Novel insect inhibitory proteins

Abstract

Pesticidal proteins exhibiting toxic activity against Lepidopteran pest species are disclosed, and include, but are not limited to, TIC8643 and related pesticidal proteins. DNA constructs are provided which encode the disclosed pesticidal proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran infestation are provided which contain recombinant nucleic acid sequences encoding the pesticidal proteins of the present invention, and vectors are described which contain at least a coding sequence for expression and the delivery of the encoded toxin proteins. Methods for detecting the presence of the recombinant nucleic acid sequences or the proteins of the present invention in a biological sample, and methods of controlling Lepidopteran species pests using TIC8643 and related pesticidal proteins are also provided.

Description

TITLE OF THE INVENTIONNOVEL INSECT INHIBITORY PROTEINSREFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States provisional application No. 63 / 515,052, filed July 21, 2023, which is herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The file named “BCS236201_01_US_SEQLISTING_ST26.xml” containing a computer- readable form of the Sequence Listing was created on May 31, 2024. This file is 8,558 bytes (measured in MS-Windows®), filed contemporaneously by electronic submission (using the United States Patent Office Patent Center), and incorporated by reference in its entirety.FIELD

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

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

[0005] Insects, particularly insects within the order Lepidoptera, are a major cause of damage to field crops, thereby decreasing crop yields in infested areas. Lcpidoptcran pest species which negatively impact agriculture include, but are not limited to, Black armyworm (Spodoptera cosmioides), Black cutworm (Agrotis ipsilon ), Corn earworm (Helicoverpa zea), Cotton leaf worm (Alabama argillacea), Diamondback moth (Plutella xyloslella), European com borer (Ostrinia nubilalis), Fall armyworm (Spodoptera frugiperda), CrylFal resistant Fall armyworm (Spodoptera frugiperda), Old World bollworm (Helicoverpa armigera), Southern armyworm (Spodoptera eridania), Soybean looper (Chrysodeixis includens), Spotted bollworm (Earias vittella), Southwestern com borer (Diatraea gr audio sella), Sunflower looper (Rachiplusia nu), Tobacco budworm (Heliothis virescens), Tobacco cutworm (Spodoptera litura, also known as cluster caterpillar), Western bean cutworm (Striacosta albicosta), and Velvet bean caterpillar (Anticarsia gemmatalis).

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

[0007] The biological control paradigm shifted when the potential of entomopathogenic bacteria, especially bacteria belonging to the genus Bacillus, were discovered and developed as a biological pest control agent. Strains of the bacterium Bacillus thuringiensis (Bt) have been used as a source for pesticidal proteins since it was discovered that Bt strains show a high toxicity against specific insects. Bt strains produce delta-endotoxins, which are localized within parasporal crystalline inclusion bodies at the onset of sporulation and during the stationary growth phase (e.g., Cry proteins), and also secreted insecticidal proteins. Upon ingestion by a susceptible insect, the deltaendotoxin^) as well as secreted toxin(s) exert their effects at the surface of the midgut epithelium, disrupting the cell membrane, leading to cell disruption and death. Genes encoding insecticidal proteins have been identified in bacterial species other than Bt, including other Bacillus and adiversity of additional bacterial species, such as Brevibacillus laterosporus, Lysinibacillus sphaericus (“Ls” formerly known as Bacillus sphaericus), Pseudomonas species, Paenibacillus popilliae, Paenibacillus lentimorbus, and Streptomyces purpeofuscus . In addition, insecticidal toxins have also been identified from a variety of non-bacterial sources, including ferns, arachnid venoms, and by delivery of dsRNA in a diet of a pest, wherein the dsRNA targets for suppression an essential gene of the pest. Each of these toxins or approaches have been found to provide an effective pest management strategy in various cases.

[0008] Crystalline and secreted soluble insecticidal toxins are highly specific for their hosts and have gained worldwide acceptance as alternatives to chemical insecticides. For example, insecticidal toxin proteins have been employed in various agricultural applications to protect agriculturally important plants from insect infestations, decrease the need for chemical pesticide applications, and increase yields. Insecticidal toxin proteins are used to control agriculturally relevant pests of crop plants by mechanical methods, such as spraying to disperse microbial formulations containing various bacteria strains onto plant surfaces, and by using genetic transformation techniques to produce transgenic plants and seeds expressing insecticidal toxin protein(s). The use of transgenic plants expressing insecticidal toxin proteins has been globally adopted. 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 stacked with herbicide tolerance traits (ISAAA. 2016. Global Status of Commercialized Biotech / GM Crops: 2016. ISAAA Brief No. 52. ISAAA: Ithaca, NY).

[0009] The global use of transgenic insect-protected crops, and the limited number of insecticidal toxin proteins used in these crops, has created a selection pressure for existing insect gene alleles that can impart resistance of the insect pest to currently utilized insecticidal proteins. The development of resistance in target pests to insecticidal toxin proteins creates the continuing need for discovery and development of new forms of insecticidal toxin proteins that are useful for managing the increase in insect resistance to transgenic crops expressing insecticidal toxin proteins. New protein toxins with improved efficacy and which exhibit control over a broader spectrum of susceptible insect species will reduce the number of surviving insects which can develop resistance alleles. In addition, the use in one plant of two or more transgenic insecticidal toxin proteins that are toxic to the same insect pest but have different modes of action, oralternatively two or more different modes of toxic action (for example, a transgene encoding a dsRNA targeting an essential gene for suppression coupled with a transgcnc that encodes a peptide or protein toxin, both toxic to the same insect species), reduces the probability of resistance in any single target insect species. Additionally, use of self-limiting technologies, such as those provided by Oxitec® Ltd, together with the proteins of the present invention, should improve durability of the traits imparted to transgenic crops expressing proteins of the present invention (See, e.g., 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; and Alphey et al. 2009 Combining pest control and resistance management: synergy of engineered insects with Bl crops. Journal of Economic Entomology, 102: 717-732).

[0010] Thus, there is a need in the art for new toxin proteins that are efficacious against target pests and can provide effective control of those target pests in an agricultural field, and that are capable of being expressed in plants without causing undesirable agronomic issues and provide an alternative mode of action compared to current toxins that are used commercially in plants.SUMMARY

[0011] Disclosed herein is a novel pesticidal protein, TIC8643, which is shown to exhibit inhibitory activity against one or more Lepidopteran pests of crop plants. The TIC8643 protein, or related proteins or fragments thereof, can be used alone or in combination with other insecticidal protein(s) and / or toxic agent(s) in formulations and / or in planta, thus providing alternatives to insecticidal proteins and insecticide chemistries currently in use in agricultural systems.

[0012] In one embodiment, disclosed in this application is a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment or sequence encoding a pesticidal protein or pesticidal fragment thereof, wherein the pesticidal protein comprises the amino acid sequence of SEQ ID NOs: 2 or 4; or the pesticidal protein comprises an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91 %, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% amino acid sequence identity, or about 100% amino acid sequence identity, to SEQ ID NOs: 2 or 4; or the polynucleotide segment or sequence hybridizesunder stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NOs: 1 or 3. The recombinant nucleic acid molecule can comprise a sequence that functions to express the pesticidal protein in a plant, and which when expressed in a plant cell produces a pesticidally effective amount of the pesticidal protein or a pesticidal fragment thereof.

[0013] In another embodiment of this application, a recombinant nucleic acid molecule is present within a bacterial or plant host cell. Contemplated bacterial host cells include at least the genus of Agrobacterium, Rhiz.obium. Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia. In certain embodiments, the Bacillus species is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus is a Brevibacillus laterosporus, or the Escherichia is a Escherichia coli. Contemplated plant host cells include a dicotyledonous plant cell and a monocotyledonous plant cell. Contemplated plant cells further include an alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g. canola), carrot, cassava, cowpea, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (Gossypium sp.), a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell.

[0014] In another embodiment, the pesticidal protein exhibits activity against Lepidopteran insects, such as Black cutworm (Agrotis ipsilori), Com earworm (Helicoverpa zea), and Fall army worm (Spodoptera frugiperda) .

[0015] Also contemplated in this application are bacteria and plants and plant parts comprising a recombinant nucleic acid molecule encoding the pesticidal protein TIC8643 or a pesticidal protein from the TIC8643 toxin protein class, or fragment thereof. The recombinant molecule (e.g. construct) may comprise a heterologous promoter for expression in bacterial or plant cells of the operably linked polynucleotide segment or sequence encoding the pesticidal protein. Both dicotyledonous plants and monocotyledonous plants are contemplated. In another embodiment, the plant is further selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinesecabbage, citrus, coconut, coffee, corn, clover, cotton (i.e., Gossypium sp.), a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, com (i.e., maize) such as sweet corn or field com, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. The plant parts may for instance include, without limitation, leaves, tubers, roots, stems, seeds, embryos, flowers, inflorescences, bolls, pollen, fruit, animal feed, and biomass. Processed plant parts, for instance wood, or oil, non- viable ground seeds or fractionated seeds, flour, or starch produced from the plant leaves, flowers, roots, seeds or tubers containing the nucleic acids encoding the proteins of the present invention, and / or containing pesticidally effective amounts of the encoded toxin proteins, are also contemplated.

[0016] In certain embodiments, seeds comprising a recombinant nucleic acid molecule encoding a pesticidally active TIC8643 toxin protein or a pesticidal protein from the TIC8643 toxin protein class, or a fragment thereof, and / or a pesticidally effective amount of the TIC8643 toxin protein or a pesticidal protein from the TIC8643 toxin protein class, or a fragment thereof, are disclosed.

[0017] In still another embodiment, an insect inhibitory composition comprising a recombinant nucleic acid molecule disclosed in this application are contemplated. The insect inhibitory composition can further comprise a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. In certain embodiments, the at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, and an ancillary protein. It is also contemplated that the at least one other pesticidal agent in the insect inhibitory composition exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, and / or Hemiptera. The at least one other pesticidal agent in the insect inhibitory composition may, in some embodiments, be selected from the group consisting of a CrylA, CrylAb, CrylAc, CrylA.105, CrylAe, CrylB, CrylC, CrylC variants, CrylD, CrylE, CrylF, CrylA / F chimeras, CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry34, Cry35, Cry43A, Cry43B, Cry51Aal, ET29, ET33, ET34, ET35, ET66, ET70, TIC400,TIC407, TIC417, TIC431 , TIC800, TIC807, TIC834, TIC853, TIC900, TIC901 , TIC1201 , TIC1415, TIC2160, TIC3131, TIC836, TIC86O, 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 variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG- 10, DIG-657, DIG- 11 protein, IPD102Aa and homologs thereof, IPDl lOAa and homologs thereof, TIC868, CrylDal_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757. TIC7641, TIC5290, TIC3668, TIC3669, TIC3670, TIC2199, TIC4064, TIC4029, TIC13085, TIC13087, IPD072Aa, IPD079Ea, and IPD103 and homologs thereof, PIP-50 and PIP-65 and homologs thereof, PIP-83 and homologs thereof, and CrylB.34; and dsRNA mediated gene suppression embodiments including those targeting for suppression Diabrotica species genes Dv snf7 and Dv ssjl.

[0018] Commodity products comprising a detectable amount of a recombinant nucleic acid molecule(s) and / or toxin protein(s) disclosed in this application are also contemplated. Such commodity products include commodity corn bagged by a grain handler, corn flakes, corn cakes, com flour, com meal, corn syrup, corn oil, corn silage, corn starch, corn cereal, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity products including, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such commodity products containing a detectable amount of such polynucleotide(s) and / or polypeptide(s) of this application, whole or processed cotton seed, cotton oil, lint, seeds and plant parts processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts.

[0019] Also contemplated in this application is a method of producing seed comprising a recombinant nucleic acid molecule encoding a pesticidally active TIC8643 toxin protein or a pesticidal protein from the TIC8643 toxin protein class, or a fragment thereof, and / or a pesticidallyeffective amount of the TIC8643 toxin protein or a pesticidal protein from the TIC8643 toxin protein class, or a fragment thereof. The method comprises planting at least one seed comprising a recombinant nucleic acid molecule(s) disclosed in this application; growing a plant from the seed; and harvesting seed from the plant, wherein the harvested seed comprises the referenced recombinant nucleic acid molecule(s) and / or a pesticidally effective amount of the encoded TIC8643 toxin protein or a pesticidal protein from the TIC8643 toxin protein class, or a fragment thereof.

[0020] In another illustrative embodiment, a plant resistant to Lepidopteran insect infestation, is provided wherein the cells of said plant comprise the recombinant nucleic acid molecules disclosed herein.

[0021] Also disclosed in this application are methods for controlling a Lepidopteran species pest and controlling a Lepidopteran species pest infestation of a plant, particularly a crop plant. The method comprises, in one embodiment, first contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NOs: 2 or 4 or a related protein or fragment thereof; or contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% amino acid sequence identity, or about 100% amino acid sequence identity, to SEQ ID NOs: 2 or 4.

[0022] Further provided herein is a method of detecting the presence of a recombinant nucleic acid molecule encoding a pesticidal protein within the TIC8643 toxin protein class wherein the method comprises contacting a sample of nucleic acids with a nucleic acid probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant comprising a polynucleotide segment or sequence encoding a pesticidal protein or fragment thereof provided herein, and does not hybridize under such hybridization conditions with genomic DNA from an otherwise isogenic plant that does not comprise the segment or sequence, wherein the probe is homologous or complementary to SEQ ID NO: 3, or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%,or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91 %, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% amino acid sequence identity, or about 100% amino acid sequence identity, to SEQ ID NOs: 2 or 4; subjecting the sample and probe to stringent hybridization conditions; and detecting hybridization of the probe with DNA of the sample. In some embodiments a step of detecting the presence of a member of the TIC8643 toxin protein class may comprise an ELISA or a western blot.

[0023] Also provided herein are methods of detecting the presence of the pesticidal protein or fragment thereof from the TIC8643 toxin protein class wherein the method comprises contacting a sample with a TIC8643 toxin protein class immunoreactive antibody or recombinant protein designed for detecting the TIC8643 protein and detecting the binding of the antibody to the TIC8643 toxin protein class protein, thus confirming the presence of the protein in the sample. In some embodiments the step of detecting comprises an ELISA, or a Western blot.

[0024] Also contemplated in this application is a method for controlling a Lepidopteran pest species or pest infestation in a field wherein the method comprises growing a crop plant which expresses an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NOs: 2 or 4; or growing a crop plant which expresses an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81 %, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% amino acid sequence identity, or about 100% amino acid sequence identity, to SEQ ID NOs: 2 or 4; and releasing into the field with crops containing a gene encoding the toxin protein of the present invention, one or more transgenic Lepidopteran pest species each carrying a self-limiting gene, for the purpose of preventing or delaying the onset of resistance of the one or more Lepidopteran pest species to the toxin protein. In one embodiment, the crop plants can be monocoty ledonous or dicotyledonous. In another embodiment, the monocotyledonous crop plants can be com, wheat, sorghum, rice, rye, or millet. In yet another embodiment, the dicotyledonous crop plant can be soybean, cotton, alfalfa, cowpea, cassava or canola.BRIEF DESCRIPTION OF THE SEQUENCES

[0025] SEQ ID NO: 1 is a native / naturally occurring nucleic acid sequence obtained from Streptomyces _purpeofuscus species MDI-0021357 encoding a TIC8643 pesticidal protein.

[0026] SEQ ID NO: 2 is the amino acid sequence of the TIC8643 pesticidal protein encoded by the sequence set forth in SEQ ID NO: 1.

[0027] SEQ ID NO: 3 is an artificial sequence encoding a TIC8643PL pesticidal protein that is designed for expression in a plant cell, wherein an alanine codon is inserted at amino acid position two (2) following the methionine codon initiating translation of the open reading frame.

[0028] SEQ ID NO: 4 is the amino acid sequence of the TIC8643PL pesticidal protein encoded by the coding sequence / open reading frame set forth in SEQ ID NO: 3.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1A is a graphical representation of data showing the average leaf disc damage caused by Black cutworm (Agrotis ipsilori) when feeding on stably transformed corn plants transformed with Construct- 1 expressing TIC8643PL protein.

[0030] FIG. IB is a graphical representation of data showing the average leaf disc damage caused by Black cutworm (Agrotis ipsilori) when feeding on stably transformed corn plants transformed with Construct-2 expressing TIC8643PL protein.

[0031] FIG. 2A is a graphical representation of data showing the average leaf disc damage caused by Fall armyworm (Spodoptera frugiperda) when feeding on stably transformed com plants transformed with Construct- 1 expressing TIC8643PL protein.

[0032] FIG. 2B is a graphical representation of data showing the average leaf disc damage caused by Fall armyworm (Spodoptera frugiperda) when feeding on stably transformed com plants transformed with Construct-2 expressing T1C8643PL protein.

[0033] FIG. 3A is a graphical representation of data showing the average leaf disc damage caused by Corn earworm (Helicoverpa z.ea) when feeding on stably transformed corn plants transformed with Construct- 1 expressing TIC8643PL protein.

[0034] FIG. 3B is a graphical representation of data showing the average leaf disc damage caused by Corn carworm (Helicoverpa zea) when feeding on stably transformed corn plants transformed with Construct-2 expressing TIC8643PL protein.DETAILED DESCRIPTION

[0035] The inventors disclose herein a novel protein from Streptomyces purpeofuscus and related proteins that exhibit insecticidal activity against target Lepidopteran species, particularly against Black cutworm (Agrotis ipsilon), Com earworm (Helicoverpa zea), and / or Fall armyworm (Spodopiera frugiperda). A novel pesticidal protein identified as TIC8643 is disclosed herein, shown as having an amino acid sequence as set forth in SEQ ID NO: 2. Use of the protein, or related proteins or fragments thereof, in pcsticidally effective amounts can address insect infestations that are problems for agricultural crop plants, particularly against a broad spectrum of Lepidopteran insect pests, and more particularly against Black cutworm (Agrotis ipsilon), Corn earworm (Helicoverpa zea), and Fall armyworm (Spodoptera species).

[0036] Reference in this application to “TIC 8643 -related toxins”, “TIC8643 -related proteins”, “related proteins” in relation to T1C8643, “T1C8643 protein toxin class”, or “T1C8643 toxin protein class” is intended to refer to any pesticidal proteins or insect inhibitory proteins, that each comprises, consists of, is substantially homologous to, is similar to, or is derived from the insect inhibitory protein sequence of TIC8643 (SEQ ID NO: 2), and pesticidal or insect inhibitory segments or fragments thereof, or any combinations thereof, that confer insecticidal activity against one or more Lepidopteran pests, including any protein exhibiting pesticidal or insect inhibitory activity if alignment of such protein with TIC8643 results in an amino acid sequence identity in a range from about 65% to about 100%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or about 100% identical to TIC8643 (SEQ ID NO: 2), or any fractional percentage between any of the foregoing. The TIC8643 proteins described herein, and related proteins and fragments thereof, and intended to be within the scope of the present disclosure include both the plastid-targeted (e.g., fused with a chloroplast targeting peptide (CTP)) and non-plastid targeted forms of the proteins.

[0037] The term “segment” or “fragment” as used in this application describes consecutive or continuous amino acid or nucleic acid sequences that arc shorter than the complete amino acid or nucleic acid sequence of a TIC8643 protein or a related protein thereof, as set forth in the sequences provided herein, particularly, for example, the nucleic acid sequence as set forth in SEQ ID NO: 1 encoding the TIC8643 protein or pesticidal proteins from the TIC8643 toxin protein class, and the amino acid sequence as set forth in SEQ ID NO: 2. A segment or fragment of the TIC8643 protein exhibiting insect inhibitory activity of the present disclosure may have an amino acid sequence alignment identity with the corresponding segment or sequence of the TIC8643 amino acid sequence set forth in SEQ ID NO: 2 from about 65% to 100%, such as about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100%, or any percentage range between any of the foregoing. The segment or fragment as described herein may comprise at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 700, or at least 800 consecutive or contiguous amino acid residues of the TIC 8643 protein, or a related protein thereof.

[0038] Reference in this application to the terms “pesticidal activity” or “pesticidal” or “insecticidal activity”, “insect inhibitory”, “pesticidally effective” or “insecticidal” are intended to refer to efficacy of a toxic agent, such as a protein toxin, in inhibiting (inhibiting growth, feeding, fecundity, or viability), suppressing (suppressing growth, feeding, fecundity, or viability), controlling (controlling the pest infestation, controlling the pest feeding activities on a particular crop), or killing (causing the morbidity, mortality, or reduced fecundity of) an insect pest. Such a toxic agent or protein toxin may comprise an effective amount of the TIC8643 protein or a related protein or fragment thereof. These terms are intended to include the result of providing a pesticidally effective amount of a toxic protein to an insect pest where the exposure of the pest to the toxic protein results in inhibiting, suppressing, controlling, or killing of the pest. These terms also include repulsion of the pest from the plant, a tissue of the plant, a plant pail, seed, plant cells, or from the particular geographic location where the plant may be growing, as a result of providing a pesticidally effective amount of the toxic protein in or on the plant. In general, pesticidal activity,etc., refers to the ability of a toxic protein to be effective in inhibiting the growth, development, viability, feeding behavior, mating behavior, fecundity, or any measurable decrease in the adverse effects imposed upon a plant when caused by an insect feeding. The Lepidopteran specific toxic protein can be produced by the plant or can be applied to the plant or to the environment within the location where the plant is located. The terms “bioactivity”, “effective”, “efficacious” or variations thereof are also terms interchangeably utilized in this application to describe the effects (or effective ability) of pesticidal proteins of the present disclosure on target insect pests.

[0039] A pesticidally effective amount of a toxic agent, when provided in the diet of a target pest, exhibits pesticidal activity when the toxic agent contacts the pest. A toxic agent can be a pesticidal protein or one or more chemical agents known in the art. Pesticidal or insecticidal chemical agents can be used alone or in combinations with each other. Chemical agents include but are not limited to dsRNA molecules targeting specific genes for suppression in a target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal or insecticidal protein agents include the protein toxins set forth in this application, as well as other proteinaceous toxic agents including those that target Lepidopterans, as well as protein toxins that are used to control other plant pests such as Cry, Vip, and Cyt proteins, Pseudomonas insect toxic proteins, and insect toxin proteins derived from fem species, that are available in the ail for use in controlling Coleopteran, Hemipteran and Homopteran species.

[0040] It is intended that reference to a pest, particularly a pest of a crop plant, means insect pests of crop plants, particularly those Lepidoptera insect pests that are controlled by pesticidal proteins within the TIC8643 protein toxin class or a fragment thereof. However, reference to a pest can also include Coleopteran, Hemipteran, and Homopteran insect pests of plants, as well as nematodes and fungi when toxic agents targeting these pests are co-localized or present together with the pesticidal protein within the TIC8643 protein toxin class or a fragment thereof, such as an insecticidal protein have an amino acid sequence that is in a range from about 65% to about 100 percent identical to TIC8643 protein of SEQ ID NO: 2 or any other sequence percentage range within this range as provided herein. The phrases “present together” or “co-localized” are intended to include any instance of which a target insect pest has been contacted by a pesticidal protein in the TIC8643 protein toxin class, including a fragment or related protein of a TIC8643 protein, as well as any other toxic agent also present in a pesticidally effective amount relative to the targetinsect pest. “Contacted” in reference to a pesticidal protein of a TIC8643 protein toxin class or a fragment thereof is intended to refer to being present in the diet of the target pest, and the diet is consumed by the target pest. The diet may be a natural diet or a controlled, artificial or experimental diet, such as for a diet assay.

[0041] The insects of the order Lepidoptera that are intended to be within the scope of the present invention include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the Family Noctuidae, e.g., Fall army worm (Spodoptera frugiperda), Beet army worm (Spodoptera exigua). Black armyworm (Spodoptera cosmioides), Southern armyworm (Spodoptera eridanid), bertha army worm (Mamestra config it rat a), black cutworm (Agrotis ipsilori), cabbage looper worm (Trichoplusia ni), Sugarcane borer (Diatraea saccharalis), soybean looper (Pseudoplusia includens), Sunflower looper (Rachiplusia nu), velvetbean caterpillar (Anticarsia gemmatalis), green cloverworm (Hypena scabra). tobacco budworm (Heliothis virescens), granulate cutworm (Agrotis subterraned), armyworm (Pseudaletia unipuncta), Sunflower looper (Rachiplusia nu), South American podworm (Helicoverpa gelotopoeon) western cutworm (Agrotis orthogonid)’, borers, casebearers, webworms, coneworms, cabbageworms and skeletonizers from the Family Pyralidae, e.g., European corn borer (Ostrinia nubilalis), navel orange worm (Amyelois transitelld), com root webworm (Crambus caliginosellus), sod webworm (Herpetogramma licarsisalis). sunflower moth (Homoeosoma eleclellum). lesser cornstalk borer (Elasmopalpus Hgnosellusg, leafrollers, budworms, seed worms, and fruit worms in the Family Tortricidae, e.g., codling moth (Cydia pomonella), grape berry moth (Endopiza viteand), oriental fruit moth (Grapholita molestd), sunflower bud moth (Suleima heliant hand)’, and many other economically important Lepidoptera, e.g., diamondback moth (Plutella xylostelld), pink bollworm (Pectinophora gossypielld), and gypsy moth (Lymantria dispar). Other insect pests of order Lepidoptera include, e.g. , cotton leaf worm (Alabama argillacea), fruit tree leaf roller (Archips argyrospila), European leafroller (Archips rosana) and other Archips species, (Chilo suppressalis, Asiatic rice borer, or rice stem borer), rice leaf roller (Cnaphalocrocis medinalis), corn root webworm (Crambus caliginosellus , bluegrass webworm (Crambus teterrellus), southwestern com borer (Diatraea grandioselld), surgarcane borer (Diatraea saccharalis), spiny boll worm (Earias insuland), spotted bollworm (Earias vittelld), American bollworm (Helicoverpa armigerd), corn earworm (Helicoverpa zea, also known as soybean podworm and cottonbollworm), tobacco budworm (Pleliothis virescens), sod webworm (Herpetogramma licarsisalis), Western bean cutworm (Striacosta alhicosta). European grape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistis citrelld), large white butterfly (Pieris brassicae), small white butterfly (Pieris rapae, also known as imported cabbageworm), beet armyworm (Spodoptera exigua). tobacco cutworm (Spodoptera litura, also known as cluster caterpillar), and tomato leaf miner (Tula absoluta).

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

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

[0044] As described further in this application, an open reading frame (ORF) (SEQ ID NO: 1) encoding TIC8643 (SEQ ID NO: 2) was discovered in DNA obtained from Slreplomycesj urpeojuscus species MDI-0021357. Bioassay using microbial host cell-derived proteins of TIC8643 demonstrated activity against the Lepidopteran species Black cutworm (BCW, Agrotis ipsilou). Corn earworm (CEW, Helicoverpa zed), and Fall armyworm (FAW, Spodoptera frugiperda).

[0045] A synthetic coding sequence (SEQ ID NO: 3) designed for expression of TIC8643PL protein (SEQ ID NO: 4) in a plant cell was determined and made, and for cloning convenience purposes, contained a bonus or supplemental alanine codon inserted between the initiating methionine codon and the serine codon in the native sequence. Corn plants expressing the artificialprotein containing this supplemental alanine at position two, referred to hereinafter as TIC8643PL, demonstrated activity that was undiminished compared to the native TIC8643 toxin used in bioassay against Black cutworm (BCW, Agrotis ipsilon ) and Fall armyworm (FAW, Spodoptera frugiperda). Pesticidal activity was also observed against Com earworm (CEW, Helicoverpa zed) when provided with corn plant tissue expressing the TIC8643 toxin. Thus, based on this data, and for purposes of nomenclature, there is no insecticidal activity difference between TIC8643 and TIC8643PL.

[0046] For expression in plant cells, the TIC8643 (SEQ ID NOs: 2 or 4) protein, or a related protein or fragment thereof, such as a pesticidal protein within the TIC8643 protein toxin class or a fragment thereof, can be expressed and localized in the cytosol or targeted to various organelles of the plant cell. For example, targeting a protein to the chloroplast may result in increased levels of expressed protein in a transgenic plant while preventing off-phenotypes from occurring if the expressed protein toxin reacts with the cell biology in any unexpected manner. Targeting may also result in an increase in pest resistance efficacy in the transgenic event. A target peptide or transit peptide is a short (3-70 amino acids long) peptide chain that can be fused to a pesticidal protein sequence to become a part of a pesticidal protein and direct the transport of the protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasma membrane. A polynucleotide coding sequence for a pesticidal protein may further include a polynucleotide coding sequence for the target peptide or transit peptide such that the pesticidal protein further includes the target peptide or transit peptide. Some target peptides are cleaved from the protein by signal peptidases after the proteins are transported. For targeting to the chloroplast, proteins contain transit peptides which are around 40-50 amino acids in length. For descriptions of the use of chloroplast transit peptides, see U.S. Patent Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP), which may be fused to, and become a part of, a pesticidal protein. Examples of such isolated CTPs include, but are not limited to, those associated with the small subunit (SSU) of ribulose- 1,5, -bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the lightharvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), and transit peptides described in U.S. Patent No. 7,193,133. It has beendemonstrated in vivo and in vitro that non-chloroplast proteins may be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide such as the Arabidopsis thaliana EPSPS CTP (CTP2) (see, Klee etal.,Mol. Gen. Genet. 210:437-442, 1987) or the Petunia hybrida EPSPS CTP (CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986) has been shown to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants (see, U.S. Patent Nos. 5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP 508909; and EP 924299). For targeting the TIC8643 toxin protein to the chloroplast, a sequence encoding a chloroplast transit peptide is placed 5' in operable linkage and in frame to a synthetic / artificial nucleotide sequence, such as SEQ ID NO: 3 encoding the TIC8643PL toxin protein.

[0047] It is contemplated that additional toxin protein sequences related to TIC8643 can be created using the amino acid sequence of TIC8643, or a related protein or fragment thereof, to create novel proteins with novel properties. The TIC8643 toxin protein can be aligned to toxin proteins related to TIC8643 by from about 65% to about 99.5% or higher percentage identity and combine differences at the aligned amino acid sequence level into one or more novel amino acid sequence variations and making appropriate changes to the recombinant nucleic acid sequence encoding a pesticidal protein within the TIC8643 protein toxin class, such as for improved expression in a plant, plant part or plant cell.

[0048] Amino acid sequence of insecticidal proteins related to the TIC8643 protein are contemplated which could exhibit improved properties relative to the sequence set forth at SEQ ID NO: 2. It is contemplated that improved amino acid sequences within the TIC8643 protein toxin class can be encoded by engineered DNA molecules as transgenes or engineered in planta by using various gene editing methods known in the ail. Such technologies used for genome editing include, but are not limited to, ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activator-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) systems. These genome editing methods can be used to alter the toxin protein coding sequence transformed within a plant cell to a different toxin coding sequence. Specifically, through these methods, one or more codons within the toxin coding sequence may be altered to engineer a new protein amino acid sequence. Alternatively, afragment within the coding sequence may be replaced or deleted, or additional DNA fragments are inserted into the coding sequence, to engineer a new toxin coding sequence. The plant cell comprising the gene edited toxin coding sequence can be used by methods known in the ail to generate whole plants expressing the new toxin protein.

[0049] It is also contemplated that fragments of TIC8643 or related protein thereof can be truncated forms wherein one or more amino acids are deleted from the N-terminal end, the C- terminal end, a middle or interior portion of the protein, or combinations thereof, wherein the fragments and related proteins retain insect inhibitory activity. These protein fragments and related proteins can be naturally occurring or synthetic proteins derived from the TIC8643 protein but should retain insect inhibitory activity that is identical or similar to the TIC8643 protein.

[0050] Proteins that resemble the TIC8643 protein can be identified and compared to each other using various computer-based algorithms known in the art. Amino acid sequence identities reported in this application are a result of a Clustal W alignment using these default parameters: Weight matrix: blosum, Gap opening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps: On, Hydrophilic residues: GPSNDQERK, Residue- specific gap penalties: On (Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percent amino acid identity is further calculated by the product of 100% multiplied by (amino acid identities / length of subject protein). Other alignment algorithms are also available in the art and provide results similar to those obtained using a Clustal W alignment and are contemplated herein.

[0051] It is intended that a protein exhibiting insect inhibitory activity against a Lepidopteran insect species is related to TIC8643 if the insecticidal protein is used in a query, e.g., in a Clustal W alignment, and exhibits at least 65% to about 100% amino acid identity along the length of the query protein that is about 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100%, or any fraction percentage in this range, or is an insecticidal fragment of the foregoing.

[0052] In addition to percent identity, TIC8643 can also be related by primary structure (conserved amino acid motifs), by length and by other characteristics. Characteristics of the TIC8643 and TIC8643PL protein toxins are provided in Table 1.Table 1. Selected characteristics of TIC8643 and TIC8643PL toxin proteins.

[0053] As described further in the Examples of this application, a synthetic or artificial nucleic acid molecule, a nucleotide sequence, encoding TIC8643PL, was designed for use in plants, as set forth in SEQ ID NO: 3. In view of the redundancy of the genetic code, it is within the skill of the art to produce any number of other sequences for encoding the toxin proteins, however, it is understood that the sequences produced for expression in planta should avoid known problems in the art that hinder or limit the efficient expression of the coding sequence, particularly as described in US Patent 5,500,365 and US Patent 7,741,118.

[0054] Expression cassettes and vectors containing a recombinant nucleic acid sequence molecule can be constructed and introduced into plants, particularly such as corn, soybean, or cotton plant cells in accordance with transformation methods and techniques known in the art. For example, Agrotectermm-mediated transformation is described in U.S. Patent Application Publications 2009 / 0138985 Al (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257 Al (sugar beet), U.S. Patent Nos. 5,750,871 (canola), 7,026,528 (wheat), and 6,365,807 (rice), and in Arencibia et al. (1998) Transgenic Res. 7 :213-222 (sugarcane). Methods to transform many crop plants are known in the ail and are described further above. Examples of methods used for transforming Cowpea are provided by Bosibori et al. (Bosibori, B., Gollasch, S., Moore, A., Harding, R., and Higgins, T.J.V. (2019) An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime. Frontiers in plant Science. 10: 1-10)and Che et al. (Che, P., Chang, S., Simon, M, Zhang, Z., Shaharyar, A., Ourada, J. O’Neill, D., Torres-Mendoza, M., Guo, Y., Marasigan, K.M., Vielle-Calzada, J-P., Ozias-Akins, P., Albertsen, M.C., and Jones, T.J. (2021) Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants. The Plant Journal. 106: 817-830.) Examples of methods to transform Cassava are provided by Segatto et al. (Segatto, R., Jones, T., Stretch, D., Albin, C, Chauhan, R.D. and Taylor, N.J. (2022) Agrobacterium- mediated Genetic Transformation of Cassava. Current Protocols e620. 2: 1-36.) Transformed cells can be regenerated into transformed plants that express TIC8643, or a related protein or fragment thereof and demonstrate pesticidal activity through bioassays performed in the presence of Lepidopteran, Coleopteran, or Hemipteran pest larvae using plant leaf disks obtained from the transformed plants. Plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art. Transformed cells can be regenerated into transformed plants that express TIC8643 and demonstrate pesticidal activity through bioassays performed in the presence of Lepidopteran pest larvae, which may involve using plant leaf disks obtained from the transformed plants. Plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming and regenerating plants are known in the art.

[0055] As an alternative to traditional transformation methods, a DNA sequence, such as a transgene, expression cassette(s), etc., may be inserted or integrated into a specific site or locus within the genome of a plant or plant cell via site-directed integration. Recombinant DNA construct(s) and molecule(s) of this disclosure may thus include a donor template sequence comprising at least one transgene, expression cassette, or other DNA sequence for insertion into the genome of the plant or plant cell. Such donor template for site-directed integration may further include one or two homology arms flanking an insertion sequence (i.e., the sequence, transgene, cassette, etc., to be inserted into the plant genome). The recombinant DNA construct(s) of this disclosure may further comprise an expression cassette(s) encoding a site-specific nuclease and / or any associated protein(s) to carry out site-directed integration. These nuclease-expressing cassette(s) may be present in the same molecule or vector as the donor template (in cis) or on a separate molecule or vector (in trans). Several methods for site-directed integration are known in the art involving different proteins (or complexes of proteins and / or guide RNA) that cut thegenomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. Briefly as understood in the art, during the process of repairing the DSB or nick introduced by the nuclease enzyme, the donor template DNA may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the donor template may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion event may occur through non- homologous end joining (NHEJ). Examples of site-specific nucleases that may be used include zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, and RNA- guided endonucleases (e.g., Cas9 or Casl2a). For methods using RNA-guided site-specific nucleases e.g., Cas9 or Casl2a), the recombinant DNA construct(s) will also comprise a sequence encoding one or more guide RNAs to direct the nuclease to the desired site within the plant genome.

[0056] Recombinant nucleic acid molecule compositions that encode bacterial and plant expressed TIC8643 proteins, or related proteins or fragments thereof, as described herein can be expressed with recombinant DNA constructs in which a polynucleotide molecule with an ORF encoding the protein is operably linked to genetic expression elements such as a promoter and any other regulatory element necessary for expression in the system for which the construct is intended. Non-limiting examples include a plant-functional promoter operably linked to a TIC8643 protein, related protein or fragment encoding sequence for expression of the protein in plants or a Bt- functional promoter operably linked to a TIC8643 protein, related protein or fragment encoding sequence for expression of the protein in a Bt bacterium or other Bacillus species. A TIC8643 protein, related protein or fragment encoding sequence may include any polynucleotide sequence encoding a TIC8643 protein or a related protein or fragment thereof as described herein. Other elements can be operably linked to the TIC8643 protein encoding sequence including, but not limited to, enhancers, introns, untranslated leaders, encoded protein immobilization tags (HIS-tag), translocation peptides (i.e., plastid transit peptides, signal peptides), polypeptide sequences for post-translational modifying enzymes, ribosomal binding sites, and RNAi target sites. Exemplary recombinant polynucleotide molecules provided herewith include, but are not limited to, a heterologous promoter operably linked to a polynucleotide sequence, such as SEQ ID NOs: I or 3, that encodes TIC8643 or TIC8643PL as set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or a relatedprotein or fragment thereof. A heterologous promoter can also be operably linked to synthetic I artificial DNA coding sequences encoding a TIC8643 or TIC8643PL, or a related protein or fragment thereof, or a plastid targeted TIC8643 or TIC8643PL, or a related protein or fragment thereof. The codons of a recombinant nucleic acid molecule encoding for proteins disclosed herein can be substituted by synonymous codons (known in the art as a silent substitution), which may be optimized for expression in a plant.

[0057] A recombinant DNA construct comprising a T1C8643 protein, related protein or fragment encoding sequence can further comprise a separate region, segment or sequence of DNA that encodes for one or more insect inhibitory agents, such as an insect inhibitory dsRNA molecule or an ancillary protein, which can be configured to concomitantly express or co-express with the TIC8643 protein, related protein or fragment encoding sequence. Ancillary proteins include, but are not limited to, co-factors, enzymes, binding-partners, or other agents that function to aid in the effectiveness of an insect inhibitory agent, for example, by aiding its expression, influencing its stability in plants, optimizing free energy for oligomerization, augmenting its toxicity, and increasing its spectrum of activity. An ancillary protein may facilitate the uptake of one or more insect inhibitory agents, for example, or potentiate the toxic effects of the toxic agent.

[0058] A recombinant DNA construct can be assembled so that all proteins or dsRNA molecules are expressed from one promoter or each protein or dsRNA molecule is under separate promoter control or some combination thereof. The protein of this invention can be expressed from a multigene expression system, in which TIC8643 or a related protein or fragment thereof, is expressed from a common nucleotide segment or sequence which also contains other open reading frames and promoters, depending on the type of expression system selected. For example, a bacterial multi-gene expression system can utilize a single promoter to drive expression of multiply- linked / tandem open reading frames from within a single operon (i.e., polycistronic expression). In another example, a plant multi-gene expression system can utilize multiply-unlinked or linked expression cassettes, each cassette expressing a different protein or other agent such as one or more dsRNA molecules.

[0059] Recombinant polynucleotides or recombinant DNA constructs comprising a TIC8643 protein, related protein or fragment encoding sequence can be delivered to host cells by vectors, e.g., a plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage, or viralvector. Such vectors can be used to achieve stable or transient expression of a TIC8643 protein, related protein or fragment encoding sequence in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that comprises a TIC8643 protein, related protein or fragment encoding sequence and that is introduced into a host cell is referred in this application as a “transgene”.

[0060] Transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain a recombinant polynucleotide that expresses T1C8643 or a related sequence encoding a family toxin protein are provided herein. The term “bacterial cell” or “bacterium” can include, but is not limited to, an Agrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas, Brevibacillus, Klebsiella, Erwinia, or a Rhizobium cell. The term “plant cell” or “plant” can include but is not limited to a dicotyledonous or monocotyledonous plant. The term “plant cell” or “plant” can also include but is not limited to an alfalfa, Arabidopsis, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, cowpea, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn (i.e. maize, such as sweet com or field corn), sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell or plant. In certain embodiments, transgenic plants and transgenic plant parts regenerated from a transgenic plant cell are provided. In certain embodiments, the transgenic plants can be obtained from a transgenic seed, by cutting, snapping, grinding or otherwise disassociating the part from the plant. In certain embodiments, the plant part can be a seed, a pollen grain, a boll, a leaf, a flower, a stem, a root, or any portion thereof, or a non-regenerable portion of a transgenic plant pail. As used in this context, a “non- regenerable” portion of a transgenic plant part is a portion that can not be induced to form a whole plant or that can not be induced to form a whole plant that is capable of sexual and / or asexual reproduction. In certain embodiments, a non-regenerable portion of a plant part is a portion of a transgenic seed, pollen grain, chloroplast, boll, leaf, flower, stem, or root.

[0061] Methods of making transgenic plants that comprise insect-inhibitory or Lepidoptera- inhibitory amounts of a TIC8643 protein, or a related protein or fragment thereof, arc provided. Such plants can be made by introducing a recombinant polynucleotide that encodes the proteins provided in this application into a plant cell, and selecting a plant derived from said plant cell that expresses an insect- or Lepidoptera-inhibitory amount of the proteins. Plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0062] Processed plant products, wherein the processed product comprises a detectable amount of a TIC8643 protein, or an insect inhibitory related protein, or an insect inhibitory segment or fragment thereof, or a TIC8643 protein encoding sequence, or any distinguishing part or portion of the foregoing, are also provided herein. In certain embodiments, the processed product is selected from the group consisting of plant pails, plant biomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint, hulls, processed seed, and seed. In certain embodiments, the processed product is non-regenerable. The plant product can comprise commodity or other product of commerce derived from a transgenic plant or transgenic plant part, where the commodity or other product can be tracked through commerce by detecting a TIC8643 protein or a related protein or fragment thereof or all or part of a polynucleotide sequence or segment or an expressed RNA that encodes all or part of a TIC8643 protein or a related protein or fragment thereof, or that comprises a distinguishing portion of a TIC8643 protein, related protein or fragment encoding sequence.

[0063] Plants expressing a TIC8643 protein, or a related protein or fragment thereof, can be crossed by breeding with transgenic events expressing other toxin proteins and / or expressing other transgenic traits such as herbicide tolerance genes, genes conferring yield or stress tolerance traits, and the like, or such traits can be combined in a single stacked vector so that the traits are all linked when present within the same transgenic genome.

[0064] As further described in the Examples, TIC8643 protein-encoding sequences and sequences having a substantial percentage identity to the sequences encoding TIC8643, can be identified using methods known to those of ordinary skill in the art such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, the proteins TIC8643 or a related protein or fragment thereof can be used to produce antibodies that bind specifically to related proteins and can be used to screen for and to find other protein members that are closely related.

[0065] Furthermore, nucleotide sequences encoding all or part of TIC8643 toxin protein or a related protein or fragment thereof can be used as probes and primers for screening to identify other members of the TIC8643 protein toxin class using thermal-cycle or isothermal amplification and hybridization methods. For example, oligonucleotides derived from, or similar to, the sequence as set forth in SEQ ID NO: 3 or other transgenic sequence can be used to determine the presence or absence of a TIC8643 related transgene in a deoxyribonucleic acid sample derived from a commodity product. Given the sensitivity of certain nucleic acid detection methods that employ oligonucleotides, it is anticipated that oligonucleotides derived from sequences as set forth in SEQ ID NO: 3 or other transgenic sequence can be used to detect a TIC8643 transgene in commodity products or samples derived from pooled sources where only a fraction of the commodity product or source is derived from a transgenic plant or plant pail containing the transgene. It is further recognized that such oligonucleotides can be used to introduce nucleotide sequence variation in each of SEQ ID NOs: 1 and 3. Such “mutagenesis” oligonucleotides are useful for identification of TIC8643 related proteins or fragments exhibiting a range of insect inhibitory activity or varied expression in transgenic plant host cells.

[0066] Nucleotide sequence homologs, e.g. , insecticidal proteins encoded by nucleotide sequences that hybridize to each or any of the sequences disclosed in this application under stringent hybridization conditions, are also an embodiment of the present invention. The invention also provides a method for detecting a first nucleotide sequence that hybridizes to a second nucleotide sequence, wherein the first nucleotide sequence (or its reverse complement sequence) encodes a pesticidal protein or pesticidal fragment thereof and hybridizes to the second nucleotide sequence. In such a case, the second nucleotide sequence can be any of the nucleotide sequences presented as SEQ ID NOs: 1 or 3 or a nucleotide sequence that hybridizes to any of SEQ ID NOs: 1 or 3 under stringent hybridization conditions. Nucleotide coding sequences hybridize to one another under appropriate hybridization conditions, such as stringent hybridization conditions, and proteins encoded by these nucleotide sequences may cross react with antiserum raised against any one of the other proteins. Stringent hybridization conditions, as defined herein, comprise at least hybridization at 42°C followed by two washes for five minutes each at room temperature with 2X SSC, 0.1 % SDS, followed by two washes for thirty minutes each at 65°C in 0.5X SSC, 0.1 % SDS. Washes at higher temperatures constitute greater stringency, e.g. , hybridization conditions of 68°C,followed by washing at 68°C, in 2xSSC containing 0.1 % SDS, meaning that a higher percentage nucleotide sequence identity will be selected and detected.

[0067] One skilled in the art will recognize that, due to the redundancy of the genetic code, many other sequences are capable of encoding such related proteins, and those sequences, to the extent that they function to express pesticidal proteins either in bacterial strains, such as Bacillus strains, fungal, yeast or other host cells, or in plant cells, are embodiments of the present invention, recognizing of course that many such redundant coding sequences may not hybridize under these conditions to the native Bacillus sequences encoding TIC8643 related proteins. This application contemplates the use of these, and other identification methods known to those of ordinary skill in the art, such as percent identity, to identify sequences encoding TIC8643 related proteins and sequences having a substantial percentage identity to nucleic acid sequences encoding proteins related to TIC8643.

[0068] This disclosure also contemplates the use of molecular methods known in the art to engineer and clone commercially useful proteins comprising chimeras of proteins from pesticidal proteins; e.g., the chimeras may be assembled from segments or portions of a TIC8643 protein or a related protein or fragment thereof to derive additional useful embodiments including assembly of segments or portions of TIC8643 protein, or a related protein or fragment thereof, with segments or portions of any diverse insecticidal proteins different from TIC8643 protein and related proteins. The TIC8643 protein and related insecticidal proteins may be subjected to alignment to each other and to other Bacillus, Paenibacillus or other pesticidal proteins (whether or not these are closely or distantly related phylogenetically) or other artificial or engineered protein sequences, and segments of each of such proteins may be identified that are useful for substitution between the aligned proteins, resulting in the construction of chimeric proteins. Such chimeric proteins can be subjected to pest bioassay analysis and characterized for the presence or absence of increased bioactivity or expanded target pest spectrum compared to the parent proteins from which each such segment in the chimera was derived. The pesticidal activity of the polypeptides may be further engineered for activity to a particular pest or to a broader spectrum of pests by swapping domains or segments with other proteins or by using directed evolution methods known in the art.

[0069] Methods of controlling insects, in particular Lepidoptera, or Coleoptera, and / or Hemiptera infestations of crop plants, with the TIC8643 protein, or a related protein or fragment thereof, aredisclosed in this application. Such methods can comprise growing a plant comprising an insector Lcpidoptcra- inhibitory amount of a TIC8643 toxin protein or related insecticidal protein. In certain embodiments, such methods can further comprise any one or more of: (i) applying any composition comprising or encoding a TIC 8643 toxin protein or related insecticidal protein to a plant or a seed that gives rise to a plant; and (ii) transforming a plant or a plant cell that gives rise to a plant with a polynucleotide encoding a TIC8643 toxin protein or related insecticidal protein. In general, it is contemplated that a TIC8643 toxin protein or related insecticidal protein can be provided in a composition, provided in a microorganism, or provided in a transgenic plant to confer insect inhibitory activity against Lepidopteran insects.

[0070] In certain embodiments, a recombinant nucleic acid molecule encoding a TIC8643 toxin protein or related insecticidal protein is the insecticidally active ingredient of an insect inhibitory composition prepared by culturing recombinant Bacillus or any other recombinant bacterial cell transformed to express a TIC8643 toxin protein or related insecticidal protein or fragment thereof, under conditions suitable to express the TIC8643 toxin protein or related insecticidal protein or fragment thereof. Such a composition can be prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of such recombinant cells expressing / producing said recombinant polypeptide. Such a process can result in a Bacillus or other entomopathogenic bacterial cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet. By obtaining the recombinant polypeptides so produced, a composition that includes the recombinant polypeptides can include bacterial cells, bacterial spores, and parasporal inclusion bodies and can be formulated for various uses, including as agricultural insect inhibitory spray products or as insect inhibitory formulations in diet bioassays.

[0071] In one embodiment, to reduce the likelihood of resistance development, an insect inhibitory composition comprising a TIC8643 protein or related insecticidal protein or fragment thereof, can further comprise at least one additional polypeptide that exhibits insect inhibitory activity against the same Lepidopteran insect species, but which is different from the TIC8643 toxin protein or related insecticidal protein. Possible additional insecticidal agents for such a composition include an insect inhibitory protein and an insect inhibitory dsRNA molecule. One example for the use of such ribonucleotide sequences to control insect pests is described in Baum, et al. (U.S. PatentPublication 2006 / 0021087 Al). Such additional polypeptide for the control of Lepidopteran pests may be selected from the group consisting of an insect inhibitory protein, such as, but not limited to, CrylA (U.S. Patent No. 5,880,275), CrylAb, CrylAc, CrylA.105, CrylAe, CrylB (U.S. Patent Publication No. 10 / 525,318), CrylC (U.S. Patent No. 6,033,874), CrylD, CrylDa and variants thereof, CrylE, CrylF, and CrylA / F chimeras (U.S. Patent Nos. 7,070,982; 6,962,705; and 6,713,063), CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL, Cryl-type chimeras such as, but not limited to, TIC836, TIC860, TIC867, TIC869, and TIC 1100 (International Application Publication W02016 / 061391), TIC2160 (International Application Publication WO2016 / 061392(A2)), Cry2A, Cry2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry43A, Cry43B, Cry51Aal, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (U.S. Patent Publication 2013-0117884 Al), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI- 112, AXMI-117, AXMI-100 (U.S. Patent Publication 2013-0310543 Al), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication 2013-0104259 Al), AXMI-134 (U.S. Patent Publication 2013-0167264 Al), AXMI-150 (U.S. Patent Publication 2010-0160231 Al), AXMI-184 (U.S. Patent Publication 2010-0004176 Al), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (U.S. Patent Publication 2011-0030096 Al), AXMI-218, AXMI-220 (U.S. Patent Publication 2014- 0245491 Al), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175 Al), AXMI-238 (U.S. Patent Publication 2014-0033363 Al), AXMI- 270 (U.S. Patent Publication 2014-0223598 Al), AXMI-345 (U.S. Patent Publication 2014- 0373195 Al), AXMI-335 (International Application Publication WO2013 / 134523(A2)), DIG-3 (U.S. Patent Publication 2013-0219570 Al), DIG-5 (U.S. Patent Publication 2010-0317569 Al), DIG-11 (U.S. Patent Publication 2010-0319093 Al), AflP-lA and derivatives thereof (U.S. Patent Publication 2014-0033361 Al), AfIP-lB and derivatives thereof (U.S. Patent Publication 2014- 0033361 Al), PIP-1APIP-1B (U.S. Patent Publication 2014-0007292 Al), PSEEN3174 (U.S. Patent Publication 2014-0007292 Al), AECFG-592740 (U.S. Patent Publication 2014-0007292 Al), Pput_1063 (U.S. Patent Publication 2014-0007292 Al), DIG-657 (International Application Publication WO2015 / 195594 A2), Pput_1064 (U.S. Patent Publication 2014-0007292 Al), GS- 135 and derivatives thereof (U.S. Patent Publication 2012-0233726 Al), GS153 and derivatives thereof (U.S. Patent Publication 2012-0192310 Al), GS154 and derivatives thereof (U.S. Patent Publication 2012-0192310 Al), GS155 and derivatives thereof (U.S. Patent Publication 2012-0192310 A 1 ), SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2012-0167259 Al, 2SEQ ID NO: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2012-0047606 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2011-0154536 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2011-0112013 Al, SEQ ID NOs: 2 or 4 and 4 and derivatives thereof as described in U.S. Patent Publication 2010-0192256 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0077507 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0077508 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2009-0313721 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent Publication 2010-0269221 Al, SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent No. 7,772,465 (B2), CF161_OO85 and derivatives thereof as described in W02014 / 008054 A2, Lepidopteran toxic proteins and their derivatives as described in US Patent Publications US2008-0172762 Al, US2011-0055968 Al, and US2012-0117690 Al; SEQ ID NOs: 2 or 4 and derivatives thereof as described in US7510878(B2), SEQ ID NOs: 2 or 4 and derivatives thereof as described in U.S. Patent No. 7812129(B1), TIC6757 (U.S. Patent Publication 2017-058294 Al), TIC7941 (U.S. Patent Publication 2022-248686) Al, TIC2199 (U.S. Patent Publication 2023-013686 Al), TIC4064 (U.S. Patent Publication 2022-192200 Al), TIC4029 (U.S. Patent Publication 2022-256863 Al), TIC 13085 and TIC 13087 (U.S. Patent Publication 2022-220160 Al), IPD072Aa (International Application Publication W02020 / 076958), IPD079Ea (International Application Publication WO2017 / 023486), and IPD103 and homologs thereof (International Application Publication W02018 / 005411), PIP-50 and PIP-65 and homologs thereof (International Application Publication WO2015 / 120270), PIP-83 and homologs thereof (U.S. Patent Publication 2016- 0347799 Al), and CrylB.34 (U.S. Patent Publication 2017-0226164 Al); and the like.

[0072] In some embodiments, such composition / formulation can further comprise at least one additional insecticidal agent that exhibits insect inhibitory activity to an insect that is not inhibited by an otherwise insect inhibitory protein of the present invention to expand the spectrum of insect inhibition obtained. For example, for the control of Hemipteran pests, combinations of insect inhibitory proteins of the present invention can be used with Hemipteran-active proteins such as TIC1415 (US Patent Publication 2013-0097735 Al), TIC807 (U.S. Patent No. 8609936), TIC834(U.S. Patent Publication 2013-0269060 A l), AXMI-036 (U.S. Patent Publication 2010-0137216 Al), and AXMI-171 (U.S. Patent Publication 2013-0055469 Al). Further a polypeptide for the control of Coleopteran pests may be selected from the group consisting of an insect inhibitory protein, such as, but not limited to, Cry3Bb (U.S. Patent No. 6,501,009), CrylC variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, AXMI134 (U.S. Patent Publication 2013-0167264 Al) AXMI-184 (U.S. Patent Publication 2010-0004176 Al), AXMI-205 (U.S. Patent Publication 2014-0298538 Al), AXMI-207 (U.S. Patent Publication 2013-0303440 Al), AXMI-218, AXMI- 220 (U.S. Patent Publication 20140245491A1), AXMI-221z, AXMI-223z (U.S. Patent Publication 2014-0196175 Al), AXMI-279 (U.S. Patent Publication 2014-0223599 Al), AXMI-R1 and variants thereof (U.S. Patent Publication 2010-0197592 Al, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication 2010-0319092 Al), eHIPs (U.S. Patent Application Publication No. 2010 / 0017914), IP3 and variants thereof ( U.S. Patent Publication 2012-0210462 Al), TIC5290 (U.S. Patent Publication 2017-0044568), TIC3668, TIC3669, and TIC3670 (U.S. Patent Publication 2016-0319302 Al), and Mexatoxin-Hvl a (U.S. Patent Application Publication 2014-0366227 l).

[0073] Additional insecticidal agents for the control of Coleopteran, Lepidopteran, and Hemipteran insect pests, which can be combined with the insect inhibitory proteins of the TIC8643 protein toxin class, can be found on the Bacillus thuringiensis toxin nomenclature website maintained by Neil Crickmore (on the world wide web at btnomenclature.info). Broadly, it is contemplated that any insect inhibitory agent or protein known to those of ordinary skill in the art can be used in combination with the proteins of the TIC8643 protein toxin class both in planta (combined through breeding or molecular stacking) or in a composition or formulation as a biopesticide or combination of biopesticides, provided however that the insect inhibitory agent or protein for use with the insecticidal protein of the TIC8643 protein toxin class in a plant does not compete with the insecticidal protein of the TIC8643 protein toxin class in its mode of action against target pests and that the protein is not detrimental to the health and desirable properties of the transgenic plant and is expressed itself, at levels pesticidal to the intended target pest.

[0074] The possibility for insects to develop resistance to certain insecticides has been documented in the art. One insect resistance management strategy is to employ transgenic crops that express two distinct insect inhibitory agents that operate through different modes of action.Therefore, any insects with resistance to either one of the insect inhibitory agents can be controlled by the other insect inhibitory agent. Another insect resistance management strategy employs the use of plants that are not protected to the targeted Lepidopteran pest species to provide a refuge for such unprotected plants. One particular example is described in U.S. Patent No. 6,551,962.

[0075] Other embodiments such as topically applied pesticidal chemistries that are designed for controlling pests that are also controlled by the insecticidal proteins disclosed herein can be used in seed treatments, spray on, drip on, or wipe on formulations and applied directly to the soil (a soil drench), applied to growing plants expressing the proteins disclosed herein, or formulated to be applied to seed containing one or more transgenes encoding one or more of the proteins disclosed. Such formulations for use in seed treatments can be applied with various stickers and tackifiers known in the art. Such formulations can contain pesticides that are synergistic in mode of action with the proteins disclosed, so that the formulation pesticides act through a different mode of action to control the same or similar pests that can be controlled by the proteins disclosed, or that such pesticides act to control pests within a broader host range or plant pest species that are not effectively controlled by the TIC8643 pesticidal protein or related protein or fragment thereof.

[0076] The aforementioned composition / formulation can further comprise an agriculturally acceptable carrier, such as a bait, a powder, dust, pellet, granule, spray, emulsion, a colloidal suspension, an aqueous solution, a Bacillus spore / crystal preparation, a seed treatment, a recombinant plant cell, plant tissue, plant seed, plant part or plant transformed to express one or more of the insecticidal proteins described herein, or a bacterium transformed to express one or more of the insecticidal proteins described herein. Depending on the level of insect inhibitory or insecticidal inhibition with a recombinant polypeptide described herein and the level of formulation to be applied to a plant or diet assay, the composition / formulation can include various by weight amounts of the recombinant polypeptide, e.g., from 0.0001% to 0.001% to 0.01% to 1% to 99% by weight of the recombinant polypeptide.

[0077] In view of the foregoing, those of skill in the art should appreciate that changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Thus, specific structural and functional details disclosed herein are not to be interpreted as limiting.EXAMPLESExample 1. Discovery, cloning, expression, and purification of TIC8643 Coding Sequences and the Encoded Toxin Protein.

[0078] TIC8643 pesticidal protein was identified through sequence analysis of the genome of the species Streptomyces purpeofuscus MDI-0021357. A culture of the microbe provided DNA which was then subjected to sequence analysis, and the sequences obtained were assembled and analyzed to identify open reading frames. The TIC8643 open reading frame set forth in SEQ ID NO: 1 from nucleotide position 1 through nucleotide position 1080, including the termination codon following the complete coding sequence, was identified by pfam analysis to the protein domains: Botulinum_HA-17 I Inhibitor_I66 I Cry35 I Ricin_B_lectin I CD toxinA I RicinB_lectin_2 I Toxin_10 I Cry55. The full length TIC8643 protein amino acid sequence exhibits 64.07% identity to GenBank Accession WP_076085859, derived from an assembled sequence of Streptomyces sp. IMTB 2501, annotated as having a Ricin-type beta-trefoil lectin-like domain and pfam of Toxin_10. There is no evidence that this assembled protein has been assayed for activity against insects.

[0079] Polymerase chain reaction (PCR) primers were designed to amplify a full-length copy of the nucleotide sequence encoding TIC8643 from total genomic DNA isolated from the Sp strain, MDI-0021357, to confirm the assembly of sequenced segments were in fact representative of the naturally occurring sequence. Amplicons were cloned using methods known in the art into an Escherichia coli (Ec) expression vector in operable linkage with an Ec expressible promoter and with or without a histidine tag which is subsequently used for purification of the his-tagged TIC8643 protein. Preparations of purified TIC8643 protein derived from the vectors expressing the toxin protein in Ec were used in bioassay against various insect pest species.Example 2. TIC8643 demonstrated Lepidopteran activity in insect bioassay.

[0080] The TIC8643 protein open reading frame initially assembled was confirmed by comparison with the sequence of thermal amplified clones, and both encoded the same amino acid sequence set forth in SEQ ID NO: 2. TIC8643 expressed in recombinant Ec using the vectors described in Example 1 were assayed for toxicity to various species of Lepidoptera, Coleoptera, Hemiptera, and Diptera.

[0081] TIC8643 was assayed for toxicity to the Lepidopteran insect species Black cutworm (BCW, Agrotis ipsilon), Com carworm (CEW, Helicoverpa zea, also known as Soybean podworm), European com borer (ECB, Ostrinia lutbilali.s). Fall armyworm (FAW, Spodoptera jritgiperda). Southern armyworm (SAW, Spodoptera eridania). Soybean looper (SBL, Chrysodeixis includens), Southwestern com borer (SWC, Diatraea grandioselld), Tobacco budworm (Helioihis virescens), Western bean cutworm (Striacosta albico.sia). and Velvet bean caterpillar (Anticarsia gemmatalis)’, the Coleopteran species Western Com Rootworm (WCR, Diabrotica virgifera) and Colorado potato beetle (CPD, Leptinotarsa decemlineata); the Hemipteran species Tarnished plant bug (TPB, Lygus Uneolaris). Western tarnished plant bug (Lygus hesperus), Southern Green Stinkbug (SGB, Nezara viridula), and Neotropical Brown Stink Bug (NBSB, Euschistus he ro y and the Dipteran species (YFM, Aedes aegypti). The bioassay results are presented in Tables 2 and 3 below wherein “+” indicates activity and indicates no activity.Table 2. Activity of T1C8643 against Lepidopteran insect species.Table 3. Activity of TIC8643 against Coleopteran, Hemipteran, and Dipteran insect species.

[0082] As can be seen from the data presented in Tables 2 and 3, TIC8643 demonstrated activity against the Lepidopteran species BCW and FAW in insect bioassay. With reference to BCW and FAW only, the concentration of toxin protein provided ranged from 0.25 to 1.3 mg / ml of diet in the assay. With respect to BCW, stunting and mortality were observed beginning at 0.5 mg / ml and increased with greater concentrations. With respect to FAW, stunting and mortality were observed beginning at 0.25 mg / ml and increased with greater concentrations.Example 3. Design of artificial coding sequences for expression of TIC8643 in plants.

[0083] An artificial coding sequence, SEQ ID NO: 3 encoding TIC8643PL, was designed for expression in a plant cell. The artificial (alternatively referred to as synthetic) sequence was synthesized, according to methods generally described in U.S. Patent 5,500,365, to avoid certain inimical problem sequences such as ATTTA and A / T rich plant polyadenylation sequences, while substantially preserving the amino acid sequence of the native Bacillus protein.

[0084] The artificial sequence (SEQ ID NO: 3) encoding TIC8643PL (SEQ ID NO: 4) further comprises an additional alanine codon inserted between the first two codons of the native coding sequence so that the first three amino acids of the TIC8643PL protein are MET-ALA-SER. SEQ ID NO: 3 was cloned into two plant transformation vectors and downstream of, and in each case functionally linked to, a plant-expressible promoter for driving expression of the coding sequence when in a plant cell, using skills known in the art. The resulting transformation vector constructs, Construct- 1 and Construct-2, were used to transform corn plant cells and comprised a first transgene cassette for expression of the TIC8643PL pesticidal protein which comprised (in the 5’ to 3’ direction) a constitutive promoter, operably linked to a leader, operably linked to an intron, operably linked to an artificial coding sequence encoding T1C8643PL, operably linked to a 3' UTR, and a second transgene cassette for the selection of transformed plant cells using glyphosate selection from the expression of a microbial CP4 EPSPS protein insensitive to glyphosate inhibition. Each of the two transformation vectors comprised different high constitutive promoters to drive TIC8643PL expression.Example 4. TIC8643 Exhibits Lepidopteran Activity when Expressed in Stably Transformed Corn Plants.

[0085] The two binary plant transformation vectors (Construct- 1 and Construct-2) comprising a transgene cassette designed to express TIC8643PL were cloned using methods known in the art, each using different constitutive expression elements to drive expression of TIC8643PL as set forth in Example 3. The resulting vectors were used to stably transform corn plants. Tissues were harvested from the transformants and used in insect bioassay against various Lepidopteran insect species.

[0086] Corn plant cells were transformed with the binary transformation vector as described in Example 3 using an Agrobacterium-mcdiatcd transformation method known in the art. The transformed cells were induced to form plants by methods known in the art. Insect bioassays using plant leaf disks were performed analogous to those described in U.S. Patent No. 8,344,207. A single freshly hatched neonate larvae less than one day old was placed on each leaf disc sample and allowed to feed for approximately four days. A non-transformed near isogenic corn plant was used to obtain tissue to be used as a negative control. Multiple transformation Ro single-copy insertion events obtained from each binary vector were assessed against Black cutworm (BCW, Agrotis ipsilon), European com borer (ECB, Ostrinia nubilalis), Fall armyworm (FAW, Spodopterafrugiperdd), and Southwestern com borer (SWC, Diatraea grandiosella). The average percent leaf damage was determined for each event derived from each construct and the results are presented in Figures 1A though 3B, and the data obtained in planta against BCW and FAW are consistent with the data observed with insect bioassay evaluations. Figures 1A and IB show the average percent leaf damage caused by BCW for each constmct tested, respectively. Figures 2A and 2B show the average percent leaf damage caused by FAW for each construct tested, respectively. Figures 3A and 3B show the average percent leaf damage caused by CEW for each construct tested, respectively. This CEW data was surprising in view of the bioassay results provided in Example 2.

[0087] As can be seen in Figures 1A and IB, many of the events demonstrated activity against BCW relative to the non-transgenic control. For example, Events 11 and 28 derived from Construct-1 and Events 9, 15, 20, and 21 derived from Construct-2 had an average leaf damage of 30 percent or less. Other events derived from both constructs demonstrated a reduction in the average percent leaf damage when compared to the non-transgenic control.

[0088] As can be seen in Figures 2A and 2B, many of the events demonstrated activity against FAW relative to the non-transgenic control. Event 2, derived from Construct-2 had an average leaf damage of 3 percent. Other events derived from both constructs demonstrated a reduction in the average percent leaf damage when compared to the non-transgenic control.

[0089] As can be seen in Figures 3A and 3B, many of the events demonstrated activity against CEW relative to the non-transgenic control, showing a reduction in the average percent leaf damage when compared to the non-transgenic control.

[0090] Thus, stably transformed corn plants transformed with constructs expressing TIC8643PL arc resistant to BCW, FAW, and CEW infestation.

[0091] All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions of this invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those of skill in the art that variations, changes, modifications, and alterations may be applied to the composition described herein, without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0092] All publications and published patent documents cited in the specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS1. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a pesticidal protein, or a pesticidal fragment thereof, wherein: a. said pesticidal protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; b. said pesticidal protein comprises an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4; or c. said polynucleotide segment comprising SEQ ID NO: 1 or 3 or hybridizing under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 3 or a complement thereof.

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

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

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

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

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

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

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

9. The recombinant nucleic acid molecule of claim 8, wherein said Lepidopteran insect is selected from the group consisting of: Black cutworm (Agrotis ipsilong Corn earworm (Helicoverpa zed), and Fall armyworm Spodoptera frugiperda).

10. A plant comprising the recombinant nucleic acid molecule of any one of claims 1-9, or a plant part thereof.

11. The plant of claim 10, wherein said plant is a monocot plant or a dicot plant, or a plant part thereof.

12. The plant of claim 10, wherein the plant is selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

13. The plant part of claim 10, wherein the plant part is a seed, and wherein said seed comprises said recombinant nucleic acid molecule.

14. An insect inhibitory composition comprising the recombinant nucleic acid molecule of any one of claims 1-9.

15. The insect inhibitory composition of claim 14, further comprising a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein.

16. The insect inhibitory composition of claim 15, wherein said at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, a chemical molecule and an ancillary protein, wherein said at least one other pesticidal agent is toxic to the same pest as the pesticidal protein or pesticidal fragment thereof.

17. The insect inhibitory composition of claim 15, wherein said at least one other pesticidal agent exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, or Hemiptera.

18. The insect inhibitory composition of claim 15, wherein said at least one other pesticidal agent is selected from the group consisting of a CrylA, CrylAb, CrylAc, CrylA.105, CrylAe, CrylB, CrylC, CrylC variants, CrylD, CrylD variants, CrylE, CrylF, CrylA / F chimeras, CrylG, CrylH, Cryll, CrylJ, CrylK, CrylL, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry 15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aal, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC86O, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI- 115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI- 196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI- 22 Iz, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IPD102Aa and homologs thereof, IPDl lOAa and homologs thereof, TIC868, CrylDal_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757. TIC7941, TIC5290, TIC3668, TIC3669, TIC3670, TIC2199, TIC4064, TIC4029, TIC13085, TIC13087, IPD072Aa, IPD079Ea, and IPD103 and homologs thereof, PIP-50 and PIP-65 and homologs thereof, PIP-83 and homologs thereof, and CrylB.34.

19. The insect inhibitory composition of claim 14, defined as comprising a plant cell that expresses the pesticidal protein from the recombinant nucleic acid molecule of claim 1.

20. A commodity product produced from the plant, or plant part thereof, of any one of claims 10-13, wherein the commodity product comprises a detectable amount of said recombinant nucleic acid molecule and / or said pesticidal protein or a pesticidal fragment thereof.

21. The commodity product of claim 20, selected from the group consisting of commodity corn bagged by a grain handler, com flakes, corn cakes, com flour, corn meal, com syrup, com oil, corn silage, com starch, com cereal, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fmit, melon, and vegetable commodity products including, where applicable, juices, concentrates, jams, jellies, marmalades, and other edible forms of such commodity products containing a detectable amount of such polynucleotides and or polypeptides of this application, whole or processed cotton seed, cotton oil, lint, seeds and plant parts processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts.

22. A method of producing progeny seed comprising the recombinant nucleic acid molecule of any one of claims 1-9, the method comprising: a. planting a first seed comprising the recombinant nucleic acid molecule; b. growing a plant from the seed of step a; and c. harvesting the progeny seed from the plants, wherein said harvested seed comprises said recombinant nucleic acid molecule.

23. A plant resistant to insect infestation, wherein the cells of said plant comprise the recombinant nucleic acid molecule of any one of claims 1-9.

24. A method for controlling a Lepidopteran species pest or pest infestation, said method comprising: a. contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO: 2 or 4; orb. contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4.

25. A method of detecting the presence of the recombinant nucleic acid molecule of claim 1 in a sample comprising plant genomic DNA, comprising: a. contacting said sample with a nucleic acid probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant comprising the recombinant nucleic acid molecule of claim 1 , and does not hybridize under such hybridization conditions with genomic DNA from an otherwise isogenic plant that does not comprise the recombinant nucleic acid molecule of claim 1, wherein said probe is homologous or complementary or hybridizes under stringent conditions to SEQ ID NO: 3; or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4; b. subjecting said sample and said probe to stringent hybridization conditions; and c. detecting hybridization of said nucleic acid probe with said recombinant nucleic acid molecule.

26. A method of detecting the presence of a pesticidal protein, or a fragment thereof, in a sample comprising protein, wherein said pesticidal protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; or said pesticidal protein comprises an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%,or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4, comprising: a. contacting said sample with an immunoreactive antibody; and b. detecting the presence of said pesticidal protein, or fragment thereof.

27. The method of claim 26, wherein the step of detecting comprises an ELISA or a Western blot analysis.

28. A pesticidally effective amount of a protein comprising the amino acid sequence as set forth in SEQ ID NO: 2 or 4.

29. A method for controlling a Lepidopteran pest species or pest infestation in a field, said method comprising: a. growing a crop plant which expresses an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO: 2 or 4; or b. growing a crop plant which expresses an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 66%, or 67%, or 68%, or 69%, or 70%, or 71%, or 72%, or 73%, or 74%, or 75%, or 76%, or 77%, or 78%, or 79%, or 80%, or 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99%, or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4; and optionally c. releasing into said field transgenic Lepidopteran pest species carrying a selflimiting gene to reduce the likelihood of development of resistance of the pest species to the pesticidal protein.

30. The method of claim 29, wherein said crop plant is a monocotyledonous or dicotyledonous crop plant.

31. The method of claim 30, wherein the monocotyledonous crop plant is com, wheat, sorghum, rice, rye, sugarcane, or millet.

32. The method of claim 31, wherein the monocotyledonous crop plant is corn.

33. The method of claim 30, wherein the dicotyledonous crop plant is soybean, cotton, alfalfa, or canola.

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