEQ ID NO: 181504 (2.2 X) 617) + Bt.4Q7FIg22 (100 nM)(SEQ
ID NO: 226)
[0704] The receptor for EF-Tu polypeptides, EF-Tu Receptor (EFR)
was previously identified in the Brassica clade, of which
Arabidsopis thaliana is a model plant. Results in Table 99 indicate
that newly identified polypeptides from Bacillus cereus EF-Tu (SEQ
ID NO: 616 and SEQ ID NO: 617) can be used to elicit a ROS response
similar in magnitude to Bt.4Q7Flg22 (SEQ ID NO: 226) when each was
tested at a 100 nM concentration. In comparison to the mock-treated
control, EF-Tu N-terminal polypeptides gave a response that was
3.2- to 2.5-fold increased, while Bt.4Q7Flg22 was 3.1-fold
increased over mock control. These results suggest that 18- and
26-amino acid fragments from the N-terminus of Bacillus cereus can
be used similarly to Bt.4Q7Flg22 in the Brassica crops, including
but not limited to kale, cabbage, collard greens, cauliflower,
Brussel sprouts, savoy, kohlrabi and gai lan, to increase plant
biomass, yield and disease prevention.
[0705] Combination treatments of EF-Tu N-terminal peptides (SEQ ID
NO: 616 and SEQ ID NO: 617) and Bt.4Q7Flg22 (SEQ ID NO: 226)
resulted in similar ROS responses to the EF-Tu peptides alone,
indicating that the combination of peptides treatments in the field
would provide no interference of activity; however, due to the
shared mechanisms between downstream signaling events for EF-Tu and
Flg22 peptides, recognized by the EFR and FLS2 receptors
respectively, a staggered application of peptide treatments may
provide the greatest growth benefit to the plant.
Example 56: Disease Protection using Bt.4Q7Flg22 and Gm.RHPP Foliar
Applications on Soybean Plants to Protect from Diseases Caused by
Phakopsora pachyrhizi and Cercospora kikuchii
[0706] Foliar application of Bt.4Q7Flg22 (SEQ ID NO: 226) and
Gm.RHPP (SEQ ID NO: 600) during reproductive phases of soy
development was previously found to decrease disease symptoms
caused by Phakopsora pachyrhizi and Cercospora kikuchii infections
(Example 53). These plants were taken to yield, and Bt.4Q7Flg22
(SEQ ID NO: 226) and Gm.RHPP (SEQ ID NO: 600) foliar applications
were found to increase yield in comparison to the untreated control
plants in replicated trials in Paraguay where plants were infected
with Asian soybean rust and Cercospora leaf blight. Foliar
applications of Bt.4Q7Flg22 at 150 and 300 mL/Ha increased yield by
+342.2 kg/Ha and +427.2 kg/Ha, respectively, in trials where the
average yield for untreated plants was 1266.3 Kg/Ha. The increase
in yield for 300 mL/Ha foliar application of Bt.4Q7Flg22 (36.1%)
was comparable to FOX Fungicide alone (36.6%), demonstrating that
Bt.4Q7Flg22 is effective as an anti-fungal foliar treatment for
both reducing disease symptoms and boosting yield. The relative
yield across all three trial sites was for plants treated with a
combined application of FOX Fungicide and Bt.4Q7Flg22 was slightly
increased over FOX fungicide or Bt.4Q7Flg22 foliar application
alone, demonstrating that the treatments are compatible. Foliar
applications of Gm.RHPP at 150 and 300 mL/Ha further increased
yield by +294.2 kg/Ha and 506.8 kg/Ha, respectively, in comparison
to the untreated control. When applied in combination with FOX
Fungicide, Gm.RHPP provided the greatest protection against disease
in the trials as evidenced by increased yield of +517.6 kg/Ha and
+539.9 kg/Ha for the 150 mL/Ha and 300 mL/Ha application rates of
Gm.RHPP, respectively. Foliar application of Gm.RHPP consistently
improved plant health and increased yield, thus Gm.RHPP is an
effective treatment for growth promotion and fungal disease
resistance.
TABLE-US-00102 TABLE 100 Soybean yield for replicated field trials
infected with Phakopsora pachyrhizi and Cercospora kikuchii where
plants were treated with Bt.4Q7FIg22 or RHPP Average change in
yield in Application Use comparison to Rate Untreated Control Yield
relative Fluid ounce/acre [1266.3 to Untreated (Fl. oz/Ac)
Kilograms/Hectare Control (%); Milliliters/hectare (Kg/Ha)]; N = 12
N = 12 reps per Foliar Formulation (mL/Ha) reps per treatment
treatment Untreated Control n/a -- 100% FOX Fungicide 5.48 Fl.
oz/Ac or +445.6 kg/Ha 136.6% (Composition 12) 400 mL/Ha Bt.4Q7FIg22
(SEQ ID 2.05 Fl. oz/Ac or +342.2 kg/Ha 130.7% NO: 226) 16.7 .mu.M
150 mL/Ha 1.67 mM Sodium Phosphate Buffer, pH 5.7 PROXEL BC
preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 13) Bt.4Q7FIg22 (SEQ ID 4.11 Fl. oz/Ac or +427.2
kg/Ha 136.1% NO: 226) 16.7 .mu.M 300 mL/Ha 1.67 mM Sodium Phosphate
Buffer, pH 5.7 PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5
.mu.M (CMIT); 26.1 .mu.M (MIT) (Composition 14) Gm. RHPP (SEQ ID
NO: 2.05 Fl. oz/Ac or +294.2 kg/Ha 125.5% 600) 100 .mu.M 150 mL/Ha
PROXEL BC preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M
(MIT) (Composition 15) Gm. RHPP (SEQ ID NO: 4.11 Fl. oz/Ac or
+506.8 kg/Ha 143.5% 600) 100 .mu.M 300 mL/Ha PROXEL BC
preservative: 330.7 .mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT)
(Composition 16) FOX Fungicide + 5.48 Fl. oz/Ac or +426.5 kg/Ha
138.4% Bt.4Q7FIg22 (SEQ ID 400 mL/Ha + NO: 226) 16.7 .mu.M 2.05 Fl.
oz/Ac or 1.67 mM Sodium 150 mL/Ha Phosphate Buffer, pH 5.7 PROXEL
BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1 .mu.M
(MIT) (Composition 17) FOX Fungicide + 5.48 Fl. oz/Ac or +418.5
kg/Ha 137.2% Bt.4Q7FIg22 (SEQ ID 400 mL/Ha + NO: 226) 16.7 .mu.M
4.11 Fl. oz/Ac or 1.67 mM Sodium 300 mL/Ha Phosphate Buffer, pH 5.7
PROXEL BC preservative: 330.7 .mu.M (BIT); 53.5 .mu.M (CMIT); 26.1
.mu.M (MIT) (Composition 18) FOX Fungicide + 5.48 Fl. oz/Ac or
+517.6 kg/Ha 145.5% Gm. RHPP (SEQ ID NO: 400 mL/Ha + 600) 100 .mu.M
2.05 Fl. oz/Ac or PROXEL BC 150 mL/Ha preservative: 330.7 .mu.M;
50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 19) FOX Fungicide
+ 5.48 Fl. oz/Ac or +539.9 kg/Ha 146.9% Gm. RHPP (SEQ ID NO: 400
mL/Ha + 600) 100 .mu.M 300 mL/Ha PROXEL BC preservative: 330.7
.mu.M; 50.1 .mu.M (CMIT); 21.71 .mu.M (MIT) (Composition 20)
Example 57. Treatment of Citrus Trees Infected with Candidatus
Liberibacter Asiaticus with Flg22 Increases Fruit Set
[0707] Previous results summarized in Example 51 indicate that
Bt.4Q7Flg22 (SEQ ID NO: 226) trunk injection reduces pathogen titer
and promotes new growth in citrus trees infected with Candidatus
Liberibacter asiaticus, the causative agent of Huanglongbing (HLB).
To assess for a potential increase in fruiting and obtain early
estimates of yield, fruit set was measured in June 2018 for the
same HLB-infected `Valencia` Orange (to be harvested spring 2019)
and `Ruby Red` Grapefruit trees (to be harvested fall 2018) that
were trunk-injected with Bt.4Q7Flg22 in April 2017 at the
commercial grove orchard located in central Florida (Okeechobee
county). As described in Example 51, trees were injected in April
2017 with either a 1.times.Bt.4Q7Flg22-Low Rate (0.55 micromoles
peptide; 0.138 .mu.M estimated phloem concentration) or a
10.times.Bt.4Q7Flg22-High Rate (5.5 micromoles peptide; 1.38 .mu.M
estimated phloem concentration). In June 2018, the
Bt.4Q7Flg22-injected trees were compared to untreated control trees
within the same area of the grove using established methods for
projecting citrus tree yield ("Forecasting Florida Citrus
Production: Methodology & Development; 1971; by S. R. Williams
for Florida Crop and Livestock Reporting Service). To quantify
fruit set, three quaternary limbs at eye level were randomly chosen
on each tree (n=8 trees per treatment `Valencia` orange, n=10 trees
per treatment `Ruby Red` grapefruit). The circumference of each
quaternary limb was measured at the junction where the limb began
and used to calculate the cross-sectional area (CSA) of the limb
using the following equations (where C=circumference,
CSA=cross-sectional area, and r=radius):
r = C 2 .times. .pi. .times. .times. and .times. .times. C .times.
.times. S .times. .times. A = .pi. .times. r 2 ##EQU00003##
[0708] Then, the total number of fruit on the quaternary limb
distal to that junction were counted. To normalize for limb size,
fruit set for each quaternary limb was quantified as the number of
fruit on the limb divided by the CSA of the quaternary limb:
Fruit .times. .times. set = Total .times. .times. fruit .times.
.times. per .times. .times. limb Limb .times. .times. .times. C
.times. .times. S .times. .times. A ##EQU00004##
[0709] The fruit count per quaternary limb CSA is reported in FIG.
11 (`Valencia` orange) and FIG. 12 (Red Grapefruit) in box and
whisker plots, where the median value for each treatment is marked
as the vertical line within the box, the mean or average value is
marked by the "x", the upper and lower quartiles are marked by the
ends of the box, and the whiskers extend to the highest and lowest
observed fruit counts per limb CSA. Any outlier values are
indicated by the small circles located outside the standard error
bars for each treatment.
[0710] To further assess the size and volume of fruit setting per
tree, the fruit diameter (mm) of at least 10 randomly chosen fruit
per tree was measured using calipers placed at the widest point on
each fruit. The average fruit diameter (mm) per tree for each
treatment is reported in FIG. 13 (`Valencia` orange) and FIG. 14
(Red Grapefruit) in box and whisker plots. The average fruit
diameter was used to estimate the total fruit volume per limb for
each treatment. For these estimates, the volume in milliliters (mL)
of a theoretically spherical orange was calculated using the
following equation, where the radius (r) of the fruit is the
average diameter (measured in mm) per limb divided by 2:
Total .times. .times. Fruit .times. .times. Volume .times. .times.
per .times. .times. l .times. imb .function. ( mL ) = Total .times.
.times. fruit .times. .times. per .times. .times. limb * 4 3
.times. .times. .times. r 3 * 1 .times. .times. mL 1000 .times.
.times. cubic .times. .times. millimeters ##EQU00005##
[0711] The estimated volume of fruit normalized by limb CSA for
each treatment is reported in FIG. 15 (`Valencia` orange) and FIG.
16 (Red Grapefruit) in box and whisker plots.
[0712] The measurements collected in June 2018 to assess fruit set
in `Valencia` orange and `Ruby Red` grapefruit trees in Okeechobee,
Fla. show increased fruit per limb and increased fruit size for
trees of both varieties receiving trunk injections of 1.times. Low
and 10.times. High rates of Bt.4Q7Flg22 (SEQ ID NO: 226) in April
2017, when comparing the mean and median values for all parameters
measured versus the untreated control. The increased fruit set and
size are predicative of increased yield. These results provide
further evidence that trunk injection of citrus trees with
Bt.4Q7Flg22 can be utilized to reduce C. liberibacter bacterial
titers in orange (FIG. 9) and grapefruit (FIG. 10; Table 84) and
stimulate new shoot and fruit growth (Table 85, FIGS. 11-16) in
citrus trees.
[0713] In view of the above, it will be seen that the several
objects of the invention are achieved and other advantageous
results attained.
[0714] As various changes could be made in the above polypeptides,
recombinant organisms, methods, and seeds, without departing from
the scope of the invention, it is intended that all matter
contained in the above description shall be interpreted as
illustrative and not in a limiting sense.
Sequence CWU 1
1
7751283PRTBacillus thuringiensis 1Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Ser
Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Lys
Ala Arg Glu Gly Gly Leu Asn Val Ala Gly Arg 50 55 60Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser
Ala Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala Ser Leu Gln Lys 100 105
110Glu Phe Ala Gln Leu Thr Glu Gln Ile Asp Tyr Ile Ala Lys Asn Thr
115 120 125Gln Phe Asn Asp Gln Gln Leu Leu Gly Thr Ala Asp Lys Lys
Ile Lys 130 135 140Ile Gln Thr Leu Asp Thr Gly Ser Thr Asn Pro Ala
Gln Ile Glu Ile145 150 155 160Thr Leu Asn Ser Val Lys Ser Ala Asp
Leu Gly Leu Asp Val Gln Ile 165 170 175Gly Asp Glu Gly Asp Ala Glu
Ser Thr Ala Ala Ala Asp Pro Thr Ser 180 185 190Ala Lys Gln Ala Ile
Asp Ala Ile Asp Ala Ala Ile Thr Thr Val Ala 195 200 205Gly Gln Arg
Ala Thr Leu Gly Ala Thr Leu Asn Arg Phe Glu Phe Asn 210 215 220Ala
Asn Asn Leu Lys Ser Gln Glu Thr Ser Met Ala Asp Ala Ala Ser225 230
235 240Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr
Lys 245 250 255Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser
Gln Ala Asn 260 265 270Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln
275 2802283PRTBacillus thuringiensis 2Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys
Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn
Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met
Lys Ala Arg Glu Gly Gly Leu Asn Val Ala Gly Arg 50 55 60Asn Thr Gln
Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90
95Ser Ala Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala Ser Leu Gln Lys
100 105 110Glu Phe Ala Gln Leu Thr Glu Gln Ile Asp Tyr Ile Ala Lys
Asn Thr 115 120 125Gln Phe Asn Asp Gln Gln Leu Leu Gly Thr Ala Asp
Lys Lys Ile Lys 130 135 140Ile Gln Thr Leu Asp Thr Gly Ser Thr Asn
Pro Ala Gln Ile Glu Ile145 150 155 160Thr Leu Asn Ser Val Lys Ser
Ala Asp Leu Gly Leu Asp Val Gln Ile 165 170 175Gly Asp Glu Gly Asp
Ala Glu Ser Thr Ala Ala Ala Asp Pro Thr Ser 180 185 190Ala Lys Gln
Ala Ile Asp Ala Ile Asp Ala Ala Ile Thr Thr Val Ala 195 200 205Gly
Gln Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Phe Glu Phe Asn 210 215
220Ala Asn Asn Leu Lys Ser Gln Glu Thr Ser Met Ala Asp Ala Ala
Ser225 230 235 240Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys 245 250 255Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn 260 265 270Gln Thr Pro Gln Met Val Ser Lys
Leu Leu Gln 275 2803283PRTBacillus thuringiensis 3Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn
Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys
Arg Ile Asn Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala
Thr Arg Met Lys Ala Arg Glu Gly Gly Leu Asn Val Ala Gly Arg 50 55
60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65
70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn
Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala Ser Leu
Gln Lys 100 105 110Glu Phe Ala Gln Leu Thr Glu Gln Ile Asp Tyr Ile
Ala Lys Asn Thr 115 120 125Gln Phe Asn Asp Gln Gln Leu Leu Gly Thr
Ala Asp Lys Lys Ile Lys 130 135 140Ile Gln Thr Leu Asp Thr Gly Ser
Thr Asn Pro Ala Gln Ile Glu Ile145 150 155 160Thr Leu Asn Ser Val
Lys Ser Ala Asp Leu Gly Leu Asp Val Gln Ile 165 170 175Gly Asp Glu
Gly Asp Ala Glu Ser Thr Ala Ala Ala Asp Pro Thr Ser 180 185 190Ala
Lys Gln Ala Ile Asp Ala Ile Asp Ala Ala Ile Thr Thr Val Ala 195 200
205Gly Gln Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Phe Glu Phe Asn
210 215 220Ala Asn Asn Leu Lys Ser Gln Glu Thr Ser Met Ala Asp Ala
Ala Ser225 230 235 240Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys 245 250 255Phe Lys Ile Leu Asn Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn 260 265 270Gln Thr Pro Gln Met Val Ser
Lys Leu Leu Gln 275 2804283PRTBacillus cereus 4Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln
Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr
Arg Met Lys Ala Arg Glu Gly Gly Leu Asn Val Ala Gly Arg 50 55 60Asn
Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75
80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln
85 90 95Ser Ala Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala Ser Leu Gln
Lys 100 105 110Glu Phe Ala Gln Leu Thr Glu Gln Ile Asp Tyr Ile Ala
Lys Asn Thr 115 120 125Gln Phe Asn Asp Gln Gln Leu Leu Gly Thr Ala
Asp Lys Lys Ile Lys 130 135 140Ile Gln Thr Leu Asp Thr Gly Ser Thr
Asn Pro Ala Gln Ile Glu Ile145 150 155 160Thr Leu Asn Ser Val Lys
Ser Ala Asp Leu Gly Leu Asp Val Gln Ile 165 170 175Gly Asp Glu Gly
Asp Ala Glu Ser Thr Ala Ala Ala Asp Pro Thr Ser 180 185 190Ala Lys
Gln Ala Ile Asp Ala Ile Asp Ala Ala Ile Thr Thr Val Ala 195 200
205Gly Gln Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Phe Glu Phe Asn
210 215 220Ala Asn Asn Leu Lys Ser Gln Glu Thr Ser Met Ala Asp Ala
Ala Ser225 230 235 240Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys 245 250 255Phe Lys Ile Leu Asn Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn 260 265 270Gln Thr Pro Gln Met Val Ser
Lys Leu Leu Gln 275 2805266PRTBacillus thuringiensis 5Met Arg Ile
Gly Thr Asn Val Leu Ser Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu
Asn Glu Lys His Met Asn Val Ala Met Glu His Leu Ala Thr 20 25 30Gly
Lys Lys Leu Asn Asn Ala Ser Asp Asn Pro Ala Asn Ile Ala Ile 35 40
45Val Thr Arg Met His Ala Arg Ala Ser Gly Met Arg Val Ala Ile Arg
50 55 60Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala Glu Ala Ala
Leu65 70 75 80Gln Thr Val Thr Asn Ile Leu Gln Arg Met Arg Asp Leu
Ala Val Gln 85 90 95Ser Ala Asn Gly Thr Asn Ser Asn Lys Asn Arg His
Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser Leu Thr Glu Lys Ile Gly
Tyr Ile Gly Glu Thr Thr 115 120 125Glu Phe Asn Asp Leu Ser Val Phe
Glu Gly Gln Asn Arg Pro Ile Thr 130 135 140Leu Asp Asp Ile Gly His
Thr Ile Asn Met Met Lys His Ile Pro Pro145 150 155 160Ser Pro Thr
Gln His Asp Ile Lys Ile Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala
Ala Ile Leu Lys Ile Glu Asp Ala Leu Gln Ser Val Ser Leu His 180 185
190Arg Ala Asp Leu Gly Ala Met Ile Asn Arg Leu Gln Phe Asn Ile Glu
195 200 205Asn Leu Asn Ser Gln Ser Met Ala Leu Thr Asp Ala Ala Ser
Leu Ile 210 215 220Glu Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe
Leu Lys Phe Lys225 230 235 240Leu Leu Thr Glu Val Ala Leu Ser Met
Val Ser Gln Ala Asn Gln Ile 245 250 255Pro Gln Met Val Ser Lys Leu
Leu Gln Ser 260 2656375PRTBacillus thuringiensis 6Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn
Gln Ala Lys Met Ser Asn Ser Met Asp Arg Leu Ser Ser 20 25 30Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala
Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ala Asp 50 55
60Asn Thr Gln Asn Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65
70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn
Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn Lys Ser Ala Leu
Gln Lys 100 105 110Glu Phe Ala Gln Leu Gln Lys Gln Ile Thr Tyr Ile
Ala Glu Asn Thr 115 120 125Gln Phe Asn Asp Lys Asn Leu Leu Asn Glu
Asp Ser Glu Val Lys Ile 130 135 140Gln Thr Leu Asp Ser Ser Lys Gly
Glu Gln Gln Ile Thr Ile Asp Leu145 150 155 160Lys Ala Val Thr Leu
Glu Lys Leu Asn Ile Lys Asp Ile Ala Ile Gly 165 170 175Lys Ala Asp
Ala Ala Asp Lys Pro Val Thr Pro Gly Ala Thr Val Asp 180 185 190Gln
Lys Asp Leu Asp Ser Val Thr Asp Lys Ile Ala Ala Leu Thr Glu 195 200
205Thr Ser Ser Lys Ala Asp Ile Asp Ala Ile Gln Ser Ser Leu Asp Asn
210 215 220Phe Lys Ala Ser Met Thr Pro Glu Asp Val Lys Thr Leu Glu
Asp Ala225 230 235 240Leu Lys Gly Phe Lys Thr Gly Gln Ala Asn Pro
Ala Asp Ala Gly Val 245 250 255Asp Ala Ile Gln Asp Ala Leu Ser Lys
Val Lys Leu Pro Thr Ala Thr 260 265 270Ala Ala Ala Pro Ala Ala Asp
Ala Asp Lys Ser Asp Ala Leu Ala Ala 275 280 285Ile Ala Ala Ile Asp
Ala Ala Leu Thr Lys Val Ala Asp Asn Arg Ala 290 295 300Thr Leu Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu305 310 315
320Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp
325 330 335Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys
Ile Leu 340 345 350Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn
Gln Thr Pro Gln 355 360 365Met Val Ser Lys Leu Leu Gln 370
3757416PRTBacillus thuringiensis 7Met Thr Gly Ile Thr Ile Asn Leu
Glu Ile Asp Phe Phe Ala Tyr Tyr1 5 10 15Arg Phe Ser Ile Cys Arg Lys
Val Asn Ile Lys Lys Trp Gly Phe Leu 20 25 30Asn Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr 35 40 45Met Arg Gln Asn Gln
Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser 50 55 60Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala65 70 75 80Ile Ala
Thr Arg Met Arg Ala Arg Glu Asn Gly Leu Gly Val Ala Ala 85 90 95Asn
Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala 100 105
110Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn
115 120 125Gln Ser Ala Asn Gly Thr Asn Thr Asp Asp Asn Gln Lys Ala
Leu Asp 130 135 140Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr
Ile Ser Lys Asn145 150 155 160Thr Glu Phe Asn Asp Lys Lys Leu Leu
Asn Gly Glu Asn Lys Thr Ile 165 170 175Ala Ile Gln Thr Leu Asp Asn
Ala Asp Thr Thr Lys Gln Ile Asn Ile 180 185 190Asn Leu Ala Asp Ser
Ser Thr Ser Ala Leu Gln Ile Asp Lys Leu Thr 195 200 205Ile Ser Gly
Lys Thr Thr Asp Thr Thr Lys Thr Glu Thr Ile Thr Val 210 215 220Thr
Asp Asp Glu Ile Lys Ala Ala Lys Thr Asp Ile Asp Glu Phe Asn225 230
235 240Asp Ala Lys Lys Ala Leu Ala Asp Leu Lys Ala Glu Thr Ser Ala
Gly 245 250 255Lys Ala Asp Gly Ser Thr Asp Asp Glu Ile Lys Thr Ala
Val Ser Asn 260 265 270Phe Thr Lys Ser Phe Glu Lys Ile Gln Lys Phe
Met Asn Asp Ser Asp 275 280 285Ile Lys Thr Val Gln Thr Glu Ile Glu
Lys Phe Asp Ala Ala Ala Pro 290 295 300Ala Leu Asp Lys Ala Lys Gly
Met Gly Ile Ala Phe Thr Ser Ala Met305 310 315 320Asp Pro Lys Ala
Gly Thr Ile Thr Lys Ala Ala Thr Arg Gln Asn Ala 325 330 335Ser Asp
Ala Ile Lys Ser Ile Asp Ala Ala Leu Glu Thr Ile Ala Ser 340 345
350Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val
355 360 365Asn Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ala Ala Ala
Ser Gln 370 375 380Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu
Met Thr Lys Phe385 390 395 400Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Val 405 410 4158368PRTBacillus
thuringiensis 8Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Asn Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Gln Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Asp Glu Asn Lys Ala Ala Met Glu Lys 100 105 110Glu Phe Gly Gln
Leu Lys Asp Gln Ile Lys Tyr Ile Thr Asp Asn Thr 115 120 125Gln Phe
Asn Asp Lys Asn Leu Leu Asp Ala Ala Ser Gly Thr Thr Lys 130 135
140Ser Ile Ala Ile Gln Thr Leu Asp Ser Asp Gln Ala Ser Thr Gln
Ile145 150 155 160Glu Ile Lys Ile Ala Gly Ser Ser Leu Ala Ala Leu
Gly Leu Asp Lys 165 170 175Val Gln Ile Gly Gln Glu Thr Val Ala Gln
Lys Asp Leu Asp Val Leu 180 185 190Thr Lys Ala Met Gly Arg Leu Ala
Ala Pro Asp Ala Asp Ala Thr Thr 195 200 205Arg Asp Leu Asp Val Gln
Val Ala Lys Asp Ala Phe Asp Lys Val Lys 210 215 220Gly Phe Ile Ala
Asp Pro Ala Gln Ala Lys Ala Val Glu Arg Ala Phe225 230 235 240Glu
Asp Tyr Thr Ala Ala Glu Ala Gly Lys Glu Glu Asp Ala Ala Lys 245 250
255Ala Ile Asp Ala
Ala Tyr Lys Lys Val Thr Gly Leu Thr Ala Gly Thr 260 265 270Thr Gly
Thr Val Asp Ala His Asn Ala Val Asn Lys Ile Asp Ala Ala 275 280
285Leu Lys Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn
290 295 300Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ala
Ser Met305 310 315 320Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp
Met Ala Lys Glu Met 325 330 335Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser Met 340 345 350Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln 355 360 3659374PRTBacillus
cereus 9Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr
Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp Arg Leu
Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly
Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly
Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr
Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Asp
Lys Asn Gln Val Ala Leu Gln Lys 100 105 110Glu Phe Gly Glu Leu Gln
Lys Gln Ile Asp Tyr Ile Ala Lys Asn Thr 115 120 125Gln Phe Asn Asp
Lys Asn Leu Leu Ser Gly Lys Ala Gly Ala Pro Asp 130 135 140Gln Ala
Leu Glu Ile Asn Ile Gln Thr Leu Asp Ser Ser Asp Pro Asn145 150 155
160Gln Gln Ile Lys Ile Ser Leu Asp Ser Val Ser Thr Ala Gln Leu Gly
165 170 175Val Lys Asp Leu Gln Ile Gly Ser Ser Ser Ile Thr Gln Gln
Gln Leu 180 185 190Asp Thr Leu Asp Asn Ala Met Lys Arg Leu Glu Thr
Ala Ser Thr Thr 195 200 205Ala Ala Val Arg Asp Gln Asp Val Ala Asp
Ala Lys Ala Ala Phe Glu 210 215 220Asn Val Lys Gly Phe Phe Ser Glu
Gly Asn Val Asp Ser Ile Asn Arg225 230 235 240Ala Phe Thr Asp Phe
Ala Asn Glu Thr Thr Asn Lys Asp Asp Lys Ala 245 250 255Glu Ala Ile
Tyr Ala Leu Tyr Asn Asn Ala Thr Leu Ile Thr Lys Pro 260 265 270Thr
Pro Asp Ala Ser Asn Pro Ala Ser Val Asp Pro Ala Asn Ala Ile 275 280
285Lys Lys Ile Asp Gln Ala Ile Glu Lys Ile Ala Ser Ser Arg Ala Thr
290 295 300Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn
Leu Lys305 310 315 320Ser Gln Gln Ser Ser Met Ala Ser Ala Ala Ser
Gln Val Glu Asp Ala 325 330 335Asp Met Ala Lys Glu Met Ser Glu Met
Thr Lys Phe Lys Ile Leu Asn 340 345 350Glu Ala Gly Ile Ser Met Leu
Ser Gln Ala Asn Gln Thr Pro Gln Met 355 360 365Val Ser Lys Leu Leu
Gln 37010266PRTBacillus cereus 10Met Arg Ile Gly Thr Asn Val Leu
Ser Met Asn Ala Arg Gln Ser Phe1 5 10 15Tyr Glu Asn Glu Lys Arg Met
Asn Val Ala Ile Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn His
Ala Ser Asp Asn Pro Ala Asn Val Ala Ile 35 40 45Val Thr Arg Met His
Ala Arg Thr Ser Gly Ile His Val Ala Ile Arg 50 55 60Asn Asn Glu Asp
Ala Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr
Val Thr Asn Ile Leu Gln Arg Met Arg Asp Val Ala Val Gln 85 90 95Ser
Ala Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile Asp Glu Thr Thr
115 120 125Glu Phe Asn Asp Leu Ser Val Phe Asp Arg Gln Asn Cys Pro
Val Thr 130 135 140Leu Asp Asp Ile Gly His Thr Val Asn Val Thr Lys
His Ile Pro Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Asn Ile
Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu
Glu Thr Leu Gln Asn Val Ser Leu His 180 185 190Arg Ala Asp Leu Gly
Ala Met Ile Asn Gln Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn
Ser Gln Ser Thr Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu
Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230
235 240Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln
Ile 245 250 255Pro Gln Met Val Tyr Lys Leu Leu Gln Ser 260
26511341PRTBacillus thuringiensis 11Met Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp1 5 10 15Ala Ala Gly Leu Ala Ile Ala
Thr Arg Met Arg Ala Arg Glu Ser Gly 20 25 30Leu Gly Val Ala Ala Asn
Asn Thr Gln Asp Gly Met Ser Leu Ile Arg 35 40 45Thr Ala Asp Ser Ala
Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met 50 55 60Arg Asp Ile Ala
Asn Gln Ser Ala Asn Gly Thr Asn Thr Ala Asp Asn65 70 75 80Gln Gln
Ala Leu Gln Lys Glu Phe Gly Gln Leu Lys Glu Gln Ile Ser 85 90 95Tyr
Ile Ala Asp Asn Thr Glu Phe Asn Asp Lys Thr Leu Leu Lys Ala 100 105
110Asp Asn Ser Val Lys Ile Gln Thr Leu Asp Ser Ala Asp Thr Asn Lys
115 120 125Gln Ile Ser Ile Asp Leu Lys Gly Val Thr Leu Asn Gln Leu
Gly Leu 130 135 140Asp Thr Val Asn Ile Gly Ser Glu Lys Leu Ser Ala
Glu Ser Leu Asn145 150 155 160Val Ala Lys Ala Thr Met Ala Arg Leu
Val Lys Ala Asp Gln Asn Ala 165 170 175Asp Pro Ser Thr Phe Ala Leu
Asp Val Asn Thr Ala Lys Glu Ser Phe 180 185 190Asp Lys Ile Lys Gly
Phe Ile Ala Asn Lys Thr Asn Val Gln Asn Val 195 200 205Glu Asn Ala
Phe Asn Asp Tyr Ala Val Ala Asp Pro Ala Asp Lys Ala 210 215 220Asp
Lys Ala Asp Ala Ile Gln Ala Ala Phe Asn Thr Ala Ile Thr Gly225 230
235 240Leu Thr Ala Gly Thr Pro Asn Thr Ser Asn Pro Ser Ser Ala Val
Asp 245 250 255Ser Ile Asp Ala Ala Leu Lys Thr Val Ala Ser Asn Arg
Ala Thr Leu 260 265 270Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val
Asn Asn Leu Lys Ser 275 280 285Gln Ser Ala Ser Met Ala Ser Ala Ala
Ser Gln Ile Glu Asp Ala Asp 290 295 300Met Ala Lys Glu Met Ser Glu
Met Thr Lys Phe Lys Ile Leu Asn Glu305 310 315 320Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val 325 330 335Ser Lys
Leu Leu Gln 34012367PRTBacillus bombysepticus 12Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln
Ala Lys Met Ser Asn Ser Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr
Arg Met Arg Ser Arg Glu Gly Gly Leu Asn Val Ala Ala Arg 50 55 60Asn
Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75
80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln
85 90 95Ser Ala Ser Gly Thr Asn Thr Asp Lys Asn Gln Ala Ala Met Gln
Lys 100 105 110Glu Phe Asp Gln Leu Lys Glu Gln Ile Gln Tyr Ile Ala
Asp Asn Thr 115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asp Gly Ser
Asn Ser Thr Ile Asn 130 135 140Ile Gln Thr Leu Asp Ser His Asp Lys
Asn Lys Gln Ile Thr Ile Ser145 150 155 160Leu Asp Ser Ala Ser Leu
Lys Asn Leu Asp Ile Lys Asp Leu Ala Ile 165 170 175Gly Ser Ala Thr
Ile Asn Gln Thr Asp Leu Asp Thr Ala Thr Asn Ser 180 185 190Met Lys
Arg Leu Ala Thr Pro Ala Thr Asp Gly Lys Val Leu Ala Gln 195 200
205Asp Ile Ala Asp Ala Lys Ala Ala Phe Asn Lys Val Gln Ser Ala Tyr
210 215 220Thr Pro Ala Glu Val Asp Lys Ile Gln Asp Ala Phe Lys Ala
Tyr Asp225 230 235 240Lys Leu Ala Ala Asp Pro Ala Ser Lys Ala Thr
Asp Ile Ala Asp Ala 245 250 255Ala Lys Asn Val Asn Thr Val Phe Gly
Thr Leu Ala Thr Pro Thr Ala 260 265 270Thr Lys Phe Asp Pro Ser Ser
Ala Val Glu Lys Ile Asp Lys Ala Ile 275 280 285Glu Thr Ile Ala Ser
Ser Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg 290 295 300Leu Asp Phe
Asn Val Thr Asn Leu Lys Ser Gln Glu Asn Ser Met Ala305 310 315
320Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser
325 330 335Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu 340 345 350Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys
Leu Leu Gln 355 360 36513400PRTBacillus thuringiensis 13Met Thr Gly
Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe Ala Tyr Tyr1 5 10 15Arg Phe
Ser Ile Cys Arg Lys Val Asn Ile Lys Lys Trp Gly Phe Leu 20 25 30Asn
Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr 35 40
45Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp Arg Leu Ser
50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu
Ala65 70 75 80Ile Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn
Val Ala Ala 85 90 95Arg Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr
Ala Asp Ser Ala 100 105 110Leu Asn Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn 115 120 125Gln Ser Ala Ser Gly Thr Asn Thr
Asp Lys Asn Gln Ala Ala Met Gln 130 135 140Lys Glu Phe Asp Gln Leu
Lys Glu Gln Ile Gln Tyr Ile Ala Asp Asn145 150 155 160Thr Glu Phe
Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Ser Thr Ile 165 170 175Asn
Ile Gln Thr Leu Asp Ser His Asp Lys Asn Lys Gln Ile Thr Ile 180 185
190Ser Leu Asp Ser Ala Ser Leu Lys Asn Leu Asp Ile Lys Asp Leu Ala
195 200 205Ile Gly Ser Ala Thr Ile Asn Gln Thr Asp Leu Asp Thr Ala
Thr Asn 210 215 220Ser Met Lys Arg Leu Ala Thr Pro Ala Thr Asp Gly
Lys Val Leu Ala225 230 235 240Gln Asp Ile Ala Asp Ala Lys Ala Ala
Phe Asn Lys Val Gln Ser Ala 245 250 255Tyr Thr Pro Ala Glu Val Asp
Lys Ile Gln Asp Ala Phe Lys Ala Tyr 260 265 270Asp Lys Leu Ala Ala
Asp Pro Ala Ser Lys Asp Thr Asp Ile Ala Asp 275 280 285Ala Ala Lys
Asn Val Asn Thr Val Phe Gly Thr Leu Ala Thr Pro Thr 290 295 300Ala
Thr Lys Phe Asp Pro Ser Ser Ala Val Glu Lys Ile Asp Lys Ala305 310
315 320Ile Glu Thr Ile Ala Ser Ser Arg Ala Thr Leu Gly Ala Thr Leu
Asn 325 330 335Arg Leu Asp Phe Asn Val Thr Asn Leu Lys Ser Gln Glu
Asn Ser Met 340 345 350Ala Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp
Met Ala Lys Glu Met 355 360 365Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser Met 370 375 380Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln385 390 395 40014367PRTBacillus
thuringiensis 14Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ser Arg Glu Gly Gly
Leu Asn Val Ala Ala Arg 50 55 60Asn Thr Glu Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Ser Gly Thr Asn
Thr Asp Lys Asn Gln Ala Ala Met Gln Lys 100 105 110Glu Phe Asp Gln
Leu Lys Glu Gln Ile Gln Tyr Ile Ala Asp Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Ser Thr Ile Asn 130 135
140Ile Gln Ala Leu Asp Ser His Asp Lys Asn Lys Gln Ile Thr Ile
Ser145 150 155 160Leu Asp Ser Ala Ser Leu Lys Asn Leu Asp Ile Lys
Asp Leu Ala Ile 165 170 175Gly Ser Ala Thr Ile Asn Gln Thr Asp Leu
Asp Thr Ala Thr Asn Ser 180 185 190Met Lys Arg Leu Ala Thr Pro Ala
Thr Asp Gly Lys Val Leu Ala Gln 195 200 205Asp Ile Ala Asp Ala Lys
Ala Ala Phe Asn Lys Val Gln Ser Ala Tyr 210 215 220Thr Pro Ala Glu
Val Asp Lys Ile Gln Asp Ala Phe Lys Ala Tyr Asp225 230 235 240Lys
Leu Ala Ala Asp Pro Ala Ser Lys Asp Thr Asp Ile Ala Asp Ala 245 250
255Ala Lys Asn Val Asn Thr Val Phe Gly Thr Leu Ala Thr Pro Thr Ala
260 265 270Thr Lys Phe Asp Pro Ser Ser Ala Val Glu Lys Ile Asp Lys
Ala Ile 275 280 285Glu Thr Ile Ala Ser Ser Arg Ala Thr Leu Gly Ala
Thr Leu Asn Arg 290 295 300Leu Asp Phe Asn Val Thr Asn Leu Lys Ser
Gln Glu Asn Ser Met Ala305 310 315 320Ala Ser Ala Ser Gln Ile Glu
Asp Ala Asp Met Ala Lys Glu Met Ser 325 330 335Glu Met Thr Lys Phe
Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu 340 345 350Ser Gln Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln 355 360
36515397PRTBacillus cereus 15Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser
Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala
Arg Glu Asn Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala
Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala Leu Asp Lys 100 105
110Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr
115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Thr
Ile Ala 130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr Ser Lys Gln
Ile Asn Ile Asn145 150 155 160Leu Ala Asp Ser Ser Thr Ser Ala Leu
Lys Ile Glu Lys Leu Thr Ile 165 170 175Ser Gly Ser Thr Ala Ile Ala
Gly Lys Thr Glu Lys Val Thr Ile Thr 180 185 190Ala Glu Asp Ile Lys
Ala Ala Glu Glu Asp Ile Lys Ala Phe Thr Gln 195 200 205Ala Gln Glu
Gly Leu Ala Asn Leu Val Lys Glu Val Lys Asp Thr Asp 210 215 220Gly
Ser Val Lys Thr
Pro Gly Ser Thr Pro Asp Asp Ile Lys Lys Ala225 230 235 240Val Thr
Ala Phe Thr Glu Ser Phe Glu Lys Met Lys Lys Phe Met Asn 245 250
255Asp Glu Asp Ile Thr Lys Val Glu Glu Lys Ile Lys Ala Phe Asp Ala
260 265 270Ala Ser Pro Asp Leu Asp Ala Ala Lys Glu Met Gly Thr Ala
Phe Thr 275 280 285Ala Ala Met Lys Pro Ala Ala Gly Glu Ile Thr Lys
Ala Ala Met Lys 290 295 300Pro Asn Ala Ser Asp Ala Ile Lys Ser Ile
Asp Glu Ala Leu Glu Thr305 310 315 320Ile Ala Ser Asn Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp 325 330 335Phe Asn Val Asn Asn
Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala 340 345 350Ala Ser Gln
Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 355 360 365Thr
Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 370 375
380Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln385 390
39516266PRTBacillus cereus 16Met Arg Ile Gly Thr Asn Val Leu Ser
Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met Asn
Val Ala Met Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn Asn Ala
Ser Asp Asn Pro Ala Asn Ile Ala Ile 35 40 45Val Thr Arg Met His Ala
Arg Ala Ser Gly Met Arg Leu Ala Ile Arg 50 55 60Asn Asn Glu Asp Thr
Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr Leu
Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln 85 90 95Ser Ala
Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile Gly Glu Thr Thr
115 120 125Glu Phe Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro
Val Thr 130 135 140Leu Asp Asp Ile Asp His Thr Ile Asn Met Thr Lys
His Ile Pro Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Lys Ile
Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala Ala Ile Leu Lys Ile Glu
Glu Ala Leu Gln Ser Val Ser Ile His 180 185 190Arg Ala Asp Leu Gly
Ser Met Ile Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn
Ser Gln Ser Met Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu
Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230
235 240Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln
Ile 245 250 255Pro Gln Met Val Ser Lys Leu Leu Gln Ser 260
26517266PRTBacillus thuringiensis 17Met Arg Ile Gly Thr Asn Val Leu
Ser Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met
Asn Val Ala Met Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn His
Ala Ser Asp Asn Pro Ala Asn Val Ala Ile 35 40 45Val Thr Arg Met His
Ala Arg Ala Ser Gly Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp
Ala Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr
Val Thr Asn Val Leu Gln Arg Met Arg Asp Val Ala Val Gln 85 90 95Ser
Ala Asn Gly Thr Asn Leu Asn Lys Asn Arg Asp Ser Leu Asn Asn 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile Asp Glu Thr Thr
115 120 125Ala Phe Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro
Val Thr 130 135 140Leu Asp Asp Ile Gly His Thr Val Asn Val Thr Lys
His Ile Ser Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Asn Ile
Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu
Glu Ala Leu Gln Asn Val Ser Leu Tyr 180 185 190Arg Ala Asp Leu Gly
Ala Met Ile Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn
Ser Gln Ser Thr Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu
Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230
235 240Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln
Ile 245 250 255Pro Gln Met Val Tyr Lys Leu Leu Gln Ser 260
26518460PRTBacillus thuringiensis 18Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu Asn Val Ala Ala Asp 50 55 60Asn Thr Gln Asn
Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser
Ala Asn Gly Thr Asn Thr Asp Ser Asn Lys Ser Ala Leu Gln Lys 100 105
110Glu Phe Ala Glu Leu Gln Lys Gln Ile Thr Tyr Ile Ala Asp Asn Thr
115 120 125Gln Phe Asn Asp Lys Asn Leu Leu Lys Glu Asp Ser Glu Val
Lys Ile 130 135 140Gln Thr Leu Asp Ser Ser Lys Gly Glu Gln Gln Ile
Gly Ile Asp Leu145 150 155 160Lys Ala Val Thr Leu Glu Lys Leu Gly
Ile Asn Asn Ile Ser Ile Gly 165 170 175Lys Ala Asp Gly Thr Thr Glu
Gly Thr Lys Ala Asp Leu Thr Ala Leu 180 185 190Gln Ala Ala Ala Lys
Lys Leu Glu Lys Pro Asp Thr Gly Thr Met Glu 195 200 205Lys Asp Val
Lys Asp Ala Lys Glu Glu Phe Asp Lys Val Lys Ala Ser 210 215 220Leu
Ser Asp Glu Asp Val Lys Lys Ile Glu Ala Ala Phe Gly Glu Phe225 230
235 240Asp Lys Asp Lys Thr Asn Thr Thr Lys Ala Ser Asp Ile Phe Asn
Ala 245 250 255Ile Lys Asp Val Lys Leu Ala Asp Lys Ala Ala Ala Ala
Pro Ala Pro 260 265 270Ala Asp Leu Thr Lys Phe Lys Ala Ala Leu Asp
Lys Leu Gln Thr Pro 275 280 285Asn Ala Gly Thr Met Val Asp Asp Val
Lys Asp Ala Lys Asp Glu Phe 290 295 300Glu Lys Ile Lys Gly Ser Leu
Ser Asp Ala Asp Ala Gln Lys Ile Gln305 310 315 320Ala Ala Phe Glu
Glu Phe Glu Lys Ala Asn Thr Asp Asp Ser Lys Ala 325 330 335Ser Ala
Ile Tyr Asn Leu Ala Lys Asp Val Lys Val Asn Ala Thr Asp 340 345
350Thr Thr Thr Gly Thr Asp Lys Asp Thr Thr Thr Ser Thr Asp Lys Asp
355 360 365Ala Ala Leu Ala Ala Ile Ala Ala Ile Asp Ala Ala Leu Thr
Lys Val 370 375 380Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn
Arg Leu Asp Phe385 390 395 400Asn Val Asn Asn Leu Lys Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala 405 410 415Ser Gln Ile Glu Asp Ala Asp
Met Ala Lys Glu Met Ser Glu Met Thr 420 425 430Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala 435 440 445Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln 450 455 46019367PRTBacillus
cereus 19Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu
Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg
Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr
Ala Glu Asn Lys Ala Ala Met Gln Lys 100 105 110Glu Phe Gly Glu Leu
Lys Asp Gln Ile Lys Tyr Ile Ser Glu Asn Thr 115 120 125Gln Phe Asn
Asp Gln His Leu Leu Asn Ala Ala Lys Gly Ser Thr Asn 130 135 140Glu
Ile Ala Ile Gln Thr Leu Asp Ser Asp Ser Ser Ser Lys Gln Ile145 150
155 160Lys Ile Thr Leu Gln Gly Ala Ser Leu Asp Ser Leu Asp Ile Lys
Asp 165 170 175Leu Gln Ile Gly Ser Gly Ser Thr Val Ser Gln Thr Asp
Leu Asp Val 180 185 190Leu Asp Ala Thr Met Thr Arg Val Lys Thr Ala
Thr Gly Ala Thr Arg 195 200 205Asp Val Asp Val Gln Ala Ala Lys Ser
Ala Phe Asp Lys Val Lys Gly 210 215 220Leu Met Thr Lys Pro Ala Glu
Val Lys Ala Ile Glu Arg Ala Phe Glu225 230 235 240Asp Tyr Asn Ala
Gly Lys Thr Asp Ala Leu Ala Thr Ala Ile Glu Ala 245 250 255Ala Tyr
Thr Ala Asn Lys Thr Gly Leu Pro Ala Pro Ala Ala Ala Ala 260 265
270Gly Thr Val Asp Ala Leu Gly Ala Ile Thr Lys Ile Asp Ala Ala Leu
275 280 285Lys Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu
Asn Arg 290 295 300Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser
Ala Ser Met Ala305 310 315 320Ser Ala Ala Ser Gln Ile Glu Asp Ala
Asp Met Ala Lys Glu Met Ser 325 330 335Glu Met Thr Lys Phe Lys Ile
Leu Asn Glu Ala Gly Ile Ser Met Leu 340 345 350Ser Gln Ala Asn Gln
Thr Pro Gln Met Val Ser Lys Leu Leu Gln 355 360 36520377PRTBacillus
thuringiensis 20Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Ser Asp Asn Gln Lys Ala Leu Asp Lys 100 105 110Glu Phe Ser Ala
Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Ser Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Thr Lys Gln Ile Asn Ile
Asn145 150 155 160Leu Ala Asp Ser Ser Thr Thr Ala Leu Asn Ile Asp
Lys Leu Ser Ile 165 170 175Glu Gly Thr Gly Asn Lys Thr Ile Thr Leu
Thr Ala Ala Asp Ile Ala 180 185 190Lys Asp Lys Ala Asn Ile Asp Ala
Val Gly Thr Ala Lys Thr Ala Leu 195 200 205Ala Gly Leu Thr Gly Thr
Pro Ala Ala Ala Ala Ile Asn Ser Ala Val 210 215 220Ala Asp Phe Lys
Thr Ala Phe Ala Lys Ala Asp Lys Asn Leu Met Ser225 230 235 240Asp
Ala Gln Ile Lys Ala Val Thr Asp Ala Ile Thr Ala Phe Glu Ala 245 250
255Asp Ala Thr Pro Asp Leu Thr Lys Ala Lys Ala Ile Gly Thr Ala Tyr
260 265 270Thr Ala Pro Ala Ala Gly Asp Ile Thr Lys Ala Ser Pro Asn
Ala Ser 275 280 285Glu Ala Ile Lys Ser Ile Asp Ala Ala Leu Asp Thr
Ile Ala Ser Asn 290 295 300Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn305 310 315 320Asn Leu Lys Ser Gln Ser Ser
Ser Met Ala Ser Ala Ala Ser Gln Ile 325 330 335Glu Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 340 345 350Ile Leu Asn
Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 355 360 365Pro
Gln Met Val Ser Lys Leu Leu Gln 370 37521367PRTBacillus
thuringiensis 21Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Ala Asp Asn Gln Gln Ala Leu Gln Lys 100 105 110Glu Phe Gly Gln
Leu Lys Glu Gln Ile Ser Tyr Ile Ala Asp Asn Thr 115 120 125Glu Phe
Asn Asp Lys Thr Leu Leu Lys Ala Asp Asn Ser Val Lys Ile 130 135
140Gln Thr Leu Asp Ser Ala Asp Thr Asn Lys Gln Ile Ser Ile Asp
Leu145 150 155 160Lys Gly Val Thr Leu Asn Gln Leu Gly Leu Asp Thr
Val Asn Ile Gly 165 170 175Ser Glu Thr Leu Ser Ala Glu Ser Leu Asn
Val Ala Lys Ala Thr Met 180 185 190Ala Arg Leu Val Lys Ala Asp Gln
Asn Ala Asp Pro Ser Thr Phe Ala 195 200 205Leu Asp Val Asn Thr Ala
Lys Glu Ser Phe Asp Lys Ile Lys Gly Phe 210 215 220Ile Thr Asn Lys
Thr Asn Val Gln Asn Val Glu Asn Ala Phe Asn Asp225 230 235 240Tyr
Thr Val Ala Asp Pro Ala Asp Lys Ala Asp Lys Ala Asp Ala Ile 245 250
255Gln Ala Ala Phe Asn Thr Ala Ile Thr Gly Leu Thr Ala Gly Thr Pro
260 265 270Asn Thr Ser Asn Pro Ser Ser Ala Val Asp Ala Ile Asp Ala
Ala Leu 275 280 285Lys Thr Val Ala Ser Asn Arg Ala Thr Leu Gly Ala
Thr Leu Asn Arg 290 295 300Leu Asp Phe Asn Val Asn Asn Leu Lys Ser
Gln Ser Ala Ser Met Ala305 310 315 320Ser Ala Ala Ser Gln Ile Glu
Asp Ala Asp Met Ala Lys Glu Met Ser 325 330 335Glu Met Thr Lys Phe
Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu 340 345 350Ser Gln Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln 355 360
36522266PRTBacillus thuringiensis 22Met Arg Ile Gly Thr Asn Val Leu
Ser Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met
Asn Val Ala Met Glu His Phe Ala Thr 20 25 30Gly Lys Lys Leu Asn His
Ala Ser Asp Asn Pro Ala Asn Val Ala Ile 35 40 45Val Thr Arg Met His
Ala Arg Ala Ser Gly Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp
Ala Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr
Val Met Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln 85 90 95Ser
Ala Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile Gly Glu Thr Thr
115 120 125Glu Phe Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro
Val Thr 130 135 140Leu Asp Asp Ile Gly His Thr Val Asn Val Thr Lys
His Thr Ser Pro145 150 155 160Ser Pro Thr Lys His Asp Ile Lys Ile
Ser Thr Glu Gln Glu Ala Arg 165
170 175Ala Ala Ile Arg Lys Ile Glu Glu Ala Leu Gln Asn Val Ser Leu
His 180 185 190Arg Ala Asp Phe Gly Ala Met Ile Asn Arg Leu Gln Phe
Asn Ile Glu 195 200 205Asn Leu Asn Ser Gln Ser Met Ala Leu Thr Asp
Ala Ala Ser Arg Ile 210 215 220Glu Asp Ala Asp Met Ala Gln Glu Met
Ser Asp Phe Leu Lys Phe Lys225 230 235 240Leu Leu Thr Glu Val Ala
Leu Ser Met Val Ser Gln Ala Asn Gln Ile 245 250 255Pro Gln Met Val
Ser Lys Leu Leu Gln Ser 260 26523367PRTBacillus thuringiensis 23Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10
15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser
20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala
Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala
Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp
Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Ala Asp Asn
Gln Gln Ala Leu Gln Lys 100 105 110Glu Phe Gly Gln Leu Lys Glu Gln
Ile Ser Tyr Ile Ala Asp Asn Thr 115 120 125Glu Phe Asn Asp Lys Thr
Leu Leu Lys Ala Asp Asn Ser Val Lys Ile 130 135 140Gln Thr Leu Asp
Ser Ala Asp Thr Asn Lys Gln Ile Ser Ile Asp Leu145 150 155 160Lys
Gly Val Thr Leu Asn Gln Leu Gly Leu Asp Thr Val Asn Ile Gly 165 170
175Ser Glu Thr Leu Ser Ala Glu Ser Leu Asn Val Ala Lys Ala Thr Met
180 185 190Ala Arg Leu Val Lys Ala Asp Gln Asn Ala Asp Pro Ser Thr
Phe Ala 195 200 205Leu Asp Val Asn Thr Ala Lys Glu Ser Phe Asp Lys
Ile Lys Gly Phe 210 215 220Ile Thr Asn Lys Thr Asn Val Gln Asn Val
Glu Asn Ala Phe Asn Asp225 230 235 240Tyr Thr Val Ala Asp Pro Ala
Asp Lys Ala Asp Lys Ala Asp Ala Ile 245 250 255Gln Ala Ala Phe Asn
Thr Ala Ile Thr Gly Leu Thr Ala Gly Thr Pro 260 265 270Asn Thr Ser
Asn Pro Ser Ser Ala Val Asp Ala Ile Asp Ala Ala Leu 275 280 285Lys
Thr Val Ala Ser Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg 290 295
300Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ala Ser Met
Ala305 310 315 320Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala
Lys Glu Met Ser 325 330 335Glu Met Thr Lys Phe Lys Ile Leu Asn Glu
Ala Gly Ile Ser Met Leu 340 345 350Ser Gln Ala Asn Gln Thr Pro Gln
Met Val Ser Lys Leu Leu Gln 355 360 36524266PRTBacillus
thuringiensis 24Met Arg Ile Gly Thr Asn Val Leu Ser Met Asn Ala Arg
Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu
His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn His Ala Ser Asp Asn Pro
Ala Asn Ile Val Ile 35 40 45Val Thr Arg Met Tyr Ala Arg Ala Ser Gly
Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp Ala Ile Ser Met Leu
Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr Val Thr Asn Ile Leu
Gln His Met Arg Asp Phe Ala Ile Gln 85 90 95Ser Ala Asn Gly Thr Asn
Ser Asn Thr Asn Arg Asp Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser
Leu Thr Glu Pro Ile Gly Tyr Ile Gly Glu Thr Thr 115 120 125Glu Phe
Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro Ile Thr 130 135
140Leu Asp Asp Ile Gly His Thr Ile Asn Met Thr Lys His Ile Pro
Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Lys Ile Ser Thr Glu
Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu Glu Ala Leu
Gln Asn Val Ser Leu His 180 185 190Arg Ala Asp Leu Gly Ser Met Ile
Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn Ser Gln Ser
Met Ala Leu Ile Asp Thr Ala Ser Gln Val 210 215 220Glu Asp Ala Asp
Met Ala Gln Glu Ile Ser Asp Phe Leu Lys Phe Lys225 230 235 240Leu
Leu Thr Ala Val Ala Leu Ser Val Val Ser Gln Ala Asn Gln Ile 245 250
255Pro Gln Ile Val Ser Lys Leu Leu Gln Ser 260 26525381PRTBacillus
thuringiensis 25Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ser Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Asp Lys Asn Gln Ser Ala Leu Asp Lys 100 105 110Glu Phe Ala Ala
Leu Lys Asp Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Gln Lys Leu Leu Asp Gly Ser Lys Lys Ser Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Asn Lys Gln Ile Asp Ile
Gln145 150 155 160Leu Ser Asn Val Ser Thr Lys Glu Leu Lys Leu Asp
Thr Leu Ser Ile 165 170 175Glu Gly Ser Ser Ser Lys Thr Phe Thr Ile
Thr Ala Asp Asp Met Leu 180 185 190Ala Val Gly Thr Ala Asn Ala Thr
Ala Lys Ala Lys Ala Gly Thr Leu 195 200 205Lys Gly Leu Asn Val Thr
Thr Gly Asp Leu Thr Ala Ala Lys Thr Asp 210 215 220Val Gln Asp Phe
Arg Ala Ala Phe Asp Lys Val Lys Gly Phe Met Gly225 230 235 240Ser
Thr Glu Val Thr Asn Ile Glu Lys Ala Leu Thr Lys Phe Asp Gly 245 250
255Asp Gln Ser Leu Ala Asn Ala Lys Ala Ile Gly Asp Ala Leu Thr Ser
260 265 270Asp Leu Ala Thr Thr Ile Ala Lys Asp Gln Thr Tyr Ser Lys
Asn Val 275 280 285Ser Asn Ala Ser Ser Ala Ile Ala Ser Ile Asp Ala
Ala Leu Glu Ser 290 295 300Ile Ala Ser Asn Arg Ala Thr Leu Gly Ala
Thr Leu Asn Arg Leu Asp305 310 315 320Phe Asn Val Asn Asn Leu Lys
Ser Gln Ser Ser Ser Met Ala Ser Ala 325 330 335Ala Ser Gln Ile Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 340 345 350Thr Lys Phe
Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 355 360 365Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln 370 375
38026381PRTBacillus thuringiensis 26Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ser Asn Gln 85 90 95Ser
Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser Ala Leu Asp Lys 100 105
110Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp Tyr Ile Ser Lys Asn Thr
115 120 125Glu Phe Asn Asp Gln Lys Leu Leu Asp Gly Ser Lys Lys Ser
Ile Ala 130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr Asn Lys Gln
Ile Asp Ile Gln145 150 155 160Leu Ser Asn Val Ser Thr Lys Glu Leu
Lys Leu Asp Thr Leu Ser Ile 165 170 175Glu Gly Ser Ser Ser Lys Thr
Phe Thr Ile Thr Ala Asp Asp Met Leu 180 185 190Ala Val Gly Thr Ala
Asn Ala Thr Ala Lys Ala Lys Ala Gly Thr Leu 195 200 205Lys Gly Leu
Asn Val Thr Thr Gly Asp Leu Thr Ala Ala Lys Thr Asp 210 215 220Val
Gln Asp Phe Arg Ala Ala Phe Asp Lys Val Lys Gly Phe Met Gly225 230
235 240Ser Thr Glu Val Thr Asn Ile Glu Lys Ala Leu Thr Lys Phe Asp
Gly 245 250 255Asp Gln Ser Leu Ala Asn Ala Lys Ala Ile Gly Asp Ala
Leu Thr Ser 260 265 270Asp Leu Ala Thr Thr Ile Ala Lys Asp Gln Thr
Tyr Ser Lys Asn Val 275 280 285Ser Asn Ala Ser Ser Ala Ile Ala Ser
Ile Asp Ala Ala Leu Glu Ser 290 295 300Ile Ala Ser Asn Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp305 310 315 320Phe Asn Val Asn
Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala 325 330 335Ala Ser
Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 340 345
350Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln
355 360 365Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln 370
375 38027381PRTBacillus thuringiensis 27Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys
Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn
Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met
Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln
Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ser Asn Gln 85 90
95Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser Ala Leu Asp Lys
100 105 110Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp Tyr Ile Ser Lys
Asn Thr 115 120 125Glu Phe Asn Asp Gln Lys Leu Leu Asp Gly Ser Lys
Lys Ser Ile Ala 130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr Asn
Lys Gln Ile Asp Ile Gln145 150 155 160Leu Ser Asn Val Ser Thr Lys
Glu Leu Lys Leu Asp Thr Leu Ser Ile 165 170 175Glu Gly Ser Ser Ser
Lys Thr Phe Thr Ile Thr Ala Asp Asp Met Leu 180 185 190Ala Val Gly
Thr Ala Asn Ala Thr Ala Lys Ala Lys Ala Gly Thr Leu 195 200 205Lys
Gly Leu Asn Val Thr Thr Gly Asp Leu Thr Ala Ala Lys Thr Asp 210 215
220Val Gln Asp Phe Arg Ala Ala Phe Asp Lys Val Lys Gly Phe Met
Gly225 230 235 240Ser Thr Glu Val Thr Asn Ile Glu Lys Ala Leu Thr
Lys Phe Asp Gly 245 250 255Asp Gln Ser Leu Ala Asn Ala Lys Ala Ile
Gly Asp Ala Leu Thr Ser 260 265 270Asp Leu Ala Thr Thr Ile Ala Lys
Asp Gln Thr Tyr Ser Lys Asn Val 275 280 285Ser Asn Ala Ser Ser Ala
Ile Ala Ser Ile Asp Ala Ala Leu Glu Ser 290 295 300Ile Ala Ser Asn
Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp305 310 315 320Phe
Asn Val Asn Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala 325 330
335Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met
340 345 350Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu
Ser Gln 355 360 365Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu
Gln 370 375 38028397PRTBacillus thuringiensis 28Met Thr Gly Ile Thr
Ile Asn Leu Glu Ile Asp Phe Phe Ala Tyr Tyr1 5 10 15Arg Phe Ser Ile
Cys Arg Lys Val Asn Ile Lys Lys Trp Gly Phe Leu 20 25 30Asn Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr 35 40 45Met Arg
Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser 50 55 60Ser
Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala65 70 75
80Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ala
85 90 95Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr Ala Asp Ser
Ala 100 105 110Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
Leu Ala Asn 115 120 125Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn
Gln Ala Ala Leu Asn 130 135 140Lys Glu Phe Asp Ala Leu Lys Glu Gln
Ile Asp Tyr Ile Ser Thr Asn145 150 155 160Thr Glu Phe Asn Asp Lys
Lys Leu Leu Asp Gly Ser Asn Lys Thr Ile 165 170 175Ala Val Gln Thr
Leu Asp Asn Ala Asp Thr Ser Lys Gln Ile Asn Ile 180 185 190Asn Leu
Ser Asn Val Ser Thr Lys Glu Leu Gly Leu Asp Thr Leu Ser 195 200
205Ile Gly Thr Asp Lys Val Glu Lys Thr Val Tyr Asp Ala Thr Thr Lys
210 215 220Ala Phe Ala Asp Leu Gly Ala Lys Thr Gly Ala Asp Lys Ala
Ala Phe225 230 235 240Asp Ala Asp Val Thr Ala Ala Met Lys Glu Phe
Asp Lys Val Lys Pro 245 250 255Phe Met Ser Ala Asp Asp Val Lys Lys
Ile Glu Thr Lys Leu Glu Asp 260 265 270Tyr Asn Lys Ala Asn Asp Ala
Gly Ala Gln Thr Ala Ala Gln Ala Leu 275 280 285Gly Lys Glu Phe Ala
Thr Leu Thr Lys Leu Glu Thr Thr Asp Leu Lys 290 295 300Ala Asn Ala
Ser Gly Ala Ile Ala Ser Ile Asp Thr Ala Leu Lys Asn305 310 315
320Ile Ala Ser Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
325 330 335Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ser Ser Met Ala
Ser Ala 340 345 350Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu
Met Ser Glu Met 355 360 365Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly
Ile Ser Met Leu Ser Gln 370 375 380Ala Asn Gln Thr Pro Gln Met Val
Ser Lys Leu Leu Gln385 390 39529397PRTBacillus thuringiensis 29Met
Thr Gly Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe Ala Tyr Tyr1 5 10
15Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys Lys Trp Gly Phe Leu
20 25 30Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu
Tyr 35 40 45Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg
Leu Ser 50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
Gly Leu Ala65 70 75 80Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala 85 90 95Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile
Arg Thr Ala Asp Ser Ala 100 105 110Met Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn 115 120 125Gln Ser Ala Asn Gly Thr
Asn Thr Asn Glu Asn Gln Ala Ala Leu Asn 130 135 140Lys Glu Phe Asp
Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Thr Asn145 150 155 160Thr
Glu Phe Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Thr Ile 165 170
175Ala Val Gln Thr Leu Asp Asn Ala Asp Thr Ser Lys Gln Ile Asn
Ile
180 185 190Asn Leu Ser Asn Val Ser Thr Lys Glu Leu Gly Leu Ser Thr
Leu Ser 195 200 205Ile Gly Thr Asp Lys Val Glu Lys Thr Val Tyr Asp
Ala Thr Thr Lys 210 215 220Ala Phe Ala Asp Leu Gly Ala Lys Thr Gly
Thr Asp Lys Ala Ala Phe225 230 235 240Ala Ala Asp Val Thr Ala Ala
Met Lys Glu Phe Asp Lys Val Lys Pro 245 250 255Phe Met Ser Ala Asp
Asp Val Lys Lys Ile Glu Thr Lys Leu Glu Asp 260 265 270Tyr Asn Lys
Ala Asn Asp Ala Gly Ala Glu Ala Ala Ala Gln Ala Leu 275 280 285Gly
Lys Glu Phe Ala Thr Leu Thr Lys Leu Glu Thr Thr Asp Leu Lys 290 295
300Ala Asn Ala Ser Gly Ala Ile Ala Ser Ile Asp Thr Ala Leu Lys
Asn305 310 315 320Ile Ala Ser Asn Arg Ala Thr Leu Gly Ala Thr Leu
Asn Arg Leu Asp 325 330 335Phe Asn Val Asn Asn Leu Lys Ser Gln Ser
Ser Ser Met Ala Ser Ala 340 345 350Ala Ser Gln Ile Glu Asp Ala Asp
Met Ala Lys Glu Met Ser Glu Met 355 360 365Thr Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 370 375 380Ala Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln385 390 39530406PRTBacillus
weihenstephanensis 30Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg
Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala
Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp
Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu
Ser Gly Leu Ser Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser
Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn
Ile Leu Leu Arg Met Arg Asp Leu Ser Asn Gln 85 90 95Ser Ala Asn Gly
Thr Asn Thr Asp Glu Asn Gln Gln Ala Leu Asn Lys 100 105 110Glu Phe
Ala Ala Leu Lys Asp Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120
125Glu Phe Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Ser Ile Ala
130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr Thr Lys Gln Ile Asn
Ile Asp145 150 155 160Leu Ser Asn Val Ser Thr Asp Thr Leu Asn Ile
Ser Gly Leu Thr Ile 165 170 175Asn Gly Lys Lys Asp Ile Thr Val Thr
Ile Ser Asp Lys Asp Ile Ala 180 185 190Asn Ala Ala Thr Asp Ile Gly
Lys Ala Thr Ser Ala Gln Gln Gly Leu 195 200 205Ala Asp Leu Thr Asp
Thr Thr Pro Ala Val Pro Asp Thr Pro Ala Val 210 215 220Ile Gly Thr
Gly Thr Ala Gly Asn Pro Gln Phe Pro Ala Val Lys Gly225 230 235
240Thr Pro Glu Ile Pro Gly Ser Ser Pro Ala Glu Ile Ala Lys Ala Val
245 250 255Asp Asp Phe Lys Gln Ala Phe Asn Lys Val Lys Gly Leu Met
Ser Asp 260 265 270Ser Ala Val Ser Ala Met Glu Gln Lys Phe Ala Thr
Phe Glu Lys Asp 275 280 285Lys Ser Leu Ala Asn Ala Lys Asp Ile Gly
Thr Ala Phe Ser Ala Pro 290 295 300Ile Ala Gly Asn Ile Thr Lys Gly
Glu Gln Asn Ala Ser Gly Ala Ile305 310 315 320Lys Ser Ile Asp Ala
Ala Leu Glu Lys Ile Ala Ser Asn Arg Ala Thr 325 330 335Leu Gly Ala
Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys 340 345 350Ser
Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 355 360
365Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn
370 375 380Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro
Gln Met385 390 395 400Val Ser Lys Leu Leu Gln 40531406PRTBacillus
thuringiensis 31Met Thr Gly Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe
Ala Tyr Tyr1 5 10 15Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys Lys
Trp Gly Phe Leu 20 25 30Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met
Arg Thr Gln Glu Tyr 35 40 45Met Arg Gln Asn Gln Ala Lys Met Ser Asn
Ala Met Asp Arg Leu Ser 50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser
Asp Asp Ala Ala Gly Leu Ala65 70 75 80Ile Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Gly Val Ala Ala 85 90 95Asn Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala 100 105 110Leu Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn 115 120 125Gln Ser
Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala Leu Asp 130 135
140Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys
Asn145 150 155 160Thr Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp
Asn Lys Ser Ile 165 170 175Ala Ile Gln Thr Leu Asp Asn Ala Asp Thr
Ala Lys Gln Ile Asn Ile 180 185 190Asn Leu Ala Asp Ser Ser Thr Lys
Ala Leu Asn Ile Asp Thr Leu Ser 195 200 205Ile Ala Gly Thr Thr Asp
Lys Thr Ile Thr Ile Thr Ala Lys Asp Leu 210 215 220Thr Asp Asn Lys
Thr Thr Leu Asp Ala Leu Lys Thr Ala Lys Asp Asp225 230 235 240Leu
Ala Lys Leu Asp Asp Lys Ser Asp Gln Ala Thr Ile Asp Lys Ala 245 250
255Val Asp Ala Phe Lys Thr Ala Phe Asn Asn Val Asp Lys Asn Leu Leu
260 265 270Ser Asp Lys Ala Ile Glu Gly Ile Thr Glu Lys Met Thr Ala
Phe Asp 275 280 285Gly Thr His Thr Ala Ala Ala Ala Ile Gly Ala Ala
Tyr Thr Glu Pro 290 295 300Thr Ala Ala Asp Ile Lys Lys Ser Ala Pro
Asn Ala Ser Gly Ala Ile305 310 315 320Lys Ser Ile Asp Ala Ala Leu
Glu Thr Ile Ala Ser Asn Arg Ala Thr 325 330 335Leu Gly Ala Thr Leu
Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys 340 345 350Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 355 360 365Asp
Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 370 375
380Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln
Met385 390 395 400Val Ser Lys Leu Leu Gln 40532266PRTBacillus
thuringiensis 32Met Arg Ile Gly Thr Asn Val Leu Ser Met Asn Ala Arg
Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu
His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn His Ala Ser Asp Asn Pro
Ala Asn Val Ala Ile 35 40 45Val Thr Arg Met His Ala Arg Ala Ser Gly
Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp Ala Leu Ser Met Leu
Arg Thr Ala Glu Ala Thr Leu65 70 75 80Gln Thr Val Ala Asn Ile Leu
Gln Arg Met Arg Asp Leu Ala Val Gln 85 90 95Ser Ser Asn Asp Thr Asn
Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser
Leu Thr Glu Gln Ile Ser Tyr Ile Gly Glu Thr Thr 115 120 125Glu Phe
Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro Val Thr 130 135
140Leu Asp Asp Ile Gly His Thr Val Asn Val Thr Lys His Ile Ser
Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Lys Ile Ser Thr Glu
Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu Glu Ala Leu
Gln Asn Val Leu Leu His 180 185 190Arg Ala Asp Leu Gly Ala Met Ile
Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn Ser Gln Ser
Met Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu Asp Ala Asp
Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230 235 240Leu
Leu Ser Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln Ile 245 250
255Pro Gln Met Val Ser Glu Leu Leu Gln Ser 260 26533393PRTBacillus
thuringiensis 33Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Thr Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Asn Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Asp Asp Asn Gln Lys Ala Leu Asp Lys 100 105 110Glu Phe Ser Ala
Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asn Gly Glu Asn Lys Thr Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Thr Lys Gln Ile Asn Ile
Asn145 150 155 160Leu Ala Asp Ser Ser Thr Ser Ala Leu Gln Ile Asp
Lys Leu Thr Ile 165 170 175Ser Gly Lys Thr Thr Asp Thr Thr Lys Thr
Gln Thr Ile Thr Val Thr 180 185 190Asp Asp Glu Ile Lys Ala Ala Lys
Thr Asp Ile Asp Glu Phe Asn Asp 195 200 205Ala Lys Lys Ala Leu Ala
Asp Leu Lys Ala Glu Ser Ala Pro Ser Lys 210 215 220Gly Asp Gly Ser
Ser Asp Asp Glu Ile Lys Glu Ala Val Ser Asn Phe225 230 235 240Lys
Lys Ser Phe Glu Lys Ile Gln Lys Phe Met Asn Asp Ser Asp Ile 245 250
255Lys Thr Val Gln Thr Glu Ile Glu Lys Phe Asp Ala Ala Ala Pro Ala
260 265 270Leu Asp Lys Ala Lys Gly Met Gly Ile Ala Phe Thr Ser Ala
Met Asp 275 280 285Pro Lys Ala Gly Thr Ile Thr Lys Ala Ala Thr Arg
Gln Asn Ala Ser 290 295 300Asp Ala Ile Lys Ser Ile Asp Ala Ala Leu
Glu Thr Ile Ala Ser Asn305 310 315 320Arg Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn 325 330 335Asn Leu Lys Ser Gln
Ser Ser Ser Met Ala Ala Ala Ala Ser Gln Ile 340 345 350Glu Asp Ala
Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 355 360 365Ile
Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 370 375
380Pro Gln Met Val Ser Lys Leu Leu Gln385 39034426PRTBacillus
thuringiensis 34Met Thr Gly Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe
Ala Tyr Tyr1 5 10 15Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys Lys
Trp Gly Phe Leu 20 25 30Ile Met Arg Ile Asn Thr Asn Ile Asn Ser Met
Arg Thr Gln Glu Tyr 35 40 45Met Arg Gln Asn Gln Thr Lys Met Ser Asn
Ala Met Asp Arg Leu Ser 50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser
Asp Asp Ala Ala Gly Leu Ala65 70 75 80Ile Ala Thr Arg Met Arg Ala
Arg Glu Asn Gly Leu Gly Val Ala Ala 85 90 95Asn Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala 100 105 110Met Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn 115 120 125Gln Ser
Ala Asn Gly Thr Asn Thr Asp Asp Asn Gln Lys Ala Leu Asp 130 135
140Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys
Asn145 150 155 160Thr Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Glu
Asn Lys Thr Ile 165 170 175Ala Ile Gln Thr Leu Asp Asn Ala Asp Thr
Thr Lys Gln Ile Asn Ile 180 185 190Asn Leu Ala Asp Ser Ser Thr Ser
Ala Leu Gln Ile Asp Lys Leu Thr 195 200 205Ile Ser Gly Lys Thr Thr
Asp Thr Thr Lys Thr Gln Thr Ile Thr Val 210 215 220Thr Asp Asp Glu
Ile Lys Ala Ala Lys Thr Asp Ile Asp Glu Phe Asn225 230 235 240Asp
Ala Lys Lys Ala Leu Ala Asp Leu Lys Ala Glu Ser Ala Pro Ser 245 250
255Lys Gly Asp Gly Ser Ser Asp Asp Glu Ile Lys Glu Ala Val Ser Asn
260 265 270Phe Lys Lys Ser Phe Glu Lys Ile Gln Lys Phe Met Asn Asp
Ser Asp 275 280 285Ile Lys Thr Val Gln Thr Glu Ile Glu Lys Phe Asp
Ala Ala Ala Pro 290 295 300Ala Leu Asp Lys Ala Lys Gly Met Gly Ile
Ala Phe Thr Ser Ala Met305 310 315 320Asp Pro Lys Ala Gly Thr Ile
Thr Lys Ala Ala Thr Arg Gln Asn Ala 325 330 335Ser Asp Ala Ile Lys
Ser Ile Asp Ala Ala Leu Glu Thr Ile Ala Ser 340 345 350Asn Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val 355 360 365Asn
Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ala Ala Ala Ser Gln 370 375
380Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys
Phe385 390 395 400Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser
Gln Ala Asn Gln 405 410 415Thr Pro Gln Met Val Ser Lys Leu Leu Gln
420 42535269PRTBacillus thuringiensis 35Met Ser Ile Met Arg Ile Gly
Thr Asn Val Leu Ser Met Asn Ala Arg1 5 10 15Gln Ser Leu Tyr Glu Asn
Glu Lys Arg Met Asn Val Ala Met Glu His 20 25 30Leu Ala Thr Gly Lys
Lys Leu Asn His Ala Ser Asp Asn Pro Ala Asn 35 40 45Ile Val Ile Val
Thr Arg Met Tyr Ala Arg Ala Ser Gly Met Arg Val 50 55 60Ala Ile Arg
Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala Glu65 70 75 80Ala
Ala Leu Gln Thr Val Thr Asn Ile Leu Gln His Met Arg Asp Phe 85 90
95Ala Ile Gln Ser Ala Asn Gly Thr Asn Ser Asn Thr Asn Arg Asp Ser
100 105 110Leu Asn Lys Glu Phe Gln Ser Leu Thr Glu Pro Ile Gly Tyr
Ile Gly 115 120 125Glu Thr Thr Glu Phe Asn Asp Leu Ser Val Phe Asp
Gly Gln Asn Arg 130 135 140Pro Ile Thr Leu Asp Asp Ile Gly His Thr
Ile Asn Met Thr Lys His145 150 155 160Ile Pro Pro Ser Pro Thr Gln
His Asp Ile Lys Ile Ser Thr Glu Gln 165 170 175Glu Ala Arg Ala Ala
Ile Arg Lys Ile Glu Glu Ala Leu Gln Asn Val 180 185 190Ser Leu His
Arg Ala Asp Leu Gly Ser Met Ile Asn Arg Leu Gln Phe 195 200 205Asn
Ile Glu Asn Leu Asn Ser Gln Ser Met Ala Leu Ile Asp Thr Ala 210 215
220Ser Gln Val Glu Asp Ala Asp Met Ala Gln Glu Ile Ser Asp Phe
Leu225 230 235 240Lys Phe Lys Leu Leu Thr Ala Val Ala Leu Ser Val
Val Ser Gln Ala 245 250 255Asn Gln Ile Pro Gln Ile Val Ser Lys Leu
Leu Gln Ser 260 26536414PRTBacillus thuringiensis 36Met Ala Arg Ile
Thr Ile Asn Leu Glu Ile Asp Phe Phe Ala Tyr Tyr1 5 10 15Arg Phe Ser
Ile Cys Arg Lys Val Asn Ile Lys Lys Trp Gly Phe Leu 20 25 30Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Asp Tyr 35 40 45Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser 50 55
60Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu
Ala65
70 75 80Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala
Ala 85 90 95Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp
Ser Ala 100 105 110Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp Ile Ser Asn 115 120 125Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys
Asn Gln Ser Ala Leu Asp 130 135 140Lys Glu Phe Ala Ala Leu Lys Asp
Gln Ile Asp Tyr Ile Ser Lys Asn145 150 155 160Thr Glu Phe Asn Asp
Gln Lys Leu Leu Asp Gly Ser Lys Lys Ser Ile 165 170 175Ala Ile Gln
Thr Leu Asp Asn Ala Asp Thr Asn Lys Gln Ile Asp Ile 180 185 190Gln
Leu Ser Asn Val Ser Thr Lys Glu Leu Lys Leu Asp Thr Leu Ser 195 200
205Ile Glu Gly Ser Ser Ser Lys Thr Phe Thr Ile Thr Ala Asp Asp Met
210 215 220Leu Ala Val Gly Thr Ala Asn Ala Thr Ala Lys Ala Lys Ala
Gly Thr225 230 235 240Leu Lys Gly Leu Asn Val Thr Thr Gly Asp Leu
Thr Ala Ala Lys Thr 245 250 255Asp Val Gln Asp Phe Arg Ala Ala Phe
Asp Lys Val Lys Gly Phe Met 260 265 270Gly Ser Thr Glu Val Thr Asn
Ile Glu Lys Ala Leu Thr Lys Phe Asp 275 280 285Gly Asp Gln Ser Leu
Ala Asn Ala Lys Ala Ile Gly Asp Ala Leu Thr 290 295 300Ser Asp Leu
Ala Thr Thr Ile Ala Lys Asp Gln Thr Tyr Ser Lys Asn305 310 315
320Val Ser Asn Ala Ser Ser Ala Ile Ala Ser Ile Asp Ala Ala Leu Glu
325 330 335Ser Ile Ala Ser Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn
Arg Leu 340 345 350Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ser
Ser Met Ala Ser 355 360 365Ala Ala Ser Gln Ile Glu Asp Ala Asp Met
Ala Lys Glu Met Ser Glu 370 375 380Met Thr Lys Phe Lys Ile Leu Asn
Glu Ala Gly Ile Ser Met Leu Ser385 390 395 400Gln Ala Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln 405 41037266PRTBacillus cereus
37Met Arg Ile Gly Thr Asn Val Leu Ser Met Asn Ala Arg Gln Ser Leu1
5 10 15Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu His Leu Ala
Thr 20 25 30Gly Lys Lys Leu Asn His Ala Ser Asn Asn Pro Ala Asn Val
Ala Ile 35 40 45Val Thr Arg Met His Ala Arg Ala Ser Gly Met Arg Val
Ala Ile Arg 50 55 60Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala
Glu Ala Ala Leu65 70 75 80Gln Thr Val Thr Asn Val Leu Gln Arg Met
Arg Asp Val Ala Val Gln 85 90 95Ser Ala Asn Gly Thr Asn Ser Ser Lys
Asn Arg Asp Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser Leu Thr Glu
Gln Ile Gly Tyr Ile Asp Glu Thr Thr 115 120 125Glu Phe Asn Asp Leu
Ser Val Phe Asp Gly Gln Asn Arg Thr Val Thr 130 135 140Leu Asp Asp
Ile Gly His Thr Val Asn Val Thr Lys His Ile Pro Pro145 150 155
160Ser Pro Thr Gln His Asp Ile Asn Ile Ser Thr Glu Gln Glu Ala Arg
165 170 175Ala Ala Ile Arg Lys Ile Glu Glu Ala Leu Gln Asn Val Ser
Leu His 180 185 190Arg Ala Asp Leu Gly Ala Met Ile Asn Arg Leu Gln
Phe Asn Ile Glu 195 200 205Asn Leu Asn Ser Gln Ser Thr Ala Leu Thr
Asp Ala Ala Ser Arg Ile 210 215 220Glu Asp Ala Asp Met Ala Gln Glu
Met Ser Asp Phe Leu Lys Phe Lys225 230 235 240Leu Leu Thr Glu Val
Ala Leu Ser Met Val Ser Gln Ala Asn Gln Ile 245 250 255Pro Gln Met
Val Ser Lys Leu Leu Gln Ser 260 26538494PRTBacillus cereus 38Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10
15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser
20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala
Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Ser Val Ala
Ala Asp 50 55 60Asn Thr Gln Asn Gly Met Ser Leu Ile Arg Thr Ala Asp
Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn
Gln Val Ala Leu Gln Lys 100 105 110Glu Phe Ala Ala Leu Lys Glu Gln
Ile Thr Tyr Ile Ala Asp Asn Thr 115 120 125Gln Phe Asn Asp Lys Asn
Leu Leu Asn Gly Asn Gln Thr Ile Asn Ile 130 135 140Gln Thr Leu Asp
Ser His Asp Ser Thr Lys Gln Ile Gly Ile Asp Leu145 150 155 160Lys
Ser Ala Thr Leu Glu Ala Leu Gly Ile Lys Asp Leu Thr Val Gly 165 170
175Ala Val Gly Ser Thr Glu Ala Lys Asn Tyr Val Asp Ala Lys Glu Ala
180 185 190Leu Ala Lys Asn Val Ala Ala Asn Glu Phe Ile Asp Ala Lys
Lys Ala 195 200 205Leu Asp Gly Asn Ala Ile Ala Lys Gly Tyr Val Glu
Ala Lys Thr Ala 210 215 220Phe Asp Asp Ala Lys Pro Glu Val Lys Ala
Leu Val Ser Asn Tyr Thr225 230 235 240Asp Ala Leu Ala Ala Leu Ala
Lys Asp Asp Thr Asn Asp Asp Leu Lys 245 250 255Lys Asp Val Ala Asp
Thr Lys Ala Leu Met Asp Ala Asn Thr Val Ala 260 265 270Lys Thr Tyr
Phe Glu Ala Lys Thr Ala His Asp Gly Ala Asp Gln Ala 275 280 285Ile
Lys Asp Ile Val Thr Thr Tyr Asp Ser Lys Leu Gly Ala Leu Asp 290 295
300Asp Ala Ala Asn Lys Ala Ile Ser Asp Phe Asp Lys Ala Lys Ala
Ala305 310 315 320Phe Asp Glu Ser Pro Ala Ala Lys Glu Leu Val Lys
Thr Met Asp Asp 325 330 335Ala Lys Gln Ala Ala Thr Gln Asn Asn Thr
Ala Asn Ala Tyr Leu Val 340 345 350Ala Lys Ala Ala Ala Glu Leu Ala
Pro Asn Asp Ala Asp Lys Lys Ala 355 360 365Glu Leu Glu Asn Ala Thr
Lys Ala Leu Glu Lys Asp Asp Thr Ala Lys 370 375 380Gly Leu Val Lys
Thr Tyr Glu Asn Ala Lys Glu Ala Leu Asn Pro Ala385 390 395 400Asn
Ala Met Pro Leu Asp Ala Val Lys Gln Ile Asp Ala Ala Leu Lys 405 410
415Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu
420 425 430Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ser Ala Met
Ala Ala 435 440 445Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys
Glu Met Ser Glu 450 455 460Met Thr Lys Phe Lys Ile Leu Asn Glu Ala
Gly Ile Ser Met Leu Ser465 470 475 480Gln Ala Asn Gln Thr Pro Gln
Met Val Ser Lys Leu Leu Gln 485 49039267PRTBacillus thuringiensis
39Met Arg Ile Gly Thr Asn Phe Leu Ser Met Asn Ala Arg Gln Ser Leu1
5 10 15Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu His Leu Ala
Thr 20 25 30Gly Lys Lys Leu Asn His Ala Ser Asp Asn Pro Ala Asn Ile
Ala Ile 35 40 45Val Thr Arg Met His Ala Arg Ala Asn Gly Met Arg Val
Ala Ile Arg 50 55 60Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala
Glu Ala Ala Leu65 70 75 80Gln Thr Val Met Asn Ile Leu Gln Arg Met
Arg Asp Leu Ala Ile Gln 85 90 95Ser Ala Asn Ser Thr Asn Ser Asn Lys
Asn Arg Asp Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser Leu Thr Glu
Gln Ile Ser Tyr Ile Gly Glu Thr Thr 115 120 125Glu Phe Asn Asp Leu
Ser Val Phe Asp Gly Gln Asn Arg Pro Val Thr 130 135 140Leu Asp Asp
Ile Gly His Thr Val His Ile Ser Lys Ser Ile Pro Pro145 150 155
160Pro Ser Pro Thr Gln His Asp Ile Lys Ile Ser Thr Glu Gln Glu Ala
165 170 175Arg Ala Ala Ile Leu Lys Ile Glu Glu Ala Leu Gln Ser Val
Ser Leu 180 185 190His Arg Ala Asp Leu Gly Ala Met Ile Asn Arg Leu
His Phe Asn Ile 195 200 205Glu Asn Leu Asn Ser Gln Ser Met Ala Leu
Thr Asp Ala Ala Ser Arg 210 215 220Ile Glu Asp Ala Asp Met Ala Gln
Glu Met Ser Asp Phe Leu Lys Phe225 230 235 240Lys Leu Leu Thr Glu
Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln 245 250 255Ile Pro Gln
Met Val Ser Lys Leu Leu Gln Ser 260 26540377PRTBacillus
thuringiensis 40Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Thr Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Asn Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Ser Asp Asn Gln Lys Ala Leu Asp Lys 100 105 110Glu Phe Ser Ala
Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Ser Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Thr Lys Gln Ile Asn Ile
Asn145 150 155 160Leu Ala Asp Ser Ser Thr Ser Ala Leu Asn Ile Asp
Lys Leu Ser Ile 165 170 175Glu Gly Thr Gly Asn Lys Thr Ile Thr Leu
Thr Ala Ala Asp Ile Ala 180 185 190Lys Asp Lys Thr Asn Ile Asp Ala
Val Gly Thr Ala Lys Thr Ala Leu 195 200 205Ala Gly Leu Thr Gly Thr
Pro Ala Ala Ala Ala Ile Asn Ser Ala Val 210 215 220Ala Asp Phe Lys
Thr Ala Phe Ala Lys Ala Asp Lys Asn Leu Met Ser225 230 235 240Asp
Ala Gln Ile Lys Ser Val Thr Asp Ala Ile Thr Ala Phe Glu Ala 245 250
255Asp Ala Thr Pro Asp Leu Thr Lys Ala Lys Ala Ile Gly Thr Ala Tyr
260 265 270Thr Ala Pro Ala Ala Gly Asp Ile Thr Lys Ala Ser Pro Asn
Ala Ser 275 280 285Glu Ala Ile Lys Ser Ile Asp Ala Ala Leu Asp Thr
Ile Ala Ser Asn 290 295 300Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn305 310 315 320Asn Leu Lys Ser Gln Ser Ser
Ser Met Ala Ser Ala Ala Ser Gln Ile 325 330 335Glu Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 340 345 350Ile Leu Asn
Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 355 360 365Pro
Gln Met Val Ser Lys Leu Leu Gln 370 37541406PRTBacillus
thuringiensis 41Met Thr Gly Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe
Ala Tyr Tyr1 5 10 15Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys Lys
Trp Gly Phe Leu 20 25 30Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met
Arg Thr Gln Glu Tyr 35 40 45Met Arg Gln Asn Gln Ala Lys Met Ser Asn
Ala Met Asp Arg Leu Ser 50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser
Asp Asp Ala Ala Gly Leu Ala65 70 75 80Ile Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Gly Val Ala Ala 85 90 95Asn Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala 100 105 110Leu Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn 115 120 125Gln Ser
Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala Leu Asp 130 135
140Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys
Asn145 150 155 160Thr Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp
Asn Lys Ser Ile 165 170 175Ala Ile Gln Thr Leu Asp Asn Ala Asp Thr
Ala Lys Gln Ile Asn Ile 180 185 190Asn Leu Ala Asp Ser Ser Thr Lys
Ala Leu Asn Ile Asp Thr Leu Ser 195 200 205Ile Ala Gly Thr Thr Asp
Lys Thr Ile Thr Ile Thr Ala Lys Asp Leu 210 215 220Thr Asp Asn Lys
Ala Thr Leu Asp Ala Leu Lys Thr Ala Lys Ala Asp225 230 235 240Leu
Ala Lys Leu Asp Asp Lys Ser Asp Gln Ala Thr Ile Asp Lys Ala 245 250
255Val Asp Ala Phe Lys Thr Ala Phe Asn Asn Val Asp Lys Asn Leu Leu
260 265 270Ser Asp Lys Ala Ile Glu Gly Ile Thr Asp Lys Met Thr Ala
Phe Asp 275 280 285Gly Thr His Thr Ala Ala Ala Ala Ile Gly Thr Ala
Tyr Thr Glu Pro 290 295 300Thr Ala Gly Asp Ile Thr Lys Ser Ala Pro
Asn Ala Ser Gly Ala Ile305 310 315 320Lys Ser Ile Asp Ala Ala Leu
Glu Thr Ile Ala Ser Asn Arg Ala Thr 325 330 335Leu Gly Ala Thr Leu
Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys 340 345 350Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 355 360 365Asp
Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 370 375
380Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln
Met385 390 395 400Val Ser Lys Leu Leu Gln 40542373PRTBacillus
thuringiensis 42Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Gly Asp Asn Gln Lys Ala Leu Asp Lys 100 105 110Glu Phe Ser Ala
Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Ser Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Ala Lys Gln Ile Asn Ile
Asn145 150 155 160Leu Ala Asp Ser Ser Thr Lys Ala Leu Asn Ile Asp
Thr Leu Ser Ile 165 170 175Ala Gly Thr Thr Asp Lys Thr Ile Thr Ile
Thr Ala Lys Asp Leu Thr 180 185 190Asp Asn Lys Ala Thr Leu Asp Ala
Leu Lys Thr Ala Lys Ala Asp Leu 195 200 205Ala Lys Leu Asp Asp Lys
Ser Asp Gln Ala Thr Ile Asp Lys Ala Val 210 215 220Asp Ala Phe Lys
Thr Ala Phe Asn Asn Val Asp Lys Asn Leu Leu Ser225 230 235 240Asp
Lys Ala Ile Glu Gly Ile Thr Asp Lys Met Thr Ala Phe Asp Gly 245 250
255Thr His Thr Ala Ala Ala Ala Ile Gly Thr Ala Tyr Thr Glu Pro Thr
260 265 270Ala Gly Asp Ile Thr Lys Ser Ala Pro Asn Ala Ser Gly Ala
Ile Lys 275 280 285Ser Ile Asp Ala Ala Leu Glu Thr Ile Ala Ser Asn
Arg Ala Thr Leu 290 295 300Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser305 310 315
320Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp
325 330 335Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu 340 345 350Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met Val 355 360 365Ser Lys Leu Leu Gln 37043361PRTBacillus
thuringiensis 43Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Asn Gly Asn Gln Ala Ala Leu Asn Lys 100 105 110Glu Phe Asp Ala
Leu Lys Gln Gln Ile Asn Tyr Ile Ser Thr Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Thr Ile Ala 130 135
140Ile Gln Thr Leu Asp Asn Ala Asp Thr Ser Lys Lys Ile Asp Ile
Gln145 150 155 160Leu Ala Asp Val Ser Thr Lys Ser Leu Asn Ile Asp
Lys Leu Lys Ile 165 170 175Gly Gly Val Ser Lys Glu Thr Thr Asp Ala
Val Gly Asp Thr Phe Thr 180 185 190Lys Leu Ser Thr Thr Ala Thr Thr
Asp Met Gly Ala Leu Lys Ile Glu 195 200 205Val Glu Ala Ala Met Lys
Glu Phe Asp Lys Val Lys Gly Ala Met Ser 210 215 220Ala Glu Asp Ala
Lys Ala Val Thr Asp Lys Leu Asp Ala Phe Asn Thr225 230 235 240Ala
Ala Ala Ala Thr Asn Asp Ala Ala Thr Ile Ala Ala Ala Lys Ala 245 250
255Leu Gly Ala Ala Phe Asp Lys Thr Lys Val Glu Met Ala Asp Pro Asn
260 265 270Ala Ser Val Ala Ala Ile Asp Ser Ala Leu Glu Asn Ile Ala
Ser Asn 275 280 285Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn 290 295 300Asn Leu Lys Ser Gln Gln Ser Ser Met Ala
Ser Ala Ala Ser Gln Ile305 310 315 320Glu Asp Ala Asp Met Ala Lys
Glu Met Ser Glu Met Thr Lys Phe Lys 325 330 335Ile Leu Asn Glu Ala
Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 340 345 350Pro Gln Met
Val Ser Lys Leu Leu Gln 355 36044465PRTBacillus cereus 44Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg
Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25
30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile
35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ala
Asn 50 55 60Asn Thr Gln Asp Gly Met Ala Leu Ile Arg Thr Ala Asp Ser
Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
Ile Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln
Ala Ala Leu Gln Lys 100 105 110Glu Phe Gly Glu Leu Gln Lys Gln Ile
Asp Tyr Ile Ala Gly Asn Thr 115 120 125Gln Phe Asn Asp Lys Asn Leu
Leu Asp Gly Ser Asn Pro Ser Ile Ser 130 135 140Ile Gln Thr Leu Asp
Ser Ala Asp Gln Ser Lys Gln Ile Ser Ile Asp145 150 155 160Leu Lys
Ser Ala Thr Leu Glu Ala Leu Gly Ile Lys Asp Leu Thr Val 165 170
175Gly Ala Thr Glu Asn Thr Leu Ala Lys Ala Thr Ile Thr Ala Lys Asp
180 185 190Ala Phe Asp Ala Ala Lys Asp Ala Ser Asp Ala Ala Lys Lys
Glu Ile 195 200 205Asp Ala Ala Ala Lys Asp Thr Pro Ser Lys Asn Asp
Ala Gln Leu Ala 210 215 220Lys Glu Tyr Ile Glu Ala Lys Ala Thr Leu
Ala Thr Leu Lys Pro Thr225 230 235 240Asp Ala Thr Tyr Ala Ala Lys
Ala Ala Glu Leu Asp Ala Ala Thr Thr 245 250 255Ala Leu Asn Asp Asn
Ala Lys Val Leu Val Asp Gly Tyr Glu Lys Lys 260 265 270Leu Thr Thr
Thr Lys Thr Lys Glu Ala Glu Tyr Thr Ala Ala Lys Glu 275 280 285Gln
Ser Thr Lys Ser Thr Ala Ala Ala Asp Leu Val Thr Lys Tyr Glu 290 295
300Thr Ala Lys Ser Asn Ala Leu Gly Asn Asp Ile Ala Lys Glu Tyr
Leu305 310 315 320Glu Ala Lys Thr Ala Tyr Glu Ala Asn Lys Asn Asp
Ile Ser Ser Lys 325 330 335Ser Arg Phe Glu Ala Ala Glu Thr Glu Leu
Asn Lys Asp Ile Thr Ala 340 345 350Asn Lys Ala Ala Lys Val Leu Val
Glu Thr Tyr Glu Lys Ala Lys Thr 355 360 365Ala Gly Thr Thr Glu Lys
Ser Leu Val Ala Val Asp Lys Ile Asp Glu 370 375 380Ala Leu Lys Thr
Ile Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu385 390 395 400Asn
Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ala Ser 405 410
415Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu
420 425 430Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly
Ile Ser 435 440 445Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val
Ser Lys Leu Leu 450 455 460Gln46545394PRTBacillus thuringiensis
45Met Thr Gly Ile Thr Ile Asn Leu Glu Ile Asp Phe Phe Ala Tyr Tyr1
5 10 15Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys Lys Trp Gly Phe
Leu 20 25 30Ile Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr 35 40 45Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser 50 55 60Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala65 70 75 80Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu Gly Val Ala Ala 85 90 95Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala Asp Ser Ala 100 105 110Met Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp Leu Ala Asn 115 120 125Gln Ser Ala Asn Gly
Thr Asn Thr Asn Glu Asn Gln Ala Ala Leu Asn 130 135 140Lys Glu Phe
Asp Ala Leu Lys Glu Gln Ile Asn Tyr Ile Ser Thr Asn145 150 155
160Thr Glu Phe Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Thr Ile
165 170 175Ala Ile Gln Thr Leu Asp Asn Ala Asp Thr Ser Lys Lys Ile
Asp Ile 180 185 190Lys Leu Ala Asp Val Ser Thr Glu Ser Leu Lys Ile
Asp Lys Leu Lys 195 200 205Ile Gly Gly Val Ser Lys Glu Thr Thr Asp
Ala Val Ser Glu Thr Phe 210 215 220Thr Lys Leu Ser Thr Thr Lys Thr
Thr Asp Lys Asp Ala Leu Lys Ala225 230 235 240Glu Val Glu Ala Ala
Met Lys Glu Phe Asp Lys Val Lys Gly Ala Met 245 250 255Ser Thr Glu
Asp Ala Lys Ala Val Thr Asp Lys Leu Gly Leu Phe Asn 260 265 270Thr
Ala Ala Ala Gly Thr Asp Asp Thr Ala Ile Ala Thr Ala Ala Lys 275 280
285Asn Leu Gly Ala Ala Phe Asp Lys Thr Lys Val Asn Met Ala Asp Pro
290 295 300Asn Ala Ser Val Ala Ala Ile Asp Ser Ala Leu Glu Asn Ile
Ala Ser305 310 315 320Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val 325 330 335Asn Asn Leu Lys Ser Gln Gln Ser Ser
Met Ala Ser Ala Ala Ser Gln 340 345 350Ile Glu Asp Ala Asp Met Ala
Lys Glu Met Ser Glu Met Thr Lys Phe 355 360 365Lys Ile Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln 370 375 380Thr Pro Gln
Met Val Ser Lys Leu Leu Gln385 39046266PRTBacillus cereus 46Met Arg
Ile Gly Thr Asn Val Leu Ser Leu Asn Ala Arg Gln Ser Leu1 5 10 15Tyr
Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu His Leu Ala Thr 20 25
30Gly Lys Lys Leu Asn Asn Ala Ser Asp Asn Pro Ala Asn Ile Ala Ile
35 40 45Val Thr Arg Met His Ala Arg Ala Ser Ser Met Arg Val Ala Ile
Arg 50 55 60Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala Glu Ala
Ala Leu65 70 75 80Gln Thr Val Thr Asn Val Leu Gln Arg Met Arg Asp
Leu Ala Val Gln 85 90 95Ser Ala Asn Asp Thr Asn Ser Asn Lys Asn Arg
Asp Ser Leu Asn Lys 100 105 110Glu Phe Gln Ser Leu Thr Glu Gln Ile
Gly Tyr Ile Asp Glu Thr Thr 115 120 125Asp Phe Asn Asp Leu Ser Val
Phe Asp Gly Gln Asn Arg Thr Val Thr 130 135 140Leu Asp Asp Ile Gly
His Thr Val Asn Val Thr Lys His Ile Pro Pro145 150 155 160Ser Pro
Thr Gln His Asp Ile Asn Ile Ser Thr Glu Gln Glu Ala Arg 165 170
175Ala Ala Ile Arg Lys Ile Glu Glu Ala Leu Gln Asn Val Ser Leu His
180 185 190Arg Ala Asp Leu Gly Ala Met Ile Asn Arg Leu Gln Phe Asn
Ile Glu 195 200 205Asn Leu Asn Ser Gln Ser Thr Ala Leu Thr Asp Ala
Ala Ser Arg Ile 210 215 220Glu Asp Ala Asp Met Ala Gln Glu Met Ser
Asp Phe Leu Lys Phe Lys225 230 235 240Leu Leu Thr Glu Val Ala Leu
Ser Met Val Ser Gln Ala Asn Gln Ile 245 250 255Pro Gln Met Val Ser
Lys Leu Leu Gln Ser 260 26547373PRTBacillus cereus 47Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40
45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ser Asn
50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala
Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu
Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn Lys Ala
Ala Met Gln Lys 100 105 110Glu Phe Gly Glu Leu Lys Glu Gln Ile Lys
Tyr Ile Ala Glu Asn Thr 115 120 125Gln Phe Asn Asp Gln His Leu Leu
Asn Ala Asp Lys Gly Ile Thr Lys 130 135 140Glu Ile Ala Ile Gln Thr
Leu Asp Ser Asp Ser Asp Ser Lys Gln Ile145 150 155 160Lys Ile Lys
Leu Gln Gly Ser Ser Leu Glu Ala Leu Asp Ile Lys Asp 165 170 175Leu
Gln Ile Gly Asn Thr Glu Leu Ala Gln Lys Asp Leu Asp Leu Leu 180 185
190Asn Ala Thr Met Asp Arg Leu Asp Ala Thr Val Pro Gly Thr Arg Asp
195 200 205Val Asp Val Gln Ala Ala Lys Asp Ala Phe Asp Lys Val Lys
Gly Phe 210 215 220Tyr Thr Asn Ser Asp Ser Val Lys Ala Ile Glu Arg
Ala Phe Glu Asp225 230 235 240Tyr Ala Thr Ala Ser Thr Ala Gly Thr
Ala Lys Ala Asp Ala Ala Thr 245 250 255Ala Ile Lys Ala Ala Phe Asp
Leu Ala Ala Asn Lys Val Gly Lys Pro 260 265 270Ala Thr Gly Gly Ala
Gln Gly Ser Ala Asn Ser Leu Gly Ala Ile Thr 275 280 285Lys Ile Asp
Ala Ala Leu Lys Thr Val Ala Asp Asn Arg Ala Thr Leu 290 295 300Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser305 310
315 320Gln Ala Ser Ser Met Ala Ala Ala Ala Ser Gln Val Glu Asp Ala
Asp 325 330 335Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile
Leu Asn Glu 340 345 350Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln
Thr Pro Gln Met Val 355 360 365Ser Lys Leu Leu Gln
37048447PRTBacillus cereus 48Met Arg Ile Asn Thr Asn Ile Asn Ser
Leu Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser
Asn Ser Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Asn Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Gly Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala
Asn Gly Thr Asn Thr Ser Asp Asn Gln Ala Ala Met Gln Lys 100 105
110Glu Phe Ala Glu Leu Gln Lys Gln Ile Thr Tyr Ile Ala Asp Asn Thr
115 120 125Gln Phe Asn Asp Lys Asn Leu Leu Gln Ser Asn Ser Ser Ile
Asn Ile 130 135 140Gln Thr Leu Asp Ser Ser Asp Gly Asn Gln Gln Ile
Gly Ile Glu Leu145 150 155 160Lys Ser Ala Ser Leu Lys Ser Leu Gly
Ile Glu Asp Leu Ala Ile Gly 165 170 175Ala Ser Val Asn Pro Leu Ala
Lys Ala Thr Val Glu Ala Ser Glu Ala 180 185 190Tyr Asp Lys Ala Lys
Ala Asp Thr Ala Ala Phe Ala Lys Ser Ile Ala 195 200 205Asp Thr Ala
Ala Thr Gly Thr Gly Ala Ala Lys Ala Asp Ala Ala Ala 210 215 220Val
Asp Ala Tyr Ile Lys Glu Ala Asp Pro Thr Ala Lys Gly Asn Leu225 230
235 240Tyr Thr Gly Leu Thr Ala Asp Gln Lys Lys Leu Ala Asp Glu His
Asn 245 250 255Thr Leu Lys Ala Ala Glu Asp Gly Lys Lys Ala Glu Leu
Thr Met Ala 260 265 270Thr Thr Lys Ser Thr Ala Asp Gly Thr Ala Lys
Gly Leu Val Asp Ala 275 280 285Tyr Asp Asn Ala Lys Ser Asp Ala Met
Asn Asp Pro Lys Ala Lys Ala 290 295 300Tyr Leu Glu Ala Lys Met Ala
Tyr Glu Lys Asp Thr Ser Asn Val Ala305 310 315 320Asn Lys Gln Lys
Leu Asp Ser Thr Lys Glu Ala Met Glu Lys Asp Pro 325 330 335Ala Ser
Lys Asp Leu Val Val Lys Leu Asp Ala Ala Lys Ala Ala Ala 340 345
350Thr Asn Gly Thr Pro Leu Asp Ala Val Ser Lys Ile Asp Ala Ala Leu
355 360 365Lys Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu
Asn Arg 370 375 380Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser
Ser Ser Met Ala385 390 395 400Ser Ala Ala Ser Gln Ile Glu Asp Ala
Asp Met Ala Lys Glu Met Ser 405 410 415Glu Met Thr Lys Phe Lys Ile
Leu Asn Glu Ala Gly Ile Ser Met Leu 420 425 430Ser Gln Ala Asn Gln
Thr Pro Gln Met Val Ser Lys Leu Leu Gln 435 440 44549373PRTBacillus
cereus 49Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu
Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp Arg
Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Gly Val Ala Ser Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala Asp Ser Ala Leu65 70 75
80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln
85 90 95Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn Lys Ala Ala Met Gln
Lys 100 105 110Glu Phe Gly Glu Leu Lys Glu Gln Ile Lys Tyr Ile Ala
Glu Asn Thr 115 120 125Gln Phe Asn Asp Gln His Leu Leu Asn Ala Asp
Lys Gly Ile Thr Lys 130 135 140Glu Ile Ala Ile Gln Thr Leu Asp Ser
Asp Ser Asp Ser Lys Gln Ile145 150 155 160Lys Ile Lys Leu Gln Gly
Ser Ser Leu Glu Ala Leu Asp Ile Lys Asp 165 170 175Leu Gln Ile Gly
Asn Thr Glu Leu Ala Gln Lys Asp Leu Asp Leu Leu 180 185 190Asn Ala
Thr Met Asp Arg Leu Asp Ala Thr Val Pro Gly Thr Arg Asp 195 200
205Val Asp Val Gln Ala Ala Lys Asp Ala Phe Asp Lys Val Lys Gly Phe
210 215 220Tyr Thr Asn Ser Asp Ser Val Lys Ala Ile Glu Arg Ala Phe
Glu Asp225 230 235 240Tyr Ala Thr Ala Ser Thr Ala Gly Thr Ala Lys
Ala Asp Ala Ala Thr 245 250 255Ala Ile Lys Ala Ala Phe Asp Leu Ala
Ala Asn Lys Val Gly Lys Pro 260 265 270Ala Thr Gly Gly Ala Gln Gly
Ser Ala Asn Ser Leu Gly Ala Ile Thr 275 280 285Lys Ile Asp Ala Ala
Leu Lys Thr Val Ala Asp Asn Arg Ala Thr Leu 290 295 300Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser305 310 315
320Gln Ala Ser Ser Met Ala Ala Ala Ala Ser Gln Val Glu Asp Ala Asp
325 330 335Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu 340 345 350Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met Val 355 360 365Ser Lys Leu Leu Gln 37050397PRTBacillus
cereus 50Met Asp Phe Phe Ala Tyr Tyr Arg Phe Ser Ile Cys Arg Lys
Val Asn1 5 10 15Ile Lys Lys Trp Gly Phe Phe Tyr Met Arg Ile Asn Thr
Asn Ile Asn 20 25 30Ser Met Arg Thr Gln Glu Tyr Met Arg Gln Asn Gln
Ala Lys Met Ser 35 40 45Asn Ala Met Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser 50 55 60Asp Asp Ala Ala Gly Leu Ala Ile Ala Thr
Arg Met Arg Ala Arg Glu65 70 75 80Ser Gly Leu Gly Val Ala Ser Asn
Asn Thr Gln Asp Gly Met Ser Leu 85 90 95Ile Arg Thr Ala Asp Ser Ala
Leu Asn Ser Val Ser Asn Ile Leu Leu 100 105 110Arg Met Arg Asp Leu
Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn 115 120 125Glu Asn Lys
Ala Ala Met Gln Lys Glu Phe Gly Glu Leu Lys Glu Gln 130 135 140Ile
Lys Tyr Ile Ala Glu Asn Thr Gln Phe Asn Asp Gln His Leu Leu145 150
155 160Asn Ala Asp Lys Gly Ile Thr Lys Glu Ile Ala Ile Gln Thr Leu
Asp 165 170 175Ser Asp Ser Asp Ser Lys Gln Ile Lys Ile Lys Leu Gln
Gly Ser Ser 180 185 190Leu Glu Ala Leu Asp Ile Lys Asp Leu Gln Ile
Gly Asn Thr Glu Leu 195 200 205Ala Gln Lys Asp Leu Asp Leu Leu Asn
Ala Thr Met Asp Arg Leu Asp 210 215 220Ala Thr Val Pro Gly Thr Arg
Asp Val Asp Val Gln Ala Ala Lys Asp225 230 235 240Ala Phe Asp Lys
Val Lys Gly Phe Tyr Thr Asn Ser Asp Ser Val Lys 245 250 255Ala Ile
Glu Arg Ala Phe Glu Asp Tyr Ala Thr Ala Ser Thr Ala Gly 260 265
270Thr Ala Lys Ala Asp Ala Ala Thr Ala Ile Lys Ala Ala Phe Asp Leu
275 280 285Ala Ala Asn Lys Val Gly Lys Pro Ala Thr Gly Gly Ala Gln
Gly Ser 290 295 300Ala Asn Ser Leu Gly Ala Ile Thr Lys Ile Asp Ala
Ala Leu Lys Thr305 310 315 320Val Ala Asp Asn Arg Ala Thr Leu Gly
Ala Thr Leu Asn Arg Leu Asp 325 330 335Phe Asn Val Asn Asn Leu Lys
Ser Gln Ala Ser Ser Met Ala Ala Ala 340 345 350Ala Ser Gln Val Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 355 360 365Thr Lys Phe
Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 370 375 380Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln385 390
39551455PRTBacillus cereus 51Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser
Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Ser Ala
Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala Leu Asp Lys 100 105
110Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr
115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Glu Asn Thr Ser
Ile Ala 130 135 140Ile Gln Thr Leu Asp Ser Ala Asp Thr Ala Lys Gln
Ile Asn Ile Asn145 150 155 160Leu Ala Asp Ser Ser Thr Ser Ala Leu
Leu Ile Asp Lys Leu Ser Ile 165 170 175Ser Gly Ala Gly Ala Gly Thr
Ala Leu Ala Gly Val Ala Thr Ala Asp 180 185 190Ile Asn Ala Ala Gly
Thr Lys Gln Ala Ala Leu Ser Gly Leu Thr Gly 195 200 205Ser Lys Thr
Thr Asp Glu Leu Asp Asp Ala Val Lys Glu Phe Lys Thr 210 215 220Glu
Phe Asp Lys Val Lys Ser Gly Leu Ser Ala Glu Asn Ala Asp Lys225 230
235 240Ile Thr Ala Ala Met Asp Lys Tyr Thr Asn Asn Lys Thr Leu Asp
Asn 245 250 255Ala Lys Ala Ile Gly Asp Leu Tyr Lys Thr Met Ala Pro
Ala Asp Ser 260 265 270Thr Val Val Gly Thr Ala Gly Thr Lys Gly Gln
Ala Leu Ile Asp Leu 275 280 285Asn Ala Thr Ala Thr Gly Asp Thr Ala
Gln Lys Arg Gln Val Ala Val 290 295 300Asp Ala Phe Lys Asp Asp Phe
Asp Lys Ile Lys Gly Gly Leu Asn Ala305 310 315 320Gln Asp Ala Ala
Lys Val Thr Ala Ala Leu Asp Lys Phe Asn Lys Ala 325 330 335Asp Gly
Ser Gly Asn Thr Leu Glu Asn Ala Gln Glu Ile Gly Lys Val 340 345
350Phe Ala Glu Val Ala Ala Gly Ser Thr Lys Ser Asn Ala Ser Asp Ala
355 360 365Ile Lys Ser Ile Asp Lys Ala Leu Glu Thr Ile Ala Ser Asn
Arg Ala 370 375 380Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn
Val Asn Asn Leu385 390 395 400Lys Ser Gln Ser Ser Ser Met Ala Ser
Ala Ala Ser Gln Ile Glu Asp 405 410 415Ala Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu 420 425 430Asn Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln 435 440 445Met Val Ser
Lys Leu Leu Gln 450 45552367PRTBacillus thuringiensis 52Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln
Asn Gln Thr Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40
45Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn Val Ala Ala Arg
50 55 60Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala
Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu
Ala Asn Gln 85 90 95Ser Ala Ser Glu Thr Asn Thr Ser Lys Asn Gln Ala
Ala Met Gln Lys 100 105 110Glu Phe Asp Gln Leu Lys Glu Gln Ile Gln
Tyr Ile Ala Asp Asn Thr 115 120 125Glu Phe Asn Asp Lys Lys Leu Leu
Asp Gly Ser Asn Ser Thr Ile Asn 130 135 140Ile Gln Thr Leu Asp Ser
His Asp Lys Asn Lys Gln Ile Thr Ile Ser145 150 155 160Leu Asp Ser
Ala Ser Leu Lys Asn Leu Asp Ile Thr Asp Leu Ala Ile 165 170 175Gly
Ser Asn Thr Val Asn Lys Asn Asp Leu Asp Thr Leu Asn Asn Ser 180 185
190Met Lys Arg Leu Glu Thr Ala Ala Ala Asp Ala Ala Val Gln Ala Gln
195 200 205Asp Val Thr Asp Ala Lys Asn Ala Phe Asn Lys Val Lys Ser
Gly Tyr 210 215 220Thr Pro Ala Glu Val Glu Lys Met Glu Asp Ala Phe
Lys Ala Tyr Asp225 230 235 240Lys Val Val Ala Asp Pro Ala Lys Thr
Asp Ala Leu Leu Lys Ala Ala 245 250 255Ala Glu Lys Ile Asn Thr Glu
Phe Lys Thr Leu Thr Ala Pro Thr Ala 260 265 270Thr Ala Phe Asp Pro
Ser Ser Ser Val Glu Lys Ile Asp Lys Ala Ile 275 280 285Glu Thr Ile
Ala Ser Ser Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg 290 295 300Leu
Asp Phe Asn Val Thr Asn Leu Lys Ser Gln Glu Asn Ser Met Ala305 310
315 320Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met
Ser 325 330 335Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile
Ser Met Leu 340 345 350Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser
Lys Leu Leu Gln 355 360 36553367PRTBacillus thuringiensis 53Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg
Gln Asn Gln Thr Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25
30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile
35 40 45Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn Val Ala Ala
Arg 50 55 60Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser
Ala Leu65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
Leu Ala Asn Gln 85 90 95Ser Ala Ser Glu Thr Asn Thr Ser Lys Asn Gln
Ala Ala Met Gln Lys 100 105 110Glu Phe Asp Gln Leu Lys Glu Gln Ile
Gln Tyr Ile Ala Asp Asn Thr 115 120 125Glu Phe Asn Asp Lys Lys Leu
Leu Asp Gly Ser Asn Ser Thr Ile Asn 130 135 140Ile Gln Thr Leu Asp
Ser His Asp Lys Asn Lys Gln Ile Thr Ile Ser145 150 155 160Leu Asp
Ser Ala Ser Leu Lys Asn Leu Asp Ile Thr Asp Leu Ala Ile 165 170
175Gly Ser Asn Thr Val Asn Lys Asn Asp Leu Asp Thr Leu Asn Asn Ser
180 185 190Met Lys Arg Leu Glu Thr Ala Ala Ala Asp Ala Ala Val Gln
Ala Gln 195 200 205Asp Val Thr Asp Ala Lys Asn Ala Phe Asn Lys Val
Lys Ser Gly Tyr 210 215 220Thr Pro Ala Glu Val Glu Lys Met Glu Asp
Ala Phe Lys Ala Tyr Asp225 230 235 240Lys Val Val Ala Asp Pro Ala
Lys Thr Asp Ala Leu Leu Lys Ala Ala 245 250 255Ala Glu Lys Ile Asn
Thr Glu Phe Lys Thr Leu Thr Ala Pro Thr Ala 260 265 270Thr Ala Phe
Asp Pro Ser Ser Ser Val Glu Lys Ile Asp Lys Ala Ile 275 280 285Glu
Thr Ile Ala Ser Ser Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg 290 295
300Leu Asp Phe Asn Val Thr Asn Leu Lys Ser Gln Glu Asn Ser Met
Ala305 310 315 320Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala
Lys Glu Met Ser 325 330 335Glu Met Thr Lys Phe Lys Ile Leu Asn Glu
Ala Gly Ile Ser Met Leu 340 345 350Ser Gln Ala Asn Gln Thr Pro Gln
Met Val Ser Lys Leu Leu Gln 355 360 36554381PRTBacillus
thuringiensis 54Met Gly Val Leu Asn Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met Arg
Ala Arg Glu Asn Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Leu Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Ile Ala Asn Gln Ser
Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln 100 105 110Lys Ala Leu Asp
Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr 115 120 125Ile Ser
Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp 130 135
140Asn Lys Ser Ile Ala Ile Gln Thr Leu Asp Asn Ala Asp Thr Ser
Lys145 150 155 160Gln Ile Asn Ile Asp Leu Ala Asn Thr Ser Thr Ser
Ser Leu Lys Ile 165 170 175Asp Lys Leu Ser Ile Glu Gly Lys Gly Asn
Gln Thr Ile Ala Ile Thr 180 185 190Ala Ala Asp Ile Ala Lys Asp Thr
Asn Ile Ala Ala Leu Thr Ser Ala 195 200 205Gln Gly Lys Leu Ala Ala
Leu Thr Gly Thr Pro Ala Pro Ala Ala Leu 210 215 220Thr Thr Ala Val
Asp Glu Phe Lys Ala Ala Phe Glu Lys Val Asp Lys225 230 235 240Asn
Leu Met Ser Asp Thr Gln Ile Thr Gly Ile Glu Asn Ala Ile Lys 245 250
255Ala Tyr Asp Gly Ala Thr Thr Lys Thr Leu Ala Leu Ala Gln Ala Val
260 265 270Gly Thr Ala Tyr Thr Ala Pro Thr Pro Gly Asp Ile Thr Lys
Glu Leu 275 280 285Pro Asn Ala Ser Ser Ser Ile Lys Ser Ile Asp Ala
Ala Leu Glu Thr 290 295 300Ile Ala Ser Asn Arg Ala Thr Leu Gly Ala
Thr Leu Asn Arg Leu Asp305 310 315 320Phe Asn Val Asn Asn Leu Lys
Ser Gln Ala Ser Ser Met Ala Ser Ala 325 330 335Ala Ser Gln Ile Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 340 345 350Thr Lys Phe
Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 355 360 365Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln 370 375
38055364PRTBacillus thuringiensis 55Met Gly Val Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile
Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala
Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr65 70 75 80Ala Asp
Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp
Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Ser Glu Asn Gln 100 105
110Ala Ala Leu Asp Lys Glu Phe Gly Ala Leu Lys Glu Gln Ile Asn Tyr
115 120 125Ile Ser Thr Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu Asp
Gly Ser 130 135 140Asn Glu Thr Ile Ala Ile Gln Thr Leu Asp Asn Ala
Asp Glu Gly Lys145 150 155 160Lys Ile Asp Ile Lys Leu Ala Asn Val
Ser Thr Asp Ser Leu Lys Ile 165 170 175Asp Lys Leu Thr Ile Gly Gly
Ala Ala Gln Lys Thr Val Asp Ala Val 180 185 190Ala
Asp Lys Phe Asn Ala Leu Lys Thr Thr Thr Thr Thr Asp Lys Ala 195 200
205Ala Ile Gln Thr Glu Val Asp Ala Val Met Lys Glu Phe Asp Lys Val
210 215 220Lys Gly Ser Met Ser Ala Glu Asp Ala Lys Val Ile Thr Asp
Lys Leu225 230 235 240Lys Asp Tyr Asn Asp Ala Ala Asp Thr Asp Thr
Ala Lys Ala Thr Ala 245 250 255Ala Lys Asp Leu Gly Ala Ala Phe Asp
Lys Thr Lys Val Asn Ile Ala 260 265 270Asn Pro Asn Ala Ala Val Ala
Ala Ile Asp Ser Ala Leu Glu Asn Ile 275 280 285Ala Ser Asn Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe 290 295 300Asn Val Asn
Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala305 310 315
320Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr
325 330 335Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser
Gln Ala 340 345 350Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln
355 36056266PRTBacillus cereus 56Met Arg Ile Gly Thr Asn Val Leu
Ser Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met
Asn Val Ala Met Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn His
Ala Ser Asn Asn Pro Ala Asn Ile Ala Ile 35 40 45Val Thr Arg Met His
Ala Arg Ala Ser Gly Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp
Ala Leu Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr
Val Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln 85 90 95Ser
Ala Asn Val Thr Asn Ser Asn Lys Asn Arg Asn Ser Leu Asn Lys 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Ser Tyr Ile Gly Glu Thr Thr
115 120 125Glu Phe Asn Asp Leu Ser Val Phe Asp Gly Gln Asn Arg Pro
Val Thr 130 135 140Leu Asp Asp Ile Gly Tyr Thr Val Asn Val Thr Lys
His Thr Pro Pro145 150 155 160Ser Pro Thr Gln His Asp Ile Lys Ile
Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu
Glu Ala Leu Gln Asn Val Ser Leu His 180 185 190Arg Ala Asp Leu Gly
Ser Met Met Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn
Ser Gln Ser Met Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu
Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230
235 240Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln
Ile 245 250 255Pro Gln Met Val Ser Lys Leu Leu Gln Ser 260
26557460PRTBacillus cereus 57Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser
Thr Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly
Ile Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala
Asn Gly Thr Asn Thr Asp Lys Asn Gln Gly Ala Leu Asp Lys 100 105
110Glu Phe Ala Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr
115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Ala
Ile Ala 130 135 140Ile Gln Thr Leu Asp Ser Asp Asp Lys Gly Lys Gln
Ile Asp Ile Ser145 150 155 160Leu Ser Asp Thr Ser Thr Thr Ala Leu
Lys Ile Asn Asn Leu Ser Ile 165 170 175Ala Ala Asn Gly Leu Gly Ile
Gly Ser Gly Lys Glu Leu Val Gly Val 180 185 190Ala Asp Asn Thr Ile
Ala Asn Ala Ser Ala Glu Ala Leu Lys Lys Leu 195 200 205Asp Gly Thr
Thr Gly Asp Thr Asp Val Lys Arg Ser Asn Ala Val Lys 210 215 220Ala
Phe Thr Asp Gln Tyr Lys Asp Leu Lys Val Ala Met Asn Ala Lys225 230
235 240Asp Val Glu Thr Ile Asp Ala Ala Ile Lys Lys Phe Glu Gly Ala
Asn 245 250 255Thr Leu Glu Asn Ala Gln Ala Ile Gly Ala Ala Phe Glu
Gly Ala Ala 260 265 270Lys Ala Thr Leu Thr Thr Asp Ile Asn Asn Ala
Thr Leu Thr Ser Lys 275 280 285Ala Leu Ser Asp Leu Asp Thr Asp Ser
Thr Thr Glu Thr Arg Lys Ala 290 295 300Ala Met Lys Asp Phe Val Ala
Ala Phe Asp Lys Val Lys Gly Ser Met305 310 315 320Asn Ser Ser Asp
Val Thr Lys Ile Ser Asp Ala Ile Asp Arg Phe Ser 325 330 335Lys Thr
Asp Asp Ser Gly Asn Thr Leu Glu Ala Ala Arg Ala Ile Gly 340 345
350Asp Ala Phe Lys Ala Ala Thr Thr Asn Gly Lys Thr Ser Thr Ala Thr
355 360 365Asp Ala Asn Ser Ala Ile Lys Ala Ile Asp Glu Ala Leu Glu
Thr Ile 370 375 380Ala Ser Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn
Arg Leu Asp Phe385 390 395 400Asn Val Asn Asn Leu Lys Asn Gln Ala
Ser Ser Met Ala Ser Ala Ala 405 410 415Ser Gln Val Glu Asp Ala Asp
Met Ala Lys Glu Met Ser Glu Met Thr 420 425 430Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala 435 440 445Asn Gln Thr
Pro Gln Met Val Ser Lys Leu Leu Gln 450 455 46058399PRTBacillus
cereus 58Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu
Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Thr Ala Met Asp Arg
Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg
Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn Thr
Asp Lys Asn Gln Ala Ala Leu Asp Lys 100 105 110Glu Phe Asn Ala Leu
Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe Asn
Asp Lys Lys Leu Leu Asp Gly Ser Asn Lys Ser Ile Ala 130 135 140Val
Gln Thr Leu Asp Asn Ala Asp Thr Ser Lys Gln Ile Asn Ile Asn145 150
155 160Leu Ser Asn Thr Ser Thr Lys Ala Leu Glu Ile Asn Ser Leu Thr
Ile 165 170 175Ser Gly Thr Thr Pro Ile Ala Gly Lys Asn Glu Thr Ser
Lys Ile Thr 180 185 190Ala Glu Gln Met Thr Ala Ala Ser Asp Ala Leu
Glu Lys Phe Lys Thr 195 200 205Ala Gln Glu Gly Leu Ala Asn Leu Thr
Glu Pro Thr Lys Gly Ser Asp 210 215 220Gly Lys Pro Glu Ala Gly Thr
Gly Ser Ser Asn Glu Asp Ile Val Lys225 230 235 240Ala Val Lys Ala
Phe Lys Glu Ala Phe Lys Asn Ile Gln Pro Leu Met 245 250 255Ser Asp
Thr Asp Ile Thr Thr Val Gln Asn Lys Ile Asp Leu Phe Asp 260 265
270Glu Asp Ala Pro Asp Leu Ser Ala Ala Lys Leu Ile Gly Thr Thr Phe
275 280 285Glu Glu Ser Met Lys Pro Val Ala Asp Lys Glu Ile Thr Lys
Ala Ala 290 295 300Val Lys Pro Asn Ala Ser Asp Ala Ile Ala Ala Ile
Asp Ala Ala Leu305 310 315 320Thr Lys Val Ala Asp Asn Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg 325 330 335Leu Asp Phe Asn Val Asn Asn
Leu Lys Ser Gln Ala Ser Ser Met Ala 340 345 350Ser Ala Ala Ser Gln
Val Glu Asp Ala Asp Met Ala Lys Glu Met Ser 355 360 365Glu Met Thr
Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu 370 375 380Ser
Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln385 390
39559266PRTBacillus cereus 59Met Arg Ile Gly Thr Asn Val Leu Ser
Leu Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg Met Asn
Val Ala Met Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn Asn Ala
Ser Asp Asn Pro Ala Asn Ile Ala Ile 35 40 45Val Thr Arg Met His Ala
Arg Ala Ser Gly Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu Asp Ala
Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln Thr Val
Thr Asn Val Leu Gln Arg Met Arg Asp Leu Ala Val Gln 85 90 95Ser Ala
Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys 100 105
110Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile Asp Glu Thr Thr
115 120 125Glu Phe Asn Asn Leu Ser Val Phe Asp Gly Gln Asn Arg Pro
Val Thr 130 135 140Leu Asp Asp Ile Gly His Thr Val Asn Val Thr Lys
His Ile Pro Pro145 150 155 160Phe Pro Thr Gln His Asp Ile Asn Ile
Ser Thr Glu Gln Glu Ala Arg 165 170 175Ala Ala Ile Arg Lys Ile Glu
Glu Ala Leu Gln Asn Val Ser Leu His 180 185 190Arg Ala Asp Leu Gly
Ala Met Ile Asn Arg Leu Gln Phe Asn Ile Glu 195 200 205Asn Leu Asn
Ser Gln Ser Thr Ala Leu Thr Asp Ala Ala Ser Arg Ile 210 215 220Glu
Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys225 230
235 240Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln
Val 245 250 255Pro Gln Met Val Ser Lys Leu Leu Gln Ser 260
26560269PRTBacillus thuringiensis 60Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg
Ala His Glu Ser Gly Leu Ser Val Ala Ala Arg 50 55 60Asn Thr Ser Asp
Gly Ile Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Gln Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Thr
Ala Asn Gly Thr Asn Lys Asp Thr Asp Ile Glu Ala Leu Gly Lys 100 105
110Glu Phe Ala Ala Leu Lys Glu Gln Ile Thr Tyr Val Ser Asp Asn Thr
115 120 125Lys Phe Asn Gly Arg Glu Leu Leu Lys Gly Gly Asp Asp Ile
Asn Ile 130 135 140Gln Thr Tyr Asp Gly Ser Asp Glu Ser Gln Gln Ile
Lys Ile Lys Ile145 150 155 160Ser Glu Leu Asp Leu Ser Ser Leu Asp
Thr Gly Glu Val Thr Asp Ser 165 170 175Asp Thr Ala Arg Gly Thr Val
Ser Thr Leu Asp Asp Ala Ile Thr Asn 180 185 190Ile Ala Ser Lys Arg
Ala Glu Leu Gly Ala Thr Leu Asn Arg Leu Asp 195 200 205Tyr Asn Thr
Gln Asn Val Asn Ser Glu Ala Ala Ser Met Ala Ala Ser 210 215 220Ala
Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met225 230
235 240Thr Lys Phe Lys Ile Leu Ser Glu Ala Gly Ile Ser Met Leu Ser
Gln 245 250 255Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln
260 26561269PRTBacillus cereus 61Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg
Ala His Glu Ser Gly Leu Ser Val Ala Ala Arg 50 55 60Asn Thr Ser Asp
Gly Ile Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75 80Gln Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln 85 90 95Thr
Ala Asn Gly Thr Asn Lys Asp Thr Asp Ile Glu Ala Leu Gly Lys 100 105
110Glu Phe Ala Ala Leu Lys Glu Gln Ile Thr Tyr Val Ser Asp Asn Thr
115 120 125Lys Phe Asn Gly Arg Glu Leu Leu Lys Gly Gly Asp Asp Ile
Asn Ile 130 135 140Gln Thr Tyr Asp Gly Ser Asp Glu Ser Gln Gln Ile
Lys Ile Lys Ile145 150 155 160Ser Glu Leu Asp Leu Ser Ser Leu Asp
Thr Gly Glu Val Thr Asp Ser 165 170 175Asp Thr Ala Arg Gly Thr Val
Ser Thr Leu Asp Asp Ala Ile Thr Asn 180 185 190Ile Ala Ser Lys Arg
Ala Glu Leu Gly Ala Thr Leu Asn Arg Leu Asp 195 200 205Tyr Asn Thr
Gln Asn Val Asn Ser Glu Ala Ala Ser Met Ala Ala Ser 210 215 220Ala
Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met225 230
235 240Thr Lys Phe Lys Ile Leu Ser Glu Ala Gly Ile Ser Met Leu Ser
Gln 245 250 255Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln
260 26562267PRTBacillus thuringiensis 62Met Arg Ile Gly Thr Asn Phe
Leu Ser Met Asn Ala Arg Gln Ser Leu1 5 10 15Tyr Glu Asn Glu Lys Arg
Met Asn Val Ala Met Glu His Leu Ala Thr 20 25 30Gly Lys Lys Leu Asn
His Ala Ser Asp Asn Pro Ala Asn Ile Ala Ile 35 40 45Val Thr Arg Met
His Ala Arg Ala Asn Gly Met Arg Val Ala Ile Arg 50 55 60Asn Asn Glu
Asp Ala Ile Ser Met Leu Arg Thr Ala Glu Ala Ala Leu65 70 75 80Gln
Thr Val Met Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Ile Gln 85 90
95Ser Ala Asn Ser Thr Asn Ser Asn Lys Asn Arg Asp Ser Leu Asn Lys
100 105 110Glu Phe Gln Ser Leu Thr Glu Gln Ile Ser Tyr Ile Gly Glu
Thr Thr 115 120 125Glu Phe Asn Asp Leu Ser Val Phe Asp Gly Gln Asn
Arg Pro Val Thr 130 135 140Leu Asp Asp Ile Gly His Thr Val His Ile
Ser Lys Ser Ile Pro Pro145 150 155 160Pro Ser Pro Thr Gln His Asp
Ile Lys Ile Ser Thr Glu Gln Glu Ala 165 170 175Arg Ala Ala Ile Leu
Lys Ile Glu Glu Ala Leu Gln Ser Val Ser Leu 180 185 190His Arg Ala
Asp Leu Gly Ala Met Ile Asn Arg Leu His Phe Asn Ile 195 200 205Glu
Asn Leu Asn Ser Gln Ser Met Ala Leu Thr Asp Ala Ala Ser Arg 210 215
220Ile Glu Asp Ala Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys
Phe225 230 235 240Lys Leu Leu Thr Glu Val Ala Leu Ser Met Val Ser
Gln Ala Asn Gln 245 250 255Ile Pro Gln Met Val Ser Lys Leu Leu Gln
Ser 260 26563373PRTBacillus thuringiensis 63Met Arg Ile Asn Thr Asn
Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala
Lys Met Ser Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg
Met Arg Ala Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr
Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Leu65 70 75
80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln
85 90 95Ser Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala Leu Asp
Lys 100 105 110Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser
Lys Asn Thr 115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp
Asn Lys Ser Ile Ala 130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr
Ala Lys Gln Ile Asn Ile Asn145 150 155 160Leu Ala Asp Ser Ser Thr
Lys Ala Leu Asn Ile Asp Thr Leu Ser Ile 165 170 175Ala Gly Thr Thr
Asp Lys Thr Ile Thr Ile Thr Ala Lys Asp Leu Thr 180 185 190Asp Asn
Lys Ala Thr Leu Asp Ala Leu Lys Thr Ala Lys Ala Asp Leu 195 200
205Ala Lys Leu Asp Asp Lys Ser Asp Gln Ala Thr Ile Asp Lys Ala Val
210 215 220Asp Ala Phe Lys Thr Ala Phe Asn Asn Val Asp Lys Asn Leu
Leu Ser225 230 235 240Asp Lys Ala Ile Glu Gly Ile Thr Asp Lys Met
Thr Ala Phe Asp Gly 245 250 255Thr His Thr Ala Ala Ala Ala Ile Gly
Thr Ala Tyr Thr Glu Pro Thr 260 265 270Ala Gly Asp Ile Thr Lys Ser
Ala Pro Asn Ala Ser Gly Ala Ile Lys 275 280 285Ser Ile Asp Ala Ala
Leu Glu Thr Ile Ala Ser Asn Arg Ala Thr Leu 290 295 300Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser305 310 315
320Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp
325 330 335Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu 340 345 350Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met Val 355 360 365Ser Lys Leu Leu Gln 37064257PRTBacillus
aryabhattai 64Met Arg Ile Asn His Asn Ile Thr Ala Leu Asn Thr Tyr
Arg Gln Phe1 5 10 15Asn Asn Ala Asn Asn Ala Gln Ala Lys Ser Met Glu
Lys Leu Ser Ser 20 25 30Gly Gln Arg Ile Asn Ser Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ser Glu Lys Met Arg Gly Gln Ile Arg Gly
Leu Asp Gln Ala Ser Arg 50 55 60Asn Ala Gln Asp Gly Val Ser Leu Ile
Gln Thr Ala Glu Gly Ala Leu65 70 75 80Asn Glu Thr His Asp Ile Leu
Gln Arg Met Arg Glu Leu Val Val Gln 85 90 95Ala Gly Asn Gly Thr Asn
Lys Thr Glu Asp Leu Asp Ala Ile Gln Asp 100 105 110Glu Ile Gly Ser
Leu Ile Glu Glu Ile Gly Gly Glu Thr Asp Ser Lys 115 120 125Gly Ile
Ser Asp Arg Ala Gln Phe Asn Gly Arg Asn Leu Leu Asp Gly 130 135
140Ser Leu Asp Ile Thr Leu Gln Val Gly Ala Asn Ala Gly Gln Gln
Val145 150 155 160Asn Leu Lys Ile Gly Asp Met Ser Ala Gly Ala Leu
Gly Ala Asp Thr 165 170 175Asp Ser Asp Gly Ala Ala Asp Ala Phe Val
Asn Ser Ile Asn Val Lys 180 185 190Asp Phe Ala Thr Thr Ser Phe Asp
Asp Gln Leu Ala Ile Ile Asp Gly 195 200 205Ala Ile Asn Gln Val Ser
Glu Gln Arg Ser Gly Leu Gly Ala Thr Gln 210 215 220Asn Arg Leu Asp
His Thr Ile Asn Asn Leu Ser Thr Ser Ser Glu Asn225 230 235 240Leu
Thr Ala Ser Glu Ser Arg Ile Arg Asp Val Asp Tyr Ala Leu Ala 245 250
255Ala65270PRTBacillus manliponensis 65Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Asp Lys
Met Asn Thr Ser Met Asn Arg Leu Ser Ser 20 25 30Gly Lys Gln Ile Asn
Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met
Arg Ala Lys Glu Gly Gly Leu Asn Val Gly Ala Lys 50 55 60Asn Thr Gln
Asp Gly Met Ser Ala Leu Arg Thr Met Asp Ser Ala Leu65 70 75 80Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Thr Gln 85 90
95Ser Ala Thr Gly Thr Asn Gln Gly Asn Asp Arg Glu Ser Leu Asp Leu
100 105 110Glu Phe Gln Gln Leu Thr Glu Glu Ile Thr His Ile Ala Glu
Lys Thr 115 120 125Asn Phe Asn Gly Asn Ala Leu Leu Ser Gly Ser Gly
Ser Ala Ile Asn 130 135 140Val Gln Leu Ser Asp Ala Ala Glu Asp Lys
Leu Thr Ile Ala Ala Ile145 150 155 160Asp Ala Thr Ala Ser Thr Leu
Leu Lys Gly Ala Val Asp Val Lys Thr 165 170 175Glu Asp Lys Ala Asp
Ala Ala Ile Thr Lys Ile Asp Gln Ala Ile Gln 180 185 190Asp Ile Ala
Asp Asn Arg Ala Thr Tyr Gly Ser Gln Leu Asn Arg Leu 195 200 205Asp
His Asn Leu Asn Asn Val Asn Ser Gln Ala Thr Asn Met Ala Ala 210 215
220Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser
Glu225 230 235 240Met Thr Lys Phe Lys Ile Leu Ser Glu Ala Gly Val
Ser Met Leu Ser 245 250 255Gln Ala Asn Gln Thr Pro Gln Met Val Ser
Lys Leu Leu Gln 260 265 27066273PRTLysinibacillus sp. 66Met Arg Ile
Gly Ser Trp Thr Ala Thr Gly Met Ser Ile Val Asn His1 5 10 15Met Asn
Arg Asn Trp Asn Ala Ala Ser Lys Ser Met Leu Arg Leu Ser 20 25 30Ser
Gly Tyr Arg Ile Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala 35 40
45Ile Ser Glu Lys Met Arg Gly Gln Ile Arg Gly Leu Thr Met Ala Ser
50 55 60Lys Asn Ile Met Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly
Ala65 70 75 80Leu Asn Glu Thr His Ala Ile Val Gln Arg Met Arg Glu
Leu Ala Val 85 90 95Gln Ala Ala Thr Asp Thr Asn Thr Asp Asp Asp Arg
Ala Lys Leu Asp 100 105 110Leu Glu Phe Gln Glu Leu Lys Lys Glu Ile
Asp Arg Ile Ser Thr Asp 115 120 125Thr Glu Phe Asn Thr Arg Thr Leu
Leu Asn Gly Asp Tyr Lys Asp Asn 130 135 140Gly Leu Lys Ile Gln Val
Gly Ala Asn Ser Gly Gln Ala Ile Glu Val145 150 155 160Lys Ile Gly
Asp Ala Gly Leu Ala Gly Ile Gly Leu Ser Thr Glu Ser 165 170 175Ile
Ala Thr Arg Glu Gly Ala Asn Ala Ala Leu Gly Lys Leu Asp Glu 180 185
190Ala Thr Lys Asn Val Ser Met Glu Arg Ser Arg Leu Gly Ala Tyr Gln
195 200 205Asn Arg Leu Glu His Ala Tyr Asn Val Ala Glu Asn Thr Ala
Ile Asn 210 215 220Leu Gln Asp Ala Glu Ser Arg Ile Arg Asp Val Asp
Ile Ala Lys Glu225 230 235 240Met Met Asn Met Val Lys Ser Gln Ile
Leu Ala Gln Val Gly Gln Gln 245 250 255Val Leu Ala Met His Met Gln
Gln Ala Gln Gly Ile Leu Arg Leu Leu 260 265
270Gly67273PRTLysinibacillus sp. 67Met Lys Ile Gly Ser Trp Thr Ala
Thr Gly Met Ser Ile Val Asn His1 5 10 15Met Asn Arg Asn Trp Asn Ala
Ala Ser Lys Ser Met Leu Arg Leu Ser 20 25 30Ser Gly Tyr Arg Ile Asn
Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala 35 40 45Ile Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Thr Met Ala Ser 50 55 60Lys Asn Ile Met
Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala65 70 75 80Leu Asn
Glu Thr His Ala Ile Val Gln Arg Met Arg Glu Leu Ala Val 85 90 95Gln
Ala Ala Thr Asp Thr Asn Thr Asp Asp Asp Arg Ala Lys Leu Asp 100 105
110Leu Glu Phe Gln Glu Leu Lys Lys Glu Ile Asp Arg Ile Ser Thr Asp
115 120 125Thr Ala Phe Asn Thr Arg Thr Leu Leu Asn Gly Asp Tyr Lys
Asp Asn 130 135 140Gly Leu Lys Ile Gln Val Gly Ala Asn Ser Gly Gln
Ala Ile Glu Val145 150 155 160Lys Ile Gly Asp Ala Gly Leu Ala Gly
Ile Gly Leu Ser Thr Glu Ser 165 170 175Ile Ala Thr Arg Glu Gly Ala
Asn Ala Ala Leu Gly Lys Leu Asp Glu 180 185 190Ala Thr Lys Asn Val
Ser Met Glu Arg Ser Arg Leu Gly Ala Tyr Gln 195 200 205Asn Arg Leu
Glu His Ala Tyr Asn Val Ala Glu Asn Thr Ala Ile Asn 210 215 220Leu
Gln Asp Ala Glu Ser Arg Ile Arg Asp Val Asp Ile Ala Lys Glu225 230
235 240Met Met His Met Val Lys Ser Gln Ile Leu Ala Gln Val Gly Gln
Gln 245 250 255Val Leu Ala Met His Ile Gln Gln Ala Gln Gly Ile Leu
Arg Leu Leu 260 265 270Gly68418PRTPaenibacillus sp. 68Met Ile Ile
Ser His Asn Leu Thr Ala Leu Asn Thr Met Asn Lys Leu1 5 10 15Lys Gln
Lys Asp Leu Ala Val Ser Lys Ser Leu Gly Lys Leu Ser Ser 20 25 30Gly
Leu Arg Ile Asn Gly Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40
45Ser Glu Lys Met Arg Gly Gln Ile Arg Gly Leu Asn Gln Ala Ser Arg
50 55 60Asn Ile Gln Asp Gly Ile Ser Leu Ile Gln Val Ala Asp Gly Ala
Met65 70 75 80Gln Glu Ile His Ser Met Leu Gln Arg Met Asn Glu Leu
Ala Val Gln 85 90 95Ala Ser Asn Gly Thr Tyr Ser Gly Ser Asp Arg Leu
Asn Ile Gln Ser 100 105 110Glu Val Glu Gln Leu Ile Glu Glu Ile Asp
Glu Ile Ala Gly Asn Thr 115 120 125Gly Phe Asn Gly Ile Lys Leu Leu
Asn Gly Asn Asn Glu Lys Thr Glu 130 135 140Lys Thr Glu Lys Thr Gly
Ser Val Val Ser Val Asn Asn Pro Pro Asn145 150 155 160Asn Lys Leu
Ile Thr Ile Ser Ser Pro Val Gly Thr Ser Val Ser Glu 165 170 175Ile
Leu Asn Asn Leu Leu Thr Val Phe Asn Glu Ala Lys Asn Gly Gln 180 185
190Val Gly Asp Ser Asp Ser Lys Arg Val Ser Ser Lys Phe Thr Leu Ser
195 200 205Ile Asn Asn Asp Glu Leu Ser Ile Val Cys Asp Thr Gly Asp
Gly Phe 210 215 220Leu Leu Ser Gly Gly Ser Pro Asn Leu Phe Tyr Gln
Gly Tyr Ile Gly225 230 235 240Gly Ser Tyr Lys Tyr Lys Phe Thr Glu
Phe Ile Asn Glu Asn Asp Phe 245 250 255Ile Asn Ile Met Asp Ile Gly
Gly Ala Asn Gly Gly Asp Thr Leu Lys 260 265 270Phe Asn Phe Ser Ser
Ile Ser Lys Glu Pro Glu Glu Gln Lys Glu Gln 275 280 285Lys Gly Leu
Thr Leu Gln Ile Gly Ala Asn Ser Gly Glu Thr Leu Asn 290 295 300Ile
Lys Leu Pro Asn Val Thr Thr Ser Ala Ile Gly Ile Ser Ser Ile305 310
315 320Asp Val Ser Thr Ile Pro Asn Ala Glu Ser Ser Leu Ser Ser Ile
Ser 325 330 335Ala Ala Ile Asp Lys Val Ser Ala Glu Arg Ala Arg Met
Gly Ala Tyr 340 345 350Gln Asn Arg Leu Glu His Ser Arg Asn Asn Val
Val Thr Tyr Ala Glu 355 360 365Asn Leu Thr Ala Ala Glu Ser Arg Ile
Arg Asp Val Asp Met Ala Lys 370 375 380Glu Met Met Glu Leu Met Lys
Asn Gln Ile Phe Thr Gln Ala Gly Gln385 390 395 400Ala Met Leu Leu
Gln Thr Asn Thr Gln Pro Gln Ala Ile Leu Gln Leu 405 410 415Leu
Lys69387PRTBacillus anthracis 69Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser
Asn Ala Met Asp Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala
Asn Gly Thr Asn Thr Lys Glu Asn Gln Asp Ala Leu Asp Lys 100 105
110Glu Phe Gly Ala Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr
115 120 125Glu Phe Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Ser
Ile Ala 130 135 140Ile Gln Thr Leu Asp Asn Ala Asp Thr Ala Lys Gln
Ile Asn Ile Asn145 150 155 160Leu Ala Asp Ser Ser Thr Lys Ala Leu
Asn Ile Asp Ser Leu Thr Ile 165 170 175Ser Gly Ser Lys Asp Ala Thr
Ile Thr Ile Thr Ala Glu Asp Ile Thr 180 185 190Ala Ala Ser Ala Glu
Ile Thr Ala Ala Lys Gly Ala Arg Thr Ala Leu 195 200 205Ala Asn Leu
Lys Asp Thr Pro Ala Asp Pro Thr Lys Asp Pro Ala Ala 210 215 220Ser
Thr Pro Ala Glu Ile Lys Ala Ala Val Asp Asp Phe Lys Gly Lys225 230
235 240Phe Glu Lys Ile Lys Gly Leu Met Asn Asp Thr Asp Val Lys Ala
Val 245 250 255Glu Glu Lys Ile Lys Glu Phe Glu Thr Thr Ser Thr Leu
Ala Lys Ala 260 265 270Gln Ala Ile Gly Thr Ala Phe Thr Thr Gly Met
Glu Pro Lys Ala Gly 275 280 285Asn Ile Thr Lys Asn Val Pro Ala Ala
Ser Ser Ser Ile Lys Ala Ile 290 295 300Asp Ser Ala Leu Glu Thr Ile
Ala Ser Asn Arg Ala Thr Leu Gly Ala305 310 315 320Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser 325 330 335Ser Ala
Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala 340 345
350Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly
355 360 365Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val
Ser Lys 370 375 380Leu Leu Gln38570300PRTBacillus anthracis 70Met
Gln Lys Ser Gln Tyr Lys Lys Met Gly Val Leu Lys Met Arg Ile1 5 10
15Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met Arg Gln Asn
20 25 30Gln Asp Lys Met Asn Val Ser Met Asn Arg Leu Ser Ser Gly Lys
Arg 35 40 45Ile Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile Ala
Thr Arg 50 55 60Met Arg Ala Arg Gln Ser Gly Leu Glu Lys Ala Ser Gln
Asn Thr Gln65 70 75 80Asp Gly Met Ser Leu Ile Arg Thr Ala Glu Ser
Ala Met Asn Ser Val 85 90 95Ser Asn Ile Leu Thr Arg Met Arg Asp Ile
Ala Val Gln Ser Ser Asn 100 105 110Gly Thr Asn Thr Ala Glu Asn Gln
Ser Ala Leu Gln Lys Glu Phe Ala 115 120 125Glu Leu Gln Glu Gln Ile
Asp Tyr Ile Ala Lys Asn Thr Glu Phe Asn 130 135 140Asp Lys Asn Leu
Leu Ala Gly Thr Gly Ala Val Thr Ile Gly Ser Thr145 150 155 160Ser
Ile Ser Gly Ala Glu Ile Ser Ile Glu Thr Leu Asp Ser Ser Ala 165 170
175Thr Asn Gln Gln Ile Thr Ile Lys Leu Ala Asn Thr Thr Ala Glu Lys
180 185 190Leu Gly Ile Asp Ala Thr Thr Ser Asn Ile Ser Ile Ser Gly
Ala Ala 195 200 205Ser Ala Leu Ala Ala Ile Ser Ala Leu Asn Thr Ala
Leu Asn Thr Val 210 215 220Ala Gly Asn Arg Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Arg225 230 235 240Asn Val Glu Asn Leu Asn Asn
Gln Ala Thr Asn Met Ala Ser Ala Ala 245 250 255Ser Gln Ile Glu Asp
Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr 260 265 270Lys Phe Lys
Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala 275 280 285Asn Gln Thr Pro Gln Met Val Ser Lys
Leu Leu Gln 290 295 30071287PRTBacillus anthracis 71Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn
Gln Asp Lys Met Asn Val Ser Met Asn Arg Leu Ser Ser 20 25 30Gly Lys
Arg Ile Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala
Thr Arg Met Arg Ala Arg Gln Ser Gly Leu Glu Lys Ala Ser Gln 50 55
60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Glu Ser Ala Met65
70 75 80Asn Ser Val Ser Asn Ile Leu Thr Arg Met Arg Asp Ile Ala Val
Gln 85 90 95Ser Ser Asn Gly Thr Asn Thr Ala Glu Asn Gln Ser Ala Leu
Gln Lys 100 105 110Glu Phe Ala Glu Leu Gln Glu Gln Ile Asp Tyr Ile
Ala Lys Asn Thr 115 120 125Glu Phe Asn Asp Lys Asn Leu Leu Ala Gly
Thr Gly Ala Val Thr Ile 130 135 140Gly Ser Thr Ser Ile Ser Gly Ala
Glu Ile Ser Ile Glu Thr Leu Asp145 150 155 160Ser Ser Ala Thr Asn
Gln Gln Ile Thr Ile Lys Leu Ala Asn Thr Thr 165 170 175Ala Glu Lys
Leu Gly Ile Asp Ala Thr Thr Ser Asn Ile Ser Ile Ser 180 185 190Gly
Ala Ala Ser Ala Leu Ala Ala Ile Ser Ala Leu Asn Thr Ala Leu 195 200
205Asn Thr Val Ala Gly Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg
210 215 220Leu Asp Arg Asn Val Glu Asn Leu Asn Asn Gln Ala Thr Asn
Met Ala225 230 235 240Ser Ala Ala Ser Gln Ile Lys Asp Ala Asp Lys
Ala Lys Glu Met Ser 245 250 255Glu Met Thr Lys Phe Lys Ile Leu Asn
Glu Ala Gly Ile Ser Met Leu 260 265 270Ser Gln Ala Asn Gln Thr Pro
Gln Met Val Ser Lys Leu Leu Gln 275 280 28572282PRTBacillus
anthracis 72Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln Glu
Tyr Met1 5 10 15Arg Gln Asn Gln Asp Lys Met Asn Val Ser Met Asn Arg
Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Ser Ala Ala Asp Asp Ala Ala
Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Gln Ser Gly Leu
Glu Lys Ala Ser Gln 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala Glu Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu Thr
Arg Met Arg Asp Ile Ala Val Gln 85 90 95Ser Ser Asn Gly Thr Asn Thr
Ala Glu Asn Gln Ser Ala Leu Gln Lys 100 105 110Glu Phe Ala Glu Leu
Gln Glu Gln Ile Asp Tyr Ile Ala Lys Asn Thr 115 120 125Glu Phe Asn
Asp Lys Asn Leu Leu Ala Gly Thr Gly Ala Val Thr Ile 130 135 140Gly
Ser Thr Ser Ile Ser Gly Ala Glu Ile Ser Ile Glu Thr Leu Asp145 150
155 160Ser Ser Ala Thr Asn Gln Gln Ile Thr Ile Lys Leu Ala Asn Thr
Thr 165 170 175Ala Glu Lys Leu Gly Ile Asp Ala Thr Thr Ser Asn Ile
Ser Ile Ser 180 185 190Gly Ala Ala Ser Ala Leu Ala Ala Ile Ser Ala
Leu Asn Thr Ala Leu 195 200 205Asn Thr Val Ala Gly Asn Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg 210 215 220Leu Asp Arg Asn Val Glu Asn
Leu Asn Asn Gln Ala Thr Asn Met Ala225 230 235 240Ser Ala Ala Ser
Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser 245 250 255Glu Met
Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu 260 265
270Ser Gln Ala Asn Gln Thr Pro Gln Met Val 275 28073265PRTBacillus
anthracis 73Met Asn Val Ser Met Asn Arg Leu Ser Ser Gly Lys Arg Ile
Asn Ser1 5 10 15Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile Ala Thr Arg
Met Arg Ala 20 25 30Arg Gln Ser Gly Leu Glu Lys Ala Ser Gln Asn Thr
Gln Asp Gly Met 35 40 45Ser Leu Ile Arg Thr Ala Glu Ser Ala Met Asn
Ser Val Ser Asn Ile 50 55 60Leu Thr Arg Met Arg Asp Ile Ala Val Gln
Ser Ser Asn Gly Thr Asn65 70 75 80Thr Ala Glu Asn Gln Ser Ala Leu
Gln Lys Glu Phe Ala Glu Leu Gln 85 90 95Glu Gln Ile Asp Tyr Ile Ala
Lys Asn Thr Glu Phe Asn Asp Lys Asn 100 105 110Leu Leu Ala Gly Thr
Gly Ala Val Thr Ile Gly Ser Thr Ser Ile Ser 115 120 125Gly Ala Glu
Ile Ser Ile Glu Thr Leu Asp Ser Ser Ala Thr Asn Gln 130 135 140Gln
Ile Thr Ile Lys Leu Ala Asn Thr Thr Ala Glu Lys Leu Gly Ile145 150
155 160Asp Ala Thr Thr Ser Asn Ile Ser Ile Ser Gly Ala Ala Ser Ala
Leu 165 170 175Ala Ala Ile Ser Ala Leu Asn Thr Ala Leu Asn Thr Val
Ala Gly Asn 180 185 190Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu
Asp Arg Asn Val Glu 195 200 205Asn Leu Asn Asn Gln Ala Thr Asn Met
Ala Ser Ala Ala Ser Gln Ile 210 215 220Glu Asp Ala Asp Met Ala Lys
Glu Met Ser Glu Met Thr Lys Phe Lys225 230 235 240Ile Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 245 250 255Pro Gln
Met Val Ser Lys Leu Leu Gln 260 26574257PRTBacillus megaterium
74Met Arg Ile Asn His Asn Ile Thr Ala Leu Asn Thr Tyr Arg Gln Phe1
5 10 15Asn Asn Ala Asn Asn Ala Gln Ala Lys Ser Met Glu Lys Leu Ser
Ser 20 25 30Gly Gln Arg Ile Asn Ser Ala Ser Asp Asp Ala Ala Gly Leu
Ala Ile 35 40 45Ser Glu Lys Met Arg Gly Gln Ile Arg Gly Leu Asp Gln
Ala Ser Arg 50 55 60Asn Ala Gln Asp Gly Val Ser Leu Ile Gln Thr Ala
Glu Gly Ala Leu65 70 75 80Asn Glu Thr His Asp Ile Leu Gln Arg Met
Arg Glu Leu Val Val Gln 85 90 95Ala Gly Asn Gly Thr Asn Lys Thr Glu
Asp Leu Asp Ala Ile Gln Asp 100 105 110Glu Ile Gly Ser Leu Ile Glu
Glu Ile Gly Gly Glu Ala Asp Ser Lys 115 120 125Gly Ile Ser Asp Arg
Ala Gln Phe Asn Gly Arg Asn Leu Leu Asp Gly 130 135 140Ser Leu Asp
Ile Thr Leu Gln Val Gly Ala Asn Ala Gly Gln Gln Val145 150 155
160Asn Leu Lys Ile Gly Asp Met Ser Ala Gly Ala Leu Gly Ala Asp Thr
165 170 175Asn Ser Asp Gly Ala Ala Asp Ala Phe Val Asn Ser Ile Asn
Val Lys 180 185 190Asp Phe Thr Ala Thr Ser Phe Asp Asp Gln Leu Ala
Ile Ile Asp Gly 195 200 205Ala Ile Asn Gln Val Ser Glu Gln Arg Ser
Gly Leu Gly Ala Thr Gln 210 215 220Asn Arg Leu Asp His Thr Ile Asn
Asn Leu Ser Thr Ser Ser Glu Asn225 230 235 240Leu Thr Ala Ser Glu
Ser Arg Ile Arg Asp Val Asp Tyr Ala Leu Ala 245 250
255Ala75286PRTAneurinibacillus sp. 75Met Arg Ile Asn His Asn Leu
Pro Ala Leu Asn Ala Tyr Arg Asn Leu1 5 10 15Ala Gln Asn Gln Ile Gly
Thr Ser Lys Ile Leu Glu Arg Leu Ser Ser 20 25 30Gly Tyr Arg Ile Asn
Arg Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Glu Gln Gly Gln Arg 50 55 60Asn Thr Met
Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu65 70 75 80Gln
Glu Ile His Glu Met Leu Gln Arg Met Arg Glu Leu Ala Val Gln 85 90
95Ala Ala Asn Gly Thr Tyr Ser Asp Lys Asp Lys Lys Ala Ile Glu Asp
100 105 110Glu Ile Asn Gln Leu Thr Ala Gln Ile Asp Gln Ile Ala Lys
Thr Thr 115 120 125Glu Phe Asn Gly Ile Gln Leu Ile Gly Asp Ser Asp
Ser Thr Ser Leu 130 135 140Gln Asp Val Lys Ile Gln Tyr Gly Pro Lys
Lys Glu Asp Ser Leu Thr145 150 155 160Leu Glu Leu Thr Thr Gln Pro
Glu Ala Asp Pro Pro Phe Ala Ala Gly 165 170 175Cys Lys Ala Asp Lys
Ala Ser Leu Lys Ile Asp Asn Val Asp Val Ile 180 185 190Ser Asp Pro
Glu Gly Ala Ile Glu Thr Phe Lys Ala Ala Ile Asp Gln 195 200 205Val
Ser Arg Ile Arg Ser Tyr Phe Gly Ala Ile Gln Asn Arg Leu Glu 210 215
220His Val Val Asn Asn Leu Ser Asn Tyr Thr Glu Asn Leu Thr Gly
Ala225 230 235 240Glu Ser Arg Ile Arg Asp Ala Asp Met Ala Lys Glu
Met Thr Glu Phe 245 250 255Thr Arg Phe Asn Ile Ile Asn Gln Ser Ala
Thr Ala Met Leu Ala Gln 260 265 270Ala Asn Gln Leu Pro Gln Gly Val
Leu Gln Leu Leu Lys Gly 275 280 28576152PRTBacillus thuringiensis
76Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Lys Ala Arg Glu Gly
Gly Leu Asn 50 55 60Val Ala Gly Arg Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Lys Gly Asn Gln Ala 100 105 110Ser Leu Gln Lys Glu Phe Ala
Gln Leu Thr Glu Gln Ile Asp Tyr Ile 115 120 125Ala Lys Asn Thr Gln
Phe Asn Asp Gln Gln Leu Leu Gly Thr Ala Asp 130 135 140Lys Lys Ile
Lys Ile Gln Thr Leu145 1507784PRTBacillus thuringiensis 77Ile Asp
Ala Ala Ile Thr Thr Val Ala Gly Gln Arg Ala Thr Leu Gly1 5 10 15Ala
Thr Leu Asn Arg Phe Glu Phe Asn Ala Asn Asn Leu Lys Ser Gln 20 25
30Glu Thr Ser Met Ala Asp Ala Ala Ser Gln Ile Glu Asp Ala Asp Met
35 40 45Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu
Ala 50 55 60Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met
Val Ser65 70 75 80Lys Leu Leu Gln78151PRTBacillus thuringiensis
78Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Lys Ala Arg Glu Gly
Gly Leu Asn 50 55 60Val Ala Gly Arg Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Lys Gly Asn Gln Ala 100 105 110Ser Leu Gln Lys Glu Phe Ala
Gln Leu Thr Glu Gln Ile Asp Tyr Ile 115 120 125Ala Lys Asn Thr Gln
Phe Asn Asp Gln Gln Leu Leu Gly Thr Ala Asp 130 135 140Lys Lys Ile
Lys Ile Gln Thr145 1507984PRTBacillus thuringiensis 79Ile Asp Ala
Ala Ile Thr Thr Val Ala Gly Gln Arg Ala Thr Leu Gly1 5 10 15Ala Thr
Leu Asn Arg Phe Glu Phe Asn Ala Asn Asn Leu Lys Ser Gln 20 25 30Glu
Thr Ser Met Ala Asp Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40
45Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala
50 55 60Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val
Ser65 70 75 80Lys Leu Leu Gln80152PRTBacillus thuringiensis 80Gly
Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10
15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala
Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Lys Ala Arg Glu Gly Gly
Leu Asn 50 55 60Val Ala Gly Arg Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn
Thr Lys Gly Asn Gln Ala 100 105 110Ser Leu Gln Lys Glu Phe Ala Gln
Leu Thr Glu Gln Ile Asp Tyr Ile 115 120 125Ala Lys Asn Thr Gln Phe
Asn Asp Gln Gln Leu Leu Gly Thr Ala Asp 130 135 140Lys Lys Ile Lys
Ile Gln Thr Leu145 1508184PRTBacillus thuringiensis 81Ile Asp Ala
Ala Ile Thr Thr Val Ala Gly Gln Arg Ala Thr Leu Gly1 5 10 15Ala Thr
Leu Asn Arg Phe Glu Phe Asn Ala Asn Asn Leu Lys Ser Gln 20 25 30Glu
Thr Ser Met Ala Asp Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40
45Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala
50 55 60Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val
Ser65 70 75 80Lys Leu Leu Gln82152PRTBacillus cereus 82Gly Phe Leu
Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu
Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg
Leu Ser Ser Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala Ala 35 40
45Gly Leu Ala Ile Ala Thr Arg Met Lys Ala Arg Glu Gly Gly Leu Asn
50 55 60Val Ala Gly Arg Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr
Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Lys
Gly Asn Gln Ala 100 105 110Ser Leu Gln Lys Glu Phe Ala Gln Leu Thr
Glu Gln Ile Asp Tyr Ile 115 120 125Ala Lys Asn Thr Gln Phe Asn Asp
Gln Gln Leu Leu Gly Thr Ala Asp 130 135 140Lys Lys Ile Lys Ile Gln
Thr Leu145 1508384PRTBacillus cereus 83Ile Asp Ala Ala Ile Thr Thr
Val Ala Gly Gln Arg Ala Thr Leu Gly1 5 10 15Ala Thr Leu Asn Arg Phe
Glu Phe Asn Ala Asn Asn Leu Lys Ser Gln 20 25 30Glu Thr Ser Met Ala
Asp Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40 45Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 50 55 60Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser65 70 75 80Lys
Leu Leu Gln84151PRTBacillus thuringiensis 84Gly Phe Leu Asn Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg
Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp 20 25 30Arg Leu Ser Ser
Gly Lys Arg Ile Asn Ser Ala Ala Asp Asp Ala Ala 35 40 45Gly Leu Ala
Ile Ala Thr Arg Met Lys Ala Arg Glu Gly Gly Leu Asn 50 55 60Val Ala
Ala Arg Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90
95Leu Ala Asn Gln Ser Ala Thr Gly Thr Asn Thr Thr Lys Asn Gln Val
100 105 110Ala Leu Asn Lys Glu Phe Ala Ala Leu Lys Glu Gln Ile Thr
Tyr Ile 115 120 125Ala Asp Asn Thr Gln Phe Asn Asp Lys Asn Leu Leu
Lys Ser Thr Gln 130 135 140Glu Ile Lys Ile Gln Thr Leu145
1508585PRTBacillus thuringiensis 85Gln Leu Asp Ala Ala Leu Thr Lys
Val Ala Asp Asn Arg Ala Thr Leu1 5 10 15Gly Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Asn Asn Leu Lys Ser 20 25 30Gln Glu Asn Ser Met Ala
Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp 35 40 45Met Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu 50 55 60Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val65 70 75 80Ser Lys
Leu Leu Gln 8586154PRTBacillus thuringiensis 86Trp Gly Phe Leu Ile
Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr
Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met 20 25 30Asp Arg Leu
Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly
Val Ala Ala Asp Asn Thr Gln Asn Gly Met Ser Leu Ile Arg Thr65 70 75
80Ala Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
85 90 95Asp Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn
Lys 100 105 110Ser Ala Leu Gln Lys Glu Phe Ala Gln Leu Gln Lys Gln
Ile Thr Tyr 115 120 125Ile Ala Glu Asn Thr Gln Phe Asn Asp Lys Asn
Leu Leu Asn Glu Asp 130 135 140Ser Glu Val Lys Ile Gln Thr Leu Asp
Ser145 1508788PRTBacillus thuringiensis 87Ala Ile Ala Ala Ile Asp
Ala Ala Leu Thr Lys Val Ala Asp Asn Arg1 5 10 15Ala Thr Leu Gly Ala
Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln
Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp
Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn
Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75
80Gln Met Val Ser Lys Leu Leu Gln 8588140PRTBacillus thuringiensis
88Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Asn
Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Asp Asp Asn Gln Lys 100 105 110Ala Leu Asp Lys Glu Phe Ser
Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu
Phe Asn Asp Lys Lys Leu Leu 130 135 1408962PRTBacillus
thuringiensis 89Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe
Asn Val Asn1 5 10 15Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ala Ala
Ala Ser Gln Ile 20 25 30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu
Met Thr Lys Phe Lys 35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu
Ser Gln Ala Asn 50 55 6090142PRTBacillus thuringiensis 90Gly Phe
Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln
Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25
30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Asn Gly Leu
Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala65 70 75 80Asp Ser Ala Leu Gln Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr
Asp Glu Asn Lys Ala 100 105 110Ala Met Glu Lys Glu Phe Gly Gln Leu
Lys Asp Gln Ile Lys Tyr Ile 115 120 125Thr Asp Asn Thr Gln Phe Asn
Asp Lys Asn Leu Leu Asp Ala 130 135 1409184PRTBacillus
thuringiensis 91Ile Asp Ala Ala Leu Lys Thr Val Ala Asp Asn Arg Ala
Thr Leu Gly1 5 10 15Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn
Leu Lys Ser Gln 20 25 30Ser Ala Ser Met Ala Ser Ala Ala Ser Gln Ile
Glu Asp Ala Asp Met 35 40 45Ala Lys Glu Met Ser Glu Met Thr Lys Phe
Lys Ile Leu Asn Glu Ala 50 55 60Gly Ile Ser Met Leu Ser Gln Ala Asn
Gln Thr Pro Gln Met Val Ser65 70 75 80Lys Leu Leu
Gln92137PRTBacillus cereus 92Met Gly Val Leu Asn Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn
Gln Ala Lys Met Ser Asn Ser Met 20 25 30Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala
Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala Asn
Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser
Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Ile
Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln 100 105
110Val Ala Leu Gln Lys Glu Phe Gly Glu Leu Gln Lys Gln Ile Asp Tyr
115 120 125Ile Ala Lys Asn Thr Gln Phe Asn Asp 130
1359373PRTBacillus cereus 93Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn1 5 10 15Asn Leu Lys Ser Gln Gln Ser Ser Met
Ala Ser Ala Ala Ser Gln Val 20 25 30Glu Asp Ala Asp Met Ala Lys Glu
Met Ser Glu Met Thr Lys Phe Lys 35 40 45Ile Leu Asn Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn Gln Thr 50 55 60Pro Gln Met Val Ser Lys
Leu Leu Gln65 7094137PRTBacillus cereus 94Met Gly Val Leu Asn Met
Arg Ile Gly Thr Asn Val Leu Ser Met Asn1 5 10 15Ala Arg Gln Ser Phe
Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Ile 20 25 30Glu His Leu Ala
Thr Gly Lys Lys Leu Asn His Ala Ser Asp Asn Pro 35 40 45Ala Asn Val
Ala Ile Val Thr Arg Met His Ala Arg Thr Ser Gly Ile 50 55 60His Val
Ala Ile Arg Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr65 70 75
80Ala Glu Ala Ala Leu Gln Thr Val Thr Asn Ile Leu Gln Arg Met Arg
85 90 95Asp Val Ala Val Gln Ser Ala Asn Gly Thr Asn Ser Asn Lys Asn
Arg 100 105 110Asp Ser Leu Asn Lys Glu Phe Gln Ser Leu Thr Glu Gln
Ile Gly Tyr 115 120 125Ile Asp Glu Thr Thr Glu Phe Asn Asp 130
1359573PRTBacillus cereus 95Arg Ala Asp Leu Gly Ala Met Ile Asn Gln
Leu Gln Phe Asn Ile Glu1 5 10 15Asn Leu Asn Ser Gln Ser Thr Ala Leu
Thr Asp Ala Ala Ser Arg Ile 20 25 30Glu Asp Ala Asp Met Ala Gln Glu
Met Ser Asp Phe Leu Lys Phe Lys 35 40 45Leu Leu Thr Glu Val Ala Leu
Ser Met Val Ser Gln Ala Asn Gln Ile 50 55 60Pro Gln Met Val Tyr Lys
Leu Leu Gln65 7096140PRTBacillus thuringiensis 96Gly Phe Leu Asn
Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr
Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu
Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55
60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65
70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Ala Asp Asn
Gln Gln 100 105 110Ala Leu Gln Lys Glu Phe Gly Gln Leu Lys Glu Gln
Ile Ser Tyr Ile 115 120 125Ala Asp Asn Thr Glu Phe Asn Asp Lys Thr
Leu Leu 130 135 1409788PRTBacillus thuringiensis 97Ala Val Asp Ser
Ile Asp Ala Ala Leu Lys Thr Val Ala Ser Asn Arg1 5 10 15Ala Thr Leu
Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys
Ser Gln Ser Ala Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp
Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55
60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65
70 75 80Gln Met Val Ser Lys Leu Leu Gln 8598128PRTBacillus
bombysepticus 98Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser
Asn Ser Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ser
Arg Glu Gly Gly Leu Asn 50 55 60Val Ala Ala Arg Asn Thr Glu Asp Gly
Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala
Ser Gly Thr Asn Thr Asp Lys Asn Gln Ala 100 105 110Ala Met Gln Lys
Glu Phe Asp Gln Leu Lys Glu Gln Ile Gln Tyr Ile 115 120
1259970PRTBacillus bombysepticus 99Leu Gly Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Thr Asn Leu Lys1 5 10 15Ser Gln Glu Asn Ser Met Ala
Ala Ser Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser Lys Leu
Leu Gln65 70100128PRTBacillus thuringiensis 100Gly Phe Leu Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp 20 25 30Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu
Ala Ile Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn 50 55 60Val
Ala Ala Arg Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Leu Ala Asn Gln Ser Ala Ser Gly Thr Asn Thr Asp Lys Asn Gln
Ala 100 105 110Ala Met Gln Lys Glu Phe Asp Gln Leu Lys Glu Gln Ile
Gln Tyr Ile 115 120 12510173PRTBacillus thuringiensis 101Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Thr1 5 10 15Asn
Leu Lys Ser Gln Glu Asn Ser Met Ala Ala Ser Ala Ser Gln Ile 20 25
30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys
35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln
Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65
70102128PRTBacillus thuringiensis 102Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ser Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn 50 55 60Val Ala Ala
Arg Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Leu Ala Asn Gln Ser Ala Ser Gly Thr Asn Thr Asp Lys Asn Gln Ala
100 105 110Ala Met Gln Lys Glu Phe Asp Gln Leu Lys Glu Gln Ile Gln
Tyr Ile 115 120 12510373PRTBacillus thuringiensis 103Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Thr1 5 10 15Asn Leu
Lys Ser Gln Glu Asn Ser Met Ala Ala Ser Ala Ser Gln Ile 20 25 30Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 35 40
45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65 70104140PRTBacillus
cereus 104Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met
Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn
Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser
Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg
Glu Asn Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met
Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser
Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn
Gly Thr Asn Thr Gly Asp Asn Gln Lys 100 105 110Ala Leu Asp Lys Glu
Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn
Thr Glu Phe Asn Asp Lys Lys Leu Leu 130 135 14010573PRTBacillus
cereus 105Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn
Val Asn1 5 10 15Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala
Ser Gln Ile 20 25 30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met
Thr Lys Phe Lys 35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser
Gln Ala Asn Gln Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65
70106136PRTBacillus cereus 106Gly Phe Leu Asn Met Arg Ile Gly Thr
Asn Val Leu Ser Met Asn Ala1 5 10 15Arg Gln Ser Leu Tyr Glu Asn Glu
Lys Arg Met Asn Val Ala Met Glu 20 25 30His Leu Ala Thr Gly Lys Lys
Leu Asn Asn Ala Ser Asp Asn Pro Ala 35 40 45Asn Ile Ala Ile Val Thr
Arg Met His Ala Arg Ala Ser Gly Met Arg 50 55 60Leu Ala Ile Arg Asn
Asn Glu Asp Thr Ile Ser Met Leu Arg Thr Ala65 70 75 80Glu Ala Ala
Leu Gln Thr Leu Thr Asn Ile Leu Gln Arg Met Arg Asp 85 90 95Leu Ala
Val Gln Ser Ala Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp 100 105
110Ser Leu Asn Lys Glu Phe Gln Ser Leu Thr Glu Gln Ile Gly Tyr Ile
115 120 125Gly Glu Thr Thr Glu Phe Asn Asp 130 13510773PRTBacillus
cereus 107Arg Ala Asp Leu Gly Ser Met Ile Asn Arg Leu
Gln Phe Asn Ile Glu1 5 10 15Asn Leu Asn Ser Gln Ser Met Ala Leu Thr
Asp Ala Ala Ser Arg Ile 20 25 30Glu Asp Ala Asp Met Ala Gln Glu Met
Ser Asp Phe Leu Lys Phe Lys 35 40 45Leu Leu Thr Glu Val Ala Leu Ser
Met Val Ser Gln Ala Asn Gln Ile 50 55 60Pro Gln Met Val Ser Lys Leu
Leu Gln65 70108153PRTBacillus thuringiensis 108Gly Val Leu Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu
Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Asn 50 55 60Val
Ala Ala Asp Asn Thr Gln Asn Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Ser Asn Lys
Ser 100 105 110Ala Leu Gln Lys Glu Phe Ala Glu Leu Gln Lys Gln Ile
Thr Tyr Ile 115 120 125Ala Asp Asn Thr Gln Phe Asn Asp Lys Asn Leu
Leu Lys Glu Asp Ser 130 135 140Glu Val Lys Ile Gln Thr Leu Asp
Ser145 15010985PRTBacillus thuringiensis 109Ala Ile Asp Ala Ala Leu
Thr Lys Val Ala Asp Asn Arg Ala Thr Leu1 5 10 15Gly Ala Thr Leu Asn
Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser 20 25 30Gln Ser Ser Ser
Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp 35 40 45Met Ala Lys
Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu 50 55 60Ala Gly
Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val65 70 75
80Ser Lys Leu Leu Gln 85110153PRTBacillus thuringiensis 110Gly Val
Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln
Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25
30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Asn 50 55 60Val Ala Ala Asp Asn Thr Gln Asn Gly Met Ser Leu Ile Arg
Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr
Asp Ser Asn Lys Ser 100 105 110Ala Leu Gln Lys Glu Phe Ala Glu Leu
Gln Lys Gln Ile Thr Tyr Ile 115 120 125Ala Asp Asn Thr Gln Phe Asn
Asp Lys Asn Leu Leu Lys Glu Asp Ser 130 135 140Glu Val Lys Ile Gln
Thr Leu Asp Ser145 15011186PRTBacillus thuringiensis 111Ala Ala Ile
Asp Ala Ala Leu Thr Lys Val Ala Asp Asn Arg Ala Thr1 5 10 15Leu Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys 20 25 30Ser
Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 35 40
45Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn
50 55 60Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln
Met65 70 75 80Val Ser Lys Leu Leu Gln 85112140PRTBacillus cereus
112Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Ala Glu Asn Lys Ala 100 105 110Ala Met Gln Lys Glu Phe Gly
Glu Leu Lys Asp Gln Ile Lys Tyr Ile 115 120 125Ser Glu Asn Thr Gln
Phe Asn Asp Gln His Leu Leu 130 135 14011378PRTBacillus cereus
113Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu1
5 10 15Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ala Ser Met Ala
Ser 20 25 30Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met
Ser Glu 35 40 45Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser 50 55 60Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu
Leu Gln65 70 75114140PRTBacillus thuringiensis 114Gly Phe Leu Asn
Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr
Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu
Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55
60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65
70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Ser Asp Asn
Gln Lys 100 105 110Ala Leu Asp Lys Glu Phe Ser Ala Leu Lys Glu Gln
Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys
Leu Leu 130 135 14011588PRTBacillus thuringiensis 115Ala Ile Lys
Ser Ile Asp Ala Ala Leu Asp Thr Ile Ala Ser Asn Arg1 5 10 15Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu
Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40
45Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile
50 55 60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro65 70 75 80Gln Met Val Ser Lys Leu Leu Gln 85116137PRTBacillus
thuringiensis 116Trp Gly Phe Leu Ile Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys
Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn
Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met
Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln
Asp Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Leu Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Ile Ala Asn Gln
Ser Ala Asn Gly Thr Asn Thr Ala Asp Asn Gln 100 105 110Gln Ala Leu
Gln Lys Glu Phe Gly Gln Leu Lys Glu Gln Ile Ser Tyr 115 120 125Ile
Ala Asp Asn Thr Glu Phe Asn Asp 130 13511788PRTBacillus
thuringiensis 117Ala Val Asp Ala Ile Asp Ala Ala Leu Lys Thr Val
Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ser Ala Ser Met Ala Ser
Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu Ala Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln Met Val Ser Lys Leu
Leu Gln 85118141PRTBacillus thuringiensis 118Trp Gly Phe Leu Ile
Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr
Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met 20 25 30Asp Arg Leu
Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly
Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65 70 75
80Ala Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
85 90 95Asp Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Ala Asp Asn
Gln 100 105 110Gln Ala Leu Gln Lys Glu Phe Gly Gln Leu Lys Glu Gln
Ile Ser Tyr 115 120 125Ile Ala Asp Asn Thr Glu Phe Asn Asp Lys Thr
Leu Leu 130 135 14011988PRTBacillus thuringiensis 119Ala Val Asp
Ala Ile Asp Ala Ala Leu Lys Thr Val Ala Ser Asn Arg1 5 10 15Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu
Lys Ser Gln Ser Ala Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40
45Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile
50 55 60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro65 70 75 80Gln Met Val Ser Lys Leu Leu Gln 85120141PRTBacillus
thuringiensis 120Trp Gly Phe Leu Ile Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys
Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn
Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met
Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln
Asp Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Met Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Ile Ser Asn Gln
Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln 100 105 110Ser Ala Leu
Asp Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp Tyr 115 120 125Ile
Ser Lys Asn Thr Glu Phe Asn Asp Gln Lys Leu Leu 130 135
14012188PRTBacillus thuringiensis 121Ala Ile Ala Ser Ile Asp Ala
Ala Leu Glu Ser Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln
Met Val Ser Lys Leu Leu Gln 85122140PRTBacillus thuringiensis
122Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Ile Ser Asn Gln Ser Ala Asn Gly Thr
Asn Thr Asp Lys Asn Gln Ser 100 105 110Ala Leu Asp Lys Glu Phe Ala
Ala Leu Lys Asp Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu
Phe Asn Asp Gln Lys Leu Leu 130 135 14012388PRTBacillus
thuringiensis 123Ala Ile Ala Ser Ile Asp Ala Ala Leu Glu Ser Ile
Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ser Ser Ser Met Ala Ser
Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu Ala Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln Met Val Ser Lys Leu
Leu Gln 85124140PRTBacillus thuringiensis 124Gly Phe Leu Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu
Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val
Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Ile Ser Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln
Ser 100 105 110Ala Leu Asp Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile
Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Gln Lys Leu
Leu 130 135 14012588PRTBacillus thuringiensis 125Ala Ile Ala Ser
Ile Asp Ala Ala Leu Glu Ser Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu
Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys
Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp
Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55
60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65
70 75 80Gln Met Val Ser Lys Leu Leu Gln 85126140PRTBacillus
thuringiensis 126Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Ile Ser Asn Gln Ser
Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser 100 105 110Ala Leu Asp
Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp Tyr Ile 115 120 125Ser
Lys Asn Thr Glu Phe Asn Asp Gln Lys Leu Leu 130 135
14012788PRTBacillus thuringiensis 127Ala Ile Ala Ser Ile Asp Ala
Ala Leu Glu Ser Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln
Met Val Ser Lys Leu Leu Gln 85128140PRTBacillus thuringiensis
128Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35
40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp 85 90 95Ile Ser Asn Gln Ser Ala Asn Gly Thr Asn Thr
Asp Lys Asn Gln Ser 100 105 110Ala Leu Asp Lys Glu Phe Ala Ala Leu
Lys Asp Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn
Asp Gln Lys Leu Leu 130 135 14012988PRTBacillus thuringiensis
129Ala Ile Ala Ser Ile Asp Ala Ala Leu Glu Ser Ile Ala Ser Asn Arg1
5 10 15Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn
Asn 20 25 30Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln
Ile Glu 35 40 45Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys
Phe Lys Ile 50 55 60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala
Asn Gln Thr Pro65 70 75 80Gln Met Val Ser Lys Leu Leu Gln
85130140PRTBacillus thuringiensis 130Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala
Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn Gln Ala
100 105 110Ala Leu Asn Lys Glu Phe Asp Ala Leu Lys Glu Gln Ile Asp
Tyr Ile 115 120 125Ser Thr Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu
130 135 14013173PRTBacillus thuringiensis 131Arg Ala Thr Leu Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn1 5 10 15Asn Leu Lys Ser
Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile 20 25 30Glu Asp Ala
Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 35 40 45Ile Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 50 55 60Pro
Gln Met Val Ser Lys Leu Leu Gln65 70132140PRTBacillus thuringiensis
132Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Ile Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Asn Glu Asn Gln Ala 100 105 110Ala Leu Asn Lys Glu Phe Asp
Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser Thr Asn Thr Glu
Phe Asn Asp Lys Lys Leu Leu 130 135 14013373PRTBacillus
thuringiensis 133Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn1 5 10 15Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser
Ala Ala Ser Gln Ile 20 25 30Glu Asp Ala Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys 35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu
Gln65 70134140PRTBacillus weihenstephanensis 134Gly Val Leu Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu
Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Ser 50 55 60Val
Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Leu Ser Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Glu Asn Gln
Gln 100 105 110Ala Leu Asn Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile
Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu
Leu 130 135 14013573PRTBacillus weihenstephanensis 135Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn1 5 10 15Asn Leu
Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile 20 25 30Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 35 40
45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65 70136140PRTBacillus
thuringiensis 136Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser
Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys 100 105 110Ala Leu Asp
Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser
Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu 130 135
14013785PRTBacillus thuringiensis 137Ile Asp Ala Ala Leu Glu Thr
Ile Ala Ser Asn Arg Ala Thr Leu Gly1 5 10 15Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Asn Asn Leu Lys Ser Gln 20 25 30Ser Ser Ser Met Ala
Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40 45Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 50 55 60Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser65 70 75 80Lys
Leu Leu Gln Ser 85138141PRTBacillus thuringiensis 138Trp Gly Phe
Leu Ile Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln
Glu Tyr Met Arg Gln Asn Gln Thr Lys Met Ser Asn Ala Met 20 25 30Asp
Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40
45Ala Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Asn Gly Leu
50 55 60Gly Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr65 70 75 80Ala Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr
Asp Asp Asn Gln 100 105 110Lys Ala Leu Asp Lys Glu Phe Ser Ala Leu
Lys Glu Gln Ile Asp Tyr 115 120 125Ile Ser Lys Asn Thr Glu Phe Asn
Asp Lys Lys Leu Leu 130 135 14013970PRTBacillus thuringiensis
139Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys1
5 10 15Ser Gln Ser Ser Ser Met Ala Ala Ala Ala Ser Gln Ile Glu Asp
Ala 20 25 30Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile
Leu Asn 35 40 45Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
Pro Gln Met 50 55 60Val Ser Lys Leu Leu Gln65 70140141PRTBacillus
thuringiensis 140Trp Gly Phe Leu Ile Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Thr Lys
Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn
Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met
Arg Ala Arg Glu Asn Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln
Asp Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Met Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Leu Ala Asn Gln
Ser Ala Asn Gly Thr Asn Thr Asp Asp Asn Gln 100 105 110Lys Ala Leu
Asp Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr 115 120 125Ile
Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu 130 135
14014170PRTBacillus thuringiensis 141Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn Asn Leu Lys1 5 10 15Ser Gln Ser Ser Ser Met
Ala Ala Ala Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala Lys Glu
Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser Lys
Leu Leu Gln65 70142141PRTBacillus thuringiensis 142Trp Gly Phe Leu
Ile Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu
Tyr Met Arg Gln Asn Gln Thr Lys Met Ser Asn Ala Met 20 25 30Asp Arg
Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala
Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Asn Gly Leu 50 55
60Gly Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65
70 75 80Ala Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met
Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Asp
Asn Gln 100 105 110Lys Ala Leu Asp Lys Glu Phe Ser Ala Leu Lys Glu
Gln Ile Asp Tyr 115 120 125Ile Ser Lys Asn Thr Glu Phe Asn Asp Lys
Lys Leu Leu 130 135 14014373PRTBacillus thuringiensis 143Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn1 5 10 15Asn
Leu Lys Ser Gln Ser Ser Ser Met Ala Ala Ala Ala Ser Gln Ile 20 25
30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys
35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln
Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65
70144140PRTBacillus thuringiensis 144Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Asp Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala
Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Ile Ser Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser
100 105 110Ala Leu Asp Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp
Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Gln Lys Leu Leu
130 135 14014588PRTBacillus thuringiensis 145Ala Ile Ala Ser Ile
Asp Ala Ala Leu Glu Ser Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser
Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala
Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75
80Gln Met Val Ser Lys Leu Leu Gln 85146173PRTBacillus thuringiensis
146Gly Phe Leu Asn Met Ala Arg Ile Thr Ile Asn Leu Glu Ile Asp Phe1
5 10 15Phe Ala Tyr Tyr Arg Phe Ser Ile Cys Arg Lys Val Asn Ile Lys
Lys 20 25 30Trp Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg 35 40 45Thr Gln Asp Tyr Met Arg Gln Asn Gln Ala Lys Met Ser
Asn Ala Met 50 55 60Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala65 70 75 80Ala Gly Leu Ala Ile Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu 85 90 95Gly Val Ala Ala Asn Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr 100 105 110Ala Asp Ser Ala Met Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg 115 120 125Asp Ile Ser Asn Gln
Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln 130 135 140Ser Ala Leu
Asp Lys Glu Phe Ala Ala Leu Lys Asp Gln Ile Asp Tyr145 150 155
160Ile Ser Lys Asn Thr Glu Phe Asn Asp Gln Lys Leu Leu 165
17014788PRTBacillus thuringiensis 147Ala Ile Ala Ser Ile Asp Ala
Ala Leu Glu Ser Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ser
Ser Ser Met Ala Ser Ala Ala Ser Gln Ile Glu 35 40 45Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln
Met Val Ser Lys Leu Leu Gln 85148157PRTBacillus cereus 148Gly Val
Leu Tyr Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln
Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25
30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu
Ser 50 55 60Val Ala Ala Asp Asn Thr Gln Asn Gly Met Ser Leu Ile Arg
Thr Ala65 70 75 80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr
Asp Lys Asn Gln Val 100 105 110Ala Leu Gln Lys Glu Phe Ala Ala Leu
Lys Glu Gln Ile Thr Tyr Ile 115 120 125Ala Asp Asn Thr Gln Phe Asn
Asp Lys Asn Leu Leu Asn Gly Asn Gln 130 135 140Thr Ile Asn Ile Gln
Thr Leu Asp Ser His Asp Ser Thr145 150 15514978PRTBacillus cereus
149Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu1
5 10 15Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ser Ser Ala Met Ala
Ala 20 25 30Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu Met
Ser Glu 35 40 45Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser 50 55 60Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu
Leu Gln65 70
75150157PRTBacillus cereus 150Gly Val Leu Tyr Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln
Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr
Arg Met Arg Ala Arg Glu Ser Gly Leu Ser 50 55 60Val Ala Ala Asp Asn
Thr Gln Asn Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala
Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Ile Ala
Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Val 100 105
110Ala Leu Gln Lys Glu Phe Ala Ala Leu Lys Glu Gln Ile Thr Tyr Ile
115 120 125Ala Asp Asn Thr Gln Phe Asn Asp Lys Asn Leu Leu Asn Gly
Asn Gln 130 135 140Thr Ile Asn Ile Gln Thr Leu Asp Ser His Asp Ser
Thr145 150 15515178PRTBacillus cereus 151Thr Val Ala Asp Asn Arg
Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu1 5 10 15Asp Phe Asn Val Asn
Asn Leu Lys Ser Gln Ser Ser Ala Met Ala Ala 20 25 30Ser Ala Ser Gln
Ile Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu 35 40 45Met Thr Lys
Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser 50 55 60Gln Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln65 70
75152140PRTBacillus thuringiensis 152Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala
Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys
100 105 110Ala Leu Asp Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp
Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu
130 135 14015370PRTBacillus thuringiensis 153Leu Gly Ala Thr Leu
Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys1 5 10 15Ser Gln Ser Ser
Ser Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala
Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala
Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val
Ser Lys Leu Leu Gln65 70154140PRTBacillus thuringiensis 154Gly Phe
Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln
Glu Tyr Met Arg Gln Asn Gln Thr Lys Met Ser Asn Ala Met Asp 20 25
30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala
35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Asn Gly Leu
Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg
Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr
Ser Asp Asn Gln Lys 100 105 110Ala Leu Asp Lys Glu Phe Ser Ala Leu
Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn
Asp Lys Lys Leu Leu 130 135 14015588PRTBacillus thuringiensis
155Ala Ile Lys Ser Ile Asp Ala Ala Leu Asp Thr Ile Ala Ser Asn Arg1
5 10 15Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn
Asn 20 25 30Leu Lys Ser Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln
Ile Glu 35 40 45Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys
Phe Lys Ile 50 55 60Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala
Asn Gln Thr Pro65 70 75 80Gln Met Val Ser Lys Leu Leu Gln
85156140PRTBacillus thuringiensis 156Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln
Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala
Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys
100 105 110Ala Leu Asp Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp
Tyr Ile 115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu
130 135 14015773PRTBacillus thuringiensis 157Arg Ala Thr Leu Gly
Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn1 5 10 15Asn Leu Lys Ser
Gln Ser Ser Ser Met Ala Ser Ala Ala Ser Gln Ile 20 25 30Glu Asp Ala
Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 35 40 45Ile Leu
Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr 50 55 60Pro
Gln Met Val Ser Lys Leu Leu Gln65 70158140PRTBacillus thuringiensis
158Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1
5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Gly Asp Asn Gln Lys 100 105 110Ala Leu Asp Lys Glu Phe Ser
Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120 125Ser Lys Asn Thr Glu
Phe Asn Asp Lys Lys Leu Leu 130 135 14015973PRTBacillus
thuringiensis 159Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn1 5 10 15Asn Leu Lys Ser Gln Ser Ser Ser Met Ala Ser
Ala Ala Ser Gln Ile 20 25 30Glu Asp Ala Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys 35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu
Gln65 70160153PRTBacillus thuringiensis 160Gly Phe Leu Asn Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg
Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser
Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala
Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala
Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Ile Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Gly Asn Gln
Ala 100 105 110Ala Leu Asn Lys Glu Phe Asp Ala Leu Lys Gln Gln Ile
Asn Tyr Ile 115 120 125Ser Thr Asn Thr Glu Phe Asn Asp Lys Lys Leu
Leu Asp Gly Ser Asn 130 135 140Lys Thr Ile Ala Ile Gln Thr Leu
Asp145 15016170PRTBacillus thuringiensis 161Leu Gly Ala Thr Leu Asn
Arg Leu Asp Phe Asn Val Asn Asn Leu Lys1 5 10 15Ser Gln Gln Ser Ser
Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala Lys
Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly
Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser
Lys Leu Leu Gln65 70162136PRTBacillus cereus 162Gly Val Leu Asn Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu
Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val
Ala Ala Asn Asn Thr Gln Asp Gly Met Ala Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Arg Asp Ile
85 90 95Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ala
Ala 100 105 110Leu Gln Lys Glu Phe Gly Glu Leu Gln Lys Gln Ile Asp
Tyr Ile Ala 115 120 125Gly Asn Thr Gln Phe Asn Asp Lys 130
13516386PRTBacillus cereus 163Asp Lys Ile Asp Glu Ala Leu Lys Thr
Ile Ala Asp Asn Arg Ala Thr1 5 10 15Leu Gly Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Asn Asn Leu Lys 20 25 30Ser Gln Ser Ala Ser Met Ala
Ser Ala Ala Ser Gln Ile Glu Asp Ala 35 40 45Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 50 55 60Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met65 70 75 80Val Ser Lys
Leu Leu Gln 85164141PRTBacillus thuringiensis 164Trp Gly Phe Leu
Ile Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu
Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met 20 25 30Asp Arg
Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala
Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55
60Gly Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65
70 75 80Ala Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met
Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu
Asn Gln 100 105 110Ala Ala Leu Asn Lys Glu Phe Asp Ala Leu Lys Glu
Gln Ile Asn Tyr 115 120 125Ile Ser Thr Asn Thr Glu Phe Asn Asp Lys
Lys Leu Leu 130 135 14016573PRTBacillus thuringiensis 165Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Asn1 5 10 15Asn
Leu Lys Ser Gln Gln Ser Ser Met Ala Ser Ala Ala Ser Gln Ile 20 25
30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys
35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln
Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65
70166141PRTBacillus cereus 166Trp Gly Phe Phe Tyr Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn
Gln Ala Lys Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala
Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ser Asn
Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser
Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Leu
Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn Lys 100 105
110Ala Ala Met Gln Lys Glu Phe Gly Glu Leu Lys Glu Gln Ile Lys Tyr
115 120 125Ile Ala Glu Asn Thr Gln Phe Asn Asp Gln His Leu Leu 130
135 14016778PRTBacillus cereus 167Thr Val Ala Asp Asn Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu1 5 10 15Asp Phe Asn Val Asn Asn Leu
Lys Ser Gln Ala Ser Ser Met Ala Ala 20 25 30Ala Ala Ser Gln Val Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu 35 40 45Met Thr Lys Phe Lys
Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser 50 55 60Gln Ala Asn Gln
Thr Pro Gln Met Val Ser Lys Leu Leu Gln65 70 75168141PRTBacillus
cereus 168Trp Gly Phe Phe Tyr Met Arg Ile Asn Thr Asn Ile Asn Ser
Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser
Asn Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala
Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met Arg Ala
Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ser Asn Asn Thr Gln Asp Gly
Met Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Leu Asn Ser Val
Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala
Asn Gly Thr Asn Thr Asn Glu Asn Lys 100 105 110Ala Ala Met Gln Lys
Glu Phe Gly Glu Leu Lys Glu Gln Ile Lys Tyr 115 120 125Ile Ala Glu
Asn Thr Gln Phe Asn Asp Gln His Leu Leu 130 135 14016978PRTBacillus
cereus 169Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn
Arg Leu1 5 10 15Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ala Ser Ser
Met Ala Ala 20 25 30Ala Ala Ser Gln Val Glu Asp Ala Asp Met Ala Lys
Glu Met Ser Glu 35 40 45Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly
Ile Ser Met Leu Ser 50 55 60Gln Ala Asn Gln Thr Pro Gln Met Val Ser
Lys Leu Leu Gln65 70 75170140PRTBacillus cereus 170Gly Val Leu Asn
Met Arg Ile Asn Thr Asn Ile Asn Ser Leu Arg Thr1 5 10 15Gln Glu Tyr
Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ser Met Asp 20 25 30Arg Leu
Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu Asn 50 55
60Val Ala Ala Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65
70 75 80Asp Ser Ala Leu Gly Ser Val Ser Asn Ile Leu Leu Arg Met Arg
Asp 85 90 95Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Ser Asp Asn
Gln Ala 100 105 110Ala Met Gln Lys Glu Phe Ala Glu Leu Gln Lys Gln
Ile Thr Tyr Ile 115 120 125Ala Asp Asn Thr Gln Phe Asn Asp Lys Asn
Leu Leu 130 135 14017184PRTBacillus cereus 171Ile Asp Ala Ala Leu
Lys Thr Val Ala Asp Asn Arg Ala Thr Leu Gly1 5 10 15Ala Thr Leu Asn
Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln 20 25 30Ser Ser Ser
Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40 45Ala Lys
Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 50 55 60Gly
Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met
Val Ser65 70 75 80Lys Leu Leu Gln172141PRTBacillus cereus 172Trp
Gly Phe Phe Tyr Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10
15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met
20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp
Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser
Gly Leu 50 55 60Gly Val Ala Ser Asn Asn Thr Gln Asp Gly Met Ser Leu
Ile Arg Thr65 70 75 80Ala Asp Ser Ala Leu Asn Ser Val Ser Asn Ile
Leu Leu Arg Met Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr
Asn Thr Asn Glu Asn Lys 100 105 110Ala Ala Met Gln Lys Glu Phe Gly
Glu Leu Lys Glu Gln Ile Lys Tyr 115 120 125Ile Ala Glu Asn Thr Gln
Phe Asn Asp Gln His Leu Leu 130 135 14017378PRTBacillus cereus
173Thr Val Ala Asp Asn Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu1
5 10 15Asp Phe Asn Val Asn Asn Leu Lys Ser Gln Ala Ser Ser Met Ala
Ala 20 25 30Ala Ala Ser Gln Val Glu Asp Ala Asp Met Ala Lys Glu Met
Ser Glu 35 40 45Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser
Met Leu Ser 50 55 60Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu
Leu Gln65 70 75174141PRTBacillus cereus 174Trp Gly Phe Phe Tyr Met
Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met
Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met 20 25 30Asp Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu
Ala Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val
Ala Ser Asn Asn Thr Gln Asp Gly Met Ser Leu Ile Arg Thr65 70 75
80Ala Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg
85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asn Glu Asn
Lys 100 105 110Ala Ala Met Gln Lys Glu Phe Gly Glu Leu Lys Glu Gln
Ile Lys Tyr 115 120 125Ile Ala Glu Asn Thr Gln Phe Asn Asp Gln His
Leu Leu 130 135 14017577PRTBacillus cereus 175Thr Val Ala Asp Asn
Arg Ala Thr Leu Gly Ala Thr Leu Asn Arg Leu1 5 10 15Asp Phe Asn Val
Asn Asn Leu Lys Ser Gln Ala Ser Ser Ala Ala Ala 20 25 30Ala Ser Gln
Val Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met 35 40 45Thr Lys
Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln 50 55 60Ala
Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu Gln65 70
75176140PRTBacillus cereus 176Gly Phe Leu Asn Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln
Ala Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr
Arg Met Arg Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala Asn Asn
Thr Gln Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala
Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Ile Ala
Asn Gln Ser Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys 100 105
110Ala Leu Asp Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile
115 120 125Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu 130 135
14017770PRTBacillus cereus 177Leu Gly Ala Thr Leu Asn Arg Leu Asp
Phe Asn Val Asn Asn Leu Lys1 5 10 15Ser Gln Ser Ser Ser Met Ala Ser
Ala Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser Lys Leu Leu
Gln65 70178128PRTBacillus thuringiensis 178Gly Phe Leu Asn Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg
Gln Asn Gln Thr Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser
Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala
Ile Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn 50 55 60Val Ala
Ala Arg Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75
80Asp Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
85 90 95Leu Ala Asn Gln Ser Ala Ser Glu Thr Asn Thr Ser Lys Asn Gln
Ala 100 105 110Ala Met Gln Lys Glu Phe Asp Gln Leu Lys Glu Gln Ile
Gln Tyr Ile 115 120 12517973PRTBacillus thuringiensis 179Arg Ala
Thr Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Thr1 5 10 15Asn
Leu Lys Ser Gln Glu Asn Ser Met Ala Ala Ser Ala Ser Gln Ile 20 25
30Glu Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys
35 40 45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln
Thr 50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65
70180128PRTBacillus thuringiensis 180Gly Phe Leu Asn Met Arg Ile
Asn Thr Asn Ile Asn Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln
Asn Gln Thr Lys Met Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile
Ala Thr Arg Met Arg Ser Arg Glu Gly Gly Leu Asn 50 55 60Val Ala Ala
Arg Asn Thr Glu Asp Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp
Ser Ala Leu Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90
95Leu Ala Asn Gln Ser Ala Ser Glu Thr Asn Thr Ser Lys Asn Gln Ala
100 105 110Ala Met Gln Lys Glu Phe Asp Gln Leu Lys Glu Gln Ile Gln
Tyr Ile 115 120 12518173PRTBacillus thuringiensis 181Arg Ala Thr
Leu Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val Thr1 5 10 15Asn Leu
Lys Ser Gln Glu Asn Ser Met Ala Ala Ser Ala Ser Gln Ile 20 25 30Glu
Asp Ala Asp Met Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys 35 40
45Ile Leu Asn Glu Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr
50 55 60Pro Gln Met Val Ser Lys Leu Leu Gln65 70182141PRTBacillus
thuringiensis 182Met Gly Val Leu Asn Met Arg Ile Asn Thr Asn Ile
Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys
Met Ser Thr Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn
Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met
Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln
Asp Gly Ile Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Met Asn
Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Leu Ala Asn Gln
Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln 100 105 110Gly Ala Leu
Asp Lys Glu Phe Ala Ala Leu Lys Glu Gln Ile Asp Tyr 115 120 125Ile
Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu 130 135
14018388PRTBacillus thuringiensis 183Ala Ile Lys Ala Ile Asp Glu
Ala Leu Glu Thr Ile Ala Ser Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Asn Gln Ala
Ser Ser Met Ala Ser Ala Ala Ser Gln Val Glu 35 40 45Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln
Met Val Ser Lys Leu Leu Gln 85184141PRTBacillus thuringiensis
184Met Gly Val Leu Asn Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg1
5 10 15Thr Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met Ser Asn Ala
Met 20 25 30Asp Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp
Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala Thr Arg Met Arg Ala Arg Glu
Ser Gly Leu 50 55 60Gly Val Ala Ala Asn Asn Thr Gln Asp Gly Ile Ser
Leu Ile Arg Thr65 70 75 80Ala Asp Ser Ala Met Asn Ser Val Ser Asn
Ile Leu Leu Arg Met Arg 85 90 95Asp Leu Ala Asn Gln Ser Ala Asn Gly
Thr Asn Thr Ser Glu Asn Gln 100 105 110Ala Ala Leu Asp Lys Glu Phe
Gly Ala Leu Lys Glu Gln Ile Asn Tyr 115 120 125Ile Ser Thr Asn Thr
Glu Phe Asn Asp Lys Lys Leu Leu 130 135 14018585PRTBacillus
thuringiensis 185Ala Ile Asp Ser Ala Leu Glu Asn Ile Ala Ser Asn
Arg Ala Thr Leu1 5 10 15Gly Ala Thr Leu Asn Arg Leu Asp Phe Asn Val
Asn Asn Leu Lys Ser 20 25 30Gln Ser Ser Ser Met Ala Ser Ala Ala Ser
Gln Ile Glu Asp Ala Asp 35 40 45Met Ala Lys Glu Met Ser Glu Met Thr
Lys Phe Lys Ile Leu Asn Glu 50 55 60Ala Gly Ile Ser Met Leu Ser Gln
Ala Asn Gln Thr Pro Gln Met Val65 70 75 80Ser Lys Leu Leu Gln
85186141PRTBacillus cereus 186Met Gly Val Leu Asn Met Arg Ile Asn
Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg Gln Asn
Gln Ala Lys Met Ser Thr Ala Met 20 25 30Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala Ile Ala
Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala Ala Asn
Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr65 70 75 80Ala Asp Ser
Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90 95Asp Leu
Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln 100 105
110Gly Ala Leu Asp Lys Glu Phe Ala Ala Leu Lys Glu Gln Ile Asp Tyr
115 120 125Ile Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu Leu 130
135 14018770PRTBacillus cereus 187Leu Gly Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Asn Asn Leu Lys1 5 10 15Asn Gln Ala Ser Ser Met Ala
Ser Ala Ala Ser Gln Val Glu Asp Ala 20 25 30Asp Met Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser Lys Leu
Leu Gln65 70188141PRTBacillus cereus 188Met Gly Val Leu Asn Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg
Gln Asn Gln Ala Lys Met Ser Thr Ala Met 20 25 30Asp Arg Leu Ser Ser
Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala
Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala
Ala Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr65 70 75 80Ala
Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90
95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln
100 105 110Gly Ala Leu Asp Lys Glu Phe Ala Ala Leu Lys Glu Gln Ile
Asp Tyr 115 120 125Ile Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu
Leu 130 135 14018968PRTBacillus cereus 189Ala Thr Leu Asn Arg Leu
Asp Phe Asn Val Asn Asn Leu Lys Asn Gln1 5 10 15Ala Ser Ser Met Ala
Ser Ala Ala Ser Gln Val Glu Asp Ala Asp Met 20 25 30Ala Lys Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 35 40 45Gly Ile Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser 50 55 60Lys Leu
Leu Gln65190140PRTBacillus cereus 190Trp Gly Phe Phe Tyr Met Arg
Ile Asn Thr Asn Ile Asn Ser Met Arg1 5 10 15Thr Gln Glu Tyr Met Arg
Gln Asn Gln Ala Lys Met Ser Thr Ala Met 20 25 30Asp Arg Leu Ser Ser
Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala 35 40 45Ala Gly Leu Ala
Ile Ala Thr Arg Met Arg Ala Arg Glu Ser Gly Leu 50 55 60Gly Val Ala
Ala Asn Asn Thr Gln Asp Gly Ile Ser Leu Ile Arg Thr65 70 75 80Ala
Asp Ser Ala Met Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg 85 90
95Asp Leu Ala Asn Gln Ser Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln
100 105 110Ala Ala Leu Asp Lys Glu Phe Asn Ala Leu Lys Glu Gln Ile
Asp Tyr 115 120 125Ile Ser Lys Asn Thr Glu Phe Asn Asp Lys Lys Leu
130 135 14019188PRTBacillus cereus 191Ala Ile Ala Ala Ile Asp Ala
Ala Leu Thr Lys Val Ala Asp Asn Arg1 5 10 15Ala Thr Leu Gly Ala Thr
Leu Asn Arg Leu Asp Phe Asn Val Asn Asn 20 25 30Leu Lys Ser Gln Ala
Ser Ser Met Ala Ser Ala Ala Ser Gln Val Glu 35 40 45Asp Ala Asp Met
Ala Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile 50 55 60Leu Asn Glu
Ala Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro65 70 75 80Gln
Met Val Ser Lys Leu Leu Gln 85192137PRTBacillus cereus 192Trp Gly
Phe Phe Tyr Met Arg Ile Gly Thr Asn Val Leu Ser Leu Asn1 5 10 15Ala
Arg Gln Ser Leu Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met 20 25
30Glu His Leu Ala Thr Gly Lys Lys Leu Asn Asn Ala Ser Asp Asn Pro
35 40 45Ala Asn Ile Ala Ile Val Thr Arg Met His Ala Arg Ala Ser Gly
Met 50 55 60Arg Val Ala Ile Arg Asn Asn Glu Asp Ala Ile Ser Met Leu
Arg Thr65 70 75 80Ala Glu Ala Ala Leu Gln Thr Val Thr Asn Val Leu
Gln Arg Met Arg 85 90 95Asp Leu Ala Val Gln Ser Ala Asn Gly Thr Asn
Ser Asn Lys Asn Arg 100 105 110Asp Ser Leu Asn Lys Glu Phe Gln Ser
Leu Thr Glu Gln Ile Gly Tyr 115 120 125Ile Asp Glu Thr Thr Glu Phe
Asn Asn 130 13519388PRTBacillus cereus 193Ala Ile Arg Lys Ile Glu
Glu Ala Leu Gln Asn Val Ser Leu His Arg1 5 10 15Ala Asp Leu Gly Ala
Met Ile Asn Arg Leu Gln Phe Asn Ile Glu Asn 20 25 30Leu Asn Ser Gln
Ser Thr Ala Leu Thr Asp Ala Ala Ser Arg Ile Glu 35 40 45Asp Ala Asp
Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys Leu 50 55 60Leu Thr
Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln Val Pro65 70 75
80Gln Met Val Ser Lys Leu Leu Gln 85194106PRTBacillus thuringiensis
194Leu Val Pro Phe Ala Val Trp Leu Ala Met Ser Arg Ile Arg Arg Arg1
5 10 15Ile Leu Asp Thr Asp Cys Lys Ala Glu Ser Ala Val
Arg Ile Lys Glu 20 25 30Ile Pro Ser Asp Val Leu Arg Ala Ala Thr Glu
Arg Pro Leu Ser Cys 35 40 45Ala Arg Ile Arg Val Ala Ile Ala Arg Pro
Ala Ala Ser Ser Glu Ala 50 55 60Leu Leu Ile Arg Leu Pro Leu Asp Lys
Arg Ser Ile Ala Leu Leu Ile65 70 75 80Leu Ala Trp Phe Trp Arg Met
Tyr Ser Cys Val Arg Met Leu Leu Met 85 90 95Phe Val Leu Ile Leu Met
Leu Arg Thr Pro 100 10519549PRTBacillus thuringiensis 195Met Ala
Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys Glu1 5 10 15Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Ser Glu Ala Gly Ile Ser 20 25
30Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu
35 40 45Gln196102PRTBacillus cereus 196Ala Val Trp Leu Ala Met Ser
Arg Ile Arg Arg Arg Ile Leu Asp Thr1 5 10 15Asp Cys Lys Ala Glu Ser
Ala Val Arg Ile Lys Glu Ile Pro Ser Asp 20 25 30Val Leu Arg Ala Ala
Thr Glu Arg Pro Leu Ser Cys Ala Arg Ile Arg 35 40 45Val Ala Ile Ala
Arg Pro Ala Ala Ser Ser Glu Ala Leu Leu Ile Arg 50 55 60Leu Pro Leu
Asp Lys Arg Ser Ile Ala Leu Leu Ile Leu Ala Trp Phe65 70 75 80Trp
Arg Met Tyr Ser Cys Val Arg Met Leu Leu Met Phe Val Leu Ile 85 90
95Leu Met Leu Arg Thr Pro 10019750PRTBacillus cereus 197Ser Met Ala
Ala Ser Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys1 5 10 15Glu Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Ser Glu Ala Gly Ile 20 25 30Ser
Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu 35 40
45Leu Gln 50198128PRTBacillus thuringiensis 198Gly Phe Leu Asn Met
Arg Ile Gly Thr Asn Phe Leu Ser Met Asn Ala1 5 10 15Arg Gln Ser Leu
Tyr Glu Asn Glu Lys Arg Met Asn Val Ala Met Glu 20 25 30His Leu Ala
Thr Gly Lys Lys Leu Asn His Ala Ser Asp Asn Pro Ala 35 40 45Asn Ile
Ala Ile Val Thr Arg Met His Ala Arg Ala Asn Gly Met Arg 50 55 60Val
Ala Ile Arg Asn Asn Glu Asp Ala Ile Ser Met Leu Arg Thr Ala65 70 75
80Glu Ala Ala Leu Gln Thr Val Met Asn Ile Leu Gln Arg Met Arg Asp
85 90 95Leu Ala Ile Gln Ser Ala Asn Ser Thr Asn Ser Asn Lys Asn Arg
Asp 100 105 110Ser Leu Asn Lys Glu Phe Gln Ser Leu Thr Glu Gln Ile
Ser Tyr Ile 115 120 12519970PRTBacillus thuringiensis 199Leu Gly
Ala Met Ile Asn Arg Leu His Phe Asn Ile Glu Asn Leu Asn1 5 10 15Ser
Gln Ser Met Ala Leu Thr Asp Ala Ala Ser Arg Ile Glu Asp Ala 20 25
30Asp Met Ala Gln Glu Met Ser Asp Phe Leu Lys Phe Lys Leu Leu Thr
35 40 45Glu Val Ala Leu Ser Met Val Ser Gln Ala Asn Gln Ile Pro Gln
Met 50 55 60Val Ser Lys Leu Leu Gln65 70200128PRTBacillus
thuringiensis 200Gly Phe Leu Asn Met Arg Ile Asn Thr Asn Ile Asn
Ser Met Arg Thr1 5 10 15Gln Glu Tyr Met Arg Gln Asn Gln Ala Lys Met
Ser Asn Ala Met Asp 20 25 30Arg Leu Ser Ser Gly Lys Arg Ile Asn Asn
Ala Ser Asp Asp Ala Ala 35 40 45Gly Leu Ala Ile Ala Thr Arg Met Arg
Ala Arg Glu Ser Gly Leu Gly 50 55 60Val Ala Ala Asn Asn Thr Gln Asp
Gly Met Ser Leu Ile Arg Thr Ala65 70 75 80Asp Ser Ala Leu Asn Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp 85 90 95Ile Ala Asn Gln Ser
Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys 100 105 110Ala Leu Asp
Lys Glu Phe Ser Ala Leu Lys Glu Gln Ile Asp Tyr Ile 115 120
12520170PRTBacillus thuringiensis 201Leu Gly Ala Thr Leu Asn Arg
Leu Asp Phe Asn Val Asn Asn Leu Lys1 5 10 15Ser Gln Ser Ser Ser Met
Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala 20 25 30Asp Met Ala Lys Glu
Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn 35 40 45Glu Ala Gly Ile
Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met 50 55 60Val Ser Lys
Leu Leu Gln65 70202154PRTBacillus aryabhattai 202Met Arg Ile Asn
His Asn Ile Thr Ala Leu Asn Thr Tyr Arg Gln Phe1 5 10 15Asn Asn Ala
Asn Asn Ala Gln Ala Lys Ser Met Glu Lys Leu Ser Ser 20 25 30Gly Gln
Arg Ile Asn Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ser
Glu Lys Met Arg Gly Gln Ile Arg Gly Leu Asp Gln Ala Ser Arg 50 55
60Asn Ala Gln Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu65
70 75 80Asn Glu Thr His Asp Ile Leu Gln Arg Met Arg Glu Leu Val Val
Gln 85 90 95Ala Gly Asn Gly Thr Asn Lys Thr Glu Asp Leu Asp Ala Ile
Gln Asp 100 105 110Glu Ile Gly Ser Leu Ile Glu Glu Ile Gly Gly Glu
Thr Asp Ser Lys 115 120 125Gly Ile Ser Asp Arg Ala Gln Phe Asn Gly
Arg Asn Leu Leu Asp Gly 130 135 140Ser Leu Asp Ile Thr Leu Gln Val
Gly Ala145 15020352PRTBacillus aryabhattai 203Ile Asp Gly Ala Ile
Asn Gln Val Ser Glu Gln Arg Ser Gly Leu Gly1 5 10 15Ala Thr Gln Asn
Arg Leu Asp His Thr Ile Asn Asn Leu Ser Thr Ser 20 25 30Ser Glu Asn
Leu Thr Ala Ser Glu Ser Arg Ile Arg Asp Val Asp Tyr 35 40 45Ala Leu
Ala Ala 50204148PRTBacillus manliponensis 204Met Arg Ile Asn Thr
Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln
Asp Lys Met Asn Thr Ser Met Asn Arg Leu Ser Ser 20 25 30Gly Lys Gln
Ile Asn Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr
Arg Met Arg Ala Lys Glu Gly Gly Leu Asn Val Gly Ala Lys 50 55 60Asn
Thr Gln Asp Gly Met Ser Ala Leu Arg Thr Met Asp Ser Ala Leu65 70 75
80Asn Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Thr Gln
85 90 95Ser Ala Thr Gly Thr Asn Gln Gly Asn Asp Arg Glu Ser Leu Asp
Leu 100 105 110Glu Phe Gln Gln Leu Thr Glu Glu Ile Thr His Ile Ala
Glu Lys Thr 115 120 125Asn Phe Asn Gly Asn Ala Leu Leu Ser Gly Ser
Gly Ser Ala Ile Asn 130 135 140Val Gln Leu Ser14520584PRTBacillus
manliponensis 205Ile Asp Gln Ala Ile Gln Asp Ile Ala Asp Asn Arg
Ala Thr Tyr Gly1 5 10 15Ser Gln Leu Asn Arg Leu Asp His Asn Leu Asn
Asn Val Asn Ser Gln 20 25 30Ala Thr Asn Met Ala Ala Ala Ala Ser Gln
Ile Glu Asp Ala Asp Met 35 40 45Ala Lys Glu Met Ser Glu Met Thr Lys
Phe Lys Ile Leu Ser Glu Ala 50 55 60Gly Val Ser Met Leu Ser Gln Ala
Asn Gln Thr Pro Gln Met Val Ser65 70 75 80Lys Leu Leu
Gln206151PRTLysinibacillus sp. 206Met Arg Ile Gly Ser Trp Thr Ala
Thr Gly Met Ser Ile Val Asn His1 5 10 15Met Asn Arg Asn Trp Asn Ala
Ala Ser Lys Ser Met Leu Arg Leu Ser 20 25 30Ser Gly Tyr Arg Ile Asn
Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala 35 40 45Ile Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Thr Met Ala Ser 50 55 60Lys Asn Ile Met
Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala65 70 75 80Leu Asn
Glu Thr His Ala Ile Val Gln Arg Met Arg Glu Leu Ala Val 85 90 95Gln
Ala Ala Thr Asp Thr Asn Thr Asp Asp Asp Arg Ala Lys Leu Asp 100 105
110Leu Glu Phe Gln Glu Leu Lys Lys Glu Ile Asp Arg Ile Ser Thr Asp
115 120 125Thr Glu Phe Asn Thr Arg Thr Leu Leu Asn Gly Asp Tyr Lys
Asp Asn 130 135 140Gly Leu Lys Ile Gln Val Gly145
15020784PRTLysinibacillus sp. 207Leu Asp Glu Ala Thr Lys Asn Val
Ser Met Glu Arg Ser Arg Leu Gly1 5 10 15Ala Tyr Gln Asn Arg Leu Glu
His Ala Tyr Asn Val Ala Glu Asn Thr 20 25 30Ala Ile Asn Leu Gln Asp
Ala Glu Ser Arg Ile Arg Asp Val Asp Ile 35 40 45Ala Lys Glu Met Met
Asn Met Val Lys Ser Gln Ile Leu Ala Gln Val 50 55 60Gly Gln Gln Val
Leu Ala Met His Met Gln Gln Ala Gln Gly Ile Leu65 70 75 80Arg Leu
Leu Gly208151PRTLysinibacillus sp. 208Met Lys Ile Gly Ser Trp Thr
Ala Thr Gly Met Ser Ile Val Asn His1 5 10 15Met Asn Arg Asn Trp Asn
Ala Ala Ser Lys Ser Met Leu Arg Leu Ser 20 25 30Ser Gly Tyr Arg Ile
Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala 35 40 45Ile Ser Glu Lys
Met Arg Gly Gln Ile Arg Gly Leu Thr Met Ala Ser 50 55 60Lys Asn Ile
Met Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala65 70 75 80Leu
Asn Glu Thr His Ala Ile Val Gln Arg Met Arg Glu Leu Ala Val 85 90
95Gln Ala Ala Thr Asp Thr Asn Thr Asp Asp Asp Arg Ala Lys Leu Asp
100 105 110Leu Glu Phe Gln Glu Leu Lys Lys Glu Ile Asp Arg Ile Ser
Thr Asp 115 120 125Thr Ala Phe Asn Thr Arg Thr Leu Leu Asn Gly Asp
Tyr Lys Asp Asn 130 135 140Gly Leu Lys Ile Gln Val Gly145
15020984PRTLysinibacillus sp. 209Leu Asp Glu Ala Thr Lys Asn Val
Ser Met Glu Arg Ser Arg Leu Gly1 5 10 15Ala Tyr Gln Asn Arg Leu Glu
His Ala Tyr Asn Val Ala Glu Asn Thr 20 25 30Ala Ile Asn Leu Gln Asp
Ala Glu Ser Arg Ile Arg Asp Val Asp Ile 35 40 45Ala Lys Glu Met Met
His Met Val Lys Ser Gln Ile Leu Ala Gln Val 50 55 60Gly Gln Gln Val
Leu Ala Met His Ile Gln Gln Ala Gln Gly Ile Leu65 70 75 80Arg Leu
Leu Gly210148PRTPaenibacillus sp. 210Met Ile Ile Ser His Asn Leu
Thr Ala Leu Asn Thr Met Asn Lys Leu1 5 10 15Lys Gln Lys Asp Leu Ala
Val Ser Lys Ser Leu Gly Lys Leu Ser Ser 20 25 30Gly Leu Arg Ile Asn
Gly Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Asn Gln Ala Ser Arg 50 55 60Asn Ile Gln
Asp Gly Ile Ser Leu Ile Gln Val Ala Asp Gly Ala Met65 70 75 80Gln
Glu Ile His Ser Met Leu Gln Arg Met Asn Glu Leu Ala Val Gln 85 90
95Ala Ser Asn Gly Thr Tyr Ser Gly Ser Asp Arg Leu Asn Ile Gln Ser
100 105 110Glu Val Glu Gln Leu Ile Glu Glu Ile Asp Glu Ile Ala Gly
Asn Thr 115 120 125Gly Phe Asn Gly Ile Lys Leu Leu Asn Gly Asn Asn
Glu Lys Thr Glu 130 135 140Lys Thr Glu Lys14521184PRTPaenibacillus
sp. 211Ile Ser Ala Ala Ile Asp Lys Val Ser Ala Glu Arg Ala Arg Met
Gly1 5 10 15Ala Tyr Gln Asn Arg Leu Glu His Ser Arg Asn Asn Val Val
Thr Tyr 20 25 30Ala Glu Asn Leu Thr Ala Ala Glu Ser Arg Ile Arg Asp
Val Asp Met 35 40 45Ala Lys Glu Met Met Glu Leu Met Lys Asn Gln Ile
Phe Thr Gln Ala 50 55 60Gly Gln Ala Met Leu Leu Gln Thr Asn Thr Gln
Pro Gln Ala Ile Leu65 70 75 80Gln Leu Leu Lys212148PRTBacillus
anthracis 212Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Ala Lys Met Ser Asn Ala Met Asp
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Glu Ser Gly
Leu Gly Val Ala Ala Asn 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Asp Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn Gln 85 90 95Ser Ala Asn Gly Thr Asn
Thr Lys Glu Asn Gln Asp Ala Leu Asp Lys 100 105 110Glu Phe Gly Ala
Leu Lys Glu Gln Ile Asp Tyr Ile Ser Lys Asn Thr 115 120 125Glu Phe
Asn Asp Lys Lys Leu Leu Asn Gly Asp Asn Lys Ser Ile Ala 130 135
140Ile Gln Thr Leu14521384PRTBacillus anthracis 213Ile Asp Ser Ala
Leu Glu Thr Ile Ala Ser Asn Arg Ala Thr Leu Gly1 5 10 15Ala Thr Leu
Asn Arg Leu Asp Phe Asn Val Asn Asn Leu Lys Ser Gln 20 25 30Ser Ser
Ala Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40 45Ala
Lys Glu Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 50 55
60Gly Ile Ser Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser65
70 75 80Lys Leu Leu Gln214172PRTBacillus anthracis 214Met Gln Lys
Ser Gln Tyr Lys Lys Met Gly Val Leu Lys Met Arg Ile1 5 10 15Asn Thr
Asn Ile Asn Ser Met Arg Thr Gln Glu Tyr Met Arg Gln Asn 20 25 30Gln
Asp Lys Met Asn Val Ser Met Asn Arg Leu Ser Ser Gly Lys Arg 35 40
45Ile Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile Ala Thr Arg
50 55 60Met Arg Ala Arg Gln Ser Gly Leu Glu Lys Ala Ser Gln Asn Thr
Gln65 70 75 80Asp Gly Met Ser Leu Ile Arg Thr Ala Glu Ser Ala Met
Asn Ser Val 85 90 95Ser Asn Ile Leu Thr Arg Met Arg Asp Ile Ala Val
Gln Ser Ser Asn 100 105 110Gly Thr Asn Thr Ala Glu Asn Gln Ser Ala
Leu Gln Lys Glu Phe Ala 115 120 125Glu Leu Gln Glu Gln Ile Asp Tyr
Ile Ala Lys Asn Thr Glu Phe Asn 130 135 140Asp Lys Asn Leu Leu Ala
Gly Thr Gly Ala Val Thr Ile Gly Ser Thr145 150 155 160Ser Ile Ser
Gly Ala Glu Ile Ser Ile Glu Thr Leu 165 17021581PRTBacillus
anthracis 215Ala Leu Asn Thr Val Ala Gly Asn Arg Ala Thr Leu Gly
Ala Thr Leu1 5 10 15Asn Arg Leu Asp Arg Asn Val Glu Asn Leu Asn Asn
Gln Ala Thr Asn 20 25 30Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala
Asp Met Ala Lys Glu 35 40 45Met Ser Glu Met Thr Lys Phe Lys Ile Leu
Asn Glu Ala Gly Ile Ser 50 55 60Met Leu Ser Gln Ala Asn Gln Thr Pro
Gln Met Val Ser Lys Leu Leu65 70 75 80Gln216159PRTBacillus
anthracis 216Met Arg Ile Asn Thr Asn Ile Asn Ser Met Arg Thr Gln
Glu Tyr Met1 5 10 15Arg Gln Asn Gln Asp Lys Met Asn Val Ser Met Asn
Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile Asn Ser Ala Ala Asp Asp Ala
Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg Met Arg Ala Arg Gln Ser Gly
Leu Glu Lys Ala Ser Gln 50 55 60Asn Thr Gln Asp Gly Met Ser Leu Ile
Arg Thr Ala Glu Ser Ala Met65 70 75 80Asn Ser Val Ser Asn Ile Leu
Thr Arg Met Arg Asp Ile Ala Val Gln 85 90 95Ser Ser Asn Gly Thr Asn
Thr Ala Glu Asn Gln Ser Ala Leu Gln Lys 100 105
110Glu Phe Ala Glu Leu Gln Glu Gln Ile Asp Tyr Ile Ala Lys Asn Thr
115 120 125Glu Phe Asn Asp Lys Asn Leu Leu Ala Gly Thr Gly Ala Val
Thr Ile 130 135 140Gly Ser Thr Ser Ile Ser Gly Ala Glu Ile Ser Ile
Glu Thr Leu145 150 15521781PRTBacillus anthracis 217Ala Leu Asn Thr
Val Ala Gly Asn Arg Ala Thr Leu Gly Ala Thr Leu1 5 10 15Asn Arg Leu
Asp Arg Asn Val Glu Asn Leu Asn Asn Gln Ala Thr Asn 20 25 30Met Ala
Ser Ala Ala Ser Gln Ile Lys Asp Ala Asp Lys Ala Lys Glu 35 40 45Met
Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly Ile Ser 50 55
60Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser Lys Leu Leu65
70 75 80Gln218159PRTBacillus anthracis 218Met Arg Ile Asn Thr Asn
Ile Asn Ser Met Arg Thr Gln Glu Tyr Met1 5 10 15Arg Gln Asn Gln Asp
Lys Met Asn Val Ser Met Asn Arg Leu Ser Ser 20 25 30Gly Lys Arg Ile
Asn Ser Ala Ala Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ala Thr Arg
Met Arg Ala Arg Gln Ser Gly Leu Glu Lys Ala Ser Gln 50 55 60Asn Thr
Gln Asp Gly Met Ser Leu Ile Arg Thr Ala Glu Ser Ala Met65 70 75
80Asn Ser Val Ser Asn Ile Leu Thr Arg Met Arg Asp Ile Ala Val Gln
85 90 95Ser Ser Asn Gly Thr Asn Thr Ala Glu Asn Gln Ser Ala Leu Gln
Lys 100 105 110Glu Phe Ala Glu Leu Gln Glu Gln Ile Asp Tyr Ile Ala
Lys Asn Thr 115 120 125Glu Phe Asn Asp Lys Asn Leu Leu Ala Gly Thr
Gly Ala Val Thr Ile 130 135 140Gly Ser Thr Ser Ile Ser Gly Ala Glu
Ile Ser Ile Glu Thr Leu145 150 15521976PRTBacillus anthracis 219Ala
Leu Asn Thr Val Ala Gly Asn Arg Ala Thr Leu Gly Ala Thr Leu1 5 10
15Asn Arg Leu Asp Arg Asn Val Glu Asn Leu Asn Asn Gln Ala Thr Asn
20 25 30Met Ala Ser Ala Ala Ser Gln Ile Glu Asp Ala Asp Met Ala Lys
Glu 35 40 45Met Ser Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala Gly
Ile Ser 50 55 60Met Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val65
70 75220137PRTBacillus anthracis 220Met Asn Val Ser Met Asn Arg Leu
Ser Ser Gly Lys Arg Ile Asn Ser1 5 10 15Ala Ala Asp Asp Ala Ala Gly
Leu Ala Ile Ala Thr Arg Met Arg Ala 20 25 30Arg Gln Ser Gly Leu Glu
Lys Ala Ser Gln Asn Thr Gln Asp Gly Met 35 40 45Ser Leu Ile Arg Thr
Ala Glu Ser Ala Met Asn Ser Val Ser Asn Ile 50 55 60Leu Thr Arg Met
Arg Asp Ile Ala Val Gln Ser Ser Asn Gly Thr Asn65 70 75 80Thr Ala
Glu Asn Gln Ser Ala Leu Gln Lys Glu Phe Ala Glu Leu Gln 85 90 95Glu
Gln Ile Asp Tyr Ile Ala Lys Asn Thr Glu Phe Asn Asp Lys Asn 100 105
110Leu Leu Ala Gly Thr Gly Ala Val Thr Ile Gly Ser Thr Ser Ile Ser
115 120 125Gly Ala Glu Ile Ser Ile Glu Thr Leu 130
13522184PRTBacillus anthracis 221Leu Asn Thr Ala Leu Asn Thr Val
Ala Gly Asn Arg Ala Thr Leu Gly1 5 10 15Ala Thr Leu Asn Arg Leu Asp
Arg Asn Val Glu Asn Leu Asn Asn Gln 20 25 30Ala Thr Asn Met Ala Ser
Ala Ala Ser Gln Ile Glu Asp Ala Asp Met 35 40 45Ala Lys Glu Met Ser
Glu Met Thr Lys Phe Lys Ile Leu Asn Glu Ala 50 55 60Gly Ile Ser Met
Leu Ser Gln Ala Asn Gln Thr Pro Gln Met Val Ser65 70 75 80Lys Leu
Leu Gln222154PRTBacillus megaterium 222Met Arg Ile Asn His Asn Ile
Thr Ala Leu Asn Thr Tyr Arg Gln Phe1 5 10 15Asn Asn Ala Asn Asn Ala
Gln Ala Lys Ser Met Glu Lys Leu Ser Ser 20 25 30Gly Gln Arg Ile Asn
Ser Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Asp Gln Ala Ser Arg 50 55 60Asn Ala Gln
Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu65 70 75 80Asn
Glu Thr His Asp Ile Leu Gln Arg Met Arg Glu Leu Val Val Gln 85 90
95Ala Gly Asn Gly Thr Asn Lys Thr Glu Asp Leu Asp Ala Ile Gln Asp
100 105 110Glu Ile Gly Ser Leu Ile Glu Glu Ile Gly Gly Glu Ala Asp
Ser Lys 115 120 125Gly Ile Ser Asp Arg Ala Gln Phe Asn Gly Arg Asn
Leu Leu Asp Gly 130 135 140Ser Leu Asp Ile Thr Leu Gln Val Gly
Ala145 15022353PRTBacillus megaterium 223Ile Ile Asp Gly Ala Ile
Asn Gln Val Ser Glu Gln Arg Ser Gly Leu1 5 10 15Gly Ala Thr Gln Asn
Arg Leu Asp His Thr Ile Asn Asn Leu Ser Thr 20 25 30Ser Ser Glu Asn
Leu Thr Ala Ser Glu Ser Arg Ile Arg Asp Val Asp 35 40 45Tyr Ala Leu
Ala Ala 50224148PRTAneurinibacillus sp. 224Met Arg Ile Asn His Asn
Leu Pro Ala Leu Asn Ala Tyr Arg Asn Leu1 5 10 15Ala Gln Asn Gln Ile
Gly Thr Ser Lys Ile Leu Glu Arg Leu Ser Ser 20 25 30Gly Tyr Arg Ile
Asn Arg Ala Ser Asp Asp Ala Ala Gly Leu Ala Ile 35 40 45Ser Glu Lys
Met Arg Gly Gln Ile Arg Gly Leu Glu Gln Gly Gln Arg 50 55 60Asn Thr
Met Asp Gly Val Ser Leu Ile Gln Thr Ala Glu Gly Ala Leu65 70 75
80Gln Glu Ile His Glu Met Leu Gln Arg Met Arg Glu Leu Ala Val Gln
85 90 95Ala Ala Asn Gly Thr Tyr Ser Asp Lys Asp Lys Lys Ala Ile Glu
Asp 100 105 110Glu Ile Asn Gln Leu Thr Ala Gln Ile Asp Gln Ile Ala
Lys Thr Thr 115 120 125Glu Phe Asn Gly Ile Gln Leu Ile Gly Asp Ser
Asp Ser Thr Ser Leu 130 135 140Gln Asp Val
Lys14522585PRTAneurinibacillus sp. 225Phe Lys Ala Ala Ile Asp Gln
Val Ser Arg Ile Arg Ser Tyr Phe Gly1 5 10 15Ala Ile Gln Asn Arg Leu
Glu His Val Val Asn Asn Leu Ser Asn Tyr 20 25 30Thr Glu Asn Leu Thr
Gly Ala Glu Ser Arg Ile Arg Asp Ala Asp Met 35 40 45Ala Lys Glu Met
Thr Glu Phe Thr Arg Phe Asn Ile Ile Asn Gln Ser 50 55 60Ala Thr Ala
Met Leu Ala Gln Ala Asn Gln Leu Pro Gln Gly Val Leu65 70 75 80Gln
Leu Leu Lys Gly 8522622PRTBacillus thuringiensis 226Asp Arg Leu Ser
Ser Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu
Ala Ile Ala 2022722PRTBacillus thuringiensis 227Asp Arg Leu Ser Ser
Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala
Ile Ala 2022822PRTBacillus thuringiensis 228Asp Arg Leu Ser Ser Gly
Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile
Ala 2022922PRTBacillus cereus 229Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023022PRTBacillus thuringiensis 230Glu His Leu Ala Thr Gly Lys Lys
Leu Asn Asn Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile Val
2023123PRTBacillus thuringiensis 231Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala Thr
2023222PRTBacillus thuringiensis 232Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023322PRTBacillus thuringiensis 233Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023422PRTBacillus cereus 234Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023522PRTBacillus cereus 235Glu His Leu Ala Thr Gly Lys Lys Leu
Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Val Ala Ile Val
2023622PRTBacillus thuringiensis 236Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023722PRTBacillus bombysepticus 237Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023822PRTBacillus thuringiensis 238Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2023922PRTBacillus thuringiensis 239Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024022PRTBacillus cereus 240Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024122PRTBacillus cereus 241Glu His Leu Ala Thr Gly Lys Lys Leu
Asn Asn Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile Val
2024222PRTBacillus thuringiensis 242Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Val Ala Ile Val
2024322PRTBacillus thuringiensis 243Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024422PRTBacillus cereus 244Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024522PRTBacillus thuringiensis 245Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024622PRTBacillus thuringiensis 246Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024722PRTBacillus thuringiensis 247Glu His Phe Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Val Ala Ile Val
2024822PRTBacillus thuringiensis 248Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2024922PRTBacillus thuringiensis 249Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Val Ile Val
2025022PRTBacillus thuringiensis 250Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025122PRTBacillus thuringiensis 251Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025222PRTBacillus thuringiensis 252Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025322PRTBacillus thuringiensis 253Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025422PRTBacillus thuringiensis 254Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025522PRTBacillus weihenstephanensis 255Asp Arg Leu Ser Ser Gly
Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile
Ala 2025622PRTBacillus thuringiensis 256Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025722PRTBacillus thuringiensis 257Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Val Ala Ile Val
2025822PRTBacillus thuringiensis 258Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2025922PRTBacillus thuringiensis 259Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026022PRTBacillus thuringiensis 260Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Val Ile Val
2026122PRTBacillus thuringiensis 261Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026222PRTBacillus cereus 262Glu His Leu Ala Thr Gly Lys Lys Leu
Asn His Ala Ser Asn Asn Pro1 5 10 15Ala Asn Val Ala Ile Val
2026322PRTBacillus cereus 263Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026422PRTBacillus thuringiensis 264Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile Val
2026522PRTBacillus thuringiensis 265Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026622PRTBacillus thuringiensis 266Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026722PRTBacillus thuringiensis 267Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026822PRTBacillus thuringiensis 268Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2026922PRTBacillus cereus 269Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027022PRTBacillus thuringiensis 270Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027122PRTBacillus cereus 271Glu His Leu Ala Thr Gly Lys Lys Leu
Asn Asn Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile Val
2027222PRTBacillus cereus 272Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027322PRTBacillus cereus 273Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027422PRTBacillus cereus 274Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027522PRTBacillus cereus 275Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027622PRTBacillus cereus 276Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027722PRTBacillus thuringiensis 277Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027822PRTBacillus thuringiensis 278Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2027922PRTBacillus thuringiensis 279Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2028022PRTBacillus thuringiensis 280Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2028122PRTBacillus cereus
281Glu His Leu Ala Thr Gly Lys Lys Leu Asn His Ala Ser Asn Asn Pro1
5 10 15Ala Asn Ile Ala Ile Val 2028222PRTBacillus cereus 282Asp Arg
Leu Ser Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala
Gly Leu Ala Ile Ala 2028322PRTBacillus cereus 283Asp Arg Leu Ser
Ser Gly Lys Arg Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu
Ala Ile Ala 2028422PRTBacillus cereus 284Glu His Leu Ala Thr Gly
Lys Lys Leu Asn Asn Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile
Val 2028522PRTBacillus thuringiensis 285Asp Arg Leu Ser Ser Gly Lys
Arg Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2028622PRTBacillus cereus 286Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2028722PRTBacillus thuringiensis 287Glu His Leu Ala Thr Gly Lys Lys
Leu Asn His Ala Ser Asp Asn Pro1 5 10 15Ala Asn Ile Ala Ile Val
2028822PRTBacillus thuringiensis 288Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2028922PRTBacillus aryabhattai 289Glu Lys Leu Ser Ser Gly Gln Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2029022PRTBacillus manliponensis 290Asn Arg Leu Ser Ser Gly Lys Gln
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029122PRTLysinibacillus sp. 291Leu Arg Leu Ser Ser Gly Tyr Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2029222PRTLysinibacillus sp. 292Leu Arg Leu Ser Ser Gly Tyr Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2029322PRTPaenibacillus sp. 293Gly Lys Leu Ser Ser Gly Leu Arg Ile
Asn Gly Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2029422PRTBacillus anthracis 294Asp Arg Leu Ser Ser Gly Lys Arg Ile
Asn Asn Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029522PRTBacillus anthracis 295Asn Arg Leu Ser Ser Gly Lys Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029622PRTBacillus anthracis 296Asn Arg Leu Ser Ser Gly Lys Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029722PRTBacillus anthracis 297Asn Arg Leu Ser Ser Gly Lys Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029822PRTBacillus anthracis 298Asn Arg Leu Ser Ser Gly Lys Arg Ile
Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2029922PRTBacillus megaterium 299Glu Lys Leu Ser Ser Gly Gln Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2030022PRTAneurinibacillus sp. 300Glu Arg Leu Ser Ser Gly Tyr Arg
Ile Asn Arg Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2030128PRTBacillus thuringiensis 301Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Lys
Gly Asn Gln Ala Ser 20 2530228PRTBacillus thuringiensis 302Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala Ser 20 2530328PRTBacillus
thuringiensis 303Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu
Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Lys Gly Asn Gln Ala
Ser 20 2530428PRTBacillus cereus 304Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Lys
Gly Asn Gln Ala Ser 20 2530528PRTBacillus thuringiensis 305Thr Val
Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Ser Asn Lys Asn Arg His Ser 20 2530628PRTBacillus
thuringiensis 306Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile
Ala Asn Gln Ser1 5 10 15Ala Asn Ile Thr Asn Thr Asn Glu Asn Lys Ser
Ala 20 2530728PRTBacillus thuringiensis 307Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Asp Asp Asn Gln Lys Ala 20 2530828PRTBacillus thuringiensis
308Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1
5 10 15Ala Asn Gly Thr Asn Thr Asp Glu Asn Lys Ala Ala 20
2530928PRTBacillus cereus 309Ser Val Ser Asn Ile Leu Leu Arg Met
Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp Lys
Asn Gln Val Ala 20 2531028PRTBacillus cereus 310Thr Val Thr Asn Ile
Leu Gln Arg Met Arg Asp Val Ala Val Gln Ser1 5 10 15Ala Asn Gly Thr
Asn Ser Asn Lys Asn Arg Asp Ser 20 2531128PRTBacillus thuringiensis
311Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1
5 10 15Ala Asn Gly Thr Asn Thr Ala Asp Asn Gln Gln Ala 20
2531228PRTBacillus bombysepticus 312Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Ser Gly Thr Asn Thr Asp
Lys Asn Gln Ala Ala 20 2531328PRTBacillus thuringiensis 313Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala
Ser Gly Thr Asn Thr Asp Lys Asn Gln Ala Ala 20 2531428PRTBacillus
thuringiensis 314Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu
Ala Asn Gln Ser1 5 10 15Ala Ser Gly Thr Asn Thr Asp Lys Asn Gln Ala
Ala 20 2531528PRTBacillus cereus 315Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Gly
Asp Asn Gln Lys Ala 20 2531628PRTBacillus cereus 316Thr Asn Ile Leu
Gln Arg Met Arg Asp Leu Ala Val Gln Ser Ala Asn1 5 10 15Gly Thr Asn
Ser Asn Lys Asn Arg Asp Ser Leu Asn 20 2531728PRTBacillus
thuringiensis 317Thr Asn Val Leu Gln Arg Met Arg Asp Val Ala Val
Gln Ser Ala Asn1 5 10 15Gly Thr Asn Leu Asn Lys Asn Arg Asp Ser Leu
Asn 20 2531828PRTBacillus thuringiensis 318Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Asp Ser Asn Lys Ser Ala 20 2531928PRTBacillus cereus 319Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Ala Glu Asn Lys Ala Ala 20 2532028PRTBacillus
thuringiensis 320Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile
Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Ser Asp Asn Gln Lys
Ala 20 2532128PRTBacillus thuringiensis 321Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Ala Asp Asn Gln Gln Ala 20 2532228PRTBacillus thuringiensis
322Thr Val Met Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln Ser1
5 10 15Ala Asn Gly Thr Asn Ser Asn Lys Asn Arg Asp Ser 20
2532328PRTBacillus thuringiensis 323Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Ala
Asp Asn Gln Gln Ala 20 2532428PRTBacillus thuringiensis 324Thr Val
Thr Asn Ile Leu Gln His Met Arg Asp Phe Ala Ile Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Ser Asn Thr Asn Arg Asp Ser 20 2532528PRTBacillus
thuringiensis 325Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile
Ser Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser
Ala 20 2532628PRTBacillus thuringiensis 326Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ser Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Asp Lys Asn Gln Ser Ala 20 2532728PRTBacillus thuringiensis
327Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ser Asn Gln Ser1
5 10 15Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ser Ala 20
2532828PRTBacillus thuringiensis 328Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asn
Glu Asn Gln Ala Ala 20 2532928PRTBacillus thuringiensis 329Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Asn Glu Asn Gln Ala Ala 20 2533028PRTBacillus
weihenstephanensis 330Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp
Leu Ser Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp Glu Asn Gln
Gln Ala 20 2533128PRTBacillus thuringiensis 331Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr
Asn Thr Gly Asp Asn Gln Lys Ala 20 2533228PRTBacillus thuringiensis
332Thr Val Ala Asn Ile Leu Gln Arg Met Arg Asp Leu Ala Val Gln Ser1
5 10 15Ser Asn Asp Thr Asn Ser Asn Lys Asn Arg Asp Ser 20
2533328PRTBacillus thuringiensis 333Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp
Asp Asn Gln Lys Ala 20 2533428PRTBacillus thuringiensis 334Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Asp Asp Asn Gln Lys Ala 20 2533528PRTBacillus
thuringiensis 335Thr Val Thr Asn Ile Leu Gln His Met Arg Asp Phe
Ala Ile Gln Ser1 5 10 15Ala Asn Gly Thr Asn Ser Asn Thr Asn Arg Asp
Ser 20 2533628PRTBacillus thuringiensis 336Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ser Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Asp Lys Asn Gln Ser Ala 20 2533728PRTBacillus cereus 337Thr Val
Thr Asn Val Leu Gln Arg Met Arg Asp Val Ala Val Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Ser Ser Lys Asn Arg Asp Ser 20 2533828PRTBacillus
cereus 338Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn
Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Val Ala 20
2533928PRTBacillus thuringiensis 339Thr Val Met Asn Ile Leu Gln Arg
Met Arg Asp Leu Ala Ile Gln Ser1 5 10 15Ala Asn Ser Thr Asn Ser Asn
Lys Asn Arg Asp Ser 20 2534028PRTBacillus thuringiensis 340Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Ser Asp Asn Gln Lys Ala 20 2534128PRTBacillus
thuringiensis 341Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile
Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys
Ala 20 2534228PRTBacillus thuringiensis 342Ser Val Ser Asn Ile Leu
Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn
Thr Gly Asp Asn Gln Lys Ala 20 2534328PRTBacillus thuringiensis
343Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1
5 10 15Ala Asn Gly Thr Asn Thr Asn Gly Asn Gln Ala Ala 20
2534428PRTBacillus cereus 344Ser Val Ser Asn Ile Leu Leu Arg Met
Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asp Lys
Asn Gln Ala Ala 20 2534528PRTBacillus thuringiensis 345Ser Val Ser
Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn
Gly Thr Asn Thr Asn Glu Asn Gln Ala Ala 20 2534628PRTBacillus
cereus 346Thr Val Thr Asn Val Leu Gln Arg Met Arg Asp Leu Ala Val
Gln Ser1 5 10 15Ala Asn Asp Thr Asn Ser Asn Lys Asn Arg Asp Ser 20
2534728PRTBacillus cereus 347Ser Val Ser Asn Ile Leu Leu Arg Met
Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asn Glu
Asn Lys Ala Ala 20 2534828PRTBacillus cereus 348Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr
Asn Thr Ser Asp Asn Gln Ala Ala 20 2534928PRTBacillus cereus 349Ser
Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10
15Ala Asn Gly Thr Asn Thr Asn Glu Asn Lys Ala Ala 20
2535028PRTBacillus cereus 350Ser Val Ser Asn Ile Leu Leu Arg Met
Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Asn Glu
Asn Lys Ala Ala 20 2535128PRTBacillus cereus 351Ser Val Ser Asn Ile
Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr
Asn Thr Gly Asp Asn Gln Lys Ala 20 2535228PRTBacillus thuringiensis
352Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1
5 10 15Ala Ser Glu Thr Asn Thr Ser Lys Asn Gln Ala Ala 20
2535328PRTBacillus thuringiensis 353Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Ser Glu Thr Asn Thr Ser
Lys Asn Gln Ala Ala 20 2535428PRTBacillus thuringiensis 354Ser Val
Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala
Asn Gly Thr Asn Thr Gly Asp Asn Gln Lys Ala 20 2535528PRTBacillus
thuringiensis 355Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu
Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr Ser Glu Asn Gln Ala
Ala 20 2535628PRTBacillus cereus 356Thr Val Thr Asn Ile Leu Gln Arg
Met Arg Asp Leu Ala Val Gln Ser1 5 10 15Ala Asn Val Thr Asn Ser Asn
Lys Asn Arg Asn Ser 20 2535728PRTBacillus cereus 357Ser Val Ser Asn
Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly
Thr Asn Thr Asp Lys Asn Gln Gly Ala 20 2535828PRTBacillus cereus
358Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1
5 10 15Ala Asn Gly Thr Asn Thr Asp Lys Asn Gln Ala Ala 20
2535928PRTBacillus cereus 359Thr Val Thr Asn Val Leu Gln Arg Met
Arg Asp Leu Ala Val Gln Ser1 5 10 15Ala Asn Gly Thr Asn Ser Asn Lys
Asn Arg Asp Ser 20 2536028PRTBacillus thuringiensis 360Ser Val Ser
Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn Gln Thr1 5 10 15Ala Asn
Gly Thr Asn Lys Asp Thr Asp Ile Glu Ala 20 2536128PRTBacillus
cereus 361Ser Val Ser Asn Ile Leu Leu Arg Met Arg Asp Ile Ala Asn
Gln Thr1 5 10 15Ala Asn Gly Thr Asn Lys Asp Thr Asp Ile Glu Ala 20
2536228PRTBacillus thuringiensis 362Thr Val Met Asn Ile Leu Gln Arg
Met Arg Asp Leu Ala Ile Gln Ser1 5 10 15Ala Asn Ser Thr Asn Ser Asn
Lys Asn Arg Asp Ser
20 2536328PRTBacillus thuringiensis 363Ser Val Ser Asn Ile Leu Leu
Arg Met Arg Asp Ile Ala Asn Gln Ser1 5 10 15Ala Asn Gly Thr Asn Thr
Gly Asp Asn Gln Lys Ala 20 2536428PRTBacillus aryabhattai 364Glu
Thr His Asp Ile Leu Gln Arg Met Arg Glu Leu Val Val Gln Ala1 5 10
15Gly Asn Gly Thr Asn Lys Thr Glu Asp Leu Asp Ala 20
2536528PRTBacillus manliponensis 365Ser Val Ser Asn Ile Leu Leu Arg
Met Arg Asp Leu Ala Thr Gln Ser1 5 10 15Ala Thr Gly Thr Asn Gln Gly
Asn Asp Arg Glu Ser 20 2536628PRTLysinibacillus sp. 366Glu Thr His
Ala Ile Val Gln Arg Met Arg Glu Leu Ala Val Gln Ala1 5 10 15Ala Thr
Asp Thr Asn Thr Asp Asp Asp Arg Ala Lys 20 2536728PRTLysinibacillus
sp. 367Glu Thr His Ala Ile Val Gln Arg Met Arg Glu Leu Ala Val Gln
Ala1 5 10 15Ala Thr Asp Thr Asn Thr Asp Asp Asp Arg Ala Lys 20
2536828PRTPaenibacillus sp. 368Glu Ile His Ser Met Leu Gln Arg Met
Asn Glu Leu Ala Val Gln Ala1 5 10 15Ser Asn Gly Thr Tyr Ser Gly Ser
Asp Arg Leu Asn 20 2536928PRTBacillus anthracis 369Ser Val Ser Asn
Ile Leu Leu Arg Met Arg Asp Leu Ala Asn Gln Ser1 5 10 15Ala Asn Gly
Thr Asn Thr Lys Glu Asn Gln Asp Ala 20 2537028PRTBacillus anthracis
370Ser Val Ser Asn Ile Leu Thr Arg Met Arg Asp Ile Ala Val Gln Ser1
5 10 15Ser Asn Gly Thr Asn Thr Ala Glu Asn Gln Ser Ala 20
2537128PRTBacillus anthracis 371Ser Val Ser Asn Ile Leu Thr Arg Met
Arg Asp Ile Ala Val Gln Ser1 5 10 15Ser Asn Gly Thr Asn Thr Ala Glu
Asn Gln Ser Ala 20 2537228PRTBacillus anthracis 372Ser Val Ser Asn
Ile Leu Thr Arg Met Arg Asp Ile Ala Val Gln Ser1 5 10 15Ser Asn Gly
Thr Asn Thr Ala Glu Asn Gln Ser Ala 20 2537328PRTBacillus anthracis
373Ser Val Ser Asn Ile Leu Thr Arg Met Arg Asp Ile Ala Val Gln Ser1
5 10 15Ser Asn Gly Thr Asn Thr Ala Glu Asn Gln Ser Ala 20
2537428PRTBacillus megaterium 374Glu Thr His Asp Ile Leu Gln Arg
Met Arg Glu Leu Val Val Gln Ala1 5 10 15Gly Asn Gly Thr Asn Lys Thr
Glu Asp Leu Asp Ala 20 2537528PRTAneurinibacillus sp. 375Glu Ile
His Glu Met Leu Gln Arg Met Arg Glu Leu Ala Val Gln Ala1 5 10 15Ala
Asn Gly Thr Tyr Ser Asp Lys Asp Lys Lys Ala 20 2537622PRTBacillus
thuringiensis 376Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Ser
Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2037722PRTBacillus
thuringiensis 377Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Ser
Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2037821PRTBacillus
thuringiensis 378Ala Ile Ala Leu Gly Ala Ala Asp Asp Ala Ser Asn
Ile Arg Lys Gly1 5 10 15Ser Ser Leu Arg Asp 2037922PRTBacillus
cereus 379Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Ser Asn Ile
Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2038021PRTBacillus
thuringiensis 380Val Ile Ala Asn Ala Pro Asn Asp Ser Ala Asn Asn
Leu Lys Lys Gly1 5 10 15Thr Ala Leu His Glu 2038122PRTBacillus
thuringiensis 381Thr Ala Ile Ala Gly Ala Ala Asp Asp Ser Ala Asn
Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2038222PRTBacillus
thuringiensis 382Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn
Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2038322PRTBacillus
thuringiensis 383Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn
Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2038422PRTBacillus
cereus 384Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile
Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp 2038522PRTBacillus cereus
385Val Ile Ala Val Asn Ala Pro Asn Asp Ser Ala His Asn Leu Lys Lys1
5 10 15Gly Thr Ala Leu His Glu 2038622PRTBacillus thuringiensis
386Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1
5 10 15Gly Ser Ser Leu Arg Asp 2038722PRTBacillus bombysepticus
387Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1
5 10 15Gly Ser Ser Leu Arg Asp 2038822PRTBacillus thuringiensis
388Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1
5 10 15Gly Ser Ser Leu Arg Asp 2038922PRTBacillus thuringiensis
389Ala Ile Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1
5 10 15Gly Ser Ser Leu Arg Asp 2039022PRTBacillus cereus 390Ala Ile
Ala Leu Gly Ala Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly
Ser Ser Leu Arg Asp 2039122PRTBacillus cereus 391Val Ile Ala Ile
Asn Ala Pro Asn Asp Ala Ser Asn Asn Leu Lys Lys1 5 10 15Gly Thr Ala
Leu His Glu 2039221PRTBacillus thuringiensis 392Val Ile Ala Asn Ala
Pro Asn Asp Ser Ala His Asn Leu Lys Lys Gly1 5 10 15Thr Ala Leu His
Glu 2039322PRTBacillus thuringiensis 393Ala Ile Ala Leu Gly Ala Ala
Asp Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2039422PRTBacillus cereus 394Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2039522PRTBacillus thuringiensis 395Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2039622PRTBacillus thuringiensis 396Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2039721PRTBacillus thuringiensis 397Val Ile Ala Asn Ala Pro Asn Asp
Ser Ala His Asn Leu Lys Lys Gly1 5 10 15Thr Ala Phe His Glu
2039821PRTBacillus thuringiensis 398Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Arg Lys Gly1 5 10 15Ser Ser Leu Arg Asp
2039922PRTBacillus thuringiensis 399Val Ile Val Ile Asn Ala Pro Asn
Asp Ser Ala His Asn Leu Lys Lys1 5 10 15Gly Thr Ala Leu His Glu
2040022PRTBacillus thuringiensis 400Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040122PRTBacillus thuringiensis 401Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040222PRTBacillus thuringiensis 402Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040322PRTBacillus thuringiensis 403Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040422PRTBacillus thuringiensis 404Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040522PRTBacillus weihenstephanensis 405Ala Ile Ala Leu Gly Ala
Ala Asp Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg
Asp 2040622PRTBacillus thuringiensis 406Ala Ile Ala Leu Gly Ala Ala
Asp Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040721PRTBacillus thuringiensis 407Val Ile Ala Asn Ala Pro Asn Asp
Ser Ala His Asn Leu Lys Lys Gly1 5 10 15Thr Ala Leu His Glu
2040822PRTBacillus thuringiensis 408Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2040922PRTBacillus thuringiensis 409Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041022PRTBacillus thuringiensis 410Val Ile Val Ile Asn Ala Pro Asn
Asp Ala Ser His Asn Leu Lys Lys1 5 10 15Gly Thr Ala Leu His Glu
2041122PRTBacillus thuringiensis 411Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041222PRTBacillus cereus 412Val Ile Ala Val Ala Asn Pro Asn Asn
Ser Ala His Asn Leu Lys Lys1 5 10 15Gly Thr Ala Leu His Glu
2041322PRTBacillus cereus 413Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041421PRTBacillus thuringiensis 414Val Ile Ala Asn Ala Pro Asn Asp
Ser Ala His Asn Leu Lys Lys Gly1 5 10 15Thr Ala Leu His Glu
2041522PRTBacillus thuringiensis 415Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041622PRTBacillus thuringiensis 416Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041722PRTBacillus thuringiensis 417Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041822PRTBacillus thuringiensis 418Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2041922PRTBacillus cereus 419Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042022PRTBacillus thuringiensis 420Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ala Ser Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042121PRTBacillus cereus 421Val Ile Ala Asn Ala Pro Asn Asp Ser
Ala Asn Asn Leu Lys Lys Gly1 5 10 15Thr Ala Leu His Glu
2042222PRTBacillus cereus 422Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042322PRTBacillus cereus 423Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042422PRTBacillus cereus 424Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042522PRTBacillus cereus 425Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042622PRTBacillus cereus 426Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042722PRTBacillus thuringiensis 427Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2042821PRTBacillus thuringiensis 428Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ala Asn Asn Ile Arg Lys Gly1 5 10 15Ser Ser Leu Arg Asp
2042922PRTBacillus thuringiensis 429Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043022PRTBacillus thuringiensis 430Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043122PRTBacillus cereus 431Val Ile Ala Ile Asn Ala Pro Asn Asn
Ser Ala His Asn Leu Lys Lys1 5 10 15Gly Thr Ala Leu His Glu
2043222PRTBacillus cereus 432Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043322PRTBacillus cereus 433Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043422PRTBacillus cereus 434Val Ile Ala Ile Asn Ala Pro Asn Asp
Ser Ala Asn Asn Leu Lys Lys1 5 10 15Gly Thr Ala Leu His Glu
2043522PRTBacillus thuringiensis 435Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043621PRTBacillus cereus 436Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ala Asn Asn Ile Arg Lys Gly1 5 10 15Ser Ser Leu Arg Asp
2043721PRTBacillus thuringiensis 437Val Ile Ala Asn Ala Pro Asn Asp
Ser Ala His Asn Leu Lys Lys Gly1 5 10 15Thr Ala Leu His Glu
2043822PRTBacillus thuringiensis 438Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2043922PRTBacillus aryabhattai 439Ser Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Ser Asn Ile Arg Gln1 5 10 15Gly Ser Ser Leu Lys Glu
2044022PRTBacillus manliponensis 440Ala Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Ser Asn Ile Gln Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2044122PRTLysinibacillus sp. 441Ser Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Tyr1 5 10 15Gly Ser Ser Leu Arg Leu
2044222PRTLysinibacillus sp. 442Ser Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Tyr1 5 10 15Gly Ser Ser Leu Arg Leu
2044322PRTPaenibacillus sp. 443Ser Ile Ala Gly Leu Ala Ala Asp Asp
Ser Ala Gly Asn Ile Arg Leu1 5 10 15Gly Ser Ser Leu Lys Gly
2044422PRTBacillus anthracis 444Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ser Ala Asn Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asp
2044522PRTBacillus anthracis 445Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2044622PRTBacillus anthracis 446Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2044722PRTBacillus anthracis 447Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2044822PRTBacillus anthracis 448Ala Ile Ala Leu Gly Ala Ala Asp Asp
Ala Ala Ser Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2044922PRTBacillus megaterium 449Ser Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Ser Asn Ile Arg Gln1 5 10 15Gly Ser Ser Leu Lys Glu
2045022PRTAneurinibacillus sp. 450Ser Ile Ala Leu Gly Ala Ala Asp
Asp Ser Ala Arg Asn Ile Arg Tyr1 5 10 15Gly Ser Ser Leu Arg Glu
2045128PRTBacillus thuringiensis 451Ser Ala Gln Asn Gly Lys Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2545228PRTBacillus thuringiensis 452Ser Ala
Gln Asn Gly Lys Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2545328PRTBacillus
thuringiensis 453Ser Ala Gln Asn Gly Lys Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2545428PRTBacillus cereus 454Ser Ala Gln Asn Gly Lys Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg
Met Arg Leu Leu Ile Asn Ser Val Ser 20 2545528PRTBacillus
thuringiensis 455Ser His Arg Asn Lys Asn Ser Asn Thr Gly Asn Ala
Ser Gln Val Ala1 5 10 15Leu Asp Arg Met Arg Gln Leu Ile Asn Thr Val
Thr 20 2545628PRTBacillus thuringiensis 456Ala Ser Lys Asn Glu Asn
Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2545728PRTBacillus thuringiensis
457Ala Lys Gln Asn Asp Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1
5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2545828PRTBacillus thuringiensis 458Ala Ala Lys Asn Glu Asp Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2545929PRTBacillus cereus 459Leu Ala Val Gln
Asn Lys Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn1 5 10 15Ala Ile Asp
Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2546028PRTBacillus
cereus 460Ser Asp Arg Asn Lys Asn Ser Asn Thr Gly Asn Ala Ser Gln
Val Ala1 5 10 15Val Asp Arg Met Arg Gln Leu Ile Asn Thr Val Thr 20
2546128PRTBacillus thuringiensis 461Ala Gln Gln Asn Asp Ala Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2546228PRTBacillus bombysepticus 462Ala Ala
Gln Asn Lys Asp Thr Asn Thr Gly Ser Ala Ser Gln Asn Ala1 5 10 15Leu
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2546328PRTBacillus
thuringiensis 463Ala Ala Gln Asn Lys Asp Thr Asn Thr Gly Ser Ala
Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2546428PRTBacillus thuringiensis 464Ala Ala Gln Asn Lys Asp
Thr Asn Thr Gly Ser Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2546528PRTBacillus cereus 465Ala Lys
Gln Asn Asp Gly Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2546628PRTBacillus
cereus 466Asn Leu Ser Asp Arg Asn Lys Asn Ser Asn Thr Gly Asn Ala
Ser Gln1 5 10 15Val Ala Leu Asp Arg Met Arg Gln Leu Ile Asn Thr 20
2546728PRTBacillus thuringiensis 467Asn Leu Ser Asp Arg Asn Lys Asn
Leu Asn Thr Gly Asn Ala Ser Gln1 5 10 15Val Ala Val Asp Arg Met Arg
Gln Leu Val Asn Thr 20 2546828PRTBacillus thuringiensis 468Ala Ser
Lys Asn Ser Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2546928PRTBacillus
cereus 469Ala Ala Lys Asn Glu Ala Thr Asn Thr Gly Asn Ala Ser Gln
Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2547028PRTBacillus thuringiensis 470Ala Lys Gln Asn Asp Ser Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2547128PRTBacillus thuringiensis 471Ala Gln
Gln Asn Asp Ala Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2547228PRTBacillus
thuringiensis 472Ser Asp Arg Asn Lys Asn Ser Asn Thr Gly Asn Ala
Ser Gln Val Ala1 5 10 15Leu Asp Arg Met Arg Gln Leu Ile Asn Met Val
Thr 20 2547328PRTBacillus thuringiensis 473Ala Gln Gln Asn Asp Ala
Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2547428PRTBacillus thuringiensis
474Ser Asp Arg Asn Thr Asn Ser Asn Thr Gly Asn Ala Ser Gln Ile Ala1
5 10 15Phe Asp Arg Met His Gln Leu Ile Asn Thr Val Thr 20
2547528PRTBacillus thuringiensis 475Ala Ser Gln Asn Lys Asp Thr Asn
Thr Gly Asn Ala Ser Gln Asn Ser1 5 10 15Ile Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2547628PRTBacillus thuringiensis 476Ala Ser
Gln Asn Lys Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ser1 5 10 15Ile
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2547728PRTBacillus
thuringiensis 477Ala Ser Gln Asn Lys Asp Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ser1 5 10 15Ile Ser Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2547828PRTBacillus thuringiensis 478Ala Ala Gln Asn Glu Asn
Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2547928PRTBacillus thuringiensis
479Ala Gln Gln Asn Glu Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ser1
5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2548028PRTBacillus weihenstephanensis 480Ala Gln Gln Asn Glu Asp
Thr Asn Thr Gly Asn Ala Ser Gln Asn Ser1 5 10 15Leu Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2548128PRTBacillus thuringiensis
481Ala Lys Gln Asn Asp Gly Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1
5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2548228PRTBacillus thuringiensis 482Ser Asp Arg Asn Lys Asn Ser Asn
Thr Asp Asn Ser Ser Gln Val Ala1 5 10 15Leu Asp Arg Met Arg Gln Leu
Ile Asn Ala Val Thr 20 2548328PRTBacillus thuringiensis 483Ala Lys
Gln Asn Asp Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2548428PRTBacillus
thuringiensis 484Ala Lys Gln Asn Asp Asp Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2548528PRTBacillus thuringiensis 485Ser Asp Arg Asn Thr Asn
Ser Asn Thr Gly Asn Ala Ser Gln Ile Ala1 5 10 15Phe Asp Arg Met His
Gln Leu Ile Asn Thr Val Thr 20 2548628PRTBacillus thuringiensis
486Ala Ser Gln Asn Lys Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ser1
5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2548728PRTBacillus cereus 487Ser Asp Arg Asn Lys Ser Ser Asn Thr
Gly Asn Ala Ser Gln Val Ala1 5 10 15Val Asp Arg Met Arg Gln Leu Val
Asn Thr Val Thr 20 2548827PRTBacillus cereus 488Ala Val Gln Lys Asp
Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala Ile1 5 10 15Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2548928PRTBacillus thuringiensis
489Ser Asp Arg Asn Lys Asn Ser Asn Thr Ser Asn Ala Ser Gln Ile Ala1
5 10 15Leu Asp Arg Met Arg Gln Leu Ile Asn Met Val Thr 20
2549028PRTBacillus thuringiensis 490Ala Lys Gln Asn Asp Ser Thr Asn
Ile Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2549128PRTBacillus thuringiensis 491Ala Lys
Gln Asn Asp Gly Thr Asn Thr Phe Asn Ala Ser Gln Asn Ala1 5 10 15Ile
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2549228PRTBacillus
thuringiensis 492Ala Lys Gln Asn Asp Gly Thr Asn Thr Phe Asn Ala
Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2549328PRTBacillus thuringiensis 493Ala Ala Gln Asn Gly Asn
Thr Asn Thr Phe Asn Ala Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg
Leu Leu Ile Asn Ser Val Ser 20 2549428PRTBacillus cereus 494Ala Ala
Gln Asn Lys Asp Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Ile
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2549528PRTBacillus
thuringiensis 495Ala Ala Gln Asn Glu Asn Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2549628PRTBacillus cereus 496Ser Asp Arg Asn Lys Asn Ser Asn
Thr Asp Asn Ala Ser Gln Val Ala1 5 10 15Leu Asp Arg Met Arg Gln Leu
Val Asn Thr Val Thr 20 2549728PRTBacillus cereus 497Ala Ala Lys Asn
Glu Asn Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg
Met Arg Leu Leu Ile Asn Ser Val Ser 20 2549828PRTBacillus cereus
498Ala Ala Gln Asn Asp Ser Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1
5 10 15Leu Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2549928PRTBacillus cereus 499Ala Ala Lys Asn Glu Asn Thr Asn Thr
Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile
Asn Ser Val Ser 20 2550028PRTBacillus cereus 500Ala Ala Lys Asn Glu
Asn Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met
Arg Leu Leu Ile Asn Ser Val Ser 20 2550128PRTBacillus cereus 501Ala
Lys Gln Asn Asp Gly Thr Asn Thr Gly Asn Ala Ser Gln Asn Ala1 5 10
15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2550228PRTBacillus thuringiensis 502Ala Ala Gln Asn Lys Ser Thr Asn
Thr Glu Ser Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu
Ile Asn Ser Val Ser 20 2550328PRTBacillus thuringiensis 503Ala Ala
Gln Asn Lys Ser Thr Asn Thr Glu Ser Ala Ser Gln Asn Ala1 5 10 15Leu
Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20 2550428PRTBacillus
thuringiensis 504Ala Lys Gln Asn Asp Gly Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2550527PRTBacillus thuringiensis 505Ala Ala Gln Asn Glu Ser
Thr Asn Thr Gly Asn Ala Gln Asn Ala Leu1 5 10 15Asp Arg Met Arg Leu
Leu Ile Asn Ser Val Ser 20 2550628PRTBacillus cereus 506Ser Asn Arg
Asn Lys Asn Ser Asn Thr Val Asn Ala Ser Gln Val Ala1 5 10 15Leu Asp
Arg Met Arg Gln Leu Ile Asn Thr Val Thr 20 2550727PRTBacillus
cereus 507Ala Gly Gln Asn Lys Asp Thr Asn Thr Asn Ala Ser Gln Asn
Ala Leu1 5 10 15Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2550828PRTBacillus cereus 508Ala Ala Gln Asn Lys Asp Thr Asn Thr
Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile
Asn Ser Val Ser 20 2550928PRTBacillus cereus 509Ser Asp Arg Asn Lys
Asn Ser Asn Thr Gly Asn Ala Ser Gln Val Ala1 5 10 15Leu Asp Arg Met
Arg Gln Leu Val Asn Thr Val Thr 20 2551028PRTBacillus thuringiensis
510Ala Glu Ile Asp Thr Asp Lys Asn Thr Gly Asn Ala Thr Gln Asn Ala1
5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2551128PRTBacillus cereus 511Ala Glu Ile Asp Thr Asp Lys Asn Thr
Gly Asn Ala Thr Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu Ile
Asn Ser Val Ser 20 2551227PRTBacillus thuringiensis 512Ser Asp Arg
Asn Lys Asn Ser Asn Thr Ser Asn Ala Ser Gln Ile Ala1 5 10 15Leu Asp
Arg Met Arg Gln Leu Ile Asn Met Thr 20 2551328PRTBacillus
thuringiensis 513Ala Lys Gln Asn Asp Gly Thr Asn Thr Gly Asn Ala
Ser Gln Asn Ala1 5 10 15Ile Asp Arg Met Arg Leu Leu Ile Asn Ser Val
Ser 20 2551428PRTBacillus aryabhattai 514Ala Asp Leu Asp Glu Thr
Lys Asn Thr Gly Asn Gly Ala Gln Val Val1 5 10 15Leu Glu Arg Met Arg
Gln Leu Ile Asp His Thr Glu 20 2551527PRTBacillus manliponensis
515Ser Glu Arg Asp Asn Gly Gln Asn Thr Gly Thr Ala Gln Thr Ala Leu1
5 10 15Asp Arg Met Arg Leu Leu Ile Asn Ser Val Ser 20
2551628PRTLysinibacillus sp. 516Lys Ala Arg Asp Asp Asp Thr Asn Thr
Asp Thr Ala Ala Gln Val Ala1 5 10 15Leu Glu Arg Met Arg Gln Val Ile
Ala His Thr Glu 20 2551728PRTLysinibacillus sp. 517Lys Ala Arg Asp
Asp Asp Thr Asn Thr Asp Thr Ala Ala Gln Val Ala1 5 10 15Leu Glu Arg
Met Arg Gln Val Ile Ala His Thr Glu 20 2551828PRTPaenibacillus sp.
518Asn Leu Arg Asp Ser Gly Ser Tyr Thr Gly Asn Ser Ala Gln Val Ala1
5 10 15Leu Glu Asn Met Arg Gln Leu Met Ser His Ile Glu 20
2551928PRTBacillus anthracis 519Ala Asp Gln Asn Glu Lys Thr Asn Thr
Gly Asn Ala Ser Gln Asn Ala1 5 10 15Leu Asp Arg Met Arg Leu Leu Ile
Asn Ser Val Ser 20 2552028PRTBacillus anthracis 520Ala Ser Gln Asn
Glu Ala Thr Asn Thr Gly Asn Ser Ser Gln Val Ala1 5 10 15Ile Asp Arg
Met Arg Thr Leu Ile Asn Ser Val Ser 20 2552128PRTBacillus anthracis
521Ala Ser Gln Asn Glu Ala Thr Asn Thr Gly Asn Ser Ser Gln Val Ala1
5 10 15Ile Asp Arg Met Arg Thr Leu Ile Asn Ser Val Ser 20
2552228PRTBacillus anthracis 522Ala Ser Gln Asn Glu Ala Thr Asn Thr
Gly Asn Ser Ser Gln Val Ala1 5 10 15Ile Asp Arg Met Arg Thr Leu Ile
Asn Ser Val Ser 20 2552328PRTBacillus anthracis 523Ala Ser Gln Asn
Glu Ala Thr Asn Thr Gly Asn Ser Ser Gln Val Ile1 5 10 15Ala Asp Arg
Met Arg Thr Leu Ile Asn Ser Val Ser 20 2552428PRTBacillus
megaterium 524Ala Asp Leu Asp Glu Thr Lys Asn Thr Gly Asn Gly Ala
Gln Val Val1 5 10 15Leu Glu Arg Met Arg Gln Leu Ile Asp His Thr Glu
20 2552527PRTAneurinibacillus sp. 525Ala Lys Lys Asp Lys Ser Tyr
Thr Gly Asn Ala Ala Gln Val Ala Leu1 5 10 15Glu Arg Met Arg Gln Leu
Met Glu His Ile Glu 20 2552622PRTEscherichia coli 526Glu Arg Leu
Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly
Gln Ala Ile Ala 2052722PRTEscherichia coli 527Ala Ile Ala Gln Gly
Ala Ala Asp Asp Lys Ala Ser Asn Ile Arg Leu1 5 10 15Gly Ser Ser Leu
Arg Glu 2052815PRTEscherichia coli 528Arg Ile Asn Ser Ala Lys Asp
Asp Ala Ala Gly Gln Ala Ile Ala1 5 10 1552915PRTEscherichia coli
529Ala Ile Ala Gln Gly Ala Ala Asp Asp Lys Ala Ser Asn Ile Arg1 5
10 1553022PRTPseudomonas aeruginosa 530Gln Arg Leu Ser Thr Gly Ser
Arg Ile Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Leu Gln Ile Ala
2053122PRTPseudomonas aeruginosa 531Ala Ile Gln Leu Gly Ala Ala Asp
Asp Lys Ala Ser Asn Ile Arg Ser1 5 10 15Gly Thr Ser Leu Arg Gln
2053222PRTXanthomonas spp. 532Gln Arg Leu Ser Ser Gly Leu Arg Ile
Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2053322PRTXanthomonas spp. 533Ser Ile Ala Leu Gly Ala Ala Asp Asp
Lys Ala Ser Asn Ile Arg Leu1 5 10 15Gly Ser Ser Leu Arg Gln
2053422PRTErwinia amylovora 534Gln Arg Leu Ser Ser Gly Leu Arg Ile
Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Gln
Ala Ile Ser 2053522PRTErwinia amylovora 535Ser Ile Ala Gln Gly Ala
Ala Asp Asp Lys Ala Ser Asn Ile Arg Leu1 5 10 15Gly Ser Ser Leu Arg
Gln 2053622PRTBurkholderia phytofirmans 536Thr Arg Leu Ser Ser Gly
Lys Arg Ile Asn Ser Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile
Ser 2053722PRTBurkholderia phytofirmans 537Ser Ile Ala Leu Gly Ala
Ala Asp Asp Ala Ala Ser Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg
Thr 2053822PRTBurkholderia ubonensis 538Asn Arg Leu Ser Ser Gly Lys
Arg Ile Asn Thr Ala Ala Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2053922PRTBurkholderia ubonensis 539Ser Ile Ala Leu Gly Ala Ala Asp
Asp Ala Ala Thr Asn Ile Arg Lys1 5 10 15Gly Ser Ser Leu Arg Asn
2054022PRTPseudomonas syringae 540Thr Arg Leu Ser Ser Gly Leu Lys
Ile Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Leu Gln Ile Ala
2054122PRTPseudomonas syringae 541Ala Ile Gln Leu Gly Ala Ala Asp
Asp Lys Ala Ser Asn Ile Lys Leu1 5 10 15Gly Ser Ser Leu Arg Thr
205424PRTArtificial SequenceSynthetic construct 542Gly Phe Leu
Asn15435PRTArtificial SequenceSynthetic construct 543Trp Gly Phe
Leu Ile1 55445PRTArtificial SequenceSynthetic construct 544Met Gly
Val Leu Asn1 55454PRTArtificial SequenceSynthetic construct 545Gly
Val Leu Asn15465PRTArtificial SequenceSynthetic construct 546Trp
Gly Phe Phe Tyr1 55479PRTArtificial SequenceSynthetic construct
547Leu Val Pro Phe Ala Val Trp Leu Ala1 55485PRTArtificial
SequenceSynthetic construct 548Ala Val Trp Leu Ala1
554912PRTArtificial SequenceSynthetic construct 549Leu Leu Gly Thr
Ala Asp Lys Lys Ile Lys Ile Gln1 5 1055011PRTArtificial
SequenceSynthetic construct 550Leu Leu Lys Ser Thr Gln Glu Ile Lys
Ile Gln1 5 1055111PRTArtificial SequenceSynthetic construct 551Leu
Leu Asn Glu Asp Ser Glu Val Lys Ile Gln1 5 105529PRTArtificial
SequenceSynthetic construct 552Leu Gly Val Ala Ala Asn Asn Thr Gln1
555310PRTArtificial SequenceSynthetic construct 553Leu Leu Arg Met
Arg Asp Leu Ala Asn Gln1 5 1055410PRTArtificial SequenceSynthetic
construct 554Leu Gln Arg Met Arg Asp Val Ala Val Gln1 5
1055510PRTArtificial SequenceSynthetic construct 555Leu Leu Arg Met
Arg Asp Ile Ser Asn Gln1 5 1055610PRTArtificial SequenceSynthetic
contstruct 556Leu Leu Arg Met Arg Asp Ile Ala Asn Gln1 5
1055713PRTArtificial SequenceSynthetic construct 557Leu Gln Lys Gln
Ile Asp Tyr Ile Ala Gly Asn Thr Gln1 5 105588PRTArtificial
SequenceSynthetic construct 558Leu Leu Ile Arg Leu Pro Leu Asp1
55599PRTArtificial SequenceSynthetic construct 559Gln Arg Met Arg
Glu Leu Ala Val Gln1 55609PRTArtificial SequenceSynthetic construct
560Thr Arg Met Arg Asp Ile Ala Val Gln1 55619PRTArtificial
SequenceSynthetic construct 561Thr Arg Met Arg Asp Ile Ala Val Gln1
55629PRTArtificial SequenceSynthetic construct 562Gln Arg Met Arg
Glu Leu Val Val Gln1 55636PRTArtificial SequenceSynthetic construct
563Leu Gly Ala Thr Leu Asn1 55646PRTArtificial SequenceSynthetic
construct 564Leu Gly Ala Thr Gln Asn1 55656PRTArtificial
SequenceSynthetic construct 565Leu Ala Gln Ala Asn Gln1
55666PRTArtificial SequenceSynthetic construct 566Leu Gly Ala Met
Ile Asn1 55676PRTArtificial SequenceSynthetic construct 567Leu Gly
Ser Met Ile Asn1 55686PRTArtificial SequenceSynthetic construct
568Met Gly Ala Tyr Gln Asn1 55696PRTArtificial SequenceSynthetic
construct 569Leu Gly Ala Tyr Gln Asn1 55706PRTArtificial
SequenceSynthetic construct 570Tyr Gly Ser Gln Leu Asn1
557122PRTBacillus thuringiensis 571Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2057222PRTBacillus thuringiensis 572Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Gln Ile Ala
2057322PRTBacillus thuringiensis 573Gln Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2057422PRTBacillus thuringiensis 574Asn Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2057522PRTCaballeronia megalochromosomata 575Thr Arg Leu Ser Ser
Gly Lys Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala
Ile Ala 2057622PRTBacillus thuringiensis 576Asp Arg Leu Ser Ser Gly
Tyr Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile
Ala 2057722PRTBacillus thuringiensis 577Asp Arg Leu Ser Ser Gly Phe
Arg Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2057822PRTBacillus thuringiensis 578Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Asp Pro1 5 10 15Ala Gly Leu Ala Ile Ala
2057922PRTBacillus thuringiensis 579Asp Arg Leu Ser Ser Gly Gln Arg
Ile Asn Ser Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ala
2058022PRTLysinibacillus spp. 580Glu Lys Leu Ser Ser Gly Leu Arg
Ile Asn Arg Ala Gly Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2058122PRTLysinibacillus spp. 581Glu Lys Leu Ser Ser Gly Tyr Lys
Ile Asn Arg Ala Ser Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2058222PRTLysinibacillus spp. 582Leu Arg Ile Ser Ser Gly Tyr Arg
Ile Asn Ser Ala Ala Asp Asp Pro1 5 10 15Ala Gly Leu Ala Ile Ser
2058322PRTLysinibacillus fusiformis 583Leu Arg Ile Ser Thr Gly Tyr
Arg Ile Asn Ser Ala Ala Asp Asp Pro1 5 10 15Ala Gly Leu Ala Ile Ser
2058422PRTLysinibacillus macroides 584Glu Lys Leu Ser Ser Gly Phe
Arg Ile Asn Arg Ala Gly Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile Ser
2058522PRTLysinibacillus xylanilyticus 585Glu Lys Leu Ser Ser Gly
Tyr Lys Ile Asn Arg Ala Gly Asp Asp Ala1 5 10 15Ala Gly Leu Ala Ile
Ser 2058622PRTEscherichia coli 586Glu Arg Leu Ser Ser Gly Leu Arg
Ile Asn Ser Ala Lys Asp Asp Ala1 5 10 15Ala Gly Gln Ala Ile Ala
2058723PRTXanthomonas species 587Asn Gln Gly Ile Ser Glu Lys Gln
Leu Asp Gln Leu Leu Thr Gln Leu1 5 10 15Ile Met Ala Leu Leu Gln Gln
2058823PRTXanthomonas species 588Gln Gln Leu Leu Ala Met Ile Leu
Gln Thr Leu Leu Gln Asp Leu Gln1 5 10 15Lys Glu Ser Ile Gly Gln Asn
2058912PRTXanthomonas species 589Leu Asp Gln Leu Leu Thr Gln Leu
Ile Met Ala Leu1 5 1059012PRTXanthomonas species 590Leu Ala Met Ile
Leu Gln Thr Leu Leu Gln Asp Leu1 5 1059119PRTXanthomonas species
591Ser Glu Lys Gln Leu Asp Gln Leu Leu Thr Gln Leu Ile Met Ala Leu1
5 10 15Leu Gln Gln59219PRTXanthomonas species 592Gln Gln Leu Leu
Ala Met Ile Leu Gln Thr Leu Leu Gln Asp Leu Gln1 5 10 15Lys Glu
Ser593143PRTXanthamonas citri 593Met Met Asn Ser Leu Asn Thr Gln
Leu Gly Ala Asn Ser Ser Phe Phe1 5 10 15Gln Val Asp Pro Ser Gln Asn
Thr Gln Ser Gly Ser Asn Gln Gly Asn 20 25 30Gln Gly Ile Ser Glu Lys
Gln Leu Asp Gln Leu Leu Thr Gln Leu Ile 35 40 45Met Ala Leu Leu Gln
Gln Ser Asn Asn Ala Glu Gln Gly Gln Gly Gln 50 55 60Gly Gln Gly Gly
Asp Ser Gly Gly Gln Gly Gly Asn Arg Gln Gln Ala65 70 75 80Gly Gln
Ser Asn Gly Ser Pro Ser Gln Tyr Thr Gln Met Leu Met Asn 85 90 95Ile
Val Gly Asp Ile Leu Gln Ala Gln Asn Gly Gly Gly Phe Gly Gly 100 105
110Gly Phe Gly Gly Gly Phe Gly Gly Gly Gly Leu Gly Thr Ser Leu Gly
115 120 125Thr Ser Leu Gly Thr Ser Leu Ala Ser Asp Thr Gly Ser Met
Gln 130 135 14059419PRTPantoea sesami 594Gln Leu Glu Gln Leu Met
Thr Gln Leu Arg Ala Arg Leu Cys Arg Leu1 5 10 15Met Ala
Met59519PRTErwinia gerudensis 595Gln Leu Glu Gln Leu Met Thr Gln
Leu Arg Ala Arg Leu Lys Arg Leu1 5 10 15Met Ala Met59619PRTPantoea
sesami 596Met Ala Met Leu Arg Cys Leu Arg Ala Arg Leu Gln Thr Met
Leu Gln1 5 10 15Glu Leu Gln59719PRTErwinia gerudensis 597Met Ala
Met Leu Arg Lys Leu Arg Ala Arg Leu Gln Thr Met Leu Gln1 5 10 15Glu
Leu Gln5985PRTArabidopsis thalianaMISC_FEATURE(1)..(1)Xaa =
sulfonated tyrosineMISC_FEATURE(3)..(3)Xaa = sulfonated tyrosine
598Xaa Ile Xaa Thr Gln1 55995PRTArabidopsis
thalianaMISC_FEATURE(3)..(3)Xaa = sulfonated
tyrosineMISC_FEATURE(5)..(5)Xaa = sulfonated tyrosine 599Gln Thr
Xaa Ile Xaa1 560012PRTGlycine max 600Gly Gly Ile Arg Ala Ala Pro
Thr Gly Asn Glu Arg1 5 1060112PRTGlycine max 601Arg Glu Asn Gly Thr
Pro Ala Ala Arg Ile Gly Gly1 5 10602217PRTGlycine max 602Met Lys
Ser Thr Ile Phe Phe Ala Leu Phe Leu Phe Cys Ala Phe Thr1 5 10 15Thr
Ser Tyr Leu Pro Ser Ala Ile Ala Asp Phe Val Leu Asp Asn Glu 20 25
30Gly Asn Pro Leu Glu Asn Gly Gly Thr Tyr Tyr Ile Leu Ser Asp Ile
35 40 45Thr Ala Phe Gly Gly Ile Arg Ala Ala Pro Thr Gly Asn Glu Arg
Cys 50 55 60Pro Leu Thr Val Val Gln Ser Arg Asn Glu Leu Asp Lys Gly
Ile Glu65 70 75 80Thr Ile Ile Ser Ser Pro Tyr Arg Ile Arg Phe Ile
Ala Glu Gly His 85 90 95Pro Leu Ser Leu Lys Phe Asp Ser Phe Ala Val
Ile Met Leu Cys Val 100 105 110Gly Ile Pro Thr Glu Trp Ser Val Val
Glu Asp Leu Pro Glu Gly Pro 115 120 125Ala Val Lys Ile Gly Glu Asn
Lys Asp Ala Met Asp Gly Trp Phe Arg 130 135 140Leu Glu Arg Val Ser
Asp Asp Glu Phe Asn Asn Tyr Lys Leu Val Phe145 150 155 160Cys Pro
Gln Gln Ala Glu Asp Asp Lys Cys Gly Asp Ile Gly Ile Ser 165 170
175Ile Asp His Asp Asp Gly Thr Arg Arg Leu Val Val Ser Lys Asn Lys
180 185 190Pro Leu Val Val Gln Phe Gln Lys Leu Asp Lys Glu Ser Leu
Ala Lys 195 200 205Lys Asn His Gly Leu Ser Arg Ser Glu 210
21560312PRTGlycine max 603Gly Gly Ile Arg Ala Thr Pro Thr Glu Asn
Glu Arg1 5 1060412PRTGlycine max 604Gly Gly Ile Arg Val Ala Ala Thr
Gly Lys Glu Arg1 5 1060512PRTGlycine max 605Arg Glu Asn Glu Thr Pro
Thr Ala Arg Ile Gly Gly1 5 1060612PRTGlycine max 606Arg Glu Lys Gly
Thr Ala Ala Val Arg Ile Gly Gly1 5 1060718PRTArabidopsis lyrata
607Ala Arg Gly Lys Phe Glu Arg Lys Lys Pro His Val Asn Ile Gly Thr1
5 10 15Ile Gly60826PRTArabidopsis lyrata 608Ala Arg Gly Lys Phe Glu
Arg Lys Lys Pro His Val Asn Ile Gly Thr1 5 10 15Ile Gly His Val Asp
His Gly Lys Thr Thr 20 2560950PRTArabidopsis lyrata 609Glu Lys Pro
Asn Val Lys Arg Gly Glu Asn Lys Trp Val Asp Lys Ile1 5 10 15Tyr Glu
Leu Met Asp Ser Val Asp Ser Tyr Ile Pro Ile Pro Thr Arg 20 25 30Gln
Thr Glu Leu Pro Phe Leu Leu Ala Val Glu Asp Val Phe Ser Ile 35 40
45Thr Gly 5061018PRTEuglena gracilis 610Ala Arg Gln Lys Phe Glu Arg
Thr Lys Pro His Ile Asn Ile Gly Thr1 5 10 15Ile Gly61126PRTEuglena
gracilis 611Ala Arg Gln Lys Phe Glu Arg Thr Lys Pro His Ile Asn Ile
Gly Thr1 5 10 15Ile Gly His Val Asp His Gly Lys Thr Thr 20
2561250PRTEuglena gracilis 612Lys Asn Pro Lys Ile Thr Lys Gly Glu
Asn Lys Trp Val Asp Lys Ile1 5 10 15Leu Asn Leu Met Asp Gln Val Asp
Ser Tyr Ile Pro Thr Pro Thr Arg 20 25 30Asp Thr Glu Lys Asp Phe Leu
Met Ala Ile Glu Asp Val Leu Ser Ile 35 40 45Thr Gly
5061318PRTAcidovorax avenae 613Ala Lys Gly Lys Phe Glu Arg Thr Lys
Pro His Val Asn Val Gly Thr1 5 10 15Ile Gly61426PRTAcidovorax
avenae 614Ala Lys Gly Lys Phe Glu Arg Thr Lys Pro His Val Asn Val
Gly Thr1 5 10 15Ile Gly His Val Asp His Gly Lys Thr Thr 20
2561550PRTAcidovorax spp. 615Lys Leu Ala Leu Glu Gly Asp Lys Gly
Pro Leu Gly Glu Gln Ala Ile1 5 10 15Asp Lys Leu Ala Glu Ala Leu Asp
Thr Tyr Ile Pro Thr Pro Glu Arg 20 25 30Ala Val Asp Gly Ala Phe Leu
Met Pro Val Glu Asp Val Phe Ser Ile 35 40 45Ser Gly
5061618PRTBacillus cereus 616Ala Lys Ala Lys Phe Glu Arg Ser Lys
Pro His Val Asn Ile Gly Thr1 5 10 15Ile Gly61726PRTBacillus cereus
617Ala Lys Ala Lys Phe Glu Arg Ser Lys Pro His Val Asn Ile Gly Thr1
5 10 15Ile Gly His Val Asp His Gly Lys Thr Thr 20
2561850PRTBacillus cereus 618Ser Ala Leu Lys Ala Leu Gln Gly Glu
Ala Glu Trp Glu Glu Lys Ile1 5 10 15Ile Glu Leu Met Ala Glu Val Asp
Ala Tyr Ile Pro Thr Pro Glu Arg 20 25 30Glu Thr Asp Lys Pro Phe Leu
Met Pro Ile Glu Asp Val Phe Ser Ile 35 40 45Thr Gly
5061926PRTBurkholderia spp. 619Ala Lys Gly Lys Phe Glu Arg Thr Lys
Pro His Val Asn Val Gly Thr1 5 10 15Ile Gly His Val Asp His Gly Lys
Thr Thr 20 2562050PRTBurkholderia spp. 620Lys Leu Ala Leu Glu Gly
Asp Thr Gly Glu Leu Gly Glu Val Ala Ile1 5 10 15Met Asn Leu Ala Asp
Ala Leu Asp Thr Tyr Ile Pro Thr Pro Glu Arg 20 25 30Ala Val Asp Gly
Ala Phe Leu Met Pro Val Glu Asp Val Phe Ser Ile 35 40 45Ser Gly
5062150PRTXanthomonas campestris 621Arg Leu Ala Leu Asp Gly Asp Gln
Ser Glu Ile Gly Val Pro Ala Ile1 5 10 15Leu Lys Leu Val Asp Ala Leu
Asp Thr Phe Ile Pro Glu Pro Thr Arg 20 25 30Asp Val Asp Arg Pro Phe
Leu Met Pro Val Glu Asp Val Phe Ser Ile 35 40 45Ser Gly
5062226PRTPseudomonas spp. 622Ala Lys Glu Lys Phe Glu Arg Ser Lys
Pro His Val Asn Val Gly Thr1 5 10 15Ile Gly His Val Asp His Gly Lys
Thr Thr 20 2562350PRTPseudomonas spp. 623Met Ala Leu Glu Gly Lys
Asp Asp Asn Glu Met Gly Thr Thr Ala Val1 5 10 15Lys Lys Leu Val Glu
Thr Leu Asp Ser Tyr Ile Pro Glu Pro Glu Arg 20 25 30Ala Ile Asp Lys
Pro Phe Leu Met Pro Ile Glu Asp Val Phe Ser Ile 35 40 45Ser Gly
5062418PRTArabidopsis lyrata 624Gly Ile Thr Gly Ile Asn Val His Pro
Lys Lys Arg Glu Phe Lys Gly1 5 10 15Arg Ala62526PRTArabidopsis
lyrata 625Thr Thr Lys Gly His Asp Val His Gly Ile Thr Gly Ile Asn
Val His1 5 10 15Pro Lys Lys Arg Glu Phe Lys Gly Arg Ala 20
2562650PRTArabidopsis lyrata 626Gly Thr Ile Ser Phe Val Asp Glu Val
Ala Leu Leu Phe Pro Leu Glu1 5 10 15Thr Gln Arg Thr Pro Ile Pro Ile
Tyr Ser Asp Val Ser Asp Met Leu 20 25 30Glu
Tyr Ile Lys Asp Val Trp Lys Asn Glu Gly Arg Lys Val Asn Pro 35 40
45Lys Glu 5062718PRTEuglena gracilis 627Gly Ile Thr Gly Ile Asn Ile
His Pro Lys Thr Arg Glu Phe Lys Gln1 5 10 15Arg Ala62826PRTEuglena
gracilis 628Thr Thr Lys Gly His Asp Val His Gly Ile Thr Gly Ile Asn
Ile His1 5 10 15Pro Lys Thr Arg Glu Phe Lys Gln Arg Ala 20
2562950PRTEuglena gracilis 629Gly Thr Ile Ser Leu Val Asp Glu Ile
Ala Met Leu Phe Asp Lys Glu1 5 10 15Thr Asp Arg Thr Pro Thr Pro Ile
Tyr Ser Asp Val Gln Asp Met Leu 20 25 30Asn Leu Ile Lys Asp Val Trp
Lys Asn Glu Gly Lys Thr Ile Lys Pro 35 40 45Asn Lys
5063018PRTAcidovorax avenae 630Gly Ile Thr Gly Val Asn Val His Pro
Lys Thr Arg Glu Phe Lys Gly1 5 10 15Lys Ala63126PRTAcidovorax
avenae 631Thr Thr Lys Gly His Asp Val His Gly Ile Thr Gly Val Asn
Val His1 5 10 15Pro Lys Thr Arg Glu Phe Lys Gly Lys Ala 20
2563250PRTAcidovorax spp. 632Gly Ser Ile Ser Phe Val Asp Glu Val
Pro Met Leu Phe Ala Gly Asp1 5 10 15Val Ala Arg Glu Pro Thr Pro Ile
Tyr Thr Asp Leu Ala Glu Ala Leu 20 25 30Lys Asp Ile Ala Gln Glu Gly
Leu Pro Gly Lys Asp Gly Glu Leu Ala 35 40 45Leu Lys
5063318PRTBacillus cereus 633Gly Ile Thr Gly Ile Asn Val His Pro
Lys Ser Arg Glu Phe Lys Ala1 5 10 15Lys Ala63426PRTBacillus cereus
634Thr Thr Lys Gly His Asp Val His Gly Ile Thr Gly Ile Asn Val His1
5 10 15Pro Lys Ser Arg Glu Phe Lys Ala Lys Ala 20
2563550PRTBacillus cereus 635Gly Ile Thr Ser Phe Val Asp Glu Ile
Pro Met Leu Phe Pro Lys Asp1 5 10 15Thr Glu Arg Glu Pro Thr Pro Ile
Tyr Ala Asp Val Glu Ala Met Leu 20 25 30Glu Ile Ile Lys Glu Glu Trp
Glu Ala Glu Gly Gln Leu Ala Lys Leu 35 40 45Ala Ser
5063626PRTBurkholderia spp. 636Thr Thr Lys Gly His Asp Val His Gly
Ile Thr Gly Val Asn Val His1 5 10 15Pro Lys Thr Arg Glu Phe Lys Gly
Lys Ala 20 2563750PRTBurkholderia spp. 637Gly Ser Ile Ser Phe Val
Asp Glu Val Pro Met Leu Phe Ala Gly Asp1 5 10 15Val Ala Arg Glu Pro
Thr Pro Ile Tyr Thr Asp Leu Ala Asp Ala Leu 20 25 30Asn Met Ile Ala
Val Glu Gly Leu Glu Gly Thr Asp Gly Glu Leu Ala 35 40 45Leu Lys
5063850PRTXanthomonas campestris 638Gly Ser Ile Ser Phe Val Asp Glu
Val Pro Met Leu Phe Pro Arg Asp1 5 10 15Val Asp Arg Thr Pro Glu Pro
Ile Phe Thr Asp Leu Ala Asp Val Leu 20 25 30Lys Leu Ile Ala Pro Val
Gly Ile Glu Ser Gln Asp Gly Asp Leu Ala 35 40 45Leu Arg
5063926PRTPseudomonas spp. 639Thr Thr Lys Gly His Asp Val His Gly
Ile Thr Gly Val Asn Val His1 5 10 15Pro Lys Ser Arg Glu Phe Lys Glu
Lys Ala 20 2564050PRTPseudomonas spp. 640Gly Ser Ile Ser Phe Val
Asp Glu Ile Pro Met Leu Phe Pro Lys Asp1 5 10 15Ile Ala Arg Glu Pro
Glu Pro Ile Tyr Ser Asp Leu Thr Glu Val Leu 20 25 30Lys Lys Val Ala
Thr Thr Gly Met Glu Asn Asp Asp Lys Gly Glu Leu 35 40 45Ala Met
5064154PRTArtificial SequenceSynthetic construct 641Met Ser Thr Ala
Thr Phe Val Asp Ile Ile Ile Ala Ile Leu Leu Pro1 5 10 15Pro Leu Gly
Val Phe Leu Arg Phe Gly Cys Gly Val Glu Phe Trp Ile 20 25 30Cys Leu
Val Leu Thr Leu Leu Gly Tyr Ile Pro Gly Ile Ile Tyr Ala 35 40 45Ile
Tyr Val Leu Thr Lys 5064254PRTCitrus clementina 642Met Gly Ser Glu
Thr Phe Leu Glu Val Ile Leu Ala Ile Leu Leu Pro1 5 10 15Pro Val Gly
Val Phe Leu Arg Tyr Gly Cys Gly Val Glu Phe Trp Ile 20 25 30Cys Leu
Leu Leu Thr Val Leu Gly Tyr Ile Pro Gly Ile Ile Tyr Ala 35 40 45Ile
Tyr Val Leu Val Gly 5064354PRTCitrus trifoliata 643Met Gly Thr Ala
Thr Cys Val Asp Ile Ile Leu Ala Val Ile Leu Pro1 5 10 15Pro Leu Gly
Val Phe Leu Lys Phe Gly Cys Lys Ala Glu Phe Trp Ile 20 25 30Cys Leu
Leu Leu Thr Ile Leu Gly Tyr Ile Pro Gly Ile Ile Tyr Ala 35 40 45Val
Tyr Val Ile Thr Lys 5064458PRTCitrus sinensis 644Met Ala Asp Glu
Gly Thr Ala Thr Cys Ile Asp Ile Ile Leu Ala Ile1 5 10 15Ile Leu Pro
Pro Leu Gly Val Phe Leu Lys Phe Gly Cys Lys Val Glu 20 25 30Phe Trp
Ile Cys Leu Leu Leu Thr Ile Phe Gly Tyr Ile Pro Gly Ile 35 40 45Ile
Tyr Ala Val Tyr Ala Ile Thr Lys Asn 50 5564558PRTCitrus sinensis
645Met Ala Asp Gly Ser Thr Ala Thr Cys Val Asp Ile Leu Leu Ala Val1
5 10 15Ile Leu Pro Pro Leu Gly Val Phe Leu Lys Phe Gly Cys Lys Ala
Glu 20 25 30Phe Trp Ile Cys Leu Leu Leu Thr Ile Leu Gly Tyr Ile Pro
Gly Ile 35 40 45Ile Tyr Ala Val Tyr Ala Ile Thr Lys Lys 50
55646104PRTCitrus clementina 646Phe Tyr Lys Gln Lys Tyr Gln Val Gln
Ile Thr Lys Ala Val Thr Gln1 5 10 15Asn Pro Lys His Phe Phe Asn Gln
Ser Ser Cys Phe Leu Thr Leu Asn 20 25 30Phe Ile Leu Phe His Phe Thr
Leu Phe Lys Asn Gln Ser Lys Met Ala 35 40 45Asp Gly Ser Thr Ala Thr
Cys Val Asp Ile Leu Leu Ala Val Ile Leu 50 55 60Pro Pro Leu Gly Val
Phe Leu Lys Phe Gly Cys Lys Ala Glu Phe Trp65 70 75 80Ile Cys Leu
Leu Leu Thr Ile Leu Gly Tyr Ile Pro Gly Ile Ile Tyr 85 90 95Ala Val
Tyr Ala Ile Thr Lys Lys 10064754PRTArabidopsis thaliana 647Met Ser
Thr Ala Thr Phe Val Asp Ile Ile Ile Ala Ile Leu Leu Pro1 5 10 15Pro
Leu Gly Val Phe Leu Arg Phe Gly Cys Gly Val Glu Phe Trp Ile 20 25
30Cys Leu Val Leu Thr Leu Leu Gly Tyr Ile Pro Gly Ile Ile Tyr Ala
35 40 45Ile Tyr Val Leu Thr Lys 5064854PRTCamelina sativa 648Met
Ser Thr Ala Thr Phe Val Asp Ile Ile Ile Ala Val Leu Leu Pro1 5 10
15Pro Leu Gly Val Phe Leu Arg Phe Gly Cys Gly Val Glu Phe Trp Ile
20 25 30Cys Leu Val Leu Thr Leu Leu Gly Tyr Ile Pro Gly Ile Ile Tyr
Ala 35 40 45Ile Tyr Val Leu Thr Lys 5064954PRTArabidopsis lyrata
649Met Gly Thr Ala Thr Cys Val Asp Ile Ile Ile Ala Ile Leu Leu Pro1
5 10 15Pro Leu Gly Val Phe Leu Arg Phe Gly Cys Gly Val Glu Phe Trp
Ile 20 25 30Cys Leu Val Leu Thr Leu Leu Gly Tyr Ile Pro Gly Ile Leu
Tyr Ala 35 40 45Leu Tyr Val Leu Thr Lys 5065052PRTArtificial
SequenceSynthetic construct 650Arg Thr Cys Glu Ser Gln Ser His Arg
Phe Lys Gly Pro Cys Ser Arg1 5 10 15Asp Ser Asn Cys Ala Thr Val Cys
Leu Thr Glu Gly Phe Ser Gly Gly 20 25 30Asp Cys Arg Gly Phe Arg Arg
Arg Cys Arg Cys Thr Arg Pro Cys Val 35 40 45Phe Asp Glu Lys
5065147PRTCitrus sinensis 651Arg Val Cys Gln Ser Gln Ser His His
Phe His Gly Ala Cys Phe Ser1 5 10 15His His Asn Cys Ala Phe Val Cys
Arg Asn Glu Gly Phe Ser Gly Gly 20 25 30Lys Cys Arg Gly Val Arg Arg
Arg Cys Phe Cys Ser Lys Leu Cys 35 40 4565246PRTAvena sativa 652Lys
Ser Cys Cys Lys Asp Ile Met Ala Arg Asn Cys Tyr Asn Val Cys1 5 10
15Arg Ile Pro Gly Thr Pro Arg Pro Val Cys Ala Thr Thr Cys Arg Cys
20 25 30Lys Ile Ile Ser Gly Asn Lys Cys Pro Lys Asp Tyr Pro Lys 35
40 4565352PRTArtificial SequenceSynthetic construct 653Arg Thr Cys
Glu Ser Gln Ser His Arg Phe Lys Gly Pro Cys Ser Arg1 5 10 15Asp Ser
Asn Cys Ala Thr Val Cys Leu Thr Glu Gly Phe Ser Gly Gly 20 25 30Asp
Cys Arg Gly Phe Arg Arg Arg Cys Arg Cys Thr Arg Pro Cys Val 35 40
45Phe Asp Glu Lys 5065475PRTCitrus sinensis 654Met Asp Ser Arg Ser
Phe Gly Leu Leu Pro Leu Leu Leu Leu Ile Leu1 5 10 15Leu Thr Ser Gln
Met Thr Val Leu Gln Thr Glu Ala Arg Leu Cys Glu 20 25 30Ser Gln Ser
His Arg Phe His Gly Thr Cys Val Arg Ser His Asn Cys 35 40 45Asp Leu
Val Cys Arg Thr Glu Gly Phe Thr Gly Gly Arg Cys Arg Gly 50 55 60Phe
Arg Arg Arg Cys Phe Cys Thr Arg Ile Cys65 70 7565573PRTCitrus
paradise 655Met Lys Ser Phe Phe Gly Ile Phe Leu Leu Leu Leu Ile Leu
Phe Ala1 5 10 15Ser Gln Glu Ile Met Val Pro Ala Glu Gly Arg Val Cys
Gln Ser Gln 20 25 30Ser His His Phe His Gly Ala Cys Phe Ser His His
Asn Cys Ala Phe 35 40 45Val Cys Arg Asn Glu Gly Phe Ser Gly Gly Lys
Cys Arg Gly Val Arg 50 55 60Arg Arg Cys Phe Cys Ser Lys Leu Cys65
7065672PRTCitrus clementina 656Met Lys Ser Phe Phe Gly Ile Phe Leu
Leu Leu Leu Ile Leu Phe Ala1 5 10 15Ser Gln Met Met Val Pro Ala Glu
Gly Arg Val Cys Gln Ser Gln Ser 20 25 30His His Phe His Gly Ala Cys
Phe Ser His His Asn Cys Ala Phe Val 35 40 45Cys Arg Asn Glu Gly Phe
Ser Gly Gly Lys Cys Arg Gly Ala Arg Arg 50 55 60Arg Cys Phe Cys Ser
Lys Leu Cys65 7065773PRTCitrus clementina 657Met Lys Ser Phe Phe
Gly Ile Phe Leu Leu Leu Leu Ile Leu Phe Ala1 5 10 15Ser Gln Glu Met
Met Val Pro Ala Glu Gly Arg Val Cys Gln Ser Gln 20 25 30Ser His His
Phe His Gly Ala Cys Phe Ser His His Asn Cys Ala Phe 35 40 45Val Cys
Arg Asn Glu Gly Phe Ser Gly Gly Lys Cys Arg Gly Ala Arg 50 55 60Arg
Arg Cys Phe Cys Ser Lys Leu Cys65 7065872PRTCitrus clementina
658Met Lys Ser Phe Phe Gly Ile Phe Leu Leu Leu Leu Ile Leu Phe Ala1
5 10 15Ser Gln Met Met Val Pro Ala Glu Gly Arg Val Cys Gln Ser Gln
Ser 20 25 30His His Phe His Gly Ala Cys Phe Ser His His Asn Cys Ala
Phe Val 35 40 45Cys Arg Asn Glu Gly Phe Ser Gly Gly Lys Cys Arg Gly
Ala Arg Arg 50 55 60Arg Cys Phe Cys Ser Lys Leu Cys65
7065978PRTNicotiana benthamiana 659Met Ala Asn Ser Met Arg Phe Phe
Ala Thr Val Leu Leu Leu Ala Leu1 5 10 15Leu Val Met Ala Thr Glu Met
Gly Pro Met Thr Ile Ala Glu Ala Arg 20 25 30Thr Cys Glu Ser Gln Ser
His Arg Phe Lys Gly Pro Cys Ser Arg Asp 35 40 45Ser Asn Cys Ala Thr
Val Cys Leu Thr Glu Gly Phe Ser Gly Gly Asp 50 55 60Cys Arg Gly Phe
Arg Arg Arg Cys Phe Cys Thr Arg Pro Cys65 70 7566078PRTNicotiana
sylvestris 660Met Ala Lys Ser Met Arg Phe Phe Ala Thr Val Leu Leu
Leu Ala Leu1 5 10 15Leu Val Met Ala Thr Glu Met Gly Pro Thr Thr Ile
Ala Glu Ala Arg 20 25 30Thr Cys Glu Ser Gln Ser His Arg Phe Lys Gly
Pro Cys Ser Arg Asp 35 40 45Ser Asn Cys Ala Thr Val Cys Leu Thr Glu
Gly Phe Ser Gly Gly Asp 50 55 60Cys Arg Gly Phe Arg Arg Arg Cys Phe
Cys Thr Arg Pro Cys65 70 7566178PRTNicotiana tabacum 661Met Ala Asn
Ser Met Arg Phe Phe Ala Thr Val Leu Leu Leu Thr Leu1 5 10 15Leu Val
Met Ala Thr Glu Met Gly Pro Met Thr Ile Ala Glu Ala Arg 20 25 30Thr
Cys Glu Ser Gln Ser His Arg Phe Lys Gly Pro Cys Ser Arg Asp 35 40
45Ser Asn Cys Ala Thr Val Cys Leu Thr Glu Gly Phe Ser Gly Gly Asp
50 55 60Cys Arg Gly Phe Arg Arg Arg Cys Phe Cys Thr Arg Pro Cys65
70 7566278PRTNicotiana tomentosiformis 662Met Ala Asn Ser Met Arg
Phe Phe Ala Thr Val Leu Leu Ile Ala Leu1 5 10 15Leu Val Met Ala Thr
Glu Met Gly Pro Met Thr Ile Ala Glu Ala Arg 20 25 30Thr Cys Glu Ser
Gln Ser His Arg Phe Lys Gly Pro Cys Ser Arg Asp 35 40 45Ser Asn Cys
Ala Thr Val Cys Leu Thr Glu Gly Phe Ser Gly Gly Asp 50 55 60Cys Arg
Gly Phe Arg Arg Arg Cys Phe Cys Thr Arg Pro Cys65 70
7566378PRTNicotiana tabacum 663Met Ala Asn Ser Met Arg Phe Phe Ala
Thr Val Leu Leu Ile Ala Leu1 5 10 15Leu Val Thr Ala Thr Glu Met Gly
Pro Met Thr Ile Ala Glu Ala Arg 20 25 30Thr Cys Glu Ser Gln Ser His
Arg Phe Lys Gly Pro Cys Ser Arg Asp 35 40 45Ser Asn Cys Ala Thr Val
Cys Leu Thr Glu Gly Phe Ser Gly Gly Asp 50 55 60Cys Arg Gly Phe Arg
Arg Arg Cys Phe Cys Thr Arg Pro Cys65 70 7566478PRTNicotiana alata
664Met Ala Asn Ser Met Arg Phe Phe Ala Thr Val Leu Leu Leu Thr Leu1
5 10 15Leu Phe Met Ala Thr Glu Met Gly Pro Met Thr Ile Ala Glu Ala
Arg 20 25 30Thr Cys Glu Ser Gln Ser His Arg Phe Lys Gly Pro Cys Ala
Arg Asp 35 40 45Ser Asn Cys Ala Thr Val Cys Leu Thr Glu Gly Phe Ser
Gly Gly Asp 50 55 60Cys Arg Gly Phe Arg Arg Arg Cys Phe Cys Thr Arg
Pro Cys65 70 7566580PRTAvena sativa 665Met Gly Ser Ile Lys Gly Leu
Lys Ser Val Val Ile Cys Val Leu Val1 5 10 15Leu Gly Ile Val Leu Glu
Gln Val Gln Val Glu Gly Lys Ser Cys Cys 20 25 30Lys Asp Ile Met Ala
Arg Asn Cys Tyr Asn Val Cys Arg Ile Pro Gly 35 40 45Thr Pro Arg Pro
Val Cys Ala Thr Thr Cys Arg Cys Lys Ile Ile Ser 50 55 60Gly Asn Lys
Cys Pro Lys Asp Tyr Pro Lys Leu His Gly Asp Pro Asp65 70 75
8066680PRTAvena sativa 666Met Gly Ser Ile Lys Gly Leu Lys Ser Val
Val Ile Cys Val Leu Val1 5 10 15Leu Gly Ile Val Leu Glu His Val Gln
Val Glu Gly Lys Ser Cys Cys 20 25 30Lys Asp Thr Thr Ala Arg Asn Cys
Tyr Asn Val Cys Arg Ile Pro Gly 35 40 45Thr Pro Arg Pro Val Cys Ala
Thr Thr Cys Arg Cys Lys Ile Ile Ser 50 55 60Gly Asn Lys Cys Pro Lys
Asp Tyr Pro Lys Leu His Gly Asp Leu Asp65 70 75 80667112PRTTulipa
gesneriana 667Leu Gly Leu Val Val Ala Gln Thr Gln Val Asp Ala Lys
Ser Cys Cys1 5 10 15Pro Ser Thr Ala Ala Arg Asn Cys Tyr Asn Val Cys
Arg Phe Pro Gly 20 25 30Thr Pro Arg Pro Val Cys Ala Ala Thr Cys Gly
Cys Lys Ile Ile Thr 35 40 45Gly Thr Lys Cys Pro Pro Asp Tyr Pro Lys
Leu Gly Trp Ser Thr Phe 50 55 60Gln Asn Ser Asp Val Ala Asp Lys Ala
Leu Asp Val Val Asp Glu Ala65 70 75 80Leu His Val Ala Lys Glu Val
Met Lys Glu Ala Val Glu Arg Cys Asn 85 90 95Asn Ala Cys Ser Glu Val
Cys Thr Lys Gly Ser Tyr Ala Val Thr Ala 100 105 11066875PRTVitis
vinifera 668Met Glu Arg Lys Ser Leu Gly Phe Phe Phe Phe Leu Leu Leu
Ile Leu1
5 10 15Leu Ala Ser Gln Glu Met Val Val Pro Ser Glu Ala Arg Val Cys
Glu 20 25 30Ser Gln Ser His Lys Phe Glu Gly Ala Cys Met Gly Asp His
Asn Cys 35 40 45Ala Leu Val Cys Arg Asn Glu Gly Phe Ser Gly Gly Lys
Cys Lys Gly 50 55 60Leu Arg Arg Arg Cys Phe Cys Thr Lys Leu Cys65
70 7566974PRTVitis vinifera 669Met Glu Arg Lys Ser Leu Gly Phe Phe
Phe Phe Leu Leu Leu Ile Leu1 5 10 15Leu Ala Ser Gln Met Val Val Pro
Ser Glu Ala Arg Val Cys Glu Ser 20 25 30Gln Ser His Lys Phe Glu Gly
Ala Cys Met Gly Asp His Asn Cys Ala 35 40 45Leu Val Cys Arg Asn Glu
Gly Phe Ser Gly Gly Lys Cys Lys Gly Leu 50 55 60Arg Arg Arg Cys Phe
Cys Thr Lys Leu Cys65 7067076PRTCitrus sinensis 670Met Glu Arg Ser
Val Arg Leu Phe Ser Thr Val Leu Leu Val Leu Leu1 5 10 15Leu Leu Ala
Ser Glu Met Gly Leu Arg Ala Ala Glu Ala Arg Ile Cys 20 25 30Glu Ser
Gln Ser His Arg Phe Lys Gly Pro Cys Val Ser Lys Ser Asn 35 40 45Cys
Ala Ala Val Cys Gln Thr Glu Gly Phe His Gly Gly His Cys Arg 50 55
60Gly Phe Arg Arg Arg Cys Phe Cys Thr Lys Arg Cys65 70
7567150PRTAesculus hippocastanum 671Leu Cys Asn Glu Arg Pro Ser Gln
Thr Trp Ser Gly Asn Cys Gly Asn1 5 10 15Thr Ala His Cys Asp Lys Gln
Cys Gln Asp Trp Glu Lys Ala Ser His 20 25 30Gly Ala Cys His Lys Arg
Glu Asn His Trp Lys Cys Phe Cys Tyr Phe 35 40 45Asn Cys
5067281PRTDacus carota 672Met Ala Lys Asn Ser Thr Ser Pro Val Ser
Leu Phe Ala Ile Ser Leu1 5 10 15Ile Phe Phe Leu Leu Ala Asn Ser Gly
Ser Ile Thr Glu Val Asp Gly 20 25 30Lys Val Cys Glu Lys Pro Ser Leu
Thr Trp Ser Gly Lys Cys Gly Asn 35 40 45Thr Gln His Cys Asp Lys Gln
Cys Gln Asp Trp Glu Gly Ala Lys His 50 55 60Gly Ala Cys His Ser Arg
Gly Gly Trp Lys Cys Phe Cys Tyr Phe Glu65 70 75
80Cys67349PRTClitoria ternatea 673Asn Leu Cys Glu Arg Ala Ser Leu
Thr Trp Thr Gly Asn Cys Gly Asn1 5 10 15Thr Gly His Cys Asp Thr Gln
Cys Arg Asn Trp Glu Ser Ala Lys His 20 25 30Gly Ala Cys His Lys Arg
Gly Asn Trp Lys Cys Phe Cys Tyr Phe Asn 35 40 45Cys67480PRTDacus
carota 674Met Ala Lys Lys Ser Ser Ser Phe Cys Leu Ser Ala Ile Phe
Leu Val1 5 10 15Leu Leu Leu Val Ala Asn Thr Gly Met Val Arg Glu Val
Asp Gly Ala 20 25 30Leu Cys Glu Lys Pro Ser Leu Thr Trp Ser Gly Asn
Cys Arg Asn Thr 35 40 45Gln His Cys Asp Lys Gln Cys Gln Ser Trp Glu
Gly Ala Lys His Gly 50 55 60Ala Cys His Lys Arg Gly Asn Trp Lys Cys
Phe Cys Tyr His Ala Cys65 70 75 8067592PRTBupleurum kaoi 675Met Ala
Lys Lys Leu Asn Ala Val Thr Val Ser Ala Ile Phe Leu Val1 5 10 15Val
Phe Leu Ile Ala Ser Tyr Ser Val Gly Ala Ala Lys Glu Ala Gly 20 25
30Ala Glu Gly Glu Val Val Phe Pro Glu Gln Leu Cys Glu Arg Ala Ser
35 40 45Gln Thr Trp Ser Gly Asp Cys Lys Asn Thr Lys Asn Cys Asp Asn
Gln 50 55 60Cys Ile Gln Trp Glu Lys Ala Arg His Gly Ala Cys His Lys
Arg Gly65 70 75 80Gly Lys Trp Met Cys Phe Cys Tyr Phe Asp Lys Cys
85 9067650PRTDahlia merckii 676Glu Leu Cys Glu Lys Ala Ser Lys Thr
Trp Ser Gly Asn Cys Gly Asn1 5 10 15Thr Gly His Cys Asp Asn Gln Cys
Lys Ser Trp Glu Gly Ala Ala His 20 25 30Gly Ala Cys His Val Arg Asn
Gly Lys His Met Cys Phe Cys Tyr Phe 35 40 45Asn Cys
50677108PRTHelianthus annuus 677Met Ala Lys Ile Ser Val Ala Phe Asn
Ala Phe Leu Leu Leu Leu Phe1 5 10 15Val Leu Ala Ile Ser Glu Ile Gly
Ser Val Lys Gly Glu Leu Cys Glu 20 25 30Lys Ala Ser Gln Thr Trp Ser
Gly Thr Cys Gly Lys Thr Lys His Cys 35 40 45Asp Asp Gln Cys Lys Ser
Trp Glu Gly Ala Ala His Gly Ala Cys His 50 55 60Val Arg Asp Gly Lys
His Met Cys Phe Cys Tyr Phe Asn Cys Ser Lys65 70 75 80Ala Gln Lys
Leu Ala Gln Asp Lys Leu Arg Ala Glu Glu Leu Ala Lys 85 90 95Glu Lys
Ile Glu Pro Glu Lys Ala Thr Ala Lys Pro 100 105678117PRTCynara
cardunculus 678Met Ala Lys Asn Ser Val Ala Phe Phe Ala Leu Leu Leu
Leu Ile Cys1 5 10 15Ile Leu Thr Ile Ser Glu Phe Ala Val Val Lys Gly
Glu Leu Cys Glu 20 25 30Lys Ala Ser Lys Thr Trp Ser Gly Asn Cys Gly
Asn Thr Arg His Cys 35 40 45Asp Asp Gln Cys Lys Ala Trp Glu Gly Ala
Ala His Gly Ala Cys His 50 55 60Thr Arg Asn Lys Lys His Met Cys Phe
Cys Tyr Phe Asn Cys Pro Lys65 70 75 80Ala Glu Lys Leu Ala Gln Asp
Lys Leu Lys Ala Glu Glu Leu Ala Arg 85 90 95Asp Lys Val Glu Ala Lys
Glu Val Pro His Phe Lys His Pro Ile Glu 100 105 110Pro Ile His His
Pro 115679117PRTCynara cardunculus 679Met Ala Lys Gln Trp Val Ser
Phe Phe Ala Leu Ala Phe Ile Val Phe1 5 10 15Val Leu Ala Ile Ser Glu
Thr Gln Thr Val Lys Gly Glu Leu Cys Glu 20 25 30Lys Ala Ser Lys Thr
Trp Ser Gly Asn Cys Gly Asn Thr Lys His Cys 35 40 45Asp Asp Gln Cys
Lys Ser Trp Glu Gly Ala Ala His Gly Ala Cys His 50 55 60Val Arg Asn
Gly Lys His Met Cys Phe Cys Tyr Phe Asn Ser Cys Ala65 70 75 80Glu
Ala Asp Lys Leu Ser Glu Asp Gln Ile Glu Ala Gly Lys Leu Ala 85 90
95Phe Glu Lys Ala Glu Lys Leu Asp Arg Asp Val Lys Lys Ala Val Pro
100 105 110Asn Val Asp His Pro 11568094PRTDaucus carota 680Met Ala
Gln Lys Val Asn Ser Ala Leu Ile Phe Ser Ala Ile Phe Val1 5 10 15Leu
Phe Leu Val Ala Ser Tyr Ser Val Thr Val Ala Glu Gly Ala Arg 20 25
30Ala Gly Ala Glu Gly Glu Val Val Tyr Pro Glu Ala Leu Cys Glu Arg
35 40 45Ala Ser Gln Thr Trp Thr Gly Lys Cys Gln His Thr Asp His Cys
Asp 50 55 60Asn Gln Cys Ile Gln Trp Glu Asn Ala Arg His Gly Ala Cys
His Lys65 70 75 80Arg Gly Gly Asn Trp Lys Cys Phe Cys Tyr Phe Asp
His Cys 85 9068178PRTArabidopsis lyrata 681Met Ala Ser Ser Tyr Thr
Leu Met Leu Phe Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Ala Ser Thr
Glu Met Met Ala Val Glu Ala Arg Ile Cys Glu 20 25 30Arg Arg Ser Lys
Thr Trp Thr Gly Phe Cys Gly Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln
Cys Lys Ser Trp Glu Arg Ala Ser His Gly Ala Cys His 50 55 60Ala Gln
Phe Pro Gly Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70
7568280PRTParthenium hysterophorus 682Met Ala Lys Ser Ser Thr Ser
Tyr Leu Val Phe Leu Leu Leu Val Leu1 5 10 15Val Val Ala Ile Ser Glu
Ile Ala Ser Val Asn Gly Lys Val Cys Glu 20 25 30Lys Pro Ser Lys Thr
Trp Phe Gly Asn Cys Lys Asp Thr Glu Lys Cys 35 40 45Asp Lys Arg Cys
Met Glu Trp Glu Gly Ala Lys His Gly Ala Cys His 50 55 60Gln Arg Glu
Ser Lys Tyr Met Cys Phe Cys Tyr Phe Asp Cys Asp Pro65 70 75
8068378PRTArabidopsis thaliana 683Met Ala Ser Ser Tyr Thr Leu Met
Leu Phe Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Ala Ser Thr Glu Met
Met Ala Val Glu Gly Arg Ile Cys Glu 20 25 30Arg Arg Ser Lys Thr Trp
Thr Gly Phe Cys Gly Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys Lys
Arg Trp Glu Arg Ala Ser His Gly Ala Cys His 50 55 60Ala Gln Phe Pro
Gly Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7568478PRTEutrema
salsugineum 684Met Ala Ser Ser Tyr Thr Leu Leu Leu Phe Val Cys Leu
Ser Ile Phe1 5 10 15Phe Ile Ala Ser Thr Glu Met Met Met Val Glu Gly
Arg Val Cys Glu 20 25 30Arg Arg Ser Lys Thr Trp Thr Gly Phe Cys Gly
Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys Lys Arg Trp Glu Arg Ala
Ser His Gly Ala Cys His 50 55 60Ala Gln Phe Pro Gly Phe Ala Cys Phe
Cys Tyr Phe Asn Cys65 70 7568582PRTVitis vinifera 685Met Ala Lys
Leu Leu Gly Tyr Leu Leu Ser Tyr Ala Leu Ser Phe Leu1 5 10 15Thr Leu
Phe Ala Leu Leu Val Ser Thr Glu Met Val Met Leu Glu Ala 20 25 30Lys
Val Cys Gln Arg Pro Ser Lys Thr Trp Ser Gly Phe Cys Gly Ser 35 40
45Ser Lys Asn Cys Asp Arg Gln Cys Lys Asn Trp Glu Gly Ala Lys His
50 55 60Gly Ala Cys His Ala Lys Phe Pro Gly Val Ala Cys Phe Cys Tyr
Phe65 70 75 80Asn Cys68682PRTCorchorus olitorius 686Met Ala Lys Ser
Leu Ser Ser Phe Ala Thr Phe Leu Ala Leu Leu Cys1 5 10 15Leu Phe Phe
Leu Leu Ser Thr Pro Asn Glu Met Lys Met Ala Glu Ala 20 25 30Lys Ile
Cys Glu Lys Arg Ser Gln Thr Trp Ser Gly Trp Cys Gly Asn 35 40 45Ser
Ser His Cys Asp Arg Gln Cys Lys Asn Trp Glu Asn Ala Arg His 50 55
60Gly Ser Cys His Ala Asp Gly Leu Gly Trp Ala Cys Phe Cys Tyr Phe65
70 75 80Asn Cys68758PRTCorchorus olitorius 687Met Glu Met Lys Met
Ala Glu Gly Lys Ile Cys Glu Lys Arg Ser Gln1 5 10 15Thr Trp Ser Gly
Trp Cys Gly Asn Ser Ser His Cys Asp Arg Gln Cys 20 25 30Lys Asn Trp
Glu Asn Ala Arg His Gly Ser Cys His Ala Asp Gly Leu 35 40 45Gly Trp
Ala Cys Phe Cys Tyr Phe Asn Cys 50 5568878PRTCamelina sativa 688Met
Ala Ser Ser Leu Lys Leu Met Leu Phe Leu Cys Leu Ser Ile Phe1 5 10
15Leu Ile Ala Ser Thr Glu Met Met Thr Val Glu Gly Arg Thr Cys Glu
20 25 30Arg Arg Ser Lys Thr Trp Thr Gly Phe Cys Gly Asn Thr Arg Gly
Cys 35 40 45Asp Ser Gln Cys Arg Ser Trp Glu Gly Ala Ser His Gly Ala
Cys His 50 55 60Ala Gln Phe Pro Gly Phe Ala Cys Phe Cys Tyr Phe Asn
Cys65 70 7568984PRTCucumis sativus 689Met Ala Lys Val Val Gly Asn
Ser Ala Lys Met Ile Val Ala Leu Leu1 5 10 15Phe Leu Leu Ala Leu Met
Leu Ser Met Asn Glu Lys Gln Gly Val Val 20 25 30Glu Ala Lys Val Cys
Glu Arg Arg Ser Lys Thr Trp Ser Gly Trp Cys 35 40 45Gly Asn Thr Lys
His Cys Asp Arg Gln Cys Lys Asn Trp Glu Gly Ala 50 55 60Thr His Gly
Ala Cys His Ala Gln Phe Pro Gly Arg Ala Cys Phe Cys65 70 75 80Tyr
Phe Asn Cys69080PRTCynara cardunculus 690Met Ile Asp Ala Phe Asn
Tyr Lys Gln Phe Ser Thr Val Lys Gly Lys1 5 10 15Ile Cys Glu Lys Pro
Ser Lys Thr Trp Phe Gly Lys Cys Gln Asp Thr 20 25 30Thr Lys Cys Asp
Lys Gln Cys Ile Glu Trp Glu Asp Ala Lys His Gly 35 40 45Ala Cys His
Glu Arg Glu Ser Lys Leu Met Cys Phe Cys Tyr Tyr Asn 50 55 60Cys Gly
Pro Pro Lys Asn Thr Pro Pro Gly Thr Pro Pro Ser Pro Pro65 70 75
8069178PRTCapsella rubella 691Met Ala Ser Ser Tyr Lys Leu Ile Leu
Phe Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Ala Ser Phe Glu Met Met
Ala Val Glu Gly Arg Ile Cys Gln 20 25 30Arg Arg Ser Lys Thr Trp Thr
Gly Phe Cys Gly Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys Lys Arg
Trp Glu Arg Ala Ser His Gly Ala Cys His 50 55 60Ala Gln Phe Pro Gly
Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7569256PRTArabidopsis
thaliana 692Met Met Ala Val Glu Gly Arg Ile Cys Glu Arg Arg Ser Lys
Thr Trp1 5 10 15Thr Gly Phe Cys Gly Asn Thr Arg Gly Cys Asp Ser Gln
Cys Lys Arg 20 25 30Trp Glu Arg Ala Ser His Gly Ala Cys His Ala Gln
Phe Pro Gly Phe 35 40 45Ala Cys Phe Cys Tyr Phe Asn Cys 50
5569378PRTBrassica napus 693Met Ala Ser Ser Tyr Thr Arg Leu Leu Leu
Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Ala Ser Thr Glu Val Met Met
Val Glu Gly Arg Val Cys Gln 20 25 30Arg Arg Ser Lys Thr Trp Thr Gly
Phe Cys Gly Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys Lys Arg Trp
Glu Arg Ala Ser His Gly Ala Cys His 50 55 60Ala Gln Phe Pro Gly Phe
Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7569478PRTBrassica rapa 694Met
Ala Ser Ser Tyr Ala Arg Leu Leu Leu Leu Cys Leu Ser Ile Phe1 5 10
15Leu Ile Ala Ser Thr Glu Val Met Met Val Glu Gly Arg Val Cys Gln
20 25 30Arg Arg Ser Lys Thr Trp Thr Gly Phe Cys Gly Asn Thr Arg Gly
Cys 35 40 45Asp Ser Gln Cys Lys Arg Trp Glu Arg Ala Ser His Gly Ala
Cys His 50 55 60Ala Gln Phe Pro Gly Phe Ala Cys Phe Cys Tyr Phe Asn
Cys65 70 7569578PRTCamelina sativa 695Met Ala Ser Ser Leu Lys Leu
Met Leu Phe Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Ala Ser Thr Glu
Met Met Thr Val Glu Gly Arg Thr Cys Glu 20 25 30Arg Arg Ser Lys Thr
Trp Thr Gly Phe Cys Gly Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys
Arg Arg Trp Glu His Ala Ser His Gly Ala Cys His 50 55 60Ala Gln Phe
Pro Gly Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7569677PRTBrassica
napus 696Met Ala Ser Tyr Thr Arg Leu Leu Leu Leu Cys Leu Ser Ile
Phe Leu1 5 10 15Ile Ala Ser Thr Glu Val Met Met Val Glu Gly Arg Val
Cys Gln Arg 20 25 30Arg Ser Lys Thr Trp Thr Gly Phe Cys Gly Asn Thr
Arg Gly Cys Asp 35 40 45Ser Gln Cys Lys Arg Trp Glu Arg Ala Ser His
Gly Ala Cys His Ala 50 55 60Gln Phe Pro Gly Phe Ala Cys Phe Cys Tyr
Phe Asn Cys65 70 7569756PRTVitis vinifera 697Met Val Met Leu Glu
Ala Lys Val Cys Gln Arg Pro Ser Lys Thr Trp1 5 10 15Ser Gly Phe Cys
Gly Ser Ser Lys Asn Cys Asp Arg Gln Cys Lys Asn 20 25 30Trp Glu Gly
Ala Lys His Gly Ala Cys His Ala Lys Phe Pro Gly Val 35 40 45Ala Cys
Phe Cys Tyr Phe Asn Cys 50 5569888PRTBrassica napus 698Met Thr Lys
Ser Phe Ile Leu Val Ala Leu Leu Cys Ile Cys Phe Ile1 5 10 15Leu Leu
Ser Pro Thr Glu Met Arg Leu Thr Leu Asn Ala Cys Leu Lys 20 25 30Leu
Ala Glu Ala Lys Ile Cys Glu Lys Tyr Ser Gln Thr Trp Ser Gly 35 40
45Arg Cys Thr Lys Thr Ser His Cys Asp Arg Gln Cys Ile Asn Trp Glu
50 55 60Asp Ala Arg His Gly Ala Cys His Gln Asp Lys His Gly Arg Ala
Cys65 70
75 80Phe Cys Tyr Phe Asn Cys Lys Lys 8569978PRTRaphanus sativus
699Met Ala Ser Ser Tyr Thr Val Phe Leu Leu Leu Cys Leu Ser Ile Phe1
5 10 15Leu Ile Ala Ser Thr Glu Val Met Met Val Glu Gly Arg Val Cys
Gln 20 25 30Arg Arg Ser Lys Thr Trp Thr Gly Phe Cys Gly Asn Thr Arg
Gly Cys 35 40 45Asp Ser Gln Cys Lys Arg Trp Glu His Ala Ser His Gly
Ala Cys His 50 55 60Ala Gln Phe Pro Gly Phe Ala Cys Phe Cys Tyr Phe
Asn Cys65 70 7570078PRTArabis alpine 700Met Ala Ser Ser Tyr Thr Leu
Leu Leu Phe Leu Cys Leu Ser Ile Phe1 5 10 15Leu Ile Val Ser Thr Glu
Met Met Met Val Glu Gly Arg Ile Cys Glu 20 25 30Arg Arg Ser Lys Thr
Trp Thr Gly Phe Cys Ala Asn Thr Arg Gly Cys 35 40 45Asp Ser Gln Cys
Lys Arg Trp Glu Arg Ala Ser His Gly Ala Cys His 50 55 60Ala Gln Phe
Pro Gly Val Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7570184PRTCucumis
melo 701Met Ala Lys Val Val Gly Asn Ser Ala Lys Met Ile Val Ala Phe
Leu1 5 10 15Phe Leu Leu Ala Leu Thr Leu Ser Met Asn Glu Lys Gln Gly
Val Val 20 25 30Glu Ala Lys Val Cys Glu Arg Arg Ser Lys Thr Trp Ser
Gly Trp Cys 35 40 45Gly Asp Thr Lys His Cys Asp Arg Gln Cys Lys Asn
Trp Glu Gly Ala 50 55 60Lys His Gly Ala Cys His Ala Gln Phe Pro Gly
Arg Ala Cys Phe Cys65 70 75 80Tyr Phe Asn Cys70282PRTErythranthe
guttate 702Met Ala Ala Ser Leu Val Tyr Arg Leu Ser Ser Val Ile Leu
Ile Val1 5 10 15Leu Leu Leu Phe Ile Met Leu Asn Asn Glu Val Met Val
Val Glu Ser 20 25 30Arg Leu Cys Glu Arg Arg Ser Lys Thr Trp Thr Gly
Phe Cys Gly Ser 35 40 45Ser Asn Asn Cys Asn Asn Gln Cys Arg Asn Trp
Glu Arg Ala Ser His 50 55 60Gly Ala Cys His Ala Gln Phe Pro Gly Phe
Ala Cys Phe Cys Tyr Phe65 70 75 80Asn Cys70376PRTSesamum indicum
703Met Ala Lys Phe Gln Val Ser Ser Thr Ile Phe Phe Ala Leu Phe Phe1
5 10 15Cys Phe Leu Leu Leu Ala Ser Asn Glu Ala Lys Ile Cys Gln Arg
Met 20 25 30Ser Lys Thr Trp Ser Gly Val Cys Leu Asn Ser Gly Asn Cys
Asp Arg 35 40 45Gln Cys Arg Asn Trp Glu Arg Ala Gln His Gly Ala Cys
His Arg Arg 50 55 60Gly Leu Gly Phe Ala Cys Leu Cys Tyr Phe Lys
Cys65 70 75704110PRTEclipta prostrata 704Met Ala Lys Asn Ser Val
Ala Phe Phe Ala Phe Leu Leu Ile Leu Phe1 5 10 15Val Leu Ala Ile Ser
Glu Ile Gly Ser Val Lys Gly Glu Leu Cys Glu 20 25 30Lys Ala Ser Gln
Thr Trp Ser Gly Thr Cys Arg Ile Thr Ser His Cys 35 40 45Asp Asn Gln
Cys Lys Ser Trp Glu Gly Ala Ala His Gly Ala Cys His 50 55 60Val Arg
Gly Gly Lys His Met Cys Phe Cys Tyr Phe Ser His Cys Ala65 70 75
80Lys Ala Glu Lys Leu Thr Gln Asp Lys Leu Lys Ala Gly His Leu Val
85 90 95Asn Glu Lys Ser Glu Ala Asp Gln Lys Val Pro Val Thr Pro 100
105 110705105PRTCynara cardunculus 705Met Ala Lys Asn Thr Lys Val
Ser Ala Phe Leu Phe Val Phe Leu Phe1 5 10 15Val Phe Phe Leu Val Val
His Ser Val Thr Ala Phe Ala Ile Arg Phe 20 25 30Lys Cys Phe Asp Thr
Asp Met Leu Leu Lys Val Ile Ala Asp Met Val 35 40 45Val Gly Met Lys
Gly Ile Glu Lys Val Cys Arg Arg Arg Ser Lys Thr 50 55 60Trp Ser Gly
Tyr Cys Gly Asp Ser Lys His Cys Asp Gln Gln Cys Arg65 70 75 80Glu
Trp Glu Gly Ala Glu His Gly Ala Cys His His Glu Gly Leu Gly 85 90
95Arg Ala Cys Phe Cys Tyr Phe Asn Cys 100 105706110PRTAmbrosia
artemisiifolia 706Met Ala Ala Gly Leu Leu Val Phe Val Leu Ala Ile
Ser Glu Ile Ala1 5 10 15Ser Val Lys Gly Lys Leu Cys Glu Lys Pro Ser
Val Thr Trp Ser Gly 20 25 30Lys Cys Lys Val Lys Gln Thr Asp Lys Cys
Asp Lys Arg Cys Ile Glu 35 40 45Trp Glu Gly Ala Lys His Gly Ala Cys
His Lys Arg Asp Ser Lys Ala 50 55 60Ser Cys Phe Cys Tyr Phe Asp Cys
Asp Pro Thr Lys Asn Pro Gly Pro65 70 75 80Pro Pro Gly Ala Pro Lys
Gly Lys Ala Pro Ala Pro Ser Pro Pro Ser 85 90 95Gly Gly Gly Gly Glu
Gly Gly Gly Glu Gly Gly Gly Glu Arg 100 105 110707111PRTAmbrosia
artemisiifolia 707Met Ala Ala Gly Leu Leu Val Phe Val Leu Ala Ile
Ser Glu Ile Ala1 5 10 15Ser Val Lys Gly Lys Leu Cys Glu Lys Pro Ser
Leu Thr Trp Ser Gly 20 25 30Lys Cys Lys Val Lys Gln Thr Asp Lys Cys
Asp Lys Arg Cys Ile Glu 35 40 45Trp Glu Gly Ala Lys His Gly Ala Cys
His Lys Arg Asp Ser Lys Ala 50 55 60Thr Cys Phe Cys Tyr Phe Asp Cys
Asp Pro Thr Lys Asn Pro Gly Pro65 70 75 80Pro Pro Gly Ala Pro Lys
Gly Lys Ala Pro Ala Pro Ser Pro Pro Ser 85 90 95Gly Gly Gly Ala Pro
Pro Pro Ser Gly Gly Glu Gly Gly Glu Arg 100 105 11070879PRTJatropha
curcas 708Met Ala Lys Leu His Ser Ser Ala Leu Cys Phe Leu Ile Ile
Phe Leu1 5 10 15Phe Leu Leu Val Ser Lys Glu Met Ala Val Thr Glu Ala
Lys Leu Cys 20 25 30Gln Arg Arg Ser Lys Thr Trp Ser Gly Phe Cys Gly
Asp Pro Gly Lys 35 40 45Cys Asn Arg Gln Cys Arg Asn Trp Glu Gly Ala
Ser His Gly Ala Cys 50 55 60His Ala Gln Phe Pro Gly Phe Ala Cys Phe
Cys Tyr Phe Lys Cys65 70 7570979PRTNelumbo nucifera 709Met Ala Lys
Ala Pro Lys Ser Val Ser Tyr Phe Ala Phe Phe Phe Ile1 5 10 15Leu Phe
Leu Leu Ala Ser Ser Glu Ile Gln Lys Thr Lys Lys Leu Cys 20 25 30Glu
Arg Arg Ser Lys Thr Trp Ser Gly Arg Cys Thr Lys Thr Gln Asn 35 40
45Cys Asp Lys Gln Cys Lys Asp Trp Glu Tyr Ala Lys His Gly Ala Cys
50 55 60His Gly Ser Trp Phe Asn Lys Lys Cys Tyr Cys Tyr Phe Asp
Cys65 70 7571082PRTPyrus x bretschneideri 710Met Ala Lys Leu Leu
Ser Arg Leu Ser Ile Pro Leu Ile Val Phe Val1 5 10 15Phe Leu Leu Ile
Leu Leu Ala Ser Thr Glu Val Ala Met Val Glu Ala 20 25 30Arg Ile Cys
Gln Arg Arg Ser Lys Thr Trp Ser Gly Phe Cys Ala Asn 35 40 45Thr Gly
Asn Cys Asn Arg Gln Cys Thr Asn Trp Glu Gly Ala Leu His 50 55 60Gly
Ala Cys His Ala Gln Phe Pro Gly Val Ala Cys Phe Cys Tyr Phe65 70 75
80Arg Cys71183PRTRicinus communis 711Met Ala Lys Leu His Phe Pro
Thr Leu Leu Cys Leu Phe Ile Phe Leu1 5 10 15Phe Leu Leu Val Ser Thr
Glu Met Gln Val Thr Gln Ala Lys Val Cys 20 25 30Gln Arg Arg Ser Lys
Thr Trp Ser Gly Phe Cys Gly Ser Thr Lys Asn 35 40 45Cys Asp Arg Gln
Cys Lys Asn Trp Glu Gly Ala Leu His Gly Ala Cys 50 55 60His Ala Gln
Phe Pro Gly Val Ala Cys Phe Cys Tyr Phe Lys Cys Gly65 70 75 80Gly
Glu Arg71291PRTAmbrosia artemisiifolia 712Lys Leu Cys Glu Lys Pro
Ser Val Thr Trp Ser Gly Lys Cys Lys Val1 5 10 15Lys Gln Thr Asp Lys
Cys Asp Lys Arg Cys Ile Glu Trp Glu Gly Ala 20 25 30Lys His Gly Ala
Cys His Lys Arg Asp Ser Lys Ala Ser Cys Phe Cys 35 40 45Tyr Phe Asp
Cys Asp Pro Thr Lys Asn Pro Gly Pro Pro Pro Gly Ala 50 55 60Pro Lys
Gly Lys Ala Pro Ala Pro Ser Pro Pro Ser Gly Gly Gly Ala65 70 75
80Pro Pro Pro Ser Gly Gly Glu Gly Gly Gly Asp 85 9071396PRTAmbrosia
artemisiifolia 713Lys Leu Cys Glu Lys Pro Ser Val Thr Trp Ser Gly
Asn Lys Val Lys1 5 10 15Gln Thr Asp Lys Cys Asp Lys Arg Cys Ile Glu
Trp Glu Gly Ala Lys 20 25 30His Gly Ala Cys His Lys Arg Asp Ser Lys
Ala Ser Cys Phe Cys Tyr 35 40 45Phe Asp Cys Asp Pro Thr Lys Asn Pro
Gly Pro Pro Pro Gly Ala Pro 50 55 60Lys Gly Lys Ala Pro Ala Pro Ser
Pro Pro Ser Gly Gly Gly Ala Pro65 70 75 80Pro Pro Ser Gly Gly Glu
Gly Gly Gly Asp Gly Gly Gly Gly Arg Arg 85 90 9571482PRTPrunus mume
714Met Ala Lys Leu Leu Ser His Leu Leu Phe Tyr Pro Ile Leu Phe Leu1
5 10 15Phe Leu Phe Ile Phe Leu Ala Ser Thr Glu Val Ala Ile Leu Glu
Ala 20 25 30Arg Ile Cys Gln Arg Arg Ser Lys Thr Trp Ser Gly Phe Cys
Gly Asn 35 40 45Thr Arg Asn Cys Asn Arg Gln Cys Arg Asn Trp Glu Gly
Ala Leu Arg 50 55 60Gly Ala Cys His Ala Gln Phe Pro Gly Phe Ala Cys
Phe Cys Tyr Phe65 70 75 80Arg Cys71580PRTCorchorus olitorius 715Met
Ala Lys Thr Leu Gln Leu Phe Ala Leu Phe Phe Ile Val Ile Leu1 5 10
15Leu Ala Asn Gln Glu Ile Pro Val Ala Glu Ala Lys Leu Cys Gln Lys
20 25 30Arg Ser Lys Thr Trp Thr Gly Ile Cys Ile Lys Thr Lys Asn Cys
Asp 35 40 45Asn Gln Cys Lys Lys Trp Glu Lys Ala Glu His Gly Ala Cys
His Arg 50 55 60Gln Gly Ile Gly Phe Ala Cys Phe Cys Tyr Phe Asn Gln
Lys Lys Cys65 70 75 8071681PRTCorchorus olitorius 716Met Ala Lys
Phe Val Ser Thr Val Ala Leu Leu Phe Ala Leu Phe Ile1 5 10 15Leu Leu
Ala Ser Phe Asp Glu Gly Met Met Pro Met Ala Glu Ala Lys 20 25 30Val
Cys Ser Lys Arg Ser Lys Thr Trp Ser Gly Phe Cys Asn Ser Ser 35 40
45Ala Asn Cys Asn Lys Gln Cys Arg Glu Trp Glu Asp Ala Lys His Gly
50 55 60Ala Cys His Phe Glu Phe Pro Gly Phe Ala Cys Phe Cys Tyr Phe
Asn65 70 75 80Cys71776PRTSolanum pennellii 717Met Asn Ser Lys Val
Ile Leu Ala Leu Leu Val Cys Phe Leu Leu Ile1 5 10 15Ala Ser Asn Glu
Met Gln Gly Gly Glu Ala Lys Val Cys Gly Arg Arg 20 25 30Ser Ser Thr
Trp Ser Gly Leu Cys Leu Asn Thr Gly Asn Cys Asn Thr 35 40 45Gln Cys
Ile Lys Trp Glu His Ala Ser Ser Gly Ala Cys His Arg Asp 50 55 60Gly
Phe Gly Phe Ala Cys Phe Cys Tyr Phe Asn Cys65 70 7571882PRTFragaria
vesca 718Met Ala Lys Leu Leu Gly Tyr His Leu Val Tyr Pro Ile Leu
Phe Leu1 5 10 15Phe Ile Phe Leu Leu Leu Ala Ser Thr Glu Met Gly Met
Leu Glu Ala 20 25 30Arg Ile Cys Gln Arg Arg Ser Lys Thr Trp Thr Gly
Leu Cys Ala Asn 35 40 45Thr Gly Asn Cys His Arg Gln Cys Arg Asn Trp
Glu Gly Ala Gln Arg 50 55 60Gly Ala Cys His Ala Gln Phe Pro Gly Phe
Ala Cys Phe Cys Tyr Phe65 70 75 80Asn Cys71980PRTCorchorus
capsularis 719Met Ala Lys Phe Val Ser Val Ala Leu Leu Leu Ala Leu
Phe Ile Leu1 5 10 15Val Ala Ser Phe Asp Glu Gly Met Val Pro Met Ala
Glu Ala Lys Leu 20 25 30Cys Ser Lys Arg Ser Lys Thr Trp Ser Gly Phe
Cys Asn Ser Ser Ala 35 40 45Asn Cys Asn Arg Gln Cys Arg Glu Trp Glu
Asp Ala Lys His Gly Ala 50 55 60Cys His Phe Glu Phe Pro Gly Phe Ala
Cys Phe Cys Tyr Phe Asp Cys65 70 75 8072056PRTSolanum tuberosum
720Met Gln Gly Gly Glu Ala Arg Val Cys Glu Arg Arg Ser Ser Thr Trp1
5 10 15Ser Gly Pro Cys Phe Asp Thr Gly Asn Cys Asn Arg Gln Cys Ile
Asn 20 25 30Trp Glu His Ala Ser Ser Gly Ala Cys His Arg Glu Gly Ile
Gly Ser 35 40 45Ala Cys Phe Cys Tyr Phe Asn Cys 50
5572180PRTDimocarpus longan 721Met Ala Lys Thr Leu Lys Ser Val Gln
Phe Phe Ala Leu Phe Phe Leu1 5 10 15Val Ile Leu Leu Ala Gly Ser Glu
Met Thr Ala Val Glu Ala Leu Cys 20 25 30Ser Lys Arg Ser Lys Thr Trp
Ser Gly Pro Cys Phe Ile Thr Ser Arg 35 40 45Cys Asp Arg Gln Cys Lys
Arg Trp Glu Asn Ala Lys His Gly Ala Cys 50 55 60His Arg Ser Gly Trp
Gly Phe Ala Cys Phe Cys Tyr Phe Asn Lys Cys65 70 75
8072280PRTCamelina sativa 722Met Ala Lys Ala Ala Thr Ile Val Thr
Leu Leu Phe Ala Ala Leu Val1 5 10 15Phe Phe Ala Ala Leu Glu Thr Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Ser Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8072380PRTArabis alpine 723Met Ala Lys Phe Ala Ser Ile Ile Ala Phe
Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Ser Phe Glu Ala Pro Thr
Met Val Glu Ala Gln Lys Tyr 20 25 30Cys Glu Lys Pro Ser Gly Thr Trp
Ser Gly Val Cys Gly Asn Ser Asn 35 40 45Ala Cys Asn Asn Gln Cys Ile
Asn Leu Glu Gly Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro
Tyr Tyr Arg Cys Ile Cys Tyr Phe Gln Cys65 70 75 8072480PRTTheobroma
cacao 724Met Ala Met Ser Leu Lys Ser Val His Phe Phe Ala Leu Phe
Phe Ile1 5 10 15Val Val Leu Leu Ala Asn Gln Glu Met Pro Val Ala Glu
Ala Lys Leu 20 25 30Cys Gln Lys Arg Ser Lys Thr Trp Thr Gly Pro Cys
Ile Lys Thr Lys 35 40 45Asn Cys Asp His Gln Cys Arg Lys Trp Glu Lys
Ala Gln His Gly Ala 50 55 60Cys His Trp Gln Trp Pro Gly Phe Ala Cys
Phe Cys Tyr Val Asn Cys65 70 75 8072580PRTAmborella trichopoda
725Met Ala Lys Leu Val Ser Pro Lys Ala Phe Phe Val Phe Leu Phe Val1
5 10 15Phe Leu Leu Ile Ser Ala Ser Glu Phe Ser Gly Ser Glu Ala Lys
Leu 20 25 30Cys Gln Lys Arg Ser Arg Thr Trp Ser Gly Phe Cys Ala Asn
Ser Asn 35 40 45Asn Cys Ser Arg Gln Cys Lys Asn Leu Glu Gly Ala Arg
Phe Gly Ala 50 55 60Cys His Arg Gln Arg Ile Gly Leu Ala Cys Phe Cys
Tyr Phe Asn Cys65 70 75 8072680PRTArabidopsis thaliana 726Met Ala
Lys Ser Ala Thr Ile Val Thr Leu Phe Phe Ala Ala Leu Val1 5 10 15Phe
Phe Ala Ala Leu Glu Ala Pro Met Val Val Glu Ala Gln Lys Leu 20 25
30Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Ser Asn
35 40 45Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala Arg His Gly
Ser 50 55 60Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe
Pro Cys65 70 75 8072780PRTArabis alpine 727Met Ala Lys Phe Ala Ser
Ile Ile Thr Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Ser Leu
Glu Ala Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Gln Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Gly 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Tyr His Arg Cys Ile Cys Tyr Phe Pro Cys65 70 75
8072880PRTBrassica juncea 728Met Ala Lys Val Ala Ser Ile Ile Ala
Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Ala Phe Glu Ala Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8072980PRTBrassica oleracea 729Met Ala Lys Phe Ala Ser Ile Ile Ala
Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Ala Leu Glu Ala Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8073080PRTCamelina sativa 730Met Ala Lys Pro Ala Thr Ile Val Thr
Leu Leu Phe Ala Ala Leu Val1 5 10 15Phe Phe Ala Ala Leu Glu Thr Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8073180PRTCamelina sativa 731Met Ala Lys Ser Ala Thr Ile Val Thr
Leu Leu Phe Ala Ala Leu Val1 5 10 15Phe Phe Ala Ala Leu Glu Thr Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8073280PRTBrassica napus 732Met Ala Lys Phe Ala Ser Ile Ile Ala Pro
Leu Phe Ala Val Leu Val1 5 10 15Leu Phe Ala Ala Phe Glu Ala Pro Thr
Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr Trp
Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys Ile
Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro
Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75 8073380PRTEutrema
salsugineum 733Met Ala Lys Phe Ala Ser Ile Ile Thr Leu Leu Phe Ala
Ala Leu Val1 5 10 15Leu Phe Ala Val Phe Glu Gly Pro Thr Met Val Glu
Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val
Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu
Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro Ala His Lys
Cys Ile Cys Tyr Phe Pro Cys65 70 75 8073480PRTRaphanus sativus
734Met Ala Lys Phe Ala Ser Ile Ile Ala Leu Leu Phe Ala Ala Leu Val1
5 10 15Leu Phe Ala Ala Phe Glu Ala Pro Thr Met Val Glu Ala Gln Lys
Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn
Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala Arg
His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys
Tyr Phe Pro Cys65 70 75 8073580PRTRaphanus sativus 735Met Ala Lys
Phe Ala Ser Ile Val Ser Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe
Thr Ala Phe Glu Ala Pro Ala Met Val Glu Ala Gln Lys Leu 20 25 30Cys
Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40
45Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala Arg His Gly Ser
50 55 60Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe Pro
Cys65 70 75 8073676PRTRaphanus sativus 736Met Asn Thr Lys Val Ile
Leu Ala Leu Leu Phe Cys Phe Leu Leu Val1 5 10 15Ala Ser Asn Glu Met
Gln Val Gly Glu Ala Lys Val Cys Gln Arg Arg 20 25 30Ser Lys Thr Trp
Ser Gly Pro Cys Ile Asn Thr Gly Asn Cys Ser Arg 35 40 45Gln Cys Lys
Gln Gln Glu Asp Ala Arg Phe Gly Ala Cys His Arg Ser 50 55 60Gly Phe
Gly Phe Ala Cys Phe Cys Tyr Phe Lys Cys65 70 7573780PRTBrassica
rapa 737Met Ala Lys Phe Ala Ser Ile Ile Ala Pro Leu Phe Ala Ala Leu
Val1 5 10 15Leu Phe Ala Ala Phe Glu Ala Pro Thr Met Val Glu Ala Gln
Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Lys Ala
Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro Ala His Lys Cys Ile
Cys Tyr Phe Pro Cys65 70 75 8073876PRTSolanum pennellii 738Met Asn
Thr Lys Leu Ile Leu Ala Leu Met Phe Cys Phe Leu Leu Ile1 5 10 15Ala
Ser Asn Glu Met Gln Val Gly Glu Ala Lys Val Cys Gln Arg Arg 20 25
30Ser Lys Thr Trp Ser Gly Pro Cys Ile Asn Thr Gly Asn Cys Ser Arg
35 40 45Gln Cys Lys Gln Gln Glu Asp Ala Arg Phe Gly Ala Cys His Arg
Ser 50 55 60Gly Phe Gly Phe Ala Cys Phe Cys Tyr Phe Lys Cys65 70
7573978PRTCitrus clementina 739Met Ala Lys Phe Thr Thr Thr Phe Ala
Leu Leu Phe Ala Phe Phe Ile1 5 10 15Leu Phe Ala Ala Phe Asp Val Pro
Met Ala Glu Ala Lys Val Cys Gln 20 25 30Arg Arg Ser Lys Thr Trp Ser
Gly Leu Cys Leu Asn Thr Gly Asn Cys 35 40 45Ser Arg Gln Cys Lys Gln
Gln Glu Asp Ala Arg Phe Gly Ala Cys His 50 55 60Arg Gln Gly Ile Gly
Phe Ala Cys Phe Cys Tyr Phe Lys Cys65 70 7574080PRTBrassica rapa
740Met Ala Lys Phe Thr Ser Ile Ile Val Leu Leu Phe Ala Ala Leu Val1
5 10 15Leu Phe Ala Gly Phe Glu Ala Pro Thr Met Val Glu Ala Gln Lys
Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn
Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg
His Gly Ser 50 55 60Cys Asn Tyr Val Phe Pro Ala Arg Lys Cys Ile Cys
Tyr Phe Pro Cys65 70 75 8074178PRTEutrema salsugineum 741Met Ala
Lys Phe Ala Ser Ile Ile Thr Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu
Phe Ala Thr Phe Ala Pro Thr Met Val Glu Ala Lys Leu Cys Glu 20 25
30Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn Ala Cys
35 40 45Lys Ser Gln Cys Gln Arg Leu Glu Gly Ala Arg His Gly Ser Cys
Asn 50 55 60Tyr Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe Pro
Cys65 70 7574279PRTEutrema salsugineum 742Met Ala Lys Phe Ala Ser
Ile Ile Thr Leu Leu Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Thr Phe
Glu Ala Pro Thr Met Val Glu Ala Lys Leu Cys 20 25 30Glu Arg Pro Ser
Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn Ala 35 40 45Cys Lys Ser
Gln Cys Gln Arg Leu Glu Gly Ala Arg His Gly Ser Cys 50 55 60Asn Tyr
Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70
7574380PRTHeliophila coronopifolia 743Met Ala Lys Phe Ala Ser Ile
Ile Ala Phe Phe Phe Ala Ala Leu Val1 5 10 15Leu Phe Ala Ala Phe Glu
Ala Pro Thr Ile Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser
Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Arg Asn
Gln Cys Ile Asn Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr
Val Phe Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75
8074480PRTBrassica oleracea 744Met Ala Lys Val Ala Ser Ile Val Ala
Leu Leu Phe Pro Ala Leu Val1 5 10 15Ile Phe Ala Ala Phe Glu Ala Pro
Thr Met Val Glu Ala Gln Lys Leu 20 25 30Cys Glu Arg Pro Ser Gly Thr
Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45Ala Cys Lys Asn Gln Cys
Ile Arg Leu Glu Lys Ala Arg His Gly Ser 50 55 60Cys Asn Tyr Val Phe
Pro Ala His Lys Cys Ile Cys Tyr Phe Pro Cys65 70 75 8074576PRTCicer
arietinum 745Met Ser Lys Phe Tyr Thr Val Phe Met Phe Leu Cys Leu
Ala Leu Leu1 5 10 15Leu Ile Ser Ser Trp Glu Val Glu Ala Lys Leu Cys
Gln Arg Arg Ser 20 25 30Lys Thr Trp Ser Gly Pro Cys Ile Ile Thr Gly
Asn Cys Lys Asn Gln 35 40 45Cys Lys Asn Val Glu His Ala Thr Phe Gly
Ala Cys His Arg Gln Gly 50 55 60Phe Gly Phe Ala Cys Phe Cys Tyr Phe
Asn Cys His65 70 7574682PRTCitrus clementina 746Met Ala Lys Ser Val
Ala Ser Ile Thr Thr Ala Phe Ala Leu Ile Phe1 5 10 15Ala Phe Phe Ile
Leu Phe Ala Ser Phe Gly Val Pro Met Ala Glu Ala 20 25 30Lys Val Cys
Gln Arg Arg Ser Lys Thr Trp Ser Gly Pro Cys Leu Asn 35 40 45Thr Gly
Lys Cys Ser Arg Gln Cys Lys Gln Gln Glu Tyr Ala Arg Tyr 50 55 60Gly
Ala Cys Tyr Arg Gln Gly Ala Gly Tyr Ala Cys Tyr Cys Tyr Phe65 70 75
80Asn Cys74782PRTCitrus sinensis 747Met Ala Lys Ser Val Ala Ser Ile
Thr Thr Ala Phe Ala Leu Ile Phe1 5 10 15Ala Phe Phe Ile Leu Phe Ala
Ser Phe Glu Val Pro Met Ala Glu Ala 20 25 30Lys Val Cys Gln Arg Arg
Ser Lys Thr Trp Ser Gly Pro Cys Leu Asn 35 40 45Thr Gly Lys Cys Ser
Arg His Cys Lys Gln Gln Glu Asp Ala Arg Tyr 50 55 60Gly Ala Cys Tyr
Arg Gln Gly Thr Gly Tyr Ala Cys Phe Cys Tyr Phe65 70 75 80Glu
Cys74878PRTCitrus sinensis 748Met Ala Lys Phe Thr Thr Thr Phe Ala
Leu Leu Phe Ala Phe Phe Ile1 5 10 15Leu Phe Ala Ala Phe Asp Val Pro
Met Ala Glu Ala Lys Val Cys Gln 20 25 30Leu Arg Ser Lys Thr Trp Ser
Gly Leu Cys Leu Asn Thr Gly Asn Cys 35 40 45Ser Arg Gln Cys Lys Gln
Gln Glu Asp Ala Arg Phe Gly Ala Cys His 50 55 60Arg Gln Gly Ile Gly
Phe Ala Cys Phe Cys Tyr Phe Lys Cys65 70 7574976PRTCitrus sinensis
749Met Glu Arg Ser Val Arg Leu Phe Ser Thr Val Leu Leu Val Leu Leu1
5 10 15Leu Leu Ala Ser Glu Met Gly Leu Arg Ala Ala Glu Ala Arg Ile
Cys 20 25 30Glu Ser Gln Ser His Arg Phe Lys Gly Pro Cys Val Ser Lys
Ser Asn 35 40 45Cys Ala Ala Val Cys Gln Thr Glu Gly Phe His Gly Gly
His Cys Arg 50 55 60Gly Phe Arg Arg Arg Cys Phe Cys Thr Lys Arg
Cys65 70 75750106PRTArtificial SequenceSynthetic construct 750Met
Ser Thr Ala Thr Phe Val Asp Ile Ile Ile Ala Ile Leu Leu Pro1 5 10
15Pro Leu Gly Val Phe Leu Arg Phe Gly Cys Gly Val Glu Phe Trp Ile
20 25 30Cys Leu Val Leu Thr Leu Leu Gly Tyr Ile Pro Gly Ile Ile Tyr
Ala 35 40 45Ile Tyr Val Leu Thr Lys Arg Thr Cys Glu Ser Gln Ser His
Arg Phe 50 55 60Lys Gly Pro Cys Ser Arg Asp Ser Asn Cys Ala Thr Val
Cys Leu Thr65 70 75 80Glu Gly Phe Ser Gly Gly Asp Cys Arg Gly Phe
Arg Arg Arg Cys Arg 85 90 95Cys Thr Arg Pro Cys Val Phe Asp Glu Lys
100 10575128PRTPseudomonas syringae 751Glu Ser Thr Asn Ile Leu Gln
Arg Met Arg Glu Leu Ala Val Gln Ser1 5 10 15Arg Asn Asp Ser Asn Ser
Ala Thr Asp Arg Glu Ala 20 2575215PRTBacillus thuringiensis 752Arg
Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu Ala Ile Ala1 5 10
1575322PRTBacillus thuringiensis 753Asp Arg Leu Ser Ser Gly Lys Arg
Ile Asn Ser Ala Ser Asp Pro Ala1 5 10 15Ala Gly Leu Ala Ile Ala
207548PRTBacillus thuringiensis 754Arg Ile Asn Ser Ala Ser Asp Asp1
57558PRTBacillus thuringiensis 755Arg Ile Asn Asn Ala Ser Asp Asp1
57568PRTBacillus manliponensis 756Gln Ile Asn Ser Ala Ser Asp Asp1
57578PRTLysinibacillus sp. 757Arg Ile Asn Ser Ala Ala Asp Asp1
57588PRTPaenibacillus sp. 758Arg Ile Asn Gly Ala Ser Asp Asp1
57598PRTAneurinibacillus sp. 759Arg Ile Asn Arg Ala Ser Asp Asp1
57608PRTEscherichia coli 760Arg Ile Asn Ser Ala Lys Asp Asp1
57618PRTBurkholderia ubonensis 761Arg Ile Asn Thr Ala Ala Asp Asp1
57628PRTPseudomonas syringae 762Lys Ile Asn Ser Ala Lys Asp Asp1
57638PRTLysinibacillus spp. 763Arg Ile Asn Arg Ala Gly Asp Asp1
57648PRTLysinibacillus spp. 764Lys Ile Asn Arg Ala Ser Asp Asp1
57658PRTLysinibacillus xylanilyticus 765Lys Ile Asn Arg Ala Gly Asp
Asp1 57668PRTBacillus thuringiensisMISC_FEATURE(1)..(1)Xaa = Arg or
Gln or LysMISC_FEATURE(4)..(4)Xaa = Ser or Asn or Gly or Arg or
ThrMISC_FEATURE(6)..(6)Xaa = Ser or Ala or Lys or Gly 766Xaa Ile
Asn Xaa Ala Xaa Asp Asp1 576715PRTBacillus thuringiensis 767Ala Ile
Ala Leu Gly Ala Ala Asp Asp Lys Ala Ser Asn Ile Arg1 5 10
1576828PRTPseudomonas syringae 768Ala Glu Arg Asp Thr Ala Ser Asn
Ser Asp Asn Arg Ser Gln Val Ala1 5 10 15Leu Glu Arg Met Arg Gln Leu
Ile Asn Thr Ser Glu 20 2576931PRTBacillus amyloliquefaciens 769Met
Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met1 5 10
15Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala 20 25
30770218PRTSchistosoma japonicum 770Met Ser Pro Ile Leu Gly Tyr Trp
Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr
Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly
Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro
Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser
Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu
Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly
Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105
110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr
Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr
Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys
Leu Asp Ala Phe Pro Lys Leu
165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp
Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln
Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys
210 2157717PRTArtificial SequenceSynthetic construct 771Gly Gly Gly
Gly Gly Gly Ser1 57725PRTArtificial SequenceSynthetic construct
772Asp Asp Asp Asp Lys1 577320DNAArtificial SequenceSynthetic
construct (primer) 773tcgagcgcgt atgcaatacg 2077424DNAArtificial
SequenceSynthetic construct (primer) 774gcgttatccc gtagaaaaag gtag
2477518PRTArtificial SequenceSynthetic construct (primer) 775Ala
Gly Ala Cys Gly Gly Phe Thr Gly Ala Gly Thr Ala Ala Cys Gly1 5 10
15Cys Gly
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