U.S. patent application number 13/643048 was filed with the patent office on 2013-07-11 for combinations including cry34ab/35ab and cry3ba proteins to prevent development of resistance in corn rootworms (diabrotica spp.).
This patent application is currently assigned to DOW AGROSCIENCES LLC. The applicant listed for this patent is Kristin Fencil, Timothy D. Hey, Huarong Li, Thomas Meade, Kenneth Narva, Monica B. Olson, Aaron T. Woosley. Invention is credited to Kristin Fencil, Timothy D. Hey, Huarong Li, Thomas Meade, Kenneth Narva, Monica B. Olson, Aaron T. Woosley.
Application Number | 20130180016 13/643048 |
Document ID | / |
Family ID | 44834532 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130180016 |
Kind Code |
A1 |
Narva; Kenneth ; et
al. |
July 11, 2013 |
COMBINATIONS INCLUDING CRY34AB/35AB AND CRY3BA PROTEINS TO PREVENT
DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.)
Abstract
The subject invention relates in part to Cry34Ab/35Ab in
combination with Cry3Ba. The subject invention relates in part to
the surprising discovery that Cry34Ab/Cry35Ab and Cry3Ba are useful
for preventing development of resistance (to either insecticidal
protein system alone) by a corn rootworm (Diabrotica spp.)
population. As one skilled in the art will recognize with the
benefit of this disclosure, plants producing these insecticidal Cry
proteins will be useful to mitigate concern that a corn rootworm
population could develop that would be resistant to either of these
insecticidal protein systems alone. The subject invention is
supported in part by the discovery that components of these Cry
protein systems do not compete with each other for binding corn
rootworm gut receptors. The subject invention also relates in part
to triple stacks or "pyramids" of three (or more) toxin systems,
with Cry34Ab/Cry35Ab and Cry3Ba being the base pair. Thus, plants
(and acreage planted with such plants) that produce these two
insecticidal protein systems are included within the scope of the
subject invention.
Inventors: |
Narva; Kenneth; (Zionsville,
IN) ; Meade; Thomas; (Zionsville, IN) ;
Fencil; Kristin; (San Diego, CA) ; Li; Huarong;
(Zionsville, IN) ; Hey; Timothy D.; (Zionsville,
IN) ; Woosley; Aaron T.; (Fishers, IN) ;
Olson; Monica B.; (Lebanon, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Narva; Kenneth
Meade; Thomas
Fencil; Kristin
Li; Huarong
Hey; Timothy D.
Woosley; Aaron T.
Olson; Monica B. |
Zionsville
Zionsville
San Diego
Zionsville
Zionsville
Fishers
Lebanon |
IN
IN
CA
IN
IN
IN
IN |
US
US
US
US
US
US
US |
|
|
Assignee: |
DOW AGROSCIENCES LLC
Indianapolis
IN
|
Family ID: |
44834532 |
Appl. No.: |
13/643048 |
Filed: |
April 22, 2011 |
PCT Filed: |
April 22, 2011 |
PCT NO: |
PCT/US11/33618 |
371 Date: |
March 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61477447 |
Apr 20, 2011 |
|
|
|
61476005 |
Apr 15, 2011 |
|
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61388273 |
Sep 30, 2010 |
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61327240 |
Apr 23, 2010 |
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Current U.S.
Class: |
800/302 ;
435/419; 435/468; 514/4.5 |
Current CPC
Class: |
Y02A 40/146 20180101;
A01G 22/00 20180201; A01N 43/50 20130101; Y02A 40/162 20180101;
A01H 5/10 20130101; B65D 85/00 20130101; B32B 1/02 20130101; C07K
14/325 20130101; A01N 37/18 20130101; Y10T 428/13 20150115; C12N
15/8286 20130101 |
Class at
Publication: |
800/302 ;
435/419; 514/4.5; 435/468 |
International
Class: |
A01N 43/50 20060101
A01N043/50; A01H 5/10 20060101 A01H005/10 |
Claims
1. A transgenic plant that produces a Cry34 protein, a Cry35
protein, and a Cry3B insecticidal protein.
2. The transgenic plant of claim 1, said plant further produces a
fourth insecticidal protein selected from the group consisting of
Cry3A and Cry6A.
3. Seed of a plant according to claim 1, wherein said seed
comprises DNA encoding said proteins.
4. A field of plants comprising a plurality of plants according to
claim 1.
5. The field of plants of claim 4, said field further comprising
non-Bt refuge plants, wherein said refuge plants comprise less than
40% of all crop plants in said field.
6. The field of plants of claim 5, wherein said refuge plants
comprise less than 30% of all crop plants in said field.
7. The field of plants of claim 5, wherein said refuge plants
comprise less than 20% of all crop plants in said field.
8. The field of plants of claim 5, wherein said refuge plants
comprise less than 10% of all crop plants in said field.
9. The field of plants of claim 5, wherein said refuge plants
comprise less than 5% of all crop plants in said field.
10. The field of plants of claim 4, wherein said field lacks refuge
plants.
11. The field of plants of claim 5, wherein said refuge plants are
in blocks or strips.
12. A mixture of seeds comprising refuge seeds from non-Bt refuge
plants, and a plurality of seeds of claim 3, wherein said refuge
seeds comprise less than 40% of all the seeds in the mixture.
13. The mixture of seeds of claim 12, wherein said refuge seeds
comprise less than 30% of all the seeds in the mixture.
14. The mixture of seeds of claim 12, wherein said refuge seeds
comprise less than 20% of all the seeds in the mixture.
15. The mixture of seeds of claim 12, wherein said refuge seeds
comprise less than 10% of all the seeds in the mixture.
16. The mixture of seeds of claim 12, wherein said refuge seeds
comprise less than 5% of all the seeds in the mixture.
17. A seed bag or container comprising a plurality of seeds of
claim 3, said bag or container having zero refuge seed.
18. A method of managing development of resistance to a Cry protein
by an insect, said method comprising planting seeds to produce a
field of plants of claim 4.
19. A field of 4, wherein said plants occupy more than 10
acres.
20. A plant of claim 1, wherein said plant is a maize plant.
21. A plant cell of a plant of claim 1, wherein said Cry35 protein
is at least 95% identical with a sequence selected from the group
consisting of SEQ ID NO:1 and SEQ ID NO:2, said Cry3B insecticidal
protein is at least 95% identical with a sequence selected from the
group consisting of SEQ ID NO:3 and SEQ ID NO:4, and said Cry34
protein is at least 95% identical with SEQ ID NO:5 .
22. A plant of claim 1, wherein said Cry35 protein comprises a
sequence selected from the group consisting of SEQ ID NO:1 and SEQ
ID NO:2, said Cry3B insecticidal protein comprises a sequence
selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4,
and said Cry34 protein comprises SEQ ID NO:5.
23. A method of producing the plant cell of claim 21.
24. A method of controlling a rootworm insect by contacting said
insect with a Cry34 protein, a Cry35 protein, and a Cry3B
insecticidal protein.
25. The plant of claim 1 wherein said Cry34 protein is a Cry34A
protein, said Cry35 protein is a Cry35A protein, and said Cry3B
protein is a Cry3Ba protein.
26. The plant of claim 1 wherein said Cry34 protein is a Cry34Ab
protein and said Cry35 protein is a Cry35Ab protein.
27. The plant of claim 2 wherein said Cry3A protein is a Cry3Aa
protein and said Cry6A protein is a Cry6Aa protein.
28. The method of claim 24 wherein said Cry34 protein is a Cry34A
protein, said Cry35 protein is a Cry35A protein, and said Cry3B
protein is a Cry3Ba protein.
29. The method of claim 24 wherein said Cry34 protein is a Cry34Ab
protein and said Cry35 protein is a Cry35Ab protein.
Description
BACKGROUND
[0001] Humans grow corn for food and energy applications. Corn is
an important crop. It is an important source of food, food
products, and animal feed in many areas of the world. Insects eat
and damage plants and thereby undermine these human efforts.
Billions of dollars are spent each year to control insect pests and
additional billions are lost to the damage they inflict.
[0002] Damage caused by insect pests is a major factor in the loss
of the world's corn crops, despite the use of protective measures
such as chemical pesticides. In view of this, insect resistance has
been genetically engineered into crops such as corn in order to
control insect damage and to reduce the need for traditional
chemical pesticides.
[0003] Over 10 million acres of U.S. corn are infested with corn
rootworm species complex each year. The corn rootworm species
complex includes the northern corn rootworm (Diabrotica barberi),
the southern corn rootworm (D. undecimpunctata howardi), and the
western corn rootworm (D. virgifera virgifera). (Other species
include Diabrotica virgifera zeae (Mexican corn rootworm),
Diabrotica balteata (Brazilian corn rootworm), and Brazilian corn
rootworm complex (Diabrotica viridula and Diabrotica
speciosa).)
[0004] The soil-dwelling larvae of these Diabrotica species feed on
the root of the corn plant, causing lodging. Lodging eventually
reduces corn yield and often results in death of the plant. By
feeding on cornsilks, the adult beetles reduce pollination and,
therefore, detrimentally affect the yield of corn per plant. In
addition, adults and larvae of the genus Diabrotica attack cucurbit
crops (cucumbers, melons, squash, etc.) and many vegetable and
field crops in commercial production as well as those being grown
in home gardens.
[0005] Synthetic organic chemical insecticides have been the
primary tools used to control insect pests but biological
insecticides, such as the insecticidal proteins derived from
Bacillus thuringiensis (Bt), have played an important role in some
areas. The ability to produce insect-resistant plants through
transformation with Bt insecticidal protein genes has
revolutionized modern agriculture and heightened the importance and
value of insecticidal proteins and their genes.
[0006] Insecticidal crystal proteins from some strains of Bacillus
thuringiensis (B.t.) are well-known in the art. See, e.g., Hofte et
al., Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These
proteins are typically produced by the bacteria as approximately
130 kDa protoxins that are then cleaved by proteases in the insect
midgut, after ingestion by the insect, to yield a roughly 60 kDa
core toxin. These proteins are known as crystal proteins because
distinct crystalline inclusions can be observed with spores in some
strains of B.t. These crystalline inclusions are often composed of
several distinct proteins.
[0007] One group of genes which have been utilized for the
production of transgenic insect resistant crops are the
delta-endotoxins from Bacillus thuringiensis (B.t.).
Delta-endotoxins have been successfully expressed in crop plants
such as cotton, potatoes, rice, sunflower, as well as corn, and
have proven to provide excellent control over insect pests.
(Perlak, F. J et al. (1990) Bio/Technology 8, 939-943; Perlak, F.
J. et al. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al.
(1993) Bio/Technology 11: 1151-1155; Tu et al. (2000) Nature
Biotechnology 18:1101-1104; PCT publication number WO 01/13731; and
Bing J W et al. (2000) Efficacy of Cry1F Transgenic Maize,
14.sup.th Biennial International Plant Resistance to Insects
Workshop, Fort Collins, Colo.)
[0008] Several Bt proteins have been used to create the
insect-resistant transgenic plants that have been successfully
registered and commercialized to date. These include Cry1Ab,
Cry1Ac, Cry1F, Cry3Aa, and Cry3Bb in corn, Cry1Ac and Cry2Ab in
cotton, and Cry3A in potato. There is also SMART STAX in corn,
which comprises Cry1A.105 and Cry2Ab.
[0009] The commercial products expressing these proteins express a
single protein except in cases where the combined insecticidal
spectrum of 2 proteins is desired (e.g., Cry1Ab and Cry3Bb in corn
combined to provide resistance to lepidopteran pests and rootworm,
respectively) or where the independent action of the proteins makes
them useful as a tool for delaying the development of resistance in
susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton
combined to provide resistance management for tobacco budworm).
[0010] Some of the qualities of insect-resistant transgenic plants
that have led to rapid and widespread adoption of this technology
also give rise to the concern that pest populations will develop
resistance to the insecticidal proteins produced by these plants.
Several strategies have been suggested for preserving the utility
of Bt-based insect resistance traits which include deploying
proteins at a high dose in combination with a refuge, and
alternation with, or co-deployment of, different toxins (McGaughey
et al. (1998), "B.t. Resistance Management," Nature Biotechnol.
16:144-146).
[0011] The proteins selected for use in an Insect Resistance
Management (IRM) stack should be active such that resistance
developed to one protein does not confer resistance to the second
protein (i.e., there is not cross resistance to the proteins). If,
for example, a pest population selected for resistance to "Protein
A" is sensitive to "Protein B", one would conclude that there is
not cross resistance and that a combination of Protein A and
Protein B would be effective in delaying resistance to Protein A
alone.
[0012] In the absence of resistant insect populations, assessments
can be made based on other characteristics presumed to be related
to cross-resistance potential. The utility of receptor-mediated
binding in identifying insecticidal proteins likely to not exhibit
cross resistance has been suggested (van Mellaert et al. 1999). The
key predictor of lack of cross resistance inherent in this approach
is that the insecticidal proteins do not compete for receptors in a
sensitive insect species.
[0013] In the event that two Bt toxins compete for the same
receptor, then if that receptor mutates in that insect so that one
of the toxins no longer binds to that receptor and thus is no
longer insecticidal against the insect, it might be the case that
the insect will also be resistant to the second toxin (which
competitively bound to the same receptor). That is, the insect is
said to be cross-resistant to both Bt toxins. However, if two
toxins bind to two different receptors, this could be an indication
that the insect would not be simultaneously resistant to those two
toxins.
[0014] A relatively newer insecticidal protein system was
discovered in Bacillus thuringiensis as disclosed in WO 97/40162.
This system comprises two proteins--one of approximately 15 kDa and
the other of about 45 kDa. See also U.S. Pat. Nos. 6,083,499 and
6,127,180. These proteins have now been assigned to their own
classes, and accordingly received the Cry designations of Cry34 and
Cry35, respectively. See Crickmore et al. website
(biols.susx.ac.uk/home/Neil_Crickmore/Bt/). Many other related
proteins of this type of system have now been disclosed. See e.g.
U.S. Pat. No. 6,372,480; WO 01/14417; and WO 00/66742.
Plant-optimized genes that encode such proteins, wherein the genes
are engineered to use codons for optimized expression in plants,
have also been disclosed. See e.g. U.S. Pat. No. 6,218,188.
[0015] The exact mode of action of the Cry34/35 system has yet to
be determined, but it appears to form pores in membranes of insect
gut cells. See Moellenbeck et al., Nature Biotechnology, vol. 19,
p. 668 (July 2001); Masson et al., Biochemistry, 43 (12349-12357)
(2004). The exact mechanism of action remains unclear despite 3D
atomic coordinates and crystal structures being known for a Cry34
and a Cry35 protein. See U.S. Pat. Nos. 7,524,810 and 7,309,785.
For example, it is unclear if one or both of these proteins bind a
typical type of receptor, such as an alkaline phosphatase or an
aminopeptidase.
[0016] Furthermore, because there are different mechanisms by which
an insect can develop resistance to a Cry protein (such as by
altered glycosylation of the receptor [see Jurat-Fuentes et al.
(2002) 68 AEM 5711-5717], by removal of the receptor protein [see
Lee et al. (1995) 61 AEM 3836-3842], by mutating the receptor, or
by other mechanisms [see Heckel et al., J. Inv. Pathol. 95 (2007)
192-197]), it was impossible to a priori predict whether there
would be cross-resistance between Cry34/35 and other Cry proteins.
Predicting competitive binding for the Cry34/35 system is also
further complicated by the fact that two proteins are involved in
the Cry34/35 binary system. Again, it is unclear if and how these
proteins effectively bind the insect gut/gut cells, and if and how
they interact with or bind with each other.
[0017] Other options for controlling coleopterans include the
following proteins: Cry3Bb, Cry3C, Cry6B, ET29, ET33 with ET34,
TIC407, TIC435, TIC417, TIC901, TIC1201, ET29 with TIC810, ET70,
ET76 with ET80, TIC851, and others. RNAi approaches have also been
proposed. See e.g. Baum et al., Nature Biotechnology, vol. 25, no.
11 (November 2007) pp. 1322-1326.
BRIEF SUMMARY
[0018] The subject invention relates in part to Cry34Ab/35Ab in
combination with Cry3Ba. The subject invention relates in part to
the surprising discovery that Cry34Ab/Cry35Ab and Cry3Ba are useful
for preventing development of resistance (to either insecticidal
protein system alone) by a corn rootworm (Diabrotica spp.)
population. As one skilled in the art will recognize with the
benefit of this disclosure, plants producing these insecticidal Cry
proteins will be useful to mitigate concern that a corn rootworm
population could develop that would be resistant to either of these
insecticidal protein systems alone.
[0019] The subject invention is supported in part by the discovery
that components of these Cry protein systems do not compete with
each other for binding corn rootworm gut receptors.
[0020] The subject invention also relates in part to triple stacks
or "pyramids" of three (or more) toxin systems, with
Cry34Ab/Cry35Ab and Cry3Ba being the base pair. Thus, plants (and
acreage planted with such plants) that produce these two
insecticidal protein systems are included within the scope of the
subject invention.
BRIEF DESCRIPTION OF THE FIGURES
[0021] The detailed description of the drawings particularly refers
to the accompanying figures in which:
[0022] FIG. 1A. Binding of .sup.125I-Cry35Ab1 as a function of
input radio-labeled Cry toxins to BBMV prepared from western corn
rootworm larvae. Specific binding=total binding-non-specific
binding, error bar=SEM (standard error of mean).
[0023] FIG. 1B. Binding of .sup.125I-Cry3BAa1 as a function of
input radio-labeled Cry toxins to BBMV prepared from western corn
rootworm larvae. Specific binding=total binding-non-specific
binding, error bar=SEM (standard error of mean).
[0024] FIG. 2. Binding of .sup.125I-Cry35Ab1 to BBMV prepared from
western corn rootworm larvae at different concentrations of
non-labeled competitor (log 0.1=-1.0, log 10=1.0, log 100=2.0, log
1,000=3.0).
[0025] FIG. 3A. Percent binding of .sup.125I-Cry35Ab1 to BBMV
prepared from western corn rootworm larvae in absence of
Cry34Ab1.
[0026] FIG. 3B. Percent binding of .sup.125I-Cry35Ab1 to BBMV
prepared from western corn rootworm larvae in presence of
Cry34Ab1.
[0027] FIG. 4. Percent binding of .sup.125I-Cry3Ba1 to BBMV
prepared from western corn rootworm larvae in presence of various
concentrations of varying non-labeled competitors.
BRIEF DESCRIPTION OF THE SEQUENCES
[0028] SEQ ID NO:1: Full length, native Cry35Ab1 protein
sequence.
[0029] SEQ ID NO:2: Chymotrypsin-truncated Cry35Ab1 core protein
sequence.
[0030] SEQ ID NO:3: Full length, native Cry3Ba1 protein
sequence.
[0031] SEQ ID NO:4: Cry3Ba1 trypsin core protein sequence.
[0032] SEQ ID NO:5: Full length, native Cry34Ab1 protein
sequence.
DETAILED DESCRIPTION
[0033] Sequences for the Cry34Ab/35Ab protein are obtainable from
Bacillus thruingiensis isolate PS149B1, for example. For other
genes, protein sequences, and source isolates for use according to
the subject invention, see the Crickmore et al. website
(lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html), for
example.
[0034] The subject invention includes the use of Cry34Ab/35Ab
insecticidal proteins in combination with a Cry3Ba toxin to protect
corn from damage and yield loss caused by corn rootworm feeding by
corn rootworm populations that might develop resistance to either
of these Cry protein systems alone (without the other).
[0035] The subject invention thus teaches Insect Resistance
Management (IRM) stacks to prevent the development of resistance by
corn rootworm to Cry3Ba and/or Cry34Ab/35Ab.
[0036] The present invention provides compositions for controlling
rootworm pests comprising cells that produce a Cry3Ba toxin protein
and a Cry34Ab/35Ab toxin system.
[0037] The invention further comprises a host transformed to
produce both a Cry3Ba protein and a Cry34Ab/35Ab binary toxin,
wherein said host is a microorganism or a plant cell.
[0038] It is additionally intended that the invention provides a
method of controlling rootworm pests comprising contacting said
pests or the environment of said pests with an effective amount of
a composition that contains a Cry3Ba protein and further contains a
Cry34Ab/35Ab binary toxin.
[0039] An embodiment of the invention comprises a maize plant
comprising a plant-expressible gene encoding a Cry34Ab/35Ab binary
toxin and a plant-expressible gene encoding a Cry3Ba protein, and
seed of such a plant.
[0040] A further embodiment of the invention comprises a maize
plant wherein a plant-expressible gene encoding a Cry34Ab/35Ab
binary toxin and a plant-expressible gene encoding a Cry3Ba protein
have been introgressed into said maize plant, and seed of such a
plant.
[0041] As described in the Examples, competitive receptor binding
studies using radiolabeled Cry35Ab core toxin protein show that the
Cry3Ba core toxin protein does not compete for binding in CRW
insect tissue samples to which Cry35Ab binds. See FIG. 2. These
results indicate that the combination of Cry3Ba and Cry34Ab/35Ab
proteins is an effective means to mitigate the development of
resistance in CRW populations to either protein system alone.
[0042] Thus, based in part on the data described above and
elsewhere herein, Cry34Ab/35Ab and Cry3Ba proteins can be used to
produce IRM combinations for prevention or mitigation of resistance
development by CRW. Other proteins can be added to this combination
to expand insect-control spectrum, for example. The subject
combination (of Cry34Ab/35Ab and Cry3Ba proteins) can also be used
in some preferred "triple stacks" or "pyramids" in combination with
yet another protein for controlling rootworms, such as Cry3Aa
and/or Cry6Aa; such additional combinations would thus provide
multiple modes of action against a rootworm. RNAi against rootworms
is a still further option. See e.g. Baum et al., Nature
Biotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.
[0043] In light of the disclosure of U.S. Ser. No. 61/327,240
(filed Apr. 23, 2010) relating to combinations of Cry34Ab/35Ab and
Cry3Aa proteins, U.S. Ser. No. 61/388,273 (filed Sep. 30, 2010)
relating to combinations of Cry34Ab/35Ab and Cry6Aa proteins, and
U.S. Ser. No. 61/477,447 (filed Sep. 20, 2011) relating to
combinations of Cry3Aa and Cry6Aa proteins, some preferred "triple
stacks" or "multiple modes of action stacks" of the subject
invention include a Cry3Ba protein combined with Cry34Ab/35Ab
proteins, together with a Cry6Aa protein and/or a Cry3Aa protein.
Transgenic plants, including corn, comprising a cry3Ba gene,
cry34Ab/35Ab genes, and a third or fourth toxin system (e.g.,
cry3Aa and/or cry6Aa gene(s)) are included within the scope of the
subject invention. Thus, such embodiments target the insect with at
least three modes of action.
[0044] Deployment options of the subject invention include the use
of Cry3Ba and Cry34Ab/35Ab proteins in corn-growing regions where
Diabrotica spp. are problematic. Another deployment option would be
to use one or both of the Cry3Ba and Cry34Ab/35Ab proteins in
combination with other traits.
[0045] A person skilled in this art will appreciate that Bt toxins,
even within a certain class such as Cry3Ba and Cry34Ab/35Ab can
vary to some extent.
[0046] Genes and toxins. The term "isolated" refers to a
polynucleotide in a non-naturally occurring construct, or to a
protein in a purified or otherwise non-naturally occurring state.
The genes and toxins useful according to the subject invention
include not only the full length sequences disclosed but also
fragments of these sequences, variants, mutants, and fusion
proteins which retain the characteristic pesticidal activity of the
toxins specifically exemplified herein. As used herein, the terms
"variants" or "variations" of genes refer to nucleotide sequences
which encode the same toxins or which encode equivalent toxins
having pesticidal activity. As used herein, the term "equivalent
toxins" refers to toxins having the same or essentially the same
biological activity against the target pests as the claimed toxins.
This applies to Cry3's and Cry34/35's, as well as Cry6's (if used
in triple/multiple stacks) according to the subject invention.
Domains/subdomains of these proteins can be swapped to make
chimeric proteins. See e.g. U.S. Pat. Nos. 7,309,785 and 7,524,810
regarding Cry34/35 proteins. The '785 patent also teaches truncated
Cry35 proteins. Truncated toxins are also exemplified herein.
[0047] As used herein, the boundaries represent approximately 95%
(Cry3Ba's and Cry34Ab's and Cry35Ab's), 78% (Cry3B's and Cry 34A's
and Cry35A's), and 45% (Cry6's and Cry 34's and Cry 35's) sequence
identity, per "Revision of the Nomenclature for the Bacillus
thuringiensis Pesticidal Crystal Proteins," N. Crickmore, D. R.
Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J.
Baum, and D. H. Dean. Microbiology and Molecular Biology Reviews
(1998) Vol 62: 807-813. The same applies to Cry3A's and/or Cry6's
if used in triple/multiple stacks, for example, according to the
subject invention.
[0048] It should be apparent to a person skilled in this art that
genes encoding active toxins can be identified and obtained through
several means. The specific genes or gene portions exemplified
herein may be obtained from the isolates deposited at a culture
depository. These genes, or portions or variants thereof, may also
be constructed synthetically, for example, by use of a gene
synthesizer. Variations of genes may be readily constructed using
standard techniques for making point mutations. Also, fragments of
these genes can be made using commercially available exonucleases
or endonucleases according to standard procedures. For example,
enzymes such as Ba131 or site-directed mutagenesis can be used to
systematically cut off nucleotides from the ends of these genes.
Genes that encode active fragments may also be obtained using a
variety of restriction enzymes. Proteases may be used to directly
obtain active fragments of these protein toxins.
[0049] Fragments and equivalents which retain the pesticidal
activity of the exemplified toxins would be within the scope of the
subject invention. Also, because of the redundancy of the genetic
code, a variety of different DNA sequences can encode the amino
acid sequences disclosed herein. It is well within the skill of a
person trained in the art to create these alternative DNA sequences
encoding the same, or essentially the same, toxins. These variant
DNA sequences are within the scope of the subject invention. As
used herein, reference to "essentially the same" sequence refers to
sequences which have amino acid substitutions, deletions,
additions, or insertions which do not materially affect pesticidal
activity. Fragments of genes encoding proteins that retain
pesticidal activity are also included in this definition.
[0050] A further method for identifying the genes encoding the
toxins and gene portions useful according to the subject invention
is through the use of oligonucleotide probes. These probes are
detectable nucleotide sequences. These sequences may be detectable
by virtue of an appropriate label or may be made inherently
fluorescent as described in International Application No.
WO93/16094. As is well known in the art, if the probe molecule and
nucleic acid sample hybridize by forming a strong bond between the
two molecules, it can be reasonably assumed that the probe and
sample have substantial homology. Preferably, hybridization is
conducted under stringent conditions by techniques well-known in
the art, as described, for example, in Keller, G. H., M. M. Manak
(1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
Some examples of salt concentrations and temperature combinations
are as follows (in order of increasing stringency): 2.times. SSPE
or SSC at room temperature; 1.times. SSPE or SSC at 42.degree. C.;
0.1.times. SSPE or SSC at 42.degree. C.; 0.1.times. SSPE or SSC at
65.degree. C. Detection of the probe provides a means for
determining in a known manner whether hybridization has occurred.
Such a probe analysis provides a rapid method for identifying
toxin-encoding genes of the subject invention. The nucleotide
segments which are used as probes according to the invention can be
synthesized using a DNA synthesizer and standard procedures. These
nucleotide sequences can also be used as PCR primers to amplify
genes of the subject invention.
[0051] Variant toxins. Certain toxins of the subject invention have
been specifically exemplified herein. Since these toxins are merely
exemplary of the toxins of the subject invention, it should be
readily apparent that the subject invention comprises variant or
equivalent toxins (and nucleotide sequences coding for equivalent
toxins) having the same or similar pesticidal activity of the
exemplified toxin. Equivalent toxins will have amino acid homology
with an exemplified toxin. This amino acid identity will typically
be greater than 75%, or preferably greater than 85%, preferably
greater than 90%, preferably greater than 95%, preferably greater
than 96%, preferably greater than 97%, preferably greater than 98%,
or preferably greater than 99% in some embodiments. The amino acid
identity will typically be highest in critical regions of the toxin
which account for biological activity or are involved in the
determination of three-dimensional configuration which ultimately
is responsible for the biological activity. In this regard, certain
amino acid substitutions are acceptable and can be expected if
these substitutions are in regions which are not critical to
activity or are conservative amino acid substitutions which do not
affect the three-dimensional configuration of the molecule. For
example, amino acids may be placed in the following classes:
non-polar, uncharged polar, basic, and acidic. Conservative
substitutions whereby an amino acid of one class is replaced with
another amino acid of the same type fall within the scope of the
subject invention so long as the substitution does not materially
alter the biological activity of the compound. Table 1 provides a
listing of examples of amino acids belonging to each class.
TABLE-US-00001 TABLE 1 Classes of amino acids with examples of
amino acids belonging to each class Class of Amino Acid Examples of
Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp
Uncharged Gly, Ser, Thr, Cys, Tyr, Asn, Gln Polar Acidic Asp, Glu
Basic Lys, Arg, His
[0052] In some instances, non-conservative substitutions can also
be made. The critical factor is that these substitutions must not
significantly detract from the biological activity of the
toxin.
[0053] Recombinant hosts. The genes encoding the toxins of the
subject invention can be introduced into a wide variety of
microbial or plant hosts. Expression of the toxin gene results,
directly or indirectly, in the intracellular production and
maintenance of the pesticide. Conjugal transfer and recombinant
transfer can be used to create a Bt strain that expresses both
toxins of the subject invention. Other host organisms may also be
transformed with one or both of the toxin genes then used to
accomplish the synergistic effect. With suitable microbial hosts,
e.g., Pseudomonas, the microbes can be applied to the situs of the
pest, where they will proliferate and be ingested. The result is
control of the pest. Alternatively, the microbe hosting the toxin
gene can be treated under conditions that prolong the activity of
the toxin and stabilize the cell. The treated cell, which retains
the toxic activity, then can be applied to the environment of the
target pest. Non-regenerable/non-totipotent plant cells from a
plant of the subject invention (comprising at least one of the
subject IRM genes) are included within the subject invention.
[0054] Plant transformation. A preferred embodiment of the subject
invention is the transformation of plants with genes encoding the
subject insecticidal protein or its variants. The transformed
plants are resistant to attack by an insect target pest by virtue
of the presence of controlling amounts of the subject insecticidal
protein or its variants in the cells of the transformed plant. By
incorporating genetic material that encodes the insecticidal
properties of the B.t. insecticidal toxins into the genome of a
plant eaten by a particular insect pest, the adult or larvae would
die after consuming the food plant. Numerous members of the
monocotyledonous and dicotyledonous classifications have been
transformed. Transgenic agronomic crops as well as fruits and
vegetables are of commercial interest. Such crops include, but are
not limited to, maize, rice, soybeans, canola, sunflower, alfalfa,
sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like.
Several techniques exist for introducing foreign genetic material
into plant cells, and for obtaining plants that stably maintain and
express the introduced gene. Such techniques include acceleration
of genetic material coated onto microparticles directly into cells
(U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may
be transformed using Agrobacterium technology, see U.S. Pat. No.
5,177,010, U.S. Pat. No. 5,104,310, European Patent Application No.
0131624B1, European Patent Application No. 120516, European Patent
Application No. 159418B1, European Patent Application No. 176112,
U.S. Pat. No. 5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No.
5,464,763, U.S. Pat. No. 4,940,838, U.S. Pat. No. 4,693,976,
European Patent Application No. 116718, European Patent Application
No. 290799, European Patent Application No. 320500, European Patent
Application No. 604662, European Patent Application No. 627752,
European Patent Application No. 0267159, European Patent
Application No. 0292435, U.S. Pat. No. 5,231,019, U.S. Pat. No.
5,463,174, U.S. Pat. No. 4,762,785, U.S. Pat. No. 5,004,863, and
U.S. Pat. No. 5,159,135. Other transformation technology includes
WHISKERS.TM. technology, see U.S. Pat. No. 5,302,523 and U.S. Pat.
No. 5,464,765. Electroporation technology has also been used to
transform plants, see WO 87/06614, U.S. Pat. No. 5,472,869, U.S.
Pat. No. 5,384,253, WO 9209696, and WO 9321335. All of these
transformation patents and publications are incorporated by
reference. In addition to numerous technologies for transforming
plants, the type of tissue which is contacted with the foreign
genes may vary as well. Such tissue would include but would not be
limited to embryogenic tissue, callus tissue types I and II,
hypocotyl, meristem, and the like. Almost all plant tissues may be
transformed during dedifferentiation using appropriate techniques
within the skill of an artisan.
[0055] Genes encoding any of the subject toxins can be inserted
into plant cells using a variety of techniques which are well known
in the art as disclosed above. For example, a large number of
cloning vectors comprising a marker that permits selection of the
transformed microbial cells and a replication system functional in
Escherichia coli are available for preparation and modification of
foreign genes for insertion into higher plants. Such manipulations
may include, for example, the insertion of mutations, truncations,
additions, or substitutions as desired for the intended use. The
vectors comprise, for example, pBR322, pUC series, M13mp series,
pACYC184, etc. Accordingly, the sequence encoding the Cry protein
or variants can be inserted into the vector at a suitable
restriction site. The resulting plasmid is used for transformation
of cells of E. coli, the cells of which are cultivated in a
suitable nutrient medium, then harvested and lysed so that workable
quantities of the plasmid are recovered. Sequence analysis,
restriction fragment analysis, electrophoresis, and other
biochemical-molecular biological methods are generally carried out
as methods of analysis. After each manipulation, the DNA sequence
used can be cleaved and joined to the next DNA sequence. Each
manipulated DNA sequence can be cloned in the same or other
plasmids.
[0056] The use of T-DNA-containing vectors for the transformation
of plant cells has been intensively researched and sufficiently
described in EP 120516; Lee and Gelvin (2008), Fraley et al.
(1986), and An et al. (1985), and is well established in the
field.
[0057] Once the inserted DNA has been integrated into the plant
genome, it is relatively stable throughout subsequent generations.
The vector used to transform the plant cell normally contains a
selectable marker gene encoding a protein that confers on the
transformed plant cells resistance to a herbicide or an antibiotic,
such as bialaphos, kanamycin, G418, bleomycin, or hygromycin, inter
alia. The individually employed selectable marker gene should
accordingly permit the selection of transformed cells while the
growth of cells that do not contain the inserted DNA is suppressed
by the selective compound.
[0058] A large number of techniques are available for inserting DNA
into a host plant cell. Those techniques include transformation
with T-DNA delivered by Agrobacterium tumefaciens or Agrobacterium
rhizogenes as the transformation agent. Additionally, fusion of
plant protoplasts with liposomes containing the DNA to be
delivered, direct injection of the DNA, biolistics transformation
(microparticle bombardment), or electroporation, as well as other
possible methods, may be employed.
[0059] In a preferred embodiment of the subject invention, plants
will be transformed with genes wherein the codon usage of the
protein coding region has been optimized for plants. See, for
example, U.S. Pat. No. 5,380,831, which is hereby incorporated by
reference. Also, advantageously, plants encoding a truncated toxin
will be used. The truncated toxin typically will encode about 55%
to about 80% of the full length toxin. Methods for creating
synthetic B.t. genes for use in plants are known in the art
(Stewart, 2007).
[0060] Regardless of transformation technique, the gene is
preferably incorporated into a gene transfer vector adapted to
express the B.t insecticidal toxin genes and variants in the plant
cell by including in the vector a plant promoter. In addition to
plant promoters, promoters from a variety of sources can be used
efficiently in plant cells to express foreign genes. For example,
one may use promoters of bacterial origin, such as the octopine
synthase promoter, the nopaline synthase promoter, and the
mannopine synthase promoter. Non-Bacillus-thuringiensis promoters
can be used in some preferred embodiments. Promoters of plant virus
origin may be used, for example, the 35S and 19S promoters of
Cauliflower Mosaic Virus, a promoter from Cassava Vein Mosaic
Virus, and the like. Plant promoters include, but are not limited
to, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit
(ssu), beta-conglycinin promoter, phaseolin promoter, ADH (alcohol
dehydrogenase) promoter, heat-shock promoters, ADF (actin
depolymerization factor) promoter, ubiquitin promoter, actin
promoter, and tissue specific promoters. Promoters may also contain
certain enhancer sequence elements that may improve the
transcription efficiency. Typical enhancers include but are not
limited to ADH1-intron 1 and ADH1-intron 6. Constitutive promoters
may be used. Constitutive promoters direct continuous gene
expression in nearly all cells types and at nearly all times (e.g.,
actin, ubiquitin, CaMV 35S). Tissue specific promoters are
responsible for gene expression in specific cell or tissue types,
such as the leaves or seeds (e.g. zein, oleosin, napin, ACP (Acyl
Carrier Protein) promoters), and these promoters may also be used.
Promoters may also be used that are active during a certain stage
of the plants' development as well as active in specific plant
tissues and organs. Examples of such promoters include but are not
limited to promoters that are root specific, pollen-specific,
embryo specific, corn silk specific, cotton fiber specific, seed
endosperm specific, phloem specific, and the like.
[0061] Under certain circumstances it may be desirable to use an
inducible promoter. An inducible promoter is responsible for
expression of genes in response to a specific signal, such as:
physical stimulus (e.g. heat shock genes); light (e.g. RUBP
carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g.
tetracycline); metabolites; and stress (e.g. drought). Other
desirable transcription and translation elements that function in
plants may be used, such as 5' untranslated leader sequences, RNA
transcription termination sequences and poly-adenylate addition
signal sequences. Numerous plant-specific gene transfer vectors are
known to the art.
[0062] Transgenic crops containing insect resistance (IR) traits
are prevalent in corn and cotton plants throughout North America,
and usage of these traits is expanding globally. Commercial
transgenic crops combining IR and herbicide tolerance (HT) traits
have been developed by multiple seed companies. These include
combinations of IR traits conferred by B.t. insecticidal proteins
and HT traits such as tolerance to Acetolactate Synthase (ALS)
inhibitors such as sulfonylureas, imidazolinones,
triazolopyrimidine, sulfonanilides, and the like, Glutamine
Synthetase (GS) inhibitors such as bialaphos, glufosinate, and the
like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such as
mesotrione, isoxaflutole, and the like,
5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such
as glyphosate and the like, and Acetyl-Coenzyme A Carboxylase
(ACCase) inhibitors such as haloxyfop, quizalofop, diclofop, and
the like. Other examples are known in which transgenically provided
proteins provide plant tolerance to herbicide chemical classes such
as phenoxy acids herbicides and pyridyloxyacetates auxin herbicides
(see WO 2007/053482 A2), or phenoxy acids herbicides and
aryloxyphenoxypropionates herbicides (see WO 2005/107437 A2, A3).
The ability to control multiple pest problems through IR traits is
a valuable commercial product concept, and the convenience of this
product concept is enhanced if insect control traits and weed
control traits are combined in the same plant. Further, improved
value may be obtained via single plant combinations of IR traits
conferred by a B.t. insecticidal protein such as that of the
subject invention, with one or more additional HT traits such as
those mentioned above, plus one or more additional input traits
(e.g. other insect resistance conferred by B.t.-derived or other
insecticidal proteins, insect resistance conferred by mechanisms
such as RNAi and the like, nematode resistance, disease resistance,
stress tolerance, improved nitrogen utilization, and the like), or
output traits (e.g. high oils content, healthy oil composition,
nutritional improvement, and the like). Such combinations may be
obtained either through conventional breeding (breeding stack) or
jointly as a novel transformation event involving the simultaneous
introduction of multiple genes (molecular stack). Benefits include
the ability to manage insect pests and improved weed control in a
crop plant that provides secondary benefits to the producer and/or
the consumer. Thus, the subject invention can be used in
combination with other traits to provide a complete agronomic
package of improved crop quality with the ability to flexibly and
cost effectively control any number of agronomic issues.
[0063] The transformed cells grow inside the plants in the usual
manner. They can form germ cells and transmit the transformed
trait(s) to progeny plants.
[0064] Such plants can be grown in the normal manner and crossed
with plants that have the same transformed hereditary factors or
other hereditary factors. The resulting hybrid individuals have the
corresponding phenotypic properties.
[0065] In a preferred embodiment of the subject invention, plants
will be transformed with genes wherein the codon usage has been
optimized for plants. See, for example, U.S. Pat. No. 5,380,831. In
addition, methods for creating synthetic Bt genes for use in plants
are known in the art (Stewart and Burgin, 2007). One non-limiting
example of a preferred transformed plant is a fertile maize plant
comprising a plant expressible gene encoding a Cry3Ba protein, and
further comprising a second set of plant expressible genes encoding
Cry34Ab/35Ab proteins.
[0066] Transfer (or introgression) of the Cry3Ba- and
Cry34Ab/35Ab-determined trait(s) into inbred maize lines can be
achieved by recurrent selection breeding, for example by
backcrossing. In this case, a desired recurrent parent is first
crossed to a donor inbred (the non-recurrent parent) that carries
the appropriate gene(s) for the Cry-determined traits. The progeny
of this cross is then mated back to the recurrent parent followed
by selection in the resultant progeny for the desired trait(s) to
be transferred from the non-recurrent parent. After three,
preferably four, more preferably five or more generations of
backcrosses with the recurrent parent with selection for the
desired trait(s), the progeny will be heterozygous for loci
controlling the trait(s) being transferred, but will be like the
recurrent parent for most or almost all other genes (see, for
example, Poehlman & Sleper (1995) Breeding Field Crops, 4th
Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol.
1: Theory and Technique, 360-376).
[0067] Insect Resistance Management (IRM) Strategies. Roush et al.,
for example, outlines two-toxin strategies, also called
"pyramiding" or "stacking," for management of insecticidal
transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B.
(1998) 353, 1777-1786).
[0068] On their website, the United States Environmental Protection
Agency (epa.gov/oppbppd1/biopesticides/pips/bt corn refuge
2006.htm) publishes the following requirements for providing
non-transgenic (i.e., non-B.t.) refuges (a block of non-Bt
crops/corn) for use with transgenic crops producing a single Bt
protein active against target pests. [0069] "The specific
structured requirements for corn borer-protected Bt (Cry1Ab or
Cry1F) corn products are as follows: [0070] Structured refuges: 20%
non-Lepidopteran Bt corn refuge in Corn Belt; [0071] 50%
non-Lepidopteran Bt refuge in Cotton Belt [0072] Blocks [0073]
Internal (i.e., within the Bt field) [0074] External (i.e.,
separate fields within 1/2 mile (1/4 mile if possible) of the Bt
field to maximize random mating) [0075] In-field Strips [0076]
Strips must be at least 4 rows wide (preferably 6 rows) to reduce
the effects of larval movement"
[0077] In addition, the National Corn Growers Association, on their
website: [0078]
(ncga.com/insect-resistance-management-fact-sheet-bt-corn) also
provides similar guidance regarding the refuge requirements. For
example: [0079] "Requirements of the Corn Borer IRM: [0080] Plant
at least 20% of your corn acres to refuge hybrids [0081] In cotton
producing regions, refuge must be 50% [0082] Must be planted within
1/2 mile of the refuge hybrids [0083] Refuge can be planted as
strips within the Bt field; the refuge strips must be at least 4
rows wide [0084] Refuge may be treated with conventional pesticides
only if economic thresholds are reached for target insect [0085]
Bt-based sprayable insecticides cannot be used on the refuge corn
[0086] Appropriate refuge must be planted on every farm with Bt
corn"
[0087] As stated by Roush et al. (on pages 1780 and 1784 right
column, for example), stacking or pyramiding of two different
proteins each effective against the target pests and with little or
no cross-resistance can allow for use of a smaller refuge. Roush
suggests that for a successful stack, a refuge size of less than
10% refuge, can provide comparable resistance management to about
50% refuge for a single (non-pyramided) trait. For currently
available pyramided Bt corn products, the U.S. Environmental
Protection Agency requires significantly less (generally 5%)
structured refuge of non-Bt corn be planted than for single trait
products (generally 20%).
[0088] There are various ways of providing the IRM effects of a
refuge, including various geometric planting patterns in the fields
(as mentioned above) and in-bag seed mixtures, as discussed further
by Roush et al. (supra), and U.S. Pat. No. 6,551,962.
[0089] The above percentages, or similar refuge ratios, can be used
for the subject double or triple stacks or pyramids. Because the
subject invention provides multiple, non-competitive modes of
action against a rootworm target insect, the subject invention
could provide "zero refuge", that is, a field that lacks refuge
plants (because they are not required). A permit is typically
required for typical B.t. transgenic fields of above about 10
acres. Thus, the subject invention includes a field of 10 acres or
more with "zero refuge" or no Bt plants; fields of this size would
previously have been required to have a significant non-Bt
refuge.
[0090] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety to the extent they are not inconsistent
with the explicit teachings of this specification.
[0091] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted. All temperatures
are in degrees Celsius.
[0092] Unless specifically indicated or implied, the terms "a",
"an", and "the" signify "at least one" as used herein.
EXAMPLES
Example 1
Construction of Expression Plasmids Encoding Cry34Ab1, Cry35Ab1,
and Cry3Ba1 Full-Length Toxins
[0093] Standard cloning methods were used in the construction of
Pseudomonas fluorescens (Pf) expression plasmids engineered to
produce full-length Cry34Ab1, Cry35Ab1, and Cry3Ba1 Cry proteins
respectively. Restriction endonucleases from New England BioLabs
(NEB; Ipswich, Mass.) were used for DNA digestion and T4 DNA Ligase
from Invitrogen was used for DNA ligation. Plasmid preparations
were performed using the Plasmid Midi Kit (Qiagen), following the
instructions of the supplier. DNA fragments were purified using the
Millipore Ultrafree.RTM.-DA cartridge (Billerica, Mass.) after
agarose Tris-acetate gel electrophoresis. The basic cloning
strategy entailed subcloning the coding sequences (CDS) of a
full-length Cry34Ab1 and Cry35Ab1 proteins into pMYC 1803 at SpeI
and XhoI (or XbaI) restriction sites, and the CDS of a full-length
Cry3Ba1 protein into pMYC1050 at KpnI and XbaI restriction sites,
respectively, whereby they were placed under the expression control
of the Ptac promoter and the rrnBT1T2 terminator from plasmid
pKK223-3 (PL Pharmacia, Milwaukee, Wis.), respectively. pMYC1803 is
a medium copy plasmid with the RSF1010 origin of replication, a
tetracycline resistance gene, and a ribosome binding site preceding
the restriction enzyme recognition sites into which DNA fragments
containing protein coding regions may be introduced (U.S. Patent
Application No. 2008/0193974). The expression plasmid was
transformed by electroporation into a P. fluorescens strain MB214,
recovered in SOC-Soy hydrolysate medium, and plated on Lysogeny
broth (LB) medium containing 20 .mu.g/ml tetracycline. Details of
the microbiological manipulations are available U.S. Patent
Application No. 2006/0008877, U.S. Patent Application No.
2008/0193974, and U.S. Patent Application No. 2008/0058262,
incorporated herein by reference. Colonies were screened by
restriction digestion of miniprep plasmid DNA. Plasmid DNA of
selected clones containing inserts was sequenced by contract with a
commercial sequencing vendor such as MWG Biotech (Huntsville,
Ala.). Sequence data were assembled and analyzed using the
Sequencher.TM. software (Gene Codes Corp., Ann Arbor, Mich.).
Example 2
Growth and Expression
[0094] Growth and expression analysis in shake flasks production of
Cry34Ab1, Cry35Ab1, and Cry3Ba1 toxins for characterization
including Bt receptor binding and insect bioassay was accomplished
by shake-flask-grown P. fluorescens strains harboring expression
constructs (e.g. clone pMYC2593 for Cry34Ab1, pMYC3122 for
Cry35Ab1, and pMYC1177 for Cry3Ba1). Seed cultures grown in LB
medium supplemented with 20 .mu.g/ml tetracycline were used to
inoculate 200 mL of the same medium with 20 .mu.g/ml tetracycline.
Expression of Cry34Ab1, Cry35Ab1, and Cry3Ba1 toxins via the Ptac
promoter was induced by addition of
isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) after an initial
incubation of 24 hours at 30.degree. C. with shaking Cultures were
sampled at the time of induction and at various times
post-induction. Cell density was measured by optical density at 600
nm (OD.sub.600).
Example 3
Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples
[0095] At each sampling time, the cell density of samples was
adjusted to OD.sub.600=20 and 1 mL aliquots are centrifuged at
14,000.times.g for five minutes. The cell pellets were frozen at
-80.degree. C. Soluble and insoluble fractions from frozen shake
flask cell pellet samples were generated using EasyLyse.TM.
Bacterial Protein Extraction Solution (EPICENTRE.RTM.
Biotechnologies, Madison, Wis.). Each cell pellet was resuspended
in 1 mL EasyLyse.TM. solution and further diluted 1:4 in lysis
buffer and incubated with shaking at room temperature for 30
minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at
4.degree. C. and the supernatant was recovered as the soluble
fraction. The pellet (insoluble fraction) was then resuspended in
an equal volume of phosphate buffered saline (PBS; 11.9 mM
Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl, pH7.4). Samples were
mixed 1:1 with 2.times. Laemmli sample buffer containing
.beta.-mercaptoethanol and boiled for 5 minutes prior to loading
onto NuPAGE Novex 4-20% Bis-Tris gels (Invitrogen, Carlsbad,
Calif.). Electrophoresis was performed in the recommended XT MOPS
buffer. Gels were stained with the SimplyBlue.TM. Safe Stain
according to the manufacturer's (Invitrogen) protocol and imaged
using the Typhoon imaging system (GE Healthcare Life Sciences,
Pittsburgh, Pa.).
Example 4
Inclusion Body Preparation
[0096] Cry protein inclusion body (IB) preparations were performed
on cells from P. fluorescens fermentations that produced insoluble
B.t. insecticidal protein, as demonstrated by SDS-PAGE and MALDI-MS
(Matrix Assisted Laser Desorption/Ionization Mass Spectrometry). P.
fluorescens fermentation pellets are thawed in a 37.degree. C.
water bath. The cells were resuspended to 25% w/v in lysis buffer
[50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt
(Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mM
Dithiothreitol (DTT)] and 5 mL/L of bacterial protease inhibitor
cocktail (P8465 Sigma-Aldrich, St. Louis, Mo.) was added just prior
to use. The cells were suspended using a homogenizer at lowest
setting (Tissue Tearor, BioSpec Products, Inc., Bartlesville,
Okla.). Lysozyme (25 mg of Sigma L7651, from chicken egg white) was
added to the cell suspension by mixing with a metal spatula, and
the suspension was incubated at room temperature for one hour. The
suspension was cooled on ice for 15 minutes, then sonicated using a
Branson Sonifier 250 (two 1-minute sessions, at 50% duty cycle, 30%
output). Cell lysis was checked by microscopy. An additional 25 mg
of lysozyme was added if necessary, and the incubation and
sonication were repeated. When cell lysis was confirmed via
microscopy, the lysate was centrifuged at 11,500.times.g for 25
minutes (4.degree. C.) to form the IB pellet, and the supernatant
was discarded. The IB pellet was resuspended with 100 mL lysis
buffer, homogenized with the hand-held mixer and centrifuged as
above. The IB pellet was repeatedly washed by resuspension (in 50
mL lysis buffer), homogenization, sonication, and centrifugation
until the supernatant became colorless and the IB pellet became
firm and off-white in color. For the final wash, the IB pellet was
resuspended in sterile-filtered (0.22 .mu.m) distilled water
containing 2 mM EDTA, and centrifuged. The final pellet was
resuspended in sterile-filtered distilled water containing 2 mM
EDTA, and stored in 1 mL aliquots at -80.degree. C.
Example 5
SDS-PAGE Analysis and Quantification
[0097] SDS-PAGE analysis and quantification of protein in IB
preparations were done by thawing a 1 mL aliquot of IB pellet and
diluting 1:20 with sterile-filtered distilled water. The diluted
sample was then boiled with 4.times. reducing sample buffer [250 mM
Tris, pH6.8, 40% glycerol (v/v), 0.4% Bromophenol Blue (w/v), 8%
SDS (w/v) and 8% .beta.-Mercapto-ethanol (v/v)] and loaded onto a
Novex.RTM. 4-20% Tris-Glycine, 12+2 well gel (Invitrogen) run with
1.times. Tris/Glycine/SDS buffer (Invitrogen). The gel was run for
approximately 60 min at 200 volts then stained and destained by
following the SimplyBlue.TM. Safe Stain (Invitrogen) procedures.
Quantification of target bands was done by comparing densitometric
values for the bands against Bovine Serum Albumin (BSA) samples run
on the same gel to generate a standard curve using the Bio-Rad
Quantity One.RTM. software.
Example 6
Solubilization of Inclusion Bodies
[0098] Ten mL of inclusion body suspensions from P. fluoresces
clones MR1253, MR1636, and MR816 (containing 50-70 mg/mL of
Cry34Ab1, Cry35Ab1, and Cry3Ba1 proteins respectively) were
centrifuged at the highest setting of an Eppendorf model 5415C
microfuge (approximately 14,000.times.g) to pellet the inclusions.
The storage buffer supernatant was removed and replaced with 25 mL
of 100 mM sodium acetate buffer, pH 3.0, for both Cry34Ab1 and
Cry35Ab1, and 100 mM sodium carbonate buffer, pH11, for Cry3Ba1, in
a 50 mL conical tube, respectively. Inclusions were resuspended
using a pipette and vortexed to mix thoroughly. The tubes were
placed on a gently rocking platform at 4.degree. C. overnight to
extract full-length Cry34Ab1, Cry35Ab1, and Cry3Ba1 proteins. The
extracts were centrifuged at 30,000.times.g for 30 min at 4.degree.
C., and the resulting supernatants (containing solubilized
full-length Cry proteins) were saved.
Example 7
Truncation of Full-Length Protoxins
[0099] Full-length Cry35Ab1 and Cry3Ba1 were truncated or digested
with chymotrypsin or trypsin to produce chymotrypsin or trypsin
core fragments that are an active form of the proteins. Specially,
the solubilized full-length Cry35Ab1 was incubated with
chymotrypsin (bovine pancreas) (Sigma, St. MO) (at 50:1 =Cry
protein: enzyme, w/w) in the 100 mM sodium acetate buffer, pH 3.0
(Example 6), at 4.degree. C. with gentle shaking for 2-3 days,
while full-length Cry3Ba1 was incubated with trypsin (bovine
pancreas) (Sigma, St. MO) (at 20:1=Cry protein: enzyme, w/w) in the
100 mM sodium carbonate buffer, pH11 (Example 6), at room
temperature for 1-3 hours. Complete proteolytic preocessing was
confirmed by SDS-PAGE analysis. The molecular mass of the
full-length Cry35Ab1 and Cry3Ba1 was approximately equal to 44 and
approximately equal to 73 kDa, and their chymotrypsin or trypsin
core was approximately equal to 40 and approximately equal to 55
kDa, respectively. The amino acid sequences of full-length and
chymotrypsin core of Cry35Ab1 are provided as SEQ ID 1 and SEQ ID
2, and the amino acid sequences of full-length and trypsin core of
Cry3Ba1 are provide as SEQ ID 3 and SEQ ID 4. Either chymotrypsin
or trypsin core is not available for Cry34Ab1, and thus the
full-length Cry34Ab1 was used for binding assays. The amino acid
sequence of the full-length Cry34Ab1 is provided as SEQ ID 5.
Example 8
Purification of Truncated Toxins
[0100] The chymotrypsinized Cry35Ab1 and trypsinized Cry3Ba1 core
fragments were purified. Specifically, the digestion reactions were
centrifuged at 30,000.times.g for 30 min at 4.degree. C. to remove
lipids, and the resulting supernatant were concentrated 5-fold
using an Amicon Ultra-15 regenerated cellulose centrifugal filter
device (10,000 Molecular Weight Cutoff; Millipore). The sample
buffers were then changed to 20 mM sodium acetate buffer, pH 3.5,
for both Cry34Ab1 and Cry35Ab1, and to 10 mM CAPS
[3-(cyclohexamino)1-propanesulfonic acid], pH 10.5, for Cry3Ba1,
using disposable PD-10 columns (GE Healthcare, Piscataway, N.J.) or
dialysis. The final volumes were adjusted to 15 ml using the
corresponding buffer for purification using ATKA Explorer liquid
chromatography system (Amersham Biosciences). For Cry35Ab1, buffer
A was 20 mM sodium acetate buffer, pH 3.5, and buffer B was buffer
A+1 M NaCl, pH 3.5. A HiTrap SP (5 ml) column (GE) was used. After
the column was fully equilibrated using the buffer A, the Cry35Ab1
solution was injected into the column at a flow rate of 5 ml/min.
Elution was performed using gradient 0-100% of buffer B at 5 ml/min
with 1 ml/fraction. For Cry3Ba1, buffer A was 10 mM CAPS buffer, pH
10.5, and buffer B was 10 mM CAPS buffer, pH 10.5+1 M NaCl. A Capto
Q, 5 ml (5 ml) column (GE) was used and all other procedures were
similar to that for Cry35Ab1. After SDS-PAGE analysis of the
selected fractions to further select fractions containing the best
quality target protein, pooled those fractions. The buffer was
changed for the purified Cry35Ab1 chymotrypsin core with 20 mM
Bist-Tris, pH 6.0, as described above. For the purified Cry3Ba1
trypsin core, the salt was removed using disposable PD-10 columns
(GE Healthcare, Piscataway, N.J.) or dialysis. The samples were
saved at 4.degree. C. for later binding assays after being
quantified using SDS-PAGE and the Typhoon imaging system (GE)
analysis with BSA as a standard.
Example 9
BBMV Preparation
[0101] Brush border membrane vesicle (BBMV) preparations of insects
have been widely used for Cry toxin receptor binding assays. The
BBMV preparations used in this invention were prepared from
isolated midguts of third instars of the western corn rootworm
(Diabrotica virgifera virgifera LeConte) using the method described
by Wolferberger et al. (1987). Leucine aminopeptidase was used as a
marker of membrane proteins in the preparation and Leucine
aminopeptidase activities of crude homogenate and BBMV preparations
were determined as previously described (Li et al. 2004a). Protein
concentration of the BBMV preparations were measured using the
Bradford method (1976).
Example 10
.sup.125I Labeling
[0102] Purified full-length Cry34Ab1, chymotrypsinized Cry35Ab1,
and trypsinized Cry3Ba1 were labeled using .sup.125I for saturation
and homologous competition binding assays. To ensure the
radio-labeling does not abolish the biological activity of the Cry
toxins, cold iodination was conducted using NaI by following the
instructions of Pierce.RTM. Iodination Beads (Pierce Biotechnology,
Thermo Scientific, Rockford Ill.). Bioassay results indicated that
iodinated Cry35Ab1 chymotrypsin core remained active against the
larvae of the western corn rootworm, but iodination inactivated
Cry34Ab1. The specific binding of radiolabeled .sup.125I-Cry34Ab1
to the insect BBMV was not able to be detected, and thus requiring
another labeling method to assess membrane receptor binding of
Cry34Ab1. Since trypsinized Cry3Ba1 had limited activity against
western corn rootworm, and thus the bioassay with the corn rootworm
using cold iodinated Cry3Ba1 trypsin core was thought difficult to
assess the activity change. In addition, the specific binding of
.sup.125I-Cry3Ba1 to the BBMV was detected even though the level
was low. Cold-iodination of Cry3Ba1 and its toxicity assay were
ignored. Radiolabeled .sup.125I-Cry35Ab1 and .sup.125I-Cry3Ba1 were
obtained through iodination with Pierce.RTM. Iodination Beads
(Pierce) and Na.sup.125I. Zeba.TM. Desalt Spin Columns (Pierce)
were used to remove unincorporated or free Na.sup.125I from the
iodinated protein. The specific radioactivities of the iodinated
Cry proteins ranged from 1-5 uCi/ug. Multiple batches of labeling
and binding assays were conducted.
Example 11
Saturation Binding Assays
[0103] Specific or saturation binding assays were performed using
.sup.125I-labeled Cry toxins as described previously (Li et al.
2004b). To determine specific binding and estimate the binding
affinity (disassociation constant, Kd) and binding site
concentration (Bmax) of Cry35Ab1 and Cry3Ba1 to the insect BBMV, a
series of increasing concentrations of either .sup.125I-Cry35Ab1 or
.sup.125I-Cry3Ba1 were incubated with a given concentration (0.1
mg/ml) of the insect BBMV, respectively, in 150 ul of 20 mM
Bis-Tris, pH 6.0, 150 mM KCl, supplemented with 0.1% BSA at room
temperature for 1 hour with gentle shaking Toxin bound to BBMV was
separated from free toxin in the suspension by centrifugation at
20,000.times.g at room temperature for 8 min. The pellet was washed
twice with (ice-cold) 900 ul of the same buffer containing 0.1%
BSA. The radioactivity remaining in the pellet was measured with a
COBRAII Auto-Gamma counter (Packard, a Canberra company) and
considered total binding. Another series of binding reactions were
setup as side by side, and a 500-1,000-fold excess of unlabeled
corresponding toxin was included in each of the binding reactions
to fully occupy all specific binding sites on the BBMV, which was
used to determine non-specific binding. Specific binding was
estimated by subtracting the non-specific binding from the total
binding. The Kd and Bmax values of these toxins were estimated
using the specific binding against the concentrations of the
labeled toxin used by running GraphPad Prism 5.01 (GraphPad
Software, San Diego, Calif.). The charts were made using either
Microsoft Excel or GraphPad Prism software. The experiments were
replicated at least four times and the result plotted in the graphs
of FIG. 1A (binding of .sup.125I-Cry35Ab1 to BBMV) and FIG. 1B
(binding of .sup.125I-Cry3Ba1 to BBMV). These binding experiments
demonstrated that both .sup.125I-Cry35Ab1 and .sup.125I-Cry3Ba1
were able to specifically bind to the BBMV (FIGS. 1A and 1B).
.sup.125I-Cry35Ab1 and .sup.125I-Cry3Ba1 had a binding affinity
Kd=11.66.+-.11.44, 7.35.+-.3.81 (nM), respectively, and a binding
site concentration Bmax=0.78.+-.0.46, 0.55.+-.0.13 (pmol/mg BBMV),
respectively.
[0104] The specific binding of .sup.125I-Cry35Ab1 was carried out
at the presence of unlabeled Cry34Ab1
(1:50=.sup.125I-Cry35Ab1:Cry34Ab1, molar ratio). The binding
parameters (Kd and Bmax) were not obtained because the specific
binding of .sup.125I-Cry35Ab1 was not saturated (FIG. 2). However,
the specific binding of .sup.125I-Cry35Ab1 accounted for
approximately 90% of the total binding at the presence of unlabeled
Cry34Ab1.
Example 12
Competition Binding Assays
[0105] Competition binding assays were conducted to determine if
Cry34Ab1 and Cry35Ab1 separately, plus their mixture as a binary
toxin, share a same set of binding sites with Cry3Ba1. For
homologous competition binding assays of Cry3Ba1, increasing
amounts (0-2,500 nM) of unlabeled Cry3Ba1 were first mixed with 5
nM .sup.125I-Cry3Ba1, and then incubated the insect BBMV at with
0.1 mg/ml at room temperature for 1 hour, respectively, to allow
them compete for the putative receptor(s) on the BBMV. Similarly,
Cry35Ab1 homologous competition was completed with 5 nM
.sup.125I-Cry35Ab1 at the absence or presence of unlabeled Cry34Ab1
(1:50=.sup.125I-Cry35Ab1:Cry34Ab1, molar ratio), and with the BBMV
at 0.03 mg/ml, respectively. The percentages of bound
.sup.125I-Cry3Ba1 or .sup.125I-Cry35Ab1 to the BBMV were determined
for each of the reactions as compared to the initial total (or
specific) binding at absence of unlabeled competitor.
[0106] Heterologous competition binding assays between
.sup.125I-Cry35Ab1 and unlabeled Cry3Ba1 were conducted at absence
or presence of unlabeled Cry34Ab1 to identify if they share a same
set of binding site(s). This was achieved by increasing the amount
of unlabeled Cry3Ba1 as a competitor to compete for the binding
with .sup.125I-Cry35Ab1 alone or .sup.125I-Cry35Ab1+Cry34Ab1
(1:50=.sup.125I-Cry35Ab1:Cry34Ab1, molar ratio). Similarly,
reciprocal heterologous competition binding assays were also
conducted, which was achieved by increasing the amount of unlabeled
Cry35Ab1 and Cry34Ab1 separately, or the Cry35Ab1+Cry34Ab1
(1:50=Cry35Ab1:Cry34Ab1, molar ratio) mixture, as one or two
competitors included in the reactions to compete for the binding
with the labeled Cry3Ba1, respectively. The experiments were
replicated at least three times and the result plotted in the
graphs of FIG. 3A (percent binding of .sup.125I-Cry35Ab alone) and
FIG. 3B (percent binding of .sup.125I-Cry35Ab1 in the presence of
Cry34Ab1).
[0107] The experimental results demonstrated that Cry35Ab1 was able
to compete off the specific binding of .sup.125I-Cry35Ab1
regardless of absence (FIG. 3A) or presence (FIG. 3B) of Cry34Ab1.
However, Cry3Ba1 was unable to compete off the specific binding of
.sup.125I-Cry35Ab1 at either absence or presence of Cry34Ab1. In
reciprocal competition binding assays, Cry3Ba was also able to
displace itself over 20% of the total binding, which reflects it
completely competed off its specific binding because the specific
binding accounts only a small fraction (see FIG. 1B). However,
either Cry34Ab1, or Cry35Ab1 alone, or the mixture of
Cry35Ab1+Cry34Ab1 (1:10) was not able to displace
.sup.125I-Cry3Ba1. These data indicated that Cry35Ab1 alone or
mixture of Cry35Ab1+Cry34Ab1 does not share a receptor binding site
with Cry3Ba1.
REFERENCES
[0108] Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding, Anal. Biochem. 72, 248-254.
[0109] Li, H., Oppert, B., Higgins, R. A., Huang, F., Zhu, K. Y.,
Buschman, L. L., 2004a. Comparative analysis of proteinase
activities of Bacillus thuringiensis-resistant and -susceptible
Ostrinia nubilalis (Lepidoptera: Crambidae). Insect Biochem. Mol.
Biol. 34, 753-762. [0110] Li, H., Oppert, B., Gonzalez-Cabrera, J.,
Ferre, J., Higgins, R. A., Buschman, L. L. and Zhu, K. Y. and
Huang, F. 2004b. Binding analysis of Cry1Ab and Cry1Ac with
membrane vesicles from Bacillus thuringiensis-resistant and
-susceptible Ostrinia nubilalis (Lepidoptera: Crambidae). Biochem.
Biophys. Res. Commun. 323, 52-57. [0111] Wolfersberger, M. G.,
Luthy, P., Maurer, A., Parenti, P., Sacchi, F., Giordana, B.,
Hanozet, G. M., 1987. Preparation and partial characterization of
amino acid transporting brush border membranevesicles from the
larval midgut of the cabbage butterfly (Pieris brassicae). Comp.
Biochem. Physiol. 86A, 301-308. [0112] US Patent Application No.
20080193974.2008. BACTERIAL LEADER SEQUENCES FOR INCREASED
EXPRESSION [0113] US Patent Application No. 20060008877,2006.
Expression systems with sec-system secretion. [0114] US Patent
Application No. 20080058262,2008. rPA optimization.
Sequence CWU 1
1
51383PRTBacillus thuringiensis 1Met Leu Asp Thr Asn Lys Val Tyr Glu
Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu
Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp
Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro
Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn
Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70
75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala
Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val
Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu
Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu
Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile
Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr
Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp
Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190
Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195
200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro
His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile
Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln
Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val
Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu
Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys
Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser
Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315
320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser
325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln
Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val
Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu
Lys Lys Tyr Tyr Phe 370 375 380 2354PRTBacillus thuringiensis 2Met
Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10
15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu
20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys
Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr
Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn
Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile
Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile
Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln
Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn
Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140
Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145
150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln
Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp
Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr
Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly
Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr
Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile
Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met
Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265
270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys
275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro
Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr
Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg
Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val
Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu
3659PRTBacillus thuringiensis 3Met Ile Arg Met Gly Gly Arg Lys Met
Asn Pro Asn Asn Arg Ser Glu 1 5 10 15 Tyr Asp Thr Ile Lys Val Thr
Pro Asn Ser Glu Leu Pro Thr Asn His 20 25 30 Asn Gln Tyr Pro Leu
Ala Asp Asn Pro Asn Ser Thr Leu Glu Glu Leu 35 40 45 Asn Tyr Lys
Glu Phe Leu Arg Met Thr Ala Asp Asn Ser Thr Glu Val 50 55 60 Leu
Asp Ser Ser Thr Val Lys Asp Ala Val Gly Thr Gly Ile Ser Val 65 70
75 80 Val Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe Ala Gly Ala
Leu 85 90 95 Thr Ser Phe Tyr Gln Ser Phe Leu Asn Ala Ile Trp Pro
Ser Asp Ala 100 105 110 Asp Pro Trp Lys Ala Phe Met Ala Gln Val Glu
Val Leu Ile Asp Lys 115 120 125 Lys Ile Glu Glu Tyr Ala Lys Ser Lys
Ala Leu Ala Glu Leu Gln Gly 130 135 140 Leu Gln Asn Asn Phe Glu Asp
Tyr Val Asn Ala Leu Asp Ser Trp Lys 145 150 155 160 Lys Ala Pro Val
Asn Leu Arg Ser Arg Arg Ser Gln Asp Arg Ile Arg 165 170 175 Glu Leu
Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser 180 185 190
Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr Ala Gln 195
200 205 Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln Val Phe
Gly 210 215 220 Glu Glu Trp Gly Tyr Ser Ser Glu Asp Ile Ala Glu Phe
Tyr Gln Arg 225 230 235 240 Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp
His Cys Val Asn Trp Tyr 245 250 255 Asn Val Gly Leu Asn Ser Leu Arg
Gly Ser Thr Tyr Asp Ala Trp Val 260 265 270 Lys Phe Asn Arg Phe Arg
Arg Glu Met Thr Leu Thr Val Leu Asp Leu 275 280 285 Ile Val Leu Phe
Pro Phe Tyr Asp Val Arg Leu Tyr Ser Lys Gly Val 290 295 300 Lys Thr
Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro Ile Phe Thr Leu 305 310 315
320 Asn Ala Leu Gln Glu Tyr Gly Pro Thr Phe Ser Ser Ile Glu Asn Ser
325 330 335 Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Arg Gly Ile Glu
Phe His 340 345 350 Thr Arg Leu Arg Pro Gly Tyr Ser Gly Lys Asp Ser
Phe Asn Tyr Trp 355 360 365 Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser
Ile Gly Ser Asn Asp Thr 370 375 380 Ile Thr Ser Pro Phe Tyr Gly Asp
Lys Ser Ile Glu Pro Ile Gln Lys 385 390 395 400 Leu Ser Phe Asp Gly
Gln Lys Val Tyr Arg Thr Ile Ala Asn Thr Asp 405 410 415 Ile Ala Ala
Phe Pro Asp Gly Lys Ile Tyr Phe Gly Val Thr Lys Val 420 425 430 Asp
Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu Thr Ser Thr Gln Thr 435 440
445 Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr Leu Gly Ala Gln Asp Ser Ile
450 455 460 Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys
Ala Tyr 465 470 475 480 Ser His Gln Leu Asn Tyr Ala Glu Cys Phe Leu
Met Gln Asp Arg Arg 485 490 495 Gly Thr Ile Pro Phe Phe Thr Trp Thr
His Arg Ser Val Asp Phe Phe 500 505 510 Asn Thr Ile Asp Ala Glu Lys
Ile Thr Gln Leu Pro Val Val Lys Ala 515 520 525 Tyr Ala Leu Ser Ser
Gly Ala Ser Ile Ile Glu Gly Pro Gly Phe Thr 530 535 540 Gly Gly Asn
Leu Leu Phe Leu Lys Glu Ser Ser Asn Ser Ile Ala Lys 545 550 555 560
Phe Lys Val Thr Leu Asn Ser Ala Ala Leu Leu Gln Arg Tyr Arg Val 565
570 575 Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Leu Phe Val Gln
Asn 580 585 590 Ser Asn Asn Asp Phe Leu Val Ile Tyr Ile Asn Lys Thr
Met Asn Ile 595 600 605 Asp Gly Asp Leu Thr Tyr Gln Thr Phe Asp Phe
Ala Thr Ser Asn Ser 610 615 620 Asn Met Gly Phe Ser Gly Asp Thr Asn
Asp Phe Ile Ile Gly Ala Glu 625 630 635 640 Ser Phe Val Ser Asn Glu
Lys Ile Tyr Ile Asp Lys Ile Glu Phe Ile 645 650 655 Pro Val Gln
4489PRTBacillus thuringiensis 4Ser Gln Asp Arg Ile Arg Glu Leu Phe
Ser Gln Ala Glu Ser His Phe 1 5 10 15 Arg Asn Ser Met Pro Ser Phe
Ala Val Ser Lys Phe Glu Val Leu Phe 20 25 30 Leu Pro Thr Tyr Ala
Gln Ala Ala Asn Thr His Leu Leu Leu Leu Lys 35 40 45 Asp Ala Gln
Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp Ile 50 55 60 Ala
Glu Phe Tyr Gln Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp 65 70
75 80 His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Ser Leu Arg Gly
Ser 85 90 95 Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg
Glu Met Thr 100 105 110 Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro
Phe Tyr Asp Val Arg 115 120 125 Leu Tyr Ser Lys Gly Val Lys Thr Glu
Leu Thr Arg Asp Ile Phe Thr 130 135 140 Asp Pro Ile Phe Thr Leu Asn
Ala Leu Gln Glu Tyr Gly Pro Thr Phe 145 150 155 160 Ser Ser Ile Glu
Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr Leu 165 170 175 Arg Gly
Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Ser Gly Lys 180 185 190
Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser 195
200 205 Ile Gly Ser Asn Asp Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys
Ser 210 215 220 Ile Glu Pro Ile Gln Lys Leu Ser Phe Asp Gly Gln Lys
Val Tyr Arg 225 230 235 240 Thr Ile Ala Asn Thr Asp Ile Ala Ala Phe
Pro Asp Gly Lys Ile Tyr 245 250 255 Phe Gly Val Thr Lys Val Asp Phe
Ser Gln Tyr Asp Asp Gln Lys Asn 260 265 270 Glu Thr Ser Thr Gln Thr
Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr Leu 275 280 285 Gly Ala Gln Asp
Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu 290 295 300 Pro Leu
Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys Phe 305 310 315
320 Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr His
325 330 335 Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile
Thr Gln 340 345 350 Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly
Ala Ser Ile Ile 355 360 365 Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu
Leu Phe Leu Lys Glu Ser 370 375 380 Ser Asn Ser Ile Ala Lys Phe Lys
Val Thr Leu Asn Ser Ala Ala Leu 385 390 395 400 Leu Gln Arg Tyr Arg
Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 405 410 415 Arg Leu Phe
Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr Ile 420 425 430 Asn
Lys Thr Met Asn Ile Asp Gly Asp Leu Thr Tyr Gln Thr Phe Asp 435 440
445 Phe Ala Thr Ser Asn Ser Asn Met Gly Phe Ser Gly Asp Thr Asn Asp
450 455 460 Phe Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile
Tyr Ile 465 470 475 480 Asp Lys Ile Glu Phe Ile Pro Val Gln 485
5123PRTBacillus thuringiensis 5Met Ser Ala Arg Glu Val His Ile Asp
Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys
Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn
Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn
Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile
Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70
75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu
Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr
Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser
115 120
* * * * *