Novel insect-inhibitory protein

Abstract

Insecticidal proteins are disclosed that exhibit toxic activity against lepidopteran and hemipteran pest species, including but not limited to TIC2199. DNA constructs are provided that contain recombinant nucleic acid sequences encoding the insecticidal proteins of the present disclosure. Transgenic plants, plant cells, seeds, and plant parts that are resistant to lepidopteran and hemipteran infestations are provided that contain recombinant nucleic acid sequences encoding the insecticidal proteins of the present invention. Methods for detecting the presence of the recombinant nucleic acid sequences or proteins of the present invention in biological samples and methods for using the TIC2199 insecticidal protein to combat lepidopteran and hemipteran pest species are also provided.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 219,604, filed July 8, 2021, and U.S. Provisional Application No. 63 / 348,278, filed June 2, 2022, all of which are incorporated by reference in their entireties herein.

[0002] Incorporation of sequence listing A file named "MONS530WO.txt" containing a computer readable form of the Sequence Listing was created on June 13, 2022. This file is 72,115 bytes (measured in MS-Windows), was filed simultaneously by electronic filing (using the U.S. Patent Office EFS-Web Filing System), and is incorporated by reference in its entirety.

[0003] The present invention relates generally to the field of insect inhibitor proteins. A novel class of toxin proteins is disclosed that exhibits insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly against insect pests of the Lepidopteran order. Plants, plant parts, seeds, cells, including plant and microbial cells, and vectors are provided that contain recombinant polynucleotide constructs encoding one or more of the disclosed toxin proteins. [Background technology]

[0004] It is becoming increasingly important to improve crop yields from agriculturally important plants, including maize, soybean, sugarcane, rice, wheat, cotton, vegetables, pearl millet, pigeon pea, peanut, potato, barley, oats, and fruit trees, among others. The amount of arable land available for cultivation is projected to decrease due to climate-related impacts and pressure from a growing population to use land for non-agricultural purposes, in addition to the growing need for agricultural products to provide food, clothing, and energy to a growing population. These factors have led to grim predictions for food security, especially in the absence of significant improvements in plant biotechnology and agricultural practices. In light of these factors, environmentally sustainable improvements in technology, agricultural techniques, and pest management are important tools to expand crop production where the amount of arable land available for cultivation is increasingly limited.

[0005] Insects, especially lepidopteran insects, are a major cause of damage to field crops, thereby reducing crop yields in infested areas. Lepidopteran pests that have a detrimental effect on agriculture include Spodoptera cosmioides, Agrotis ipsilon, Helicoverpa zea, Alabama argillacea, Plutella xylostella, Ostrinia nubilalis, Spodoptera frugiperda, Cry1Fa1-resistant Spodoptera frugiperda, Helicoverpa armigera, Spodoptera eridania, Chrysodeixis includens, Earias vittella, Diatraea arborescens, Spodoptera spp., Spodoptera nigricans ... grandiosella), sunflower looper (Rachiplusia nu), tobacco budworm (Heliothis virescens), common cutworm (Spodoptera litura, also known as cluster caterpillar), western bean cutworm (Striacosta albicosta), and velvet bean caterpillar (Anticarsia gemmatalis).

[0006] Historically, intensive application of synthetic chemical insecticides has been relied upon as pest control agents in agriculture. Environmental and human health concerns, along with emerging resistance problems and the fact that such pest control agents do not differentiate and target beneficial insects and other organisms, have stimulated research and development of biological insecticides specifically targeted to control pests that cause crop losses. This research effort has led to the gradual discovery and use of various insect pathogenic microbial species, including bacteria.

[0007] The discovery of the potential of entomopathogenic bacteria, particularly those belonging to the genus Bacillus, and their development as biological pest control agents has shifted the paradigm of biological control. Strains of the bacterium Bacillus thuringiensis (Bt) have been used as a source of insecticidal proteins since it was discovered that Bt strains are highly toxic to certain insects. Bt strains are known to produce delta-endotoxins (e.g., Cry proteins) that are localized within parasporal crystalline inclusions at the onset of sporulation and during stationary growth phase, and are also known to produce secreted insecticidal proteins. Upon ingestion by susceptible insects, the delta-endotoxins and secreted toxins exert their effects at the surface of the midgut epithelium, disrupting cell membranes and resulting in cell destruction and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including other Bacillus and a variety of additional bacterial species such as Brevibacillus laterosporus, Lysinibacillus sphaericus ("Ls" was previously known as Bacillus sphaericus), Pseudomonas species, Paenibacillus popilliae, and Paenibacillus lentimorbus. In addition, insecticidal toxins have been identified from a variety of non-bacterial sources, including fern and spider venom, and delivery of dsRNA in pest feed to target the silencing of essential genes has been identified as an effective pest management strategy.

[0008] Soluble insecticidal toxins, both crystalline and secreted, are highly specific to their hosts and have been accepted worldwide as alternatives to chemical pesticides. For example, insecticidal toxin proteins have been used in various agricultural applications to protect agriculturally important plants from insect infestations, reduce the need for chemical pesticide applications, and increase yields. Insecticidal toxin proteins are used to control agriculturally relevant pests of crop plants by mechanical methods such as spraying to distribute microbial preparations, including various bacterial strains, on plant surfaces, and by using recombinant genetic techniques to produce transgenic plants and seeds that express the insecticidal toxin protein(s).

[0009] The use of transgenic plants expressing insecticidal toxin proteins has become widespread worldwide. For example, in 2016, 23.1 million hectares were planted with transgenic crops expressing Bt toxins and 75.4 million hectares were planted with transgenic crops expressing Bt toxins with added herbicide resistance traits (ISAAA. 2016. Global Status of Commercialized Biotech / GM Crops: 2016. ISAAA Brief No. 52. ISAAA: Ithaca, NY). The global use of transgenic insect protection crops and the limited number of insecticidal toxin proteins used in these crops has created selection pressure for existing insect alleles that confer resistance to currently utilized insecticidal proteins.

[0010] The development of resistance in target pests to insecticidal toxin proteins creates a continuing need to discover and develop 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 showing control of a wider range of susceptible insect species will reduce the number of insects that can develop resistance alleles. In addition, the use of two or more transgenic insecticidal toxin proteins (e.g., transgenes encoding dsRNAs targeting essential genes for inhibition combined with transgenes encoding peptide or protein toxins toxic to the same insect species) in one plant that are toxic to the same insect pest and show different modes of action, or alternatively, two or more different modes of toxic action, reduces the probability of resistance in any single target insect species. In addition, the use of self-limiting technologies such as those provided by Oxitec Ltd, when used in conjunction with the proteins of the invention, will improve the durability of the traits conferred to transgenic crops expressing the proteins of the invention (Zhou et al. 2018. Combining the high-dose / refuge strategy and self-limiting transgenic insects in resistance management-a test in experimental mesocosms. Evol Appl 11(5):727-738; Alphey et al. 2009. Combining pest control and resistance management: synergy of engineered insects with Bt crops. Journal of Economic Entomology,102:717-732).

[0011] Accordingly, the inventors disclose herein novel proteins from Bacillus thuringiensis, as well as improved and engineered proteins that exhibit modified amino acid sequences compared to the native toxin, and exemplary recombinant proteins, each of which exhibit insecticidal activity against the following targets: Lepidoptera species, in particular the cutworm moth (Agrotis ipsilon), tobacco budworm (Helicoverpa zea), nettle looper (Trichoplusia ni), European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), southern armyworm (Spodoptera eridania), soybean looper (Chrysodeixis includens), southwestern corn borer (Diatraea grandiosella), velvet bean caterpillar (Anticarsia gemmatalis), and western bean cutworm (Striacosta albicosta, as well as the Hemipteran species Lygus lineolaris and Euschistus heros. Summary of the Invention

[0012] Disclosed herein is a novel insecticidal protein TIC2199, which has been shown to exhibit inhibitory activity against one or more pests of crop plants. The TIC2199 protein can be used alone or in combination with other insecticidal proteins and poisons in formulations and plants, thus providing an alternative to insecticidal proteins and insecticidal chemicals currently used in agricultural systems.

[0013] In one embodiment, the present application discloses a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding an insecticidal protein or an insecticidal fragment thereof, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17, or the insecticidal protein comprises an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17, or the polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 8, 9, 11, 12, 14, 16, or 18. The recombinant nucleic acid molecule functions to express the insecticidal protein in a plant and may comprise a sequence that, when expressed in a plant cell, produces a pesticidally effective amount of the insecticidal protein or an insecticidal fragment thereof.

[0014] In another embodiment of the present application, the recombinant nucleic acid molecule is present in a bacterial or plant host cell. Contemplated bacterial host cells include at least the genera 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 Brevibacillus laterosporus, or the Escherichia is Escherichia coli. Contemplated plant host cells include dicotyledonous and monocotyledonous plant cells. Further contemplated plant cells include those from alfalfa, banana, barley, bean, broccoli, cabbage, Brassica (e.g., canola), carrot, cassava, castor bean, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (Gossypium sp.), cucurbits, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, lettuce, loblolly pine, foxtail millet, melon, nuts, oats, olives, onions, ornamentals, palms, pastures, peas, peanuts, peppers, pigeon peas, pine, potatoes, poplars, pumpkins, radiata pine, radish, rapeseed, rice, rhizomes, rye, safflower, shrubs, sorghum, southern pine, soybeans, spinach, squash, strawberries, sugar beets, sugarcane, sunflowers, sweet corn, sweetgum, sweet potatoes, switchgrass, tea, tobacco, tomatoes, triticale, turfgrass, watermelon, and wheat.

[0015] In another embodiment, the insecticidal protein exhibits activity against lepidopteran insects including at least Agrotis ipsilon, Helicoverpa zea, Trichoplusia ni, Ostrinia nubilalis, Spodoptera frugiperda, Spodoptera eridania, Chrysodeixis includens, Diatraea grandiosella, Diatraea saccharalis, Anticarsia gemmatalis, Striacostra albicosta, and Elasmopalpus lignosellus.

[0016] In another embodiment, the insecticidal protein exhibits activity against hemipteran insects, including at least Lygus lineolaris and Euschistus heros.

[0017] The present application also contemplates bacteria and plants and plant parts comprising a recombinant nucleic acid molecule encoding the insecticidal protein TIC2199 or a fragment thereof. The recombinant molecule (e.g., construct) may include a heterologous promoter for expression in a bacterial or plant cell of an operably linked polynucleotide segment encoding an insecticidal protein. Both dicotyledonous and monocotyledonous plants are contemplated. In another embodiment, the plant is further selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, Brassica (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (e.g., Gossypium sp.), cucurbits, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, lettuce, loblolly pine, foxtail millet, melons, nuts, oats, olives, onions, ornamentals, palms, pasture grasses, peas, peanuts, peppers, pigeon peas, pine, potatoes, poplars, pumpkins, radiata pine, radish, rapeseed, rice, roots, rye, safflower, shrubs, sorghum, southern pine, soybeans, spinach, squash, strawberries, sugar beets, sugar cane, sunflowers, corn (i.e., maize) (e.g., sweet corn or field corn), maple, sweet potato, switchgrass, tea, tobacco, tomatoes, triticale, wheatgrass, watermelon, and wheat. Plant parts can include, for example, but are not limited to, leaves, tubers, roots, stems, seeds, embryos, flowers, inflorescences, pods, pollen, fruits, animal feed, and biomass. Treated plant parts, such as wood, or oil, non-viable ground or sorted seeds, flour, or starch produced from the leaves, flowers, roots, seeds, or tubers of the plant, that contain a nucleic acid encoding a protein of the invention and / or contain an insecticidally effective amount of the encoded toxin protein, are also contemplated.

[0018] In a particular embodiment, a seed comprising a recombinant nucleic acid molecule and an insecticidally effective amount of a TIC2199 toxin protein is disclosed.

[0019] In yet another embodiment, an insect inhibitor composition is contemplated that includes the recombinant nucleic acid molecule disclosed in the present application. The insect inhibitor composition may further include a nucleotide sequence encoding at least one other insecticide different from the insecticidal protein. In certain embodiments, the at least one other insecticide is selected from the group consisting of an insect inhibitor protein, an insect inhibitor dsRNA molecule, and an auxiliary protein. It is also contemplated that the at least one other insecticide in the insect inhibitor composition exhibits activity against one or more pest species of the order Lepidoptera, Coleoptera, or Hemiptera. The at least one other insecticide in the insect inhibitor composition, in one embodiment, is selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variant, Cry1D, Cry1E, Cry1F, Cry1A / F chimera, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variant, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and its variants, IP3 and its variants, DIG-3, DIG-5, DI G-10, DIG-657, DIG-11 protein, IDP102Aa and its homologs, IDP110Aa and its homologs, TIC868, Cry1Da1_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757, TIC7641, IDP072Aa, TIC5290, TIC3668, TIC3669, TIC3670, IDP072Aa and IDP103 and their homologs, PIP-50 and PIP-65 and their homologs, PIP-83 and its homologs, and Cry1B.34, and embodiments of dsRNA-mediated gene silencing include those that target silencing of Diabrotica species genes Dv snf7 and Dv ssj1.

[0020] Also contemplated are commercial products containing detectable amounts of the recombinant nucleic acid molecules and toxin proteins disclosed herein, including corn products (such as corn flakes, corn cake, corn flour, corn meal, corn syrup, corn oil, corn silage, corn starch, corn cereals, etc.) bagged by grain handlers, as well as corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable products, where applicable, juices, concentrates, jams, jellies, marmalades, and other products containing detectable amounts of such polynucleotides and / or polypeptides of the present application. Included are other edible forms, whole or processed cottonseed, cottonseed oil, lint, seeds, and plant parts processed for feed or food, fiber, paper, biomass, fuel products such as fuel derived from cotton oil or pellets derived from cotton ginning waste, whole or processed soybean seeds, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soy cheese, soybean wine, animal feeds containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass, and fuel products made using soybean plants and parts of soybean plants.

[0021] The present application also contemplates a method of producing seeds comprising a recombinant nucleic acid molecule and an insecticidally effective amount of the encoded TIC2199 toxin protein, the method comprising planting at least one seed comprising a recombinant nucleic acid molecule disclosed herein, growing a plant from the seed, and harvesting seeds from the plant, the harvested seeds comprising the referenced recombinant nucleic acid molecule and / or an insecticidally effective amount of the encoded TIC2199 toxin protein.

[0022] In another exemplary embodiment, a plant that is resistant to lepidopteran infestation is provided, wherein cells of the plant comprise a recombinant nucleic acid molecule disclosed herein.

[0023] The present application also discloses a method for controlling lepidopteran pests and for controlling the infestation of lepidopteran pests in plants, particularly crop plants. The method, in one embodiment, comprises first contacting the pest with an insecticidally effective amount of an insecticidal protein as set forth in SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17, or contacting the pest with an insecticidally effective amount of one or more insecticidal proteins comprising an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17.

[0024] Further provided herein is a method of detecting the presence of a recombinant nucleic acid molecule of the TIC2199 toxin protein class, comprising contacting a sample of nucleic acid with a nucleic acid probe that hybridizes under stringent hybridization conditions to genomic DNA from a plant comprising a polynucleotide segment encoding an insecticidal protein or fragment thereof provided herein, but does not hybridize under such hybridization conditions to genomic DNA from an otherwise isogenic plant that does not comprise the segment, wherein the probe is a sequence encoding an insecticidal protein comprising an amino acid sequence that is homologous or complementary to SEQ ID NO:3, 6, 9, 12, 14, 16, or 18, or has at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:2, 4, 7, 10, 13, 15, or 17; subjecting the sample and probe to stringent hybridization conditions; and detecting hybridization of the probe to the DNA of the sample. In some embodiments, detecting the presence of a member of the TIC2199 toxin protein class may comprise an ELISA or a Western blot.

[0025] Also provided herein is a method for detecting the presence of an insecticidal protein or fragment thereof from the TIC2199 toxin protein class, comprising contacting a sample with an immunoreactive antibody or recombinant protein of the TIC2199 toxin protein class designed to detect the TIC2199 protein, and detecting binding of the antibody to the TIC2199 toxin protein class protein, thereby confirming the presence of the protein in the sample. In some embodiments, the detecting step comprises an ELISA or Western blot.

[0026] Also contemplated in the present application is a method for combating lepidopteran pest species or pest infestations in the field, comprising growing a crop plant expressing an insecticidally effective amount of an insecticidal protein as set forth in SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17, or growing a crop plant expressing an insecticidally effective amount of one or more insecticidal proteins comprising an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17, and releasing into the field one or more transgenic lepidopteran pest species, each having a self-limiting gene, into the field, the crop plant comprising a gene encoding a toxin protein of the present invention, for the purpose of preventing or delaying the development of resistance to the toxin protein in one or more lepidopteran pest species. In one embodiment, the crop plant can be monocotyledonous or dicotyledonous. In another embodiment, the monocotyledonous crop plant can be corn, wheat, sorghum, rice, rye, or foxtail millet. In yet another embodiment, the dicotyledonous crop plant can be soybean, cotton, or canola.

[0027] A brief description of the sequence SEQ ID NO:1 is a nucleic acid sequence encoding the TIC2199 insecticidal protein obtained from Bacillus thuringiensis species EG8639.

[0028] SEQ ID NO:2 is the amino acid sequence of the TIC2199 insecticidal protein encoded by the sequence shown in SEQ ID NO:1.

[0029] SEQ ID NO:3 is a synthetic coding sequence that encodes TIC2199, which can be used in plant cells.

[0030] SEQ ID NO: 4 is the amino acid sequence of TIC2199_3, a variant of TIC2199 in which the N-terminal 44 amino acids containing the secretory peptide have been removed, and the C-terminal 64 amino acids have also been removed.

[0031] SEQ ID NO:5 is a DNA sequence encoding the TIC2199_3 variant of SEQ ID NO:4.

[0032] SEQ ID NO:6 is a synthetic coding sequence that encodes the TIC2199_3 variant of SEQ ID NO:4, which can be used in plant cells.

[0033] SEQ ID NO: 7 is the amino acid sequence of TIC2199_1, a variant of TIC2199 in which the N-terminal 44 amino acids containing the secretory peptide have been removed.

[0034] SEQ ID NO:8 is a DNA sequence encoding the TIC2199_1 variant of SEQ ID NO:7.

[0035] SEQ ID NO:9 is a synthetic coding sequence that encodes the TIC2199_1 variant of SEQ ID NO:7, which can be used in plant cells.

[0036] SEQ ID NO:10 is the amino acid sequence of a variant of TIC2199, TIC2199_2, in which the C-terminal 64 amino acids have also been removed.

[0037] SEQ ID NO:11 is a DNA sequence encoding the TIC2199_2 variant of SEQ ID NO:10.

[0038] SEQ ID NO: 12 is a synthetic coding sequence that encodes the TIC2199_2 variant of SEQ ID NO: 10, which can be used in plant cells.

[0039] SEQ ID NO:13 is the amino acid sequence of the TIC2199_1 variant, a variant in which the initiating methionine has been removed and used to operably link to the chloroplast transit peptide.

[0040] SEQ ID NO:14 is a synthetic coding sequence encoding a TIC2199_1 variant in which the initiation methionine codon has been removed and which can be used to operably link to a chloroplast transit peptide and used in plant cells.

[0041] SEQ ID NO:15 is the amino acid sequence of the TIC2199_2 variant, a variant in which the initiating methionine has been removed and used to operably link the chloroplast transit peptide.

[0042] SEQ ID NO:16 is a synthetic coding sequence encoding a TIC2199_2 variant in which the initiation methionine codon has been removed and which can be used to operably link to a chloroplast transit peptide and used in plant cells.

[0043] SEQ ID NO:17 is the amino acid sequence of the TIC2199_3 variant, a variant in which the initiating methionine has been removed and used to operably link the chloroplast transit peptide.

[0044] SEQ ID NO:18 is a synthetic coding sequence encoding a TIC2199_3 variant in which the initiation methionine codon has been removed and which can be used to operably link to a chloroplast transit peptide and used in plant cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] One problem in the field of agricultural pest control can be characterized as the need for new toxin proteins that are effective against target pests, exhibit a broad range of toxicity against target pest species, can be expressed in plants without causing undesirable agronomic problems, and offer an alternative mode of action compared to current toxins used commercially in plants.

[0046] Disclosed herein are novel insecticidal proteins, exemplified by TIC2199. The use of proteins in insecticidally effective amounts is directed to the control of insects that are a problem in the art, particularly a wide range of lepidopteran insect pests, more specifically Agrotis ipsilon, Helicoverpa zea, Trichoplusia ni, Ostrinia nubilalis, Spodoptera frugiperda, Spodoptera eridania, Chrysodeixis includens, Diatraea grandiosella, Velvet bean caterpillar (Anticarsia gemmatalis), and Western bean cutworm (Striacosta albicosta), as well as the hemipteran species Lygus lineolaris and Euschistus albicosta. This can be used to combat the spread of heroes.

[0047] References in this application to TIC2199, "TIC2199 protein", "TIC2199 protein toxin", "TIC2199 insecticidal protein", "TIC2199 related toxin", "TIC2199 related toxins", "TIC2199 protein toxin class", "TIC2199 toxin protein class" and the like refer to any novel insecticidal or insect inhibitory protein comprising, consisting of, substantially homologous to, similar to, or derived from any insecticidal or insect inhibitory protein sequence of TIC2199 (SEQ ID NO: 2), and insecticidal or insect inhibitory segments thereof, or combinations thereof, that confer activity against lepidopteran pests, including any protein that exhibits insecticidal or insect inhibitory activity, where alignment of such protein with TIC2199 results in an amino acid sequence identity of any percentage from about 96% to about 100%. The TIC2199 protein includes both plastid-targeted and non-plastid-targeted forms of the protein.

[0048] The term "segment" or "fragment" is used in the present application to describe a contiguous amino acid or nucleic acid sequence that is shorter than the complete amino acid or nucleic acid sequence describing the TIC2199 protein. A segment or fragment that exhibits insect inhibitory activity is also disclosed in the present application if alignment of such a segment or fragment with the corresponding section of the TIC2199 protein set forth in SEQ ID NO:2 results in any percentage of amino acid sequence identity between the segment or fragment and the corresponding segment of amino acids in the TIC2199 protein, from about 96% to about 100%. A fragment described herein may contain at least 50, at least 100, at least 250, at least 400, at least 500, at least 600, or at least 800 contiguous amino acid residues of the TIC2199 protein. For example, a fragment of SEQ ID NO:2 is presented as SEQ ID NO:4. The amino acid sequence of SEQ ID NO:4 includes domains I, II, and III of TIC2199. The N-terminal 44 amino acids of the proposed secretory signal peptide were removed based on alignment with other Cry1I insecticidal proteins (Ruiz de Escudero et al. 2006. Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. Applied and Environmental Microbiology, 72(7):4796-4804), retaining the N-terminal methionine residue. In addition, the predicted trypsin-cleaved carboxy-terminal pro-toxin domain was also removed in SEQ ID NO:4.The trypsin cleavage site is predicted to be the first lysine (K) after the highly conserved "DRIEF" sequence at the end of block 5, domain III near the start of the pro-toxin domain (Schnepf et al. 1998. Bacillus thuringiensis and Its Pesticidal Crystal Proteins. Microbiol. Mol. Biol. Rev. 62(3):775-806). SEQ ID NO:4 can be encoded, for example, by SEQ ID NO:5 for expression in bacteria or by SEQ ID NO:6 for expression in plant cells.

[0049] In addition, a fragment of SEQ ID NO:2 is presented as SEQ ID NO:7. The amino acids of SEQ ID NO:7 include domains I, II, and III of TIC2199. The N-terminal 44 amino acids of the proposed secretory signal peptide have been removed based on alignment with other Cry1I insecticidal proteins. SEQ ID NO:7 can be encoded, for example, by SEQ ID NO:8 for expression in bacteria or by SEQ ID NO:9 for expression in plant cells. A fragment of SEQ ID NO:2 is presented as SEQ ID NO:10. SEQ ID NO:10 includes domains I, II, and III of TIC2199. The predicted trypsin-cleaved carboxy-terminal pro-toxin domain has also been removed in SEQ ID NO:10. SEQ ID NO:10 can be encoded, for example, by SEQ ID NO:11 for expression in bacteria or by SEQ ID NO:12 for expression in plant cells. A fragment of SEQ ID NO:2 is presented as SEQ ID NO:13. The amino acids of SEQ ID NO:13 include domains I, II, and III of TIC2199. The N-terminal 44 amino acids of the proposed secretory signal peptide have been removed based on alignment with other Cry1I insecticidal proteins and lack the initiation methionine. SEQ ID NO:13 is used to operably link the TIC2199_1 amino acid sequence to the chloroplast transit peptide. SEQ ID NO:13 is encoded by SEQ ID NO:14 for expression in plant cells. A fragment of SEQ ID NO:2 is presented as SEQ ID NO:15. The amino acids of SEQ ID NO:15 comprise domains I, II, and III of TIC2199. The predicted trypsin-cleaved carboxy-terminal pro-toxin domain has also been removed in SEQ ID NO:15 along with the initiation methionine and is used to operably link the TIC2199_2 amino acid sequence to the chloroplast transit peptide. SEQ ID NO:15 is encoded by SEQ ID NO:16 for expression in plant cells. The amino acids of SEQ ID NO:17 comprise domains I, II, and III of TIC2199.The N-terminal 44 amino acids of the proposed secretory signal peptide have been removed based on alignments with other Cry1I insecticidal proteins, and the predicted trypsin-cleaved carboxy-terminal pro-toxin domain has also been removed along with the initiating methionine in SEQ ID NO:17 and is used to operably link the TIC2199_3 amino acid sequence to the chloroplast transit peptide.

[0050] In this application, references to the terms "active" or "activity", "pesticidal activity" or "pesticidal" or "insecticidal activity", "insect inhibitory", "pesticidally effective", or "insecticidal" refer to the effectiveness of a toxic agent, such as a protein toxin containing an effective amount of a TIC2199 protein, in inhibiting (inhibiting growth, feeding, fertility, or viability), suppressing (inhibiting growth, feeding, fertility, or viability), eradicating (reducing pest infestations, eradicating pest feeding activity on a particular crop), or killing (causing morbidity, mortality, or reduced reproduction) of a pest. These terms are intended to include the result of providing a pest with an insecticidally effective amount of a toxic protein where exposure of the pest to the toxic protein results in morbidity, mortality, reduced reproduction, or stunting. These terms also include the repulsion of pests from plants, plant tissues, plant parts, seeds, plant cells, or from a particular geographic location where the plant may grow, as a result of providing an insecticidally effective amount of the toxic protein in or to the plant. In general, insecticidal activity refers to the ability of the toxic protein to be effective in inhibiting growth, development, survival rate, feeding behavior, mating behavior, fecundity, or to reduce any measurable adverse effect caused by insect feeding. The toxic protein may be produced by the plant or applied to the plant or to the environment within the location where the plant is located. The terms "biologically active", "effective", "effective", or variations thereof are also used interchangeably in this application to describe the effect of the protein of the present invention on the target insect pest.

[0051] When an insecticidally effective amount of the toxic agent is provided to the diet of a target pest, the toxic agent exhibits insecticidal activity when it contacts the pest. The toxic agent can be an insecticidal protein or one or more chemicals known in the art. The insecticides or insecticidal chemicals can be used alone or in combination with each other. Chemicals include, but are not limited to, dsRNA molecules that target specific genes for the suppression of the target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoides. Pesticidal or insecticidal protein agents include the protein toxins presented in this application as well as other proteinaceous toxic agents, including those that target Lepidoptera, and protein toxins such as Cry, Vip, and Cyt proteins used to control other plant pests, Pseudomonas insect toxic proteins, and insect toxic proteins from fern species that are available in the art for use in controlling Coleoptera, Hemiptera, and Homoptera species.

[0052] Reference to pests, particularly pests of crop plants, is intended to mean insect pests of crop plants, particularly those lepidopteran insect pests that are controlled by the TIC2199 protein toxin class. However, reference to pests can also include plant Coleoptera, Hemiptera (e.g., Lygus lineolaris) and Euschistus heros, and Homoptera insect pests, as well as nematodes and fungi, when the toxic agent targeted to these pests is co-localized or co-localized with the TIC2199 protein or a protein that is 95 to about 100 percent identical to the TIC2199 protein. The phrase "co-localized" or "co-localized" is intended to include any case in which the target insect pest is contacted with the TIC2199 toxin protein, as well as any other toxic agent that is also present in an insecticidal amount against the target insect pest. "Contacted" is intended, in certain embodiments, to refer to being present in the diet of a target pest, where the diet is consumed by the target pest.

[0053] Lepidoptera insects include armyworms, cutworms, loopers, and Heliothines of the Noctuidae family, such as Spodoptera frugiperda, Spodoptera exigua, Spodoptera cosmioides, Spodoptera eridania, Mamestra configurata, Agrotis ipsilon, Trichoplusia ni, Diatraea saccharalis, Pseudoplusia includens, Rachiplusia nu, Velvet bean caterpillar, Hypena scabra), tobacco budworm (Heliothis virescens), granulated cutworm (Agrotis subterranea), armyworm (Pseudaletia unipuncta), sunflower looper (Rachiplusia nu), South American podworm (Helicoverpa gelotopoeon), western cutworm (Agrotis orthogonia), borers, case bearers, webworms, cornworms, cabbageworms and skeletonizers of the family Pyralidae, such as European corn borer (Ostrinia nubilalis), navel orangeworm (Amyelois transitella), corn root webworm (Crambus caliginosellus), black banded black borer (Herpetogramma licarsisalis), sunflower moth (Homoeosoma electellum), corn moth (Elasmopalpus lignosellus), leaf rollers, budworms, seedworms, and fruitworms of the Tortricidae family, such as the codling moth (Cydia pomonella), the grape borer moth (Endopizaviteana, Grapholita molesta, Sunflower bud moth (Suleima helianthana), and many other economically important Lepidoptera, such as, but not limited to, Plutella xylostella, Pink bollworm (Pectinophora gossypiella), and Gypsy moth (Lymantria dispar). Other insect pests of the order Lepidoptera include, for example, the cotton leafworm (Alabama argillacea), fruit tree leaf roller (Archips argyrospila), European leaf roller (Archips rosana) and other Archips species (Chilo suppressalis, rice stem borer, or rice stem borer), rice leaf borer (Cnaphalocrocis medinalis), corn root webworm (Crambus caliginosellus), bluegrass webworm (Crambus teterrellus), southwestern corn borer (Diatraea grandiosella), sugarcane borer (Diatraea saccharalis), spiny ballworm (Earias insulana), spotted ballworm (Earias vittella), cotton bollworm (Helicoverpa armigera), tobacco budworm (Helicoverpa zea, also known as the soybean podworm and cotton bollworm, tobacco budworm (Heliothis virescens), black banded grain moth (Herpetogramma licarsisalis), western bean cutworm (Striacosta albicosta), European grapevine moth (Lobesia botrana), citrus leafminer (Phyllocnistis citrella), large cabbage white butterfly (Pieris brassicae), cabbage white butterfly (Pieris rapae, also known as the imported cabbageworm), beet armyworm (Spodoptera exigua), common cutworm (Spodopteralitura, also known as the cluster caterpillar, and the tomato bush moth (Tuta absoluta).

[0054] Insects of the order Hemiptera include bugs of the family Pentatomidae: the green grass bugs of the genus Chinavia (Chinavia hilaris, Chinavia marginata, and Chinavia pensylvanica), the 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 osborni, Chlorochroa saucia, and Chlorochroa senilis), the southern green stink bug (Nezara viridula), the stink bugs of the genus Edessa (Edessa meditabunda, Edessa bifida, and Edessa florida), Neotropical brown stink bug (Euschistus heros), Euschistus genus stink bugs (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 marmorated stink bug (Halyomorpha halys), red-shouldered stink bug (Thyanta accerra), Thyanta genus stink bugs (Thyanta calceata, Thyanta custator, Thyantapallidovirens, Thyanta perditor, Thyanta maculate, and Thyanta pseudocasta), the greenberry stink bug (Dichelops melacanthus) and other bugs of the Dichelop genus (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) and Piezodorus lituratus, and insects of the family Plataspidae (e.g., Megacopta cribraria, Lygus serrata, and others), Examples of insecticides that may be used include, but are not limited to, the Japanese grass cricket (Lygus hesperus) and the rusty-colored grass cricket (Lygus lineolaris).

[0055] Reference in this application to an "isolated DNA molecule" or equivalent term or expression is intended to mean that the DNA molecule is present alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements naturally found in the DNA of the genome of an organism, such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc., are not considered to be "isolated" as long as the elements are in the genome of the organism and in the location in the genome where the elements are found in nature. However, each of these elements, and subportions of these elements, are "isolated" within the scope of this disclosure as long as the elements are not in the genome of the organism and in the location in the genome where the elements are found in nature. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein is considered an isolated nucleotide sequence as long as the nucleotide sequence is not in the DNA of a bacterium in which the sequence encoding the protein is found in nature. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered to be isolated for the purposes of this disclosure. For purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, is considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used to transform a cell, present in the genome of the plant or bacteria, or present in detectable amounts in tissue, progeny, biological sample, or commercial product derived from the plant or bacteria.

[0056] References to the term "self-limiting gene" in this application refer to genes that limit the survival of the host, resulting in a reduction in the host population. Such technology is provided by Oxitech Ltd. Transgenic male insects carrying a transgenic self-limiting gene are released and bred with wild females. As a result, the offspring inherit a copy of the self-limiting gene. The self-limiting gene interferes with the proper functioning of the insect's cells by overproducing a protein within the insect's cells and interfering with the cell's ability to produce other essential proteins required for development. The gene prevents the insect from surviving to adulthood by interfering with normal development. For example, the self-limiting diamondback moth (Plutellidae xylostella) strain OX4319L was developed by Oxitech Ltd. and carries a male-selection gene that utilizes sequences from the sex-determining gene doublesex (dsx). This gene expresses sex-selective splicing to engineer female-specific expression of the self-limiting gene, preventing female offspring from surviving beyond the larval stage and allowing the production of a cohort of male-only self-limiting moths. After release, the males mate with the pest females, reducing the number of female offspring in the next generation, thereby locally suppressing the population of P. xylostella. To facilitate rearing of a large number of males for release in diamondback moth production facilities, the expression of female-specific dsx in the OX4319L strain is suppressed by adding tetracycline or a suitable analogue to the larval diet. OX4319L also expresses the fluorescent protein DsRed to allow for 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). When applied in the field using plants containing the toxin genes of the present invention, this technology can delay or prevent the development of resistance in pest species targeted for control by the toxin genes and proteins of the present invention, thus enhancing the durability of any plant product containing the toxin genes and proteins of the present invention.

[0057] As further described in this application, an open reading frame (ORF) encoding TIC2199 (SEQ ID NO:1) was discovered in DNA obtained from Bacillus thuringiensis species EG8639. Bioassays using microbial host cell-derived proteins of TIC2199 have been performed on the lepidopteran species Agrotis ipsilon (BCW), Helicoverpa zea (CEW), Trichoplusia ni (CLW), Ostrinia nubilalis (ECB), Spodoptera frugiperda (FAW), Spodoptera eridania (SAW), Chrysodeixis includens (SBL), Diatraea grandiosella (SWC), Diatraea saccharalis (SCB), Anticarsia gemmatalis (VBC), and Striacosta serrata (WBC). albicosta), as well as the hemipteran species TPB (Lygus lineolaris) and NBSB (Euschistus heros).

[0058] Synthetic coding sequences designed for use in plant cells were generated to express TIC2199 (SEQ ID NO:3), TIC2199_1 (SEQ ID NOs:9 and 14), TIC2199_2 (SEQ ID NOs:12 and 16), and TIC2199_3 (SEQ ID NOs:6 and 18), particularly when operatively / functionally linked to a plant functional promoter and other elements that function to mediate the desired level and spatial characteristics for expression of the protein in plants. Corn plants expressing TIC2199, TIC2199_1, TIC2199_2, and TIC2199_3 showed effective activity against the lepidopteran species European Corn Borer (ECB, Ostrinia nubilalis) and Southwestern Corn Borer (SWC, Diatraea grandiosella). In Brazil, corn plants expressing TIC2199 were effective against SCB and highly effective against LCSB.

[0059] For expression in plant cells, the TIC2199 (SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17) protein can be expressed and localized to the cytoplasm or targeted to various organelles of the plant cell. For example, targeting the protein to the chloroplast can increase the levels of protein expressed in the 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 increased pest resistance efficacy in the transgenic event. Targeting or transit peptides are short (3-70 amino acids long) peptide chains that direct the transport of a protein to a specific region within the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisomes, and plasma membrane. Some targeting peptides are cleaved from the protein by a signal peptidase after the protein is transported. When targeting the chloroplast, the protein contains a transit peptide of about 40-50 amino acids long. For a description 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 as precursors from nuclear genes and targeted to the chloroplast by chloroplast transit peptides (CTPs). Examples of such isolated CTPs include, but are not limited to, the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvylshikimate phosphate synthase (EPSPS), and those related to the transit peptides described in U.S. Patent No. 7,193,133. Non-chloroplast proteins can be targeted to the chloroplast by using protein fusions with heterologous CTPs, and it has been demonstrated in vivo and in vitro that CTPs are sufficient to target proteins to the chloroplasts.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 the chloroplasts in transgenic plants (see U.S. Pat. Nos. 5,627,061, 5,633,435, and 5,312,910, as well as EP 0218571, EP 189707, EP 508909, and EP 924299). To target the TIC2199 toxin protein to chloroplasts, a sequence encoding a chloroplast transit peptide is placed 5' in operable linkage to, and in frame with, a synthetic coding sequence encoding a TIC2199 toxin protein designed for expression in plant cells.

[0060] It is contemplated that additional toxin protein sequences related to TIC2199 can be generated using the amino acid sequence of TIC2199 to generate novel proteins with novel properties. The TIC2199 toxin proteins can be aligned and differences at the amino acid sequence level can be combined into novel amino acid sequence variants, with appropriate changes made to recombinant nucleic acid sequences encoding the variants.

[0061] It is contemplated that improved variants of the TIC2199 protein toxin class can be engineered in plants by using various gene editing methods known in the art. Such techniques used for genome editing include, but are not limited to, ZFN (zinc finger nuclease), meganuclease, TALEN (transcription activator-like effector nuclease), and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) systems. These genome editing methods can be used to modify the toxin protein coding sequence transformed in plant cells into a different toxin coding sequence. Specifically, through these methods, one or more codons in the toxin coding sequence can be modified to engineer the amino acid sequence of a new protein. Alternatively, fragments in the coding sequence can be replaced or deleted, or additional DNA fragments can be inserted into the coding sequence to engineer the coding sequence of a new toxin. The new coding sequence can encode a toxin protein with new properties, such as increased activity or spectrum against insect pests, as well as provide activity against one or more insect pest species that have developed or are likely to develop resistance to the original insect toxin protein. Plant cells containing the genetically edited toxin coding sequence can be used to generate whole plants that express the new toxin protein by methods known in the art.

[0062] It is also contemplated that fragments of TIC2199 or its protein variants may be truncated forms in which one or more amino acids are deleted from the N-terminus, C-terminus, the middle of the protein, or combinations thereof, such that the fragments and variants retain insect inhibitory activity. These fragments may be naturally occurring or synthetic variants of TIC2199 or derived protein variants, but must retain at least the insect inhibitory activity of TIC2199.

[0063] Proteins similar to TIC2199 protein can be identified and compared with each other using various computer-based algorithms known in the art. The amino acid sequence identity reported in this application is the result of Clustal W alignment using the following default parameters: weight matrix: blosum, gap opening penalty: 10.0, gap extension penalty: 0.05, hydrophilic gaps: on, hydrophilic residues: GPSNDQERK, residue-specific gap penalty: on (Thompson, et al (1994) Nucleic Acids Research, 22: 4673-4680). Percentage amino acid identity is further calculated by multiplying by 100% (amino acid identity / length of the subject protein). Other alignment algorithms are also available in the art that provide similar results to those obtained using Clustal W alignment and are contemplated herein.

[0064] Proteins that exhibit insect inhibitory activity against lepidopteran insect species are intended to be related to TIC2199 and proteins of the invention shown as SEQ ID NO: 2 or 4 when the protein is used in a query (e.g., a Clustal W alignment) such that the query protein is identified as a hit in such an alignment (exhibiting at least 96% to about 100% amino acid identity over the entire length of the query protein, which is about 96%, 97%, 98%, 99%, 100%, or any percentage within this range).

[0065] In addition to percent identity, TIC2199 and variants of TIC2199 can also be related by primary structure (conserved amino acid motifs), length, and other features. Table 1 reports the characteristics of TIC2199 and variants of the TIC2199 protein toxin. [Table 1]

[0066] As further described in the Examples of this application, synthetic nucleic acid molecule sequences encoding TIC2199 and variants of TIC2199 encoded by SEQ ID NOs: 3, 6, 9, 12, 14, 16, and 18 have been designed for use in plants. Given the redundancy of the genetic code, it is within the skill of the art to produce any number of other sequences encoding toxin proteins, however, it is understood that sequences produced for expression in plants must avoid problems known in the art that prevent or limit efficient expression of coding sequences, particularly as described in U.S. Patent No. 5,500,365.

[0067] Expression cassettes and vectors containing the recombinant nucleic acid sequence molecules can be constructed and introduced into plants (such as corn, soybean, or cotton plant cells, among others) according to transformation methods and techniques known in the art. For example, Agrobacterium-mediated transformation is described in U.S. Patent Application Publication Nos. 2009 / 0138985A1 (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257A1 (sugar beet), U.S. Patent Nos. 5,750,871 (canola), 7,026,528 (wheat), and 6,365,807 (rice), as well as Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugarcane), all of which are incorporated herein by reference in their entirety. The transformed cells can be regenerated into transformed plants which express TIC2199 and exhibit insecticidal activity through bioassays conducted in the presence of lepidopteran pest larvae using plant leaf discs obtained from the transformed plants. Plants can be derived from plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0068] As an alternative to traditional transformation methods, DNA sequences such as transgenes, expression cassette(s) can be inserted or integrated into a specific site or locus within the genome of a plant or plant cell via site-specific integration. Thus, the recombinant DNA construct(s) and molecule(s) of the present disclosure may include a donor template sequence that includes at least one transgene, expression cassette, or other DNA sequence for insertion into the genome of a plant or plant cell. Such donor templates for site-specific integration may further include one or two homology arms flanking the insertion sequence (i.e., the sequence, transgene, cassette, etc., to be inserted into the plant genome). The recombinant DNA construct(s) of the present disclosure may further include expression cassette(s) encoding a site-specific nuclease and / or any associated protein(s) for performing site-specific integration. These nuclease expression cassette(s) may be present in the same molecule or vector as the donor template (cis) or on a separate molecule or vector (trans). Several methods for site-specific integration are known in the art, involving different proteins (or complexes of proteins and / or guide RNAs) that cleave genomic DNA to generate double-strand breaks (DSBs) or nicks at desired genomic sites or loci. Briefly, as understood in the art, during the process of repairing the DSB or nick introduced by a nuclease enzyme, the donor template DNA can be integrated into the genome at the site of the DSB or nick. The presence of homology arm(s) in the donor template can facilitate the introduction and targeting of the insertion sequence into the plant genome during the repair process via homologous recombination, although the insertion event can also occur via non-homologous end joining (NHEJ). Examples of site-specific nucleases that can be used include zinc finger nucleases, engineered or natural meganucleases, TALE endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cas12a).For methods using an RNA-guided site-specific nuclease (e.g., Cas9 or Cas12a), the recombinant DNA construct(s) also include a sequence encoding one or more guide RNAs to guide the nuclease to a desired site within the plant genome.

[0069] Recombinant nucleic acid molecule compositions encoding TIC2199 proteins expressed in bacteria and plants can be expressed using recombinant DNA constructs in which a polynucleotide molecule having an ORF encoding the protein is operably linked to gene expression elements such as promoters and any other regulatory elements necessary for expression in the system for which the construct is intended. Non-limiting examples include a plant functional promoter operably linked to the coding sequence of the TIC2199 protein for expression of the protein in plants, or a Bt functional promoter operably linked to the coding sequence of the TIC2199 protein for expression of the protein in Bt bacteria or other Bacillus species. Other elements can be operably linked to the coding sequence of the TIC2199 protein, including, but not limited to, enhancers, introns, non-translated leaders, encoded protein immobilization tags (HIS tags), transit peptides (i.e., plastid transit peptides, signal peptides), polypeptide sequences for post-translational modification enzymes, ribosome binding sites, and RNAi target sites. Exemplary recombinant polynucleotide molecules provided herein include, but are not limited to, a heterologous promoter operably linked to a polynucleotide such as SEQ ID NO: 1, 3, 5, 6, 8, 9, 11, 12, 14, 16, or 18 encoding TIC2199 or a truncated variant of TIC2199, or a protein having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 7, 10, 13, 15, or 17. A heterologous promoter may also be operably linked to a synthetic DNA coding sequence encoding a plastid-targeted TIC2199. Codons of recombinant nucleic acid molecules encoding proteins disclosed herein may be replaced by synonymous codons (known in the art as silent substitutions).

[0070] The recombinant DNA construct comprising the coding sequence of the TIC2199 protein may further comprise a DNA region encoding one or more insect inhibitors that may be configured to simultaneously express or co-express the DNA sequence encoding the TIC2199 protein, the insect inhibitory dsRNA molecule, or the auxiliary protein. The auxiliary protein may include, but is not limited to, a cofactor, enzyme, binding partner, or other agent that functions to assist the effectiveness of the insect inhibitor, for example, by assisting the expression of the insect inhibitor, affecting its stability in the plant, optimizing the free energy for oligomerization, enhancing its toxicity, and increasing its spectrum of activity. The auxiliary protein may, for example, facilitate the uptake of one or more insect inhibitors or enhance the toxic effect of the poison agent.

[0071] The recombinant DNA construct can be constructed so that all proteins or dsRNA molecules are expressed from one promoter, or each protein or dsRNA molecule is under the control of a separate promoter or some combination thereof. The proteins of the present invention can be expressed from a multigene expression system, where TIC2199 is expressed from a common nucleotide segment that also contains other open reading frames and promoters, depending on the type of expression system selected. For example, a bacterial multigene expression system can utilize a single promoter to drive the expression of multiple linked / tandem open reading frames from within a single operon (i.e., polycistronic expression). In another example, a plant multigene expression system can utilize multiple unlinked or linked expression cassettes, each cassette expressing a different protein or other agent, such as one or more dsRNA molecules.

[0072] A recombinant polynucleotide or recombinant DNA construct comprising the coding sequence of a TIC2199 protein can be delivered to a host cell by a vector (e.g., a plasmid, a baculovirus, a synthetic chromosome, a virion, a cosmid, a phagemid, a phage, or a viral vector). Such vectors can be used to achieve stable or transient expression of the coding sequence of a TIC2199 protein in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct comprising the coding sequence of a TIC2199 protein and introduced into a host cell is referred to as a "transgene" in this application.

[0073] Provided herein are transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts comprising a recombinant polynucleotide expressing a coding sequence for TIC2199 or a related family toxin protein. The term "bacterial cell" or "bacteria" can include, but is not limited to, Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, Brevibacillus, Klebsiella, Erwinia, or Rhizobium cells. The term "plant cell" or "plant" can include, but is not limited to, dicotyledonous or monocotyledonous plants. The term "plant cell" or "plant" includes, but is not limited to, alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassava, castor bean, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, chive, lettuce, loblolly pine, foxtail millet, melon, nuts, oats, olives, onions, ornamentals, palm, pasture grass, peas, peanuts, and the like. The plant cells or plants of the transgenic plants may include, but are not limited to, plant cells or plants of: corn, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn (i.e., maize) (e.g., sweet corn or field corn), sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, wheatgrass, watermelon, and wheat. In certain embodiments, transgenic plants and transgenic plant parts regenerated from transgenic plant cells are provided. In certain embodiments, transgenic plants can be obtained from transgenic seeds by cutting, snapping, grinding, or otherwise dissociating the parts from the plant.In certain embodiments, the plant part may be a seed, pod, leaf, flower, stem, root, or any part thereof, or a non-regenerable part of a transgenic plant part. As used in this context, a "non-regenerable" part of a transgenic plant part is a part that cannot be induced to form a whole plant or a part that cannot be induced to form a whole plant capable of sexual and / or asexual reproduction. In certain embodiments, the non-regenerable part of a plant part is a transgenic seed, pod, leaf, flower, stem, or root part.

[0074] Methods are provided for producing transgenic plants that contain an insect lepidopteran-inhibitory or hemipteran-inhibitory amount of TIC2199 protein. Such plants can be produced by introducing a recombinant polynucleotide encoding any of the proteins provided in the present application into a plant cell and selecting plants derived from the plant cell that express an insect lepidopteran-inhibitory amount of the protein. Plants can be derived from plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0075] Also disclosed herein are processed plant products, the processed products comprising detectable amounts of TIC2199, insect inhibitory segments or fragments thereof, or any characteristic portion thereof. In certain embodiments, the processed products are selected from the group consisting of plant parts, plant biomass, oils, meals, sugars, animal feed, flours, debris, bran, lint, husks, processed seeds, and seeds. In certain embodiments, the processed products are non-renewable. Plant products can include commercial products or other products of commerce derived from transgenic plants or transgenic plant parts, which can be tracked through commerce by detecting nucleotide segments or expressed RNA or proteins that code for or contain characteristic portions of TIC2199.

[0076] Plants expressing TIC2199 proteins can be crossed by mating with transgenic events expressing other toxin proteins and / or expressing other transgenic traits, such as herbicide resistance genes, genes that confer yield or stress tolerance traits, or such traits can be combined such that the traits are all linked in a single stacked vector.

[0077] As further described in the Examples, coding sequences for TIC2199 protein and sequences having a significant percentage of identity to TIC2199 can be identified using methods known to those skilled in the art, such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, protein TIC2199 can be used to generate antibodies that specifically bind to related proteins, and can be used to screen for and detect other closely related protein members.

[0078] Additionally, the nucleotide sequences encoding the TIC2199 toxin protein can be used as probes and primers for screening to identify other members of the class using thermal cycling or isothermal amplification and hybridization methods. For example, oligonucleotides derived from the sequences shown in SEQ ID NO: 3, 6, 9, or 12 can be used to determine the presence or absence of the TIC2199 transgene in deoxyribonucleic acid samples derived from commercial products. Given the sensitivity of certain nucleic acid detection methods using oligonucleotides, it is expected that oligonucleotides derived from the sequence shown in SEQ ID NO: 3 can be used to detect the TIC2199 transgene in commercial products derived from pooled sources, where a small proportion of the commercial products are derived from transgenic plants containing any of the transgenes. It is further recognized that such oligonucleotides can be used to introduce nucleotide sequence variations in each of SEQ ID NO: 1, 3, 5, 6, 8, 9, 11, 12, 14, 16, and 18. Such "mutagenizing" oligonucleotides are useful for identifying TIC2199 amino acid sequence variants that exhibit insect inhibitory activity or a range of diverse expression in transgenic plant host cells.

[0079] Nucleotide sequence homologues, such as insecticidal proteins encoded by nucleotide sequences that hybridize to each or any of the sequences disclosed in the present application under stringent hybridization conditions, are also an embodiment of the present invention. The present invention also provides a method for detecting a first nucleotide sequence that hybridizes to a second nucleotide sequence, where the first nucleotide sequence (or its reverse complement) encodes an insecticidal protein or an insecticidal fragment thereof and hybridizes to a second nucleotide sequence. In such a case, the second nucleotide sequence can be any of the nucleotide sequences shown as SEQ ID NO: 1, 3, 5, 6, 8, 9, 11, 12, 14, 16, or 18 under stringent hybridization conditions. The nucleotide coding sequences hybridize to each other under appropriate hybridization conditions, such as stringent hybridization conditions, and the proteins encoded by these nucleotide sequences cross-react with antisera raised against any one of the other proteins. Stringent hybridization conditions, as defined herein, include hybridization at at least 42° C., followed by two washes for 5 minutes each in 2×SSC, 0.1% SDS at room temperature, followed by two washes for 30 minutes each in 0.5×SSC, 0.1% SDS at 65° C. Washes at even higher temperatures constitute even more stringent conditions, for example hybridization conditions of 68° C., followed by washes in 2×SSC containing 0.1% SDS at 68° C.

[0080] Those skilled in the art will recognize that due to redundancy in the genetic code, many other sequences can encode such related proteins, and that these sequences are embodiments of the invention insofar as they function to express insecticidal proteins in either Bacillus strains or plant cells, and of course many such redundant coding sequences will not hybridize under these conditions to native Bacillus sequences encoding TIC2199 variants. The present application contemplates the use of these and other identification methods known to those skilled in the art to identify sequences encoding TIC2199 variant proteins and sequences having a significant percentage of identity to sequences encoding TIC2199 variant proteins.

[0081] The present disclosure also contemplates the use of molecular methods known in the art for engineering and cloning commercially useful proteins, including chimeras of proteins derived from insecticidal proteins. For example, chimeras can be assembled from segments of TIC2199 proteins, including the assembly of segments of TIC2199 proteins with various protein segments that differ from TIC2199 proteins and related proteins, to derive additional useful embodiments. TIC2199 proteins may be subjected to alignment with each other and with other Bacillus, Paenibacillus, or other insecticidal proteins (whether these are closely or distantly related phylogenetically), and segments of each such protein may be identified that are useful for substitution between the aligned proteins, resulting in the construction of chimeric proteins. Such chimeric proteins may be subjected to pest bioassay analysis and characterized for the presence or absence of increased biological activity 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 can be further engineered for activity against specific pests or a broader range of pests by swapping domains or segments with other proteins or by using directed evolution methods known in the art.

[0082] The present application discloses a method of using TIC2199 protein to control insects, particularly lepidopteran infestations of crop plants. Such a method may include growing a plant containing an insect or lepidopteran inhibitory amount of TIC2199 toxin protein. In certain embodiments, such a method may further include any one or more of the following: (i) applying any composition containing or encoding TIC2199 toxin protein to a plant or a seed that produces a plant, and (ii) transforming the plant or a plant cell that produces a plant with a polynucleotide that encodes TIC2199 toxin protein. In general, it is contemplated that TIC2199 toxin protein may be provided in a composition, provided in a microorganism, or provided in a transgenic plant to provide insect inhibitory activity against lepidopteran insects.

[0083] In certain embodiments, the recombinant nucleic acid molecule of the TIC2199 toxin protein is an insecticidal active ingredient of an insect inhibitor composition prepared by culturing recombinant Bacillus or any other recombinant bacterial cell transformed to express the TIC2199 toxin protein under conditions suitable for expressing the TIC2199 toxin protein. Such compositions can be prepared by drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of such recombinant cells expressing / producing the recombinant polypeptide. Such processes can yield cell extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, or cell pellets of Bacillus or other insect pathogenic bacteria. By obtaining the recombinant polypeptide so produced, compositions containing the recombinant polypeptide can include bacterial cells, bacterial spores, and parasporular inclusions, which can be formulated for various uses, including as agricultural insect inhibitor spray products or as insect inhibitory formulations in bait bioassays.

[0084] In one embodiment, to reduce the possibility of resistance development, an insect inhibitor composition comprising the TIC2199 protein can further comprise at least one additional polypeptide that exhibits insect inhibitory activity against the same lepidopteran insect species, but is different from the TIC2199 toxin protein. Possible additional polypeptides for such compositions include insect inhibitory proteins and insect inhibitory dsRNA molecules. An example of the use of such ribonucleotide sequences to control insect pests is described in Baum, et al. (US Patent Publication No. 2006 / 0021087A1). Such additional polypeptides for the control of lepidopteran pests can be selected, for example, from the group consisting of, but not limited to, the following insect inhibitory proteins: Cry1A (US Patent Publication No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (US Patent Publication No. 10 / 525,318), Cry1C (US Patent Publication No. 6,033,874), Cry1D, Cr y1Da and variants thereof, Cry1E, Cry1F, and Cry1A / F chimeras (U.S. Pat. Nos. 7,070,982, 6,962,705, and 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras, including, but not limited to, TIC836, TIC860, TIC867, TIC869, and TIC1100 (International Publication No. No. 2016 / 061391(A2)), TIC2160 (International Application Publication No. 2016 / 061392(A2)), Cry2A, Cry2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 (U.S. Patent Publication No. 2013-0117884A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication No. 2013-0310543A1), AXMI-115,AXMI-113, AXMI-005 (U.S. Patent Publication No. 2013-0104259A1), AXMI-134 (U.S. Patent Publication No. 2013-0167264A1), AXMI-150 (U.S. Patent Publication No. 2010-0160231A1), AXMI-184 (U.S. Patent Publication No. 2010-0004176A1), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (U.S. Patent Publication No. 2011-0030096A1), AXMI-218, AXMI-220 (U.S. Patent Publication No. 2014-0245491A1), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication No. 2014-0196175A1), AXMI-238 (U.S. Patent Publication No. 2014-0033363A1), AXMI-270 (U.S. Patent Publication No. 2014-0223598A1), AXMI-345 (U.S. Patent Publication No. 2014-0373195A1), AXMI-335 (International Application Publication No. 2013 / 134523(A2)), DIG-3 (U.S. Patent Publication No. 2013-0219570A1), DIG-5 (U.S. Patent Publication No. 2013-0219570A1), US Patent Publication No. 2010-0317569A1), DIG-11 (US Patent Publication No. 2010-0319093A1), AfIP-1A and its derivatives (US Patent Publication No. 2014-0033361A1), AfIP-1B and its derivatives (US Patent Publication No. 2014-0033361A1), PIP-1APIP-1B (US Patent Publication No. 2014-0007292A1), PSEEN3174 (US Patent Publication No. 2014-0007292A1), AECFG-592740 (US Patent Publication No. 2014-0007292A1), Pput _1063 (U.S. Patent Publication No. 2014-0007292A1), DIG-657 (International Application Publication No. 2015 / 195594A2), Pput_1064 (U.S. Patent Publication No. 2014-0007292A1), GS-135 and its derivatives (U.S. Patent Publication No. 2012-0233726A1), GS153 and its derivatives (U.S. Patent Publication No. 2012-0192310A1), GS154 and its derivatives (U.S. Patent Publication No. 2012-0192310A1), GS155 and its derivatives (U.S. Patent Publication No. 2012-0192310A1),SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2012-0167259A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2012-0047606A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2011-0154536A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2011-0112013A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2010-0192256A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2010-0077507A1), SEQ ID NO:2 or 4 and derivatives thereof (described in U.S. Patent Publication No. 2010-0077508A1), SEQ ID NO:2 or 4 and derivatives thereof (described in US Patent Publication No. 2009-0313721A1), SEQ ID NO:2 or 4 and derivatives thereof (described in US Patent Publication No. 2010-0269221A1), SEQ ID NO:2 or 4 and derivatives thereof (described in US Patent Publication No. 7,772,465(B2)), CF161_0085 and derivatives thereof (described in WO2014 / 008054A2), lepidopteran toxic proteins and their derivatives (described in US Patent Publication Nos. 2008-0172762A1, 2011-0055968A1, and 2012-0117690A1), SEQ ID NO:2 or 4 and derivatives thereof (described in US Patent Publication No. 7510878(B2)), SEQ ID NO:2 or 4 and derivatives thereof (described in US Patent No. 7812129(B1)), etc.

[0085] In other embodiments, such compositions / formulations can further comprise at least one additional polypeptide that exhibits insect inhibitory activity against insects that are not otherwise inhibited by the insect inhibitor protein of the present invention, in order to expand the range of insect inhibition obtained.For example, for the control of hemipteran pests, the combination of insect inhibitor proteins of the present invention can be used with hemipteran active proteins, such as TIC1415 (US Patent Publication No. 2013-0097735A1), TIC807 (US Patent No. 8609936), TIC834 (US Patent Publication No. 2013-0269060A1), AXMI-036 (US Patent Publication No. 2010-0137216A1), and AXMI-171 (US Patent Publication No. 2013-0055469A1). Furthermore, the polypeptide for the control of coleopteran pests can be selected from the group consisting of, but not limited to, the following insect inhibitor proteins: Cry3Bb (US Pat. No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, AXMI134 (US Pat. Pub. No. 2013-0167264A1), AXMI-184 (US Pat. Pub. No. 2010-0004176A1), AXMI-205 (US Pat. Pub. No. 2014-0298538A1), AXMI-207 (US Pat. Pub. No. 2013-0303440A1), AXMI-218, AXMI-220 (US Pat. Pub. No. 2014-0245491A1), AXM I-221z, AXMI-223z (U.S. Patent Publication No. 2014-0196175A1), AXMI-279 (U.S. Patent Publication No. 2014-0223599A1), AXMI-R1 and its variants (U.S. Patent Publication No. 2010-0197592A1), TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, T IC1201, TIC3131, DIG-10 (U.S. Patent Publication No. 2010-0319092A1), eHIP (U.S. Patent Application Publication No. 2010 / 0017914), IP3 and its variants (U.S. Patent Publication No. 2012-0210462A1), and ω-Hexatoxin-Hv1a (U.S. Patent Application Publication No. 2014-0366227A1).

[0086] Additional polypeptides for the control of Coleopteran, Lepidopteran, and Hemiptera insect pests that can be combined with the TIC2199 class of insect inhibitor proteins can be found at the Bacillus thuringiensis toxin nomenclature website maintained by Neil Crickmore (on the World Wide Web at btnomenclature.info). In general, it is contemplated that any insect inhibitor protein known to those of skill in the art can be used in combination with the TIC2199 family of proteins, both in plants (combined through breeding or molecular stacking) or in compositions or formulations as bioinsecticides or bioinsecticide combinations.

[0087] The possibility that insects develop resistance to certain insecticides has been documented in the art. One insect resistance management strategy is to use transgenic crops that express two different insect inhibitors that act through different modes of action. Thus, any insect that is resistant to any one of the insect inhibitors can be eradicated by the other insect inhibitor. Another insect resistance management strategy uses plants that are not protected from the targeted lepidopteran pest species to provide relief for such unprotected plants. One particular example is described in U.S. Patent No. 6,551,962 (incorporated by reference in its entirety).

[0088] In other embodiments, such as topical application pesticidal chemicals designed to control pests that are also controlled by the proteins disclosed herein, used with the proteins in seed treatment, spray-on, drip-on, or wipe-on formulations, can be formulated to be applied directly to the soil (soil drench), to growing plants expressing the proteins disclosed herein, or to seeds containing one or more transgenes encoding one or more of the proteins disclosed. Such formulations for use in seed treatment can be applied with various adhesives and tackifiers known in the art. Such formulations can contain insecticides that are synergistic in mode of action with the disclosed proteins, so that the formulated insecticides act through different modes of action to control the same or similar pests that can be controlled by the disclosed proteins, or such insecticides act to control pests or plant pest species within a broader host range that are not effectively controlled by the TIC2199 insecticidal proteins.

[0089] The aforementioned compositions / formulations may further comprise an agriculturally acceptable carrier such as a bait, powder, dust, pellet, granule, spray, emulsion, colloidal suspension, aqueous solution, Bacillus spore / crystal preparation, seed treatment, recombinant plant cells / plant tissues / seeds / plants transformed to express one or more proteins, or bacteria transformed to express one or more proteins. Depending on the level of insect inhibition or insecticidal inhibition inherent to the recombinant polypeptide and the level of formulation applied to the plant or feeding assay, the composition / formulation may comprise various amounts (by weight) of the recombinant polypeptide, e.g., 0.0001% to 0.001%, to 0.01%, to 1%, to 99% by weight of the recombinant polypeptide.

[0090] In view of the above, it should be understood that those skilled in the art can make modifications in the specific aspects disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting. It should be understood that the entire disclosure of each reference cited herein is incorporated within the disclosure of this application. EXAMPLES

[0091] Example 1 Discovery, cloning, and expression of TIC2199 The TIC2199 insecticidal protein was identified by sequence analysis of the genome of Bacillus thuringiensis (Bt) strain EG8639. Bt strain EG8639 was first identified as a strain of Bt or Bt-like bacteria that is sporulating, crystalline, and contains a plasmid. DNA was isolated from EG8639 and sequenced. The assembled sequence was then biologically analyzed. The TIC2199 protein was identified by pfam analysis for endotoxin domain hits and identity to known Cry1Ib3 toxin (GenBank accession number ACD75515). The amino acid sequence of the full-length TIC2199 protein shows 95.13% identity to the amino acid sequence of the CryIb3 protein of GenBank accession number ACD75515.

[0092] Polymerase chain reaction (PCR) primers were designed to amplify a full-length copy of the coding region of TIC2199 from total genomic DNA isolated from Bt strain EG8639. The PCR amplicons were cloned into two plasmid constructs, one an Escherichia coli (Ec) expression vector operably linked to an Ec-expressible promoter and a histidine tag used for protein purification, and the other a Bt expression vector operably linked to a Bt-expressible promoter, using methods known in the art. Preparations of TIC2199 from both Ec and Bt were used in the bioassays.

[0093] Example 2 TIC2199 demonstrated lepidopteran activity in insect bioassays The insecticidal protein TIC2199 was expressed in recombinant Ec and Bt using the vector described in Example 1, and the resulting proteins expressed in these systems were assayed for toxicity against various species of Lepidoptera, Coleoptera, and Hemiptera.

[0094] Lepidoptera species include the common cutworm moth (Agrotis ipsilon, BCW), the tobacco budworm (Helicoverpa zea, CEW, also known as the soybean podworm), the nettle looper (Trichoplusia ni, CLW), the European corn borer (Ostrinia nubilalis, ECB), the fall armyworm (Spodoptera frugiperda, FAW), the southern armyworm (Spodoptera eridania, SAW), the soybean looper (Chrysodeixis, SBL), the sugarcane borer (Diatraea saccharalis, SCB), the southwestern corn borer (Diatraea grandiosella, SWC), and the velvet bean caterpillar (Anticarsia gemmatalis, VBC), and the coleoptera species the western corn rootworm (Diabrotica TIC2199 was assayed for toxicity against the hemipteran species Lygus virgifera (SCR), Diabrotica undecimpunctata howardii (SCR), and the hemipteran species Lygus lineolaris (TPB), Lygus hesperus (Lygus hesperus), and Euschistus heros (NBSB). The bioassay results are shown in Table 2 below, where "+" indicates activity and "-" indicates inactivity. [Table 2]

[0095] As can be seen from the data presented in Table 2, TIC2199 demonstrated activity against the lepidopteran species BCW, CLW, ECB, FAW, SAW, SBL, SCB, SWC, VBC, and WBC, and the hemipteran species TPB and NBSB.

[0096] Example 3 Design of an artificial coding sequence of TIC2199 for use in plant expression. Artificial coding sequences of SEQ ID NOs: 3, 6, 9, 12, 14, 16, and 18 encoding TIC2199, as well as truncated forms of TIC2199, were designed for expression in plant cells. The artificial (or otherwise referred to as synthetic) sequences were synthesized according to methods generally described in U.S. Patent No. 5,500,365 to preserve the amino acid sequence of the native Bacillus protein while avoiding certain disadvantageous and problematic sequences such as ATTTA and A / T-rich plant polyadenylation sequences.

[0097] Artificial sequences encoding TIC2199, TIC2199_1, TIC2199_2, and TIC2199_3 were cloned into plant transformation vectors using techniques known in the art, operably linked downstream of a plant promoter driving expression of the coding sequence in plant cells. Five of the resulting transformation vectors used to transform corn plants included a first transgene cassette for expression of the TIC2199 insecticidal protein, the first transgene cassette including a constitutive promoter and operably linked 5' to a leader, operably linked 5' to an intron, operably linked 5' to an artificial coding sequence encoding TIC2199, which in turn was operably linked 5' to a 3'UTR, and a second transgene cassette for selecting transformed plant cells using glyphosate selection. Nine plant transformation vectors also contained a chloroplast transit peptide (CTP) operably linked and cloned in frame 5' to the TIC2199 coding sequence, which directs accumulation of the protein in chloroplasts. Six of the nine plant transformation vectors containing a chloroplast transit peptide contained the coding sequence of SEQ ID NO:3, 6, 9, or 12. Three of the nine plant transformation vectors containing a chloroplast transit peptide contained the coding sequence of SEQ ID NO:14, 16, or 18.

[0098] Example 4 TIC2199 is When expressed in stably transformed maize plants it exhibits lepidopteran activity. Binary plant transformation vectors containing transgene cassettes designed to express the insecticidal proteins of TIC2199 and TIC2199 truncation variants were cloned using methods known in the art. The resulting vectors were used to stably transform maize plants. Tissues were harvested from transformants and used in insect bioassays against various lepidopteran insect species.

[0099] Corn plants were transformed with the six binary transformation vectors described in Example 3 using Agrobacterium-mediated transformation methods. The transformed cells were induced to form plants by methods known in the art. Bioassays using plant leaf discs were performed similarly to those described in U.S. Pat. No. 8,344,207. A single newly hatched neonate larva less than one day old was placed on each leaf disc sample and allowed to feed for approximately four days. Non-transformed corn plants were used to obtain tissue and serve as negative controls. Multiple transformed R0 single copy insertion events from each binary vector were evaluated against Corn Cutworm (BCW, Agrotis ipsilon), European Corn Borer (ECB, Ostrinia nubilalis), Fall Armyworm (FAW, Spodoptera frugiperda), and Southwestern Corn Borer (SWC, Diatraea grandiosella). Based on the percentage of leaf damage in the bioassay, an efficacy rating score ranging from 0 to 3 (0 being no effect and 3 being very effective) was assigned to each event.

[0100] Based on the efficacy score, TIC2199, TIC2199_1, TIC2199_2, and TIC2199_3 showed efficacy scores of 3 in transformed R0 single copy events against ECB and SWC with or without CTP, and were highly effective against these two pest species.

[0101] Selected R0 plants derived from the three transformation vectors expressing TIC2199 were crossed with untransformed wild-type elite cultivar plants to produce hybrid seeds. Heterozygous F1 seeds containing TIC2199 were grown and assayed against corn cutworm (BCW, Agrotis ipsilon), European corn borer (ECB, Ostrinia nubilalis), and Southwestern corn borer (SWC, Diatraea grandiosella) as described above. Based on efficacy evaluation scores, stably transformed heterozygous F1 corn plants expressing TIC2199 were highly effective against ECB and SWC with or without CTP.

[0102] Plants expressing TIC2199 were assayed against the sugarcane stem borer (SCB, Diatraea saccharalis) and the common corn moth (LCSB, Elasmopalpus lignosellus) using a leaf disc assay similar to that described above. TIC2199 was effective against the SCB and highly effective against the LCSB.

[0103] Example 5 Assaying the activity of TIC2199 against lepidopteran pests in stably transformed soybean plants The binary plant transformation vector containing the transgene cassette designed to express the TIC2199 insecticidal protein is cloned using methods known in the art. The resulting vector is used to transform stably transformed soybean plants. Tissues are harvested from the transformants and used in insect bioassays against various lepidopteran insect species.

[0104] The artificial coding sequences encoding TIC2199 and TIC2199 truncation variants are cloned into a plant transformation vector construct using techniques known in the art. The resulting transformation vector construct comprises a first transgene cassette for expression of the TIC2199 insecticidal protein, the first transgene cassette comprising a plant-expressible promoter and operably linked 5' to a leader, optionally operably linked 5' to an intron, operably linked 5' to an artificial coding sequence encoding TIC2199, which is in turn operably linked 5' to a 3'UTR, and a second transgene cassette for selecting transformed plant cells using spectinomycin selection. A chloroplast transit peptide (CTP) can optionally be cloned 5' and in frame with the artificial TIC2199 coding sequence.

[0105] Soybean plants are transformed using Agrobacterium-mediated transformation methods. Transformed cells are induced to form plants by methods known in the art. Bioassays using plant leaf discs are performed similarly to those described in U.S. Patent No. 8,344,207. A single newly hatched neonate larva less than one day old is placed on each leaf disc sample and fed for approximately four days. Non-transformed corn plants are used to obtain tissue and serve as negative controls. Multiple transformed R0 single copy insertion events from each binary vector are evaluated against lepidopteran insect species, including but not limited to, common cutworm (BAW, Spodoptera cosmioides), southern armyworm (SAW, Spodoptera eridania), soybean looper (SBL, Chrysodeixis includens), tobacco budworm (SPW, Helicoverpa zea), sunflower looper (SFL, Rachiplusia nu), and velvet bean caterpillar (VBC, Anticarsia gemmatalis). An efficacy rating score ranging from 0 to 3 is assigned to each event based on the percentage of leaf damage in the bioassay for each event and the percentage of events that shared the lowest percentage of damage coverage (penetration rate) as shown in Table 3. [Table 3]

[0106] Example 6 Hemipteran insect pests in stably transformed soybean plants Assay of activity of TIC2199 against Binary plant transformation vectors containing transgene cassettes designed to express the TIC2199 insecticidal protein are cloned using methods known in the art. The resulting vectors are used to transform stably transformed soybean plants. Tissues are harvested from the transformants and used in insect bioassays against various lepidopteran insect species.

[0107] The artificial coding sequence encoding TIC2199 is cloned into a plant transformation vector construct using techniques known in the art. The resulting transformation vector construct is similar to that described above in Example 5. A chloroplast transit peptide (CTP) can optionally be cloned 5' and in frame with the artificial TIC2199 coding sequence.

[0108] Soybean plants are transformed using Agrobacterium-mediated transformation methods. Transformed cells are induced to form plants by methods known in the art. Assays for activity against hemipteran pests are performed using a variety of techniques, which will depend on the species of hemipteran pest and the preferred target tissue of the pest. For example, hemipteran pest species of stink bugs typically feed on the developing seeds and pods of soybean plants. To assay activity against stink bugs, R5 stage pods are taken from transgenic soybean plants expressing TIC2199 and placed in a covered Petri dish or large multi-well plate containing a layer of either agar or wet paper to provide humidity to the feeding environment. Second instar stink bug nymphs are placed in the Petri dish or large multi-well plate. A cover is placed over the feeding environment to provide oxygen exchange while preventing desiccation. The stink bug nymphs are allowed to feed for several days. Stunting and mortality measurements are made and compared to stink bug nymphs feeding on pods of non-transformed soybean plants.

[0109] Alternatively, activity assays can also be performed on whole stably transformed plants. Transformed plants expressing TIC2199 are grown in growth chambers or greenhouses. At the R5 stage, the plants are placed in cages made of breathable plastic "pollination" sheeting (Vilutis and Company Inc, Frankfort, IL). A Velcro® tie is used to secure the sheet sleeve to the main stem just above the soil surface. Each plant is infested with a specific number of second instar stink bug nymphs. The nymphs are released into individual cages through small slits in the cage side, and the cages are tightly closed to prevent insect escape. The nymphs are allowed to feed on soybean pods for several days to a week or more. Daily observations are made to determine stunting and mortality measurements. At the end of the feeding period, live and dead nymphs are collected. The plants are cut under the cage and transferred to the laboratory, and the insects are collected per plant. Before opening the cage, the plants are shaken vigorously to ensure that all insects have fallen from the feeding site to the base of the cage. The base of the cage is then opened and all plant material is removed and placed on a black sheet. An aspirator or some other means can be used to collect the insects. The number of insects and their developmental stage are recorded for each plant. Also, the number and developmental stage of dead nymphs are recorded. These measurements are compared to those of negative controls obtained from non-transformed plants.

[0110] Delay in development of stink bug nymphs (stunting) or mortality is interpreted as an indication of toxicity if significantly different when compared to untransformed controls.

[0111] Example 7 Hemipteran insect pests in stably transformed cotton plants Assay of activity of TIC2199 against This example describes assays of activity against hemipteran insect pests in cotton plants stably transformed to express TIC2199.

[0112] Binary plant transformation vectors containing transgene cassettes designed to express both plastid-targeted and non-targeted TIC2199 are cloned using methods known in the art.

[0113] Cotton plants are transformed using the binary plant transformation vector. Transformed cotton plant cells are induced to form whole plants. Assays for activity against hemipteran pests are performed using a variety of techniques, which will depend on the species of hemipteran pest and the preferred target tissue of the pest. For example, hemipteran pest species of stink bugs are typically seed feeding, and therefore damage to cotton bolls is a primary concern. They primarily damage cotton by drilling holes in the cotton seed and eating the seeds. Their feeding activity can result in black spots approximately 1 / 16 inch in diameter on the outside of larger cotton seeds where feeding has taken place. Seed feeding can result in reduced lint production and lint staining near the feeding site. Adults and 4th and 5th instar nymphs have the most potential to damage cotton seeds due to their size, therefore it is important to kill insects at the earlier nymphal stages. Hemiptera pest species of Lygus feed primarily on squares and young cottonseeds. Nymphs are the more voracious feeders and tend to cause the most severe damage. When feeding on squares, the Lygus targets developing anthers, often causing the squares to wither and fall off the plant. For those squares that develop on the cottonseed, the cottonseed may have anthers that cannot form pollen, unfertilized seeds, and empty locules. When feeding on cottonseed, the Lygus targets developing seeds, causing small sunken black spots on the outside of the cottonseed.

[0114] One method to assay the activity of TIC2199 in stably transformed cotton plants is to use squares in an insect bioassay. Squares are taken from transformed cotton plants expressing TIC2199. The squares can be placed in Petri dishes or each square can be placed in a well of a large well plate. Young neonate mirid bugs or stink bug nymphs are placed in the Petri dishes or large well plates and allowed to feed for a given period of time. Stunting and mortality measurements are made over the period of feeding and compared to a control (squares from untransformed cotton plants are used in the assay).

[0115] Alternatively, assays for activity may be performed on whole transformed cotton plants. For example, for assays against mirid bug species, R1 seeds from one TIC2199 expressing plant are sown in 10-inch pots. Non-transformed cotton plants, preferably from the same variety as the transformed plants, are used as negative controls. Plants are maintained in an environmental chamber with 16 hours of light at 32°C, 8 hours of darkness at 23°C, and a light intensity of 800-900 micro-Einsteins. 40-45 days after planting, individual plants are placed into cages made of breathable plastic "pollination" sheeting (Vilutis and Company Inc, Frankfort, IL). Velcro® ties are used to secure the sheet sleeve to the main stem just above the soil surface. Two pairs of sexually mature male and female Lygus lineolaris or Lygus hesperus adults (6 days old) from laboratory cultures are collected into 14 milliliter round-bottom plastic tubes (Becton Dickinson Labware, Franklin Lakes, NJ) for each plant. Adults are released into individual cages through a small slit in the cage side, and the cages are tightly closed to prevent insects from escaping. The insects are allowed to mate, and the plants are maintained in the cages for 21 days.

[0116] After 21 days, the plants are then cut under the cage and transferred to the laboratory, and the insects are collected and counted per plant. Before opening the cage, the plants are vigorously shaken to ensure that all insects have fallen from the feeding site to the base of the cage. The base of the cage is then opened and all plant material is removed and placed on a black sheet. An aspirator is used to collect the insects. The plants are then thoroughly inspected to collect any remaining insects. The number of insects collected and their developmental stage are recorded per plant. The number of insects is divided into several groups based on the maturity of the mirid bugs (nymphs up to 3rd instar, nymphs up to 4th instar, nymphs up to 5th instar, adults).

[0117] To assay for stink bug species, R1 seeds from plants expressing TIC2199 are sown in pots, grown and caged as above. Non-transformed cotton plants are also used as negative controls. Second instar stink bug nymphs are used to infest the plants and allowed to feed on the squares and cottonseeds for several days or weeks. Caged plants are harvested as above and the harvested stink bugs are inspected and scored for mortality, as well as the maturity of the nymphs. These scores are then compared to the negative control plants.

[0118] All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions of the present invention have been described with respect to the above exemplary embodiments, it will be apparent to those skilled in the art that variations, changes, modifications, and alterations may be applied to the compositions 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 chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0119] All publications and published patent documents cited in this specification are herein incorporated 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

**Claim 1** A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding an insecticidal protein or an insecticidal fragment thereof, optionally a. the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2, or b. the insecticidal protein comprises an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2, or c. the polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 18, 6, 5, 8, 9, 14, 11, 12, 16, 3 or 1, said recombinant nucleic acid molecule. **Claim 2** a. the recombinant nucleic acid molecule is expressed in a plant cell to produce an insecticidally effective amount of the insecticidal protein or insecticidal fragment, or b. the recombinant nucleic acid molecule is operably linked to a vector, said vector being selected from the group consisting of plasmids, phagemids, bacmids, cosmids, and bacterial or yeast artificial chromosomes, the recombinant nucleic acid molecule according to claim 1. **Claim 3** The recombinant nucleic acid molecule according to claim 1, which is present in a host cell, said host cell being selected from the group consisting of bacterial cells and plant cells. **Claim 4** The recombinant nucleic acid molecule according to claim 3, wherein the bacterial host cell is derived from a bacterial genus selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia. **Claim 5** The recombinant nucleic acid molecule according to claim 4, wherein the Bacillus is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus is Brevibacillus laterosporus, and the Escherichia is Escherichia coli. **Claim 6** The recombinant nucleic acid according to claim 2, wherein the plant cell is a dicotyledonous plant cell or a monocotyledonous plant cell. **Claim 7** The recombinant nucleic acid according to claim 6, wherein the plant cell is selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, Brassica, canola, carrot, cassava, castor bean, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucumber family, cucumber, white pine, eggplant, eucalyptus, flax, garlic, grape, hop, chive, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, forage grass, pea, peanut, pepper, kidney bean, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn, Japanese maple, sweet potato, switchgrass, tea, tobacco, tomato, triticale, Japanese mugwort, watermelon, and wheat plant cells.

8. The recombinant nucleic acid molecule according to claim 1, wherein the protein exhibits activity against Lepidoptera insects.

9. The recombinant nucleic acid molecule according to claim 8, wherein the Lepidoptera insect is selected from the group consisting of Agrotis ipsilon, Helicoverpa zea, Trichoplusia ni, Ostrinia nubilalis, Spodoptera frugiperda, Spodoptera eridania, Chrysodeixis includens, Diatraea grandiosella, Diatraea saccharalis, Anticarsia gemmatalis, Striacosta albicosta, and Elasmopalpus lignosellus.

10. The recombinant nucleic acid molecule according to claim 1, wherein the protein exhibits activity against Hemiptera insects.

11. The recombinant nucleic acid molecule according to claim 10, wherein the hemipteran insect is selected from the group consisting of Lygus lineolaris and Euschistus heros.

12. A plant comprising the recombinant nucleic acid molecule according to claim 1 or a portion thereof.

13. The plant according to claim 12, wherein the plant is a monocotyledon or a dicotyledon, or a portion thereof.

14. The plant according to claim 12, wherein the plant is selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, Brassica, canola, carrot, cassava, castor bean, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucumber family, cucumber, fir, eggplant, eucalyptus, flax, garlic, grape, hop, leek, lettuce, loblolly pine, millet, melon, nut, oats, olive, onion, ornamental plant, palm, pasture, pea, peanut, pepper, kidney bean, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, tanoak, sweet potato, switchgrass, tea, tobacco, tomato, triticale, wormwood, watermelon, and wheat.

15. The plant according to claim 12, wherein the portion of the plant is a seed, and the seed contains the recombinant nucleic acid molecule.

16. An insect-inhibiting composition comprising the recombinant nucleic acid molecule according to claim 1.

17. The insect-inhibiting composition according to claim 16, further comprising a nucleotide sequence encoding at least one other insecticide different from the insecticidal protein.

18. The at least one other insecticide is selected from the group consisting of an insect-inhibiting protein, an insect-inhibiting dsRNA molecule, a chemical molecule, and an auxiliary protein, and the at least one other insecticide is toxic to the same pest as the insecticidal protein or an insecticidal fragment thereof. The insect-inhibiting composition according to claim 17.

19. The insect-inhibiting composition according to claim 17, wherein the at least one other insecticide exhibits activity against one or more pest species of Lepidoptera, Coleoptera, or Hemiptera.

20. The at least one other pesticidal protein is Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variant, Cry1D, Cry1D variant, Cry1E, Cry1F, Cry1A / F chimeric, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variant, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and its variants, IP3 and its variants, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IDP102Aa and its homologs, IDP110Aa and its homologs, TIC868, Cry1Da1_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757, TIC7641, IDP072Aa, TIC5290, TIC3668, TIC3669, TIC3670, IDP103 and its homologs, PIP-50 and PIP-65 and their homologs, PIP-83 and its homologs,The insect-inhibiting composition according to claim 17, which is selected from the group consisting of Cry1B.34.,

21. The insect-inhibiting composition according to claim 16, defined as comprising a plant cell expressing said insecticidal protein from the recombinant nucleic acid molecule according to claim 1.

22. A product produced from the plant or a part thereof according to claim 12, said product comprising a detectable amount of said recombinant nucleic acid molecule, said insecticidal protein, or an insecticidal fragment thereof.

23. Products of corn bagged by grain handlers (corn flakes, corn cakes, corn flour, corn meal, corn syrup, corn oil, corn silage, corn starch, corn cereal, etc.), as well as corresponding soybean, rice, wheat, sorghum, chickpea, peanut, fruit, melon, and vegetable products, and, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such products containing a detectable amount of such polynucleotides and / or polypeptides of the present application, whole or processed cottonseed, cottonseed oil, lint, seeds, and plant parts processed for feed or food, fibers, paper, biomass, and fuel products such as fuel derived from cottonseed oil or pellets derived from cotton gin waste, whole or processed soybean seeds, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean hulls, soy milk, soybean cheese, soybean wine, animal feed containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass, and fuel products manufactured using soybean plants and parts of soybean plants, said product selected from the group consisting of said products according to claim 22.

24. A method for producing progeny seeds comprising the recombinant nucleic acid molecule according to claim 1, said method comprising: a. planting a first seed comprising said recombinant nucleic acid molecule; b. growing a plant from said seed of step a; c. collecting said progeny seeds from said plant, said collecting being such that the collected seeds comprise said recombinant nucleic acid molecule.

25. A plant resistant to the spread of insects, the cells of said plant comprising the recombinant nucleic acid molecule according to claim 1.

26. A method for controlling pests of Lepidoptera species or the spread of pests, said method comprising: a. contacting said pests with an insecticidally effective amount of an insecticidal protein shown in SEQ ID NO: 17, 4, 13, 7, 15, 10, or 2, or b. contacting the pest with an insecticidally effective amount of one or more insecticidal proteins comprising an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2;

27. A method for detecting the presence of the recombinant nucleic acid molecule according to claim 1 in a sample containing plant genomic DNA, comprising: a. contacting the sample with a nucleic acid probe that hybridizes with genomic DNA derived from a plant comprising the recombinant nucleic acid molecule according to claim 1 under stringent hybridization conditions, but does not hybridize with genomic DNA derived from an otherwise isogenic plant that does not comprise the recombinant nucleic acid molecule according to claim 1 under such hybridization conditions, wherein the probe is homologous or complementary to SEQ ID NO: 18, 6, 9, 14, 12, 16 or 3, or is a sequence encoding an insecticidal protein comprising an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2; b. subjecting the sample and the probe to stringent hybridization conditions; c. detecting hybridization between the nucleic acid probe and the recombinant nucleic acid molecule.

28. A method for detecting the presence of an insecticidal protein or a fragment thereof in a sample containing a protein, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 4 or 2, or the insecticidal protein comprises an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2, the method comprising: a. contacting the sample with an immunoreactive antibody; b. detecting the presence of the insecticidal protein or a fragment thereof.

29. The method according to claim 26, wherein the detecting step comprises ELISA or Western blot.

30. A protein in an insecticidally effective amount comprising the amino acid sequence set forth in SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2.

31. A method for controlling the spread of lepidopteran pest species or pests in the field, said method comprising: a. growing a crop plant that expresses an insecticidally effective amount of an insecticidal protein shown in SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2; or b. growing a crop plant that expresses an insecticidally effective amount of one or more insecticidal proteins comprising an amino acid sequence having at least 96%, or 97%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 17, 4, 13, 7, 15, 10 or 2; and optionally c. releasing a transgenic lepidopteran pest species having a self-limiting gene into the field to reduce the likelihood of development of resistance of the pest species to the insecticidal protein.

32. The method according to claim 31, wherein the crop plant is a monocotyledonous crop plant or a dicotyledonous crop plant.

33. The method according to claim 32, wherein the monocotyledonous crop plant is maize, wheat, sorghum, rice, rye or oats.

34. The method according to claim 33, wherein the monocotyledonous crop plant is maize.

35. The method according to claim 31, wherein the dicotyledonous crop plant is soybean, cotton or canola.