Novel insect inhibitory proteins

Abstract

Disclosed herein are pesticidal proteins that exhibit toxic activity against lepidoptera pest species, and that include, but are not limited to, TIC8643 and related pesticidal proteins. DNA constructs encoding the disclosed pesticidal proteins are provided. Transgenic plants, plant cells, seeds and plant parts that are resistant to lepidoptera insect infestation are provided, which contain recombinant nucleic acid sequences encoding insecticidal proteins of the invention, and vectors containing at least one coding sequence for expressing and delivering the encoded toxin proteins are also described. Also provided are methods for detecting the presence of a recombinant nucleic acid sequence or protein of the invention in a biological sample, as well as methods of using TIC8643 and related pesticidal proteins to control pests of Lepidoptera species.

Description

Citation of relevant applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 515,052, filed July 21, 2023, which is incorporated herein by reference in its entirety.

[0002] Merging of sequence lists A file named "BCS236201_01_US_SEQLISTING_ST26.xml" containing a sequence list in computer-readable form was created on May 31, 2024. This file is 8,558 bytes (in MS-Windows). ® The measurement (in the middle) is submitted simultaneously via electronic submission (using the US Patent and Trademark Office Patent Centre) and incorporated in its entirety by reference. Technical Field

[0003] This invention generally relates to the field of insect inhibitory proteins. This document discloses a novel class of toxin proteins that exhibit insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly insect pests of Lepidoptera species. Plants, plant parts, seeds, cells including plant and microbial cells are provided, as well as vectors containing recombinant polynucleotide constructs encoding one or more of the disclosed toxin proteins. Background Technology

[0004] Increasing crop yields of key agricultural plants, including maize, soybeans, sugarcane, rice, wheat, cotton, vegetables, pearl millet, pigeon pea, peanuts, potatoes, barley, oats, and fruit trees, has become increasingly important. In addition to the growing demand from a burgeoning population for agricultural products that provide food, clothing, and energy, anticipated climate-related impacts and pressure from a growing population using land for non-agricultural purposes will further reduce the amount of arable land available for agriculture. These factors lead to grim predictions of food security, particularly given the lack of significant improvements in plant biotechnology and agronomic practices. In light of these factors, environmentally sustainable improvements in technology, agricultural techniques, and pest management are crucial tools for expanding crop production on the increasingly limited amount of arable land available for agriculture.

[0005] Insects, particularly lepidopteran insects, are a major cause of damage to field crops, thereby reducing crop yields in affected areas. Lepidopteran pests that negatively impact agriculture include, but are not limited to, the black armyworm (…). Spodoptera cosmioides ), small cutworm ( Agrotis ipsilon ), corn ear worm ( Helicoverpa zea ), cotton leafworm ( Alabama argillacea Diamondback moth ( Plutella xylostella ), European corn borer ( Ostrinianubilalis Fall armyworm ( Spodoptera frugiperda Cry1Fa1 resistant fall armyworm ( Spodoptera frugiperda ), Old World cotton bollworm ( Helicoverpa armigera Southern armyworm () Spodoptera eridania ), soybean looper ( Chrysodeixis includens ), spotted bollworm ( Earias vittella ), Southwest corn borer ( Diatraea grandiosella ), sunflower inchworm ( Rachiplusia nu ), smoke moth ( Heliothis virescens ), Spodoptera litura ( Spodoptera litura Also known as tea worm (cluster caterpillar) or western bean worm ( Striacosta albicosta ) and fluffy bean caterpillar ( Anticarsia gemmatalis ).

[0006] Historically, pest control in agriculture relied on the intensive application of synthetic chemical pesticides. These chemicals typically do not target specific insects, acting indiscriminately and sometimes persisting in the environment and accumulating at excessively high levels in apexpreders. Concerns about the environment and human health, the emergence of pesticide resistance, and the potential indiscriminate effects of these substances on non-target insects and other organisms have spurred research and development into biopesticides specifically designed to control crop-damaging pests. This has led to the gradual discovery and utilization of various insect pathogenic microorganisms, including bacteria.

[0007] When insect pathogens, especially those belonging to the genus Bacillus ( Bacillus The biological control paradigm shifted when the potential of Bacillus thuringiensis (Bt) was discovered and developed into a biopesticide. Bacillus thuringiensis ) ( Bt This strain has been used as a source of insecticidal proteins because it has been found... Bt The strain exhibits high toxicity to certain insects. Bt The strains produce delta-endotoxins, which are located within parasporal crystal inclusions (e.g., Cry proteins) at the onset of sporulation and during the stationary growth phase. These strains also produce secreted insecticidal proteins. Both the delta-endotoxins and the secreted toxins exert their effects on the midgut epithelial surface after ingestion by susceptible insects, disrupting cell membranes and causing cell damage and death. [Further details omitted] Bt Genes encoding insecticidal proteins have been identified in bacterial species outside of Bacillus species, including other Bacillus species and several other bacterial species, such as Bacillus laterosporus (B. brevis). Brevibacillus laterosporus ), Bacillus spheroides ( Lysinibacillus sphaericus ) (“Ls Previously also known as Bacillus spheroidae ( Bacillus sphaericus )), Pseudomonas ( Pseudomonas ) species, Japanese beetle spores ( Paenibacillus popilliae ) and slow-acting spore-forming bacteria ( Paenibacillus lentimorbus Furthermore, insecticidal toxins have been identified from a variety of non-bacterial sources, including fern and arachnid venoms, as well as through the delivery of dsRNAs into the pests' diets, wherein the dsRNAs are used to suppress essential genes of the pests. These toxins or methods have been found to provide effective pest control strategies under various conditions.

[0008] Crystallized and secreted soluble insecticides are highly specific to their hosts and have gained worldwide recognition as an alternative to chemical pesticides. For example, insecticide proteins have been used in various agricultural applications to protect agriculturally important plants from insect infestation, reduce the need for chemical pesticides, and increase yields. Insecticidal proteins are used to control agriculturally relevant pests on crops through mechanical methods, such as dispersing microbial preparations containing various bacterial strains onto the plant surface by spraying; and through genetic transformation technologies to produce transgenic plants and seeds expressing insecticide proteins. The use of transgenic plants expressing insecticide proteins has been implemented globally. For example, in 2016, 23.1 million hectares were planted with plants expressing insecticide proteins. Bt Genetically modified crops containing toxins, and 75.4 million hectares have been planted with crops expressing these toxins. Bt Genetically modified crops that are toxic and have 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 places a selective pressure on existing insect alleles that confer resistance to the insecticidal proteins currently used. The development of resistance to insecticidal toxin proteins in target pests requires the constant discovery and development of new forms of insecticidal toxin proteins that can be used to manage the increase in insect resistance to transgenic crops expressing insecticidal toxin proteins. New protein toxins with improved efficacy and that exhibit control over a broader spectrum of susceptible insect species will reduce the number of surviving insects that can give rise to resistant alleles. Furthermore, the use of two or more transgenic insecticidal toxin proteins in one plant that are toxic to the same insect pest class but have different modes of action or have two or more different modes of toxic action (e.g., a transgene encoding a dsRNA targeting an essential gene to be suppressed combined with a transgene encoding a peptide or protein toxin, both of which are toxic to the same insect species) will reduce the likelihood of resistance development by any single target insect species. Additionally, the use of self-limiting technologies (such as those provided by Oxitec® Ltd) with the proteins of the present application will increase the durability of the trait conferred to transgenic crops expressing the proteins of the present application (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 Bt crops. Journal of EconomicEntomology, 102 : 717-732).

[0010] Accordingly, there is a need in the art for new toxin proteins that are effective against target pests, can provide effective control of those target pests in the agricultural field, and can be expressed in plants without causing undesirable agronomic problems, and provide an alternative mode of action to the toxins currently used in commerce in plants. SUMMARY

[0011] This paper discloses a novel insecticidal protein, TIC8643, which has been shown to exhibit inhibitory activity against one or more lepidopteran pests on crop plants. TIC8643 protein, or related proteins or fragments thereof, can be used alone or in combination with other insecticidal proteins and / or toxicants in formulations and / or plants. in planta This provides an alternative to the insecticidal proteins and pesticides currently used in agricultural systems.

[0012] In one embodiment, this application discloses a recombinant nucleic acid molecule comprising a heteropromoter operably linked to a multinucleotide segment or sequence encoding an insecticidal protein or an insecticidal fragment thereof, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; or the insecticidal protein comprises an amino acid sequence of SEQ ID NO: 2 or 4. Amino acid sequence 2 or 4 has 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 an amino acid sequence having approximately 100% amino acid sequence identity; or the polynucleotide segment or sequence hybridizes with a polynucleotide sequence having SEQ ID NO: 1 or 3 under strict hybridization conditions. The recombinant nucleic acid molecule may contain a sequence for expressing an insecticidal protein in plants, and the sequence, when expressed in plant cells, produces an insecticidally effective amount of the insecticidal protein or its insecticidal fragment.

[0013] In another embodiment of this application, the recombinant nucleic acid molecule is present within bacterial or plant host cells. The considered bacterial host cells include at least *Agrobacterium* (…). Agrobacterium)、 Rhizobium ( Rhizobium ), Bacillus spp. Bacillus ), Bacillus brevis ( Brevibacillus Escherichia coli spp. Escherichia ), Pseudomonas spp. Pseudomonas Klebsiella spp. Klebsiella ), Pantotheca ( Pantoea ) and Erwinia spp. Erwinia In some implementations, the Bacillus species is Bacillus cereus (…). Bacillus cereus ) or Bacillus thuringiensis, the genus Bacillus is Bacillus laterosporus, or the genus Escherichia coli is Escherichia coli ( Escherichia coliThe plant host cells considered include both dicotyledonous and monocotyledonous plant cells. Other plant cells considered include alfalfa, banana, barley, legumes, broccoli, cabbage, brassica (e.g., rapeseed), carrots, cassava, cowpeas, castor beans, cauliflower, celery, chickpeas, Chinese cabbage, citrus, coconut, coffee, corn, clover, and cotton (Cotton genus). Gossypium (Species), gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, slash pine, millet, melons, nuts, oats, olives, onions, ornamental plants, palm, pasture, peas, peanuts, pepper, pigeon pea, pine, potato, poplar, pumpkin, radish, rapeseed, rice, rootstock, rye, safflower, shrubs, sorghum, southern pine, soybean, spinach, squash, strawberry, beet, sugarcane, sunflower, sweet corn, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon, and wheat plant cells.

[0014] In another embodiment, the insecticidal protein is effective against lepidopteran insects, such as cutworms (…). Agrotis ipsilon ), corn ear worm ( Helicoverpa zea ) and fall armyworm ( Spodoptera frugiperda It exhibits activity.

[0015] The application also encompasses bacteria and plants and plant parts comprising a recombinant nucleic acid molecule encoding a pesticidal protein TIC8643 or a pesticidal protein from the TIC8643 toxin protein class. The recombinant molecule (e.g., construct) can comprise a heterologous promoter for expressing the operably linked polynucleotide segment or sequence encoding the pesticidal protein in a bacterial or plant cell. Both dicot and monocot plants are encompassed. In another embodiment, the plant is additionally selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, brassica (e.g., canola), carrot, cassaya, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (i.e., Gossypium spp.), cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn (i.e., maize) (e.g., sweet corn or fodder corn), sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. Plant parts can include, for example, but are not limited to, leaves, tubers, roots, stems, seeds, embryos, flowers, inflorescences, bolls, pollen, fruits, animal feed, and biomass. Processed plant parts are also encompassed, such as wood or oil produced from plant leaves, flowers, roots, seeds, or tubers containing nucleic acids encoding a protein of the application and / or containing a pesticidally effective amount of an encoded toxin protein, inactivated milled seed or graded seed, flour, or starch.

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

[0017] In yet another embodiment, an insect inhibiting composition comprising a recombinant nucleic acid molecule disclosed herein is contemplated. The insect inhibiting composition can further comprise a nucleotide sequence encoding at least one other insecticidal agent that is different from the insecticidal protein. In certain embodiments, the at least one other insecticidal agent is selected from the group consisting of an insect inhibiting protein, an insect inhibiting dsRNA molecule, and a helper protein. Additionally, it is contemplated that the at least one other insecticidal agent in the insect inhibiting composition exhibits activity against one or more pest species in the orders Lepidoptera, Coleoptera, and / or Hemiptera. In some embodiments, the at least one other insecticidal agent in the insect inhibiting composition can be selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105、Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A / F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-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 proteins, IPD102Aa and homologs thereof, IPD110Aa and homologs thereof, TIC868, Cry1Dai_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 Cry1B.34; and dsRNA-mediated gene suppression embodiments, including for suppression of Diabrotica spp. Diabrotica) those of insect genes Dv snf7 and Dv ssj 1.

[0018] Also encompassed are commodity products comprising a detectable amount of a recombinant nucleic acid molecule and / or toxin protein disclosed herein. Such commodity products include commodity corn, corn flakes, corn tortillas, corn meal, corn flour, corn syrup, corn oil, corn silage, corn starch, corn flakes, and the like, as well as corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity products, where applicable, including juices, concentrates, jams, jellies, preserves, and other edible forms, whole or processed cottonseed, cotton oil, lint, seeds, and processed plant parts for feed or food, fiber, paper, biomass, and fuel products, such as fuel derived from cotton oil or pellets derived from gin waste, whole or processed soybean seeds, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soy cheese, soy wine, animal feed comprising soybean, paper comprising soybean, butter comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts, that contain a detectable amount of such polynucleotide and / or polypeptide of the application.

[0019] Also encompassed in the present application is a method of producing a seed comprising a recombinant nucleic acid molecule encoding a TIC8643 toxin protein having pesticidal activity or a pesticidal protein from the class of TIC8643 toxin proteins, or a fragment thereof, and / or a pesticidally effective amount of a TIC8643 toxin protein having pesticidal activity or a pesticidal protein from the class of TIC8643 toxin proteins, or a fragment thereof. The method comprises planting at least one seed comprising a recombinant nucleic acid molecule disclosed herein; growing a plant from the seed; and harvesting a seed from the plant, wherein the harvested seed comprises the mentioned recombinant nucleic acid molecule and / or a pesticidally effective amount of the encoded TIC8643 toxin protein having pesticidal activity or a pesticidal protein from the class of TIC8643 toxin proteins, or a fragment thereof.

[0020] In another exemplary embodiment, a plant is provided that is resistant to infestation by a Lepidopteran insect, wherein a cell of the plant comprises a recombinant nucleic acid molecule disclosed herein.

[0021] The present application also discloses methods for controlling lepidopteran species pests and for controlling infestation of lepidopteran species pests of plants, particularly crop plants. In one embodiment, the method comprises: first contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO: 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 having about 100% amino acid sequence identity, to SEQ ID NO: 2 or 4.

[0022] Also 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 nucleic acid sample with a nucleic acid probe that hybridizes under stringent hybridization conditions to genomic DNA of a plant comprising a polynucleotide segment or sequence encoding a pesticidal protein provided herein or a fragment thereof, and does not hybridize under such conditions to genomic DNA of another 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 encoding 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 having about 100% amino acid sequence identity, to SEQ ID NO: 2 or 4; subjecting the sample and probe to stringent hybridization conditions; and detecting hybridization of the probe to the sample DNA. In some embodiments, the step of detecting the presence of a member of the TIC8643 toxin protein class can comprise ELISA or Western blot.

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

[0024] The present application also encompasses a method for controlling a lepidopteran pest species or infestation in a field, wherein the method comprises growing a crop plant expressing an insecticidally effective amount of an insecticidal protein as set forth in SEQ ID NO: 2 or 4; or growing a crop plant expressing an insecticidally effective amount of one or more insecticidal 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 having about 100% amino acid sequence identity, to SEQ ID NO: 2 or 4; and releasing one or more transgenic lepidopteran pest species each carrying a self-limiting gene into a field containing a crop plant encoding a gene for a toxin protein of the present application to prevent or delay the development of resistance to the toxin protein by the one or more lepidopteran pest species. In one embodiment, the crop plant can be monocotyledonous or dicotyledonous. In another embodiment, the monocotyledonous crop plant can be corn, wheat, sorghum, rice, rye, or millet. In yet another embodiment, the dicotyledonous crop plant can be soybean, cotton, alfalfa, cowpea, cassava, or canola.

[0025] Sequence Summary SEQ ID NO: 1 is a native / naturally occurring nucleic acid sequence obtained from Streptomyces rubiginosus (strain MDI-0021357) encoding a TIC8643 insecticidal protein. Streptomyces_purpeofuscus ) strain MDI-0021357 encoding a TIC8643 insecticidal protein.

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

[0027] SEQ ID NO: 3 is an artificial sequence encoding a TIC8643PL insecticidal protein designed for expression in plant cells with an alanine codon inserted at the second (2) amino acid position following the methionine codon at the start of the open reading frame translation.

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

[0029] Figure 1A is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Agrotis ipsilon ) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein.

[0030] Figure 1B is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Agrotis ipsilon

[0031] Figure 2A is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Spodoptera frugiperda

[0032] Figure 2B is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Spodoptera frugiperda

[0033] Figure 3A is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Helicoverpa zea

[0034] Figure 3B is a graphical representation of data showing the average leaf damage caused by the European corn borer (Ostrinia nubilalis) when feeding on corn plants stably transformed with Construct-1, which expresses the TIC8643PL protein. Helicoverpa zea DETAILED DESCRIPTION

[0035] ​​​​​The present inventors disclose herein a novel protein from Streptomyces rubiginosus and related proteins that exhibit insecticidal activity against target Lepidoptera species, particularly against Agrotis ipsilon ), European corn borer ( Helicoverpa zea ), and / or fall armyworm ( Spodoptera frugiperda ). Disclosed herein is a novel pesticidal protein identified as TIC8643, the amino acid sequence of which is set forth in SEQ ID NO: 2. Use of a pesticidally effective amount of the protein or related proteins or fragments thereof can address the problem of insect infestation of crop plants, particularly against a broad spectrum of Lepidoptera insect pests, more particularly against Agrotis ipsilon ), European corn borer ( Helicoverpa zea ), and fall armyworm ( Spodoptera Spodoptera ) species.

[0036] Reference to a "TIC8643-related toxin," "TIC8643-related protein," "related protein" in relation to TIC8643, "TIC8643 protein toxin class," or "TIC8643 toxin protein class" in this application is intended to refer to any pesticidal protein or insect inhibitory protein 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 combination thereof, that confer insecticidal activity against one or more Lepidoptera pests, including any protein that exhibits pesticidal or insect inhibitory activity, provided that such protein is within about 65% to about 100% identity to the amino acid sequence of TIC8643 (SEQ ID NO: 2) when aligned to TIC8643, for example 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% identity, or any fractional percentage between any of the foregoing. The TIC8643 proteins and related proteins and fragments thereof described herein and intended to be within the scope of the present disclosure include plastid-targeted forms of the proteins (e.g., fused to a chloroplast targeting peptide (CTP)) and non-plastid-targeted forms.

[0037] The term "segment" or "fragment" as used in the present application describes a contiguous or consecutive amino acid or nucleic acid sequence that is 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, in particular, for example, the nucleic acid sequence encoding a TIC8643 protein or an insecticidal protein from the TIC8643 toxin protein class set forth in SEQ ID NO: 1, and the amino acid sequence set forth in SEQ ID NO: 2. Segments or fragments of the TIC8643 proteins of the present disclosure that exhibit insect inhibitory activity can have from about 65% to 100%, for example, 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 range of percent identity in amino acid sequence alignment to the corresponding segment or sequence of the TIC8643 amino acid sequence set forth in SEQ ID NO: 2. Segments or fragments described herein can 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 contiguous or consecutive amino acid residues of a TIC8643 protein or a related protein thereof.

[0038] The terms "pesticidal activity" or "pesticidal" or "insecticidal activity," "insect inhibitory," "pesticidally effective," or "insecticidal" as referred to in this application are intended to refer to the 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 pest infestation, controlling pest feeding activity on a particular crop), or killing (causing morbidity, mortality, or reduction in fecundity) an insect pest. Such a toxic agent or protein toxin can comprise an effective amount of a TIC8643 protein or related protein or fragment thereof. These terms are intended to include the result of providing an insect pest with a pesticidally effective amount of a toxic protein, wherein exposure of the pest to the toxic protein will inhibit, suppress, control, or kill the pest. These terms also include the repelling of a pest from a plant, plant tissue, plant part, seed, plant cell, or from a particular geographic location in which a plant can grow as a result of providing a pesticidally effective amount of a toxic protein within or on the plant. In general, pesticidal activity and the like refer to the ability of a toxic protein to effectively inhibit growth, development, viability, feeding behavior, mating behavior, fecundity, or to effectively cause any measurable reduction in the adverse effects on a plant caused by insect feeding. A lepidopteran-specific toxic protein can be produced by a plant or can be applied to a plant or the environment within which the plant is located. The terms "bioactivity," "effective," "efficacious," or variations thereof are also terms that are used interchangeably in this application to describe the effect (or effective ability) of the pesticidal proteins of the present disclosure on a target insect pest.

[0039] An insecticidally effective amount of a toxic agent, when provided in the diet of a target pest, will exhibit insecticidal activity upon contact of the toxic agent with the pest. The toxic agent can be an insecticidal protein or one or more chemical agents known in the art. Insecticidal or insecticidal chemical agents can be used alone or in combination with one another. Chemical agents include, but are not limited to, dsRNA molecules targeting specific genes to be suppressed in the target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Insecticidal or insecticidal protein agents include the protein toxins set forth in this application, as well as other proteinaceous toxic agents, including those targeting Lepidoptera, as well as protein toxins for control of other plant pests such as Cry, Vip, and Cyt proteins, Pseudomonas insecticidal proteins, and insecticidal proteins derived from fern species, which are useful in the art for control of Coleoptera, Hemiptera, and Homoptera species.

[0040] Reference to a pest, particularly a pest of a crop plant, means an insect class pest of a crop plant, particularly those Lepidoptera insect class pests controlled by insecticidal proteins within the TIC8643 protein toxin class or fragments thereof. However, reference to a pest can also include plant insect class pests of Coleoptera, Hemiptera, and Homoptera, as well as nematodes and fungi, when a toxic agent against these pests is co-located or co-present with an insecticidal protein within the TIC8643 protein toxin class or fragments thereof, for example, an amino acid sequence of an insecticidal protein having an identity within the range of about 65% to about 100% to the TIC8643 protein of SEQ ID NO: 2, or any other sequence percentage range within this range provided herein. The phrases "co-present" or "co-located" are intended to include any situation in which a target insect class pest has been exposed to an insecticidal protein in the TIC8643 toxin protein class (including fragments of the TIC8643 protein or related proteins), as well as any other toxic agent is also present in insecticidally effective amounts relative to the target insect class pest. "Exposure" to an insecticidal protein or fragment thereof of the TIC8643 protein toxin class is intended to mean present in the diet of the target pest, and the diet is consumed by the target pest. The diet can be a natural diet or a controlled, artificial, or experimental diet, for example, a diet used in diet assays.

[0041] Lepidoptera insects intended to be within the scope of the present application include, but are not limited to, armyworms, sugar moths, geometer moths, and noctuid moths of the family Noctuidae, for example, Spodoptera eridania (Cabo Negro) ( Spodoptera frugiperda ), Spodoptera exigua (beet armyworm) Spodoptera exigua ), Spodoptera frugiperda (fall armyworm) Spodoptera cosmioides ), Spodoptera littoralis (Egyptian cotton leafworm) Spodoptera eridania ), Spodoptera ornithogalli (omithogalloides) Mamestra configurata ), Phyllophaga sp. (white grubs) Agrotis ipsilon ), Plutella xylostella (diamondback moth) Trichoplusia ni ), Scirpophaga incertulas (sugarcane borer) Diatraea saccharalis), soybean looper ( Pseudoplusia includens ), sunflower inchworm ( Rachiplusia nu ), fluffy bean caterpillar ( Anticarsia gemmatalis ), alfalfa green moth ( Hypena scabra ), smoke moth ( Heliothis virescens ), Particle moth ( Agrotis subterranea ), armyworms ( Pseudaletia unipuncta ), sunflower inchworm ( Rachiplusia nu ), South American bean worm ( Helicoverpa gelotopoeon ), Western root-cutting worm ( Agrotis orthogonia ); Pyralids include stem borers, leafminers, web-forming caterpillars, trypanosomes, cabbage borers, and leaf-carving insects, such as the European corn borer ( Ostrinia nubilalis ), navel orange moth ( Amyelois transitella Corn root-knot caterpillar ( Crambus caliginosellus Grassland web-weaving caterpillars ( Herpetogramma licarsisalis ), sunflower borer ( Homoeosoma electellum ), small corn stem borer ( Elasmopalpus lignosellus ); leafrollers, bud borers, seed borers, and fruit borers of the family Helicidae, such as the apple leafroller ( Cydia pomonella Grape leafroller ( Endopiza viteana ), pear fruit moth ( Grapholita molesta ), sunflower bud moth ( Suleima helianthana ); and many other economically important Lepidoptera, such as the diamondback moth ( Plutella xylostella ), pink bollworm ( Pectinophora gossypiella ) and gypsy moth ( Lymantria dispar Other insect pests of the Lepidoptera order include, for example, the cotton leaf beetle (…). Alabama argillacea Fruit tree leafroller ( Archips argyrospila ), European leafroller ( Archips rosana ) and other leafroller species (rice stem borer ( Chilo suppressalis) , Asiatic rice borer or rice stem borer, rice leaf roller ( Cnaphalocrocis medinalis Corn root-knot caterpillar ( Crambus caliginosellus ), Bluegrass web-weaving caterpillar ( Crambus teterrellus ), Southwest corn borer ( Diatraea grandiosella ), sugarcane borer ( Diatraea saccharalis Dingdian Diamond () Earias insulana ), Jadeite Diamond ( Earias vittella ), American cotton bollworm ( Helicoverpa armigera ), corn ear worm ( Helicoverpa zea Also known as soybean pod borer and cotton bollworm), tobacco budworm ( Heliothis virescens Grassland web-weaving caterpillars ( Herpetogramma licarsisalis ), Western bean worm (Striacosta albicosta ), European grape moth ( Eupoecilia ambiguella Denis & Schiffermuller), Lobesia botrana ), citrus leafminer ( Phyllocnistis citrella Stainton), Phyllocnistis citrella ), small white butterfly ( Pieris rapae L.), Pieris brassicae ), beet armyworm ( Spodoptera exigua Hubner), Pieris rapae ), cabbage moth ( Pieris rapae L.), Beet armyworm ), cabbage moth ( Pieris rapae L.), Tobacco cutworm ), cabbage moth ( Pieris rapae L.), Tomato leafminer ).

[0042] Reference in this application to an "isolated DNA molecule" or equivalent terms or phrases is intended to mean a DNA molecule that exists either alone or in combination with other compositions, but not in its natural environment. For example, a nucleic acid element (such as a coding sequence, an intron sequence, an untranslated leader sequence, a promoter sequence, a transcriptional termination sequence, etc.) that naturally exists in the DNA of an organism's genome is not considered to be "isolated" as long as it is located within the organism's genome and at its naturally occurring location within the genome. However, each of these elements and sub-portions of these elements are "isolated" within the scope of the present disclosure as long as they are not within the organism's genome and are not at their naturally occurring location within the genome. Similarly, a nucleotide sequence that encodes an insecticidal protein or any naturally occurring insecticidal protein associated with that protein is an isolated nucleotide sequence as long as it is not in the DNA of the bacterium in which the sequence naturally occurs that encodes the protein. For purposes of the present disclosure, a synthetic nucleotide sequence that encodes an amino acid sequence of a naturally occurring insecticidal protein will be considered to be isolated. For purposes of the present disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA that is inserted into the genome of a plant or bacterial cell or that is present in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence whether it is present in a plasmid or similar structure used to transform the cell, in the genome of the plant or bacterium, or in detectable amounts in tissue, progeny, biological samples, or commodity products derived from the plant or bacterium.

[0043] The term "self-limiting gene" as referred to in this application means a gene that limits host survival, thereby resulting in a reduction in the host population. This technology is provided by Oxitech Limited. Transgenic male insects carrying the transgenic self-limiting gene are released and mate with wild female insects. As a result, the offspring inherit a copy of the self-limiting gene. The self-limiting gene disrupts the normal functioning of the insect cell by overproducing a protein in the insect cell that interferes with the cell's ability to produce other essential proteins needed for development, thereby destroying the normal function of the insect cell. By destroying the normal development of the insect, the gene prevents the insect from surviving to adulthood. For example, the self-limiting diamondback moth ( Plutella xylostella L.) is a transgenic male that carries a transgenic self-limiting gene that prevents the insect from surviving to adulthood. Diamondback mothThe OX4319L strain was developed by Oxitech Limited and carries a male selection gene that utilizes the sequence of the sex determining gene doublesex ( dsx ) of the Lepidoptera. The gene expresses a gender-alternating splice that engineers female-specific expression of a self-limiting gene, thereby preventing female offspring from surviving past the larval stage and allowing the production of only male populations of self-limiting moths. When released, the males mate with pest females, resulting in a reduction in the number of female offspring in the next generation, thereby locally suppressing the Plodia xylostella population. To facilitate the rearing of large numbers of males in a Plodia xylostella production facility for release, expression of the female-specific dsx in the OX4319L strain is suppressed by the addition of tetracycline or a suitable analogue to the larval diet. OX4319L also expresses the fluorescent protein DsRed to allow efficient 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 to fields containing plants of the invention that contain the toxic genes and proteins of the invention, can delay or prevent the development of resistance to the toxins and proteins of the invention by the pest species intended to be controlled by the invention, thereby making any plant products containing the toxins and proteins of the invention more durable.

[0044] As further described herein, an open reading frame (ORF) (SEQ ID NO: 1) encoding TIC8643 (SEQ ID NO: 2) was discovered in DNA obtained from Streptomyces rubiginosus strain MDI-0021357. Bioassays using the microbial host cell-derived TIC8643 protein demonstrated that the protein was active against lepidopteran species of the genus Agrotis (BCW, Black cutworm ipsilon ), Helicoverpa zea (CEW, Corn earworm ), and Spodoptera frugiperda (FAW, Fall armyworm ).

[0045] A synthetic coding sequence (SEQ ID NO: 3) designed to express the TIC8643PL protein (SEQ ID NO: 4) in plant cells has been identified and manufactured, and for ease of cloning, an additional or supplemental alanine codon has been inserted between the start methionine codon and the serine codon of the native sequence. Corn plants expressing the artificial protein containing this supplemental alanine in the second position (hereinafter referred to as TIC8643PL) exhibited activity against Agrotis segetum (BCW, Black cutworm ) and Spodoptera frugiperda (FAW,Spodoptera frugiperda The natural TIC8643 toxin was not reduced. When maize plant tissues expressing the TIC8643 toxin were provided, resistance to the maize ear borer (CEW) was also observed. Corn earworm Therefore, based on these data, and for the purpose of naming, there is no difference in insecticidal activity between TIC8643 and TIC8643PL.

[0046] For expression in plant cells, the TIC8643 (SEQ ID NO: 2 or 4) protein or related proteins or fragments thereof, such as insecticidal proteins or fragments within the TIC8643 protein toxin class, can be expressed and localized in the cytoplasm or targeted to various organelles of the plant cell. For example, targeting a protein to chloroplasts may increase the level of the protein expressed in the transgenic plant while preventing off-phenotypes when the expressed protein toxin reacts to cell biology in any unexpected way. Targeting can also increase pest resistance efficacy in transgenic events. The target peptide or transport peptide is a short peptide chain (3-70 amino acids in length) that can be fused to the insecticidal protein sequence to become part of the insecticidal protein and guide the transport of said protein to specific regions of the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplasts, apoplasts, peroxisomes, and plasma membrane. The polynucleotide coding sequence of the insecticidal protein may also include the polynucleotide coding sequence of the target peptide or transport peptide, thereby further comprising the target peptide or transport peptide of the insecticidal protein. After the protein is transported, some target peptides are cleaved from the protein by signal peptidases. To target chloroplasts, the protein contains a transport peptide of approximately 40-50 amino acids in length. For a description of the use of chloroplast transport peptides, see U.S. Patent Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed as precursors of nuclear genes and target chloroplasts via chloroplast transport peptides (CTPs). These proteins can fuse with insecticidal proteins and become part of them. Examples of such isolated CTPs include, but are not limited to, those associated with: the small subunit of ribulose-1,5-bisphosphate carboxylase (SSU), ferricredoxin, ferricredoxin oxidoreductase, light-trapping complex proteins I and II, thioredoxin F, enolpyruvate shikimate phosphate synthase (EPSPS), and the transport peptide described in U.S. Patent No. 7,193,133. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to chloroplasts using protein fusions with heterologous CTPs, and that CTPs are sufficient to target proteins to chloroplasts. It has been confirmed that incorporating suitable chloroplast transport peptides, such as those found in Arabidopsis thaliana (…),… Arabidopsis thaliana EPSPS CTP (CTP2) (See Klee et al., Molecular and General Genetics210:437-442, 1987) or petunia ( Petunia hybrida EPSPS CTP (CTP4) (see della-Cioppa et al.) Proceedings of the National Academy of Sciences of the United States of America (83:6873-6877, 1986) enables heterologous EPSPS protein sequences to target chloroplasts in transgenic plants (see U.S. Patents 5,627,061, 5,633,435, and 5,312,910; and EP 0218571, EP 189707, EP 508909, and EP 924299). To target the TIC8643 toxin protein to chloroplasts, a sequence encoding a chloroplast transport peptide is placed at the 5' position of a synthetic / artificial nucleotide sequence (e.g., SEQ ID NO: 3) encoding the TIC8643PL toxin protein, operatively linked to and framed with said synthetic / artificial nucleotide sequence.

[0047] Consideration has been given to the possibility of using the amino acid sequence of TIC8643 or its associated proteins or fragments to generate additional toxin protein sequences associated with TIC8643, thereby producing novel proteins with novel properties. The TIC8643 toxin protein may share approximately 65% ​​to approximately 99.5% or higher percentage of identity with TIC8643-associated toxin proteins when aligned, and differences at the aligned amino acid sequence level may be combined into one or more novel amino acid sequence variations, and appropriate modifications may be made to the recombinant nucleic acid sequence encoding an insecticidal protein within the TIC8643 toxin protein class, for example, to enhance expression in plants, plant parts, or plant cells.

[0048] Amino acid sequences encompassing insecticidal proteins related to the TIC8643 protein that exhibit improved properties relative to the sequence set forth in SEQ ID NO: 2 are contemplated. 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 plants through the use of various gene editing methods known in the art. Such techniques for genome editing include, but are not limited to, zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) systems. These genome editing methods can be used to change a toxin protein coding sequence transformed within a plant cell to a different toxin coding sequence. Specifically, through these methods, one or more codons within a toxin coding sequence can be altered to engineer a new protein amino acid sequence. Alternatively, a fragment within a coding sequence can be replaced or deleted, or an additional DNA fragment can be inserted into a coding sequence, to engineer a new toxin coding sequence. Plant cells comprising a toxin coding sequence following gene editing can be used to produce whole plants expressing the new toxin protein through methods known in the art.

[0049] Additionally, it is contemplated that fragments of TIC8643 or related proteins thereof can be truncated forms, wherein one or more amino acids are deleted from the N-terminus, C-terminus, middle or internal portion, or a combination thereof, of the protein, wherein the fragments and related proteins retain insect inhibitory activity. These protein fragments and related proteins can be naturally occurring proteins, or synthetic proteins derived from the TIC8643 protein, but should retain the same or similar insect inhibitory activity as the TIC8643 protein.

[0050] Various computer-based algorithms known in the art can be used to identify proteins similar to the TIC8643 protein and compare to one another. The amino acid sequence identity reported in this application is the 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). The percent amino acid identity is further calculated by multiplying 100% by the product of (number of identical amino acids of the subject protein / length). Other alignment algorithms are also available in the art and provide results similar to those obtained using Clustal W alignment and are encompassed herein.

[0051] If, in a query, for example in a Clustal W alignment, a protein exhibiting insect-inhibiting activity against lepidopteran insect species is used, and the protein shows at least 65% to approximately 100% amino acid identity along the length of the query protein, i.e., approximately 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 approximately 100%, or any fractional percentage of amino acid identity within this range, or the aforementioned insecticidal fragment, then the insecticidal protein is considered to be associated with TIC8643.

[0052] In addition to the percentage of identity, TIC8643 can also be correlated based on its primary structure (conserved amino acid motif), length, and other characteristics. Table 1 provides the characteristics of TIC8643 and TIC8643PL proteases.

[0053] Table 1. Selected characteristics of TIC8643 and TIC8643PL toxin proteins.

[0054] As further described in the embodiments of this application, synthetic or artificial nucleic acid molecules (i.e., nucleotide sequences) encoding TIC8643PL are designed for use in plants, as shown in SEQ ID NO: 3. Given the redundancy of the genetic code, generating any number of other sequences encoding toxin proteins is within the scope of the art; however, it should be understood that generating sequences for expression in plants should avoid problems known in the art that hinder or limit the efficient expression of coding sequences, particularly those described in U.S. Patents 5,500,365 and 7,741,118.

[0055] Expression cassettes and vectors containing recombinant nucleic acid sequences can be constructed and introduced into plants, particularly plant cells such as maize, soybean, or cotton, using transformation methods and techniques known in the art. For example, Agrobacterium (… AgrobacteriumTransformation mediated by [unspecified species] is described in U.S. Patent Application Publications 2009 / 0138985A1 (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257A1 (sugar beet), U.S. Patent Nos. 5,750,871 (canola), 7,026,528 (wheat), and 6,365,807 (rice), and Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugarcane). Various methods for transforming crops are known in the art, and these have been described in more detail above. Examples of methods for transforming cowpeas are provided in Bosibori et al. ([unspecified species]). 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 for converting cassava are provided in 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 The transformed cells can be regenerated into transformed plants expressing TIC8643 or related proteins or fragments, and their insecticidal activity is demonstrated by bioassays performed in the presence of lepidopteran, coleopteran, or hemiptera pest larvae using plant leaves obtained from the transformed plants. Plants can be obtained from plant cells through regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming and regenerating plants are known in the art. The transformed cells can be regenerated into transformed plants expressing TIC8643, and their insecticidal activity is demonstrated by bioassays performed in the presence of lepidopteran pest larvae using plant leaves obtained from the transformed plants. Plants can be obtained from plant cells through regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming and regenerating plants are known in the art.

[0056] As an alternative to traditional transformation methods, DNA sequences such as transgenes, expression cassettes, or the like can be inserted or integrated into a plant or plant cell genome via site-directed integration into a specific site or locus within the genome. Thus, one or more recombinant DNA constructs and one or more molecules of the present disclosure can include a donor template sequence comprising at least one transgene, expression cassette, or other DNA sequence for insertion into the genome of a plant or plant cell. Such a donor template for site-directed integration can also include one or two homology arms flanking the insert sequence, i.e., the sequence, transgene, cassette, or the like intended for insertion into the plant genome. One or more recombinant DNA constructs of the present disclosure can also comprise one or more expression cassettes encoding a site-specific nuclease and / or any one or more associated proteins to perform site-directed integration. These nuclease expression cassettes can be present in the same molecule or vector as the donor template (cis), or in different molecules or vectors (trans). Several methods for site-directed integration are known in the art, which involve different proteins (or complexes of proteins and / or guide RNAs) that cleave genomic DNA to create a double-strand break (DSB) or a nick at a desired genomic site or locus. Briefly, as understood in the art, during the process of repairing a DSB or nick introduced by a nuclease, a donor template DNA can be integrated into the genome at the site of the DSB or nick during the repair process by homologous recombination. The presence of one or more homology arms in the donor template can facilitate the adoption of the insert sequence and targeting it into the plant genome during the repair process by homologous recombination, but insert events can also occur by non-homologous end joining (NHEJ). Examples of site-specific nucleases that can be used include zinc finger nucleases, engineered or natural meganucleases, TALE-endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cas12a). For methods using RNA-guided site-specific nucleases (e.g., Cas9 or Cas12a), the one or more recombinant DNA constructs will also comprise sequences encoding one or more guide RNAs to direct the nuclease to a desired site within the plant genome.

[0057] The recombinant nucleic acid molecule compositions described herein encoding a TIC8643 protein or a related protein or fragment thereof for bacterial and plant expression can be expressed with a recombinant DNA construct, wherein the polynucleotide molecule having the ORF encoding the protein is operably linked to genetic expression elements such as a promoter and any other regulatory elements required for expression in the system in which the construct is to be used. Non-limiting examples include a plant functional promoter operably linked to the TIC8643 protein, related protein, or fragment coding sequence for expression of the protein in a plant, or a plant functional promoter operably linked to the TIC8643 protein, related protein, or fragment coding sequence for expression of the protein in a bacterium. The recombinant nucleic acid molecule compositions described herein can be expressed with a recombinant DNA construct, wherein the polynucleotide molecule having the ORF encoding the protein is operably linked to genetic expression elements such as a promoter and any other regulatory elements required for expression in the system in which the construct is to be used. Non-limiting examples include a plant functional promoter operably linked to the TIC8643 protein, related protein, or fragment coding sequence for expression of the protein in a plant, or a plant functional promoter operably linked to the TIC8643 protein, related protein, or fragment coding sequence for expression of the protein in a bacterium. BtBacteria or other Bacillus bacteria in which the protein is expressed Bt A functional promoter. The TIC8643 protein, related protein, or fragment-encoding sequence can include any polynucleotide sequence encoding a TIC8643 protein or a related protein or fragment thereof, as described herein. Other elements can also be operably linked to the TIC8643 protein-encoding sequence, including but not limited to enhancers, introns, untranslated leader sequences, encoded protein immobilization tags (HIS-tags), translocation peptides (i.e., plastid transit peptides, signal peptides), polypeptide sequences of post-translational modification enzymes, ribosome binding sites, and RNAi target sites. Exemplary recombinant polynucleotide molecules provided herein include, but are not limited to, a heterologous promoter operably linked to a polynucleotide encoding a TIC8643 or TIC8643PL or a related protein or fragment thereof as set forth in SEQ ID NO: 2 or SEQ ID NO: 4, such as SEQ ID NO: 1 or 3. A heterologous promoter can also be operably linked to a synthetic / artificial DNA-encoding sequence 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 the recombinant nucleic acid molecules encoding the proteins disclosed herein can be replaced with synonymous codons (referred to in the art as silent substitutions), whereby they can be optimized for expression in plants.

[0058] A recombinant DNA construct comprising a TIC8643 protein, related protein, or fragment-encoding sequence can also comprise a separate DNA region, segment, or sequence encoding one or more insect inhibitors, such as insect-inhibiting dsRNA molecules or helper proteins, which can be configured to be expressed simultaneously or co-expressed with the TIC8643 protein, related protein, or fragment-encoding sequence. Helper proteins include, but are not limited to, cofactors, enzymes, binding partners, or other agents that aid in the effectiveness of the insect inhibitors, such as by helping with the expression of the insect inhibitors, affecting the stability of the insect inhibitors in the plant, optimizing the free energy of oligomerization, enhancing the toxicity of the insect inhibitors, and increasing the spectrum of activity of the insect inhibitors. Helper proteins can facilitate the uptake of, for example, one or more insect inhibitors, or enhance the toxic effects of toxic agents.

[0059] Recombinant DNA constructs can be assembled such that all proteins or dsRNA molecules are expressed from a single promoter, or each protein or dsRNA molecule is under the control of an independent promoter, or some combination thereof. The proteins of the present invention can be expressed by a multi-gene expression system, wherein TIC8643 or its associated proteins or fragments are expressed by a common nucleotide segment or sequence, depending on the type of expression system chosen, which also contains other open reading frames and promoters. For example, a bacterial multi-gene expression system can utilize a single promoter to drive the expression of multiple linked / tandem open reading frames from a single operon (i.e., polycistronic expression). In another example, a plant multi-gene expression system can utilize multiple unlinked or linked expression cassettes, each expressing a different protein or other agent, such as one or more dsRNA molecules.

[0060] Recombinant polynucleotides or recombinant DNA constructs containing the coding sequence of the TIC8643 protein, related proteins, or fragments can be delivered to host cells via vectors, such as plasmids, baculoviruses, synthetic chromosomes, viral particles, granules, phage particles, bacteriophages, or viral vectors. These vectors can be used to achieve stable or transient expression of the coding sequence of the TIC8643 protein, related proteins, or fragments in host cells, or subsequent expression of the encoded polypeptide. Exogenous recombinant polynucleotides or recombinant DNA constructs containing the coding sequence of the TIC8643 protein, related proteins, or fragments and introduced into host cells are referred to herein as "transgenic" structures.

[0061] This article provides information on transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts containing recombinant polynucleotides expressing TIC8643 or related sequences encoding a family of toxin proteins. The terms "bacterial cell" or "bacteria" may include, but are not limited to, *Agrobacterium*, *Bacillus*, *Escherichia*, and *Salmonella*. Salmonella), Pseudomonas, Brevibacillus, Klebsiella, Erwinia, or Rhizobium cells. The term "plant cell" or "plant" can include, but is not limited to, a dicotyledonous or monocotyledonous plant. The term "plant cell" or "plant" can also include, but is not limited to, 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, cucurbit, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn (i.e., maize) such as sweet corn or field corn, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cells or plants. In certain embodiments, transgenic plants and transgenic plant parts are provided that are regenerated from the transgenic plant cells. In certain embodiments, the transgenic plants can be obtained from transgenic seeds by cutting, breaking, grinding, or otherwise separating a portion from the plant. In certain embodiments, the plant part can be a seed, pollen grain, boll, leaf, flower, stem, root, or any portion thereof, or a non-regenerable portion of a transgenic plant part. As used herein, a "non-regenerable" portion of a transgenic plant part is a portion that cannot be induced to form a whole plant, or a portion that cannot be induced to form a whole plant that is capable of sexual and / or asexual reproduction. In certain embodiments, a non-regenerable portion of a plant part is a portion of a transgenic seed, pollen grain, chloroplast, boll, leaf, flower, stem, or root.

[0062] Methods of making a transgenic plant comprising an insect-inhibiting or lepidopteran-inhibiting amount of a TIC8643 protein or a related protein or fragment thereof are provided. Such plants can be obtained by introducing a recombinant polynucleotide encoding a protein provided herein into a plant cell, and selecting a plant obtained from the plant cell that expresses an insect- or lepidopteran-inhibiting amount of the protein. Plants can be obtained from plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0063] Also provided herein are processed plant products, wherein the processed products comprise a detectable amount of a TIC8643 protein or insect inhibiting related protein thereof, or an insect inhibiting segment or fragment, or a TIC8643 protein coding sequence, or any distinctive portion or part of the foregoing. In certain embodiments, the processed products are selected from the group consisting of plant parts, plant biomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint, hulls, processed seeds, and seed. In certain embodiments, the processed products are non-regenerable. Plant products can comprise commodities or other commercial products derived from a transgenic plant or transgenic plant parts, wherein the commodities or other products can be commercially tracked by detecting a TIC8643 protein or related protein or fragment thereof, or a polynucleotide sequence or segment encoding all or part of a TIC8643 protein or related protein or fragment thereof, or a distinctive portion comprising a TIC8643 protein, related protein or fragment coding sequence, or expressed RNA.

[0064] Plants expressing a TIC8643 protein or related protein or fragment thereof can be crossed 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, either by breeding together or these traits can be combined in a single stacked vector, such that the traits are linked together when present within the same transgenic genome.

[0065] As further described in the Examples, TIC8643 protein coding sequences and sequences having a substantial percentage of identity to TIC8643 coding sequences 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, a protein TIC8643 or related protein or fragment thereof can be used to generate antibodies that specifically bind to the related protein, and can be used to screen and find other protein members that are closely related.

[0066] In addition, nucleotide sequences encoding all or part of a TIC8643 toxin protein or related proteins or fragments thereof can be used as probes and primers for screening to identify other members of the TIC8643 protein toxin class using thermal cycling or isothermal amplification and hybridization methods. For example, oligonucleotides derived from or analogous to the sequence set forth in SEQ ID NO: 3 or other transgenic sequences can be used to determine whether a TIC8643 related transgene is present in a deoxyribonucleic acid sample derived from a commodity product. Given the sensitivity of certain nucleic acid detection methods employing oligonucleotides, it is expected that oligonucleotides derived from the sequence set forth in SEQ ID NO: 3 or other transgenic sequences can be used to detect a TIC8643 transgene in a commodity product or sample derived from a mixed source, where only a small fraction of the commodity product or source is derived from a transgenic plant or plant part containing the transgene. Furthermore, it is also recognized that these oligonucleotides can be used to introduce nucleotide sequence variations in each of SEQ ID NOs: 1 and 3. Such "mutagenic" oligonucleotides can be used to identify TIC8643 related proteins or fragments that exhibit a range of insect inhibitory activities or exhibit different expression profiles in transgenic plant host cells.

[0067] Nucleotide sequence homologs, e.g., insecticidal proteins encoded by nucleotide sequences that hybridize under stringent hybridization conditions to each or any of the sequences disclosed herein, are also embodiments of the present application. The present application 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) encodes a pesticidal protein or a pesticidal fragment thereof and hybridizes to the second nucleotide sequence. In this case, the second nucleotide sequence can be the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or any of the nucleotide sequences that hybridize under stringent hybridization conditions to either of SEQ ID NOs: 1 or 3. The nucleotide coding sequences hybridize to each other under appropriate hybridization conditions, such as stringent hybridization conditions, and the proteins encoded by these nucleotide sequences can cross-react with antisera raised against any one of the other proteins. As defined herein, stringent hybridization conditions include at least hybridization at 42°C followed by two washes in 2X SSC, 0.1% SDS at room temperature for five minutes each, followed by two washes in 0.5X SSC, 0.1% SDS at 65°C for thirty minutes each. Washing at higher temperatures constitutes higher stringency, for example, hybridization conditions at 68°C followed by washing in 2x SSC containing 0.1% SDS at 68°C means that a higher percentage of nucleotide sequence identity will be selected and detected.

[0068] One skilled in the art will recognize that, due to the redundancy of the genetic code, many other sequences are also capable of encoding such related proteins, and that those sequences are embodiments of the present invention in the sense that they are useful for expressing pesticidal proteins in bacterial strains such as Bacillus strains, fungi, yeast, or other host cells, or in plant cells, and it will of course be recognized that many such redundantly encoding sequences will not hybridize under these conditions to the native Bacillus sequence encoding a TIC8643 related protein. This application encompasses the use of these and other methods of identification known to one of ordinary skill in the art to identify sequences encoding TIC8643 related proteins as well as sequences having a substantial percentage of identity to nucleic acid sequences encoding TIC8643 related proteins.

[0069] The present disclosure also encompasses the use of molecular methods known in the art to engineer and clone commercially useful proteins comprising chimeras of proteins from pesticidal proteins; for example the chimeras can be assembled from segments or portions of the TIC8643 protein or related proteins or fragments thereof to yield additional useful embodiments, including the assembly of segments or portions of the TIC8643 protein or related proteins or fragments thereof with segments or portions of any different insecticidal protein than the TIC8643 protein and related proteins. The TIC8643 protein and related insecticidal proteins can be subjected to mutual alignment and alignment with other Bacillus, Paenibacillus, or other pesticidal proteins, whether these proteins are closely or distantly related phylogenetically, or other artificial or engineered protein sequences, and segments of each of the proteins can be identified that are useful for substitution between the aligned proteins, thereby constructing chimeric proteins. These chimeric proteins can be subjected to pest bioassay analysis and characterized for the presence of increased biological activity or expanded target pest spectrum compared to the parent proteins from which each such segment in the chimera is derived. The insecticidal activity of the polypeptides can be further engineered for activity against specific pests or a broader spectrum of pests by exchanging domains or segments with other proteins or by using methods of directed evolution known in the art.

[0070] The present application discloses methods of preventing infestation of crop plants by insects, particularly Lepidoptera, Coleoptera, and / or Hemipteran insects, with TIC8643 proteins or related proteins or fragments thereof. These methods can include growing a plant comprising an insect- or Lepidoptera-inhibiting amount of a TIC8643 toxin protein or related insecticidal protein. In certain embodiments, these methods can further include any one or more of: (i) applying to a plant or seed producing a plant any composition comprising or encoding a TIC8643 toxin protein or related insecticidal protein; and (ii) transforming a plant or plant cell producing a plant with a polynucleotide encoding a TIC8643 toxin protein or related insecticidal protein. Generally, it is contemplated that a TIC8643 toxin protein or related insecticidal protein can be provided in a composition, in a microorganism, or in a transgenic plant to impart insect-inhibitory activity against Lepidopteran insects.

[0071] In certain embodiments, a recombinant nucleic acid molecule encoding a TIC8643 toxin protein or related insecticidal protein is an insecticidal active ingredient of an insect-inhibiting composition prepared by culturing a 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 for expression of the TIC8643 toxin protein or related insecticidal protein or fragment thereof. Such a composition can be prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting, or concentrating a culture of these recombinant cells expressing / producing the 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 polypeptide so produced, a composition including the recombinant polypeptide can include bacterial cells, bacterial spores, and parasporal inclusion bodies, and can be formulated for various uses, including as an agricultural insect-inhibiting spray product or as an insect-inhibiting formulation in diet bioassays.

[0072] In one embodiment, to reduce the likelihood of resistance development, the insect-inhibiting composition comprising the TIC8643 protein or its associated insecticidal protein or fragment may further comprise at least one additional polypeptide that exhibits insect-inhibiting activity against the same Lepidoptera species, but is different from the TIC8643 toxin protein or associated insecticidal protein. Possible additional insecticides for such compositions include insect-inhibiting proteins and insect-inhibiting dsRNA molecules. An example of using such ribonucleotide sequences to control insect pests is described in Baum et al. (US Patent Publication 2006 / 0021087A1). This additional polypeptide for controlling lepidopteran pests can be selected from the group consisting of insect inhibitory proteins, such as, but not limited to, Cry1A (US Patent No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (US Patent Publication No. 10 / 525,318), Cry1C (US Patent No. 6,033,874), Cry1D, Cry1Da and its variants, Cry1E, Cry1F and Cry1A / F chimeras (US Patent Nos. 7,070,982, 6,962,705 and 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, and Cry1-type chimeras, such as, but not limited to, TIC836, TIC860, TIC867, TIC869 and TIC1100. (International Application Publication WO2016 / 061391), TIC2160 (International Application Publication WO2016 / 061392(A2)), Cry2A, Cry2Ab (US Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (US Patent Publication 2013-0117884) AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (US Patent Publication 2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (US Patent Publication 2013-0104259 A1), AXMI-134 (US Patent Publication 2013-0167264 A1), AXMI-150 (US Patent Publication 2010-0160231)AXMI-184 (US Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (US Patent Publication 2011-0030096 A1), AXMI-218, AXMI-220 (US Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (US Patent Publication 2014-0196175 A1), AXMI-238 (US Patent Publication 2014-0033363 A1), AXMI-270 (US Patent Publication 2014-0223598 A1), AXMI-345 (US Patent Publication 2014-0373195) A1), AXMI-335 (International Application Publication WO2013 / 134523(A2)), DIG-3 (US Patent Publication 2013-0219570 A1), DIG-5 (US Patent Publication 2010-0317569 A1), DIG-11 (US Patent Publication 2010-0319093 A1), AfIP-1A and its derivatives (US Patent Publication 2014-0033361 A1), AfIP-1B and its derivatives (US Patent Publication 2014-0033361A1), PIP-1APIP-1B (US Patent Publication 2014-0007292 A1), PSEEN3174 (US Patent Publication 2014-0007292 A1), AECFG-592740 (US Patent Publication 2014-0007292 A1), Pput_1063 (US Patent Publication 2014-0007292 A1), DIG-657 (International Application Publication WO2015 / 195594 A2), Pput_1064 (US Patent Publication 2014-0007292 A1), GS-135 and its derivatives (US Patent Publication 2012-0233726 A1), GS153 and its derivatives (US Patent Publication 2012-0192310 A1), GS154 and its derivatives (US Patent Publication 2012-0192310 A1), GS155 and its derivatives (US Patent Publication 2012-0192310 A1), SEQ ID NO: 2 or 4 and its derivatives (as described in US Patent Publication 2012-0167259 A1), 2SEQ ID NO: 2 or 4 and its derivatives (as described in US Patent Publication 2012-0047606 A1), SEQ ID NO: 2 or 4 and their derivatives (such as those described in U.S. Patent Publication 2011-0154536 A1), SEQID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent Publication 2011-0112013 Al), SEQ ID NO: 2 or 4 and 4 and derivatives thereof (as described in U.S. Patent Publication 2010-0192256 Al), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent Publication 2010-0077507 Al), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent Publication 2010-0077508 Al), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent Publication 2009-0313721 Al), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent Publication 2010-0269221 Al), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent No. 7,772,465 (B2), CF161_0085 and derivatives thereof (as described in WO2014 / 008054 A2), lepidopteran toxic proteins and derivatives thereof (as described in U.S. Patent Publications US2008-0172762 Al, US2011-0055968 Al, and US2012-0117690 Al); SEQ ID NO: 2 or 4 and derivatives thereof (as described in US7510878 (B2), SEQ ID NO: 2 or 4 and derivatives thereof (as described in U.S. Patent No. 7812129 (Bl), 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), TIC13085 and TIC13087 (U.S. Patent Publication 2022-220160 Al), IPD072Aa (International Application Publication WO2020 / 076958), IPD079Ea (International Application Publication WO2017 / 023486), and IPD103 and homologs thereof (International Application Publication WO2018 / 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 Cry1B.34 (U.S. Patent Publication 2017-0226164 Al), and the like.

[0073] In some embodiments, the composition / formulation can also comprise at least one additional insecticidal agent that exhibits insect inhibitory activity against insects not inhibited by the other insect inhibitory proteins of the present application, thereby expanding the insect inhibitory spectrum achieved. For example, to control Hemipteran pests, combinations of insect inhibitory proteins of the present application can be used with Hemipteran active proteins such as TIC1415 (U.S. Patent Publication 2013-0097735 Al), TIC807 (U.S. Patent No. 8609936), TIC834 (U.S. Patent Publication 2013-0269060 Al), AXMI-036 (U.S. Patent Publication 2010-0137216 Al), and AXMI-171 (U.S. Patent Publication 2013-0055469 Al). In addition, polypeptides for controlling Coleopteran pests can be selected from the group consisting of insect inhibitory proteins 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 20140245491 Al), 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 omega-hexatoxin-Hvl a (U.S. Patent Application Publication 2014-0366227 Al).

[0074] Additional insecticidal agents for use in controlling Coleopteran, Lepidopteran and Hemipteran insect pests that can be combined with an insect inhibitory protein of the TIC8643 protein class can be found on the B. thuringiensis toxin nomenclature website maintained by Neil Crickmore (World Wide Web address: btnomenclature.info). In general, any insect inhibitor or protein known to one of ordinary skill in the art can be combined with a protein of the TIC8643 protein toxin class for use in a plant (by breeding or molecular stacking) or in a composition or formulation in a biopesticide or biopesticide combination, provided that the insect inhibitor or protein used in a plant with an insecticidal protein of the TIC8643 protein toxin class does not compete with the insecticidal protein of the TIC8643 protein toxin class in its mode of action against the target pest, and the protein does not impair the health and desirable traits of the transgenic plant, and its own expression level is insecticidal to the intended target pest.

[0075] The potential for insects to develop resistance to certain pesticides has been documented in the art. One insect resistance management strategy is to employ a transgenic crop that expresses two different insect inhibitors that act by different modes of action. Thus, any insect that is resistant to either insect inhibitor can be controlled by the other. Another insect resistance management strategy is to employ a plant that is not protected against the target Lepidopteran pest species to provide a refuge for such unprotected plants. One specific example is described in U.S. Patent No. 6,551,962.

[0076] Other embodiments designed to control pests that are also controlled by the insecticidal proteins disclosed herein, such as topically applied insecticidal chemicals, can be used in seed treatments, spray formulations, drip-on formulations, or wipe-on formulations and applied directly to the soil (soil drenching), to growing plants expressing the proteins disclosed herein, or formulated for application to seeds containing one or more transgenes encoding one or more of the disclosed proteins. Such formulations for seed treatment can be applied with various stickers and tackifiers known in the art. These formulations can contain pesticides that are synergistic with the disclosed proteins in mode of action, such that the formulation pesticides act by different modes of action to control the same or similar pests that the disclosed proteins can control, or these pesticides are used to control pests in a broader host range or plant pest species that the TIC8643 insecticidal protein or its related proteins or fragments cannot effectively control.

[0077] The above compositions / formulations can also comprise an agriculturally acceptable carrier, such as a bait, a dust, a powder, a pellet, a granule, a spray, an emulsion, a colloidal suspension, an aqueous solution, a Bacillus spore / crystal formulation, a seed treatment, a recombinant plant cell transformed to express one or more of the insecticidal proteins described herein, a plant tissue, a plant seed, a plant part, or a plant, or a bacterium transformed to express one or more of the insecticidal proteins described herein. Depending on the level of insect inhibition or insecticidal inhibition of the recombinant polypeptides described herein and the level of the formulation applied to the plant or diet assay, the compositions / formulations can comprise various amounts of the recombinant polypeptides by weight, for example, 0.0001% to 0.001% to 0.01% to 1% to 99% of the recombinant polypeptides by weight.

[0078] In view of the foregoing, those skilled in the art will appreciate that changes can be made in the specifically disclosed aspects without departing from the spirit and scope of the application. Accordingly, the specific structural and functional details disclosed herein are not to be interpreted as limiting.

[0079] Examples Example 1. Discovery, cloning, expression, and purification of TIC8643 coding sequence and encoded toxin protein.

[0080] A TIC8643 insecticidal protein was identified by sequence analysis of the genome of Streptomyces rubiginosus MDI-0021357. DNA was extracted from the microbial culture, then subjected to sequence analysis, and the resulting sequences were assembled and analyzed to identify open reading frames. The following protein domains were analyzed by pfam to identify the TIC8643 open reading frame shown in SEQ ID NO: 1 from nucleotide position 1 to the stop codon after nucleotide position 1080, including the entire coding sequence: botulinum_HA-17 | inhibin_I66 | Cry35 | ricin_B_ lecithin | CD_toxinA | ricin_B_ lecithin_2 | toxin_10 | Cry55. The amino acid sequence of the full-length TIC8643 protein exhibits 64.07% identity to GenBank Accession No. WP_076085859 (derived from Streptomyces IMTB 2501 assembly sequence), annotated with ricin-type beta-dandelion agglutinin-like domain and toxin_10 pfam. There is no evidence that the activity of this assembly protein against insects has been determined.

[0081] Polymerase chain reaction (PCR) primers were designed from the SpTotal genomic DNA isolated from strain MDI-0021357 was amplified to obtain a full-length copy encoding the nucleotide sequence of TIC8643 to confirm that the assembled sequenced region indeed represents a naturally occurring sequence. The amplicon was cloned into *E. coli* using methods known in the art. Ec In the expression carrier, with Ec The expression promoter is operablely linked and may or may not contain a histidine tag for subsequent purification of the histidine-tagged TIC8643 protein. (The remaining text appears to be incomplete and requires further context.) Ec The purified TIC8643 protein formulation obtained from the vector expressing the toxin protein was used in bioassays against various insect pests.

[0082] Example 2. TIC8643 showed Lepidoptera activity in insect bioassays.

[0083] The open reading frame of the initially assembled TIC8643 protein was confirmed by comparison with the sequence of the thermally amplified clone, and both encoded amino acid sequences identical to those shown in SEQ ID NO: 2. The assay was performed using the vector described in Example 1 in the recombinant... Ec The TIC8643 expressed in the sample is toxic to various Lepidoptera, Coleoptera, Hemiptera, and Diptera insects.

[0084] The toxicity of TIC8643 was determined against the following lepidopteran insect species: the small cutworm (BCW). Agrotis ipsilon ), corn ear borer (CEW, Helicoverpa zea Also known as the soybean pod borer), the European corn borer (ECB, Ostrinia nubilalis Fall armyworm (FAW) Spodoptera frugiperda Southern armyworm (SAW) Spodoptera eridania ), soybean looper (SBL, Chrysodeixis includens ), Southwest Corn Borer (SWC, Diatraea grandiosella Tobacco moth () Heliothis virescens ), Western bean moth ( Striacosta albicosta ) and fluffy bean caterpillar ( Anticarsia gemmatalis ); Coleoptera species Western Corn Rootworm (WCR, Diabrotica virgifera ) and the Colorado potato beetle (CPD); Leptinotarsa decemlineata ); Tarnished plant bug (TPB), a species of hemipteran. Lygus lineolaris ), Western pasture mirid bug ( Lygus hesperus ) 、Southern Green Stink Bug (SGB, Nezara viridula), Nezara viridula Neotropical Brown Stink Bug (NBSB, Euschistus heros); Euschistus heros and Dipteran species (YFM, Aedes aegypti Aedes aegypti ). Bioassay results are presented in Tables 2 and 3 below, where "+" indicates activity and "-" indicates no activity.

[0085] Table 2. Activity of TIC8643 against Lepidopteran insect species.

[0086] Table 3. Activity of TIC8643 against Coleopteran, Hemipteran and Dipteran insect species.

[0087] As can be seen from the data presented in Tables 2 and 3, TIC8643 exhibited activity in insect bioassays against the Lepidopteran insects BCW and FAW. By way of example only, for BCW and FAW, the concentration range of toxin protein provided in the assays was 0.25 to 1.3 mg per ml of diet. For BCW, developmental delays and mortality were observed starting at 0.5 mg / ml and intensified as the concentration increased. For FAW, developmental delays and mortality were observed starting at 0.25 mg / ml and intensified as the concentration increased.

[0088] Example 3. Artificial coding sequence designed for expression of TIC8643 in plants.

[0089] An artificial coding sequence encoding TIC8643PL, SEQ ID NO: 3, was designed for expression in plant cells. The artificial (or synthetic) sequence was synthesized according to methods generally described in U.S. Patent 5,500,365 to avoid certain problematic sequences, such as ATTTA and A / T-rich plant polyadenylation sequences, while generally preserving the amino acid sequence of the native Bacillus protein.

[0090] The artificial sequence (SEQ ID NO: 3) encoding TIC8643PL (SEQ ID NO: 4) also contains an additional alanine codon inserted between the first two codons of the naturally encoded sequence, such that the first three amino acids of the TIC8643PL protein are MET-ALA-SER. Using techniques known in the art, SEQ ID NO: 3 was cloned into two plant transformation vectors and downstream of a plant expressible promoter and in each case functionally linked to the promoter so as to drive expression of the coding sequence when in a plant cell. The resulting transformation vector constructs, Construct-1 and Construct-2, were used to transform corn plant cells and the constructs contained: a first transgene cassette for expressing a TIC8643PL pesticidal protein, the expression cassette comprising (in the 5' to 3' direction) a constitutive promoter, operably linked to a leader sequence, operably linked to an intron, operably linked to the artificial coding sequence encoding TIC8643PL, operably linked to a 3' UTR; and a second transgene cassette, the expression cassette selected using glyphosate selection from expression of a microorganism CP4 EPSPS protein that is insensitive to glyphosate inhibition to select the transformed plant cells. The two transformation vectors each contained a different high constitutive promoter for driving TIC8643PL expression.

[0091] Example 4. TIC8643 exhibits Lepidoptera activity when expressed in stably transformed corn plants.

[0092] Two binary plant transformation vectors (Construct-1 and Construct-2) were cloned using methods known in the art, the transformation vectors containing a transgene cassette designed for expression of TIC8643PL, each vector using a different constitutive expression element to drive expression of TIC8643PL as described in Example 3. The resulting vectors were used to stably transform corn plants. Tissue was harvested from the transformants and used for insect bioassays against various Lepidoptera insect species.

[0093] Corn plant cells were transformed with the binary transformation vectors using Agrobacterium-mediated transformation methods known in the art as described in Example 3. The transformed cells were induced to form plants by methods known in the art. Insect bioassays were performed using plant leaf disks in a similar manner to the methods described in U.S. Patent No. 8,344,207. Individual newly hatched less than one day old neonate larvae were placed on each leaf disk sample and allowed to feed for approximately four days. Untransformed near-isogenic corn plants were used to obtain tissue used as negative controls. The insects were the black cutworm (BCW, Agrotis ipsilon), Agrotis ipsilon ), the European corn borer (ECB, Ostrinia nubilalis), Ostrinia nubilalis ), the fall armyworm (FAW, Spodoptera frugiperda), Spodoptera frugiperda ), and the southwestern corn borer (SWC, Diatraea grandiosella). Diatraea grandiosellaEvaluation was performed on the multiple transformation R0 single copy insertion events obtained from each binary vector. The average percent leaf injury for each event produced by each construct was determined and the results are shown in Tables 1-3, below, and in Figures 1-3, respectively. Figures 1A to 3B The data obtained in the plants for BCW and FAW are consistent with the data observed in the insect bioassay evaluations. Figure 1A and 1B show the average percent leaf injury caused by BCW in the presence of each test construct, respectively. Figure 2A and 2B show the average percent leaf injury caused by FAW in the presence of each test construct, respectively. Figure 3A and 3B show the average percent leaf injury caused by CEW in the presence of each test construct, respectively. This CEW data is surprising in light of the bioassay results provided in Example 2.

[0094] As can be seen from Tables 1-3, many of the events exhibited 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 all had an average percent leaf injury of 30% or less. Other events derived from both constructs exhibited a decrease in average percent leaf injury when compared to the non-transgenic control. Figure 1A 1B As can be seen from Tables 1-3, many of the events exhibited activity against FAW relative to the non-transgenic control. Event 2 derived from Construct-2 had an average percent leaf injury of 3%. Other events derived from both constructs exhibited a decrease in average percent leaf injury when compared to the non-transgenic control.

[0095] As can be seen from Tables 1-3, many of the events exhibited activity against CEW relative to the non-transgenic control, showing a decrease in average percent leaf injury when compared to the non-transgenic control. Figure 2A 2B As can be seen from Tables 1-3, many of the events exhibited activity against CEW relative to the non-transgenic control, showing a decrease in average percent leaf injury when compared to the non-transgenic control.

[0096] As can be seen from Tables 1-3, many of the events exhibited activity against CEW relative to the non-transgenic control, showing a decrease in average percent leaf injury when compared to the non-transgenic control. Figure 3A 3B Thus, the stably transformed corn plants transformed with the constructs expressing TIC8643PL were resistant to infestation by BCW, FAW and CEW.

[0097] Thus, the stably transformed corn plants transformed with the constructs expressing TIC8643PL were resistant to infestation by BCW, FAW and CEW.

[0098] ​​​All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While this application has been described with respect to the illustrative embodiments and implementations shown and described, one skilled in the art will readily appreciate that various modifications can be made to the techniques and methodologies here described without departing from the spirit and scope of the application. More specifically, it will be apparent to one of ordinary skill in the art that certain agents described herein can be substituted for other agents described herein while achieving the same or similar results. All such similar substitutes and modifications are considered to be within the scope of the present application as defined by the following claims.

[0099] All publications and patent documents cited in this specification are incorporated by reference herein in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. 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. the pesticidal protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; b. 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% or about 100% amino acid sequence identity to SEQ ID NO: 2 or 4; or c. the polynucleotide segment comprises SEQ ID NO: 1 or 3, or hybridizes 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. the 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. the recombinant nucleic acid molecule is operably linked to a vector, and the vector is selected from the group consisting of a plasmid, a phagemid, a bacmid, a cosmid, and a bacterial or yeast artificial chromosome.

3. The recombinant nucleic acid molecule of claim 1, which is present within a host cell, wherein the 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 the bacterial host cell is from a bacterial genus selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia.

5. The recombinant nucleic acid molecule of claim 4, wherein the Bacillus genus is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus genus is Brevibacillus laterosporus, and the Escherichia genus is Escherichia coli.

6. The recombinant nucleic acid of any one of claims 2 to 5, wherein the host or plant cell is a dicotyledonous plant cell or a monocotyledonous plant cell. ​ ​ ​ ​ ​ 7. The recombinant nucleic acid of claim 6, wherein the plant cell is selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetgum, 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 to 7, wherein the protein exhibits activity against a lepidopteran insect.

9. The recombinant nucleic acid molecule of claim 8, wherein the Lepidopteran insect is selected from the group consisting of: Agrotis ipsilon ( Agrotis ipsilon ), Helicoverpa zea ( Helicoverpa zea ), and Spodoptera frugiperda ( Spodoptera frugiperda ).

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

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

12. The plant of claim 10, wherein the plant is selected from the group consisting of alfalfa, banana, barley, bean, broccoli, cabbage, brassica, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetgum, 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 the seed comprises the recombinant nucleic acid molecule.

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

15. The insect inhibiting composition of claim 14, further comprising a nucleotide sequence encoding at least one other insecticide different from the insecticidal protein.

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

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

18. The insect inhibiting composition of claim 15, wherein the at least one other insecticide is selected from the group consisting of CrylA, CrylAb, CrylAc, CrylA.105、Cry1Ae, Cry1B, Cry1C, Cry1C variant, Cry1D, Cry1D variant, Cry1E, Cry1F, Cry1A / F chimera, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variant, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IPD102Aa and homologs thereof, IPD110Aa and homologs thereof, TIC868, Cry1Dal_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 Cry1B.

34.

19. The insect inhibitory composition of claim 14, defined as comprising a plant cell expressing an insecticidal 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 the recombinant nucleic acid molecule and / or the insecticidal protein or insecticidal fragment thereof.

21. The commodity product of claim 20, selected from the group consisting of commodity corn bagged with a grain handler, corn flakes, corn tortillas, corn meal, corn flour, corn syrup, corn oil, corn silage, corn starch, corn flakes, and the like, and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodity products, where applicable, including juice, concentrate, jam, jelly, chutney, and other edible forms of the commodity product containing a detectable amount of the polynucleotide and or polypeptide of the present application, whole or processed cottonseed, cotton oil, lint, seed, and plant parts processed for feed or food, fiber, paper, biomass, and fuel products such as fuel derived from cottonseed oil or pellets derived from gin trash, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soy cheese, soy wine, animal feed comprising soybean, paper comprising soybean, butter comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts.

22. A method of producing progeny seed comprising a 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 plant, wherein the harvested seed comprises the recombinant nucleic acid molecule.

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

24. A method for controlling a Lepidoptera species pest or infestation, the method comprising: a. contacting the pest with an insecticidally effective amount of an insecticidal protein as set forth in SEQ ID NO: 2 or 4; or b. contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 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 the sample with a nucleic acid probe that hybridizes under stringent hybridization conditions to genomic DNA in a plant comprising the recombinant nucleic acid molecule of claim 1 and does not hybridize under the hybridization conditions to genomic DNA in another isogenic plant that does not comprise the recombinant nucleic acid molecule of claim 1, wherein the probe is homologous or complementary to, or hybridizes under stringent conditions to, SEQ ID NO: 3 or a sequence encoding 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. placing the sample and the probe under stringent hybridization conditions; and c. detecting hybridization of the nucleic acid probe to the recombinant nucleic acid molecule.

26. A method of detecting the presence of an insecticidal protein or fragment thereof in a sample comprising a protein, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2 or 4; or the insecticidal 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; the method comprising: a. contacting the sample with an immunoreactive antibody; and b. detecting the presence of the insecticidal protein or fragment thereof.

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

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

29. A method for controlling a Lepidopteran pest species or infestation in a field, the method comprising: a. growing a crop plant expressing an insecticidally effective amount of an insecticidal protein as set forth in SEQ ID NO: 2 or 4; or b. growing a crop plant expressing an insecticidally effective amount of one or more insecticidal 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 a transgenic Lepidopteran pest species carrying a self-limiting gene into the field to reduce the likelihood of the pest species developing resistance to the insecticidal protein.

30. The method of claim 29, wherein the crop plant is a monocot crop plant or a dicot crop plant.

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

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

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

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