U.S. patent application number 16/002994 was filed with the patent office on 2018-12-06 for novel insect inhibitory proteins.
The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to David J. Bowen, Catherine A. Chay, Stanislaw Flasinski, Yong Yin.
Application Number | 20180346925 16/002994 |
Document ID | / |
Family ID | 57885446 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180346925 |
Kind Code |
A1 |
Bowen; David J. ; et
al. |
December 6, 2018 |
NOVEL INSECT INHIBITORY PROTEINS
Abstract
A pesticidal protein class exhibiting toxic activity against
Coleopteran, Lepidopteran, and Hemipteran pest species is
disclosed, and includes, but is not limited to, TIC5290. DNA
constructs are provided which contain a recombinant nucleic acid
sequence encoding the TIC5290 pesticidal protein. Transgenic
plants, plant cells, seed, and plant parts resistant to
Lepidopteran, Coleopteran and Hemipteran infestation are provided
which contain recombinant nucleic acid sequences encoding the
TIC5290 pesticidal protein of the present invention. Methods for
detecting the presence of the recombinant nucleic acid sequences or
the protein of the present invention in a biological sample, and
methods of controlling Coleopteran, Lepidopteran, and Hemipteran
species pests using the TIC5290 pesticidal protein are also
provided.
Inventors: |
Bowen; David J.; (Wildwood,
MO) ; Chay; Catherine A.; (Ballwin, MO) ;
Flasinski; Stanislaw; (Ballwin, MO) ; Yin; Yong;
(Creve Coeur, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
57885446 |
Appl. No.: |
16/002994 |
Filed: |
June 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15221544 |
Jul 27, 2016 |
10100330 |
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16002994 |
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62199024 |
Jul 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6897 20130101;
C07K 14/325 20130101; C12N 15/8286 20130101; A01N 37/46 20130101;
Y02A 40/146 20180101; A01N 63/10 20200101; Y02A 40/162
20180101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01N 63/02 20060101 A01N063/02 |
Claims
1. A recombinant nucleic acid molecule comprising a heterologous
promoter operably linked to a polynucleotide segment encoding a
pesticidal protein or pesticidal fragment thereof, wherein: a. said
pesticidal protein comprises the amino acid sequence of SEQ ID
NO:2; or b. said pesticidal protein comprises an amino acid
sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or
90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence
identity to SEQ ID NO:2; or c. said polynucleotide segment
hybridizes under stringent hybridization conditions to a
polynucleotide having the nucleotide sequence of SEQ ID NO:1 or SEQ
ID NO:3.
2. The recombinant nucleic acid molecule of claim 1, wherein: a.
the recombinant nucleic acid molecule comprises a sequence that
functions to express the pesticidal protein in a plant; or b. the
recombinant nucleic acid molecule is expressed in a plant cell to
produce a pesticidally effective amount of the pesticidal protein;
or c. the recombinant nucleic acid molecule is in operable linkage
with a vector, and said vector is selected from the group
consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial
or yeast artificial chromosome.
3. The recombinant nucleic acid molecule of claim 1, defined as
present within a host cell, wherein said host cell is selected from
the group consisting of a bacterial and a plant cell.
4. The recombinant nucleic acid molecule of claim 3, wherein the
bacterial host cell is from a genus of bacteria selected from the
group consisting of: Agrobacterium, Rhizobium, Bacillus,
Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and
Erwinia.
5. The recombinant nucleic acid molecule of claim 4, wherein the
Bacillus species is Bacillus cereus or Bacillus thuringiensis, said
Brevibacillus is Brevibacillus laterosperus, or said Escherichia is
Escherichia coli.
6. The recombinant nucleic acid molecule of claim 3, wherein said
plant cell is a dicotyledonous or a monocotyledonous plant
cell.
7. The recombinant nucleic acid molecule of claim 6, wherein said
plant host cell is selected from the group consisting of an
alfalfa, banana, barley, bean, broccoli, cabbage, Brassica, carrot,
cassava, castor, cauliflower, celery, chickpea, Chinese cabbage,
citrus, coconut, coffee, corn, clover, cotton, a cucurbit,
cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape,
hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat,
olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,
pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish,
rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,
Southern pine, soybean, spinach, squash, strawberry, sugar beet,
sugarcane, sunflower, sweet corn, sweet gum, sweet potato,
switchgrass, tea, tobacco, tomato, triticale, turf grass,
watermelon, and wheat plant cell.
8. The recombinant nucleic acid molecule of claim 1, wherein said
pesticidal protein exhibits activity against a Coleopteran
insect.
9. The recombinant nucleic acid molecule of claim 8, wherein said
insect is Western Corn Rootworm, Southern Corn Rootworm, Northern
Corn Rootworm, Mexican Corn Rootworm, Brazilian Corn Rootworm, or
Brazilian Corn Rootworm complex consisting of Diabrotica viridula
and Diabrotica speciosa.
10. The recombinant nucleic acid molecule of claim 1, wherein said
pesticidal protein exhibits activity against an insect species of
the order of Lepidoptera.
11. The recombinant nucleic acid molecule of claim 10, wherein said
insect is Velvet bean caterpillar, Sugarcane borer, Lesser
cornstalk borer, Corn earworm, Tobacco budworm, Soybean looper,
Black armyworm, Southern armyworm, Fall armyworm, Beet armyworm,
Old World bollworm, Oriental leaf worm, Pink bollworm, Black
cutworm, Southwestern Corn Borer, or European corn borer.
12. The recombinant nucleic acid molecule of claim 1, wherein said
pesticidal protein exhibits activity against a Hemipteran
insect.
13. The recombinant nucleic acid molecule of claim 12, wherein said
insect is a Western tarnished plant bug, a Tarnished plant bug, or
a Cotton fleahopper.
14. A plant or part thereof comprising the recombinant nucleic acid
molecule of claim 1.
15. The plant or part thereof of claim 14, wherein said plant is a
monocot plant or a dicot plant.
16. The plant or part thereof of claim 14, wherein the plant is
selected from the group consisting of an alfalfa, banana, barley,
bean, broccoli, cabbage, Brassica, carrot, cassava, castor,
cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut,
coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir,
eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce,
Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental,
palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato,
poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks,
rye, safflower, shrub, sorghum, Southern pine, soybean, spinach,
squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn,
sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,
triticale, turf grass, watermelon, and wheat.
17. A seed of the plant of claim 14, wherein said seed comprises
said recombinant nucleic acid molecule.
18. An insect inhibitory composition comprising the recombinant
nucleic acid molecule of claim 1.
19. The insect inhibitory composition of claim 18, further
comprising a nucleotide sequence encoding at least one other
pesticidal agent that is different from said pesticidal
protein.
20. The insect inhibitory composition of claim 19, wherein said at
least one other pesticidal agent is selected from the group
consisting of an insect inhibitory protein, an insect inhibitory
dsRNA molecule, and an ancillary protein.
21. The insect inhibitory composition of claim 19, wherein said at
least one other pesticidal agent exhibits activity against one or
more pest species of the orders Lepidoptera, Coleoptera, or
Hemiptera.
22. The insect inhibitory composition of claim 21, wherein said at
least one other pesticidal protein is selected from the group
consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B,
Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras,
Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae,
Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15,
Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35,
ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834,
TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836,
TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-,
AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115,
AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184,
AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218,
AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z,
AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and
variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10,
DIG-657 and a DIG-11protein.
23. The insect inhibitory composition of claim 18, defined as
comprising a plant cell that expresses said recombinant nucleic
acid molecule.
24. A commodity product produced from the plant or part thereof of
claim 14, wherein the commodity product comprises a detectable
amount of said recombinant nucleic acid molecule or a pesticidal
protein encoded thereby.
25. The commodity product of claim 24, selected from the group
consisting of commodity corn bagged by a grain handler, corn
flakes, corn cakes, corn flour, corn meal, corn syrup, corn oil,
corn silage, corn starch, corn cereal, and the like, whole or
processed cotton seed, cotton oil, lint, seeds and plant parts
processed for feed or food, fiber, paper, biomasses, and fuel
products such as fuel derived from cotton oil or pellets derived
from cotton gin waste, whole or processed soybean seed, soybean
oil, soybean protein, soybean meal, soybean flour, soybean flakes,
soybean bran, soybean milk, soybean cheese, soybean wine, animal
feed comprising soybean, paper comprising soybean, cream comprising
soybean, soybean biomass, and fuel products produced using soybean
plants and soybean plant parts.
26. A method of producing seed comprising: a. planting at least a
first seed according to claim 17; b. growing a plant from the seed;
and c. harvesting seed from the plant, wherein said harvested seed
comprises said recombinant nucleic acid molecule.
27. A plant resistant to insect infestation, wherein the cells of
said plant comprise the recombinant nucleic acid molecule of claim
1.
28. A method for controlling a Coleopteran or Lepidopteran or
Hemipteran species pest or pest infestation, said method
comprising: a. contacting the pest with an insecticidally effective
amount of a pesticidal protein as set forth in SEQ ID NO:2; or b.
contacting the pest with an insecticidally effective amount of one
or more pesticidal proteins comprising an amino acid sequence
having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or
95%, or 98%, or 99%, or about 100% amino acid sequence identity to
SEQ ID NO:2.
29. A method of detecting the presence of the recombinant nucleic
acid molecule of claim 1 in a sample comprising plant genomic DNA,
comprising: a. contacting the sample with a nucleic acid probe that
hybridizes under stringent hybridization conditions with genomic
DNA from a plant comprising the DNA molecule of claim 1, and does
not hybridize under such hybridization conditions with genomic DNA
from an otherwise isogenic plant that does not comprise the
recombinant nucleic acid molecule of claim 1, wherein the probe is
homologous or complementary to SEQ ID NO:1, SEQ ID NO:3, or a
sequence that encodes a pesticidal protein comprising an amino acid
sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or
90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence
identity to SEQ ID NO:2; b. subjecting the sample and probe to
stringent hybridization conditions; and c. detecting hybridization
of the probe with DNA of the sample.
30. A method of detecting the presence of a pesticidal protein or
fragment thereof in a sample comprising protein, wherein said
pesticidal protein comprises the amino acid sequence of SEQ ID
NO:2; or said pesticidal protein comprises an amino acid sequence
having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or
95%, or 98%, or 99% or about 100% amino acid sequence identity to
SEQ ID NO:2; comprising: a. contacting the sample with an
immunoreactive antibody; and b. detecting the presence of the
protein.
31. The method of claim 30, wherein the step of detecting comprises
an ELISA, or a Western blot.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/199,024, filed Jul. 30, 2015, which is herein
incorporated by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The file named "MONS394US_sequence_listing.txt" containing a
computer-readable form of the Sequence Listing was created on Jul.
19, 2016. This file is 16,077 bytes (measured in MS-Windows.RTM.),
is contemporaneously filed by electronic submission (using the
United States Patent Office EFS-Web filing system), and is
incorporated into this application by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention generally relates to the field of insect
inhibitory proteins. A novel class of proteins exhibiting insect
inhibitory activity against agriculturally-relevant pests of crop
plants and seeds is disclosed. In particular, the disclosed protein
is insecticidally active against agriculturally-relevant pests of
crop plants and seeds, particularly Coleopteran, Lepidopteran, and
Hemipteran species of insect pests. Plants, plant parts, and seeds
containing a recombinant polynucleotide construct encoding one or
more of the disclosed toxin proteins are provided.
BACKGROUND OF THE INVENTION
[0004] Improving crop yield from agriculturally significant plants
including, among others, corn, soybean, sugarcane, rice, wheat,
vegetables, and cotton, has become increasingly important. In
addition to the growing need for agricultural products to feed,
clothe and provide energy for a growing human population,
climate-related effects and pressure from the growing population to
use land other than for agricultural practices are predicted to
reduce the amount of arable land available for farming. These
factors have led to grim forecasts of food security, particularly
in the absence of major improvements in plant biotechnology and
agronomic practices. In light of these pressures, environmentally
sustainable improvements in technology, agricultural techniques,
and pest management are vital tools to expand crop production on
the limited amount of arable land available for farming.
[0005] Insects, particularly insects within the Lepidoptera,
Coleoptera and Hemipteran orders, are considered a major cause of
damage to field crops, thereby decreasing crop yields over infested
areas. Lepidopteran pest species which negatively impact
agriculture include, but are not limited to, Helicoverpa zea,
Ostrinia nubilalis, Diatraea saccharalis, Diatraea grandiosella,
Anticarsia gemmatalis, Spodoptera frugiperda, Spodoptera exigua,
Agrotis ipsilon, Trichoplusia ni, Chrysodeixis includens, Heliothis
virescens, Plutella xylostella, Pectinophora gossypiella,
Helicoverpa armigera, Elasmopalpus lignosellus, Striacosta
albicosta and Phyllocnistis citrella. Coleopteran pest species
which negatively impact agriculture include, but are not limited
to, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema
tibialis, Cosmopolites spp., Curculio spp., Dermestes spp.,
Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa
decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus
spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes
spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga
spp., Tenebrio spp., Tribolium spp. and Trogoderma spp,
particularly wherein the pest is Diabrotica virgifera virgifera
(Western Corn Rootworm, WCR), Diabrotica barberi (Northern Corn
Rootworm, NCR), Diabrotica virgifera zeae (Mexican Corn Rootworm,
MCR), Diabrotica balteata (Brazilian Corn Rootworm (BZR),
Diabrotica undecimpunctata howardii (Southern Corn Rootworm, SCR),
and a Brazilian Corn Rootworm complex (BCR) consisting of
Diabrotica viridula and Diabrotica speciosa). Hemipteran pest
species which negatively impact agriculture include, but are not
limited to, Lygus hesperus, Lygus lineolaris, and Pseudatomoscelis
seriatus.
[0006] Historically, the intensive application of synthetic
chemical insecticides was relied upon as the pest control agent in
agriculture. Concerns for the environment and human health, in
addition to emerging resistance issues, stimulated the research and
development of biological pesticides. This research effort led to
the progressive discovery and use of various entomopathogenic
microbial species, including bacteria.
[0007] The biological control paradigm shifted when the potential
of entomopathogenic bacteria, especially bacteria belonging to the
genus Bacillus, was discovered and developed as a biological pest
control agent. Strains of the bacterium Bacillus thuringiensis (Bt)
have been used as a source for pesticidal proteins since it was
discovered that Bt strains show a high toxicity against specific
insects. Bt strains are known to produce delta-endotoxins that are
localized within parasporal crystalline inclusion bodies at the
onset of sporulation and during the stationary growth phase (e.g.,
Cry proteins), and are also known to produce secreted insecticidal
protein. Upon ingestion by a susceptible insect, delta-endotoxins
as well as secreted toxins exert their effects at the surface of
the midgut epithelium, disrupting the cell membrane, leading to
cell disruption and death. Genes encoding insecticidal proteins
have also been identified in bacterial species other than Bt,
including other Bacillus and a diversity of additional bacterial
species, such as Brevibacillus laterosporus, Lysinibacillus
sphaericus ("Ls" formerly known as Bacillus sphaericus) and
Paenibacillus popilliae.
[0008] Crystalline and secreted soluble insecticidal toxins are
highly specific for their hosts and have gained worldwide
acceptance as alternatives to chemical insecticides. For example,
insecticidal toxin proteins have been employed in various
agricultural applications to protect agriculturally important
plants from insect infestations, decrease the need for chemical
pesticide applications, and increase yields. Insecticidal toxin
proteins are used to control agriculturally-relevant pests of crop
plants by mechanical methods, such as spraying to disperse
microbial formulations containing various bacteria strains onto
plant surfaces, and by using genetic transformation techniques to
produce transgenic plants and seeds expressing insecticidal toxin
protein.
[0009] The use of transgenic plants expressing insecticidal toxin
proteins has been globally adapted. For example, in 2012, 26.1
million hectares were planted with transgenic crops expressing Bt
toxins (James, C., Global Status of Commercialized Biotech/GM
Crops: 2012. ISAAA Brief No. 44). The global use of transgenic
insect-protected crops and the limited number of insecticidal toxin
proteins used in these crops has created a selection pressure for
existing insect alleles that impart resistance to the
currently-utilized insecticidal proteins.
[0010] The development of resistance in target pests to
insecticidal toxin proteins creates the continuing need for
discovery and development of new forms of insecticidal toxin
proteins that are useful for managing the increase in insect
resistance to transgenic crops expressing insecticidal toxin
proteins. New protein toxins with improved efficacy and which
exhibit control over a broader spectrum of susceptible insect
species will reduce the number of surviving insects which can
develop resistance alleles. In addition, the use in one plant of
two or more transgenic insecticidal toxin proteins toxic to the
same insect pest and displaying different modes of action reduces
the probability of resistance in any single target insect
species.
[0011] Thus the inventors herein disclose a novel protein toxin
family from Bacillus thuringiensis along with similar toxin
proteins, variant proteins, and exemplary recombinant proteins that
exhibit insecticidal activity against target Lepidopteran,
Coleopteran and Hemipteran pest species, particularly against
Western Corn Rootworm.
SUMMARY OF THE INVENTION
[0012] Disclosed herein is a novel group of pesticidal proteins
with insect inhibitory activity (toxin proteins), referred to
herein as TIC5290, which are shown to exhibit inhibitory activity
against one or more pests of crop plants. The TIC5290 protein and
proteins in the TIC5290 protein toxin class can be used alone or in
combination with other insecticidal proteins and toxic agents in
formulations and in planta, thus providing alternatives to
insecticidal proteins and insecticide chemistries currently in use
in agricultural systems.
[0013] In one embodiment, disclosed in this application is a
recombinant nucleic acid molecule comprising a heterologous
promoter operably linked to a polynucleotide segment encoding a
pesticidal protein or fragment thereof, wherein: (a) said
pesticidal protein comprises the amino acid sequence of SEQ ID
NO:2; or (b) said pesticidal protein comprises an amino acid
sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or
90%, or 95%, or 98%, or 99% or about 100% amino acid sequence
identity to SEQ ID NO:2; or (c) said polynucleotide segment
hybridizes to a polynucleotide having the nucleotide sequence of
SEQ ID NO:1 or SEQ ID NO:3; or (d) said polynucleotide segment
encoding a pesticidal protein or fragment thereof comprises a
polynucleotide sequence having at least 65%, or 70%, or 75%, or
80%, or 85%, or 90%, or 95%, or 98%, or 99% or about 100% sequence
identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3;
or (e) said recombinant nucleic acid molecule is in operable
linkage with a vector, and said vector is selected from the group
consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial
or yeast artificial chromosome. The recombinant nucleic acid
molecule can comprise a sequence that functions to express the
pesticidal protein in a plant; or is expressed in a plant cell to
produce a pesticidally effective amount of pesticidal protein.
[0014] In another embodiment of this application are host cells
comprising a recombinant nucleic acid molecule of the application,
wherein the host cell is selected from the group consisting of a
bacterial and a plant cell. Contemplated host cells include
Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia,
Pseudomonas, Klebsiella, Pantoea, and Erwinia. In certain
embodiments said Bacillus species is Bacillus cereus or Bacillus
thuringiensis, said Brevibacillus is Brevibacillus laterosperus, or
said Escherichia is Escherichia coli. Contemplated plant host cells
include a dicotyledonous cell and a monocotyledonous cell. Further
contemplated plant host cells include an alfalfa, banana, barley,
bean, broccoli, cabbage, Brassica, carrot, cassava, castor,
cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut,
coffee, corn, clover, cotton (Gossypium sp.), a cucurbit, cucumber,
Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek,
lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion,
ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea,
pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed,
rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine,
soybean, spinach, squash, strawberry, sugar beet, sugarcane,
sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,
tobacco, tomato, triticale, turf grass, watermelon, and wheat plant
cell.
[0015] In yet another embodiment, the pesticidal protein exhibits
activity against Coleopteran insect, including Western Corn
Rootworm, Southern Corn Rootworm, Northern Corn Rootworm, Mexican
Corn Rootworm, Brazilian Corn Rootworm, or Brazilian Corn Rootworm
complex consisting of Diabrotica viridula and Diabrotica
speciosa.
[0016] In another embodiment, the pesticidal protein exhibits
activity against a Lepidopteran insect, including Velvet bean
caterpillar, Sugarcane borer, Lesser cornstalk borer, Corn earworm,
Tobacco budworm, Soybean looper, Black armyworm, Southern armyworm,
Fall armyworm, Beet armyworm, Old World bollworm, Oriental leaf
worm, Pink bollworm, Black cutworm, Southwestern Corn Borer,
Diamondback moth, or European corn borer.
[0017] In yet another embodiment, the pesticidal protein exhibits
activity against a Hemipteran insect, including Western tarnished
plant bug, Tarnished plant bug, or Cotton fleahopper.
[0018] Also contemplated in this application are plants comprising
a recombinant nucleic acid molecule comprising a heterologous
promoter operably linked to a polynucleotide segment encoding a
pesticidal protein or fragment thereof, wherein: (a) said
pesticidal protein comprises the amino acid sequence of SEQ ID
NO:2; or (b) said pesticidal protein comprises an amino acid
sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or
90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence
identity to SEQ ID NO:2; or (c) said polynucleotide segment
hybridizes under stringent hybridization conditions to the
compliment of the nucleotide sequence of SEQ ID NO:1 or SEQ ID
NO:3; or (d) said plant exhibits a detectable amount of said
pesticidal protein. In certain embodiments the pesticidal protein
comprises SEQ ID NO:2. In one embodiment, the plant is either a
monocot or a dicot. In another embodiment, the plant is selected
from the group consisting of an alfalfa, banana, barley, bean,
broccoli, cabbage, Brassica, carrot, cassava, castor, cauliflower,
celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,
clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant,
eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly
pine, millets, melons, nut, oat, olive, onion, ornamental, palm,
pasture grass, pea, peanut, pepper, pigeon pea, pine, potato,
poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks,
rye, safflower, shrub, sorghum, Southern pine, soybean, spinach,
squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn,
sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,
triticale, turf grass, watermelon, and wheat.
[0019] In further embodiments, seeds comprising the recombinant
nucleic acid molecules are disclosed.
[0020] In another embodiment, an insect inhibitory composition
comprising the recombinant nucleic acid molecules disclosed in this
application are contemplated. The insect inhibitory composition can
further comprise a nucleotide sequence encoding at least one other
pesticidal agent that is different from said pesticidal protein.
The at least one other pesticidal agent is selected from the group
consisting of an insect inhibitory protein, an insect inhibitory
dsRNA molecule, and an ancillary protein. The at least one other
pesticidal agent in the insect inhibitory composition exhibits
activity against one or more pest species of the orders
Lepidoptera, Coleoptera, or Hemiptera. The at least one other
pesticidal agent in the insect inhibitory composition is in one
embodiment selected from the group consisting of: a Cry1A, Cry1Ab,
Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D,
Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K,
Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B,
Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B,
Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407,
TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901,
TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869,
TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-, AXMI-88, AXMI-97,
AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and
AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196,
AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220,
AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238,
AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants
thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657
and a DIG-11protein.
[0021] Commodity products comprising a detectable amount of the
recombinant nucleic acid molecules disclosed in this application
are contemplated. Such commodity products include commodity corn
bagged by a grain handler, corn flakes, corn cakes, corn flour,
corn meal, corn syrup, corn oil, corn silage, corn starch, corn
cereal, and the like, and corresponding cotton commodity products
such as whole or processed cotton seed, cotton oil, lint, seeds and
plant parts processed for feed or food, fiber, paper, biomasses,
and fuel products such as fuel derived from cotton oil or pellets
derived from cotton gin waste, and corresponding soybean commodity
products such as whole or processed soybean seed, soybean oil,
soybean protein, soybean meal, soybean flour, soybean flakes,
soybean bran, soybean milk, soybean cheese, soybean wine, animal
feed comprising soybean, paper comprising soybean, cream comprising
soybean, soybean biomass, and fuel products produced using soybean
plants and soybean plant parts, and corresponding rice, wheat,
sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity
products including where applicable, juices, concentrates, jams,
jellies, marmalades, and other edible forms of such commodity
products containing a detectable amount of such polynucleotides and
or polypeptides of this application.
[0022] Also contemplated in this application is a method of
producing seed comprising the recombinant nucleic acid molecules
disclosed in this application. The method comprises planting at
least one of the seed comprising the recombinant nucleic acid
molecules disclosed in this application; growing plant from the
seed; and harvesting seed from the plants, wherein the harvested
seed comprises the recombinant nucleic acid molecules in this
application.
[0023] In another illustrative embodiment, a plant resistant to
insect infestation is provided, wherein the cells of said plant
comprise: (a) a recombinant nucleic acid molecule encoding an
insecticidally effective amount of a pesticidal protein as set
forth in SEQ ID NO:2; or (b) an insecticidally effective amount of
a protein comprising an amino acid sequence having at least 65%, or
70%, or 75%, or 80%, or 85%, or 90%, or 95%, or about 100% amino
acid sequence identity to SEQ ID NO:2.
[0024] Also disclosed in this application are methods for
controlling a Coleopteran or Lepidopteran or Hemipteran species
pest, and controlling a Coleopteran or Lepidopteran or Hemipteran
species pest infestation of a plant, particularly a crop plant. The
method comprises, in one embodiment, (a) contacting the pest with
an insecticidally effective amount of one or more pesticidal
proteins as set forth in SEQ ID NO:2; or (b) contacting the pest
with an insecticidally effective amount of one or more pesticidal
proteins comprising an amino acid sequence having at least 65%, or
70%, or 75%, or 80%, or 85%, or 90%, or 95%, or about 100% amino
acid sequence identity to SEQ ID NO:2.
[0025] Further provided herein is a method of detecting the
presence of a recombinant nucleic acid molecule comprising a
polynucleotide segment encoding a pesticidal protein or fragment
thereof, wherein: (a) said pesticidal protein comprises the amino
acid sequence of SEQ ID NO:2; or (b) said pesticidal protein
comprises an amino acid sequence having at least 65%, or 70%, or
75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100%
amino acid sequence identity to SEQ ID NO:2; or (c) said
polynucleotide segment hybridizes to a polynucleotide having the
nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In one
embodiment of the invention, the method comprises contacting a
sample of nucleic acids with a nucleic acid probe that hybridizes
under stringent hybridization conditions with genomic DNA from a
plant comprising a polynucleotide segment encoding a pesticidal
protein or fragment thereof provided herein, and does not hybridize
under such hybridization conditions with genomic DNA from an
otherwise isogenic plant that does not comprise the segment,
wherein the probe is homologous or complementary to SEQ ID NO:1,
SEQ ID NO:3, or a sequence that encodes a pesticidal protein
comprising an amino acid sequence having at least 65%, or 70%, or
75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100%
amino acid sequence identity to SEQ ID NO:2. The method may further
comprise (a) subjecting the sample and probe to stringent
hybridization conditions; and (b) detecting hybridization of the
probe with DNA of the sample.
[0026] Also provided by the invention are methods of detecting the
presence of a pesticidal protein or fragment thereof in a sample
comprising protein, wherein said pesticidal protein comprises the
amino acid sequence of SEQ ID NO:2; or said pesticidal protein
comprises an amino acid sequence having at least 65%, or 70%, or
75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100%
amino acid sequence identity to SEQ ID NO:2. In one embodiment, the
method comprises: (a) contacting a sample with an immunoreactive
antibody; and (b) detecting the presence of the protein. In some
embodiments the step of detecting comprises an ELISA, or a Western
blot.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 illustrates in planta Western Corn Rootworm (WCR)
inhibitory activity of exemplary chloroplast targeted and
non-targeted TIC5290 proteins.
BRIEF DESCRIPTION OF THE SEQUENCES
[0028] SEQ ID NO:1 is a nucleic acid encoding a TIC5290 pesticidal
protein sequence obtained from Bacillus thuringiensis species
EG6657.
[0029] SEQ ID NO:2 is the amino acid sequence of the TIC5290
protein.
[0030] SEQ ID NO:3 is a synthetic coding sequence encoding a
TIC5290 pesticidal protein used for expression in plant cells.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The problem in the art of agricultural pest control can be
characterized as a need for new toxin proteins that are efficacious
against target pests, exhibit broad spectrum toxicity against
target pest species, are capable of being expressed in plants
without causing undesirable agronomic issues, and provide an
alternative mode of action compared to current toxins that are used
commercially in plants.
[0032] Novel insecticidal proteins are disclosed herein,
exemplified by TIC5290 and related family members that provide
resistance against Coleopteran, Lepidopteran, and Hemipteran insect
pests, and more particularly against corn rootworm pest species.
Also disclosed are synthetic coding sequences designed for
expression in a plant cell that encode TIC5290. Further disclosed
are recombinant nucleic acid molecules comprising a promoter in
operable linkage to a coding sequence encoding a TIC5290 toxin
protein, or related family members, or fragments thereof.
[0033] Reference in this application to TIC5290, "TIC5290 protein",
"TIC5290 protein toxin", "TIC5290 toxin protein", "TIC5290
pesticidal protein", "TIC5290-related toxins", or "TIC5290-related
toxin protein", and the like, refer to any novel pesticidal protein
or insect inhibitory protein, that comprises, that consists of,
that is substantially homologous to, that is similar to, or that is
derived from any pesticidal protein or insect inhibitory protein
sequence of TIC5290 (SEQ ID NO:2) and pesticidal or insect
inhibitory segments thereof, or combinations thereof, that confer
activity against Coleopteran pests, Lepidopteran pests, and
Hemipteran pests, including any protein exhibiting pesticidal or
insect inhibitory activity if alignment of such protein with
TIC5290 results in amino acid sequence identity of any fraction
percentage from about 65 to about 100 percent.
[0034] The term "segment" or "fragment" is used in this application
to describe consecutive amino acid or nucleic acid sequences that
are shorter than the complete amino acid or nucleic acid sequence
describing the TIC5290 protein or related family member
insecticidal protein. A segment or fragment exhibiting insect
inhibitory activity is also disclosed in this application if
alignment of such segment or fragment, with the corresponding
section of the TIC5290 protein set forth in SEQ ID NO:2, results in
amino acid sequence identity of any fraction percentage from about
65 to about 100 percent between the segment or fragment and the
corresponding section of the TIC5290 protein.
[0035] Reference in this application to the terms "active" or
"activity", "pesticidal activity" or "pesticidal" or "insecticidal
activity", "insect inhibitory" or "insecticidal" refer to efficacy
of a toxic agent, such as a protein toxin, in inhibiting
(inhibiting growth, feeding, fecundity, or viability), suppressing
(suppressing growth, feeding, fecundity, or viability), controlling
(controlling the pest infestation, controlling the pest feeding
activities on a particular crop containing an effective amount of
the TIC5290 protein) or killing (causing the morbidity, mortality,
or reduced fecundity of) a pest. These terms are intended to
include the result of providing a pesticidally effective amount of
a toxic protein to a pest where the exposure of the pest to the
toxic protein results in morbidity, mortality, reduced fecundity,
or stunting. These terms also include repulsion of the pest from
the plant, a tissue of the plant, a plant part, seed, plant cells,
or from the particular geographic location where the plant may be
growing, as a result of providing a pesticidally effective amount
of the toxic protein in or on the plant. In general, pesticidal
activity refers to the ability of a toxic protein to be effective
in inhibiting the growth, development, viability, feeding behavior,
mating behavior, fecundity, or any measurable decrease in the
adverse effects caused by an insect feeding on this protein,
protein fragment, protein segment or polynucleotide of a particular
target pest, including but not limited to insects of the order
Lepidoptera, Coleoptera or Hemiptera. The toxic protein can be
produced by the plant or can be applied to the plant or to the
environment within the location where the plant is located. The
terms "bioactivity", "effective", "efficacious" or variations
thereof are also terms interchangeably utilized in this application
to describe the effects of proteins of the present invention on
target insect pests.
[0036] A pesticidally effective amount of a toxic agent, when
provided in the diet of a target pest, exhibits pesticidal activity
when the toxic agent contacts the pest. A toxic agent can be a
pesticidal protein or one or more chemical agents known in the art.
Pesticidal or insecticidal chemical agents and pesticidal or
insecticidal protein agents can be used alone or in combinations
with each other. Chemical agents include, but are not limited to,
dsRNA molecules targeting specific genes for suppression in a
target pest, organochlorides, organophosphates, carbamates,
pyrethroids, neonicotinoids, and ryanoids. Pesticidal or
insecticidal protein agents include the protein toxins set forth in
this application, as well as other proteinaceous toxic agents
including those that target Lepidopteran, Coleopteran and
Hemipteran pest species, as well as protein toxins that are used to
control other plant pests such as Cry proteins available in the art
for use in controlling Homopteran species.
[0037] It is intended that reference to a pest, particularly a pest
of a crop plant, means insect pests of crop plants, particularly
those that are controlled by the TIC5290 protein. However,
reference to a pest can also include Homopteran insect pests of
plants, as well as nematodes and fungi when toxic agents targeting
these pests are co-localized or present together with the TIC5290
protein, or a protein that is about 65 to about 100 percent
identical to TIC5290.
[0038] The insecticidal proteins of the TIC5290 protein toxin class
are related by common function and exhibit insecticidal activity
towards insect pests from the Coleopteran and Lepidopteran insect
species, including adults, pupae, larvae, and neonates, as well as
Hemipteran insect species, including adults and nymphs.
[0039] The insects of the order Lepidoptera include, but are not
limited to, armyworms, cutworms, loopers, and heliothines in the
Family Noctuidae, e.g., fall armyworm (Spodoptera frugiperda), beet
armyworm (Spodoptera exigua), bertha armyworm (Mamestra
configurata), Southern armyworm (Spodoptera eridania), black
cutworm (Agrotis ipsilon), cabbage looper (Trichoplusia ni),
soybean looper (Pseudoplusia includens), velvetbean caterpillar
(Anticarsia gemmatalis), green cloverworm (Hypena scabra), tobacco
budworm (Heliothis virescens), granulate cutworm (Agrotis
subterranea), armyworm (Pseudaletia unipuncta), western cutworm
(Agrotis orthogonia); borers, casebearers, webworms, coneworms,
cabbageworms and skeletonizers from the Family Pyralidae, e.g.,
European corn borer (Ostrinia nubilalis), navel orangeworm
(Amyelois transitella), corn root webworm (Crambus caliginosellus),
sod webworm (Herpetogramma licarsisalis), sunflower moth
(Homoeosoma electellum), lesser cornstalk borer (Elasmopalpus
lignosellus); leafrollers, budworms, seed worms, and fruit worms in
the Family Tortricidae, e.g., codling moth (Cydia pomonella), grape
berry moth (Endopiza viteana), oriental fruit moth (Grapholita
molesta), sunflower bud moth (Suleima helianthana); and many other
economically important Lepidoptera, e.g., diamondback moth
(Plutella xylostella), pink bollworm (Pectinophora gossypiella) and
gypsy moth (Lymantria dispar). Other insect pests of order
Lepidoptera include, e.g., cotton leaf worm (Alabama argillacea),
fruit tree leaf roller (Archips argyrospila), European leafroller
(Archips rosana) and other Archips species, (Chilo suppressalis,
Asiatic rice borer, or rice stem borer), rice leaf roller
(Cnaphalocrocis medinalis), corn root webworm (Crambus
caliginosellus), bluegrass webworm (Crambus teterrellus),
southwestern corn borer (Diatraea grandiosella), surgarcane borer
(Diatraea saccharalis), spiny bollworm (Earias insulana), spotted
bollworm (Earias vittella), Old World bollworm (Helicoverpa
armigera), corn earworm (Helicoverpa zea, also known as soybean
podworm and cotton bollworm), tobacco budworm (Heliothis
virescens), sod webworm (Herpetogramma licarsisalis), European
grape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistis
citrella), large white butterfly (Pieris brassicae), small white
butterfly (Pieris rapae, also known as imported cabbageworm),
diamondback moth (Plutella xylostella), beet armyworm (Spodoptera
exigua), tobacco cutworm (Spodoptera litura, also known as cluster
caterpillar), and tomato leafminer (Tuta absoluta).
[0040] The insects of the order Coleoptera include, but are not
limited to, Agriotes spp., Anthonomus spp., Atomaria linearis,
Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes
spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa
decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus
spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes
spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga
spp., Tenebrio spp., Tribolium spp. and Trogoderma spp,
particularly when the pest is Western Corn Rootworm (Diabrotica
virgifera, WCR), Northern Corn Rootworm (Diabrotica barberi, NCR),
Mexican Corn Rootworm (Diabrotica virgifera zeae, MCR), Brazilian
Corn Rootworm (Diabrotica balteata, BZR), Southern Corn Rootworm
(Diabrotica undecimpunctata howardii, SCR) and a Brazilian Corn
Rootworm complex (BCR, consisting of Diabrotica viridula and
Diabrotica speciosa).
[0041] The insects of Hemiptera include, but are not limited to,
Western tarnished plant bug (Lygus hesperus), Tarnished plant bug
(Lygus lineolaris), and Cotton fleahopper (Pseudatomoscelis
seriatus).
[0042] Reference in this application to an "isolated DNA molecule",
or an equivalent term or phrase, is intended to mean that the DNA
molecule is one that is present alone or in combination with other
compositions, but not within its natural environment. For example,
nucleic acid elements such as a coding sequence, intron sequence,
untranslated leader sequence, promoter sequence, transcriptional
termination sequence, and the like, that are naturally found within
the DNA of the genome of an organism are not considered to be
"isolated" so long as the element is within the genome of the
organism and at the location within the genome in which it is
naturally found. However, each of these elements, and subparts of
these elements, would be "isolated" within the scope of this
disclosure so long as the element is not within the genome of the
organism and at the location within the genome in which it is
naturally found. Similarly, a nucleotide sequence encoding an
insecticidal protein or any naturally occurring insecticidal
variant of that protein would be an isolated nucleotide sequence so
long as the nucleotide sequence was not within the DNA of the
bacterium from which the sequence encoding the protein is naturally
found. A synthetic nucleotide sequence encoding the amino acid
sequence of the naturally occurring insecticidal protein would be
considered to be isolated for the purposes of this disclosure. For
the purposes of this disclosure, any transgenic nucleotide
sequence, i.e., the nucleotide sequence of the DNA inserted into
the genome of the cells of a plant or bacterium, or present in an
extrachromosomal vector, would be considered to be an isolated
nucleotide sequence whether it is present within the plasmid or
similar structure used to transform the cells, within the genome of
the plant or bacterium, or present in detectable amounts in
tissues, progeny, biological samples or commodity products derived
from the plant or bacterium.
[0043] As described further herein, an open reading frame (ORF)
encoding TIC5290 (SEQ ID NO:1), was discovered in DNA obtained from
Bacillus thuringiensis strain EG6657. The coding sequence was
cloned and expressed in microbial host cells to produce protein
(SEQ ID NO:2) used in bioassays. The closest toxin homolog to
TIC5290 is the Vip4Aa protein with a sequence identity of 56.9%,
indicating that TIC5290 represents a novel Vip4 subfamily. Bioassay
using microbial host cell-derived proteins of TIC5290 demonstrated
activity against the Coleopteran pest Western Corn Rootworm
(Diabrotica virgifera virgifera, WCR); the Lepidopteran species
Fall armyworm (Spodoptera frugiperda, FAW), Corn earworm
(Helicoverpa zea, CEW), European corn borer (Ostrinia nubilalis),
and Diamondback moth (Plutella xylostella); as well as the
Hemipteran pest Western tarnished plant bug (Lygus hesperus).
[0044] It is contemplated that additional toxin protein sequences
related to TIC5290 can be created by using the naturally occurring
amino acid sequence of TIC5290 to create novel proteins and with
novel properties. The TIC5290 toxin protein can be aligned with
other proteins similar to TIC5290 to combine differences at the
amino acid sequence level into novel amino acid sequence variants
and making appropriate changes to the recombinant nucleic acid
sequence encoding the variants.
[0045] It is further contemplated that improved variants of TIC5290
can be engineered in planta by using various gene editing methods
known in the art. Such technologies used for genome editing
include, but are not limited to, ZFN (zinc-finger nuclease),
meganucleases, TALEN (Transcription activator-like effector
nucleases), and CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats)/Cas (CRISPR-associated) systems. These genome
editing methods can be used to alter the toxin protein coding
sequence transformed within a plant cell to a different toxin
coding sequence. Specifically, through these methods, one or more
codons within the toxin coding sequence is altered to engineer a
new protein amino acid sequence. Alternatively, a fragment within
the coding sequence is replaced or deleted, or additional DNA
fragments are inserted into the coding sequence, to engineer a new
toxin coding sequence. The new coding sequence can encode a toxin
protein with new properties such as increased activity or spectrum
against insect pests, as well as provide activity against an insect
pest species wherein resistance has developed against the original
insect toxin protein. The plant cell comprising the gene edited
toxin coding sequence can be used by methods known in the art to
generate whole plants expressing the new toxin protein.
[0046] It is also contemplated that fragments of the TIC5290
protein or protein variants thereof can be truncated forms wherein
one or more amino acids are deleted from the N-terminal end,
C-terminal end, the middle of the protein, or combinations thereof
with insect inhibitory activity. These fragments can be naturally
occurring or synthetic variants of TIC5290 or derived protein
variants, but should retain the insect inhibitory activity of
TIC5290.
[0047] Proteins that resemble the TIC5290 protein can be identified
by comparison to each other using various computer based algorithms
known in the art. For example, amino acid sequence identities of
proteins related to TIC5290 can be analyzed using a Clustal W
alignment using these default parameters: Weight matrix: blosum,
Gap opening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic
gaps: On, Hydrophilic residues: GPSNDQERK, Residue-specific gap
penalties: On (Thompson, et al (1994) Nucleic Acids Research,
22:4673-4680). Percent amino acid identity is further calculated by
the product of 100% multiplied by (amino acid identities/length of
subject protein). Other alignment algorithms are also available in
the art and provide results similar to those obtained using a
Clustal W alignment.
[0048] It is intended that a protein exhibiting insect inhibitory
activity against a Lepidopteran, Coleopteran or Hemipteran insect
species is related to TIC5290 if alignment of such query protein
with TIC5290 exhibits at least 65% to about 100% amino acid
identity along the length of the query protein that is about 65%,
66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 100% amino acid sequence identity (or any
fraction of a percentage in this range) between query and subject
protein.
[0049] The TIC5290 protein can also be related by primary structure
(conserved amino acid motifs), by length (about 937 amino acids)
and by other characteristics. Bioinformatic analysis suggests that
TIC5290 is a pore-forming protein, has a PA14 Pfam domain (PF07691)
that is likely involved with binding functions to cell receptor(s)
on the target insect midgut is detected in amino acids 16-140,
followed by a Binary_toxB Pfam domain (PF03495) in amino acids
186-593 that might contribute to the formation of a beta barrel
transmembrane pore. Both of these Pfams likely contribute to
insecticidal activity of the protein. Characteristics of the
TIC5290 protein toxin are reported in Table 1.
TABLE-US-00001 TABLE 1 Selected characteristics of the TIC5290
protein. No. of No. of Molecular Strongly Strongly No. of No. of
Weight Amino Basic (-) Acidic Hydrophobic Polar (in Acid
Isoelectric Charge Amino Amino Amino Amino Protein Daltons) Length
Point at PH 7.0 Acids Acids Acids Acids TIC5290 104962.93 937
6.7076 1.0 116 110 440 497
[0050] As described further in the Examples of this application,
recombinant nucleic acid molecule sequences encoding TIC5290 were
designed for use in plants. An exemplary plant-optimized
recombinant nucleic acid molecule sequence that was designed for
use in plants encoding the TIC5290 protein is set forth in SEQ ID
NO:3.
[0051] Expression cassettes and vectors containing these synthetic
nucleic acid molecule sequences can be constructed and introduced
into corn, soybean, cotton or other plant cells in accordance with
transformation methods and techniques known in the art. For
example, Agrobacterium-mediated transformation is described in U.S.
Patent Application Publications 2009/0138985A1 (soybean),
2008/0280361A1 (soybean), 2009/0142837A1 (corn), 2008/0282432
(cotton), 2008/0256667 (cotton), 2003/0110531 (wheat), 2001/0042257
A1 (sugar beet), U.S. Pat. No. 5,750,871 (canola), U.S. Pat. No.
7,026,528 (wheat), and U.S. Pat. No. 6,365,807 (rice), and in
Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugarcane) all
of which are incorporated herein by reference in their entirety.
Transformed cells can be regenerated into transformed plants that
express TIC5290 and demonstrate pesticidal activity through
bioassays performed in the presence of Lepidoptera or Hemiptera
pest larvae using plant leaf disks obtained from transformed
plants. To test pesticidal activity against Coleopteran pests,
transformed plants of R.sub.o and F.sub.1 generation are used in
root worm assay as described in the example below.
[0052] As an alternative to traditional transformation methods, a
DNA sequence, such as a transgene, expression cassette(s), etc.,
may be inserted or integrated into a specific site or locus within
the genome of a plant or plant cell via site-directed integration.
Recombinant DNA construct(s) and molecule(s) of this disclosure may
thus include a donor template sequence comprising at least one
transgene, expression cassette, or other DNA sequence for insertion
into the genome of the plant or plant cell. Such donor template for
site-directed integration may further include one or two homology
arms flanking an insertion sequence (i.e., the sequence, transgene,
cassette, etc., to be inserted into the plant genome). The
recombinant DNA construct(s) of this disclosure may further
comprise an expression cassette(s) encoding a site-specific
nuclease and/or any associated protein(s) to carry out
site-directed integration. These nuclease expressing cassette(s)
may be present in the same molecule or vector as the donor template
(in cis) or on a separate molecule or vector (in trans). Several
methods for site-directed integration are known in the art
involving different proteins (or complexes of proteins and/or guide
RNA) that cut the genomic DNA to produce a double strand break
(DSB) or nick at a desired genomic site or locus. As understood in
the art, during the process of repairing the DSB or nick introduced
by the nuclease enzyme, the donor template DNA may become
integrated into the genome at the site of the DSB or nick. The
presence of the homology arm(s) in the donor template may promote
the adoption and targeting of the insertion sequence into the plant
genome during the repair process through homologous recombination,
although an insertion event may occur through non-homologous end
joining (NHEJ). Examples of site-specific nucleases that may be
used include zinc-finger nucleases, engineered or native
meganucleases, TALE-endonucleases, and RNA-guided endonucleases
(e.g., Cas9 or Cpf1). For methods using RNA-guided site-specific
nucleases (e.g., Cas9 or Cpf1), the recombinant DNA construct(s)
will also comprise a sequence encoding one or more guide RNAs to
direct the nuclease to the desired site within the plant
genome.
[0053] Recombinant nucleic acid molecule compositions that encode
TIC5290 are contemplated. For example, TIC5290 protein can be
expressed with recombinant DNA constructs in which a polynucleotide
molecule with an ORF encoding the protein is operably linked to
genetic expression elements such as a promoter and any other
regulatory element necessary for expression in the system for which
the construct is intended. Non-limiting examples include a
plant-functional promoter operably linked to the TIC5290 protein
encoding sequences for expression of the protein in plants or a
Bt-functional promoter operably linked to a TIC5290 protein
encoding sequence for expression of the protein in a Bt bacterium
or other Bacillus species. Other elements can be operably linked to
the TIC5290 protein encoding sequences including, but not limited
to, enhancers, introns, untranslated leaders, encoded protein
immobilization tags (HIS-tag), translocation peptides (i.e.,
plastid transit peptides, signal peptides), polypeptide sequences
for post-translational modifying enzymes, ribosomal binding sites,
and RNAi target sites. Exemplary recombinant polynucleotide
molecules provided herewith include, but are not limited to, a
heterologous promoter operably linked to a polynucleotide such as
SEQ ID NO:1 and SEQ ID NO:3 that encodes the polypeptide or protein
having the amino acid sequence as set forth in SEQ ID NO:2. A
heterologous promoter can also be operably linked to synthetic DNA
coding sequences encoding a plastid targeted TIC5290 and untargeted
TIC5290. The codons of a recombinant nucleic acid molecule encoding
for protein disclosed herein can be substituted by synonymous
codons (known in the art as a silent substitution).
[0054] A recombinant DNA construct comprising a TIC5290 protein
encoding sequence can further comprise a region of DNA that encodes
for one or more insect inhibitory agents which can be configured to
concomitantly express or co-express with a DNA sequence encoding a
TIC5290 protein, a protein different from a TIC5290 protein, an
insect inhibitory dsRNA molecule, or an ancillary protein.
Ancillary proteins include, but are not limited to, co-factors,
enzymes, binding-partners, or other agents that function to aid in
the effectiveness of an insect inhibitory agent, for example, by
aiding its expression, influencing its stability in plants,
optimizing free energy for oligomerization, augmenting its
toxicity, and increasing its spectrum of activity. An ancillary
protein may facilitate the uptake of one or more insect inhibitory
agents, for example, or potentiate the toxic effects of the toxic
agent.
[0055] A recombinant DNA construct can be assembled so that all
proteins or dsRNA molecules are expressed from one promoter or each
protein or dsRNA molecules is under separate promoter control or
some combination thereof. The proteins of this invention can be
expressed from a multi-gene expression system in which TIC5290 is
expressed from a common nucleotide segment which also contains
other open reading frames and promoters, depending on the type of
expression system selected. For example, a plant multi-gene
expression system can utilize a single promoter to drive expression
of multiply-linked/tandem open reading frames from within a single
operon. In another example, a plant multi-gene expression system
can utilize multiply-unlinked expression cassettes each expressing
a different protein or other agent such as one or more dsRNA
molecules.
[0056] Recombinant nucleic acid molecules or recombinant DNA
constructs comprising a TIC5290 protein encoding sequence can be
delivered to host cells by vectors, e.g., a plasmid, baculovirus,
synthetic chromosome, virion, cosmid, phagemid, phage, or viral
vector. Such vectors can be used to achieve stable or transient
expression of a TIC5290 protein encoding sequence in a host cell,
or subsequent expression of the encoded polypeptide. An exogenous
recombinant polynucleotide or recombinant DNA construct that
comprises a TIC5290 protein encoding sequence and that is
introduced into a host cell is referred herein as a
"transgene".
[0057] Transgenic bacteria, transgenic plant cells, transgenic
plants, and transgenic plant parts that contain a recombinant
polynucleotide that expresses any one or more of the TIC5290
protein encoding sequences are provided herein. The term "bacterial
cell" or "bacterium" can include, but is not limited to, an
Agrobacterium, a Bacillus, an Escherichia, a Salmonella, a
Pseudomonas, or a Rhizobium cell. The term "plant cell" or "plant"
can include but is not limited to a dicotyledonous cell or a
monocotyledonous cell. Contemplated plants and plant cells include,
but are not limited to, alfalfa, banana, barley, bean, broccoli,
cabbage, Brassica, carrot, cassava, castor, cauliflower, celery,
chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover,
cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,
flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,
melons, nut, oat, olive, onion, ornamental, palm, pasture grass,
pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,
Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower,
shrub, sorghum, Southern pine, soybean, spinach, squash,
strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet
gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale,
turf grass, watermelon, and wheat plant cell or plant. In certain
embodiments, transgenic plants and transgenic plant parts
regenerated from a transgenic plant cell are provided. In certain
embodiments, the transgenic plants can be obtained from a
transgenic seed, by cutting, snapping, grinding or otherwise
disassociating the part from the plant. In certain embodiments, the
plant part can be a seed, a boll, a leaf, a flower, a stem, a root,
or any portion thereof, or a non-regenerable portion of a
transgenic plant part. As used in this context, a "non-regenerable"
portion of a transgenic plant part is a portion that can not be
induced to form a whole plant or that can not be induced to form a
whole plant that is capable of sexual and/or asexual reproduction.
In certain embodiments, a non-regenerable portion of a plant part
is a portion of a transgenic seed, boll, leaf, flower, stem, or
root.
[0058] Methods of making transgenic plants that comprise insect,
Coleoptera-, or Lepidoptera-, or Hemiptera-inhibitory amounts of a
TIC5290 protein are provided. Such plants can be made by
introducing a recombinant polynucleotide that encodes the TIC5290
protein provided in this application into a plant cell, and
selecting a plant derived from said plant cell that expresses an
insect, Coleoptera-, or Lepidoptera-, or Hemiptera-inhibitory
amount of the TIC5290 protein. Plants can be derived from the plant
cells by regeneration, seed, pollen, or meristem transformation
techniques. Methods for transforming plants are known in the
art.
[0059] Processed plant products, wherein the processed product
comprises a detectable amount of a TIC5290 protein, an insect
inhibitory segment or fragment thereof, or any distinguishing
portion thereof, are also disclosed herein. In certain embodiments,
the processed product is selected from the group consisting of
plant parts, plant biomass, oil, meal, sugar, animal feed, flour,
flakes, bran, lint, hulls, processed seed, and seed. In certain
embodiments, the processed product is non-regenerable. The plant
product can comprise commodity or other products of commerce
derived from a transgenic plant or transgenic plant part, where the
commodity or other products can be tracked through commerce by
detecting nucleotide segments or expressed RNA or proteins that
encode or comprise distinguishing portions of a TIC5290
protein.
[0060] Plants expressing the TIC5290 protein can be crossed by
breeding with transgenic events expressing other toxin proteins
and/or expressing other transgenic traits such as herbicide
tolerance genes, genes conferring yield or stress tolerance traits,
and the like, or such traits can be combined in a single vector so
that the traits are all linked.
[0061] As described further in the Examples, synthetic or
artificial sequences encoding TIC5290 that were designed for use in
plants are set forth in SEQ ID NO:3.
[0062] For expression in plant cells, the TIC5290 protein can be
expressed to reside in the cytosol or targeted to various
organelles of the plant cell. For example, targeting a protein to
the chloroplast may result in increased levels of expressed protein
in a transgenic plant while preventing off-phenotypes from
occurring. Targeting may also result in an increase in pest
resistance efficacy in the transgenic event. A target peptide or
transit peptide is a short (3-70 amino acids long) peptide chain
that directs the transport of a protein to a specific region in the
cell, including the nucleus, mitochondria, endoplasmic reticulum
(ER), chloroplast, apoplast, peroxisome and plasma membrane. Some
target peptides are cleaved from the protein by signal peptidases
after the proteins are transported. For targeting to the
choloroplast, proteins contain transit peptides which are around
40-50 amino acids. For descriptions of the use of chloroplast
transit peptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925. Many
chloroplast-localized proteins are expressed from nuclear genes as
precursors and are targeted to the chloroplast by a chloroplast
transit peptide (CTP). Examples of such isolated chloroplast
proteins include, but are not limited to, those associated with the
small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase,
ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex
protein I and protein II, thioredoxin F, enolpyruvyl shikimate
phosphate synthase (EPSPS), and transit peptides described in U.S.
Pat. No. 7,193,133. It has been demonstrated in vivo and in vitro
that non-chloroplast proteins may be targeted to the chloroplast by
use of protein fusions with a heterologous CTP and that the CTP is
sufficient to target a protein to the chloroplast. Incorporation of
a suitable chloroplast transit peptide such as the Arabidopsis
thaliana EPSPS CTP (CTP2) (See, Klee et al., Mol. Gen. Genet.
210:437-442, 1987) or the Petunia hybrida EPSPS CTP (CTP4) (See,
della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986)
has been shown to target heterologous EPSPS protein sequences to
chloroplasts in transgenic plants (See, U.S. Pat. Nos. 5,627,061;
5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP 508909; and
EP 924299). For targeting the TIC5290 protein to the chloroplast, a
sequence encoding a chloroplast transit peptide is placed 5' in
operable linkage and in frame to a synthetic coding sequence
encoding the TIC5290 toxin protein that has been designed for
optimal expression in plant cells.
[0063] Expression cassettes and vectors containing these synthetic
or artificial nucleotide sequences can be constructed and
introduced into corn, cotton, and soybean plant cells in accordance
with transformation methods and techniques which are known in the
art. Transformed cells are regenerated into transformed plants that
are observed to be expressing TIC5290. To test pesticidal activity,
bioassays are performed in the presence of Lepidopteran,
Coleopteran and Hemipteran pests.
[0064] TIC5290 protein-encoding sequences and sequences having a
substantial percentage identity to TIC5290 can be identified using
methods known to those of ordinary skill in the art such as
polymerase chain reaction (PCR), thermal amplification and
hybridization. For example, the protein TIC5290 can be used to
produce antibodies that bind specifically to related proteins, and
can be used to screen for and to find other protein members that
are closely related.
[0065] Furthermore, nucleotide sequences encoding the TIC5290 toxin
protein can be used as probes and primers for screening to identify
other members of the class using thermal-cycle or isothermal
amplification and hybridization methods. For example,
oligonucleotides derived from the sequence as set forth as SEQ ID
NO:3, can be used to determine the presence or absence of a TIC5290
transgene in a deoxyribonucleic acid sample derived from a
commodity product. Given the sensitivity of certain nucleic acid
detection methods that employ oligonucleotides, it is anticipated
that oligonucleotides derived from the sequence as set forth as SEQ
ID NO:3 can be used to detect a TIC5290 transgene in commodity
products derived from pooled sources where only a fraction of the
commodity product is derived from a transgenic plant containing SEQ
ID NO:3. It is further recognized that such oligonucleotides can be
used to introduce nucleotide sequence variation in SEQ ID NO:3.
Such "mutagenesis" oligonucleotides are useful for identification
of TIC5290 amino acid sequence variants exhibiting a range of
insect inhibitory activity or varied expression in transgenic plant
host cells.
[0066] Nucleotide sequence homologs, e.g., insecticidal proteins
encoded by nucleotide sequences that hybridize to each or any of
the sequences disclosed in this application under hybridization
conditions, are also an embodiment of the present invention. The
invention also provides a method for detecting a first nucleotide
sequence that hybridizes to a second nucleotide sequence, wherein
the first nucleotide sequence (or its reverse complement sequence)
encodes a pesticidal protein or pesticidal fragment thereof and
hybridizes under stringent hybridization conditions to the second
nucleotide sequence. In such case, the second nucleotide sequence
can be the nucleotide sequence presented as SEQ ID NO:1 or SEQ ID
NO:3 under stringent hybridization conditions. Nucleotide coding
sequences hybridize to one another under appropriate hybridization
conditions and the proteins encoded by these nucleotide sequences
cross react with antiserum raised against any one of the other
proteins. Stringent hybridization conditions, as defined herein,
comprise at least hybridization at 42.degree. C. followed by two
washes for five minutes each at room temperature with 2.times.SSC,
0.1% SDS, followed by two washes for thirty minutes each at
65.degree. C. in 0.5.times.SSC, 0.1% SDS. Washes at even higher
temperatures constitute even more stringent conditions, e.g.,
hybridization conditions of 68.degree. C., followed by washing at
68.degree. C., in 2.times.SSC containing 0.1% SDS.
[0067] One skilled in the art will recognize that, due to the
redundancy of the genetic code, many other sequences are capable of
encoding proteins related to TIC5290, and those sequences, to the
extent that they function to express pesticidal proteins either in
Bacillus strains or in plant cells, are embodiments of the present
invention, recognizing of course that many such redundant coding
sequences will not hybridize under these conditions to the native
Bacillus sequences encoding TIC5290. This application contemplates
the use of these and other identification methods known to those of
ordinary skill in the art, to identify TIC5290 protein-encoding
sequences and sequences having a substantial percentage identity to
TIC5290 protein-encoding sequences.
[0068] Methods of controlling insects, in particular Lepidoptera,
or Coleoptera, or Hemiptera infestations of crop plants, with the
TIC5290 protein are also disclosed in this application. Such
methods can comprise growing a plant comprising an insect-,
Coleoptera-, or Lepidoptera-, or Hemiptera-inhibitory amount of a
protein of the TIC5290 toxin protein. In certain embodiments, such
methods can further comprise any one or more of: (i) applying any
composition comprising or encoding a TIC5290 toxin protein to a
plant or a seed that gives rise to a plant; and (ii) transforming a
plant or a plant cell that gives rise to a plant with a
polynucleotide encoding a TIC5290 toxin protein. In general, it is
contemplated that TIC5290 toxin protein can be provided in a
composition, provided in a microorganism, or provided in a
transgenic plant to confer insect inhibitory activity against
Lepidopteran, Coleopteran or Hemipteran insects.
[0069] In certain embodiments, a recombinant nucleic acid molecule
of TIC5290 toxin protein is the insecticidally active ingredient of
an insect inhibitory composition prepared by culturing recombinant
Bacillus or any other recombinant bacterial cell transformed to
express a TIC5290 toxin protein under conditions suitable to
express the TIC5290 toxin protein. Such a composition can be
prepared by desiccation, lyophilization, homogenization,
extraction, filtration, centrifugation, sedimentation, or
concentration of a culture of such recombinant cells
expressing/producing said recombinant polypeptide. Such a process
can result in a Bacillus or other entomopathogenic bacterial cell
extract, cell suspension, cell homogenate, cell lysate, cell
supernatant, cell filtrate, or cell pellet. By obtaining the
recombinant polypeptides so produced, a composition that includes
the recombinant polypeptides can include bacterial cells, bacterial
spores, and parasporal inclusion bodies and can be formulated for
various uses, including as agricultural insect inhibitory spray
products or as insect inhibitory formulations in diet
bioassays.
[0070] In one embodiment, to reduce the likelihood of resistance
development, an insect inhibitory composition comprising TIC5290
can further comprise at least one additional polypeptide that
exhibits insect inhibitory activity against the same Lepidopteran,
Coleopteran or Hemipteran insect species, but which is different
from the TIC5290 toxin protein. Possible additional polypeptides
for such a composition include an insect inhibitory protein and an
insect inhibitory dsRNA molecule. One example for the use of such
ribonucleotide sequences to control insect pests is described in
Baum, et al. (U.S. Patent Publication 2006/0021087 A1). Such
additional polypeptide for the control of Lepidopteran pests may be
selected from the group consisting of an insect inhibitory protein,
such as, but not limited to, Cry1A (U.S. Pat. No. 5,880,275),
Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. patent Publication
Ser. No. 10/525,318), Cry1C (U.S. Pat. No. 6,033,874), Cry1D,
Cry1Da and variants thereof, Cry1E, Cry1F, and Cry1A/F chimeras
(U.S. Pat. Nos. 7,070,982; 6,962,705; and 6,713,063), Cry1G, Cry1H,
Cry1I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras such as, but not
limited to, TIC836, TIC860, TIC867, TIC869, and TIC1100
(International Application Publication WO2016/061391 (A2)), TIC2160
(International Application Publication WO2016/061392(A2)), Cry2A,
Cry2Ab (U.S. Pat. No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8,
Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800,
TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002,
AXMI-030, AXMI-035, AND AXMI-045 (U.S. Patent Publication
2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102,
AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication 2013-0310543
A1), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication
2013-0104259 A1), AXMI-134 (U.S. Patent Publication 2013-0167264
A1), AXMI-150 (U.S. Patent Publication 2010-0160231 A1), AXMI-184
(U.S. Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204,
AXMI-207, axmi209 (U.S. Patent Publication 2011-0030096 A1),
AXMI-218, AXMI-220 (U.S. Patent Publication 2014-0245491 A1),
AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent
Publication 2014-0196175 A1), AXMI-238 (U.S. Patent Publication
2014-0033363 A1), AXMI-270 (U.S. Patent Publication 2014-0223598
A1), AXMI-345 (U.S. Patent Publication 2014-0373195 A1), AXMI-335
(International Application Publication WO2013/134523(A2)), DIG-3
(U.S. Patent Publication 2013-0219570 A1), DIG-5 (U.S. Patent
Publication 2010-0317569 A1), DIG-11 (U.S. Patent Publication
2010-0319093 A1), AfIP-1A and derivatives thereof (U.S. Patent
Publication 2014-0033361 A1), AfIP-1B and derivatives thereof (U.S.
Patent Publication 2014-0033361 A1), PIP-1APIP-1B (U.S. Patent
Publication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication
2014-0007292 A1), AECFG-592740 (U.S. Patent Publication
2014-0007292 A1), Pput_1063 (U.S. Patent Publication 2014-0007292
A1), DIG-657 (International Application Publication
WO2015/195594(A2)), Pput_1064 (U.S. Patent Publication 2014-0007292
A1), GS-135 and derivatives thereof (U.S. Patent Publication
2012-0233726 A1), GS153 and derivatives thereof (U.S. Patent
Publication 2012-0192310 A1), GS154 and derivatives thereof (U.S.
Patent Publication 2012-0192310 A1), GS155 and derivatives thereof
(U.S. Patent Publication 2012-0192310 A1), SEQ ID NO:2 and
derivatives thereof as described in U.S. Patent Publication
2012-0167259 A1, SEQ ID NO:2 and derivatives thereof as described
in U.S. Patent Publication 2012-0047606 A1, SEQ ID NO:2 and
derivatives thereof as described in U.S. Patent Publication
2011-0154536 A1, SEQ ID NO:2 and derivatives thereof as described
in U.S. Patent Publication 2011-0112013 A1, SEQ ID NO:2 and 4 and
derivatives thereof as described in U.S. Patent Publication
2010-0192256 A1, SEQ ID NO:2 and derivatives thereof as described
in U.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 and
derivatives thereof as described in U.S. Patent Publication
2010-0077508 A1, SEQ ID NO:2 and derivatives thereof as described
in U.S. Patent Publication 2009-0313721 A1, SEQ ID NO:2 or 4 and
derivatives thereof as described in U.S. Patent Publication
2010-0269221 A1, SEQ ID NO:2 and derivatives thereof as described
in U.S. Pat. No. 7,772,465 (B2), CF161_0085 and derivatives thereof
as described in WO2014/008054 A2, Lepidopteran toxic proteins and
their derivatives as described in US Patent Publications
US2008-0172762 A1, US2011-0055968 A1, and US2012-0117690 A1; SEQ ID
NO:2 and derivatives thereof as described in U.S. Pat. No.
7,510,878(B2), SEQ ID NO:2 and derivatives thereof as described in
U.S. Pat. No. 7,812,129(B1); and the like.
[0071] Such additional polypeptide for the control of Coleopteran
pests may be selected from the group consisting of an insect
inhibitory protein, such as, but not limited to, Cry3Bb (U.S. Pat.
No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B,
Cry34/35, 5307, AXMI134 (U.S. Patent Publication 2013-0167264 A1)
AXMI-184 (U.S. Patent Publication 2010-0004176 A1), AXMI-205 (U.S.
Patent Publication 2014-0298538 A1), axmi207 (U.S. Patent
Publication 2013-0303440 A1), AXMI-218, AXMI-220 (U.S. Patent
Publication 20140245491A1), AXMI-221z, AXMI-223z (U.S. Patent
Publication 2014-0196175 A1), AXMI-279 (U.S. Patent Publication
2014-0223599 A1), AXMI-R1 and variants thereof (U.S. Patent
Publication 2010-0197592 A1, TIC407, TIC417, TIC431, TIC807,
TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication
2010-0319092 A1), eHIPs (U.S. Patent Application Publication No.
2010/001791d), IP3 and variants thereof (U.S. Patent Publication
2012-0210462 A1), and .omega.-Hexatoxin-Hv1a (U.S. Patent
Application Publication US2014-0366227 A1).
[0072] Such additional polypeptides for the control of Hemipteran
pests may be selected from the group consisting of
Hemipteran-active proteins such as, but not limited to, TIC1415 (US
Patent Publication 2013-0097735 A1), TIC807 (U.S. Pat. No.
8,609,936), TIC834 (U.S. Patent Publication 2013-0269060 A1),
AXMI-036 (U.S. Patent Publication 2010-0137216 A1), and AXMI-171
(U.S. Patent Publication 2013-0055469 A1). Additional polypeptides
for the control of Coleopteran, Lepidopteran, and Hemipteran insect
pests can be found on the Bacillus thuringiensis toxin nomenclature
website maintained by Neil Crickmore (on the world wide web at
btnomenclature.info).
[0073] In other embodiments, such composition/formulation can
further comprise at least one additional polypeptide that exhibits
insect inhibitory activity to an insect that is not inhibited by an
otherwise insect inhibitory protein of the present invention to
expand the spectrum of insect inhibition obtained.
[0074] The possibility for insects to develop resistance to certain
insecticides has been documented in the art. One insect resistance
management strategy is to employ transgenic crops that express two
distinct insect inhibitory agents that operate through different
modes of action. Therefore, any insects with resistance to either
one of the insect inhibitory agents can be controlled by the other
insect inhibitory agent. Another insect resistance management
strategy employs the use of plants that are not protected to the
targeted Coleopteran, or Lepidopteran, or Hemipteran pest species
to provide a refuge for such unprotected plants. One particular
example is described in U.S. Pat. No. 6,551,962, which is
incorporated by reference in its entirety.
[0075] Other embodiments such as topically applied pesticidal
chemistries that are designed for controlling pests that are also
controlled by the proteins disclosed herein to be used with
proteins in seed treatments, spray on, drip on, or wipe on
formulations can be applied directly to the soil (a soil drench),
applied to growing plants expressing the proteins disclosed herein,
or formulated to be applied to seed containing one or more
transgenes encoding one or more of the proteins disclosed. Such
formulations for use in seed treatments can be applied with various
stickers and tackifiers known in the art. Such formulations can
contain pesticides that are synergistic in mode of action with the
proteins disclosed, so that the formulation pesticides act through
a different mode of action to control the same or similar pests
that can be controlled by the proteins disclosed, or that such
pesticides act to control pests within a broader host range or
plant pest species that are not effectively controlled by the
TIC5290 pesticidal proteins.
[0076] The aforementioned composition/formulation can further
comprise an agriculturally-acceptable carrier, such as a bait, a
powder, dust, pellet, granule, spray, emulsion, a colloidal
suspension, an aqueous solution, a Bacillus spore/crystal
preparation, a seed treatment, a recombinant plant cell/plant
tissue/seed/plant transformed to express one or more of the
proteins, or bacterium transformed to express one or more of the
proteins. Depending on the level of insect inhibitory or
insecticidal inhibition inherent in the recombinant polypeptide and
the level of formulation to be applied to a plant or diet assay,
the composition/formulation can include various by weight amounts
of the recombinant polypeptide, e.g. from 0.0001% to 0.001% to
0.01% to 1% to 99% by weight of the recombinant polypeptide.
EXAMPLES
[0077] In view of the foregoing, those of skill in the art should
appreciate that changes can be made in the specific aspects which
are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention. Thus,
specific structural and functional details disclosed herein are not
to be interpreted as limiting. It should be understood that the
entire disclosure of each reference cited herein is incorporated
within the disclosure of this application.
Example 1
Discovery of TIC5290
[0078] This Example describes the discovery of pesticidal protein
TIC5290.
[0079] A sequence encoding a novel Bacillus thuringiensis (Bt)
pesticidal protein was identified, cloned, sequence confirmed and
tested in insect bioassay. The pesticidal protein, TIC5290,
presented herein as SEQ ID NO:1 (Bt coding sequence) and 2
(protein), was isolated from Bt strain EG6657. TIC5290 is a 937
amino acid long protein and was identified based on homology to
known insecticidal protein toxins and through the use of Pfam
analysis to cluster it with known insecticidal protein families.
Bioinformatics analysis suggests TIC5290 is a pore-forming protein.
A PA14 Pfam domain (PF07691) at amino acid residues 16 through 140
is likely involved with binding functions. This domain is followed
by a Binary_toxB Pfam domain (PF03495) at amino acids 186 through
593 that might contribute to the formation of a beta-barrel
transmembrane pore. The closest Bt toxin homolog to TIC5209 is the
Vip4Aa protein, with a sequence identity of 56.9%, suggesting that
TIC5290 represents a novel Vip4 subfamily.
[0080] PCR primers were designed to amplify a full length copy of
the coding region for TIC5290 from total genomic DNA isolated from
strain EG6657. The PCR amplicon also included the start and stop
codons of the coding sequence.
[0081] The TIC5290 amplicon was cloned using methods known in the
art into a Bt plasmid expression vector in operable linkage with a
Bt expressible promoter that is on during sporulation of the
bacillus. In addition, the amplicon was cloned into a vector used
for protein expression in an Escherichia coli (E. coli) expression
system. The resulting recombinant strains were observed to express
recombinant protein.
Example 2
TIC5290 Demonstrates Coleopteran, Lepidopteran and Hemipteran
Activity in Insect Bioassay
[0082] This Example illustrates inhibitory activity exhibited by
TIC5290 proteins against various species of Coleoptera, Lepidoptera
and Hemiptera.
[0083] The novel pesticidal protein TIC5290 was expressed in Bt and
E. coli and assayed for toxicity to various species of Coleoptera,
Lepidoptera, Hemiptera, and Diptera. Preparations of each toxin
from Bt and E. coli were assayed against the Coleopteran species
Leptinotarsa decemlineata (Colorado potato beetle, CPB) and
Diabrotica virgifera virgifera (Western Corn Rootworm, WCR). The
toxin preparations were also assayed against the Lepidopteran
species Corn earworm (CEW, Helicoverpa zea), European corn borer
(ECB, Ostrinia nubilalis), Fall armyworm (FAW, Spodoptera
frugiperda), Soybean looper (SBL, Chrysodeixis includens),
Southwestern Corn Borer (SWC, Diatraea grandiosella), Tobacco
budworm (TBW, Heliothis virescens), and Diamondback moth (DBM,
(lutella xylostella). The toxin preparations were also assayed
against the Hemipteran species Tarnished plant bug (TPB, Lygus
lineolaris) and Western tarnished plant bug (WTP, Lygus hesperus);
as well as the Dipteran species, Yellow fever mosquito (YFM, Aedes
aegypti). Corn earworm (CEW, Helicoverpa zea) is also referred to
as Soybean pod worm (SPW) and Cotton bowl worm (CBW).
[0084] Assay of proteins expressed in each of the vectors required
different preparations added to an insect diet. For vectors using
the promoter active during sporulation, a crystal/spore mixture was
harvested after three days of growth in culture and used in an
insect diet (generally applied to insect artificial diets and fed
separately to various insects). Protein preparations derived from
expression in E. coli were purified and also provided in an insect
diet.
[0085] TIC5290 demonstrated activity against WCR, CEW, ECB, FAW,
DBM and WTP, as shown in Table 2 below wherein "+" indicates
activity.
TABLE-US-00002 TABLE 2 Bioassay activity of TIC5290 against
Coleopteran, Lepidopteran, Hemipteran and Dipteran insect pests.
Diet Insect Bioassay WCR + CPB - CEW + ECB + FAW + SBL - SWC - TBW
- DBM + TPB - WTP + YFM -
Example 3
Design of Synthetic Coding Sequences Encoding TIC5290 for
Expression in Plant Cells
[0086] Synthetic or artificial coding sequences were constructed
for use in expression of TIC5290 in plants, and were cloned into a
binary plant transformation vector, and used to transform plant
cells. The synthetic nucleic acid sequences were synthesized
according to methods generally described in U.S. Pat. No.
5,500,365, avoiding certain inimical problem sequences such as
ATTTA and A/T rich plant polyadenylation sequences while preserving
the amino acid sequence of the native Bt protein. The synthetic
coding sequence for the TIC5290 pesticidal protein is presented as
SEQ ID NO:3 and encodes the protein presented as SEQ ID NO:2.
Example 4
Expression Cassettes for Expression of TIC5290 in Plant Cells
[0087] A variety of plant expression cassettes were designed with
the sequences as set forth in SEQ ID NO:3. Such expression
cassettes are useful for transient expression in plant protoplasts
or transformation of plant cells. Typical expression cassettes were
designed with respect to the eventual placement of the protein
within the plant cell. For plastid targeted protein the synthetic
TIC5290 pesticidal protein coding sequence was operably linked in
frame with a chloroplast targeting signal peptide coding sequence.
The resulting plant transformation vectors comprise a first
transgene cassette for expression of the pesticidal protein which
comprises a constitutive promoter, operably linked 5' to a leader,
operably linked 5' to an intron (or optionally no intron), operably
linked 5' to a synthetic coding sequence encoding a plastid
targeted or untargeted TIC5290 protein, which is in turn operably
linked 5' to a 3' UTR and; a second transgene cassette for the
selection of transformed plant cells using glyphosate or antibiotic
selection. All of the elements described above were arranged
contiguously often with additional sequence provided for the
construction of the expression cassette such as restriction
endonuclease sites or ligation independent cloning sites.
Example 5
TIC5290 Provides Efficacious Resistance to Western Corn Rootworm
(Diabrotica virgifera Virgifera) when Expressed in Stably
Transformed Corn Plants
[0088] This Example illustrates the inhibitory activity exhibited
by TIC5290 against Coleoptera such as corn rootworm when expressed
in plants and provided as a diet to the respective insect pest.
[0089] Binary plant transformation vectors comprising transgene
cassettes designed to express both plastid targeted and untargeted
TIC5290 pesticidal proteins were cloned using methods known in the
art. The resulting vectors were used to stably transform corn
plants. Single T-DNA insertion events were selected and grown.
Pesticidal activity were assayed against the Coleopteran pest
Western Corn Rootworm (Diabrotica virgifera virgifera) feeding on
the roots of the stably transformed corn plants.
[0090] R.sub.0 stably transformed plants were used to assay for
Coleopteran resistance as well as generating F.sub.1 progeny.
Multiple single copy events were selected from each binary vector
transformation. A portion of those events arising from each binary
vector transformation were used in the Coleopteran assay, while
another portion of events were used to generate F.sub.1 progeny for
further testing.
[0091] The R.sub.0 assay plants were transplanted to eight inch
pots. The plants were inoculated with eggs from Western Corn
Rootworm (Diabrotica virgifera virgifera, WCR). The eggs were
incubated for approximately ten days prior to inoculation to allow
hatching to occur four days after inoculation to ensure a
sufficient number of larvae survive and are able to attack the corn
roots. The transformed plants were inoculated at approximately V2
to V3 stage. The plants were grown after infestation for
approximately twenty eight days. The plants were removed from the
pots with the roots being carefully washed to remove all soil. The
damage to the roots was assessed using a damage rating scale of 1-5
as presented in Table 3 below. Comparison was also made to the
negative control to assure the assay has been performed properly.
Low root damage scores indicate resistance conferred by the TIC5290
protein to the Coleopteran pest. Multiple R.sub.0 events for each
binary vector transformation were used in the WCR assay. Many of
the R.sub.0 events expressing both plastid targeted and untargeted
TIC5290 demonstrated resistance to WCR determined by the root
damage rating scores when compared to transgenic controls.
TABLE-US-00003 TABLE 3 R.sub.0 root damage rating scores. Root
Damage Score Description 1 No visible feeding 2 Some feeding; no
pruning 3 Pruning of at least one root 4 Entire node pruned 5 More
than one node pruned
[0092] A portion of the R.sub.0 stably transformed events arising
from each binary vector transformation were used to produce F.sub.1
progeny. The R.sub.0 stably transformed plants were allowed to
self-fertilize, producing F.sub.1 progeny. The F.sub.1 seed was
planted. Heterozygous plants were identified through molecular
methods known in the art and used for assay against WCR, as well as
ELISA expression measurements of TIC5290 toxin protein. A portion
of the heterozygous F.sub.1 progeny from each event was used for
insect assay, while another portion was used to measure TIC5290
expression.
[0093] Eggs from Western Corn Rootworm (Diabrotica virgifera
virgifera, WCR) were incubated for approximately ten days to allow
hatching within four days after inoculation. The plants were
inoculated at approximately V2 to V3 stage. For WCR, each pot was
inoculated with about two thousand eggs. The plants were grown
after infestation for approximately twenty eight days. The plants
were removed from the pots with the roots being carefully washed to
remove all soil. The damage to the roots was assessed using a
damage rating scale of 0-3 as presented in Table 4 below.
Comparison was made to the negative control to assure the assay has
been performed properly. Low root damage scores indicated
resistance conferred by the TIC5290 protein to the Coleopteran
pest. Many of the F.sub.1 events demonstrated efficacious
resistance to WCR when compared to the controls. FIG. 1 depicts the
average root damage rating for several events for TIC5290 when
expressed in F1 corn plants regardless of whether the protein is
targeted to the chloroplast.
TABLE-US-00004 TABLE 4 F.sub.1 root damage rating scores. Root
Damage Score Description 0 No visible feeding 0.01-0.09 Feeding
scars and tracks 0.1-0.9 Root pruning, but less than a full node
1.0-1.9 At least a full node (or equivalent) destroyed to within
1.5 inches of plant 2.0-2.9 Two or more nodes gone 3 Three or more
nodes gone
Example 5
Assay of Activity of TIC5290 Against Lepidopteran Pests when
Expressed in Stably Transformed Corn, Soybean, or Cotton Plants
[0094] Binary plant transformation vectors comprising transgene
cassettes designed to express both plastid targeted and untargeted
TIC5290 pesticidal protein are cloned using methods known in the
art.
[0095] Corn, soybean, or cotton is transformed with the binary
transformation vectors described above using an
Agrobacterium-mediated transformation method. The transformed cells
are induced to form plants by methods known in the art. Bioassays
using plant leaf disks are performed analogous to those described
in U.S. Pat. No. 8,344,207. A non-transformed corn, soybean, or
cotton plant is used to obtain tissue to be used as a negative
control. Multiple transformation events from each binary vector are
assessed against Lepidopteran pests such as, but not limited to,
Corn earworm (CEW, Helicoverpa zea), European corn borer (ECB,
Ostrinia nubilalis), Fall armyworm (FAW, Spodoptera frugiperda),
Soybean looper (SBL, Chrysodeixis includens), Southwestern Corn
Borer (SWCB, Diatraea grandiosella), Tobacco budworm (TBW,
Heliothis virescens), and Diamondback moth (DBM, (lutella
xylostella). Those insects demonstrating stunting and/or mortality
in the insect bioassay are determined to be susceptible to the
effects of the TIC5290 insect toxin.
Example 6
Assay of Activity Against Hemipteran Pests Using Stably Transformed
Cotton Plants Expressing TIC5290
[0096] Binary plant transformation vectors comprising transgene
cassettes designed to express both plastid targeted and untargeted
TIC5290 pesticidal proteins are cloned using methods known in the
art. The resulting vectors are used to stably transform cotton
plants. Pesticidal activity is assayed against Hemipteran pests
feeding on the stably transformed cotton plants.
[0097] The binary vectors described previously in Example 3 in
which plastid targeted and untargeted TIC5290 is expressed are used
to stably transform cotton plants. Single T-DNA insertion events
are selected and grown. The R.sub.0 stably transformed plants are
allowed to self-fertilize, producing R.sub.1 progeny.
[0098] R.sub.1 transgenic seeds comprising expression cassettes for
TIC5290 are sown in 10 inch pots along with seeds corresponding to
a non-transgenic control. Plants are maintained in an environment
chamber with a photoperiod of sixteen hours of light at thirty two
degrees Celsius and eight hours of dark at twenty three degrees
Celsius, and a light intensity between eight hundred to nine
hundred micro-Einsteins. At forty to forty five days after
planting, the individual plants are enclosed in a cage made from
breathable plastic "pollination" sheets (Vilutis and Company Inc,
Frankfort, Ill.). The sheet sleeves are secured to the main stem
just above the soil surface using a Velcro.RTM. tie. Two pairs of
sexually mature male and female Lygus lineolaris or Lygus hesparus
adults (six days old) from laboratory culture are collected into a
fourteen milliliter round-bottom plastic tube (Becton Dickson
Labware, Franklin Lakes, N.J.) and used for each plant. The adults
are released into each individual cage through a small slit on the
cage side and then the cage is securely closed ensuring the insects
will not escape. The insects are allowed to mate and the plants are
kept in the cage for twenty one days. After twenty one days, the
plants are then cut below the cages and moved to a laboratory where
the insects are collected for each plant and counted. Before
opening the cage, the plants are vigorously shaken to ensure all of
the insects fall off from their feeding sites to the base of the
cage. Then the cage base is opened and all plant material removed
and placed on a black sheet. The insects are collected using an
aspirator. The plant is then thoroughly inspected to recover any
remaining insects. The numbers of insects and their developmental
stage are recorded for each plant. The insect counts are divided
into several groups based upon maturity of the Lygus; nymphs up to
3.sup.rd instar, 4.sup.th instar, 5.sup.th instar and adults.
Transgenic cotton plants demonstrating reduced numbers of nymphs
and adults relative to the untransformed cotton control plants
demonstrate resistance conferred to the Hemipteran pests through
expression of the TIC5290 toxin protein.
[0099] All of the compositions disclosed and claimed herein can be
made and executed without undue experimentation in light of the
present disclosure. While the compositions of this invention have
been described in terms of the foregoing illustrative embodiments,
it will be apparent to those of skill in the art that variations,
changes, modifications, and alterations may be applied to the
composition described herein, without departing from the true
concept, spirit, and scope of the invention. More specifically, it
will be apparent that certain agents that are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope, and
concept of the invention as defined by the appended claims.
[0100] All publications and published patent documents cited in the
specification are incorporated herein by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
312814DNABacillus thuringiensismisc_feature(1)..(2814)DNA sequence
derived from Bt strain EG6657 encoding TIC5290. 1atgcaaaata
tcgtttcttc aaaaagcgaa caggcgacgg tcattggatt agttggtttt 60tactttaaag
atagtacatt taaagaattg atgtttatcc aggtgggcga aaaaagtaac
120ttaatgaata aagcgagaat aaacacagat gcgcaacaaa ttcaatctat
acgatggatg 180gggaatttga agtctcccca aacgggagag tataggcttt
ctacatcttc cgatgaaaat 240gtaattctgc aaataaatgg cgaaacagtc
attaatcaag ctagtataca aaaaaacctt 300aaactagaag caaatcaggt
atatgaaatc aagattgagt atcgtaatac atcgaataca 360ttaccagatt
tgcagttatt ctggtcaatg aacaatgcgc aaaaagaaca aatcccggag
420aaatatatac tctctccaaa tttctctgaa aaagcaaatt cacttgccga
aaaagaaaca 480caaagtttct ttccaaatta taatttattt gatagacaac
aagaaaatgg agaaaaacaa 540tccatgtcta ctcctgtaga cacagataat
gattgtattc cagatgaatg ggaagaaaag 600ggatatacat ttagaaatca
acaaattgtg ccatggaatg atgcatatag tgctgaaggc 660tacaaaaaat
atgtctcaaa tccttatcat gcacgaacag taaaagatcc ttatacagat
720tttgaaaagg taacaggaca tatgccggca gcgacgaaat acgaagcaag
agatccacta 780gtagctgcgt atccatcggt tggagttgga atggaaaaac
ttcatttttc taaaaatgat 840actgtaactg agggaaatgc tgacacaaaa
tctaaaacaa ccacaaaaac agatacgacc 900acaaatactg tagagattgg
tggatcgctc ggcttttcag ataaaggctt ttctttttca 960atttcaccaa
aatacacaca ttcttggagt agtagcacat ccgttgcaga tacggacagt
1020acaacatggt cttcacaaat tggaattaac acggcagaaa gagcatattt
aaatgcaaat 1080gttcgttatt acaatggtgg aacggcacct atttatgatc
taaaaccgac aacaaatttt 1140gtcttccaaa attcaggtga ttccattact
acgataacgg ctggacctaa tcaaattgga 1200aatagtctag gtgcaggcga
tacctatccg caaaaaggac aggccccgat ttcattagat 1260aaagcaaatg
aggctggtac agtaaaaatt gcaatcaatg ctgaacaatt agataaaata
1320caagctggta cagaaatatt aaatatagaa actacgcaaa atagaggaca
atatggaatt 1380ttagatgaaa aaggtcaagt aattccaggt ggagaatggg
atccgattcg aacaaatatt 1440gatgcggtct ctggatcact cacattaaat
cttggtacag ggaaagatag tctcgaacga 1500agagtagctg caaaaaatat
gaatgatcca gaagataaaa cacctgaaat tacaatcaaa 1560gaagcaatca
aaaaagcgtt taatgcacaa gaaaaagatg gtagattata ctatacggat
1620caaggcgaaa aagatatatt tatcgatgaa ccttctatta atttaatcac
agatgaaaat 1680acaaaaaaag aaattgagcg ccaattaaat caaatgccag
gtaaaacagt atatgatgta 1740aaatggaaac gcgggatgaa gatcacactt
catgtaccaa taaagtacta tgatttcgaa 1800acctccgaaa atctatggta
ttatacatac caagaaagcg gaggatatac gggtaaaaaa 1860cgaggaagaa
ttggtacaga tgggcatggg actgcgatgt caaatccaca attaaaaccg
1920tatacaagtt atacggtgcg tgcatacgta cgaacagcat caacaacggg
tagtaatgaa 1980gttgtatttt atgcagataa tagctccggg aatggacaag
gtgcaaaagt aagtggaaaa 2040gtcacaggtg gtaaatggaa aatagcggaa
ttttctttta atacttttaa caacccagag 2100tattttaaaa taatcggttt
gaaaaataac gggaatgcta atctccattt tgatgatgta 2160tctgtaatag
agtggaaaac aaatgaaaat cttcaaaaaa aacatatatt tgaaaaatgg
2220agttttggtt caaatgatga gatggtgata ggtgcaacgt ttactcgtgt
tccaagttcg 2280aagattcgat accaatggaa aataaatggt aggttgggaa
gtataatacc tgcaccgcca 2340ttagacgcta atggtaaaag aactgtaacc
tatggatcaa ttactgctat tactcccatg 2400gaattatatg ctgtagatga
aaaaaatgac aacctaaaag taaaagtagc tgaactcggc 2460gagagtgaga
ttgaaaaagt aatgatagat gcacataaat tttccgggtg gtggtattta
2520tctgaaaacc caaacctgta tagtggtctt agtttataca aattacctga
tatattttat 2580aataacgtat cttcttataa aattcgagtg aatggaaaaa
aagttcaaac agtctcaaaa 2640ccaagcccat ttctttttca gataacgttt
aatctaaaaa atcctaatgg tggcacttat 2700cctactaaag atgcatcagt
cgaattatgg gctacagtag gtggaaaaga tttaaaggtg 2760ttgcataagt
ggattcaaaa aagcgatgtt atgtacagtc agactaataa ttaa
28142937PRTBacillus thuringiensisMISC_FEATURE(1)..(937)Amino acid
sequence of TIC5290. 2Met Gln Asn Ile Val Ser Ser Lys Ser Glu Gln
Ala Thr Val Ile Gly 1 5 10 15 Leu Val Gly Phe Tyr Phe Lys Asp Ser
Thr Phe Lys Glu Leu Met Phe 20 25 30 Ile Gln Val Gly Glu Lys Ser
Asn Leu Met Asn Lys Ala Arg Ile Asn 35 40 45 Thr Asp Ala Gln Gln
Ile Gln Ser Ile Arg Trp Met Gly Asn Leu Lys 50 55 60 Ser Pro Gln
Thr Gly Glu Tyr Arg Leu Ser Thr Ser Ser Asp Glu Asn 65 70 75 80 Val
Ile Leu Gln Ile Asn Gly Glu Thr Val Ile Asn Gln Ala Ser Ile 85 90
95 Gln Lys Asn Leu Lys Leu Glu Ala Asn Gln Val Tyr Glu Ile Lys Ile
100 105 110 Glu Tyr Arg Asn Thr Ser Asn Thr Leu Pro Asp Leu Gln Leu
Phe Trp 115 120 125 Ser Met Asn Asn Ala Gln Lys Glu Gln Ile Pro Glu
Lys Tyr Ile Leu 130 135 140 Ser Pro Asn Phe Ser Glu Lys Ala Asn Ser
Leu Ala Glu Lys Glu Thr 145 150 155 160 Gln Ser Phe Phe Pro Asn Tyr
Asn Leu Phe Asp Arg Gln Gln Glu Asn 165 170 175 Gly Glu Lys Gln Ser
Met Ser Thr Pro Val Asp Thr Asp Asn Asp Cys 180 185 190 Ile Pro Asp
Glu Trp Glu Glu Lys Gly Tyr Thr Phe Arg Asn Gln Gln 195 200 205 Ile
Val Pro Trp Asn Asp Ala Tyr Ser Ala Glu Gly Tyr Lys Lys Tyr 210 215
220 Val Ser Asn Pro Tyr His Ala Arg Thr Val Lys Asp Pro Tyr Thr Asp
225 230 235 240 Phe Glu Lys Val Thr Gly His Met Pro Ala Ala Thr Lys
Tyr Glu Ala 245 250 255 Arg Asp Pro Leu Val Ala Ala Tyr Pro Ser Val
Gly Val Gly Met Glu 260 265 270 Lys Leu His Phe Ser Lys Asn Asp Thr
Val Thr Glu Gly Asn Ala Asp 275 280 285 Thr Lys Ser Lys Thr Thr Thr
Lys Thr Asp Thr Thr Thr Asn Thr Val 290 295 300 Glu Ile Gly Gly Ser
Leu Gly Phe Ser Asp Lys Gly Phe Ser Phe Ser 305 310 315 320 Ile Ser
Pro Lys Tyr Thr His Ser Trp Ser Ser Ser Thr Ser Val Ala 325 330 335
Asp Thr Asp Ser Thr Thr Trp Ser Ser Gln Ile Gly Ile Asn Thr Ala 340
345 350 Glu Arg Ala Tyr Leu Asn Ala Asn Val Arg Tyr Tyr Asn Gly Gly
Thr 355 360 365 Ala Pro Ile Tyr Asp Leu Lys Pro Thr Thr Asn Phe Val
Phe Gln Asn 370 375 380 Ser Gly Asp Ser Ile Thr Thr Ile Thr Ala Gly
Pro Asn Gln Ile Gly 385 390 395 400 Asn Ser Leu Gly Ala Gly Asp Thr
Tyr Pro Gln Lys Gly Gln Ala Pro 405 410 415 Ile Ser Leu Asp Lys Ala
Asn Glu Ala Gly Thr Val Lys Ile Ala Ile 420 425 430 Asn Ala Glu Gln
Leu Asp Lys Ile Gln Ala Gly Thr Glu Ile Leu Asn 435 440 445 Ile Glu
Thr Thr Gln Asn Arg Gly Gln Tyr Gly Ile Leu Asp Glu Lys 450 455 460
Gly Gln Val Ile Pro Gly Gly Glu Trp Asp Pro Ile Arg Thr Asn Ile 465
470 475 480 Asp Ala Val Ser Gly Ser Leu Thr Leu Asn Leu Gly Thr Gly
Lys Asp 485 490 495 Ser Leu Glu Arg Arg Val Ala Ala Lys Asn Met Asn
Asp Pro Glu Asp 500 505 510 Lys Thr Pro Glu Ile Thr Ile Lys Glu Ala
Ile Lys Lys Ala Phe Asn 515 520 525 Ala Gln Glu Lys Asp Gly Arg Leu
Tyr Tyr Thr Asp Gln Gly Glu Lys 530 535 540 Asp Ile Phe Ile Asp Glu
Pro Ser Ile Asn Leu Ile Thr Asp Glu Asn 545 550 555 560 Thr Lys Lys
Glu Ile Glu Arg Gln Leu Asn Gln Met Pro Gly Lys Thr 565 570 575 Val
Tyr Asp Val Lys Trp Lys Arg Gly Met Lys Ile Thr Leu His Val 580 585
590 Pro Ile Lys Tyr Tyr Asp Phe Glu Thr Ser Glu Asn Leu Trp Tyr Tyr
595 600 605 Thr Tyr Gln Glu Ser Gly Gly Tyr Thr Gly Lys Lys Arg Gly
Arg Ile 610 615 620 Gly Thr Asp Gly His Gly Thr Ala Met Ser Asn Pro
Gln Leu Lys Pro 625 630 635 640 Tyr Thr Ser Tyr Thr Val Arg Ala Tyr
Val Arg Thr Ala Ser Thr Thr 645 650 655 Gly Ser Asn Glu Val Val Phe
Tyr Ala Asp Asn Ser Ser Gly Asn Gly 660 665 670 Gln Gly Ala Lys Val
Ser Gly Lys Val Thr Gly Gly Lys Trp Lys Ile 675 680 685 Ala Glu Phe
Ser Phe Asn Thr Phe Asn Asn Pro Glu Tyr Phe Lys Ile 690 695 700 Ile
Gly Leu Lys Asn Asn Gly Asn Ala Asn Leu His Phe Asp Asp Val 705 710
715 720 Ser Val Ile Glu Trp Lys Thr Asn Glu Asn Leu Gln Lys Lys His
Ile 725 730 735 Phe Glu Lys Trp Ser Phe Gly Ser Asn Asp Glu Met Val
Ile Gly Ala 740 745 750 Thr Phe Thr Arg Val Pro Ser Ser Lys Ile Arg
Tyr Gln Trp Lys Ile 755 760 765 Asn Gly Arg Leu Gly Ser Ile Ile Pro
Ala Pro Pro Leu Asp Ala Asn 770 775 780 Gly Lys Arg Thr Val Thr Tyr
Gly Ser Ile Thr Ala Ile Thr Pro Met 785 790 795 800 Glu Leu Tyr Ala
Val Asp Glu Lys Asn Asp Asn Leu Lys Val Lys Val 805 810 815 Ala Glu
Leu Gly Glu Ser Glu Ile Glu Lys Val Met Ile Asp Ala His 820 825 830
Lys Phe Ser Gly Trp Trp Tyr Leu Ser Glu Asn Pro Asn Leu Tyr Ser 835
840 845 Gly Leu Ser Leu Tyr Lys Leu Pro Asp Ile Phe Tyr Asn Asn Val
Ser 850 855 860 Ser Tyr Lys Ile Arg Val Asn Gly Lys Lys Val Gln Thr
Val Ser Lys 865 870 875 880 Pro Ser Pro Phe Leu Phe Gln Ile Thr Phe
Asn Leu Lys Asn Pro Asn 885 890 895 Gly Gly Thr Tyr Pro Thr Lys Asp
Ala Ser Val Glu Leu Trp Ala Thr 900 905 910 Val Gly Gly Lys Asp Leu
Lys Val Leu His Lys Trp Ile Gln Lys Ser 915 920 925 Asp Val Met Tyr
Ser Gln Thr Asn Asn 930 935 32814DNAArtificialSynthetic coding
sequence encoding TIC5290 designed for expression in plants.
3atgcagaaca ttgtctcctc caagagtgag caagcgacgg tcatcggcct ggtcggcttc
60tacttcaagg actcaacctt caaggagctc atgttcatcc aggtgggcga gaagtccaac
120ctcatgaaca aggctcgcat caacacggac gcccagcaga tccagtccat
tcggtggatg 180ggcaacctga agagcccgca gaccggcgag taccgcctct
ccacatcctc cgacgagaac 240gtaatcctcc agatcaatgg cgagacggtc
atcaaccagg cgagcatcca gaagaacctc 300aaactagagg caaaccaggt
ctacgagatt aagatcgagt acaggaacac ctccaacacc 360ctgccggacc
tacagctctt ctggtcgatg aacaacgcgc agaaggaaca gatcccggag
420aagtacatac tgagcccgaa tttcagcgag aaggcgaact ctctcgcgga
gaaggagacc 480cagagcttct tcccgaacta caacctcttc gaccgccagc
aagagaacgg cgagaagcag 540tcgatgtcca cgccggtgga caccgacaac
gactgcatcc ctgatgaatg ggaagagaaa 600ggatacacct tccgtaacca
gcagatcgtg ccgtggaacg acgcctacag tgcagaaggc 660tacaagaagt
acgtgagcaa cccttaccac gcccgtacgg tcaaagaccc gtacaccgac
720ttcgagaagg tgactgggca catgcccgct gctacgaagt atgaggcgcg
cgatcctcta 780gtggccgcct atccctccgt tggcgtcgga atggagaagc
tccacttcag caagaacgat 840acggtgacgg agggcaacgc ggatacaaag
agcaagacta caactaagac cgacaccacg 900accaacaccg tcgagatcgg
cggcagcctg ggcttcagcg acaagggctt cagtttctca 960atctcaccaa
agtacaccca cagctggtcg tcgtccacaa gtgtggccga caccgactct
1020actacctgga gttcgcagat agggatcaac actgccgaga gggcgtatct
caacgcgaac 1080gtgcgctatt acaatggtgg caccgcgccc atctacgacc
tgaagccgac caccaacttc 1140gtcttccaga actcaggcga cagcatcacc
acgatcaccg ccggccctaa ccagatcggc 1200aactcgctcg gtgccggcga
cacctatccg cagaagggcc aggctcctat ctccctagac 1260aaggccaacg
aggcgggcac cgtgaagata gcgatcaacg ccgagcagct ggacaagatc
1320caggcgggca cggagattct caacatcgag actacgcaga accgcggcca
gtacggtatc 1380ctcgacgaga aaggccaggt gatacccgga ggcgagtggg
acccgatccg gacaaacatt 1440gacgctgtca gtgggagcct tactcttaac
ctcggcacgg gcaaggatag cctcgagcgc 1500cgggtcgcgg cgaagaacat
gaacgatccg gaggacaaga ctccggagat caccatcaag 1560gaggccatca
agaaggcgtt taacgctcag gagaaggacg gcagactgta ctacacggac
1620cagggtgaga aggacatctt cattgatgag ccttccatca acctcatcac
ggacgagaac 1680acgaagaaag aaatcgagcg ccagctgaac cagatgcccg
gcaagacggt gtacgacgtg 1740aagtggaagc gcggcatgaa gatcacgcta
cacgtcccga tcaagtacta cgacttcgag 1800acctcagaga acctgtggta
ctacacctac caagaatccg gaggctacac cggcaagaag 1860cgcgggcgga
tcggcactga cgggcacggc acggcgatgt caaacccgca gctgaagcca
1920tacacctcct acactgtgcg ggcgtacgtg cgcaccgcca gcaccactgg
gagcaacgag 1980gtcgtcttct acgctgacaa cagctccggc aacgggcaag
gcgcgaaggt ttccgggaag 2040gtgaccggcg ggaagtggaa gatagcggag
ttctccttca acacgttcaa taaccctgaa 2100tacttcaaga tcatcggcct
gaagaataac gggaacgcca acctgcactt cgacgatgtc 2160tccgtgatcg
agtggaagac caacgagaac ctgcagaaga aacacatctt tgagaagtgg
2220tccttcggct ccaacgacga gatggtgatc ggtgccacgt tcactcgcgt
gccgagtagc 2280aagatccgat accagtggaa gatcaacggc cgcctcggta
gcatcatccc tgcgccgcct 2340ctggacgcca acgggaagcg gacggtgacg
tacggcagca tcactgcgat cacgcctatg 2400gagctctacg ccgttgacga
gaagaacgac aacctcaaag tgaaagtcgc tgagcttggc 2460gagtccgaga
tcgagaaagt tatgatcgac gcccacaaat tctcaggctg gtggtatctc
2520tcagagaatc caaacctgta ctccggcctc agcctgtaca agctgcccga
catcttctac 2580aacaacgtgt cgagctacaa gatccgcgtg aacggcaaga
aggtccagac cgtcagcaag 2640ccgagcccgt tcttgttcca gattacgttt
aatctcaaga accctaacgg cgggacctac 2700ccgacgaaag atgctagcgt
agagctctgg gcgacggtcg gcgggaagga cctgaaggtg 2760ctacacaagt
ggattcagaa gtcggatgtc atgtacagcc agaccaacaa ctga 2814
* * * * *