Novel insect inhibitory protein

Abstract

Insecticidal proteins showing toxicity activity against coleopteran and lepidopteran pest species are disclosed and include, but are not limited to, TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863, and TIC-3668 type proteins. DNA molecules and constructs containing polynucleotide sequences encoding one or more of the disclosed TIC3668 type proteins are provided. Transgenic plants, plant cells, seeds, and plant parts containing polynucleotide sequences encoding the insecticidal proteins of the present invention and resistant to coleopteran and lepidopteran infestations are provided. Methods for detecting the presence of the polynucleotides or proteins of the present invention in biological samples are also provided, as well as methods for using any of the TIC3668 type insecticidal proteins to control pests of coleopteran and lepidopteran species.

Description

[0001] This application is a divisional application of the patent application No. 201580070268.5, with the filing date of November 18, 2015 and the invention title of "Novel Insect Inhibitory Protein".

[0002] Citation of Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 082,504, filed on November 20, 2014, which is incorporated herein by reference in its entirety.

[0004] Incorporation of Sequence Listing

[0005] A sequence listing in computer-readable form is submitted herewith by electronic submission. The sequence listing is incorporated herein by reference in its entirety and is contained in a file created on November 13, 2015, having the file name "MONS387WO_ST25.txt" and a size of 114 kb (as measured in the MS-Windows operating system). Technical Field

[0006] The present invention generally relates to the field of insect inhibitory proteins. A novel class of proteins is disclosed that exhibits insect inhibitory activity against agriculturally relevant pests of crop plants and seeds. Specifically, the proteins of the disclosed class have insecticidal activity against agriculturally relevant pests of crop plants and seeds, particularly insect pests of Lepidopteran and Coleopteran species. Plants, plant parts, and seeds containing recombinant polynucleotide constructs encoding one or more of the disclosed toxin proteins are provided. Background Art

[0007] Improving the crop yields of agriculturally important plants, especially including maize, soybean, sugarcane, rice, wheat, vegetables, and cotton, has become increasingly important. In addition to the growing need for agricultural products to provide food, clothing, and energy for an increasing population, it is predicted that climate-related impacts and the pressure caused by the use of land other than land used for agricultural practices by an increasing population will also reduce the amount of arable land available for farming. These factors have led to dire predictions for food security, especially in the absence of significant improvements in plant biotechnology and agronomic practices. Given these pressures, environmentally sustainable improvements in technology, agrotechnology, and pest management are important tools for expanding crop yields in the face of limited arable land available for farming.

[0008] Insects, especially those belonging to the orders Lepidoptera and Coleoptera, are considered to be the main cause of damage to field crops, thereby reducing crop yields in the infested areas. Lepidopteran pest species that 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 that negatively impact agriculture include, but are not limited to, certain species of Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., certain species of Eremnus, 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 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 the Brazilian corn rootworm complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa.

[0009] Historically, the widespread use of synthetic chemical insecticides has been relied upon as pest control agents in agriculture. In addition to the emerging resistance problems, concerns for the environment and human health have spurred research and development of biopesticides. This research effort has led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

[0010] The biocontrol paradigm shifted when the potential of entomopathogenic bacteria, especially those belonging to the genus Bacillus, was discovered and developed as biopesticide agents. Strains of the bacterium Bacillus thuringiensis (Bt) have been used as sources of proteins exhibiting insecticidal activity, since Bt strains were found to show high toxicity against specific insects. The main characteristic of Bt is the production of parasporal bodies containing one or more crystals that contain specific insecticidal endotoxins (Cry proteins), which act on ingestion by susceptible insects through a pore-forming mechanism that is harmful to the epithelial lining of the insect digestive tract. In addition to Bt, other Bacillus species, such as Bacillus sphaericus, and other bacterial species containing genes that promote an entomopathogenic phenotype, such as Brevibacillus laterosporus, have shown pest control potential.

[0011] Insecticidal toxin proteins have been used in various agricultural applications to protect agriculturally important plants 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 preparations containing various bacterial strains on the plant surface, and by using genetic transformation techniques to produce transgenic plants and seeds that express insecticidal toxin proteins.

[0012] The use of transgenic plants expressing insecticidal toxin proteins has been adopted globally. For example, in 2012, 26.1 million hectares of transgenic crops expressing Bt toxins were planted (James, C., Global Status of Commercialized Biotech / GM Crops: 2012. ISAAA Brief No. 44). The expanding use of transgenic insect-protected crops and the limited number of commercially available insecticidal toxin proteins are generating selection pressure for alleles that confer resistance to currently utilized insecticidal proteins. The development of resistance to insecticidal toxin proteins in target pests would undermine the effectiveness and advantages of this technology. Such advantages include increased crop yields, reduced use of chemical insecticides, and reduced costs and labor associated with chemical insecticide use.

[0013] The discovery and development of novel insecticidal toxin proteins are important for managing the increased resistance of insects to transgenic crops expressing insecticidal toxin proteins. Novel protein toxins with improved efficacy and demonstrating control of a broader spectrum of susceptible insect species will reduce the number of surviving insects that can generate resistance alleles. In addition, two or more transgenic toxins in a plant that are toxic to the same insect pest and exhibit different modes of action further reduce the likelihood of resistance development in the target insect species.

[0014] Accordingly, there is a need to discover and develop effective insecticidal proteins with improved insecticidal properties compared to proteins known in the art, such as increased efficacy against a broader spectrum of target insect pest species and different modes of action. A novel family of protein toxins from Brevibacillus laterosporus, as well as similar toxin proteins, variant proteins, and exemplary recombinant proteins, are disclosed herein that exhibit insecticidal activity against important target Lepidoptera and Coleoptera pest species, particularly against the western corn rootworm. Summary of the Invention

[0015] Disclosed herein is a group of novel insect-inhibiting recombinant polynucleotide molecules and polypeptides (toxin proteins) encoded thereby, referred to herein as TIC3668-type proteins, which are shown to exhibit inhibitory activity against one or more pests of crop plants. Each protein can be used alone or in combination with each other and with other insecticidal proteins and toxic agents in formulations and in planta, thereby providing alternatives to the insecticidal proteins and pesticidal chemicals currently used in agricultural systems.

[0016] In one aspect, the present invention provides a recombinant polynucleotide molecule encoding an insect inhibitory polypeptide, the insect inhibitory polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In one embodiment, the recombinant polynucleotide molecule encodes an insect inhibitory polypeptide comprising at least 35% identity, such as at least 40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In another embodiment, the recombinant polynucleotide molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72.In another embodiment, the recombinant polynucleotide molecule comprises a nucleotide sequence having at least 35% identity, such as at least 40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72. In another embodiment, the recombinant polynucleotide molecule comprises a sequence that hybridizes to: (i) the reverse complementary sequence of the nucleotide sequence at positions 4 - 885 of a sequence selected from the group consisting of: SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72; or (ii) the reverse complementary sequence of a sequence selected from the group consisting of: SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61. In another embodiment, the hybridization conditions are stringent conditions, for example, such stringent conditions may include hybridization for 4 to 12 hours at 37°C in 50% formamide, 1 M NaCl and 1% SDS, and washing at 60°C - 65°C in 0.1X SSC. In other embodiments, the recombinant polynucleotide molecule is operably linked to a heterologous promoter.

[0017] In another aspect, the present invention provides an insect-inhibiting recombinant polypeptide encoded by a recombinant polynucleotide molecule provided herein. In one embodiment, the insect-inhibiting recombinant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In another embodiment, the insect-inhibiting recombinant polypeptide comprises at least 35% identity, such as at least 40%, 50%, 60%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0018] In another embodiment, the insect-inhibiting recombinant polypeptide exhibits inhibitory activity against insect species of Coleoptera, such as including Western corn rootworm, Southern corn rootworm, Northern corn rootworm, Mexican corn rootworm, Brazilian corn rootworm, or the Brazilian corn rootworm complex consisting of Diabrotica speciosa and Diabrotica virgifera. In another embodiment, the insect-inhibiting recombinant polypeptide exhibits inhibitory activity against insect species of Lepidoptera, such as including European corn borer, Southwestern corn borer, Black cutworm, Fall Army Worm, Corn Earworm, and Soybean looper.

[0019] In another aspect, the present invention provides a host cell comprising the recombinant polynucleotide molecule of the present invention, wherein the host cell is selected from the group consisting of bacterial host cells and plant host cells. In certain embodiments, the bacterial host cells include Agrobacterium, Rhizobium, Bacillus thuringiensis, Brevibacillus laterosporus, Bacillus cereus, Escherichia coli, Pseudomonas, Klebsiella, and Erwinia. In other embodiments, the plant cells include alfalfa, banana, barley, bean, broccoli, cabbage, Brassica, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, pasture grass, pea, peanut, pepper, persimmon, pigeon pea, pine, pomegranate, poplar, potato, pumpkin, radiata pine, radish, rapeseed, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugar cane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea plant, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cells.

[0020] In another aspect, the present invention provides an insect-inhibiting composition that may comprise the recombinant polynucleotide molecule of the present invention. In one embodiment, the insect-inhibiting composition may further comprise a nucleotide sequence encoding at least one other insecticide. In certain embodiments, the at least one other insecticide is different from the TIC3668 type insect-inhibiting polypeptide of the present invention and is optionally selected from the group consisting of insect-inhibiting proteins, insect-inhibiting dsRNA molecules, and accessory proteins. In other embodiments, the other insecticide exhibits activity against one or more pest species of Lepidoptera, Coleoptera, or Hemiptera. In certain embodiments, the other insecticide is selected from the group consisting of: Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry3A, 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, VIP3A, and VIP3B proteins. In another aspect, the present invention provides an insect-inhibiting composition comprising an insect-inhibiting effective amount of an insect-inhibiting recombinant polypeptide of the present invention such as a TIC3668 type insect-inhibiting polypeptide.

[0021] In another aspect, the present invention provides a method for controlling pests of Coleoptera or Lepidoptera species and for controlling the infestation of plants such as crop plants by pests of Coleoptera or Lepidoptera species, wherein the method comprises contacting the pests with an insect-inhibiting amount of an insect-inhibiting recombinant polypeptide of the present invention such as a TIC3668 type insect-inhibiting polypeptide.

[0022] In another aspect, the present invention provides a seed comprising the recombinant polynucleotide molecule of the present invention or an insect-inhibiting recombinant polypeptide such as a TIC3668 type insect-inhibiting polypeptide.

[0023] In another aspect, the present invention provides a commercial product comprising a detectable amount of a recombinant polynucleotide molecule of the present invention or an insect inhibitory polypeptide such as an insect inhibitory polypeptide of the TIC3668 type. In another aspect, the commercial product of the present invention may comprise a host cell comprising a recombinant polynucleotide molecule of the present invention, wherein the commercial product comprises a detectable amount of the recombinant polynucleotide molecule or an insect inhibitory recombinant polypeptide encoded by the recombinant polynucleotide. In certain embodiments, the commercial product may include commercial corn, corn chips, tortillas, corn meal, corn flour, corn syrup, corn oil, corn silage, corn starch, corn grain, etc. bagged by a grain processor, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon and vegetable commercial products, including, when applicable, juices, concentrates, jams, jellies, marmalades and other edible forms of such commercial products containing a detectable amount of such polynucleotides and / or polypeptides of the present application.

[0024] In another aspect, the present invention provides a method for producing a seed comprising a recombinant polynucleotide of the present invention, wherein the method comprises: (a) planting at least one seed comprising the recombinant polynucleotide molecule; (b) growing a plant from the seed; and (c) harvesting a seed from the plant, wherein the harvested seed comprises the recombinant polynucleotide molecule.

[0025] In another aspect, the present invention provides a recombinant vector comprising a recombinant polynucleotide molecule of the present invention. In one embodiment, the recombinant vector is selected from the group consisting of plasmids, bacmids, phagemids and cosmids.

[0026] In another aspect, the present invention provides a plant resistant to insect infestation, wherein the cells of the plant comprise a recombinant polynucleotide molecule of the present invention or an insect inhibitory recombinant polypeptide.

[0027] In particular, the present application relates to the following:

[0028] 1. A recombinant polynucleotide molecule encoding an insect inhibitory polypeptide, the insect inhibitory polypeptide comprising:

[0029] (a) an amino acid sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; or

[0030] (b) an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0031] 2. The recombinant polynucleotide molecule according to item 1, comprising:

[0032] (a) a nucleotide sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72;

[0033] (b) a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72; or

[0034] (c) a nucleotide sequence that hybridizes under stringent conditions to:

[0035] (i) The reverse complementary sequence of the nucleotide sequence at positions 4 - 885 from a sequence selected from the group consisting of: SEQ ID NO:37, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72; or

[0036] (ii) The reverse complementary sequence of a sequence selected from the group consisting of: SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61;

[0037] wherein the stringent conditions include hybridization for 4 to 12 hours at 37°C in 50% formamide, 1 M NaCl, and 1% SDS, and washing in 0.1X SSC at 60°C - 65°C.

[0038] 3. The recombinant polynucleotide molecule according to item 1, which is operably linked to a heterologous promoter.

[0039] 4. An insect - inhibitory recombinant polypeptide encoded by the recombinant polynucleotide molecule according to item 1.

[0040] 5. The insect - inhibitory recombinant polypeptide according to item 4, wherein the insect - inhibitory recombinant polypeptide comprises:

[0041] (a) An amino acid sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; or

[0042] (b) comprising an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0043] 6. The insect inhibitory recombinant polypeptide according to item 4, wherein the insect inhibitory recombinant polypeptide exhibits inhibitory activity against insect species of Coleoptera.

[0044] 7. The insect inhibitory recombinant polypeptide according to item 6, wherein the insect species of Coleoptera is Western corn rootworm, Southern corn rootworm, Northern corn rootworm, Mexican corn rootworm, Brazilian corn rootworm, or the Brazilian corn rootworm complex consisting of Diabrotica virgifera virgifera and Diabrotica speciosa.

[0045] 8. The insect inhibitory recombinant polypeptide according to item 4, wherein the insect inhibitory recombinant polypeptide exhibits inhibitory activity against insect species of Lepidoptera.

[0046] 9. The insect inhibitory recombinant polypeptide according to item 8, wherein the species of Lepidoptera is selected from the group consisting of European corn borer, Southwestern corn borer, Black cutworm, Fall armyworm, Corn earworm, and Soybean looper.

[0047] 10. A host cell comprising the recombinant polynucleotide molecule according to item 1, wherein the host cell is selected from the group consisting of bacterial host cells and plant host cells.

[0048] 11. An insect inhibitory composition comprising the recombinant polynucleotide molecule according to item 1.

[0049] 12. The insect inhibitory composition according to item 11, further comprising a nucleotide sequence encoding at least one other insecticide different from the insect inhibitory polypeptide.

[0050] 13. The insect inhibitory composition according to item 12, wherein the at least one other insecticide is selected from the group consisting of insect inhibitory proteins, insect inhibitory dsRNA molecules, and accessory proteins.

[0051] 14. The insect inhibitory composition according to item 13, wherein the at least one other insecticide exhibits activity against one or more pest species of Lepidoptera, Coleoptera, or Hemiptera.

[0052] 15. The insect-inhibiting composition according to item 14, wherein the at least one other insecticide is selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry3A, 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, VIP3A and VIP3B proteins.

[0053] 16. An insect-inhibiting composition comprising an insect-inhibiting effective amount of the insect-inhibiting recombinant polypeptide according to item 4.

[0054] 17. A method for controlling pests of Coleoptera species, the method comprising contacting the pests with an insect-inhibiting amount of the insect-inhibiting recombinant polypeptide according to item 4.

[0055] 18. A seed comprising the recombinant polynucleotide molecule according to item 1 or the insect-inhibiting recombinant polypeptide according to item 4.

[0056] 19. A commercial product comprising the host cell according to item 10, the commercial product comprising a detectable amount of the recombinant polynucleotide or an insect-inhibiting recombinant polypeptide encoded by the recombinant polynucleotide.

[0057] 20. A method for producing a seed comprising the recombinant polynucleotide molecule according to item 1, the method comprising:

[0058] (a) planting at least one seed comprising the recombinant polynucleotide molecule;

[0059] (b) growing a plant from the seed; and

[0060] (c) harvesting seeds from the plant, wherein the harvested seeds comprise the recombinant polynucleotide molecule.

[0061] 21. A recombinant vector comprising the recombinant polynucleotide molecule according to item 3.

[0062] 22. The recombinant vector according to item 21, wherein the vector is selected from the group consisting of plasmids, bacmids, phagemids, and cosmids.

[0063] 23. A plant resistant to insect infestation, wherein the cells of the plant contain the recombinant polynucleotide molecule according to item 1 or the insect inhibitory recombinant polypeptide according to item 4. Brief Description of the Drawings

[0064] Figure 1 Illustration of the alignment of collage protein TIC4260 with five exemplary TIC3668-type proteins. In this sequence alignment, positions with sequence diversity are highlighted in grey shading.

[0065] Figure 2 Illustration of the Western corn rootworm (WCR) inhibitory activity of exemplary chloroplast-targeted and non-targeted mature-length TIC3668-type proteins in plants.

[0066] Figure 3 Illustration of the WCR inhibitory activity of exemplary chloroplast-targeted and non-targeted mature-length TIC-3668-type proteins in plants.

[0067] Sequence Summary

[0068] SEQ ID NO:1 is a recombinant polynucleotide sequence encoding the TIC3668 protein obtained from the Brevibacillus laterosporus species, with an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0069] SEQ ID NO:2 is a translation of the amino acid sequence of the TIC3668 precursor protein obtained from the open reading frame as shown in SEQ ID NO:1.

[0070] SEQ ID NO:3 is a recombinant polynucleotide sequence encoding the TIC3669 protein obtained from the Brevibacillus laterosporus species, with an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0071] SEQ ID NO:4 is a translation of the amino acid sequence of the TIC3669 protein obtained from the open reading frame as shown in SEQ ID NO:3.

[0072] SEQ ID NO:5 is a recombinant polynucleotide sequence encoding the TIC3670 protein obtained from the Brevibacillus laterosporus species, with an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0073] SEQ ID NO:6 is the amino acid sequence translation of the TIC3670 precursor protein obtained from the open reading frame shown in SEQ ID NO:5.

[0074] SEQ ID NO:7 is a recombinant polynucleotide sequence encoding the TIC4076 protein obtained from the Brevibacillus laterosporus species, which is encoded by an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0075] SEQ ID NO:8 is the amino acid sequence translation of the TIC4076 precursor protein obtained from the open reading frame shown in SEQ ID NO:7.

[0076] SEQ ID NO:9 is a recombinant polynucleotide sequence encoding the TIC4078 protein obtained from the Brevibacillus laterosporus species, which is encoded by an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0077] SEQ ID NO:10 is the amino acid sequence translation of the TIC4078 precursor protein obtained from the open reading frame shown in SEQ ID NO:9.

[0078] SEQ ID NO:11 is a recombinant polynucleotide sequence encoding the mosaic TIC4260 protein obtained from the Brevibacillus laterosporus species, which is generated by in-frame combination of DNA segments encoding each of the coding sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9 to include the sequence variations of these 5 different open reading frames.

[0079] SEQ ID NO:12 is the amino acid sequence translation of the mosaic protein TIC4260 precursor protein obtained from the open reading frame shown in SEQ ID NO:11.

[0080] SEQ ID NO:13 is a recombinant polynucleotide sequence encoding the TIC4346 protein obtained from the Brevibacillus laterosporus species, which is encoded by an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0081] SEQ ID NO:14 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:13.

[0082] SEQ ID NO:15 is a recombinant polynucleotide sequence encoding the TIC4826 protein obtained from the Brevibacillus laterosporus species, which is encoded by an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0083] SEQ ID NO:16 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:15.

[0084] SEQ ID NO:17 is a recombinant polynucleotide sequence encoding the TIC4861 protein, obtained from the Brevibacillus laterosporus species, consisting of an open reading frame at nucleotide positions 1 - 918 and a translation termination codon.

[0085] SEQ ID NO:18 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:17.

[0086] SEQ ID NO:19 is a recombinant polynucleotide sequence encoding the TIC4862 protein, obtained from the Brevibacillus laterosporus species, consisting of an open reading frame at nucleotide positions 1 - 945 and a translation termination codon.

[0087] SEQ ID NO:20 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:19.

[0088] SEQ ID NO:21 is a recombinant polynucleotide sequence encoding the TIC4863 protein, obtained from the Brevibacillus laterosporus species, consisting of an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0089] SEQ ID NO:22 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:21.

[0090] SEQ ID NO:23 is the amino acid sequence of the mature TIC3668 protein mTIC3668.

[0091] SEQ ID NO:24 is the amino acid sequence of the mature TIC3669 protein mTIC3669.

[0092] SEQ ID NO:25 is the amino acid sequence of the mature TIC3670 protein mTIC3670.

[0093] SEQ ID NO:26 is the amino acid sequence of the mature TIC4076 protein mTIC4076.

[0094] SEQ ID NO:27 is the amino acid sequence of the mature TIC4078 protein mTIC4078.

[0095] SEQ ID NO:28 is the amino acid sequence of the mature TIC4260 protein mTIC4260.

[0096] SEQ ID NO:29 is the amino acid sequence of the mature TIC4346 protein, mTIC4346.

[0097] SEQ ID NO:30 is the amino acid sequence of the mature TIC4826 protein, mTIC4826.

[0098] SEQ ID NO:31 is the amino acid sequence of the mature TIC4861 protein, mTIC4891.

[0099] SEQ ID NO:32 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC3668 protein.

[0100] SEQ ID NO:33 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC3668 protein, mTIC3668.

[0101] SEQ ID NO:34 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC3669 protein.

[0102] SEQ ID NO:35 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC3669 protein, mTIC3669.

[0103] SEQ ID NO:36 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC3670 protein.

[0104] SEQ ID NO:37 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC3670 protein, mTIC3670.

[0105] SEQ ID NO:38 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4076 protein.

[0106] SEQ ID NO:39 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4076 protein, mTIC4076.

[0107] SEQ ID NO:40 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4078 protein.

[0108] SEQ ID NO:41 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4078 protein, mTIC4078.

[0109] SEQ ID NO:42 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4260 protein.

[0110] SEQ ID NO:43 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4260 protein mTIC4260.

[0111] SEQ ID NO:44 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4346 protein.

[0112] SEQ ID NO:45 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4346 protein mTIC4346.

[0113] SEQ ID NO:46 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4826 protein.

[0114] SEQ ID NO:47 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4826 protein mTIC4826.

[0115] SEQ ID NO:48 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4861 protein.

[0116] SEQ ID NO:49 is a synthetic nucleotide sequence designed for expression in plants and encoding the mature TIC4861 protein (mTIC4861), mature TIC4862 protein (mTIC4862), and mature TIC4863 protein (mTIC4863).

[0117] SEQ ID NO:50 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4682 protein.

[0118] SEQ ID NO:51 is a synthetic nucleotide sequence designed for expression in plants and encoding the TIC4863 protein.

[0119] SEQ ID NO:52 is a nucleotide sequence representing a synthetic oligonucleotide designed to hybridize to the (-) strand of the DNA encoding the proteins disclosed in the present application, and corresponding to positions 1 to 36 of SEQ ID NO:1 (TIC3668 forward primer).

[0120] SEQ ID NO:53 is a nucleotide sequence representing a synthetic oligonucleotide designed to hybridize to the (+) strand of the DNA encoding the proteins disclosed in the present application, and corresponding to positions 920 to 954 of SEQ ID NO:1 (TIC3668 reverse primer).

[0121] SEQ ID NO:54 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (-) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 1 to 41 of SEQ ID NO:3 (TIC3669 forward primer).

[0122] SEQ ID NO:55 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (+) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 920 to 954 of SEQ ID NO:3 (TIC3669 reverse primer).

[0123] SEQ ID NO:56 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (-) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 1 to 36 of SEQ ID NO:5 (TIC3670 forward primer).

[0124] SEQ ID NO:57 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (+) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 920 to 954 of SEQ ID NO:5 (TIC3670 reverse primer).

[0125] SEQ ID NO:58 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (-) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 1 to 41 of SEQ ID NO:7 (TIC4076 forward primer).

[0126] SEQ ID NO:59 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (+) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 920 to 954 of SEQ ID NO:7 (TIC4076 reverse primer).

[0127] SEQ ID NO:60 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (-) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 1 to 36 of SEQ ID NO:9 (TIC4078 forward primer).

[0128] SEQ ID NO:61 represents the nucleotide sequence of a synthetic oligonucleotide for hybridizing with the (+) strand of the DNA encoding the protein disclosed in the present application, and corresponds to positions 920 to 954 of SEQ ID NO:9 (TIC4078 reverse primer).

[0129] SEQ ID NO:62 is a recombinant polynucleotide sequence encoding the TIC2462 protein obtained from the Brevibacillus laterosporus species, which consists of an open reading frame at nucleotide positions 1 - 951 and a translation termination codon.

[0130] SEQ ID NO:63 is the amino acid sequence translation of the open reading frame shown in SEQ ID NO:62.

[0131] SEQ ID NO:64 is a synthetic nucleotide sequence encoding the mature TIC3668 protein mTIC3668 for expression in bacteria.

[0132] SEQ ID NO:65 is a synthetic nucleotide sequence encoding the mature TIC3669 protein mTIC3669 for expression in bacteria.

[0133] SEQ ID NO:66 is a synthetic nucleotide sequence encoding the mature TIC3670 protein mTIC3670 for expression in bacteria.

[0134] SEQ ID NO:67 is a synthetic nucleotide sequence encoding the mature TIC4076 protein mTIC4076 for expression in bacteria.

[0135] SEQ ID NO:68 is a synthetic nucleotide sequence encoding the mature TIC4078 protein mTIC4078 for expression in bacteria.

[0136] SEQ ID NO:69 is a synthetic nucleotide sequence encoding the mature TIC4260 protein mTIC4260 for expression in bacteria.

[0137] SEQ ID NO:70 is a synthetic nucleotide sequence encoding the mature TIC4346 protein mTIC4346 for expression in bacteria.

[0138] SEQ ID NO:71 is a synthetic nucleotide sequence encoding the mature TIC4826 protein mTIC4826 for expression in bacteria.

[0139] SEQ ID NO:72 is a synthetic nucleotide sequence encoding the mature TIC4861 (mTIC4861), TIC4862 (mTIC4862), and TIC4863 (mTIC4863) proteins for expression in bacteria. Detailed Description of the Invention

[0140] Problems in the field of agricultural pest control can be characterized as requiring novel toxin proteins that are effective against target pests, exhibit broad-spectrum toxicity against target pest species, can be expressed in plants without causing undesirable agronomic problems, and provide an alternative mode of action compared to currently used toxins in plants. A novel insecticidal protein exemplified by TIC3668 is disclosed herein and addresses each of these needs, particularly against broad-spectrum coleopteran and lepidopteran insect pests, and more particularly against corn root pest species.

[0141] As used in this application, references to "TIC3668", "TIC3668 protein", "TIC3668 protein toxin", "TIC3668 toxin protein", "TIC3668-related toxin", "TIC3668-related protein toxin class or family", "TIC3668-related toxin protein", "TIC3668-type protein", "TIC3668-like protein", "TIC3668-related toxin polypeptide", "TIC3668-related insecticidal protein", or "TIC3668-type insect inhibitory polypeptide", etc. refer to any novel insect inhibitory protein that comprises, consists of, is substantially homologous to, is similar to, or is derived from the following: any insect inhibitory polypeptide sequence of TIC3668 (SEQ ID NO:2) that confers activity against coleopteran and lepidopteran pests and its insect inhibitory segments or combinations thereof, including any protein that exhibits insect inhibitory activity, provided that such protein has an amino acid sequence identity of from about 35% to about 100% percentage score of any percentage fraction when aligned with TIC3668 (SEQ ID NO:2), TIC3669 (SEQ ID NO:4), TIC3670 (SEQ ID NO:6), TIC4076 (SEQ ID NO:8), TIC4078 (SEQ ID NO:10), TIC4346 (SEQ ID NO:14), TIC4826 (SEQ ID NO:16), TIC4861 (SEQ ID NO:18), TIC4862 (SEQ ID NO:20), and TIC4863 (SEQ ID NO:22). TIC3668-type protein toxins disclosed in this application include TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863, and the chimeric TIC4260 protein (SEQ ID NO:12). The TIC3668-type protein class is intended to include both precursor forms and mature-length forms of the protein.

[0142] The terms "segment" or "fragment" are used in this application to describe a shorter contiguous amino acid or nucleic acid sequence as compared to the complete amino acid or nucleic acid sequence of the TIC3668 protein. Segments or fragments that exhibit insect inhibitory activity are also disclosed in this application, provided that the alignment of such segments or fragments with the corresponding segment of the TIC3668 protein shown in SEQ ID NO:2 results in an amino acid sequence identity of any percent score between about 35 and about 100 percent between the segment or fragment and the corresponding segment of the TIC3668 protein.

[0143] As used in this application, the terms "active" or "activity", "insecticidal activity" or "insecticidal activity", "insect inhibitory" or "insecticidal" refer to the efficacy of a toxicant such as a protein toxin in inhibiting (inhibiting growth, feeding, fecundity or viability), curbing (curbing growth, feeding, fecundity or viability), controlling (controlling pest infestations, controlling the feeding activities of pests on a particular crop containing an effective amount of the TIC3668 protein) or killing (causing morbidity, lethality or reduced fecundity) of pests. These terms are intended to include the results of providing an insecticidal effective amount of a toxic protein to a pest, where exposure of the pest to the toxic protein results in morbidity, lethality, reduced fecundity or stunted growth. These terms also include the repulsion of pests from a plant, plant tissue, plant part, seed, plant cell or from a particular geographical location where the plant may be growing due to the provision of an insecticidal effective amount of a toxic protein in or on the plant. Generally, insecticidal activity refers to the ability of a toxic protein to effectively inhibit the growth, development, viability, feeding behavior, mating behavior, fecundity of a specific target pest (including but not limited to insects of the order Lepidoptera or Coleoptera), or any measurable reduction in adverse effects caused by the ingestion of this protein, protein fragment, protein segment or polynucleotide by an insect. The toxic protein may be produced by a plant or may be applied to the plant or to the environment within the location where the plant is situated. The terms "bioactive", "effective", "efficacious" or variations thereof are also terms that are used interchangeably in this application to describe the action of the proteins of the present invention on target insect pests.

[0144] When provided in the diet of a target pest, an insecticidally effective amount of a toxicant exhibits insecticidal activity when the toxicant contacts the pest. The toxicant can be an insecticidal protein or one or more chemical agents known in the art. The insecticidal chemical agents and insecticidal protein agents can be used alone or in combination with each other. Chemical agents include, but are not limited to, dsRNA molecules that target specific genes to achieve suppression in target pests, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Insecticidal protein agents include the protein toxins shown in the present application, as well as other protein toxicants, including those targeting Lepidoptera and Coleoptera, and protein toxins for controlling other plant pests such as Cry proteins available in the art for controlling Hemiptera and Homoptera species.

[0145] The reference to pests, particularly pests of crop plants, is intended to mean insect pests of crop plants, particularly those controlled by the TIC3668-related protein toxin class. However, the reference to pests can also include Hemiptera and Homoptera insect pests of plants, as well as nematodes and fungi, when: the toxicants targeting these pests are co-localized or present together with one or more proteins of the TIC3668-related protein toxin class.

[0146] The individual proteins that make up the TIC3668-related protein class are related due to having a common function and exhibit insecticidal activity against insect pests from Coleoptera and Lepidoptera insect species, said insect pests including adults, pupae, larvae, and neonates. Lepidoptera insects include, but are not limited to, armyworms, cutworms, loopers, and bollworms in the family Noctuidae, such as fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), shawl caterpillar (Mamestra configurata), 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 leafminers from the family Pyralidae, such as European corn borer (Ostrinia nubilalis), navel orangeworm (Amyelois transitella), corn root webworm (Crambus caliginosellus), rice leafroller (Herpetogramma licarsisalis), sunflower moth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpus lignosellus); leafrollers, budworms, seedworms, and fruitworms in the family Tortricidae, such as 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 insects, such as diamondback moth (Plutella xylostella), pink bollworm (Pectinophora gossypiella), and gypsy moth (Lymantria dispar).Other insect pests of the Lepidoptera order include, for example, Alabama argillacea (cotton leaf worm), Archips argyrospila (fruit tree leaf roller), Archips rosana (European leaf roller) and other Archips species, Chilo suppressalis (Asian rice borer or rice stem borer), Cnaphalocrocis medinalis (rice leaf roller), Elasmopalpus lignosellus (lesser cornstalk borer), Crambus teterrellus (bluegrass webworm), Diatraea grandiosella (southwestern corn borer), Diatraea saccharalis (sugarcane borer), Earias insulana (spiny bollworm), Earias vittella (spotted bollworm), Helicoverpa armigera (American bollworm), Helicoverpa zea (corn earworm or cotton bollworm), Heliothis virescens (tobacco budworm), Herpetogramma licarsisalis (grass webworm), Lobesia botrana (European grapevine moth), Phyllocnistis citrella (citrus leafminer), Pieris brassicae (large white butterfly), Pieris rapae (imported cabbageworm or small white butterfly), Plutella xylostella (diamondback moth), Spodoptera exigua (beet armyworm), Spodoptera litura (tobacco cutworm, cluster caterpillar) and Tuta absoluta (tomato leafminer). Insects of the Coleoptera order include, but are not limited to, certain species of Agriotes, certain species of Anthonomus, Cryptocephalus pusillus, Chaetocnema concinna, certain species of Cylindrocopturus, certain species of Curculio, certain species of Dermestes, certain species of Diabrotica, certain species of Epilachna, certain species of Eremnus, Leptinotarsa decemlineata, certain species of Lissorhoptrus, certain species of Melolontha, certain species of Oryzaephilus, certain species of Otiorhynchus, certain species of Popillia, certain species of Psylliodes, certain species of Rhyzopertha, Scarabaeidae, certain species of Sitophilus, certain species of Sitotroga, certain species of Tenebrio, certain species of Tribolium and certain species of Trogoderma, especially when the pest is Diabrotica virgifera virgifera (western corn rootworm - WCR), Diabrotica barberi (northern corn rootworm - NCR), Diabrotica viridula (Mexican corn rootworm - MCR), Diabrotica balteata (Brazilian corn rootworm - BZR), Diabrotica undecimpunctata howardi (southern corn rootworm - SCR) and the Diabrotica complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa.

[0147] As used herein, the term "isolated DNA molecule", "isolated polynucleotide molecule", or equivalent terms or phrases mean a DNA molecule that is present alone or in combination with other components, but not in its natural environment. For example, nucleic acid elements such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc. that are naturally found within the DNA of an organism's genome are not considered to be "isolated" so long as the element is within the organism's genome and at the location within the genome where it is naturally found. However, within the scope of the present disclosure, each of these elements and sub-parts of these elements will be "isolated" so long as the element is not within the organism's genome and not at the location within the genome where it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of an insecticidal protein will be an isolated nucleotide sequence so long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein is naturally found. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered to be isolated. For the purposes of the present disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA that has been inserted into the genome of a plant or bacterial cell or is present in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence regardless of whether it is present within a plasmid or similar construct used to transform the cell, within the genome of the plant or bacterium, or present in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from the plant or bacterium.

[0148] As further described in this application, an open reading frame (ORF) (SEQ ID NO: 1) encoding TIC3668 (SEQ ID NO: 2) was found in the DNA obtained from the Brevibacillus laterosporus strain EG5552. Sequences encoding TIC3668-related proteins in other bacterial genomes were then screened. Several other open reading frames encoding amino acid sequences similar to the EG5552 TIC3668 protein were identified in these other bacterial genomes, including the following TIC3668-like proteins: TIC3669 (encoded by SEQ ID NO: 3 and SEQ ID NO: 4) found in the DNA obtained from the Brevibacillus laterosporus strain EG5551, TIC3670 (encoded by SEQ ID NO: 5 and SEQ ID NO: 6) found in the DNA obtained from the Brevibacillus laterosporus strain EG5553, TIC4076 (encoded by SEQ ID NO: 7 and SEQ ID NO: 8) found in the DNA obtained from the Brevibacillus laterosporus strain ATCC6456, TIC4078 (encoded by SEQ ID NO: 9 and SEQ ID NO: 10) found in the DNA obtained from the Brevibacillus laterosporus strain EG4227, TIC4346 (encoded by SEQ ID NO: 13 and SEQ ID NO: 14) found in the DNA obtained from the Brevibacillus laterosporus strain EG5551, TIC4826 (encoded by SEQ ID NO: 15 and SEQ ID NO: 16) found in the DNA obtained from the Brevibacillus laterosporus strain AG0021D10, TIC4861 (encoded by SEQ ID NO: 17 and SEQ ID NO: 18), TIC4862 (encoded by SEQ ID NO: 19 and SEQ ID NO: 20), and TIC4863 (encoded by SEQ ID NO: 21 and SEQ ID NO: 22) found in the DNA obtained from the Brevibacillus laterosporus strain EG4227. An additional TIC3668-like protein, TIC4260 (encoded by SEQ ID NO: 11 and SEQ ID NO: 12), was generated by combining naturally occurring amino acid sequence variants from 5 different native TIC3668-like proteins to produce a mosaic protein.

[0149] The corresponding coding sequences were cloned and expressed in a microbial host cell to produce recombinant proteins for use in insect bioassays. As further described in this application, these proteins were shown to exhibit biological activity against species of the genus Diabrotica, including the western corn rootworm (WCR, Diabrotica virgifera virgifera), the European corn borer (ECB, Ostrinia nubilalis), the southwestern corn borer (SWC, Diatraea grandiosella), and the soybean looper (SBL, Chrysodeixis includens).

[0150] A surprising feature of the TIC3668-type proteins is the presence of N-terminal amino acid segments corresponding to amino acid positions 1 to 23 (for TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4863); 1 to 12 (for TIC4861); and 1 to 21 (for TIC4862). These N-terminal amino acid segments can each be omitted from the respective proteins, and the polynucleotide sequences encoding the respective segments can also be omitted. When expressed in plants, omission of these respective segments surprisingly results in increased insecticidal activity against corn rootworm species compared to expression of the full-length protein toxins containing the omitted segments. The protein toxin segments lacking the above-mentioned N-terminal amino acid segments are referred to herein as "mature TIC3668-type toxin proteins". In general, reference to the mature form of the TIC3668-type protein is annotated herein with the letter "m" before the name of the toxin to distinguish the mature sequence from the full-length native sequence. For example, the mature form of the amino acid sequence of TIC3668 (SEQ ID NO:2) is mTIC3668 (SEQ ID NO:23). The mature forms of TIC3669 (SEQ ID NO:4), TIC3670 (SEQ ID NO:6), TIC4076 (SEQ ID NO:8), TIC4078 (SEQ ID NO:10), TIC4260 (SEQ ID NO:12), TIC4346 (SEQ ID NO:14), and TIC4826 (SEQ ID NO:16) are mTIC3669 (SEQ ID NO:24), mTIC3670 (SEQ ID NO:25), mTIC4076 (SEQ ID NO:26), mTIC4078 (SEQ ID NO:27), mTIC4260 (SEQ ID NO:28), mTIC4346 (SEQ ID NO:29), and mTIC4826 (SEQ ID NO:30), respectively. The full-length proteins TIC4861 (SEQ ID NO:18), TIC4862 (SEQ ID NO:20), and TIC4863 (SEQ ID NO:22) are sequence length variants of each other and differ only in the length of their N-terminal amino acid segments. Removal of the N-terminal amino acid segments in TIC4861, TIC4862, and TIC4863 results in the same mature amino acid sequence for mTIC4861, mTIC4862, and mTIC4863. Thus, the amino acid sequences of mTIC4861, mTIC4862, and mTIC4863 are encoded by the same polynucleotide sequence (mTIC4861, SEQ ID NO:31).The mature TIC3668-like protein sequences are encoded by SEQ ID NO:64 (encoding mTIC3668), SEQ ID NO:65 (encoding mTIC3669), SEQ ID NO:66 (encoding mTIC3670), SEQ ID NO:67 (encoding mTIC4076), SEQ ID NO:68 (encoding mTIC4078), SEQ ID NO:69 (encoding mTIC4260), SEQ ID NO:70 (encoding mTIC4346), SEQ ID NO:71 (encoding mTIC4826), and SEQ ID NO.72 (encoding mTIC4861, mTIC4862, and mTIC4863) for expression in a bacterial host.

[0151] Additional members of the TIC3668-type family can be generated by using naturally occurring amino acid variations from some or all of the family members to produce novel proteins with a higher level of amino acid sequence diversity and novel properties. Variants of the TIC3668-type protein toxin class are generated by aligning the amino acid sequences of the TIC3668-type family members and combining the differences at the amino acid sequence level to obtain novel amino acid sequences, and making appropriate changes to the polynucleotides encoding these variants. One such example is TIC4260. SEQ ID NO:11 is the polynucleotide sequence encoding the TIC4260 protein (SEQ ID NO:12). The mature protein (mTIC4260, SEQ ID NO:28) is encoded by the polynucleotide sequence SEQ ID NO:43.

[0152] Fragments of the TIC3668 protein toxin can be truncated forms that are missing one or more amino acids from the N-terminus, C-terminus, middle, or a combination thereof of the protein and have insect inhibitory activity. These fragments can be naturally occurring variants or synthetic variants of TIC3668, TIC3669, TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862, or TIC4863, but should retain or improve the insect inhibitory activity of TIC3668, TIC3669, TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862, or TIC4863. Truncated N-terminus or C-terminus deletion variants include, but are not limited to, TIC3668, TIC3669, TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862, or TIC4863 proteins lacking amino acid residues from the N-terminus and / or C-terminus. For example, amino acid residues 1 to 23 of the N-terminus of the TIC3668 protein can be deleted, resulting in a toxin protein having amino acids 24 - 317 of SEQ ID NO:2. Removal of 10 or 20 amino acids from the C-terminal amino terminus of the TIC3668 protein results in a loss of insecticidal activity, while removal of a single amino acid does not affect the activity.

[0153] Proteins of the TIC3668 protein class and proteins similar to proteins of the TIC3668 protein class can be identified by comparing them to each other using various computer-based algorithms known in the art (see Tables 1 and 2). The amino acid sequence identities reported herein are the result of Clustal W alignments using the following default parameters: weight matrix: blosum, gap open 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). The percentage of amino acid identity is further calculated by the product of 100% multiplied by (the number of identical amino acids / the length of the subject protein). Other alignment algorithms are also available in the art and provide results similar to those obtained using Clustal W alignments.

[0154] A protein expected to exhibit insecticidal activity against Lepidopteran insect species is a member of the TIC3668 type of protein toxin class, provided that the protein is used, for example, in a query in a Clustal W alignment, and at least one protein of the present invention, described as mTIC4260, is identified as a hit in such an alignment, wherein the query protein exhibits at least about 85% to about 100% amino acid sequence identity along the length of the query protein, i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any percentage fraction within this range; or at least one protein of the present invention, described as mTIC3668, is identified as a hit in such an alignment, wherein the query protein exhibits at least about 89% to about 100% amino acid sequence identity along the length of the query protein, i.e., 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any percentage fraction within this range; or at least one protein of the present invention, described as mTIC3669 and / or mTIC3670, is identified as a hit in such an alignment, wherein the query protein exhibits at least about 90% to about 100% amino acid sequence identity along the length of the query protein, i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any percentage fraction within this range; or at least one protein of the present invention, described as mTIC4826, is identified as a hit in such an alignment, wherein the query protein exhibits at least about 91% to about 100% amino acid sequence identity along the length of the query protein, i.e., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any percentage fraction within this range.

[0155] A protein expected to exhibit insecticidal activity against Coleoptera insect species is a member of the TIC3668 type protein toxin class, provided that the protein is used, for example, in a query in a Clustal W alignment, and at least one protein of the present invention, such as mTIC3668, mTIC3669, mTIC3670, mTIC4076, mTIC4078, mTIC4260, mTIC4346, mTIC4826, mTIC4861, mTIC4862, and mTIC4863, is identified as a hit in such an alignment, wherein the query protein exhibits at least about 35% to about 100% amino acid identity along the length of the query protein, i.e., about 35%, 40%, 50%, 60%, 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% or any percentage fraction within this range.

[0156] Exemplary proteins of the TIC3668 type protein toxin class were aligned with each other using the Clustal W algorithm. A pairwise matrix of the percentage of amino acid sequence identity of each pair of full-length proteins was created, as reported in Table 1. A pairwise matrix of the percentage of amino acid sequence identity of each pair of mature-length proteins was created, as reported in Table 2.

[0157] Table 1. Pairwise matrix of exemplary full-length proteins shows

[0158]

[0159] Table description: The Clustal W alignment between (X) and (Y) is reported in the pairwise matrix. Each column under (N) refers to SEQ ID NO. Column (M) refers to the protein name (TIC#). The percentage of amino acid identity between all pairs was calculated and is represented by the first number in each box. The second number in each box (in parentheses) represents the number of identical amino acids between the pair.

[0160] Table 2. Pairwise matrix of exemplary mature proteins shows

[0161]

[0162] Table description: The Clustal W alignment between (X) and (Y) is reported in the pairwise matrix. Each column under (N) refers to SEQ ID NO. Column (M) refers to the protein name (TIC#). The percentage of amino acid identity between all pairs is calculated and represented by the first number in each box. The second number in each box (in parentheses) represents the number of identical amino acids between the pair.

[0163] The full-length and mature proteins of the TIC3668 type protein toxin class can also be related due to primary structure (conserved amino acid motifs), length (the mature protein is approximately 295 amino acids, while the full-length protein is approximately 317 amino acids), and other characteristics. When run on a protein gel under denaturing conditions, the full-length protein of the present invention has a measured mass of approximately 35 kDa (Dalton), while the mature protein has a measured mass of approximately 32 kDa. The characteristics of the full-length and mature forms of the TIC3668 type protein toxin class are reported in Tables 3 and 4.

[0164] Table 3. Characteristics of the full-length protein

[0165]

[0166] Table 4 - Characteristics of the mature protein

[0167]

[0168] The proteins of the disclosed TIC3668 type of protein toxin represent a novel class of insecticidal proteins. Referring to Table 5, all numbers above the diagonal corresponding to 100% identity represent the number of amino acid differences between the corresponding proteins compared at the intersection of that particular row and column. The numbers below the diagonal corresponding to 100% identity represent the percentage identity of the corresponding proteins compared at the intersection of that particular row and column. The mature length members of this protein class exhibit no more than 90.54% amino acid identity with any other insecticidal protein known in the art, as shown in the alignments provided in Table 5. The insecticidal protein showing the closest identity to any mature length protein of the present invention is SEQ ID NO:50 (AXMI-209) in U.S. Patent Application Publication No. 20110030093, having 90.5% sequence identity with mTIC4076, mTIC4346, mTIC4826, and mTIC4863. This disclosure only teaches activity against Lepidoptera, while the exemplary proteins of the present invention show activity against Coleoptera. H0UDD3_BRELA, F7TVP6_BRELA, and U4WSU1_BRELA are unannotated protein sequences predicted from open reading frames in the genomic sequence and are reported to have been obtained from Brevibacillus laterosporus. The insecticidal activity of these proteins has not been reported.

[0169] Table 5 - Alignment of Mature Length TIC3886 Protein with Prior Art Proteins

[0170]

[0171] The TIC3668 protein disclosed in the present application exhibits activity against Coleoptera, including WCR, in dietary bioassays. In some cases, Lepidoptera activity has also been observed.

[0172] As further described in the embodiments of the present application, polynucleotide sequences encoding the TIC3668 toxin protein are designed for use in plants. Exemplary polynucleotides designed for expression in plants and encoding the full-length insect inhibitory TIC3668, TIC3669, TIC3670, TIC4260, TIC4076, TIC4078, TIC4346, TIC4826, TIC4861, TIC4862 and TIC4863 proteins are shown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:51. Exemplary polynucleotides designed for expression in plants and encoding the mature forms of the insect inhibitory mTIC3668, mTIC3669, mTIC3670, mTIC4260, mTIC4076, mTIC4078, mTIC4346, mTIC4826, mTIC4861, mTIC4862 and mTIC4863 proteins are shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:49.

[0173] Expression cassettes and vectors containing these polynucleotide sequences are constructed and introduced into maize plant cells according to transformation methods and techniques known in the art. The transformed cells are regenerated into transformed plants that are observed to express the TIC3668 toxin protein. To test for insecticidal activity, plant leaf discs obtained from the transformed plants are used in a bioassay in the presence of lepidopteran or coleopteran pest larvae.

[0174] The insect inhibitory activity of exemplary members of the TIC3668 type protein toxin class is described in more detail in the examples. The exemplary proteins are related due to having a common function and exhibit insecticidal activity against coleopteran and lepidopteran insect species, including adults, pupae, larvae and neonates.

[0175] Recombinant polynucleotide compositions encoding the TIC3668 protein are encompassed. For example, the TIC3668 protein can be expressed using a recombinant DNA construct, wherein a polynucleotide molecule having an open reading frame (ORF) encoding the protein is operably linked to gene expression elements such as a promoter and any other regulatory elements necessary for expression in the system for which the construct is intended. Non-limiting examples include a plant functional promoter operably linked to the TIC3668 protein coding sequence for expression of the protein in plants, or a Bt functional promoter operably linked to the TIC3668 protein coding sequence for expression of the protein in Bt bacteria or other Bacillus species. Other elements can be operably linked to the TIC3668 protein coding sequence, including but not limited to enhancers, introns, untranslated leaders, polypeptide sequences encoding protein affinity tags (HIS tags), translocation peptides (i.e., plastid transit peptides, signal peptides), polypeptide sequences of post-translational modification enzymes, ribosome binding sites, and RNAi target sites. Exemplary recombinant polynucleotide molecules provided herein include but are not limited to a heterologous promoter operably linked to a polynucleotide such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21 encoding the corresponding polypeptide or protein having the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22. The codons of the recombinant polynucleotide molecules encoding the proteins disclosed herein can be replaced by synonymous codons (referred to in the art as silent substitutions).Non-limiting examples of modified polynucleotides encoding any of the TIC3668 proteins disclosed in the present application are shown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:51 (for full-length protein sequences) and SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, and SEQ ID NO:49 (for mature protein sequences).

[0176] A recombinant DNA construct containing a TIC3668 protein coding sequence may further contain a DNA region encoding one or more insect inhibitors, which may be constructed to be expressed in parallel with the DNA sequence encoding the TIC3668 protein or to co-express a protein, an insect inhibitory dsRNA molecule, or an auxiliary protein different from the TIC3668 protein. Auxiliary proteins include, but are not limited to, cofactors, enzymes, binding partners, or other reagents that play a role in contributing to the effectiveness of the insect inhibitor, such as by assisting its expression, affecting its stability in plants, optimizing oligomeric free energy, enhancing its toxicity, and increasing its activity spectrum. The auxiliary protein may promote the uptake of one or more insect inhibitors (for example) or enhance the toxic effect of the toxic agent.

[0177] The recombinant DNA construct may be assembled such that all proteins or dsRNA molecules are expressed according to one promoter, or each protein or dsRNA molecule is under the control of a separate promoter, or in a certain combination thereof. The proteins of the present invention may be expressed by a multi-gene expression system, in which one or more of the TIC3668 proteins are expressed from a common nucleotide segment, which may also contain other open reading frames and promoters depending on the type of the selected expression system. For example, a bacterial multi-gene expression system may utilize a single promoter to drive the expression of multiple linked / tandem open reading frames from within a single operon (i.e., polycistronic expression). In another example, a plant multi-gene expression system may utilize unlinked expression cassettes, each expressing a different protein or other reagent such as one or more dsRNA molecules.

[0178] A recombinant polynucleotide or recombinant DNA construct comprising a protein-encoding sequence of type TIC3668 can be delivered to a host cell via a vector such as a plasmid, baculovirus, synthetic chromosome, virus particle, cosmid, phagemid, phage, or viral vector. Such vectors can be used to achieve stable or transient expression of the protein-encoding sequence of type TIC3668 in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that comprises a protein-encoding sequence of type TIC3668 and is introduced into a host cell is referred to herein as a "transgene".

[0179] This disclosure provides transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain a recombinant polynucleotide that expresses any one or more protein-encoding sequences of type TIC3668. The term "bacterial cell" or "bacterium" can include, but is not limited to, Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, or Rhizobium cells. The term "plant cell" or "plant" can include, but is not limited to, monocots, dicots, alfalfa, banana, barley, bean, broccoli, cabbage, Brassica, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugar cane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cells or plants. In certain embodiments, transgenic plants and transgenic plant parts regenerated from transgenic plant cells are provided. In certain embodiments, a transgenic plant can be obtained from a transgenic seed by cutting, breaking, grinding, or otherwise dissociating a part from the plant. In certain embodiments, the plant part can be a seed, pod, leaf, flower, stem, root, or any part or non-regenerable part of a transgenic plant part. As used in this context, a "non-regenerable" part of a transgenic plant part is a part that cannot be induced to form a whole plant, or cannot be induced to form a whole plant capable of sexual and / or asexual reproduction. In certain embodiments, the non-regenerable part of a plant part is a part of a transgenic seed, pod, leaf, flower, stem, or root.

[0180] A method for preparing transgenic plants comprising a TIC3668-type protein in an amount inhibitory to Coleoptera or Lepidoptera insects is provided. Such plants can be prepared by introducing a recombinant polynucleotide encoding any of the TIC3668-type proteins provided herein into a plant cell, and selecting a plant derived from said plant cell that expresses a TIC3668-type protein in an amount inhibitory to Coleoptera or Lepidoptera insects. Plants can be derived from plant cells by regeneration, sowing, pollination, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0181] Processed plant products are also disclosed herein, wherein said processed product comprises a detectable amount of a TIC3668-type protein, an insect inhibitory segment or fragment thereof, or any characteristic portion thereof. In certain embodiments, the processed product is selected from the group consisting of plant parts, plant biomass, oils, meals, sugars, animal feeds, flours, flakes, bran, linters, hulls, processed seeds, and seeds. In certain embodiments, the processed product is non-renewable. Plant products can include commodities or other commercial products derived from transgenic plants or transgenic plant parts, wherein said commodities or other products can be commercially traced by detecting a nucleotide segment encoding or comprising a characteristic portion of a TIC3668-type protein, or the expressed RNA or protein.

[0182] Plants expressing the TIC3668 protein can be bred to hybridize 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, etc.), or such traits can be combined in a single vector to associate all the traits.

[0183] Methods known to those of ordinary skill in the art, such as polymerase chain reaction (PCR), thermal amplification, and hybridization, can be used to identify TIC3668-type protein coding sequences and sequences having substantially percent identity to the TIC3668-type protein coding sequence. For example, proteins of the TIC3668-type protein toxin class can be used to generate antibodies that specifically bind proteins of this class, and can be used to screen for and discover other members of this class.

[0184] In addition, the nucleotide sequences (and reverse complementary sequences) encoding the TIC3668 type of protein toxin can be used as screening probes and primers to identify other members of the type using thermal cycling or isothermal amplification and hybridization methods. Specifically, oligonucleotides derived from the sequences shown in any of SEQ ID NOs: 52 to 61 can be used to determine the presence or absence of the TIC3668 type of transgene in a deoxyribonucleic acid sample derived from a commercial product. Given the sensitivity of certain nucleic acid detection methods using oligonucleotides, it is expected that oligonucleotides derived from the sequences shown in any of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61 can be used to detect the TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, or TIC4260 transgenes in a commercial product derived from a pooled source, in which only a portion of the commercial product is derived from transgenic plants containing any of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61. It should be further recognized that such oligonucleotides can be used to introduce nucleotide sequence variants in each of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61. Such "mutagenic" oligonucleotides are used to identify TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, or TIC4260 amino acid sequence variants that exhibit a range of insect inhibitory activities or changes in expression in transgenic plant host cells.

[0185] Nucleotide sequence homologs, such as insecticidal proteins encoded by nucleotide sequences that hybridize to each or any of the sequences disclosed in the present application under stringent hybridization conditions, are also an embodiment of the present invention. The present 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 complementary sequence) encodes an insecticidal protein or an insecticidal fragment thereof and hybridizes to the second nucleotide sequence under stringent hybridization conditions. In such cases, the second nucleotide sequence can be any nucleotide sequence disclosed in the TIC3668 type protein toxin class under stringent hybridization conditions. Nucleotide coding sequences hybridize to each other under appropriate hybridization conditions, and the proteins encoded by these nucleotide sequences cross-react with antisera raised against any of the other proteins. Stringent hybridization conditions are known in the art and can vary depending on the desired application and results, and can encompass a variety of reagents and conditions. For example, washing at a higher temperature constitutes more stringent conditions. In certain embodiments, the hybridization conditions of the present invention can include hybridizing at least at 42 °C, followed by washing twice at room temperature with 2X SSC, 0.1% SDS for 5 minutes each, and then washing twice at 65 °C in 0.5X SSC, 0.1% SDS for 30 minutes each; or hybridizing at 68 °C, followed by washing at 68 °C in 2X SSC containing 0.1% SDS; or hybridizing at 37 °C in 50% formamide, 1 M NaCl, and 1% SDS for 4 to 12 hours, and washing at 60 °C - 65 °C in 0.1X SSC.

[0186] Those skilled in the art will recognize that due to genetic code redundancy, many other sequences are capable of encoding such related proteins, and those sequences are embodiments of the present invention insofar as they function to express an insecticidal protein in a Bacillus strain or in a plant cell. It should of course be recognized that many such redundant coding sequences will not hybridize to the native Bacillus sequence encoding TIC3668 under these conditions. This application encompasses the use of these identification methods and other identification methods known to those of ordinary skill in the art to identify TIC3668 type protein coding sequences and sequences having substantially percent identity to the TIC3668 type protein coding sequence.

[0187] The present disclosure also encompasses the use of molecular methods known in the art to engineer and clone commercially useful proteins, including chimeras of proteins from pesticidal proteins; for example, chimeras can be assembled from segments of the TIC3668 type protein to obtain additional useful embodiments, including assembling segments of the TIC3668 type protein with segments of different proteins that are different from TIC3668 and related proteins. The TIC3668 type protein classes can be subjected to pairwise alignment with each other and with other Bacillus pesticidal proteins (whether these proteins are phylogenetically closely or distantly related), and segments for substitution between the aligned proteins can be identified, resulting in the construction of segments of chimeric proteins. Such chimeric proteins can be subjected to pest bioassay analysis and characterized for the presence or absence of increased biological activity and / or an expanded target pest spectrum compared to the parental proteins from which each such segment in the chimera is derived. The pesticidal activity of polypeptides can be further engineered to obtain activity against specific pests or a broader spectrum of pests by swapping domains or segments with other proteins or by using directed evolution methods known in the art.

[0188] The present application also discloses methods for controlling insect (especially Lepidoptera or Coleoptera insects) infestation of crop plants with proteins from the TIC3668 toxin protein class. Such methods can include growing plants that contain an insect inhibitory amount of a protein from the TIC3668 toxin protein class against Coleoptera or Lepidoptera insects. In certain embodiments, such methods can further include any one or more of the following operations: (i) applying any composition containing or encoding a protein from the TIC3668 type protein toxin class to the plant or the seed from which the plant is produced; and (ii) transforming the plant or the plant cell from which the plant is produced with a polynucleotide encoding a protein from the TIC3668 type protein toxin class. Generally, it is expected that any protein in the TIC3668 type protein toxin class can be provided in a composition, provided in a microorganism, or provided in a transgenic plant to confer insect inhibitory activity against Lepidoptera or Coleoptera insects.

[0189] In certain embodiments, a recombinant polypeptide of the TIC3668 type protein toxin class is the insecticidal active ingredient of an insect inhibitory composition prepared by culturing recombinant Bacillus cells or any other recombinant bacterial cells transformed to express a TIC3668 type protein toxin under conditions suitable for expressing and producing a protein of the TIC3668 type protein toxin class. Such compositions can be prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting, or concentrating the culture of such recombinant cells that express / produce the recombinant polypeptide. Such methods can yield extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, or cell precipitates of Bacillus cells or other entomopathogenic bacterial cells. By obtaining the recombinant polypeptide so produced, compositions comprising the recombinant polypeptide can include bacterial cells, bacterial spores, and parasporal inclusions, and can be formulated for various uses, including as an agricultural insect inhibitory spray product or as an insect inhibitory preparation in dietary bioassays.

[0190] In one embodiment, to reduce the likelihood of resistance formation, an insect inhibitory composition comprising one or more proteins from the TIC3668 type of protein toxin category may further comprise at least one additional polypeptide that exhibits insect inhibitory activity against the same Lepidoptera or Coleoptera insect species but is different from the TIC3668 type of protein toxin. Additional polypeptides that may be used in such compositions include insect inhibitory proteins and insect inhibitory dsRNA molecules. An example of using such ribonucleotide sequences to control insect pests is described in Baum et al. (U.S. Patent Publication 2006 / 0021087A1). Such additional polypeptides for controlling Lepidoptera pests may be selected from the group consisting of: insect inhibitory proteins such as, but not limited to, Cry1A (U.S. Patent No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. Patent Publication No. 10 / 525,318), Cry1C (U.S. Patent No. 6,033,874), Cry1D, Cry1Da and its variants, Cry1E, Cry1F, and Cry1A / F chimeras (U.S. Patent 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), Cry2A, Cry2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-184, AXMI-196, DIG-3, DIG-4, DIG-5, DIG-11, AfIP-1A and its derivatives (U.S. Patent Publication 2014-0033361A1), AfIP-1B and its derivatives (U.S. Patent Publication 2014-0033361A1), PIP-1A PIP-1B (U.S. Patent Publication 2014-0007292A1), PSEEN3174 (U.S. Patent Publication 2014-0007292A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292A1), Pput_1064 (U.S. Patent Publication 2014-0007292A1), GS-135 and its derivatives (U.S. Patent Publication 2012-0233726A1), GS153 and its derivatives (U.S. Patent Publication 2012-0192310A1), GS154 and its derivatives (U.S. Patent Publication 2012-0192310A1), GS155 and its derivatives (U.S. Patent Publication 2012-0192310A1), SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2012-0047606A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2011-0154536A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 and 4 and their derivatives as described in U.S. Patent Publication 2010-0192256A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2010-0077507A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2010-0077508A1, SEQ ID NO:2 and its derivatives as described in U.S. Patent Publication 2009-0313721A1, SEQ ID NO:2 or 4 and their derivatives as described in U.S. Patent Publication 2010-0269221A1, as U.S. Patent No. 7,772,SEQ ID NO:2 and its derivatives as described in 465(B2), CF161_0085 and its derivatives as described in WO2014 / 008054A2, lepidopteran-toxic proteins and their derivatives as described in U.S. Patent Publications US2008-0172762A1, US2011-0055968A1 and US2012-0117690A1; SEQ ID NO:2 and its derivatives as described in US7510878(B2), SEQ ID NO:2 and its derivatives as described in U.S. Patent No. 7812129(B1); and other lepidopteran inhibitory proteins known to those of ordinary skill in the art. Such additional polypeptides for controlling coleopteran pests are optionally selected from the group consisting of insect inhibitory proteins such as, but not limited to, Cry3Bb (U.S. Patent No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, Axmi184, Axmi205, AxmiR1, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10, eHIPs (U.S. Patent Application Publication No. 2010 / 0017914) and other coleopteran inhibitory proteins known to those of ordinary skill in the art. .,

[0191] In other embodiments, such compositions / formulations may further comprise at least one additional polypeptide that exhibits insect inhibitory activity against insects not inhibited by the additional insect inhibitory proteins of the invention to broaden the resulting insect inhibitory spectrum. For example, for controlling hemipteran pests, combinations of the insect inhibitory proteins of the invention can be used with hemipteran-active proteins such as TIC1415 (U.S. Patent Application Publication No. 2013 / 0097735), TIC807 (U.S. Patent No. 8609936), TIC834 (U.S. Patent Application Publication No. 2013 / 0269060) and other hemipteran-active proteins known to those of ordinary skill in the art. Additional polypeptides for controlling coleopteran, lepidopteran and hemipteran insect pests can be found on the Bacillus thuringiensis toxin nomenclature website maintained by Neil Crickmore (at btnomenclature.info on the World Wide Web).

[0192] In the art, the possibility of insects developing resistance to certain insecticides has been documented. One insect resistance management strategy involves using transgenic crops that express two different insect inhibitors that act through different modes of action. Thus, any insect that is resistant to either insect inhibitor can be controlled by the other insect inhibitor. Another insect resistance management strategy uses the use of plants that are not protected against the targeted coleopteran or lepidopteran pest species to provide a refuge for the unprotected plants. A specific example is described in U.S. Patent No. 6,551,962, which is incorporated herein by reference in its entirety.

[0193] Other embodiments, such as topically applied pesticidal chemicals, spray formulations, drip formulations, or wipe formulations to be used in combination with the proteins for seed treatment that are designed to control pests that are also controlled by the proteins disclosed herein, can be applied directly to the soil (soil drench), to growing plants expressing the proteins disclosed herein, or formulated to be applied to seeds containing one or more transgenes encoding one or more of the disclosed proteins. Such formulations for seed treatment can be applied with various adhesives and tackifiers. Such formulations can contain insecticides that are synergistic with the disclosed proteins in terms of mode of action, such that the formulated insecticides act through different modes of action to control the same or similar pests that can be controlled by the disclosed proteins, or such insecticides act to control pests within a broader host range or plant pest species that are not effectively controlled by the TIC3668 type protein toxin class.

[0194] The above-mentioned compositions / formulations can also comprise agriculturally acceptable carriers, such compositions / formulations as baits, powders, dusts, pellets, granules, sprays, emulsions, colloidal suspensions, aqueous solutions, Bacillus spore / crystal formulations, seed treatments, recombinant plant cells / plant tissues / seeds / plants transformed to express one or more proteins, or bacteria transformed to express one or more proteins. Depending on the inherent insect inhibitory or pesticidal inhibitory level of the recombinant polypeptide and the level of the formulation to be applied to the plant or used in dietary assays, the compositions / formulations can include various amounts of the recombinant polypeptide by weight, for example, from 0.0001% to 0.001% to 0.01% to 1% to 99% of the recombinant polypeptide by weight.

[0195] Examples

[0196] In view of the foregoing, those skilled in the art will appreciate that changes can be made in the specific aspects disclosed without departing from the spirit and scope of the invention and still obtain similar or like results. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting. It is understood that the entire disclosure of each reference cited herein is incorporated within the disclosure of the present application.

[0197] Example 1

[0198] Discovery of the TIC3668-related protein toxin class

[0199] Identify and prepare bacterial strains that exhibit unique properties such as inferred toxicity, proteomic diversity, and morphological variation when compared to each other for genomic sequencing using methods well known in the art. The protein TIC3668 (SEQ ID NO:2) exhibiting inhibitory activity against Coleoptera insects in in vitro bioassays was discovered from the Bacillus laterosporus strain EG5552. Other strains were also found to contain proteins similar to TIC3668. The polynucleotide segments encoding these proteins were cloned and inserted into recombinant host strains to test for expression.

[0200] Design thermal amplification primers to amplify full-length copies of the gene from the total genomic DNA of different Bacillus laterosporus bacterial strains including EG5552. Separate thermal amplification products (amplicons) were generated from each strain, and the presence of open reading frames that could encode TIC3668-related proteins in these products was analyzed. It was determined that each amplicon had a single open reading frame that contained a translation start codon, followed by an in-frame continuous open reading frame that terminated with an in-frame translation stop codon. The deduced amino acid sequences obtained from each of these additional different bacterial strains are shown as SEQ ID NO:2 (TIC3668), SEQ ID NO:4 (TIC3669), SEQ ID NO:6 (TIC3670), SEQ ID NO:8 (TIC4076), SEQ ID NO:10 (TIC4078), SEQ ID NO:14 (TIC4346), SEQ ID NO:16 (TIC4826), SEQ ID NO:18 (TIC4861), SEQ ID NO:20 (TIC4862), SEQ ID NO:22 (TIC4863), respectively. These amplicons were cloned downstream of a sporulation-specific expression promoter in a recombinant Bacillus thuringiensis (Bt) plasmid expression vector and transformed into crystal-free Bt host cells. The amplicons were also cloned into an Escherichia coli expression system. The resulting recombinant strains were observed to express the recombinant proteins.

[0201] Example 2

[0202] Coleoptera activity of the TIC3668-related protein toxin class

[0203] This example demonstrates the inhibitory activity exhibited by TIC3668-like proteins against Coleoptera.

[0204] Protein preparations of the full-length proteins of TIC3668, TIC3669, TIC3670, TIC4260, TIC4076, and TIC2462 produced by recombinant bacteria as described in Example 1 were submitted for insect diet-overlay bioassays against the Colorado potato beetle (Leptinotarsa decemlineata, CPB) and against at least one corn rootworm species. Known members of the corn rootworm species are Diabrotica virgifera virgifera (western corn rootworm, WCR), Diabrotica barberi (northern corn rootworm, NCR), Diabrotica mexicana (Mexican corn rootworm, MCR), Diabrotica balteata (Brazilian corn rootworm (BZR)), Diabrotica undecimpunctata howardi (southern corn rootworm, SCR), and the Diabrotica brasiliensis complex (BCR) consisting of Diabrotica viridula and Diabrotica speciosa.

[0205] As shown in Table 6, the results showed that TIC3668, TIC3669, TIC3670, TIC4260, and TIC4076 exhibited lethality against corn rootworms. TIC2462 (SEQ ID NO: 62 encoding SEQ ID NO: 63), a protein closely related to the AXMI-209 protein (>99% identical at the amino acid level and differing by only two amino acids compared to TIC2462), did not exhibit lethality against corn rootworms, thus distinguishing the TIC3668-like protein toxin class from the activity of proteins similar to AXMI-209. Surprisingly, no lethality of any of the tested proteins against the Colorado potato beetle, a species commonly tested as an indicator of coleopteran activity in bioassays, was observed.

[0206] Table 6. Lethality of exemplary proteins observed against coleopteran insect pests.

[0207] Toxin Corn Rootworm CPB TIC2462 - - TIC3668, TIC3669, TIC3670 + - TIC4260, TIC4076 + - TIC4078 NT - TIC4346 + + TIC4826, TIC4861, TIC4862, TIC4863 NT NT

[0208] + = Lethality observed

[0209] - = No lethality observed

[0210] NT = Not tested

[0211] Example 3

[0212] Mature form of the TIC3668 protein toxin

[0213] This example illustrates the presence of a membrane transport peptide at the amino terminus of the native protein within the TIC3668 protein toxin class and the discovery of the active mature toxin protein of the TIC3668 protein toxin class.

[0214] Bioinformatics analysis of the amino acid sequence translation product obtained from the TIC3668 coding sequence (SEQ ID NO:1) using the SignalP program (Petersen et al. (2011), Nature Methods, 8:785-786) predicted the presence of a membrane transport segment corresponding to the first 23 amino acids at the N-terminus.

[0215] Experiments were designed to confirm the presence of a membrane transport segment within each member of the TIC3668-like protein toxin class. TIC3668 was cloned into a Bt host cell behind a non-sporulation specific Bt promoter. The insecticidal activity of the resulting culture supernatant was tested. Proteins corresponding to the three forms of TIC3668 were recovered from the supernatant in a mixture. Subsequently, mass spectrometry analysis and N-terminal sequence analysis were used to determine that the different fragments of these less than full-length TIC3668 proteins contained amino acids 16, 19, or 24 as shown in SEQ ID NO:2 at their respective amino termini. Only small amounts of these three truncated forms of TIC3668 were detected in the culture medium. The most abundant form of the protein detected was observed to have serine at position 24 as shown in SEQ ID NO:2 at its amino terminus. When tested in an artificial diet bioassay, the protein concentrated and purified from the culture supernatant exhibited biological activity against WCR.

[0216] Generate different expression constructs to identify the minimal peptide segments of each TIC3668-type protein that exhibit insecticidal activity. Introduce these constructs into a crystal-free Bacillus thuringiensis strain or an Escherichia coli strain. Design a construct for expressing the full-length TIC3668 protein from amino acids 1 to 317 as shown in SEQ ID NO:2 in a crystal-free Bt strain. Design constructs for expressing the full-length TIC3668 protein and various shorter variant forms of the TIC3668 protein in an Escherichia coli expression system with a carboxyl-terminal HIS tag sequence (HHHHAHHH). The constructs designed for expression in Escherichia coli consist of: (1) a construct designed to express the full-length TIC3668 protein from amino acid positions 1 to 317 as shown in SEQ ID NO:2; (2) a construct designed to express a variant TIC3668 protein having amino acids 16 to 317 as shown in SEQ ID NO:2; (3) a construct designed to express a variant TIC3668 protein from amino acids 24 to 317 as shown in SEQ ID NO:2; (4) a construct designed to express a variant TIC3668 protein from amino acids 26 to amino acid 317 as shown in SEQ ID NO:2; (5) a construct designed to express a variant TIC3668 protein from amino acids 28 to 317 as shown in SEQ ID NO:2. Additionally, obtain a TIC3668 protein with an N-terminal 10-his tag and a TVMV (tobacco vein mottling virus) protease site (MHHHHHHHHHHGTETVRFQ) from an Escherichia coli expression system used to produce the TIC3668 protein starting at residue no. 24 as shown in SEQ ID NO:2.

[0217] Proteins were obtained from the supernatants of the Bt expression systems and the proteins were subjected to mass spectrometry analysis and N-terminal sequence analysis. The Bt expression systems produced the predicted TIC3668 mature toxin from amino acids 24 to 317 as shown in SEQ ID NO:2. No proteins were observed in the E. coli supernatants. Proteins were obtained from the respective E. coli expression constructs by osmotic shock to release the proteins from the periplasm. It was confirmed that the proteins produced by constructs designed to contain amino acids 16 or 24 at the amino terminus of less than full-length proteins contained these amino acids at their respective amino termini. The proteins produced by constructs designed to express full-length TIC3668 produced mature-length proteins with serine at position 24 as shown in SEQ ID NO:2 at the amino terminus. The proteins produced by constructs designed to contain amino acid 26 or amino acid 28 as shown in SEQ ID NO:2 as the N-terminal amino acid each surprisingly contained only amino acid 28 as the N-terminal amino acid, indicating that processing to maintain amino acid number 24 as shown in SEQ ID NO:2 at the N-terminus may be important for toxin stability.

[0218] Protein samples obtained from the analysis of these expression systems were submitted for testing against western corn rootworm larvae in an insect diet-overlay bioassay as described in Example 2. It was determined that certain N-terminal truncated forms from this study exhibited reduced bioactivity. Specifically, it was observed that when the N-terminal amino acid was 26 or 28 as shown in SEQ ID NO:2, the insecticidal activity was significantly reduced. It can be extrapolated that the N-terminally truncated other members of the TIC3668 protein family shorter than the mature protein (starting at amino acid residue no. 24 for TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826 and TIC4863; starting at amino acid 13 for TIC4861; and starting at amino acid 22 for TIC4862) are the shortest forms of the TIC3668-type proteins tested that exhibit insecticidal activity against WCR. All variants of TIC3668 that are of equal length or longer than the mature protein showed high activity against WCR even at relatively low concentrations. The data also demonstrated that the E. coli processing of TIC3668 varied with construct design.

[0219] Example 4

[0220] Genes encoding TIC3668-type proteins for expression in plants were synthesized

[0221] Design the nucleotide sequences encoding the full-length and mature forms of the TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, and TIC4863 proteins. Synthesize the nucleotide sequences encoding TIC3668, TIC3669, and TIC3670 according to the method generally described in U.S. Patent 5,500,365, avoiding certain harmful problem sequences such as plant polyadenylation sequences rich in ATTTA and A / T, while retaining the amino acid sequences of the native Brevibacillus laterosporus proteins. These nucleotide sequences are provided herein as SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:51 (for the full-length sequences) and SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, and SEQ ID NO:49 (for the mature sequences).

[0222] Example 5

[0223] Expression cassette for expressing a TIC3668-type protein in plants

[0224] Design a variety of plant expression cassettes having sequences as shown in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. Such expression cassettes are used for transient expression in plant protoplasts or transformation of plant cells. Design typical expression cassettes with respect to the ultimate placement of the protein within the cell. Design a set of expression cassettes such that the protein is translated in a manner having a native N-terminal segment. Design another set of expression cassettes such that a protein without an N-terminal segment (i.e., a mature length protein) is expressed. Design another set of expression cassettes to have in-frame expression and a transit peptide operably linked to the mature length toxin protein to allow targeting of cell organelles such as chloroplasts or plastids. All expression cassettes are designed to be initiated at the 5'-end with a promoter, which may consist of multiple contiguous promoter elements, enhancer elements, or other expression elements known to those of ordinary skill in the art to enhance transgene expression. The promoter sequence is typically followed in succession by one or more leader sequences at the 3'-end of the promoter. An intron sequence is provided at the 3'-end of the leader sequence to improve transgene expression. The coding sequence of the toxin or transit peptide and the coding sequence of the toxin are located at the 3'-end of the promoter, leader sequence, and intron configuration. A 3'-UTR sequence is provided at the 3'-end of the coding sequence to promote transcription termination and sequences important for polyadenylation of the resulting transcript. All of the above elements are arranged in succession with the sequences (such as restriction endonuclease sites or ligation-independent cloning sites) that are typically additional and provided for construction of the expression cassette.

[0225] Example 6

[0226] A transformation vector containing a protein expression cassette of type TIC3668

[0227] Construct Agrobacterium-mediated transformation vectors to deliver DNA encoding the TIC3668, mTIC3668, TIC3669, mTIC3669, TIC3670, and mTIC3670 proteins into the plant genome. Clone the expression cassettes between Agrobacterium border sequences in a suitable vector such that they will be transferred, along with a selectable marker gene, from an Agrobacterium host containing the construct vector into the genome of a host plant cell. More specifically, clone restriction fragments containing the entire cytoplasmic expression cassettes encoding one of the above-mentioned proteins into an Agrobacterium plant transformation vector. Similarly, clone restriction fragments containing the entire plastid-targeted expression cassettes into an Agrobacterium plant transformation vector. Introduce the vectors containing the TIC3668-type protein expression cassettes (i.e., non-targeted or targeted cassettes) into Agrobacterium by electroporation or by tri-parental mating.

[0228] Clone expression cassettes containing artificial genes encoding TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, and TIC4863 (each with and without a sequence encoding the N-terminal 23 amino acids (amino acids 1 - 23 of SEQ ID NO:2) present in the native Brevibacillus laterosporus open reading frame) between Agrobacterium border sequences in a suitable vector such that they are transferred into the genome of a host cell and test the expression and biological activity of the encoded proteins.

[0229] Example 7

[0230] Coleopteran activity of TIC3668-type proteins in plants

[0231] This example demonstrates the inhibitory activity exhibited by TIC3668-like proteins against Coleoptera such as corn rootworm larvae when expressed in plants and provided as a diet to the corresponding insect pests.

[0232] Generate R0 transgenic maize plants expressing the TIC3668, mTIC3668, TIC3669, mTIC3669, TIC3670, and mTIC3670 proteins using the vectors containing the expression cassettes described in Example 5

[0233] F1 transgenic maize plants were grown from seeds produced by pollinating the ears of non-transformed wild-type commercial germplasm plants with pollen from R0 transformants. After transfer to soil in cage pots, the F1 plants were infested with neonatal western corn rootworm insects and grown for 13 days under controlled conditions. The root damage rating (RDR) was determined using the 0 - 3 rating scale of Oleson et al., where 0 means no damage and 3 means 3 or more nodules were clipped within 1.5 inches of the stem (J.D. Oleson, Y-L. Park, T.M. Nowatzki, J.J. Tollefson, “Node-Injury Scale to Evaluate Root Injury by Corn Rootworms”, Journal of Economic Entomology, 98(1):1 - 8, 2005). Insect lethality was evaluated by counting the number of remaining third-instar larvae at the end of the growth period.

[0234] In a first set of experiments, plants expressing full-length TIC3668, TIC3669, and TIC3670 proteins were tested against WCR. Compared to the negative control with an average root damage rating (RDR) value between 2 and 2.5, some events showed a statistically significant reduction in nodule injury, but no commercially significant activity was observed for the full-length proteins.

[0235] In a second set of experiments, mature proteins mTIC3668 (SEQ ID NO:23), mTIC3669 (SEQ ID NO:24), and mTIC3670 (SEQ ID NO:25) with or without a chloroplast targeting peptide were expressed in maize plants and tested against WCR. Significant WCR lethality was observed for each mature protein. For each plant expressing mTIC3668, mTIC3669, and mTIC3670 in the presence and absence of the additional targeting sequence, a statistically significant reduction in nodule injury was shown compared to the negative control. Figure 2 The average RDR values for several events depicting mTIC3668 and mTIC3669 proteins are shown, and Figure 3 the average RDR values for several events depicting mTIC3670 are shown, in the case where the protein was expressed in F1 maize plants whether or not it was targeted to the chloroplast. Figure 2 and 3 “TS” in the event name indicates the presence of a targeting sequence. For many of these events expressing the mature proteins mTIC3668, mTIC3669, and mTIC3670, commercially significant activity was observed.

[0236] Surprisingly, removal of the membrane transport domain from the TIC3668-like protein (amino acids 1 - 23 as shown in SEQ ID NO:2) increases efficacy against western corn rootworm when expressed in maize plants. When expressed in plants, the mature length TIC3668-like protein shows a higher level of insecticidal activity against coleopteran pests compared to the full-length protein.

[0237] Example 8

[0238] Insecticidal Activity of TIC3668-Related Proteins Expressed in Maize against Cry3Bb1-Resistant WCR

[0239] This example demonstrates the insecticidal activity exhibited by the TIC3668-like protein against a western corn rootworm (WCR) strain that has developed resistance to the Bt toxin Cry3Bb1. F1 transgenic maize plants expressing mTIC3668, mTIC3669, or mTIC3670 generated using the method described in Example 7 were infested with 2000 eggs of the Hopkinton strain of WCR per plant.

[0240] The Hopkinton strain of western corn rootworm (Diabrotica virgifera virgifera LeConte) is a non-diapausing strain that has developed field-evolved resistance to Cry3Bb1 expressed in maize plants. The strain was derived from adult WCR samples obtained from a field that had been planted with Cry3Bb1 maize for seven consecutive years. The population was backcrossed three times with a non-diapausing WCR strain and selected three times for Cry3Bb1 resistance (Gassmann et al. (2011) PLoS ONE 6(7):e22629; Gassmann et al. (2012) GM Crops Food 3(3):235 - 244). The colony was obtained from the laboratory of Dr. Aaron Gassman at Iowa State University and maintained by the Monsanto Biotech Entomology group in Chesterfield, MO.

[0241] After infestation, the WCR-Hopkinton strain eggs hatched within 48 hours, and the neonates began feeding on the roots. After 24 days, the roots were removed from the soil, and corn root damage was evaluated using a 0 - 3 scale as described in Example 7. As shown in Table 7, plants expressing mTIC3668, mTIC3669, and mTIC3670 were highly effective in protecting corn roots from damage in the presence of WCR-Hopkinton strain neonates, thereby overcoming WCR resistance to the Cry3Bb1 toxin.

[0242] Table 7. Mean RDR of transgenic maize plants infested with Cry3Bb1-resistant WCR

[0243] Toxin N Average RDR (0 - 3) Standard Error mTIC3668 18 0.06 0.004 mTIC3669 15 0.05 1.82e-10 mTIC3670 14 0.05 1.95e-10 Negative Control 6 2.14 0.24

[0244] N: Number of plants evaluated

[0245] Example 9

[0246] Insecticidal activity of the TIC3668-related protein expressed in maize against native WCR infestation at field test sites

[0247] This example demonstrates the effectiveness of transgenic maize plants expressing a TIC3668-like protein in reducing root damage against native WCR infestation in farmland in the Midwestern United States.

[0248] During the period from late April to early May, F1 transgenic maize plants expressing mTIC3668, mTIC3669 or mTIC3670 generated using the method described in Example 7 were planted at 5 sites in the Midwestern United States. The trials at these sites relied on the existing native corn rootworm infestation pressure. Root excavation was completed by the end of July for damage assessment. Rootworm damage was determined according to the nodule damage scale as described in Example 7.

[0249] Results from the root excavation trials indicate that under actual farming conditions in open fields and in the presence of native corn rootworm pressure, plants expressing mTIC3668, mTIC3669 and mTIC3670 are highly effective in protecting corn roots from damage. Table 8 shows the number of plants evaluated (N), mean RDR and standard error of the test plants when combining all sites.

[0250] Table 8. Mean RDR of transgenic maize plants tested in farmland with native WCR infestation

[0251] Toxin N Average RDR (0 - 3) Standard Error mTIC3668 755 0.144 0.009 mTIC3669 1108 0.159 0.008 mTIC3670 1311 0.120 0.007 Negative Control 362 1.426 0.047

[0252] Example 10

[0253] Lepidopteran activity of the TIC3668-related protein toxin class

[0254] This example demonstrates the inhibitory activity exhibited by the TIC3668-like proteins against Lepidoptera. Protein formulations of the full-length TIC3668, TIC3669, TIC3670, TIC4076, and TIC4078 proteins as described in Example 1 were submitted for insect diet-overlay bioassays against black cutworm (BCW, Agrotis ipsilon), western bean cutworm (WBC, Striacosta albicosta), corn earworm (CEW, Helicoverpa zea), European corn borer (ECB, Ostrinia nubilalis), sugarcane borer (SCB, Diatraea saccharalis), southwestern corn borer (SWC, Diatraea grandiosella), cabbage looper (CLW, Trichoplusia ni), soybean looper (SBL, Chrysodeixis includens), and fall armyworm (FAW, Spodoptera frugiperda). Protocols and methods for preparing and conducting inhibitory protein bioassays are known in the art.

[0255] Activity of certain TIC3668-type proteins against certain Lepidopteran insect pests was observed, as shown in Table 9.

[0256] Table 9. Observed growth inhibition of exemplary proteins against Lepidopteran insect pests.

[0257]

[0258] + = Growth inhibition observed

[0259] ++ = Growth inhibition and lethality

[0260] - = Lethality not observed

[0261] NT = Not tested

[0262] Example 11

[0263] Lepidopteran activity of TIC3668-type proteins in plants

[0264] This example demonstrates the inhibitory activity of TIC3668-type proteins against ECB, SWC, BCW, FAW, CEW, and SBL when expressed in plants and provided as diet to the corresponding insect pests.

[0265] Bioassays against Lepidopteran pests were conducted on R0 maize plants expressing TIC3668, TIC3669, and TIC3670 in a similar manner using plant leaf discs as described in U.S. Patent 8,344,207. Based on the percentage of leaf discs eaten by insects, a leaf damage rating (LDR) was assigned a score on a scale of 0 (0% eaten) to 11 (greater than 50% eaten). The rating score step increased incrementally by 5%. R0 plants without insecticidal proteins served as negative controls. Cytoplasmic expression of the full-length TIC3668-type protein reduced feeding damage against CEW, FAW, and SWC relative to the untransformed control. Cytoplasmic expression of the TIC3670 protein reduced feeding damage against SWC relative to the untransformed control.

[0266] Example 12

[0267] Generation of the chimeric protein TIC4260

[0268] This example teaches the generation of novel gene sequences based on family members of TIC3668. Amino acid variations from 5 native TIC3668-type proteins were combined to generate the novel chimeric protein TIC4260 (SEQ ID NO:12), which exhibits different amino acid sequence diversity compared to the naturally occurring proteins. Figure 1 Alignment of 5 native TIC3668-type proteins with TIC4260 is depicted. In this sequence alignment, positions with sequence diversity are highlighted in grey shading. An artificial polynucleotide sequence encoding the TIC4260 protein (SEQ ID NO:11) was constructed. The mature TIC4260 protein (mTIC4260, SEQ ID NO:28) is encoded by the polynucleotide sequence shown in SEQ ID NO:43.

[0269] Similar alignments of other TIC3668-type proteins can be performed to generate novel proteins exhibiting Lepidopteran and / or Coleopteran toxic activity. These novel proteins are expressed, purified, and tested against Lepidopteran and Coleopteran insects in dietary bioassays. Expression cassettes for generating these novel proteins are produced and transformed into plants to express these proteins for controlling Lepidopteran and Coleopteran pests of the plants.

[0270] All compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those skilled in the art that changes, alterations, modifications, and variations can be applied to the compositions, methods, and steps of the methods described herein without departing from the true spirit, scope, and concept of the invention. More specifically, it will be apparent that certain reagents, which are both chemically and physiologically related, can replace the reagents described herein while the same or similar results will 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.

[0271] It should be apparent to those skilled in the art that these different sequences of improvements can be combined to produce variants that are also within the scope of the invention.

[0272] All publications and patent documents cited in this specification are hereby incorporated by reference herein to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant polynucleotide molecule encoding a polypeptide, said polypeptide consisting of the amino acid sequence of SEQ ID NO:

24.

2. The recombinant polynucleotide molecule according to claim 1, which consists of the nucleotide sequence of SEQ ID NO:

35.

3. The recombinant polynucleotide molecule according to claim 1, wherein the nucleotide sequence encoding said polypeptide is operably linked to a heterologous promoter.

4. A recombinant polypeptide encoded by the recombinant polynucleotide molecule according to claim 1.

5. The recombinant polypeptide according to claim 4, wherein said recombinant polypeptide exhibits inhibitory activity against insect species selected from the group consisting of European corn borer, Southwestern corn borer, and Western corn rootworm.

6. The recombinant polypeptide according to claim 4, wherein said recombinant polypeptide exhibits inhibitory activity against Western corn rootworm.

7. The recombinant polypeptide according to claim 4, wherein said recombinant polypeptide exhibits inhibitory activity against European corn borer and / or Southwestern corn borer.

8. A bacterial host cell comprising the recombinant polynucleotide molecule according to claim 1.

9. A composition comprising the recombinant polynucleotide molecule according to claim 1.

10. The composition according to claim 9, which further comprises a nucleotide sequence encoding at least one other insecticide different from said insect inhibitory polypeptide.

11. The composition according to claim 10, wherein said at least one other insecticide is selected from the group consisting of insect inhibitory proteins, insect inhibitory dsRNA molecules, and accessory proteins.

12. The composition according to claim 11, wherein said at least one other insecticide exhibits activity against one or more pest species of Lepidoptera, Coleoptera, or Hemiptera.

13. The composition according to claim 12, wherein said at least one other insecticide is selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry3A, 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, VIP3A, and VIP3B proteins.

14. A composition comprising an insect-inhibiting effective amount of the recombinant polypeptide according to claim 4.

15. A method for controlling pests, the method comprising contacting the pests with an insect-inhibiting amount of the recombinant polypeptide as claimed in claim 4, wherein the pests are selected from the group consisting of European corn borer, Southwestern corn borer, and Western corn rootworm.

16. A corn plant product comprising a detectable amount of the recombinant polynucleotide molecule as claimed in claim 1 or an insect-inhibiting recombinant polypeptide encoded by the recombinant polynucleotide molecule, wherein the corn plant product is starch.

17. A corn plant product comprising a detectable amount of the recombinant polynucleotide molecule as claimed in claim 1 or an insect-inhibiting recombinant polypeptide encoded by the recombinant polynucleotide molecule, wherein the corn plant product is flour.

18. A method for producing corn seeds comprising the recombinant polynucleotide molecule as claimed in claim 1, the method comprising: (a) planting at least one corn seed comprising the recombinant polynucleotide molecule; (b) growing a corn plant from the at least one corn seed; and (c) harvesting corn seeds from the corn plant, wherein the harvested corn seeds comprise the recombinant polynucleotide molecule.

19. A recombinant vector comprising the recombinant polynucleotide molecule as claimed in claim 3.

20. The recombinant vector as claimed in claim 19, wherein the vector is selected from the group consisting of plasmids, bacmids, phagemids, and cosmids.

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