Insect inhibitory proteins

Abstract

Pesticidal proteins exhibiting toxic activity against Lepidopteran, Coleopteran, and Hemipteran pest species are disclosed, and include, but are not limited to, TIC3410, TIC4123, and TIC4124 and related pesticidal proteins. DNA constructs are provided which encode the disclosed pesticidal proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran, Coleopteran, and Hemipteran infestation are provided which contain recombinant nucleic acid sequences encoding the pesticidal proteins of the present invention, and vectors are described which contain at least a coding sequence for expression and the delivery of the encoded toxin proteins. Methods for detecting the presence of the recombinant nucleic acid sequences or the proteins of the present invention in a biological sample, and methods of controlling Lepidopteran, Coleopteran, and Hemipteran species pests using TIC3410, TIC4123, and TIC4124 and related pesticidal proteins are also provided.

Description

[0001] TITLE OF THE INVENTION

[0002] INSECT INHIBITORY PROTEINS

[0003] REFERENCE TO RELATED APPLICATION

[0004]

[0001] This application claims the benefit of United States provisional application No. 63 / 618,849, filed January 8, 2024, which is herein incorporated by reference in its entirety.

[0005] INCORPORATION OF SEQUENCE LISTING

[0006]

[0002] The file named “MONS593WO_ST26.xml” containing a computer-readable form of the Sequence Listing was created on December 23, 2024. This file is 17.4 KB (measured in MS- Windows®), filed contemporaneously by electronic submission (using the United States Patent Office Patent Center), and incorporated by reference in its entirety.

[0007] FIELD

[0008]

[0003] The invention generally relates to the field of insect inhibitory proteins. A novel class of toxin proteins are disclosed exhibiting insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly Lepidopteran, Coleopteran, and Hemipteran species of insect pests. Plants, plant parts, seed, cells including plant and microbial cells, and vectors containing a recombinant polynucleotide construct encoding one or more of the disclosed toxin proteins are provided.

[0009] BACKGROUND

[0010]

[0004] Improving crop yield from agriculturally significant plants including, among others, corn, soybean, sugarcane, rice, wheat, cotton, vegetables, pearl millets, pigeon pea, cassava, cowpea, peanut, potato, barley, oat, fruit trees, and the like has become increasingly important. In addition to the growing need for agricultural products to feed, clothe and provide energy for a growing human population, climate-related effects and pressure from the growing population to use land for other non-agricultural practices arc predicted to reduce the amount of arable land available for farming. These factors have led to grim forecasts of food security, particularly in the absence of major improvements in plant biotechnology and agronomic practices. In light of these factors, environmentally sustainable improvements in technology, agricultural techniques, and pest management are vital tools to expand crop production on the increasingly limited amount of arable land available for farming.

[0011]

[0005] Insects, particularly insects within the order Lepidoptera, Coleoptera, and Hemiptera, are a major cause of damage to field crops, thereby decreasing crop yields in infested areas. Lepidopteran pest species which negatively impact agriculture include, but are not limited to, Black armyworm (Spodoptera cosmioides), Black cutworm (Agrotis ipsilon). Corn earworm (Helicoverpa zea), Cotton leaf worm (Alabama argillacea). Diamondback moth (Plutella xylostella). European com borer (Ostrinia nubilalls). Fall armyworm (Spodoptera frugiperda), CrylFal resistant Fall army worm (Spodoptera frugiperdd), Old World bollworm (Helicoverpa armigera), Southern armyworm (Spodoptera eridania). Soybean looper (Chrysodeixis incliidens). Spotted bollworm (Earias villella), Southwestern com borer (Diatraea grandioselki), Sunflower looper (Rachiplusia nu), Tobacco budworm (Heliothis virescens). Tobacco cutworm (Spodoptera litura. also known as cluster caterpillar), Western bean cutworm (Striacosta albicosta), and Velvet bean caterpillar (Anticarsia gemmat alls). Coleopteran insect pests which negatively impact agriculture include, but are not limited to Western Com Rootworm (Diabrotica virgiferd), Northern Com Rootworm (Diabrotica barberi). Mexican Corn Rootworm (Diabrotica virgifera zeae). Brazilian Corn Rootworm (Diabrotica ball eala), Southern Corn Rootworm (Diabrotica undecimpunctata howardii), Colorado potato beetle (Leptinotarsa decemlineata, CPB), a Brazilian Corn Rootworm complex (consisting of Diabrotica viridula and Diabrotica peciosa). Crucifer Flea Beetle (Phyllotreta cruciferae), Striped Flea Beetle (Phyllotreta striolatd), and Western Black Flea Beetle (Phyllotreta pusilia). Hemipteran pest species which negatively impact agriculture include, but are not limited to, Southern Green Stink Bug (Nezara viridula , Neotropical Brown Stink Bug (Euschistus heros), Brown Marmorated Stink Bug (Halyomorpha halys), Red- Shouldered Stink Bug (Thyanta accerra), Green Belly Stink Bug (Dichelops melacanthus), Western tarnished plant bug (Eygus he s perns), and Tarnished plant bug (Lygus lineolaris).

[0012]

[0006] Historically, the application of synthetic chemical insecticides was relied upon as the pest control agent in agriculture. Such chemicals often do not target specific insects, may be indiscriminate in effect, and can sometimes persist in the environment and accumulate to unreasonably high levels . Concerns for the environment and human health, in addition to emerging resistance issues, and potentially indiscriminate effect on non-target insects and other organisms, have stimulated research and development of biological pesticides that arc specifically targeted to control the insect pests that cause crop loss, which has led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria, as well as transgenic plants expressing insecticidal toxin proteins.

[0013]

[0007] The global use of transgenic insect-protected crops and the limited number of insecticidal toxin proteins used in these crops has created a selection pressure for existing insect alleles that impart resistance to the currently utilized insecticidal proteins. The development of resistance in target pests to insecticidal toxin proteins creates a continuing need for discovery and development of new forms of insecticidal toxin proteins that are useful for managing the increase in insect resistance to transgenic crops expressing insecticidal toxin proteins. New protein toxins with improved efficacy and which exhibit control a broader or different spectrum of susceptible insect pest species will reduce the number of surviving insects which can develop resistance alleles. In addition, the use in one plant of two or more transgenic insecticidal toxin proteins that are toxic to the same insect pest but have different modes of action, or alternatively two or more different modes of toxic action (for example, a transgene encoding a dsRNA targeting an essential gene for suppression coupled with a transgene that encodes a peptide or protein toxin, both toxic to the same insect species), reduces the probability of resistance in any single target insect species. Further, use of self-limiting technologies, such as those provided by Oxitec Ltd, when used together with the proteins of the present invention, should improve durability of the traits imparted to transgenic crops expressing insecticidal proteins (see, e.g., Zhou et al., Combining the high- dose / refuge strategy and elf- limiting transgenic insects in resistance management — a test in experimental mesocosms, Evol Appl 1 l(5):727-738 (2018); and Alphey et al., Combining pest control and resistance management: synergy of engineered insects with Bt crops, Journal of Economic Entomology, 102: 717-732 (2018)).

[0014]

[0008] Thus, there is a need in the art for new toxin proteins that are efficacious against target pests and can provide effective control of those target pests in an agricultural field, and that are capable of being expressed in plants without causing undesirable agronomic issues and provide an alternative mode of action compared to current toxins that are used commercially in plants. SUMMARY

[0015]

[0009] Disclosed herein are pesticidal proteins, TIC3410, TIC4123, and TIC4124, which are each shown to exhibit inhibitory activity against one or more Lepidopteran, Coleopteran, and Hemipteran pests of crop plants. Each of the TIC3410, TIC4123, and TIC4124 proteins, or variants or fragments thereof, can be used alone or in combination with other insecticidal protein(s) and / or toxic agent(s) in formulations and / or in planta, thus providing alternatives to insecticidal proteins and insecticide chemistries currently in use in agricultural systems. In some embodiments, each of the TIC3410, TIC4123, and TIC4124 proteins, or a pesticidal protein from the TIC3410, TIC4123, or TIC4124 toxin protein class, or a fragment thereof, can be used in combination with one or both of the other two pesticidal proteins disclosed herein or one or both of the other two toxin protein classes, or a fragment thereof, in formulations or in planta, which may include a combination of, for example, TIC3410 and TIC4123; TIC3410 and TIC4124; TIC3410 and TIC4124; TIC4123 and TIC4124; or TIC3410, TIC4123, and TIC4124, or a pesticidal protein from the TIC3410, TIC4123, and / or TIC4124 toxin protein class, or a fragment of any of the foregoing, respectively. Pesticidal proteins are provided herein may comprise the amino acid sequence of SEQ ID NO: 3, 6, or 9, or an amino acid sequence having at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% amino acid sequence identity to SEQ ID NO: 9. Further provided are compositions comprising a pesticidally effective amount of the pesticidal protein as provided herein.

[0016]

[0010] In one embodiment, disclosed in this application is a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment or sequence encoding a pesticidal protein or pesticidal fragment thereof, wherein the pesticidal protein comprises the amino acid sequence of SEQ ID NOs: 3, 6, or 9; or the pesticidal protein comprises an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9; or the polynucleotide segment or sequence hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NOs: 1, 2, 4, 5, 7, or 8. The recombinant nucleic acid molecule can comprise a sequence that functions to express the pesticidal protein in a plant, and which when expressed in a plant cell produces a pesticidally effective amount of the pesticidal protein or a pesticidal fragment thereof.

[0017] [Oil] In another embodiment of this application the recombinant nucleic acid molecule is present within a bacterial or plant host cell. Contemplated bacterial host cells include at least the genus of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia. In certain embodiments, the Bacillus species is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus is a Brevibacillus laterosporus, or the Escherichia is a Escherichia coli. Contemplated plant host cells include a dicotyledonous plant cell and a monocotyledonous plant cell. Contemplated plant cells further include an alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g. canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, cowpea, clover, cotton (Gossypium sp.), a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell.

[0018]

[0012] In another embodiment, the pesticidal protein exhibits activity against Lepidopteran insects, such as Black cutworm (Agrotis ipsilon), Com earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis). Fall armyworm (Spodopterafrugiperdd), Soybean looper (Chrysodeixis includens), Southwestern com borer (Diatraea grandioselki), and Tobacco budworm (Heliothis virescens). In another embodiment, the pesticidal protein exhibits activity against Coleopteran insects, such as Western Corn Rootworm (Diabrotica virgi / era) and Colorado potato beetle (Leptinotarsa decemlineaia). In yet another embodiment, the pesticidal protein exhibits activity against the Hemipteran insects, such as Whitefly (Bemisia tabaci). Western tarnished plant bug (Lygus hesperus), Tarnished plant bug (Lygus lineolaris), Neotropical Brown Stink Bug (Euschistus heros). and Southern Green Stink Bug (Nezara viridula).

[0019]

[0013] Also contemplated in this application are microorganisms, bacteria and / or plants and plant parts comprising a recombinant nucleic acid molecule encoding the pesticidal protein TIC3410, TIC4123, or TIC4124 or a pesticidal protein from the TIC3410, TIC4123 or TIC4124 toxin protein class, or fragment thereof. The recombinant molecule (e.g., construct) may comprise a heterologous promoter for expression in bacterial or plant cells of the operably linked polynucleotide segment or sequence encoding the pesticidal protein. Both dicotyledonous plants and monocotyledonous plants arc contemplated. In another embodiment, the plant is further selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, cowpea, clover, cotton (i.e., Gossypium sp.), a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn (i.e., maize) such as sweet corn or field com, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. The plant parts may for instance include, without limitation, leaves, tubers, roots, stems, seeds, embryos, flowers, inflorescences, bolls, pollen, fruit, animal feed, and biomass. Processed plant parts, for instance wood, or oil, non-viable ground seeds or fractionated seeds, flour, or starch produced from the plant leaves, flowers, roots, seeds or tubers containing the nucleic acids encoding the proteins of the present invention, and / or containing pesticidally effective amounts of the encoded toxin proteins, are also contemplated. Microorganisms are provided comprising a recombinant nucleic acid molecule and / or a pesticidal protein as provided herein. The microorganism may be a bacterial cell, a fungal cell or a plant cell.

[0020]

[0014] In certain embodiments, seeds comprising the recombinant nucleic acid molecules encoding pesticidally active TIC3410, TIC4123, and / or TIC4124 toxin protein(s) or pesticidal protein(s) from the TIC3410, TIC4123 and / or TIC4124 toxin protein class(es), or fragment(s) thereof, and / or pesticidally effective amounts of the TIC3410, TIC4123, or TIC4124 toxin protein(s) or pesticidal protein(s) from the TIC3410, TIC4123 and / or TIC4124 toxin protein class(es), or fragment(s) thereof, are disclosed.

[0021]

[0015] In still another embodiment, an insect inhibitory composition comprising a recombinant nucleic acid molecule(s) disclosed in this application are contemplated. The insect inhibitory composition can further comprise a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. In certain embodiments, the at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, and an ancillary protein. It is also contemplated that the at least one other pesticidal agent in the insect inhibitory composition exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, and / or Hemiptera. The at least one other pesticidal agent in the insect inhibitory composition may, in some embodiments, be selected from the group consisting of a CrylA, Cryl Ab, Cryl Ac, CrylA.105, Cryl Ae, CrylB, Cry1C, CrylC variants, CrylD, CrylE, CrylF, CrylA / F chimeras, CrylG, CrylH, Cryll, CrylJ, CrylK, Cry IL, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry34, Cry35, Cry43A, Cry43B, Cry51Aal, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC86O, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab,AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXML100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI- 204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and valiants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG- 10, DIG-657, DIG-11 protein, IPD102Aa and homologs thereof, IPDl lOAa and homologs thereof, TIC868, CrylDal_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757. TIC7641, TIC5290, TIC3668, TIC3669, TIC3670, TIC2199, TIC4064, TIC4029, TIC13085, TIC13087, IPD072Aa, IPD079Ea, and IPD103 and homologs thereof, PIP-50 and PIP-65 and homologs thereof, PIP-83 and homologs thereof, and Cry IB.34; and dsRNA mediated gene suppression embodiments including those targeting for suppression Diabrotica species genes Dv snf7 and Dv ssj l.

[0022]

[0016] Commodity products comprising a detectable amount of a recombinant nucleic acid molecule(s) and / or toxin protein(s) disclosed in this application are also contemplated. Such commodity products include commodity corn bagged by a grain handler, corn flakes, corn cakes, com flour, com meal, corn syrup, corn oil, corn silage, corn starch, corn cereal, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity products including, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such commodity products containing a detectable amount of such polynucleotide(s) and / or polypeptide(s) of this application, whole or processed cotton seed, cotton oil, lint, seeds and plant parts processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean Hour, soybean flakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts, cowpea seed, cowpea oil, cowpea protein, cowpea meal, cowpea flour, animal feed comprising cowpea, cowpea forage, cassava flour, cassava chips, cassava starch, and Garri.

[0023]

[0017] Also contemplated in this application is a method of producing seed comprising recombinant nucleic acid molecules encoding pesticidally active TIC3410, TIC4123, and / or TIC4124 toxin protein(s) or pesticidal protein(s) from the TIC3410, TIC4123 and / or TIC4124 toxin protein class(es), or fragment(s) thereof, and / or pesticidally effective amounts of the (encoded) TIC3410, TIC4123, and TIC4124 toxin protein(s) or pesticidal protein(s) from the TIC3410, TIC4123 and / or TIC4124 toxin protein class(es), or fragment(s) thereof. The method comprises planting at least one seed comprising a recombinant nucleic acid molecule(s) disclosed in this application; growing a plant from the seed; and harvesting seed from the plant, wherein the harvested seed comprises the referenced recombinant nucleic acid molecule(s) and / or a pesticidally effective amount of the encoded TIC3410, TIC4123, and / or TIC4124 toxin protein(s) or pesticidal protein(s) from the TIC3410, TIC4123 and / or TIC4124 toxin protein class(es), or fragment(s) thereof.

[0024]

[0018] In another illustrative embodiment, a plant resistant to Lepidopteran, Coleopteran, or Hemipteran insect infestation, is provided wherein the cells of said plant comprise the recombinant nucleic acid molecules disclosed herein.

[0025]

[0019] Also disclosed in this application are methods for controlling a Lepidopteran, Coleopteran, or Hemipteran species pest and controlling a Lepidopteran, Coleopteran, or Hemipteran species pest infestation of a plant, particularly a crop plant. The method comprises, in one embodiment, first contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NOs: 3, 6, or 9 or a variant or fragment thereof; or contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or about 100% amino acid sequence identity to SEQ ID NO: 9.

[0026]

[0020] Further provided herein is a method of detecting the presence of a recombinant nucleic acid molecule encoding a pesticidal protein from the TIC3410, TIC4123, or TIC4124 toxin protein class wherein the method comprises contacting a sample of nucleic acids with a nucleic acid probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant comprising a polynucleotide segment or sequence encoding a pesticidal protein or fragment thereof provided herein, and does not hybridize under such hybridization conditions with genomic DNA from an otherwise isogenic plant that does not comprise the segment or sequence, wherein the probe is homologous or complementary to SEQ ID NOs: 3, 6, or 9, or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or about 100% amino acid sequence identity to SEQ ID NO: 9; subjecting the sample and probe to stringent hybridization conditions; and detecting hybridization of the probe with DNA of the sample. In some embodiments, a step of detecting the presence of a member of the TIC3410, TIC4123, or TIC4124 toxin protein class may comprise an ELISA or a western blot.

[0027]

[0021] Also provided herein are methods of detecting the presence of the pesticidal protein or fragment thereof from the TIC3410, TIC4123, or TIC4124 toxin protein class wherein the method comprises contacting a sample with a TIC3410, TIC4123, or TIC4124 toxin protein class immunoreactive antibody or recombinant protein designed for detecting the TIC3410, TIC4123, or TIC4124 protein, and detecting the binding of the antibody to the TIC3410, TIC4123, or TIC4124 toxin protein, thus confirming the presence of the protein in the sample. In some embodiments the step of detecting comprises an ELISA, or a Western blot.

[0028]

[0022] Also contemplated in this application is a method for controlling a Lepidopteran, Coleopteran, or Hemipteran pest species or pest infestation in a field wherein the method comprises growing a crop plant which expresses an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NOs: 3, 6, or 9; or growing a crop plant which expresses an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or about 100% amino acid sequence identity to SEQ ID NO: 9; and releasing into the field with crops containing a gene encoding the toxin protein of the present invention, one or more transgenic Lepidopteran, Coleopteran, or Hemipteran pest species each carrying a self-limiting gene, for the purpose of preventing or delaying the onset of resistance of the one or more Lepidopteran, Coleopteran, or Hemipteran pest species to the toxin protein. In one embodiment, the crop plants can be monocoty ledonous or dicotyledonous. In another embodiment, the monocotyledonous crop plants can be corn, wheat, sorghum, rice, rye, or millet. In yet another embodiment, the dicotyledonous crop plant can be soybean, cotton, alfalfa, cowpea, cassava, or canola. SEQUENCE LISTING

[0029]

[0023] SEQ ID NO: 1 is a native / naturally occurring nucleic acid sequence obtained from Bacillus thurigiensis species EG10168 encoding a TIC3410 pesticidal protein.

[0030]

[0024] SEQ ID NO: 2 is an artificial sequence encoding a TIC3410 pesticidal protein and is designed for expression in a plant cell.

[0031]

[0025] SEQ ID NO: 3 is the amino acid sequence of the pesticidal protein TIC3410 encoded by the sequence set forth in SEQ ID NOs: 1 and 2.

[0032]

[0026] SEQ ID NO: 4 is a native I naturally occurring nucleic acid sequence obtained from Bacillus thurigiensis species CFB213052 encoding a TIC4123 pesticidal protein.

[0033]

[0027] SEQ ID NO: 5 is an artificial sequence encoding a TIC4123 pesticidal protein and is designed for expression in a plant cell.

[0034]

[0028] SEQ ID NO: 6 is the amino acid sequence of the pesticidal protein TIC4123 encoded by the sequence set forth in SEQ ID NOs: 4 and 5.

[0035]

[0029] SEQ ID NO: 7 is a native I naturally occurring nucleic acid sequence obtained from Bacillus thurigiensis species EG5O18 encoding a TIC4124 pesticidal protein.

[0036]

[0030] SEQ ID NO: 8 is an artificial sequence encoding a TIC4124 pesticidal protein and is designed for expression in a plant cell.

[0037]

[0031] SEQ ID NO: 9 is the amino acid sequence of the pesticidal protein TIC4124 encoded by the sequence set forth in SEQ ID NOs: 7 and 8.

[0038] DETAILED DESCRIPTION

[0039]

[0032] The inventors disclose herein novel proteins from different Bacillus thuringiensis strains and related proteins that exhibit insecticidal activity against target Lepidopteran, Coleopteran and Hemipteran species. Pesticidal proteins identified as TIC3410, TIC4123, and TIC4124 are disclosed herein, shown as having amino acid sequences set forth in SEQ ID NOs: 3, 6, and 9, respectively. Use of these proteins, or valiants or fragments thereof, in pesticidally effective amounts can address insect infestation that are problems in the art for agricultural crop plants, particularly against a broad spectrum of Lepidopteran insect pests, and more particularly against Black cutworm (Agrotis ipsilori), Com earworm Helicoverpa zea), European corn borer (Oslrinia nubilalis), Fall armyworm (Spodoptera frugiperda). Soybean looper (Chrysodeixis includens), Southwestern corn borer (Diatraea gmndiosella), Tobacco budworm (Heliolhis virescens); of Colcoptcran insect pests, and more particularly against Western Corn Rootworm (Diabrotica virgiferd), Colorado potato beetle (Leplinolarsa decemlineatd),' and of Hemipteran insect pests, and more particularly against Western tarnished plant bug (Lygus Hesperus), Tarnished plant bug (Lygus lineolaris), Neotropical Brown Stink Bug (Euschistus heros), and Southern Green Stink Bug (N ezara viridula).

[0040]

[0033] Reference in this application to “TIC3410-related toxins”, “TIC3410 protein toxin class”, or “TIC3410 toxin protein class” is intended to refer to any novel pesticidal protein or insect inhibitory protein, that comprises, that consists of, that is substantially homologous to, that is similar to, or that is derived from any pesticidal protein or insect inhibitory protein sequence of TIC3410 (SEQ ID NO: 3), and pesticidal or insect inhibitory fragments or segments thereof that confer insecticidal activity against one or more Lepidopteran, Coleopteran, and / or Hemipteran pests, including any protein exhibiting pesticidal or insect inhibitory activity if alignment of such protein with TIC3410 has a percent sequence identity in a range from about 99.5% to about 100% percent, including any fractional percentage therewithin, such as at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% identical to TIC3410 (SEQ ID NO: 3). “TIC3410-related toxins”, “TIC3410 protein toxin class”, or “TIC3410 toxin protein class” may refer to pesticidal proteins or insect inhibitory proteins belonging to the Cry 46 or Cry46Aa protein class and / or conferring activity against one or more Lepidopteran, Coleopteran, and / or Hemipteran pests, which may have activity against the same or different as the one or more Lepidopteran, Coleopteran, and / or Hemipteran pests as the TIC3410 protein. Reference in this application to “TIC4123-related toxins”, “TIC4123 protein toxin class”, or “TIC4123 toxin protein class” is intended to refer to any novel pesticidal protein or insect inhibitory protein, that comprises, that consists of, that is substantially homologous to, that is similar to, or that is derived from any pesticidal protein or insect inhibitory protein sequence of TIC4123 (SEQ ID NO: 6), and pesticidal or insect inhibitory segments thereof, or combinations thereof, that confer activity against one or more Lepidopteran, Coleopteran, and / or Hemipteran pests, including any protein exhibiting pesticidal or insect inhibitory activity if alignment of such protein with TIC4123 has a percent sequence identity in a range from about 99.5% to about 100% percent, including any fractional percentage therewithin, such as at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% identical to TIC4123 (SEQ ID NO: 6). “TIC4123-related toxins”, “TIC4123 protein toxin class”, or “TIC4123 toxin protein class” may refer to pesticidal proteins or insect inhibitory proteins belonging to the Cry46 or Cry46Ab protein class and / or conferring activity against one or more of the same Lepidopteran, Coleopteran, and / or Hemipteran pests as the TIC4123 protein. Reference in this application to “TIC4124-related toxins”, “TIC4124 protein toxin class”, or “TIC4124 toxin protein class” is intended to refer to any novel pesticidal protein or insect inhibitory protein, that comprises, that consists of, that is substantially homologous to, that is similar to, or that is derived from any pesticidal protein or insect inhibitory protein sequence of TIC4124 (SEQ ID NO: 9), and pesticidal or insect inhibitory fragments or segments thereof that confer activity against one or more Lepidopteran, Coleopteran, and / or Hemipteran pests, including any protein exhibiting pesticidal or insect inhibitory activity if alignment of such protein with TIC4124 (SEQ ID NO: 9) has a percent sequence identity in a range from about 99% to about 100% percent, including any fractional percentage therewithin, such as at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% identical to TIC4124 (SEQ ID NO: 9). “TIC4124- related toxins”, “TIC4124 protein toxin class”, or “TIC4124 toxin protein class” may refer to pesticidal proteins or insect inhibitory proteins belonging to the Cry46 or Cry 46 Ab protein class and / or conferring activity against one or more of the same Lepidopteran, Coleopteran, and / or Hemipteran pests as the TIC4124 protein. The TIC3410, TIC4123, and TIC4124 proteins described herein, and variants and fragments thereof, and intended to be within the scope of the present disclosure include both the plastid-targeted (e.g., fused with a chloroplast targeting peptide (CTP)) and non-plastid targeted forms of the proteins.

[0041]

[0034] The term “variant” as used in this application in reference to a TIC3410, TIC4123, and TIC4124 protein refers to any pesticidal protein or insect inhibitory protein within the TIC3410, TIC4123, and TIC4124 toxin protein class, respectively, but differing in amino acid sequence in comparison to the TIC3410, TIC4123, and TIC4124 protein, respectively. A variant may include conservative amino acid substitutions relative to a reference sequence based on their known chemical properties. The term “segment” or “fragment” as used in this application describes consecutive or contiguous amino acid or nucleic acid sequences that are shorter than the complete amino acid or nucleic acid sequence of a TIC3410, TIC4123, or TIC4124 protein or a variant thereof, as set forth in the sequences provided herein, particularly, for example, the nucleic acid sequences as set forth in SEQ ID NOs: 1, 2, 4, 5, 7, and 8 encoding the TIC3410, TIC4123, and TIC4124 proteins or pesticidal proteins from the TIC3410, TIC4123 and / or TIC4124 toxin protein classes, and the amino acid sequences as set forth in SEQ ID NOs: 3, 6, and 9. A segment or fragment of the TIC3410 or TIC4123 protein exhibiting insect inhibitory activity is also disclosed in this application if alignment of such segment or fragment, with the corresponding segment of the TIC4124 amino acid sequence set forth in SEQ ID NO: 3 or 6, results in amino acid sequence identity percentage in a range from about 99.5% to about 100%, such as about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or 100% between the segment or fragment and the corresponding segment of amino acids within the TIC3410 or TIC4123 protein or SEQ ID NO: 3 or 6, respectively. A segment or fragment of the TIC4124 protein exhibiting insect inhibitory activity is also disclosed in this application if alignment of such segment or fragment, with the corresponding segment of the TIC4124 amino acid sequence set forth in SEQ ID NO: 9, results in amino acid sequence identity percentage in a range from about 99% to about 100%, such as about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or 100% between the segment or fragment and the corresponding segment of amino acids within the TIC4124 protein or SEQ ID NO: 9. A fragment as described herein may comprise at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 700, or at least 800 consecutive or contiguous amino acid residues of the TIC3410, TIC4123, or TIC4124 protein or a variant thereof.

[0042]

[0035] Reference in this application to the terms “pesticidal activity” or “pesticidal” or “insecticidal activity”, “insect inhibitory”, “pesticidally effective” or “insecticidal” are intended to refer to efficacy of a toxic agent, such as a protein toxin, in inhibiting (inhibiting growth, feeding, fecundity, or viability), suppressing (suppressing growth, feeding, fecundity, or viability), controlling (controlling the pest infestation, controlling the pest feeding activities on a particular crop), or killing (causing the morbidity, mortality, or reduced fecundity of) an insect pest. Such a toxic agent or protein toxin may comprise an effective amount of the TIC8643 protein or a variant or fragment thereof. These terms are intended to include the result of providing a pesticidally effective amount of a toxic protein to an insect pest where the exposure of the pest to the toxic protein results in inhibiting, suppressing, controlling, and / or killing the pest. These terms also include repulsion of the pest from the plant, a tissue of the plant, a plant part, seed, plant cells, or from the particular geographic location where the plant may be growing, as a result of providing a pesticidally effective amount of the toxic protein in or on the plant. In general, pesticidal activity, etc., refers to the ability of a toxic protein to be effective in inhibiting the growth, development, viability, feeding behavior, mating behavior, fecundity, or any measurable decrease in the adverse effects imposed upon a plant when caused by an insect feeding. The Lepidopteran, Coleopteran, and / or Hemipteran specific toxic protein can be produced by the plant or can be applied to the plant or to the environment within the location where the plant is located. The terms “bioactivity”, “effective”, “efficacious” or variations thereof are also terms interchangeably utilized in this application to describe the effects (or effective ability) of pesticidal proteins of the present disclosure on target insect pests.

[0043]

[0036] A pesticidally effective amount of a toxic agent, when provided in the diet of a target pest, exhibits pesticidal activity when the toxic agent contacts the pest. A toxic agent can be a pesticidal protein or one or more chemical agents known in the art. Pesticidal or insecticidal chemical agents can be used alone or in combinations with each other. Chemical agents include but are not limited to dsRNA molecules targeting specific genes for suppression in a target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal or insecticidal protein agents include the protein toxins set forth in this application, as well as other proteinaceous toxic agents including those that target Lepidopterans, Coleopterans, and / or Hemipterans, as well as protein toxins that are used to control other plant pests, such as Cry, Vip, and Cyt proteins, Pseudomonas insect toxic proteins, and insect toxin proteins derived from fem species, that are available in the art for use in controlling Coleopteran, Hemipteran and Homopteran species.

[0044]

[0037] It is intended that reference to a pest, particularly a pest of a crop plant, means insect pests of crop plants, particularly those Lepidoptera, Coleoptera, and / or Hemiptera insect pests that are controlled by pesticidal proteins within the TIC3410, TIC4123, and / or TIC4124 protein toxin class(es). However, reference to a pest can also include Homopteran and Thysanopteran insect pests of plants, as well as nematodes and fungi when toxic agents targeting these pests are colocalized or present together with the pesticidal protein within the TIC3410, TIC4123, and / or TIC4124 protein toxin class(es) or a protein that is at least 99 percent, at least 99.1 percent, at least 99.2 percent, at least 99.3 percent, at least 99.4 percent, at least 99.5%, at least 99.6%, or at least 99.7 percent, at least 99.8%, at least 99.9%, or about 100 percent identical to the TIC3410, TIC4123, and / or TIC4124 protein or SEQ ID NO: 3, 6 and / or 9. The phrases “present together” or “co-localized” are intended to include any instance of which a target insect pest has been contacted by a pesticidal protein in the TIC3410, TIC4123, or TIC4124 protein toxin class or fragment, or variant thereof, as well as any other toxic agent also present in a pesticidally effective amount relative to the target insect pest. “Contacted” in reference to a pesticidal protein of a TIC3410, TIC4123, or TIC4124 protein toxin class or a fragment thereof is intended to refer to being present in the diet of the target pest, and the diet is consumed by the target pest. The diet may be a natural diet or a controlled, artificial or experimental diet, such as for a diet assay.

[0045]

[0038] The insects of the order Lepidoptera that are intended to be within the scope of the present invention include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the Family Noctuidae, e.g., Fall army worm (Spodoptera frugiperda), Beet army worm (Spodoptera. exigua). Black armyworm (Spodoptera cosmioides), Southern armyworm (Spodoptera eridania). bertha army worm (Mamestra configurata), black cutworm (Agrotis ipsilon), cabbage looper worm (Trichoplusia ni), Sugarcane borer (Diatraea saccharalis), soybean looper (Pseudoplusia includens). Sunflower looper (Rachiplusia nu), velvetbean caterpillar (Anticarsia gemmatalis), green cloverworm (Hypena scabra), tobacco budworm (Heliot is virescens), granulate cutworm (Agrotis subterranea). armyworm (Pseudaletia unipuncta), Sunflower looper (Rachiplusia nu), South American podworm (Helicoverpa gelotopoeon) western cutworm (Agrotis orlhogonia)'. borers, casebearers, webworms, coneworms, cabbageworms and skeletonizers from the Family Pyralidae, e.g., European corn borer (Ostrinia nubilalis). navel orange worm (Amyelois transitelld), com root webworm (Crambus caliginosellus). sod webworm (Herpetogramma licarsisalis), sunflower moth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpus lignosellus)'. leafrollers, budworms, seed worms, and fruit worms in the Family Tortricidae, e.g., codling moth (Cydia pomonella). grape berry moth (Endopiza viteana), oriental fruit moth (Grapholita mo lest a), sunflower bud moth (Suleima helianthana),' and many other economically important Lepidoptera, e.g., diamondback moth (Plutella xylostella), pink bollworm (Pectinophora gossypiella). and gypsy moth (Lymantria dispar). Other insect pests of order Lepidoptera include, e.g. , cotton leaf worm (Alabama argillacea), fruit tree leaf roller (Archips argyrospila), European leafroller (Archips rosana) and other Archips species, (Chilo suppressalis, Asiatic rice borer, or rice stem borer), rice leaf roller (Cnaphalocrocis medinalis), corn root webworm (Crambus caliginosellus), bluegrass webworm (Crambus teterrellus), southwestern com borer (Diatraea grandiosella), surgarcane borer (Diatraea saccharalis), spiny bollworm (Earias insulana), spotted bollworm (Earias vittella), American bollworm (Elelicoverpa armigera), corn carworm (Elelicoverpa zea, also known as soybean podworm and cotton bollworm), tobacco budworm ( l e Hot his virescens). sod webworm (Herpetogramma licarsisalis). Western bean cutworm (Striacosta albicosta), European grape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistis citrella), large white butterfly (Pieris brassicae), small white butterfly (Pieris rapae, also known as imported cabbageworm), beet armyworm (Spodoptera exigua). tobacco cutworm (Spodoptera litura, also known as cluster caterpillar), and tomato leaf miner (Tuta absoluta).

[0046]

[0039] The insects of the order Coleoptera include, but are not limited to, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeiclae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp, particularly when the pest is Western Com Rootworm (Diabrotica virgifera), Northern Corn Rootworm (Diabrotica barberi), Mexican Corn Rootworm (Diabrotica virgifera zeae). Brazilian Corn Rootworm (Diabrotica balteata). Southern Corn Rootworm (Diabrotica undecimpunctata howardii). Colorado potato beetle (Leptinotarsa decemlineata'), a Brazilian Com Rootworm complex (consisting of Diabrotica viridula and Diabrotica speciosa), Cmcifer Flea Beetle (Phyllotreta cruciferae), Striped Flea Beetle (Phyllotreta striolata), and Western Black Flea Beetle (Phyllotreta pusilia).

[0047]

[0040] The insects of the order Hemiptera include, but are not limited to, Stink Bugs of the family Pentatomidae: Green Stink Bugs from the genus Chinavia (Chinavia hilaris, Chinavia marginata, and Chinavia pensylvanica), Stink bugs of the genus Chlorochroa (Chlorochroa granulose, Chlorochroa kanei, Chlorochroa ligata, Chlorochroa lineate, Chlorochroa opuntiae, Chlorochroa persimilis, Chlorochroa rossiana, Chlorochroa sayi, Chlorochroa uhleri, Chlorochroa belfragii, Chlorochroa faceta, Chlorochroa osbomi, Chlorochroa saucia, and Chlorochroa senilis), Southern Green Stink Bug (Nez.ara viridula), Stink Bugs from the genus Edessa (Edessa meditabunda, Edessa bifida, and Edessa florida), the Neotropical Brown Stink Bug (Euschistus heros), stink bugs from the genus Euschistus (Euschistus acuminatus, Euschistus biformis, Euschistus conspersus, Euschistus crenator, Euschistus egglestoni, Euschistus ictericus, Euschistus inflatus, Euschistus latimarginatus, Euschistus obscures, Euschistus politus, Euschistus quadrator, Euschistus sevus, Euschistus strenuous, Euschistus tristigmus, and Euschistus variolarius), Brown Marmoratcd Stink Bug (Halyomorpha halys), Rcd-Shouldcrcd Stink Bug Thyanta accerra), stink bugs of the genus Thyanta Thyanta calceata, Thyanta custator, Thyanta pallidovirens, Thyanta perditor, Thyanta maculate, and Thyanta pseudocasta), the Green Belly Stink Bug (Dichelops melacanthus) and other stink bugs of the genus Dichelops (Dichelops avilapiresi, Dichelops bicolor, Dichelops dimidatus, Dichelops furcatus, Dichelops furcifrons, Dichelops lobatus, Dichelops miriamae, Dichelops nigrum, Dichelops peruanus, Dichelops phoenix, and Dichelops saltensis), the Red Banded Stink Bug (Piezodorus guildinni) as well as Piezodorus lituratus', insects of the family of Plataspidae such as, but not limited to, Kudzu Bug (Megacopta cribraria), Western tarnished plant bug (Lygus hesperus), Tarnished plant bug (Lygus lineolaris)', aphid species such as, but not limited to, Soybean aphid (Aphis glycines), Green Peach aphid (Myzus persicae), Potato aphid (Macrosiphum euphorbiae), Melon aphid (Aphis gossypii), Cabbage aphid (Brevicoryne brassicae); Planthopper species such as, but not limited to, Blue-green leafhopper (Graphocephala atropunctata) and Ligurian leafhopper (Eupteryx decemnotata)’, whitefly pest species, such as but not limited to, African cassava whitefly (Bemisia tabaci), Silverleaf whitefly (Bemisia argentifolii), Greenhouse whitefly (Trialeurodes vaporariorum), Giant whitefly (Aleurodicus dugesii), Citrus blackfly (Aleurocanthus woglumi), Cabbage whitefly (Aleyrodes proletella), other species within the Aleyrodidae family such as those belonging to the genera Acanthaleyrodes, Acanthobemisia, Acaudaleyrodes, Acutaleyrodes, Africaleurodes, Agrostaleyrodes, Aleurocanthus, Aleurocerus, Aleurochiton, Aleuroclava, Aleurocybotus, Aleurocyperus, Aleurodicus, Aleuroduplidens, Aleuroglandulus, Aleuroinanis, Aleurolobus, Aleurolonga, Aleuromarginatus, Aleuronudus, Aleuropapillatus, Aleuroparadoxus, Aleuroplatus, Aleuropleurocelus, Aleuroporosus, Aleuropteridis, Aleuroputeus, Aleurothrixus, Aleurotithius, Aleurotrachelus, Aleurotuba, Aleurotulus, Aleuroviggianus, Aleurovitreus, Aleyrodes, Aleyrodiella, Aleyrodiella, Anomaleyrodes, Anomaleyrodes, Apobemisia, Apobemisia, Arachnaleyrodes, Arachnaleyrodes, Asialeyrodes, Asialeyrodes, Asterobemisia, Asterobemisia, Asterochiton, Asterochiton, Auslroaleurodicus, Axacalia, Axacalia, Azuraleurodicus, Bakerius, Bellitudo, Bellitudo, Bemisaleyrodes, Bemisaleyrodes, Bemisia, Bemisia, Bemisiella, Bemisiella, Brazzaleyrodes, Brazzaleyrodes, Bulgarialeurodes, Bulgarialeurocles, Calluneyrodes, Calluneyrodes, Ceraleurodicus, Chitonaleyrodes, Chitonaleyrodes, Cockerelliella,

[0048] Cockerelliella, Cohicaleyrodes, Cohicaleyrodes, Combesaleyrodes, Combesaleyrodes, Corbettia, Costalimada, Crenidorsum, Crescentaleyrodes, Cryptolingula, Davidiella, Dialeurodes, Dialeurodicus, Dialeurolobus, Dialeurolonga, Dialeuropora, Dialeurotrachelus, Disiphon, Distinctaleyrodes, Dothioia, Dumbletoniella, Editaaleyrodes, Eudialeurodicus, Extensaleyrodes, Fascaleyrodes, Filicaleyrodes, Fippataleyrod.es, Gagudjuia, Gomenella, Harpaleyrodes, Harpaleyrodes,, Hesperaleyrodes, Heteraleyrodes, Heterobemisia, Indoaleyrodes, Juglasaleyrodes, Keralaleyrodes, Laingiella, Lecanoideus, Leonardius, Leucopogonella, Lipaleyrodes, Malayaleyrodes, Marginaleyrodes, Mas silieur odes, Metabemisia, Metaleurodicus, Metaleyrodes, Minutaleyrodes, Mixaleyrodes, Nealeurodicus, Nealeyrodes, Neoaleurodes, Neoaleurotrachelus, Neomaskellia, Neopealius, Nigrasialeyrodes, Nipaleyrodes, Octaleurodicus, Orchamoplatus, Orientaleyrodes, Orstomaleyrodes, Papillipes, Parabemisia, Paraleurolobus, Paraleyrodes, Paulianaleyrodes, Pealius, Pectinaleyrodes, Pentaleyrodes, Peracchius, Plataleyrodes, Pogonaleyrodes, Pseudaleurolobus, Pseudaleuroplatus, Pseudaleyrodes, Pseudosynaleurodicus, Pseudozaphanera, Ramsesseus, Rhachisphora, Rosanovia, Rugaleyrodes, Rusostigma, Russellaleyrodes, Septaleurodicus, Setaleyrodes, Simplaleurodes, Singhiella, Singhius, Siphoninus, Sphericaleyrodes, Stenaleyrodes, Synaleurodicus, Tegmaleurodes, Tetraleurodes, Tetralicia, Trialeurodes, Trialeurolonga, Trichoaleyrodes, Tuberaleyrodes, Udamoselis, Vasantharajiella, Vasdavidius, Venezale urodes, Viennotaleyrodes, Xenaleyrodes, Xenobemisia, Yleyrodes, Zaphanera.

[0049]

[0041] Whiteflies typically feed on the underside of leaves and present major problems for protecting certain crops. The principal harm is caused by their feeding behavior both as a disease vector and by causing extrusion or release of sap or plant fluid that provides a medium for fungal growth. Like many sap-sucking Hemiptera, they secrete large amounts of honeydew, a sugar-rich sticky liquid. The honeydew provides a medium that supports infestations of the crop plant by sooty mold. In addition, whiteflies are also vectors for the transmission of certain crop viruses. The most prominent disease vectors amongst the whiteflies are species from the genus Bemisia. B. tabaci and B. argentifolii transmit African cassava mosaic, bean golden mosaic, bean dwarf mosaic, bean calico mosaic, tomato yellow leaf curl, tomato mottle, and other Begomoviruses, in the family Geminiviridae . The worldwide spread of emerging biotypes, such as B. tabaci biotype B, also known as, 'B. argentifolii' , and a new biotype Q (mitochondria- type), continue to cause severe crop losses which are expected to increase, demanding matching increases in pesticide use on many crops (tomatoes, beans, cassava, cotton, cucurbits, potatoes, sweet potatoes, and cowpeas) Navas-Catillor, J., Eiallo-Olive, E., Sanchez-Campos, S., (2011 ) Emerging Virus Diseases Transmitted by Whiteflies. Annual Review of Phytopathology. Annual Reviews, 49(1): 219-248).

[0050]

[0042] Reference in this application to an “isolated DNA molecule”, or an equivalent term or phrase, is intended to mean that the DNA molecule is one that is present alone or in combination with other compositions, but not within its natural environment. For example, nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence was not within the DNA of the bacterium from which the sequence encoding the protein is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring insecticidal protein would be considered to be isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., the nucleotide sequence of the DNA inserted into the genome of the cells of a plant, plant part, microorganism or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant, plant part, microorganism or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant, plant part, microorganism or bacterium.

[0051]

[0043] Reference in this application to the term “self-limiting gene” refers to a gene that limits survival of the host, resulting in a reduction in the host population. Such technology is offered by Oxitech Ltd. Transgenic male insects carrying a transgenic self-limiting gene are released and reproduce with wild females. As a result, the progeny inherits a copy of the self-limiting gene. The self-limiting gene disrupts the proper functioning of the insects’ cells by over-producing a protein in them, interfering with the cells’ ability to produce other essential proteins needed for development. By disrupting the insect’s normal development, the gene prevents it from surviving to adulthood. For example, the self-limiting Diamondback Moth (Plutellidae xylostella) strain OX4319L was developed by Oxitcch Ltd and carries a male- selecting gene that utilizes sequences from the sex determination gene doublesex (dsx). The gene expresses sex-alternate splicing, to engineer female- specific expression of the self-limiting gene which prevents survival of female offspring beyond the larval stage and allows for production of male only cohorts of self-limiting moths. After being released, males mate with pest females, leading to a reduction in the number of female offspring in the next generation, thereby locally suppressing P. xylostella populations. To facilitate the rear ing of large numbers of males for release within diamondback moth production facilities, the expression of female- specific dsx within the 0X4319L strain is repressed by the addition of tetracycline, or suitable analogs, into the larval feed. 0X4319L also expresses the fluorescent protein, DsRed, to permit the effective monitoring of the presence of this strain in the field (Jin et al., 2013. Engineered female-specific lethality for control of pest Lepidoptera. ACS Synthetic Biology, 2: 160-166). This technology, when applied in the field with plants containing the toxin genes of the present invention, can delay or prevent the onset of resistance of pest species targeted for control by the toxin genes and proteins of the present invention, thus giving a greater durability of any plant product containing the toxin genes and proteins of the present invention.

[0052]

[0044] While self-limiting technologies have not yet been developed to control Coleopteran and Hemipteran insect pests, some of the key genes in sex determination of Hemiptera have been identified and the differential splicing of their transcripts between the sexes determined for the Hemipteran species brown planthopper (N. luyens). the whitefly (B. labaci). and the kissing bug (Rhodnius prolixus). More research is needed before sex ratios can be altered as part of a genetic- control strategy. Some hemipteran species, such as whiteflies, are haplo-diploid and others lack Y chromosomes. These alternative genetic systems will, most likely, influence the design and efficiencies of genetic-control mechanisms such as self-limiting technologies. While many of the attributes that make Diptera and Lepidoptera amenable to genetic-control mechanisms, many of these constraints are not insurmountable. Current control strategies will need to be adapted or new strategies developed to enable the field of hemipteran control. Within the past 4 years, there have been substantial advances in the field of hemipteran biotechnology. The enabling technology of CRISPR / Cas-meditated mutagenesis in the Hemiptera is emerging. The increasing numbers of annotated genome assemblies now provide essential components for the development of the genetic toolbox required for extended genetic control into Hemipteran pests (Pacheco et al., (2022 ) Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives, Frontiers in Bioengineering and Biotechnology, 10:1-26).

[0053]

[0045] As described further in this application, an open reading frame (ORF) (SEQ ID NO: 1) encoding TIC3410 (SEQ ID NO: 3) was discovered in DNA obtained from Bacillus thuringiensis species EG10168. Bioassay using microbial host cell-derived proteins of TIC3410 demonstrated activity against the Lepidopteran species Black cutworm (Agrotis ipsilon). European corn borer (Ostrinia nubilalis), Southwestern corn borer (Diat aea grandiosella), Tobacco budworm (Heliothis virescens); the Coleopteran species Colorado potato beetle (Leptinotarsa decemlineata); and the Hemipteran species Western tarnished plant bug (Lygus he s per us), Neotropical Brown Stink Bug (Euschistus heros).

[0054]

[0046] As described further in this application, an open reading frame (ORF) (SEQ ID NO: 4) encoding TIC4123 (SEQ ID NO: 6) was discovered in DNA obtained from Bacillus thuringiensis species CFB213052. Bioassay using microbial host cell-derived proteins of TIC4123 demonstrated activity against the Lepidopteran species Black cutworm (Agrotis ipsilon), Corn earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), Fall army worm (Spodoptera frugiperda), Soybean looper (Chrysodeixis includens), Southwestern com borer (Diatraea grandiosella), Tobacco budworm (Heliothis virescens) the Coleopteran species Western Com Rootworm (Diabrotica virgifera), Colorado potato beetle (Leptinotarsa decemlineata); and the Hemipteran species Tarnished plant bug Lygus lineolaris).

[0055]

[0047] As described further in this application, an open reading frame (ORF) (SEQ ID NO: 7) encoding TIC4124 (SEQ ID NO: 9) was discovered in DNA obtained from Bacillus thuringiensis species EG5018. Bioassay using microbial host cell-derived proteins of TIC4124 demonstrated activity against the Lepidopteran species Black cutworm (Agrotis ipsilon), Corn earworm (Helicoverpa zea), European com borer (Ostrinia nubilalis), Fall armyworm (Spodoptera frugiperda), Soybean looper (Chrysodeixis includens), Southwestern com borer (Diatraea grandiosella), Tobacco budworm (Heliothis virescens)', the Coleopteran species Western Com Rootworm (Diabrotica virgifera), Colorado potato beetle (Leptinotarsa decemlineata); and the Hemipteran species Tarnished plant bug (Lygus lineolaris), Neotropical Brown Stink Bug (Euschistus heros), and Southern Green Stink Bug (Nezara viridula).

[0056]

[0048] Synthetic coding sequences (SEQ ID NOs: 2, 5, and 8) designed for expression in a plant cell of the TIC3410, TIC4123, and TIC4124 proteins (SEQ ID NOs: 3, 6, and 9, respectively) were determined and made. These synthetic coding sequences can be used to express the TIC3410, TIC4123, and TIC4124 pesticidal proteins in monocot and dicot plants such as, but not limited to, com, wheat, rice, millet, soybean, canola, cowpea, and alfalfa.

[0057]

[0049] For expression in plant cells, the TIC3410, TIC4123, and TIC4124 (SEQ ID NOs: 3, 6, and 9) proteins, or a variant or fragment thereof, such as a pesticidal protein within the TIC3410, TIC4123, or TIC4124 protein toxin class or a fragment thereof, can be expressed and localized in the cytosol or targeted to various organelles of the plant cell. For example, targeting a protein to the chloroplast may result in increased levels of expressed protein in a transgenic plant while preventing off-phenotypes from occurring if the expressed protein toxin reacts with the cell biology in any unexpected manner. Targeting may also result in an increase in pest resistance efficacy in the transgenic event. A target peptide or transit peptide is a short (3-70 amino acids long) peptide chain that can be fused to a pesticidal protein sequence to become a part of a pesticidal protein and direct the transport of the protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasma membrane. A polynucleotide coding sequence for a pesticidal protein may further include a polynucleotide coding sequence for the target peptide or transit peptide such that the pesticidal protein further includes the target peptide or transit peptide. Some target peptides are cleaved from the protein by signal peptidases after the proteins are transported. For targeting to the chloroplast, proteins can contain transit peptides which are around 40-50 amino acids in length. For descriptions of the use of chloroplast transit peptides, see U.S. Patent Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP), which may be fused to, and become a part of, a pesticidal protein. Examples of such isolated CTPs include, but are not limited to, those associated with the small subunit (SSU) of ribulose- 1,5, -bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), and transit peptides described in U.S. Patent No. 7,193,133. It has been demonstrated in vivo and in vitro that nonchloroplast proteins may be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide such as the Arabidopsis thaliana EPSPS CTP (CTP2) (see, Klee et al., Mol. Gen. Genet. 210:437-442, 1987) or the Petunia hybrida EPSPS CTP (CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986) has been shown to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants see, U.S. Patent Nos. 5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP 508909; and EP 924299). For targeting the TIC3410, TIC4123, or TIC4124 toxin protein to the chloroplast, a sequence encoding a chloroplast transit peptide is placed 5 ' in operable linkage and in frame to a synthetic I artificial nucleotide sequence, such as SEQ ID NO: 2, 5, or 8 encoding the TIC3410, TIC4123, or TIC4124 toxin protein, respectively.

[0058]

[0050] It is contemplated that additional toxin protein sequences related to TIC3410, TIC4123, and TIC4124 can be created using the amino acid sequence of TIC3410, TIC4123, and TIC4124, or a variant or fragment thereof, to create novel proteins with novel properties. The TIC3410, TIC4123, and TIC4124 toxin proteins can be aligned to toxin proteins related to TIC3410, TIC4123, and TIC4124 and have a sequence identity percentage in a range from about 90% to about 99.9% identity, such as at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.1% identity, at least 99.2% identity, at least 99.3% identity, at least 99.4% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, or at least 99.9% or higher percentage identity or 100% identity to TIC3410, TIC4123, and TIC4124, and combine differences at the aligned amino acid sequence level into one or more novel amino acid sequence valiants and making appropriate changes to the recombinant nucleic acid sequence encoding variants, such as for improved expression in a plant, plant part or plant cell.

[0059]

[0051] Amino acid sequence variants are contemplated which could exhibit improved properties relative to the sequences set forth at SEQ ID NOs: 3, 6, and 9. It is contemplated that improved amino acid sequence variants of the TIC3410, TIC4123, and TIC4124 protein toxin classes can be encoded by engineered DNA molecules as transgenes or engineered in planta by using various gene editing methods known in the art. Such technologies used for genome editing include, but are not limited to, ZFN (zine-finger nuclease), meganucleases, TALEN (Transcription activatorlike effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) systems. These genome editing methods can be used to alter the toxin protein coding sequence transformed within a plant cell to a different toxin coding sequence. Specifically, through these methods, one or more codons within the toxin coding sequence may be altered to engineer a new protein amino acid sequence. Alternatively, a fragment within the coding sequence may be replaced or deleted, or additional DNA fragments arc inserted into the coding sequence, to engineer a new toxin coding sequence. The plant cell comprising the gene edited toxin coding sequence can be used by methods known in the art to generate whole plants expressing the new toxin protein.

[0060]

[0052] It is also contemplated that fragments of TIC3410, TIC4123, and TIC4124 proteins or protein variants thereof can be truncated forms wherein one or more amino acids are deleted from the N-terminal end, the C-terminal end, a middle or interior portion of the protein, or combinations thereof, wherein the fragments and variants retain insect inhibitory activity. These protein fragments can be naturally occurring or synthetic variants of TIC3410, TIC4123, and TIC4124 or derived protein variants but should retain insect inhibitory activity that is identical or similar to the TIC3410, TIC4123, and / or TIC4124 protein(s), such as the TIC3410, TIC4123, or TIC4124 protein from which the protein fragment is derived.

[0061]

[0053] Proteins that resemble the TIC3410, TIC4123, and TIC4124 proteins can be identified and compared to each other using various computer-based algorithms known in the art. Amino acid sequence identities reported in this application are a result of a Clustal W alignment using these default parameters: Weight matrix: blosum, Gap opening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps: On, Hydrophilic residues: GPSNDQERK, Residue-specific gap penalties: On (Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percent amino acid identity is further calculated by the product of 100% multiplied by (amino acid identities / length of subject protein). Other alignment algorithms are also available in the art and provide results similar to those obtained using a Clustal W alignment and are contemplated herein.

[0062]

[0054] It is intended that a protein exhibiting insect inhibitory activity against a Lepidopteran, Coleopteran, or Hemipteran insect species is related to TIC3410, TIC4123, and TIC4124 if the insecticidal protein is used in a query, e.g., in a Clustal W alignment, and exhibits at least 90% to about 100% amino acid sequence identity percentage to SEQ ID NO: 3, 6 or 9, or is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% identical to SEQ ID NO: 3, 6 or 9, or is about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 99.1%, or about 99.2%, or about 99.3%, or about 99.4%, or about 99.5%, or about 99.6%, or about 99.7%, or about 99.8%, or about 99.9%, or about 100% identical to SEQ ID NO: 3, 6 or 9, or any fraction or fragment thereof.

[0063]

[0055] Table 1 below shows a multi- sequence alignment using Clustal W for TIC3410, TIC4123, and TIC4124 wherein the numbers in parathesis are the numbers of identical amino acids.

[0064] Table 1. Percent identity between TIC3410, TIC4123, and TIC4124.

[0065]

[0056] The closest known identity match to the amino acid sequence of TIC3410 has been disclosed in GenBank Accession BAC79010 which is 100% identical to TIC3410. The closest known identity match to the amino acid sequence of TIC4123 has been disclosed in the International Application Publication, WO2023 / 107943 and is 100% identical to SEQ ID NO:2 of WO2023 / 107943. The closest known identity match to the amino acid sequence of TIC4124 has been disclosed in GenBank Accession BAD35170 and is 99.34% identical to TIC4124.

[0066]

[0057] In addition to percent sequence identity, TIC3410, TIC4123, and TIC4124 can also be related by primary structure (conserved amino acid motifs), by length and by other characteristics. Characteristics of the T1C3410, T1C4123, and T1C4124 protein toxins are reported in Table 2.

[0067] Table 2. Selected characteristics of TIC3410, TIC4123, and TIC4124 toxin proteins.

[0068]

[0058] As described further in the Examples of this application, a synthetic or artificial nucleic acid molecule, a nucleotide sequence, encoding TIC3410, TIC4123, and TIC4124, was designed for use in plants, as set forth in SEQ ID NOs: 2, 5, and 8, respectively. In view of the redundancy of the genetic code, it is within the skill of the art to produce any number of other sequences for encoding the toxin proteins, however, it is understood that the sequences produced for expression in planta should avoid known problems in the art that hinder or limit the efficient expression of the coding sequence, particularly as described in US Patent 5,500,365 and US Patent 7,741,118.

[0059] Expression cassettes and vectors containing a recombinant nucleic acid sequence molecule can be constructed and introduced into plants, particularly such as corn, soybean, or cotton plant cells in accordance with transformation methods and techniques known in the art. For example, Agro / zacterrnm-mediated transformation is described in U.S. Patent Application Publications 2009 / 0138985 Al (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257 Al (sugar beet), U.S. Patent Nos. 5,750,871 (canola), 7,026,528 (wheat), and 6,365,807 (rice), and in Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugarcane). Methods to transform many crop plants are known in the art and are described further above. Examples of methods used for transforming Cowpea are provided by Bosibori et al. (Bo ih ri, B., Gollasch, S., Moore, A., Harding, R., and Higgins, T.J.V. (2019) An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime. Frontiers in plant Science. 10: 1-10) and Che et al. (Che, P., Chang, S., Simon, M, Zhang, Z., Shaharyar, A., Ourada, J. O’Neill, D., Torres-Mendoz.a, M., Guo, K, Marasigan, K.M., Vielle-Calzada, J-P., Ozias-Akins, P., Albertsen, M.C., and Jones, T.J. (2021) Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants. The Plant Journal. 106: 817-830.) Examples of methods to transform Cassava are provided by Segatto et al. (Segatto, R., Jones, T., Stretch, D., Albin, C, Chauhan, R.D. and Taylor, N.J. (2022) Agrobacterium- mediated Genetic Transformation of Cassava. Current Protocols e620. 2: 1-36.) Transformed cells can be regenerated into transformed plants that express TIC3410, TIC4123, or TIC4124, or a variant or fragment thereof, and demonstrate pesticidal activity through bioassays performed in the presence of Lepidopteran, Coleopteran, or Hemipteran pest larvae, which may involve using plant leaf disks obtained from the transformed plants. Plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming and regenerating plants are known in the art.

[0069]

[0060] As an alternative to traditional transformation methods, a DNA sequence, such as a transgene, expression cassette(s), etc., may be inserted or integrated into a specific site or locus within the genome of a plant or plant cell via site-directed integration. Recombinant DNA construct(s) and molecule(s) of this disclosure may thus include a donor template sequence comprising at least one transgcnc, expression cassette, or other DNA sequence for insertion into the genome of the plant or plant cell. Such donor template for site-directed integration may further include one or two homology arms flanking an insertion sequence (z.e., the sequence, transgene, cassette, etc., to be inserted into the plant genome). The recombinant DNA construct(s) of this disclosure may further comprise an expression cassette(s) encoding a site-specific nuclease and / or any associated protein(s) to carry out site-directed integration. These nuclease-expressing cassette(s) may be present in the same molecule or vector as the donor template (in cis) or on a separate molecule or vector (in trans). Several methods for site-directed integration are known in the art involving different proteins (or complexes of proteins and / or guide RNA) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. Briefly as understood in the art, during the process of repairing the DSB or nick introduced by the nuclease enzyme, the donor template DNA may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the donor template may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion event may occur through non- homologous end joining (NHEJ). Examples of site-specific nucleases that may be used include zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, and RNA- guided endonucleases (e.g., Cas9 or Casl2a). For methods using RNA-guided site-specific nucleases (e.g., Cas9 or Casl2a), the recombinant DNA construct(s) will also comprise a sequence encoding one or more guide RNAs to direct the nuclease to the desired site within the plant genome.

[0070]

[0061] Recombinant nucleic acid molecule compositions that encode bacterial and plant expressed TIC3410, TIC4123, and TIC4124 proteins, or variants or fragments thereof, as described herein can be expressed with recombinant DNA constructs in which a polynucleotide molecule with an ORF encoding the protein is operably linked to genetic expression elements such as a promoter and any other regulatory element necessary for expression in the system for which the construct is intended. Non-limiting examples include a plant-functional promoter operably linked to a TIC3410, TIC4123, or TIC4124 protein, variant or fragment, encoding sequence for expression of the protein in plants or a Bt- functional promoter operably linked to a TIC3410, TIC4123, or TIC4124 protein, variant or fragment, encoding sequence for expression of the protein, variant or fragment in a Bt bacterium or other Bacillus species. Reference herein to a “TIC3410, TIC4123, or TIC4124 protein, variant or fragment” means a pesticidal protein within a TIC3410, TIC4123, or TIC4124 protein toxin class, respectively, and a “TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence” means a polynucleotide or DNA sequence or segment encoding such a pesticidal protein (z.e., a TIC3410, TIC4123, or TIC4124 protein, variant or fragment, respectively). A polynucleotide or DNA sequence or segment encoding such a pesticidal protein (z.e., a TIC3410, TIC4123, or TIC4124 protein or variant) may comprise one or more of SEQ ID NO: 1, 2, 4, 5, 7, and / or 8, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of SEQ ID NO: 1, 2, 4, 5, 7, and / or 8. A polynucleotide or DNA sequence or segment encoding a fragment of a pesticidal protein (e.g., a fragment of a TIC3410, TIC4123, or TIC4124 protein or variant) may comprise at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, or at least 2000 consecutive or contiguous nucleotides of one or more of SEQ ID NO: 1, 2, 4, 5, 7, and / or 8, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, or at least 2000 consecutive or contiguous nucleotides of one or more of SEQ ID NO: 1, 2, 4, 5, 7, and / or 8. A TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence may include any polynucleotide sequence encoding a TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, as described herein. Other elements can be operably linked to the TIC3410, TIC4123, or TIC4124 protein encoding sequence including, but not limited to, enhancers, introns, untranslated leaders, encoded protein immobilization tags (HIS- tag), translocation peptides (z'.e., plastid transit peptides, signal peptides), polypeptide sequences for post-translational modifying enzymes, ribosomal binding sites, and RNAi target sites. Exemplary recombinant polynucleotide molecules provided herewith include, but are not limited to, a heterologous promoter operably linked to a polynucleotide sequence, such as SEQ ID NOs: 1, 2, 4, 5, 7, and / or 8, that encodes TIC3410, TIC4123, or TIC4124, as set forth in SEQ ID NOs: 3, 6, or 9, or a variant or fragment thereof. A heterologous promoter can also be operably linked to synthetic / artificial DNA coding sequences encoding a TIC3410, TIC4123, or TIC4124, or a variant or fragment thereof, or a plastid targeted TIC3410, TIC4123, or TIC4124, or a variant of fragment thereof. The codons of a recombinant nucleic acid molecule encoding for proteins disclosed herein can be substituted by synonymous codons (known in the art as a silent substitution), which may be optimized for expression in a plant.

[0071]

[0062] A recombinant DNA constructs comprising a TIC3410, TIC4123, and TIC4124 protein, variant or fragment encoding sequence can further comprise a separate region, segment or sequence of DNA that encodes for one or more insect inhibitory agents, such as an insect inhibitory dsRNA molecule or an ancillary protein, which can be configured to concomitantly express or coexpress with the TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence. Ancillary proteins include, but are not limited to, co-factors, enzymes, binding-partners, or other agents that function to aid in the effectiveness of an insect inhibitory agent, for example, by aiding its expression, influencing its stability in plants, optimizing free energy for oligomerization, augmenting its toxicity, and increasing its spectrum of activity. An ancillary protein may facilitate the uptake of one or more insect inhibitory agents, for example, or potentiate the toxic effects of the toxic agent.

[0072]

[0063] A recombinant DNA construct can be assembled so that all proteins or dsRNA molecules are expressed from one promoter, or each protein or dsRNA molecule is under separate promoter control or some combination thereof. The protein of this invention can be expressed from a multigene expression system, in which TIC3410, TIC4123, or TIC4124 or a related protein, or a variant or fragment thereof, is expressed from a common nucleotide segment or sequence which also contains other open reading frames and promoters, depending on the type of expression system selected. For example, a bacterial multi-gene expression system can utilize a single promoter to drive expression of multiply linked / tandem open reading frames from within a single operon (i.e., polycistronic expression). In another example, a plant multi-gene expression system can utilize multiply unlinked or linked expression cassettes, each cassette expressing a different protein or other agent such as one or more dsRNA molecules.

[0073]

[0064] Recombinant polynucleotides or recombinant DNA constructs comprising a TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence can be delivered to host cells by vectors, e.g., a plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage, or viral vector. Indeed, a recombinant DNA molecule as provided herein may be or comprise a vector or a plasmid or extrachromosomal DNA, a chromosome or plastomic DNA, or baculovirus, virion, cosmid, phagemid, phage, virus or any other biological DNA molecule. Such vectors can be used to achieve stable or transient expression of a TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that comprises a TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence and that is introduced into a host cell is referred in this application as a “transgene”.

[0074]

[0065] Transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain a recombinant polynucleotide that expresses TIC3410, TIC4123, or TIC4124 or a related sequence encoding a family toxin protein are provided herein. The term “bacterial cell” or “bacterium” refers to a single bacterial cell and can include, but is not limited to, an Agrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas, Brevibacillus, Klebsiella, Erwinia, or aRhizobium cell, and “bacteria” refers to one or more bacterium or bacterial cells. The term “plant cell” or “plant” can include but is not limited to a dicotyledonous or monocotyledonous plant. The term “plant cell” or “plant” can also include but is not limited to an alfalfa, Arabidopsis, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, cowpea, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, com (i.e., maize, such as sweet corn or field corn), sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell or plant. In certain embodiments, transgenic plants and transgenic plant parts regenerated from a transgenic plant cell are provided. In certain embodiments, the transgenic plants can be obtained from a transgenic seed, by cutting, snapping, grinding or otherwise disassociating the part from the plant. In certain embodiments, the plant part can be a seed, a pollen grain, a boll, a leaf, a flower, a stem, a root, or any portion thereof, or a non-regenerable portion of a transgenic plant part. As used in this context, a “non-regenerable” portion of a transgenic plant part is a portion that cannot be induced to form a whole plant or that cannot be induced to form a whole plant that is capable of sexual and / or asexual reproduction. In certain embodiments, a non-regenerable portion of a plant part is a portion of a transgenic seed, pollen grain, chloroplast, boll, leaf, flower, stem, or root. According to embodiments of the present disclosure, a microorganism(s), such as a bacterium or bacteria or bacterial cell(s), fungus, fungi or fungal cell(s), archaea, algae, viruses, protozoa, or plant cell(s), are provided that comprise any recombinant nucleic acid molecule(s) and / or insecticidal or pesticidal protcin(s) as provided herein. Such a microorganism may be transgenic (e.g., containing a recombinant transgene or cassette in its genome, plastome and / or plasmid or extrachromosomal DNA), genetically modified or edited, and / or comprise or contain any recombinant nucleic acid molecule(s)and / or an insecticidal or pesticidal protein(s) as provided herein.

[0075]

[0066] Methods of making transgenic plants that comprise insect-inhibitory or Lepidoptera- inhibitory amounts of a TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, are provided. Such plants can be made by introducing a recombinant polynucleotide that encodes the proteins provided in this application into a plant cell, and selecting a plant derived from said plant cell that expresses an insect-, Lepidoptera-, Coleoptera-, or Hcmiptcra-inhibitory amount of the proteins. Plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0076]

[0067] Processed plant products, wherein the processed product comprises a detectable amount of a TIC3410, TIC4123, or TIC4124 protein, or an insect inhibitory variant or segment or fragment thereof, or a TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence, or any distinguishing part or portion of the foregoing, are also provided herein. In certain embodiments, the processed product is selected from the group consisting of plant parts, plant biomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint, hulls, processed seed, and seed. In certain embodiments, the processed product is non-regenerable. The plant product can comprise commodity or other product of commerce derived from a transgenic plant or transgenic plant part, where the commodity or other product can be tracked through commerce by detecting a TIC3410, TIC4123, or TIC4124 protein or a variant or fragment thereof or all or part of a polynucleotide sequence or segment or an expressed RNA that encodes all or part of a TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, or that comprises a distinguishing portion of a TIC3410, TIC4123, or TIC4124 protein, variant or fragment encoding sequence.

[0077]

[0068] Plants expressing a TIC3410, TIC4123, and TIC4124 protein, or a variant or fragment thereof, can be crossed by breeding with transgenic events expressing other toxin proteins and / or expressing other transgenic traits such as herbicide tolerance genes, genes conferring yield or stress tolerance traits, and the like, or such traits can be combined in a single stacked vector so that the traits are all linked when present within the same transgenic genome.

[0069] As further described in the Examples, TIC3410, TIC4123, and TTC4124 protein-encoding sequences and sequences having a substantial percentage identity to the sequences encoding TIC3410, TIC4123, and TIC4124, can be identified using methods known to those of ordinary skill in the art such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, the proteins TIC3410, TIC4123, and TIC4124, or a variant or fragment thereof, can be used to produce antibodies that bind specifically to related proteins and can be used to screen for and to find other protein members that are closely related.

[0078]

[0070] Furthermore, nucleotide sequences encoding all or part of a TIC3410, TIC4123, or TIC4124 toxin protein, or a variant or fragment thereof, can be used as probes and primers for screening to identify other members of the TIC3410, TIC4123, or TIC4124 protein toxin class using thermal-cycle or isothermal amplification and hybridization methods. For example, oligonucleotides derived from, or similar to, the sequences as set forth in SEQ ID NOs: 1, 2, 4, 5, 7 or 8 or other transgenic sequences can be used to determine the presence or absence of a TIC3410, TIC4123, or TIC4124 related transgene in a deoxyribonucleic acid sample derived from a commodity product. Given the sensitivity of certain nucleic acid detection methods that employ oligonucleotides, it is anticipated that oligonucleotides derived from sequences as set forth in SEQ ID NOs: 1, 2, 4, 5, 7 or 8 or other transgenic sequences can be used to detect a TIC3410, TIC4123, or TIC4124 transgene in commodity products or samples derived from pooled sources where only a fraction of the commodity product or source is derived from a transgenic plant or plant part containing the transgene. It is further recognized that such oligonucleotides can be used to introduce nucleotide sequence variation in each of SEQ ID NOs: 1, 2, 4, 5, 7, or 8. Such “mutagenesis” oligonucleotides are useful for identification of TIC3410, TIC4123, or TIC4124 amino acid sequence variants or fragments exhibiting a range of insect inhibitory activity or varied expression in transgenic plant host cells.

[0079]

[0071] Nucleotide sequence homologs, e.g. , insecticidal proteins encoded by nucleotide sequences that hybridize to each or any of the sequences disclosed in this application under stringent hybridization conditions, are also an embodiment of the present invention. The invention also provides a method for detecting a first nucleotide sequence that hybridizes to a second nucleotide sequence, wherein the first nucleotide sequence (or its reverse complement sequence) encodes a pesticidal protein or pesticidal fragment thereof and hybridizes to the second nucleotide sequence. In such a case, the second nucleotide sequence can be any of the nucleotide sequences presented as SEQ ID NOs: 1 , 2, 4, 5, 7, or 8 or a nucleotide sequence that hybridizes to any of SEQ ID NOs: 1, 2, 4, 5, 7, or 8 under stringent hybridization conditions. Nucleotide coding sequences hybridize to one another under appropriate hybridization conditions, such as stringent hybridization conditions, and proteins encoded by these nucleotide sequences may cross react with antiserum raised against any one of the other proteins. Stringent hybridization conditions, as defined herein, comprise at least hybridization at 42°C followed by two washes for five minutes each at room temperature with 2X SSC, 0.1% SDS, followed by two washes for thirty minutes each at 65°C in 0.5X SSC, 0.1% SDS. Washes at higher temperatures constitute greater stringency, e.g., hybridization conditions of 68°C, followed by washing at 68°C, in 2xSSC containing 0.1% SDS, meaning that a higher percentage nucleotide sequence identity will be selected and detected.

[0080]

[0072] One skilled in the art will recognize that, due to the redundancy of the genetic code, many other sequences are capable of encoding such related proteins, and those sequences, to the extent that they function to express pesticidal proteins either in bacterial strains, such as Bacillus strains, fungal, yeast or other host cells, or in plant cells, are embodiments of the present invention, recognizing of course that many such redundant coding sequences may not hybridize under these conditions to the native Bacillus sequences encoding TIC3410, TIC4123, or TIC4124 variants. This application contemplates the use of these, and other identification methods known to those of ordinary skill in the art, such as percent sequence identity or sequence identity percentage or the like, to identify TIC3410, TIC4123, or TIC4124 variant protein-encoding sequences and sequences having a substantial sequence percentage identity to nucleic acid sequences encoding proteins related to TIC3410, TIC4123, or TIC4124.

[0081]

[0073] This disclosure also contemplates the use of molecular methods known in the art to engineer and clone commercially useful proteins comprising chimeras of proteins from pesticidal proteins; e.g., the chimeras may be assembled from segments or portions of a TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, to derive additional useful embodiments including assembly of segments or portions of TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, with segments or portions of any diverse insecticidal proteins different from TIC3410, TIC4123, or TIC4124 protein and related proteins. The TIC3410, TIC4123, or TIC4124 protein and related insecticidal proteins may be subjected to alignment with each other and to other Bacillus, Paenibacillus or other pesticidal proteins (whether or not these are closely or distantly related phylogenetically) or other artificial or engineered protein sequences, and segments of each of such proteins may be identified that are useful for substitution between the aligned proteins, resulting in the construction of chimeric proteins. Such chimeric proteins can be subjected to pest bioassay analysis and characterized for the presence or absence of increased bioactivity or expanded target pest spectrum compared to the parent proteins from which each such segment in the chimera was derived. The pesticidal activity of the polypeptides may be further engineered for activity to a particular pest or to a broader spectrum of pests by swapping domains or segments with other proteins or by using directed evolution methods known in the art.

[0082]

[0074] Methods of controlling insects, in particular Lepidoptera, or Coleoptera, and / or Hemiptera infestations of crop plants, with the TIC3410, TIC4123, or TIC4124 protein, or a variant or fragment thereof, are disclosed in this application. Such methods can comprise growing a plant comprising an insect-, or Lepidoptera-, or Coleoptera-, or Hemiptera- inhibitory amount of a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein. In certain embodiments, such methods can further comprise any one or more of: (i) applying any composition comprising or encoding a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein to a plant or a seed that gives rise to a plant; and (ii) transforming a plant or a plant cell that gives rise to a plant with a polynucleotide encoding a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein. In general, it is contemplated that a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein can be provided in a composition, provided in a microorganism, or provided in a transgenic plant to confer insect inhibitory activity against Lepidopteran, Coleopteran, and / or Hemipteran insect(s).

[0083]

[0075] In certain embodiments, a recombinant nucleic acid molecule encoding a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein, or a variant or fragment thereof, is the insecticidally active ingredient of an insect inhibitory composition prepared by culturing recombinant Bacillus or any other recombinant bacterial cell transformed to express a TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein, or a variant or fragment thereof, under conditions suitable to express the TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein, or a variant or fragment thereof. Such a composition can be prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of such recombinant cells expressing / producing said recombinant polypeptide. Such a process can result in a Bacillus or other entomopathogenic bacterial cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet. By obtaining the recombinant polypeptides so produced, a composition that includes the recombinant polypeptides can include bacterial cells, bacterial spores, and parasporal inclusion bodies and can be formulated for various uses, including as agricultural insect inhibitory spray products or as insect inhibitory formulations in diet bioassays.

[0084]

[0076] In one embodiment, to reduce the likelihood of resistance development, an insect inhibitory composition comprising a TIC3410, TIC4123, or TIC4124 protein or related insecticidal protein, or a variant or fragment thereof, can further comprise at least one additional polypeptide that exhibits insect inhibitory activity against the same Lepidopteran, Coleopteran, and / or Hemipteran insect species, but which is different from the TIC3410, TIC4123, or TIC4124 toxin protein or related insecticidal protein. Possible additional insecticidal agents for such a composition include an insect inhibitory protein and an insect inhibitory dsRNA molecule. One example for the use of such ribonucleotide sequences to control insect pests is described in Baum, et al. (U.S. Patent Publication 2006 / 0021087 Al). Such additional polypeptide for the control of Lepidopteran pests may be selected from the group consisting of an insect inhibitory protein, such as, but not limited to, CrylA (U.S. Patent No. 5,880,275), CrylAb, CrylAc, CrylA.105, CrylAe, CrylB (U.S. Patent Publication No. 10 / 525,318), CrylC (U.S. Patent No. 6,033,874), CrylD, CrylDa and variants thereof, CrylE, CrylF, and CrylA / F chimeras (U.S. Patent Nos. 7,070,982; 6,962,705; and 6,713,063), CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL, Cryl-type chimeras such as, but not limited to, TIC836, TIC860, TIC867, TIC869, and TIC 1100 (International Application Publication WO20 16 / 061391), TIC2160 (International Application Publication WO2016 / 061392(A2)), Cry2A, Cry2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry43A, Cry43B, Cry51Aal, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (U.S. Patent Publication 2013-0117884 Al), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI- 112, AXMI-117, AXMI-100 (U.S. Patent Publication 2013-0310543 Al), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication 2013-0104259 Al), AXMI-134 (U.S. Patent Publication 2013-0167264 Al), AXMI-150 (U.S. Patent Publication 2010-0160231 Al), AXMI-184 (U.S. Patent Publication 2010-0004176 Al), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (U.S. Patent Publication 2011-0030096 Al), AXMI-218, AXMI-220 (U.S. Patent Publication 2014- 0245491 Al), AXMI-221Z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175 Al), AXMI-238 (U.S. Patent Publication 2014-0033363 Al), AXML 270 (U.S. Patent Publication 2014-0223598 Al), AXMI-345 (U.S. Patent Publication 2014- 0373195 Al), AXMI-335 (International Application Publication WO2013 / 134523(A2)), DIG-3 (U.S. Patent Publication 2013-0219570 Al), DIG-5 (U.S. Patent Publication 2010-0317569 Al), DIG-11 (U.S. Patent Publication 2010-0319093 Al), AfIP-lA and derivatives thereof (U.S. Patent Publication 2014-0033361 Al), AfIP-lB and derivatives thereof (U.S. Patent Publication 2014- 0033361 Al), PIP-1APIP-1B (U.S. Patent Publication 2014-0007292 Al), PSEEN3174 (U.S. Patent Publication 2014-0007292 Al), AECFG-592740 (U.S. Patent Publication 2014-0007292 Al), Pput_1063 (U.S. Patent Publication 2014-0007292 Al), DIG-657 (International Application Publication WO2015 / 195594 A2), Pput_1064 (U.S. Patent Publication 2014-0007292 Al), GS- 135 and derivatives thereof (U.S. Patent Publication 2012-0233726 Al), GS153 and derivatives thereof (U.S. Patent Publication 2012-0192310 Al), GS154 and derivatives thereof (U.S. Patent Publication 2012-0192310 Al), GS155 and derivatives thereof (U.S. Patent Publication 2012- 0192310 Al), SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2012-0167259 Al, 2SEQ ID NO:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2012-0047606 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2011-0154536 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2011-0112013 Al, SEQ ID NOs:2 or 4 and 4 and derivatives thereof as described in U.S. Patent Publication 2010-0192256 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0077507 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0077508 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2009-0313721 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0269221 Al, SEQ ID NOs:2 or 4 and derivatives thereof as described in U.S. Patent No. 7,772,465 (B2), CF161_0085 and derivatives thereof as described in WO2014 / 008054 A2, Lepidopteran toxic proteins and their derivatives as described in US Patent Publications US2008-0172762 Al, US2011-0055968 Al, and US2012-0117690 Al; SEQ ID NOs:2 or 4 and derivatives thereof as described in US7510878(B2), SEQ ID NOs;2 or 4 and derivatives thereof as described in U.S. Patent No. 7812129(B1), TIC6757 (U.S. Patent Publication 2017-058294 Al), TIC7941 (U.S. Patent Publication 2022-248686) Al, TIC2199 (U.S. Patent Publication 2023-013686 Al), TIC4064 (U.S. Patent Publication 2022-192200 Al), TIC4029 (U.S. Patent Publication 2022-256863 Al), TIC13O85 and TIC13087 (U.S. Patent Publication 2022-220160 Al), IPD103 and homologs thereof (International Application Publication WO2018 / 005411 ), PIP-50 and PIP-65 and homologs thereof (International Application Publication W02015 / 120270), PIP-83 and homologs thereof (U.S. Patent Publication 2016-0347799 Al), and Cry IB.34 (U.S. Patent Publication 2017- 0226164 Al); and the like.

[0085]

[0077] In some embodiments, such composition / formulation can further comprise at least one additional insecticidal agent that exhibits insect inhibitory activity to an insect that is not inhibited by an otherwise insect inhibitory protein of the present invention to expand the spectrum of insect inhibition obtained. For example, for the control of Hemipteran pests, combinations of insect inhibitory proteins of the present invention can be used with Hemipteran- active proteins such as TIC1415 (US Patent Publication 2013-0097735 Al), TIC807 (U.S. Patent No. 8609936), TIC834 (U.S. Patent Publication 2013-0269060 Al), AXMI-036 (U.S. Patent Publication 2010-0137216 Al), and AXMI-171 (U.S. Patent Publication 2013-0055469 Al). Further a polypeptide for the control of Coleopteran pests may be selected from the group consisting of an insect inhibitory protein, such as, but not limited to, Cry3Bb (U.S. Patent No. 6,501,009), CrylC variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, AXMI134 (U.S. Patent Publication 2013-0167264 Al) AXMI-184 (U.S. Patent Publication 2010-0004176 Al), AXMI-205 (U.S. Patent Publication 2014-0298538 Al), AXMI-207 (U.S. Patent Publication 2013-0303440 Al), AXMI-218, AXMI- 220 (U.S. Patent Publication 20140245491A1), AXMI-221z, AXMI-223z (U.S. Patent Publication 2014-0196175 Al), AXMI-279 (U.S. Patent Publication 2014-0223599 Al), AXMI-R1 and variants thereof (U.S. Patent Publication 2010-0197592 Al, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication 2010-0319092 Al), eHIPs (U.S. Patent Application Publication No. 2010 / 0017914), IP3 and variants thereof (U.S. Patent Publication 2012-0210462 Al), TIC5290 (U.S. Patent Publication 2017-0044568), TIC3668, TIC3669, and TIC3670 (U.S. Patent Publication 2016-0319302 Al), IPD072Aa and variants thereof (U.S. Patent Publication 2016-0366891), IPD079Ea and variants thereof (U.S. Patent Publication 2018-0222947), and OT-Hcxatoxin-Hv l a (U.S. Patent Application Publication 2014- 0366227 Al).

[0086]

[0078] Additional insecticidal agents for the control of Coleopteran, Lepidopteran, and Hemipteran insect pests, which can be combined with the insect inhibitory proteins of the TIC3410, TIC4123, or TIC4124 protein toxin class, can be found on the Bacillus thuringiensis toxin nomenclature website maintained by Neil Crickmore (on the world wide web at btnomenclature.info). Broadly, it is contemplated that any insect inhibitory agent or protein known to those of ordinary skill in the art can be used in combination with the proteins of the TIC3410, TIC4123, or TIC4124 protein toxin class both in planta (combined through breeding or molecular stacking) or in a composition or formulation as a biopesticide or combination of biopesticides, provided however that the insect inhibitory agent or protein for use with the insecticidal protein of the TIC3410, TIC4123, or TIC4124 protein toxin class in a plant does not compete with the insecticidal protein of the TIC3410, TIC4123, or TIC4124 protein toxin class in its mode of action against target pests and that the protein is not detrimental to the health and desirable properties of the transgenic plant and is expressed itself, at levels pesticidal to the intended target pest.

[0087]

[0079] The possibility for insects to develop resistance to certain insecticides has been documented in the ail. One insect resistance management strategy is to employ transgenic crops that express two distinct insect inhibitory agents that operate through different modes of action. Therefore, any insects with resistance to either one of the insect inhibitory agents can be controlled by the other insect inhibitory agent. Another insect resistance management strategy employs the use of plants that are not protected to the targeted Lepidopteran, Coleopteran, or Hemipteran pest species to provide a refuge for such unprotected plants. One particular example is described in U.S. Patent No. 6,551,962.

[0088]

[0080] Other embodiments such as topically applied pesticidal chemistries that are designed for controlling pests that are also controlled by the insecticidal proteins disclosed herein can be used in seed treatments, spray on, drip on, or wipe on formulations and applied directly to the soil (a soil drench), applied to growing plants expressing the proteins disclosed herein, or formulated to be applied to seed containing one or more transgenes encoding one or more of the proteins disclosed. Such formulations for use in seed treatments can be applied with various stickers and tackifiers known in the art. Such formulations can contain pesticides that are synergistic in mode of action with the proteins disclosed, so that the formulation pesticides act through a different mode of action to control the same or similar pests that can be controlled by the proteins disclosed, or that such pesticides act to control pests within a broader host range or plant pest species that are not effectively controlled by the TIC3410, TIC4123, or TIC4124 pesticidal protein or related protein, or a variant or fragment thereof.

[0089]

[0081] The aforementioned composition / formulation can further comprise an agriculturally acceptable carrier, such as a bait, a powder, dust, pellet, granule, spray, emulsion, a colloidal suspension, an aqueous solution, a Bacillus spore / crystal preparation, a seed treatment, a recombinant plant cell, plant tissue, plant seed, plant part or plant transformed to express one or more of the insecticidal proteins described herein, or a bacterium transformed to express one or more of the insecticidal proteins described herein. Depending on the level of insect inhibitory or insecticidal inhibition with a recombinant polypeptide described herein and the level of formulation to be applied to a plant or diet assay, the composition / formulation can include various by weight amounts of the recombinant polypeptide, e.g., from 0.0001% to 0.001% to 0.01% to 1% to 99% by weight of the recombinant polypeptide.

[0090]

[0082] In view of the foregoing, those of skill in the art should appreciate that changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Thus, specific structural and functional details disclosed herein are not to be interpreted as limiting.

[0091] EXAMPLES

[0092] Example 1

[0093] Discovery, cloning, expression, and purification of TIC3410, TIC4123, and TIC4124 Coding Sequences and the Encoded Toxin Protein

[0094]

[0083] TIC3410 pesticidal protein was identified through sequence analysis of the genome of the species Bacillus thuringiensis EG10168. A culture of the microbe provided DNA which was then subjected to sequence analysis, and the sequences obtained were assembled and analyzed to identify open reading frames. The TIC3410 open reading frame set forth in SEQ ID NO:1 from nucleotide position 1 through nucleotide position 1017, including the termination codon following the complete coding sequence, was identified as belonging to the Cry46Aa protein class. The closest identity match to the amino acid sequence of TIC3410 has been disclosed in GenBank Accession BAC79010 which is 100% identical to TIC3410. This sequence was demonstrated in U.S. Patent Application Publication US2014 / 0283208 to have activity against the Lepidopteran insect pest species Black cutworm (Agrotis ipsilo ), Com earworm (Helicoverpa zea), European com borer (Ostrinia nubilalis). Fall armyworm (Spodoptera frugiperda), Southern armyworm (Spodoptera eridaniap Soybean looper (Chrysodeixis includens); the Coleopteran pest species Western Com Rootworm (Diabrotica virgiferd) and the Hemipteran pest species Lygus hesperus and Lygus elisus.

[0095]

[0084] TIC4123 pesticidal protein was identified through sequence analysis of the genome of the species Bacillus thuringiensis CFB213052. A culture of the microbe provided DNA which was then subjected to sequence analysis, and the sequences obtained were assembled and analyzed to identify open reading frames. The TIC4123 open reading frame set forth in SEQ ID NO:4 from nucleotide position 1 through nucleotide position 915, including the termination codon following the complete coding sequence, was identified as belonging to the Cry46Ab protein class. The closest identity match to the amino acid sequence of TIC4123 has been disclosed in the International Application Publication, WO2023 / 107943 and is 100% identical to SEQ ID NO: 2 of WO2023 / 107943. This toxin sequence was found to have activity against the Lepidopteran pest species Black armyworm (Spodoptera cosmioides). Black cutworm (Agrotis ipsilon), Com earworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), Fall army worm (Spodoptera frugiperda). Soybean looper (Chrysodeixis includens). Southwestern com borer (Diatraea gr audio sella), Sugarcane borer (Diatraea saccharalis), Tobacco budworm (Heliothis virescens), and Velvet bean caterpillar (Anticarsia gemmatalis)', and the Coleopteran pest species Western Com Rootworm (WCR, Diabrotica virgifera) and Southern Com Rootworm (Diabrotica undecimpunctata howardii).

[0096]

[0085] TIC4124 pesticidal protein was identified through sequence analysis of the genome of the species Bacillus thuringiensis EG5018. A culture of the microbe provided DNA which was then subjected to sequence analysis, and the sequences obtained were assembled and analyzed to identify open reading frames. The TIC4124 open reading frame set forth in SEQ ID NO:7 from nucleotide position 1 through nucleotide position 915, including the termination codon following the complete coding sequence, was identified as belonging to the Cry46Ab protein class. The closest identity match to the amino acid sequence of TIC4124 has been disclosed in GenBank Accession BAD35170 and is 99.34% identical to TIC4124. There is no evidence that the protein described in GenBank Accession BAD35170 has been assayed for activity against insects.

[0097]

[0086] Polymerase chain reaction (PCR) primers were designed to amplify full-length copies of the nucleotide sequences encoding TIC3410, TIC4123, and TIC4124 from total genomic DNA isolated from the Sp strains, EG10168, CFB213052, and EG5018, respectively to confirm the assembly of sequenced segments were in fact representative of the naturally occurring sequence. Amplicons were cloned using methods known in the art into a Bacillus thuringiensis (Bf) expression vector in operable linkage with a Bt expressible promoter. Preparations of purified TIC3 10, TIC 123, and TIC 124 protein derived from the vectors expressing the toxin proteins in Bt were used in bioassay against various insect pest species.

[0098] Example 2

[0099] TIC3410, TIC4123, and TIC4124 demonstrate Lepidopteran, Coleopteran, and Hemipteran activity in insect bioassay.

[0100]

[0087] The TIC3410, TIC4123, and TIC4124 protein open reading frames initially assembled were confirmed by comparison with the sequence of thermal amplified clones, and both encoded the same amino acid sequence set forth in SEQ ID NO: 3, 6, and 9. TIC3410, TIC4123, and TIC4124 expressed in recombinant Bt using the vectors described in Example 1 were assayed for toxicity to various species of Lepidoptera, Coleoptera, Hemiptera, and Diptera.

[0101]

[0088] TIC3410, TIC4123, and TIC4124 were assayed for toxicity to the Lepidopteran insect species Black cutworm (BCW, Agrotis ipsilon), Com earworm (CEW, Helicoverpa zea, also known as Soybean podworm), European corn borer (ECB, Ostrinia nubilalis). Fall armyworm (FAW, Spodoptera frugiperda), Soybean looper (SBL, Chrysodeixis includens), Southwestern corn borer (SWC, Diatraea grandiosella), and Tobacco budworm (TBW, Heliothis virescens): the Coleopteran species Western Com Rootworm (WCR, Diabrotica virgifera) and Colorado potato beetle (CPD, Leptinotarsa decemlineata); and the Hemipteran species Western tarnished plant bug (WTP, Lygus hesperus), Tarnished plant bug (TPB, Lygus lineolaris). Neotropical Brown Stink Bug (NBSB, Euschistus hems), and Southern Green Stinkbug (SGB, Nezara viridida). The bioassay results are presented in Tables 3 and 4 below wherein “+++” indicates high stunting and / or high mortality, “++” indicates stunting and mortality, “+” indicates stunting, indicates no activity, and NT indicates not tested. Table 3. Activity of TIC3410, TIC4123, and TIC4124 against Lepidopteran insect species.

[0102] Table 4. Activity of TIC3410, T1C4123, and T1C4124 against Coleopteran and Hemipteran insect species.

[0103]

[0089] As can be seen in the data presented in Tables 3 and 4, the three proteins, TIC3410, T1C4123, and T1C4124, demonstrated activity against insect pests in the orders of Lepidoptera, Coleoptera, and Hemiptera in insect bioassay. TIC3410 demonstrated activity against the Lepidopteran species BCW, ECB, SWC and TBW; the Coleopteran species CPB, and the Hemipteran species WTP and NBSB. TIC4123 demonstrated activity against the Lepidopteran species BCW, CEW, ECB, FAW, SBL, SWC and TBW; the Coleopteran species WCR and CPB; and the Hemipteran species TPB. TIC4124 demonstrated activity against the Lepidoptera species BCW, CEW, ECB, FAW, SBL, SWC, and TBW; the Coleopteran species WCR and CPB; and the Hemipteran species TPB, NBSB, and SGB. TIC3410, TIC4123, and TIC4124 demonstrated broad activity against all three orders assayed.

[0104] Example 3 Design of artificial coding sequences for expression of TIC3410, TIC4123, and TIC4124 in plants.

[0105]

[0090] Artificial coding sequences, SEQ ID NOs: 2, 5, and 8 encoding TIC3410, TIC4123, and TIC4124, were designed for expression in a plant cell. The artificial (alternatively referred to as synthetic) sequence was synthesized, according to methods generally described in U.S. Patent 5,500,365, to avoid certain inimical problem sequences such as ATTTA and A / T rich plant poly adenylation sequences, while substantially preserving the amino acid sequence of the native Bacillus protein.

[0106]

[0091] The artificial sequences (SEQ ID NOs: 2, 5, and 8) encoding TIC3410, TIC4123, and TIC4124 (SEQ ID NOs: 3, 6, and 9, respectively) are cloned plant transformation vectors and downstream of, and in each case, functionally linked to, a plant promoter for driving expression of the coding sequence when in a plant cell, using skills known in the ail. The resulting transformation vectors are used to transform monocot and / or dicot plant cells and comprises a first transgene cassette for expression of the TIC3410, TIC4123, or TIC4124 pesticidal protein which comprises a constitutive promoter, operably linked 5 ' to a leader, optionally operably linked 5 ' to an intron, operably linked 5' to an artificial coding sequence encoding TIC3410, TIC4123, or TIC4124, which is in turn operably linked 5' to a 3' UTR, and a second transgene cassette for the selection of transformed plant cells using a gene that confers tolerance to an herbicide or resistance to an antibiotic.

[0107] Example 4

[0108] Bioassay of TIC3410, TIC4123, and TIC4124 activity against Whitefly (Bemisia tabaci).

[0109]

[0092] The pesticidal toxins TIC3410, TIC4123, and TIC4124 are assayed for activity against Whitefly (Bemisia tabaci). To assess the toxicity of the pesticidal toxins TIC3410, TIC4123, and TIC4124 to adult Whitefly, a simple bioassay system is used similar to that described by Vyas et al. (Vyas, M., Raza, A., Ali, M.Y., Ashraf, M.A., Mansoor, S., Shahid, A.a., and Brown, J.K. (2017) Knock down of Whitefuly Gut Gene Expression and Mortaility by Orally Delivered Gut Gene- Specific dsRNAs PLOS ONE, DOI:10.1371 / journal.pone.0168921). Adult whiteflies are collected from host plants using a hand-held aspirator and transferred to a glass vial covered with Parafilm™. Preparations of the bacterial expressed pesticidal toxins TIC3410, TIC4123, and TIC4124 in buffer are made in varying concentrations. Each pesticidal toxin preparation in buffer is added to 1 ml sterile 20% sucrose solution and sandwiched between two sterile layers of Parafilm™. Whiteflies are allowed to feed on the sucrose solution with the pesticidal toxin for six days. A negative experimental control consisting of 1 ml of 20% sucrose solution with an equivalent amount of buffer solution as used for the toxin is provided to assess the effects of the toxins. Observations are made at least once a day for the six-day period to assess the percentage of dead whiteflies and compared to the negative control. Replicate experiments are performed to provide reliable results.

[0110] Example 5

[0111] Assay of TIC3410, TIC4123, and TIC4124 activity against Whitefly (Bemisia t abaci) in stably transformed plants.

[0112]

[0093] The pesticidal toxins TIC3410, TIC4123, and TIC4124 are assayed for activity against Whitefly (Bemisia tabaci) using leaves of a plant transformed to express TIC3410, TIC4123, or TIC4124, or an entire plant transformed to express TIC3410, TIC4123, or TIC4124. All stages of whiteflies feed on plant sap, using their piercing- sucking mouthparts. The whitefly has a complex life cycle. It undergoes five distinct stages of development. Eggs are laid on the abaxial surface of leaves, and are at first pale yellow, but turn gray before hatching in 5 to 7 days. The immature stages (eggs, crawlers, scales, and pupae) are all yellowish and found primarily on the abaxial surface of leaves. The crawler is a small, translucent, mobile stage that actively searches for a feeding site. Within a few days, crawlers settle down and begin feeding, soon transforming to the sedentary scale stage. The scale is a highly modified sucking insect, and its out covering thickens after it feeds, giving it added protection. Adult development (pupation) occurs within the scale cover. Four days later, adults emerge. The life cycle takes about 40 days, depending upon temperature. Transformed plant assays provide a means to assay toxicity of insecticidal proteins that may have varying levels of toxicity to the whitefly, depending upon its developmental stages.

[0094] Monocot or dicot plant cells are transformed using an groZzacferzum-mediated transformation system using plasmid transformation vectors similar to those described in Example 3 which comprise an expression cassette for the expression of the pesticidal proteins TIC3410, TIC4123, or TIC4124; and an expression cassette used for selection of transformed plant cells using a selectable marker that provides tolerance to an herbicide or resistance to an antibiotic.

[0113]

[0095] Plant cells are transformed with transformation vectors used for the expression of TIC3410, TIC4123, and TIC4124 and induced to form whole plants. Whiteflies are collected from the field and raised in an insectary using a suitable host plant. Single-copy transformed events are selected for assay against whitefly. Each selected transformed event is grown in an individual pot in whitefly-proof cages consisting of large and transparent cylindrical cages (with 30 cm height and 20 cm diameter) with an organdy sleeve. Organdy is a fine translucent cotton or silk fabric typically used for clothing which permit the free exchange of air but prevents the escape of the adult whiteflies. When the transformed events have grown to a suitable growth stage of 2 or 4 leaves, the plants are infested with 150 or 200 young adult whiteflies with a sex ratio of 1:1. The adults are allowed to mate and oviposit on the leaves for 24 hours. Then, the adults are removed and the plants with whitefly eggs are transferred to other insect-free cages. After sufficient amount of time to allow for the development of whiteflies to adults, approximately 40 to 50 days, the percentage of dead nymphs and adults are determined and compared to non-transgenic control plants treated in the same manner as the transgenic events. Whitefly nymphs will appear as a color change from yellowish to dark brown or if the body appears dry. Variations of such an assay method can be designed to assess toxicity to the younger stages of development such as egg hatching and early nymphal stages by reducing the amount of time before observations are made prior to adult emergence.

[0114]

[0096] All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions of this invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those of skill in the art that variations, changes, modifications, and alterations may be applied to the composition described herein, without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the ait are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0115]

[0097] All publications and published patent documents cited in the specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS1. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a pesticidal protein, or a pesticidal fragment thereof, wherein: a. said pesticidal protein comprises the amino acid sequence of SEQ ID NO: 3, 6, or 9; b. said pesticidal protein comprises an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9; and / or c. said polynucleotide segment comprising SEQ ID NO: 1, 2, 4, 5, 7, or 8, or comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 2, 4, 5, 7, or 8, or comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, or at least 2000 contiguous nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, or 8, or hybridizing under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, or 8 or a complement thereof.

2. The recombinant nucleic acid molecule of claim 1, wherein: a. said recombinant nucleic acid molecule is expressed in a plant cell to produce a pesticidally effective amount of the pesticidal protein or pesticidal fragment; or b. said recombinant nucleic acid molecule is in operable linkage with a vector, and said vector is selected from the group consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial or yeast artificial chromosome.

3. The recombinant nucleic acid molecule of claim 1, present within a host cell, wherein said host cell is selected from the group consisting of a bacterial cell and a plant cell.

4. The recombinant nucleic acid molecule of claim 3, wherein said bacterial host cell is from a genus of bacteria selected from the group consisting of: Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia.

5. The recombinant nucleic acid molecule of claim 4, wherein said Bacillus is Bacillus cereus or Bacillus thuringiensis , said Brevibacillus is a Brevibacillus laterosperous, and said Escherichia is an Escherichia coli.

6. The recombinant nucleic acid of any one of claims 2-5, wherein said host or plant cell is a dicotyledonous or a monocotyledonous plant cell.

7. The recombinant nucleic acid of claim 6, wherein said plant cell is selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, cowpea, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell.

8. The recombinant nucleic acid molecule of any one of claims 1-7, wherein said protein exhibits activity against a Lepidopteran, Coleopteran, or Hemipteran insect.

9. The recombinant nucleic acid molecule of claim 8, wherein said Lepidopteran insect is selected from the group consisting of: Black cutworm (Agrotis ipsilon). Corn earworm (Helicoverpa z.ea). European com borer (Oslrinia nubilalis). Fall armyworm (Spodoptera frugiperda), Soybean looper (Chrysodeixis includens), Southwestern com borer (Diatraea grandio sella), and Tobacco budworm (Heliothis virescens).

10. The recombinant nucleic acid molecule of claim 8, wherein said Coleopteran insect is selected from the group consisting of: Western Corn Rootworm Diabrotica virgifera) and Colorado potato beetle (Leptinotarsa decemlineata, CPB).

11. The recombinant nucleic acid molecule of claim 8, wherein said Hemipteran insect is selected from the group consisting of: Whitefly (Bemisia labaci), Western tarnished plant bug (Lygus hesperus), Tarnished plant bug (Eygus lineolaris), Neotropical Brown Stink Bug (Euschistus heros), and Southern Green Stink Bug (N ezara viridula).

12. A plant or a plant part thereof comprising the recombinant nucleic acid molecule of any one of claims 1-11.

13. The plant of claim 12, wherein said plant is a monocot plant or a dicot plant.

14. The plant of claim 12, wherein the plant is selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, cowpea, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

15. The plant part of claim 12, wherein the plant part is a seed, and wherein said seed comprises said recombinant nucleic acid molecule.

16. An insect inhibitory composition comprising the recombinant nucleic acid molecule of any one of claims 1-11.

17. The insect inhibitory composition of claim 16, further comprising a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein.

18. The insect inhibitory composition of claim 17, wherein said at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, a chemical molecule and an ancillary protein, wherein said at least one other pesticidal agent is toxic to the same pest as the pesticidal protein or pesticidal fragment thereof.

19. The insect inhibitory composition of claim 17, wherein said at least one other pesticidal agent exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, or Hemiptera.

20. The insect inhibitory composition of claim 17, wherein said at least one other pesticidal agent is selected from the group consisting of a CrylA, CrylAb, CrylAc, CrylA.105, CrylAe, Cry IB, CrylC, CrylC variants, Cry ID, Cry ID variants, Cry IE, Cry IF, CrylA / F chimeras, CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry34, Cry35, Cry43A, Cry43B, Cry51Aal, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415,TIC2160, TIC3131 , TIC836, TTC86O, TIC867, TTC869, TIC 1 100, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI- 115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI- 196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI- 22 Iz, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IPD102Aa and homologs thereof, IPDl lOAa and homologs thereof, TIC868, CrylDal_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757. TIC7941, TIC5290, TIC3668, TIC3669, TIC3670, TIC2199, TIC4064, TIC4029, TIC13085, TIC13087, IPD072Aa, IPD079Ea, and IPD103 and homologs thereof, PIP-50 and PIP-65 and homologs thereof, PIP-83 and homologs thereof, and CrylB.34.

21. The insect inhibitory composition of claim 16, defined as comprising a plant cell that expresses the pesticidal protein from the recombinant nucleic acid molecule of claim 1.

22. A commodity product produced from the plant, or plant part thereof, of any one of claims 12-15, wherein the commodity product comprises a detectable amount of said recombinant nucleic acid molecule and / or said pesticidal protein or a pesticidal fragment thereof.

23. The commodity product of claim 22, selected from the group consisting of commodity corn bagged by a grain handler, com flakes, corn cakes, com flour, corn meal, com syrup, com oil, corn silage, com starch, com cereal, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fmit, melon, and vegetable commodity products including, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such commodity products containing a detectable amount of such polynucleotides and or polypeptides of this application, whole or processed cotton seed, cotton oil, lint, seeds and plant pails processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean Hour, soybean Hakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts,cowpea seed, cowpea oil, cowpea protein, cowpea meal, cowpea flour, animal feed comprising cowpea, cowpea forage, cassava flour, cassava chips, cassava starch, and Garri.

24. A method of producing progeny seed comprising the recombinant nucleic acid molecule of any one of claims 1-11, the method comprising: a. planting a first seed comprising the recombinant nucleic acid molecule; b. growing a plant from the seed of step a; and c. harvesting the progeny seed from the plants, wherein said harvested seed comprises said recombinant nucleic acid molecule.

25. A plant resistant to insect infestation, wherein the cells of said plant comprise the recombinant nucleic acid molecule of any one of claims 1-11.

26. A method for controlling a Lepidopteran, Coleopteran, or Hemipteran species pest or pest infestation, said method comprising: a. contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO: 3, 6, or 9; or b. contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9.

27. A method of detecting the presence of the recombinant nucleic acid molecule of claim 1 in a sample comprising plant genomic DNA, comprising: a. contacting said sample with a nucleic acid probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant comprising the recombinant nucleic acid molecule of claim 1, and does not hybridize under such hybridization conditions with genomic DNA from an otherwise isogenic plant that does not comprise the recombinant nucleic acid molecule of claim 1, wherein said probe is homologous or complementary or hybridizes under stringent conditions to SEQ ID NOs: 1, 2, 4, 5, 7 or 8; or a sequence that encodes a pesticidal protein comprising an amino acid sequence as set forth in SEQ ID NO: 3, 6, or 9; or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9; b. subjecting said sample and said probe to stringent hybridization conditions; andc. detecting hybridization of said nucleic acid probe with said recombinant nucleic acid molecule.

28. A method of detecting the presence of a pesticidal protein, or a fragment thereof, in a sample comprising protein, wherein said pesticidal protein comprises the amino acid sequence of SEQ ID NO: 3, 6, or 9; or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9, comprising: a. contacting said sample with an immunoreactive antibody; and b. detecting the presence of said pesticidal protein, or fragment thereof.

29. The method of claim 28, wherein the step of detecting comprises an ELISA or a Western blot analysis.

30. A pesticidal protein comprising the amino acid sequence of SEQ ID NO: 3, 6, or 9, or an amino acid sequence having at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or about 100% amino acid sequence identity to SEQ ID NO: 9.

31. A composition comprising a pesticidally effective amount of the pesticidal protein of claim 30.

32. A method for controlling a Lepidopteran, Coleopteran, or Hemipteran pest species or pest infestation in a field, said method comprising: a. growing a crop plant which expresses an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO: 3, 6, or 9; or b. growing a crop plant which expresses an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 99.4%, at least 99.7%, or about 100% amino acid sequence identity to SEQ ID NO: 9; and optionally c. releasing into said field transgenic Lepidopteran, Coleopteran, or Hemipteran pest species carrying a self-limiting gene to reduce the likelihood of development of resistance of the pest species to the pesticidal protein.

33. The method of claim 32, wherein said crop plant is a monocoty ledonous or dicotyledonous crop plant.

34. The method of claim 33, wherein the monocotyledonous crop plant is com, wheat, sorghum, rice, rye, sugarcane, or millet.

35. The method of claim 34, wherein the monocotyledonous crop plant is com.

36. The method of claim 33, wherein the dicotyledonous crop plant is soybean, cotton, alfalfa, cowpea, or canola.

37. A microorganism comprising the recombinant nucleic acid molecule of any one of claims 1-11 and / or the pesticidal protein of claim 30.

38. The microorganism of claim 37, wherein the microorganism is a bacterial cell, a fungal cell or a plant cell.

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