Novel insect inhibitory proteins

Abstract

Disclosed are pesticidal proteins that exhibit toxic activity against hemipteran and lepidopteran pest species, and include, but are not limited to, TIC 11207 and TIC 11304. A DNA construct containing a recombinant nucleic acid sequence encoding the disclosed pesticidal protein is provided. Transgenic plants, plant cells, seeds and plant parts that are resistant to hemiptera and lepidoptera infestation are provided, containing recombinant nucleic acid sequences encoding the pesticidal proteins of the invention. 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 for controlling pests of hemipteran and lepidoptera species using TIC11207 and TIC11304 pesticidal proteins.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 386,432, filed on December 7, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporation of Sequence Listing

[0004] A file named "MONS571WO_ST26.xml" containing a sequence listing in computer-readable form was created on November 27, 2023. This file is 37.5 KB (measured in MS- ), and was submitted electronically (using the Patent Center of the United States Patent and Trademark Office) concurrently, and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present invention generally relates to the field of insect inhibitory proteins. A novel class of toxin proteins is disclosed that exhibits insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly insect pests of Hemiptera species. Plants, plant parts, seeds, cells including plant and microbial cells, and vectors containing recombinant polynucleotide constructs encoding one or more of the disclosed toxin proteins are provided. Background Art

[0006] Increasing the crop yields of important agricultural plants, including maize, soybean, sugarcane, rice, wheat, cotton, vegetables, pearl millet, pigeon pea, peanut, potato, barley, oats, fruit trees, etc., has become increasingly important. In addition to the growing demand for agricultural products to provide food, clothing, and energy for a growing population, climate-related impacts and the pressure of a growing population to use land for non-agricultural practices are expected to reduce the amount of arable land available for agriculture. These factors have led to dire predictions for food security, especially in the absence of major improvements in plant biotechnology and agronomic practices. Given these factors, environmentally sustainable improvements in technology, agrotechnology, and pest management are important tools for expanding crop production on the increasingly limited amount of arable land available for agriculture.

[0007] Insects, particularly Hemiptera insects, are a major cause of damage to field crops, thereby reducing crop yields in infested areas. Hemiptera pest species that have a negative impact on agriculture include, but are not limited to, the Southern Green Stink Bug (Nezara viridula), the Neotropical Brown Stink Bug (Euschistus heros), the Brown Marmorated Stink Bug (Halyomorpha halys), the Red-Shouldered Stink Bug (Thyanta acerra), the Green Belly Stink Bug (Dichelops melacanthus), the Western tarnished plant bug (Lygus hesperus), or the tarnished plant bug (Lygus lineolaris).

[0008] Historically, pest control agents in agriculture have relied mainly on the intensive application of synthetic chemical insecticides. In addition to emerging resistance problems and the fact that such pest control agents do not distinguish between beneficial insects and other organisms and target beneficial insects and other organisms, concerns about the environment and human health have also stimulated the research and development of biopesticides specifically for controlling pests that cause crop losses. This research work has led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

[0009] When the potential of entomopathogenic bacteria, particularly bacteria belonging to the genus Bacillus, was discovered and developed as biopesticides, the biocontrol paradigm shifted. Bacillus thuringiensis (Bt) strains have been used as a source of insecticidal proteins because Bt strains have been found to be highly toxic to specific insects. Bt strains are known to produce δ-endotoxins, which are located within parasporal crystal inclusions (e.g., Cry proteins) at the onset of sporulation and during the stationary growth phase, and are also known to produce secreted insecticidal proteins. After ingestion by susceptible insects, the δ-endotoxins, as well as the secreted toxins, act on the midgut epithelial surface, disrupting cell membranes and leading to cell destruction and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including other Bacillus species and a variety of other bacterial species such as Brevibacillus laterosporus, Lysinibacillus sphaericus (previously also known as Bacillus sphaericus), Pseudomonas species, Paenibacillus popilliae, and Paenibacillus lentimorbus. In addition, insecticidal toxins have been identified from a variety of non-bacterial sources, including ferns and spider venoms, and the delivery of dsRNA for suppressing essential genes in the pest diet has been established as an effective pest management strategy.

[0010] The crystalline and secreted soluble insecticidal toxins are highly specific for their hosts and have gained worldwide acceptance as alternatives to chemical insecticides. For example, insecticidal toxin proteins have been used in various agricultural applications to protect agriculturally important plants from insect infestations, reduce the need for chemical pesticide applications, and increase yields. Insecticidal toxin proteins are used to control agriculturally relevant pests of crop plants by mechanical means, such as spraying to disperse microbial preparations containing various strains onto the plant surface, and by using genetic transformation techniques to produce transgenic plants and seeds that express insecticidal toxin proteins.

[0011] The use of transgenic plants expressing insecticidal toxin proteins has been applied globally. For example, in 2016, 23.1 million hectares were planted with transgenic crops expressing Bt toxin, and 75.4 million hectares were planted with transgenic crops expressing Bt toxin and having herbicide tolerance traits (ISAAA. 2016. Global Status of Commercialized Biotech / GM Crops: 2016. ISAAA Brief No. 52. ISAAA: Ithaca, NY). The global use of transgenic insect-protected crops and the limited number of insecticidal toxin proteins used in these crops have imposed a selection pressure on existing insect alleles that can confer resistance to the currently used insecticidal proteins.

[0012] The development of resistance of target pests to insecticidal toxin proteins has created a continuous demand for the discovery and development of new forms of insecticidal toxin proteins that can be used to manage the increasing resistance of insects to transgenic crops expressing insecticidal toxin proteins. New protein toxins with improved efficacy and exhibiting control effects against a broader spectrum of susceptible insect species will reduce the number of surviving insects that can generate resistant alleles. In addition, the use of two or more transgenic insecticidal toxin proteins in a plant, which are toxic to the same insect pest and exhibit different modes of action, or two or more different toxic modes of action (e.g., a transgenic encoding dsRNA targeting an essential gene to be suppressed in combination with a transgenic encoding a peptide or protein toxin, both being toxic to the same insect species), reduces the likelihood of any single target insect species developing resistance. Additionally, when used in combination with the proteins of the present invention, the use of self-limiting technologies (such as those provided by Oxitec Ltd) will enhance the durability of the traits of transgenic crops expressing the proteins of the present invention (Zhou et al. 2018. Combining the high-dose / refuge strategy and self-limiting transgenic insects in resistance management—a test in experimental mesocosms. Evol Appl 11(5): 727–738; Alphey et al. 2009. Combining pest control and resistance management: synergy of engineered insects with Bt crops. Journal of Economic Entomology, 102 : 717-732).

[0013] Accordingly, the inventors disclose herein novel proteins from Paenarthrobacter nitroguajacolicus that exhibit insecticidal activity against target Hemiptera species, particularly Nezara viridula (Southern green stink bug), Thyanta perditor (Neotropical brown stink bug), and Lygus hesperus (Western tarnished plant bug). Summary of the Invention

[0014] Disclosed herein are novel insecticidal proteins TIC11207 and TIC11304, which have been demonstrated to exhibit inhibitory activity against one or more pests of crop plants. The TIC11207 and TIC11304 proteins can be used alone or in combination with other insecticidal proteins and toxicants in formulations and in planta, thereby providing alternatives to the insecticidal proteins and insecticidal chemicals currently used in agricultural systems.

[0015] In one embodiment, the present application discloses a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding an insecticidal protein or an insecticidal fragment thereof, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2, 4, 12, 14, 17, or 19; or the insecticidal protein comprises an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 12, 14, 17, or 19; or the polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, or 18. The recombinant nucleic acid molecule may comprise a sequence having the function of expressing the insecticidal protein in a plant and, when expressed in a plant cell, produces an insecticidally effective amount of the insecticidal protein or an insecticidal fragment thereof.

[0016] In another embodiment of the present application, the recombinant nucleic acid molecule is present in a bacterial or plant host cell. The bacterial host cells contemplated include at least Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea, Paenarthrobacter, and Erwinia. In certain embodiments, the Bacillus species is Bacillus cereus or Bacillus thuringiensis, the Brevibacillus species is Brevibacillus laterosporus, or the Escherichia species is Escherichia coli. The plant host cells contemplated include dicotyledonous plant cells and monocotyledonous plant cells. The plant cells contemplated also include alfalfa, banana, barley, bean, broccoli, cabbage, Brassica plants (e.g., rapeseed), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (Gossypium species), gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hop, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, pasture, 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, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon, and wheat plant cells.

[0017] In another embodiment, the insecticidal protein exhibits activity against Hemiptera insects, which at least include Nezara viridula (Southern green stink bug), Thyanta perditor (Neotropical brown stink bug), and Lygus hesperus (Western tarnished plant bug).

[0018] In another embodiment, the insecticidal protein exhibits activity against Lepidoptera insects, which at least include Spodoptera eridania (Southern armyworm) and Chrysodeixis includens (Soybean looper).

[0019] The present application also contemplates bacteria and plants and plant parts comprising recombinant nucleic acid molecules encoding the pesticidal proteins TIC11207 and TIC11304 or fragments thereof. The recombinant molecules (e.g., constructs) can comprise a heterologous promoter for expressing operably linked polynucleotide segments encoding pesticidal proteins in bacterial or plant cells. Both dicotyledonous and monocotyledonous plants are contemplated. In another embodiment, the plant is further selected from the group consisting of: alfalfa, banana, barley, beans, broccoli, cabbage, Brassica species (e.g., canola), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton (i.e., Gossypium species), gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, pasture, 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, sugar cane, sunflower, corn (i.e., maize) such as sweet corn or feed corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, 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 contemplated, such as wood or oil, non-viable ground seeds or fractionated seeds, flour or starch produced from plant leaves, flowers, roots, seeds, or tubers containing nucleic acids encoding the proteins of the present invention and / or containing pesticidally effective amounts of the encoded toxin proteins.

[0020] In certain embodiments, seeds are disclosed that comprise a recombinant nucleic acid molecule and pesticidally effective amounts of the TIC11207 and TIC11304 toxin proteins.

[0021] In another embodiment, an insect inhibitory composition comprising the recombinant nucleic acid molecules disclosed in the present application is contemplated. The insect inhibitory composition may further comprise a nucleotide sequence encoding at least one other insecticide different from the insecticidal protein. In certain embodiments, the at least one other insecticide is selected from the group consisting of insect inhibitory proteins, insect inhibitory dsRNA molecules, and accessory proteins. It is also contemplated that at least one other insecticide in the insect inhibitory composition exhibits activity against one or more pest species of Lepidoptera, Coleoptera, or Hemiptera. In one embodiment, the at least one other insecticide in the insect inhibitory composition is selected from the group consisting of: Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variant, Cry1D, Cry1E, Cry1F, Cry1A / F chimeric, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variant, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, TIC4029, TIC4064, TIC13085, TIC13087, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113 and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and its variants, IP3 and its variants, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IPD102Aa and its homologs, IPD110Aa and its homologs, TIC868, Cry1Da1_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757, TIC7641, TIC5290, TIC3668, TIC3669, TIC3670, IPD072Aa and its homologs, IPD103 and its homologs, PIP-50 and PIP-65 and their homologs, PIP-83 and its homologs and Cry1B.34; and dsRNA-mediated gene silencing embodiments that include those targeting the suppression of the genes Dv snf7 and Dv ssj1 in Diabrotica species of corn rootworm.

[0022] Commercially available products containing a detectable amount of the recombinant nucleic acid molecules and toxin proteins disclosed in this application are also contemplated. Such commercially available products include commercially available corn in bags by grain processors, corn chips, tortillas, corn flour, cornmeal, corn syrup, corn oil, corn silage, corn starch, corn cereal, etc., as well as corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon, and vegetable commercially available products, and in applicable cases, fruit juices, concentrates, jams, jellies, marmalades, and other edible forms of such commercially available products containing a detectable amount of such polynucleotides and / or polypeptides of this application, whole or processed cottonseed, cotton oil, lint, seeds, and plant parts processed for feed or food, fibers, 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, soy protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soy cheese, soy wine, animal feed containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass, and fuel products produced using soybean plants and soybean plant parts.

[0023] This application also contemplates a method of producing seeds containing a recombinant nucleic acid molecule and an insecticidally effective amount of the encoded TIC11207 and TIC11304 toxin proteins. The method includes planting at least one seed containing the recombinant nucleic acid molecule disclosed in this application; growing a plant from the seed; and harvesting seeds from the plant, wherein the harvested seeds contain the recombinant nucleic acid molecule mentioned and / or an insecticidally effective amount of the encoded TIC11207 and TIC11304 toxin proteins.

[0024] In another exemplary embodiment, plants resistant to Hemiptera or Lepidoptera insect infestation are provided, wherein the cells of the plants contain the recombinant nucleic acid molecules disclosed herein.

[0025] This application also discloses methods for controlling pests of Hemiptera or Lepidoptera species and controlling infestation of plants, particularly crop plants, by Hemiptera or Lepidoptera species pests. In one embodiment, the method includes first contacting the pest with an insecticidally effective amount of an insecticidal protein as shown in SEQ ID NO: 2, 4, 12, or 14; or contacting the pest with an insecticidally effective amount of one or more insecticidal proteins that contain an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 12, or 14.

[0026] The present disclosure also provides a method for detecting the presence of recombinant nucleic acid molecules that detect TIC11207 and TIC11304 toxin protein classes, wherein the method comprises contacting a nucleic acid sample with a nucleic acid probe that hybridizes under stringent hybridization conditions to the genomic DNA of a plant comprising a polynucleotide segment encoding an insecticidal protein or a fragment thereof provided herein and that does not hybridize under such hybridization conditions to the genomic DNA of other isogenic plants that do not comprise the segment, wherein the probe is homologous or complementary to SEQ ID NO: 3, 5, 6, 7, 8, 9, 10, 13, or 15 or a sequence encoding an insecticidal protein that comprises an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 12, or 14; placing the sample and the probe under stringent hybridization conditions; and detecting hybridization of the probe to the sample DNA.

[0027] The present disclosure also provides methods for detecting the presence of an insecticidal protein or a fragment thereof from the TIC11207 and TIC11304 toxin protein classes, wherein the method comprises contacting a sample with an antibody immunoreactive with the TIC11207 and TIC11304 toxin protein classes or a recombinant protein designed to detect the TIC11207 and TIC11304 proteins, and detecting binding of the antibody to the TIC11207 and TIC11304 toxin protein class proteins to confirm the presence of the proteins in the sample. In some embodiments, the detecting step comprises ELISA or Western blotting.

[0028] The present application also contemplates a method for controlling hemipteran or lepidopteran pest species or pest infestations 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, 4, 12, 14, 17, or 19; or growing a crop plant expressing an insecticidally effective amount of one or more insecticidal proteins, the one or more insecticidal proteins comprising an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 12, 14, 17, or 19; and releasing one or more transgenic hemipteran or lepidopteran pest species each carrying a self-limiting gene into a field of a crop comprising a gene encoding a toxin protein of the present invention for preventing or delaying the development of resistance of the one or more hemipteran or lepidopteran pest species to the toxin protein. In one embodiment, the crop plant can be monocotyledonous or dicotyledonous. In another embodiment, the dicotyledonous crop plant can be soybean, cotton, or rapeseed, and the monocotyledonous crop plant can be corn, wheat, sorghum, rice, rye, or millet. In yet another embodiment, the monocotyledonous crop plant can be corn, wheat, sorghum, rice, rye, or millet.

[0029] Brief Description of Sequences

[0030] SEQ ID NO:1 is a nucleic acid sequence encoding the TIC11207 insecticidal protein obtained from Arthrobacter myxococcoides strain MDI-0030264.

[0031] SEQ ID NO:2 is the amino acid sequence of the TIC11207 insecticidal protein encoded by the sequence shown in SEQ ID NO:1.

[0032] SEQ ID NO:3 is a synthetic coding sequence TIC11207PL-1, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0033] SEQ ID NO:4 is the amino acid sequence of the TIC11207PL insecticidal protein encoded by SEQ ID NO:3, 5, 6, 7, 8, 9 and 10, with an additional alanine residue inserted immediately after the start methionine.

[0034] SEQ ID NO:5 is a synthetic coding sequence TIC11207PL-2, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0035] SEQ ID NO:6 is a synthetic coding sequence TIC11207PL-3, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0036] SEQ ID NO:7 is a synthetic coding sequence TIC11207PL-4, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0037] SEQ ID NO:8 is a synthetic coding sequence TIC11207PL-5, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0038] SEQ ID NO:9 is a synthetic coding sequence TIC11207PL-6, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, with an additional codon encoding an alanine residue inserted immediately after the start methionine codon.

[0039] SEQ ID NO:10 is a synthetic coding sequence TIC11207PL-7, which encodes the TIC11207PL insecticidal protein and can be used in plant cells, wherein an additional codon encoding an alanine residue is inserted immediately after the start methionine codon.

[0040] SEQ ID NO:11 is a nucleic acid sequence encoding the TIC11304 insecticidal protein obtained from Arthrobacter myxogenes strain MDI-0030376.

[0041] SEQ ID NO:12 is the amino acid sequence of the TIC11304 insecticidal protein encoded by the sequence shown in SEQ ID NO:11.

[0042] SEQ ID NO:13 is a synthetic coding sequence TIC11304PL-1, which encodes the TIC1304PL insecticidal protein and can be used in plant cells, wherein an additional codon encoding an alanine residue is inserted immediately after the start methionine codon.

[0043] SEQ ID NO:14 is the amino acid sequence of the TIC11304PL insecticidal protein encoded by SEQ ID NO:13 and 15, wherein an additional alanine residue is inserted immediately after the start methionine.

[0044] SEQ ID NO:15 is a synthetic coding sequence TIC11304PL-2, which encodes the TIC11304PL insecticidal protein and can be used in plant cells, wherein an additional codon encoding an alanine residue is inserted immediately after the start methionine codon.

[0045] SEQ ID NO:16 is a nucleic acid sequence encoding the TIC11207 insecticidal protein, which has a histidine tag operably linked to the 3' end and is referred to herein as TIC11207-His.

[0046] SEQ ID NO:17 is the amino acid sequence of the TIC11207-His protein encoded by SEQ ID NO:16.

[0047] SEQ ID NO:18 is a nucleic acid sequence encoding the TIC11304 insecticidal protein, which has a histidine tag operably linked to the 3' end and is referred to herein as TIC11304-His.

[0048] SEQ ID NO:19 is the amino acid sequence of the TIC11304-His protein encoded by SEQ ID NO:18. Description of the Drawings

[0049] Figure 1 Depicts the alignment of pesticidal toxins TIC11207, TIC11304, and APG01463 as presented in U.S. Patent Application Publication US20180066277. In the multiple alignment, identical amino acids are marked with an asterisk.

[0050] Figure 2 Is a schematic diagram of the average percentage survival of the neotropical brown stink bug (Euschistus heros) when feeding on stably transformed soybean plants expressing the TIC11207PL protein. The control plants are untransformed wild-type soybean plants.

[0051] Figure 3 Is a schematic diagram of the percentage survival of the neotropical brown stink bug (Euschistus heros) when feeding on a stably transformed soybean event expressing the TIC11207PL protein. The control plants are untransformed wild-type soybean plants. Detailed Description

[0052] A problem in the field of agricultural pest control can be characterized as the need for new toxin proteins that are effective against target pests, exhibit broad-spectrum toxicity against target pest species, can be expressed in plants without causing undesirable agronomic problems, and provide an alternative mode of action compared to toxins currently used commercially in plants.

[0053] Two novel pesticidal proteins, exemplified by TIC11207 and TIC11304, are disclosed herein. Using pesticidally effective amounts of the proteins can solve the problem of insect infestation existing in the art, particularly against a range of hemipteran insect pests, more specifically against the southern green stink bug (Nezara viridula), the neotropical brown stink bug (Euschistus heros), and the western tarnished plant bug (Lygus hesperus), as well as lepidopteran insect pests, more specifically against the fall armyworm (Spodoptera frugiperda) and the soybean looper (Chrysodeixis includens).

[0054] References in this application to TIC11207, "TIC11207 protein", "TIC11207 protein toxin", "TIC11207 insecticidal protein", "TIC11207-related toxin", "TIC11207-related toxin", "TIC11207 protein toxin class", "TIC11207 toxin protein class", etc. refer to any novel insecticidal protein or insect inhibitory protein that contains any insecticidal protein or insect inhibitory protein sequence of TIC11207 (SEQ ID NO:2), consists of it, is substantially homologous to it, is similar to it, or is derived from it. References in this application to TIC11304, "TIC11304 protein", "TIC11304 protein toxin", "TIC11304 insecticidal protein", "TIC11304-related toxin", "TIC11304-related toxin", "TIC11304 protein toxin class", "TIC11304 toxin protein class", etc. refer to any novel insecticidal protein or insect inhibitory protein that contains any insecticidal protein or insect inhibitory protein sequence of TIC11304 (SEQ ID NO:12), consists of it, is substantially homologous to it, is similar to it, or is derived from it.

[0055] The terms "segment" or "fragment" are used in this application to describe a continuous amino acid or nucleic acid sequence that is shorter than the complete amino acid or nucleic acid sequence describing the TIC11207, TIC11207PL, TIC11304, and TIC11304PL proteins. This application also discloses segments or fragments that exhibit insect inhibitory activity, provided that the alignment of such segments or fragments with the corresponding portions of the TIC11207, TIC11207PL, TIC11304, and TIC11304PL proteins shown in SEQ ID NO:2, 4, 12, and 14, respectively, results in an amino acid sequence identity of about 80% to about 100% between the segment or fragment and the corresponding amino acid segments within the TIC11207, TIC11207PL, TIC11304, and TIC11304PL proteins for any fractional percentage.

[0056] As used herein, the terms "active" or "activity", "pesticidal activity" or "pesticidal" or "insecticidal activity", "insect inhibitory", "pesticidally effective" or "insecticidal" refer to the efficacy of a toxicant (such as a protein toxin) containing an effective amount of the TIC11207, TIC11207PL, TIC11304 or TIC11304PL protein 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 pests (causing morbidity, death or reduced fecundity). These terms are intended to include the results of providing an insecticidally effective amount of a toxic protein to a pest, wherein exposure of the pest to the toxic protein results in morbidity, death, reduced fecundity or developmental retardation. These terms also include the exclusion of pests from plants, plant tissues, plant parts, seeds, plant cells or a particular geographical location where a plant may grow due to the provision of an insecticidally effective amount of a toxic protein in or on the plant. Generally speaking, insecticidal activity refers to the ability of a toxic protein to effectively inhibit growth, development, viability, feeding behavior, mating behavior, fecundity, or any measurable reduction in adverse effects caused by insect feeding. The toxic protein can be produced by a plant or can be applied to the plant or the environment within the location where the plant is located. The terms "bioactivity", "effective", "efficacious" or variants thereof are also terms that are used interchangeably herein to describe the action of the proteins of the present invention on target insect pests.

[0057] When provided in the diet of a target pest, an insecticidally effective amount of a toxicant exhibits insecticidal activity when the toxicant contacts the pest. The toxicant can be an insecticidal protein or one or more chemical agents known in the art. Insecticidal or insect-killing chemical agents can be used alone or in combination with each other. 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 insect-killing protein agents include the protein toxins presented in this application, as well as other proteinaceous toxicants, including those targeting Lepidoptera, and protein toxins for controlling other plant pests, such as Cry, Vip and Cyt proteins, Pseudomonas insecticidal proteins, and insect toxin proteins derived from fern species, which are useful in the art for controlling Lepidoptera, Coleoptera, Diptera, Hemiptera and Homoptera species.

[0058] Reference to pests, particularly pests of crop plants, means insect pests of crop plants, particularly those hemipteran and lepidopteran insect pests controlled by the TIC11207 and TIC11304 protein toxin classes. However, reference to pests can also include coleopteran, hemipteran, lepidopteran, dipteran, and homopteran plant insect pests, and can include nematodes and fungi when the toxicant directed against these pests is present together with or coexists with the TIC11207 and TIC11304 proteins or with proteins having 80% to about 100% identity to the TIC11207 and TIC11304 proteins. The phrases "present together" or "coexists" are intended to include any situation in which the target insect pest has been contacted by the TIC11207 and TIC11304 toxin proteins and any other toxicant that is also present in an insecticidally effective amount relative to the target insect pest. In certain embodiments, "contacted" is intended to refer to being present in the diet of the target pest and the diet being consumed by the target pest.

[0059] Hemipteran insects include, but are not limited to, stink bugs of the family Pentatomidae: green stink bugs of the genus Chinavia (Chinavia hilaris, Chinavia marginata, and Chinavia pensylvanica), stink bugs of the genus Chlorochroa (Chlorochroa granulose, Chlorochroa kanei, Chlorochroa ligata (also known as the cotton stainer), Chlorochroa lineate, Chlorochroa opuntiae, Chlorochroa persimilis, Chlorochroa rossiana, Chlorochroa sayi, Chlorochroa uhleri, Chlorochroa belfragii, Chlorochroa faceta, Chlorochroa osborni, Chlorochroa saucia, and Chlorochroa senilis), southern green stink bug (Nezara viridula), stink bugs of the genus Edessa (Edessa meditabunda, Edessa bifida, and Edessa florida), neotropical brown stink bug (Euschistus heros), stink bugs of the genus Euschistus (Euschistus acuminatus, Euschistus biformis, Euschistus conspersus, Euschistus crenator, Euschistus egglestoni, Euschistus ictericus, Euschistus inflatus, Euschistus latimarginatus, Euschistus obscures, Euschistus politus, Euschistus quadrator, Euschistus sevus, Euschistus strenuous, Euschistus tristigmus, and Euschistus variolarius), brown-winged stink bug (Halyomorpha halys), red-shouldered stink bug (Thyanta accerra), stink bugs of the genus Thyanta (Thyanta calceata, Thyanta custator, Thyanta pallidovirens, Thyanta perditor, Thyantamaculate and Thyanta pseudocasta), green stink bugs (Dichelops melacanthus) and other stink bugs of the genus Dichelops (Dichelops avilapiresi, Dichelops bicolor, Dichelops dimidatus, Dichelops furcatus, Dichelops furcifrons, Dichelops lobatus, Dichelops miriamae, Dichelops nigrum, Dichelops peruanus, Dichelops phoenix and Dichelops saltensis), red-banded stink bugs (Piezodorus guildinni) and Piezodorus lituratus; insects of the family Plataspidae, such as but not limited to Megacopta cribraria, western tarnished plant bugs (Lygus hesperus) and Lygus lineolaris; aphid species, such as but not limited to soybean aphids (Aphis glycines), peach aphids (Myzus persicae), potato aphids (Macrosiphum euphorbiae), melon aphids (Aphis gossypii), cabbage aphids (Brevicoryne brassicae); whitefly pest species, such as but not limited to greenhouse whiteflies (Trialeurodes vaporariorum), sweet potato whiteflies (Bemisia tabaci), giant whiteflies (Aleurodicus dugesii); and planthopper species, such as but not limited to blue-green leafhoppers (Graphocephala atropunctata) and Eupteryx decemnotata.

[0060] Lepidopteran insects include, but are not limited to, armyworms, sugar moths, inchworms, and cutworms of the family Noctuidae, such as Spodoptera frugiperda, Spodoptera exigua, Spodoptera cosmioides, southern armyworm (southern grey-winged cutworm), Mamestra configurata, Agrotis ipsilon, Trichoplusia ni, Diatraea saccharalis, soybean looper (bean silverspot), Rachiplusia nu, velvetbean caterpillar (Anticarsia gemmatalis), Hypena scabra, Heliothis virescens, Agrotis subterranea, Pseudaletia unipuncta, Rachiplusia nu, Helicoverpa gelotopoeon, Agrotis orthogonia; borers, casebearers, webworms, coneworms, cabbageworms, and leafrollers of the family Pyralidae, such as Ostrinia nubilalis, Amyelois transitella, Crambus caliginosellus, Herpetogramma licarsisalis, Homoeosoma electellum, Elasmopalpus lignosellus; leafrollers, budworms, seedworms, and fruitworms of the family Tortricidae, such as Cydia pomonella, Endopiza viteana, Grapholita molesta, Suleima helianthana; and many other economically important Lepidoptera, such as Plutella xylostella, Pectinophora gossypiella, and Lymantria dispar.Other insect pests of the order Lepidoptera include, for example, Alabama argillacea, Archips argyrospila, Archips rosana and other species of the genus Archips (Chilo suppressalis, Asiatic rice borer or rice stem borer), Cnaphalocrocis medinalis, Crambus caliginosellus, Crambus teterrellus, Diatraea grandiosella, Diatraea saccharalis, Earias insulana, Earias vittella, Helicoverpa armigera, Helicoverpa zea (also known as soybean podworm and cotton bollworm), Heliothis virescens, Herpetogramma licarsisalis, Striacosta albicosta, Lobesia botrana, Phyllocnistis citrella, Pieris brassicae, Pieris rapae (also known as imported cabbageworm), Spodoptera exigua, Spodoptera litura (also known as tea caterpillar) and Tuta absoluta.

[0061] As used herein, the term "isolated DNA molecule" or equivalent terms or phrases are intended to mean a DNA molecule that is present alone or in combination with other compositions, but not in its natural environment. For example, as long as nucleic acid elements (such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc.) that are naturally present in the DNA of an organism's genome are located within the organism's genome and at their natural genomic locations, such elements are not considered to be "isolated". However, as long as each of these elements and sub-parts of these elements are not within the organism's genome and at their natural genomic locations, such elements are "isolated" within the scope of the present disclosure. Similarly, as long as a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of the protein is not in the DNA of the bacterium in which the sequence encoding the protein is naturally present, the nucleotide sequence is an isolated nucleotide sequence. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered to be isolated. For the purposes of the present disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar construct used to transform cells, in the genome of a plant or bacterium, or present in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacterium.

[0062] References to the term "self-limiting gene" in this application refer to genes that limit host survival, resulting in a reduction in the host population. This technology is provided by Oxitech Ltd. Transgenic male insects carrying the transgenic self-limiting gene are released and breed with wild female insects. As a result, the offspring inherit a copy of the self-limiting gene. The self-limiting gene disrupts the normal function of insect cells by overproducing a protein in the insect cells, interfering with the cell's ability to produce other essential proteins required for development. By disrupting normal insect development, the gene prevents the insect from surviving to adulthood. For example, the self-limiting diamondback moth (Plutellidae xylostella) strain OX4319L was developed by Oxitech Ltd and carries a male-selective gene that utilizes the sequence of the sex-determining gene doublesex (dsx). The gene expresses sex-alternative splicing to engineer female-specific expression of the self-limiting gene, which prevents female offspring from surviving beyond the larval stage and allows for the production of only male populations of self-limiting moths. After the males are released, they mate with pest females, resulting in a reduction in the number of female offspring in the next generation, thereby locally suppressing the diamondback moth population. To facilitate the rearing of large numbers of males for release in diamondback moth production facilities, the expression of female-specific dsx in the OX4319L strain is suppressed by adding tetracycline or a suitable analog to the larval diet. OX4319L also expresses the fluorescent protein DsRed to allow for the effective monitoring of the presence of the strain in the field (Jin et al., 2013. Engineered female-specific lethality for control of pest Lepidoptera. ACS Synthetic Biology, 2:160-166). When this technology is applied to fields of plants containing the toxin gene of the present invention, the development of resistance in pest species targeted by the toxin gene and protein of the present invention can be delayed or prevented, thereby conferring greater persistence to any plant product containing the toxin gene and protein of the present invention.

[0063] Although self-limiting technologies for controlling hemipteran insect pests have not yet been developed, some key genes in sex determination have been identified, and differential splicing of transcripts between different sexes has been determined for hemipteran species such as the brown planthopper (Nilaparvata lugens), the whitefly (Bemisia tabaci), and the kissing bug (Rhodnius prolixus). More research is needed before altering sex ratios can be considered as part of a genetic control strategy. Some hemipteran species, such as whiteflies, are haplodiploid, while others lack a Y chromosome. These alternative genetic systems are likely to affect the design and efficiency of genetic control mechanisms such as self-limiting technologies. Although many of these characteristics make dipterans and lepidopterans suitable for genetic control mechanisms, many of these limitations are not insurmountable. Current control strategies need to be adjusted or new strategies developed to enable the field of hemipteran control. Substantial progress has been made in the field of hemipteran biotechnology over the past four years. Enabling technologies for CRISPR / Cas-mediated mutagenesis in hemipterans are emerging. An increasing number of annotated genome assemblies now provide the building blocks for developing the genetic toolbox required for extended genetic control of hemipteran pests (Pacheco et al., (2022) Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives, Frontiers in Bioengineering and Biotechnology, 10:1-26).

[0064] As further described herein, an open reading frame (ORF) encoding TIC11207 (SEQ ID NO:1) was found in DNA obtained from the nitropinic acid Arthrobacter species MDI-0030264. Bioassays using the microbial host cell-derived protein of TIC11207 demonstrated its activity against the hemipteran species Nezara viridula (green stink bug), Thyanta perditor (southern green stink bug), and Lygus hesperus (western tarnished plant bug). As further described herein, an open reading frame (ORF) encoding TIC11304 (SEQ ID NO:11) was found in DNA obtained from the nitropinic acid Arthrobacter species MDI-0030376. Bioassays using the microbial host cell-derived protein of TIC11304 demonstrated its activity against the hemipteran species Nezara viridula (green stink bug), Thyanta perditor (southern green stink bug), and Lygus hesperus (western tarnished plant bug), as well as the lepidopteran pest species Mythimna separata (oriental armyworm) and Pseudoplusia includens (soybean looper).

[0065] Synthetic coding sequences were generated for plant cells to express TIC11207PL, the TIC11207 amino acid sequence, with an additional alanine residue inserted immediately after the start methionine, the amino acid sequence being encoded by SEQ ID NO:3, 5, 6, 7, 8, 9, and 10. Synthetic coding sequences were generated for plant cells to express TIC11304PL, the TIC11304 amino acid sequence, with an additional alanine residue inserted immediately after the start methionine, the amino acid sequence being encoded by SEQ ID NO:13 and 15. These coding sequences were operably / functionally linked to plant functional promoters and other elements that function in plants to mediate the desired level and spatial characteristics of expression in plants. Soybean plants expressing TIC11207PL exhibited effective activity against the hemipteran species Thyanta perditor (southern green stink bug).

[0066] For expression in plant cells, the TIC11207, TIC11207PL, TIC11304, and TIC11304PL (SEQ ID NO: 2, 4, 12, 14) proteins can be expressed and localized in the cytosol or targeted to various organelles of plant cells. For example, targeting a protein to the chloroplast may result in increased levels of the protein expressed in transgenic plants and can prevent the appearance of an off-phenotype if the expressed protein toxin has a cellular biological response in any unexpected manner. Targeting can also lead to an increase in the pest resistance efficacy in transgenic events. A targeting peptide or transit peptide is a short peptide chain (3 - 70 amino acids in length) that directs the transport of a protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplasts, apoplast, peroxisomes, and plasma membrane. After the protein is transported, some targeting peptides are cleaved from the protein by signal peptidase. For targeting to the chloroplast, the protein contains a transit peptide approximately 40 - 50 amino acids in length. For a description of the use of chloroplast transit peptides, see U.S. Patent Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated CTPs include, but are not limited to, those associated with the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-harvesting complex proteins I and II, thioredoxin F, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and the transit peptides 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 the chloroplast by using a protein fusion with a heterologous CTP, and that the CTP is sufficient to target the protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide, such as the Arabidopsis EPSPS CTP (CTP2) (see, Klee et al., Mol. Gen. Genet. 210:437 - 442, 1987) or the Petunia EPSPS CTP (CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873 - 6877, 1986), has been shown to target heterologous EPSPS protein sequences to the chloroplasts in transgenic plants (see, U.S. Patent Nos. 5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP 508909; and EP 924299).To target the TIC11207, TIC11207PL, TIC11304 or TIC11304PL toxin proteins to the chloroplast, a sequence encoding a chloroplast transit peptide is placed at the 5ˊ position and operably linked and in-frame with a sequence designed to be expressed in a plant cell to encode any one of the toxin proteins TIC11207, TIC11207PL, TIC11304 or TIC11304PL.

[0067] Improved variants of the TIC11207 and TIC11304 protein toxin classes can be engineered in planta by using various gene editing methods known in the art. Such techniques for genome editing include, but are not limited to, ZFN (zinc finger nuclease), meganuclease, TALEN (transcription activator-like effector nuclease), and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) systems. These genome editing methods can be used to alter the toxin protein-encoding sequence transformed within a plant cell to a different toxin-encoding sequence. Specifically, by these methods, one or more codons within the toxin-encoding sequence can be altered to engineer a new protein amino acid sequence. Alternatively, a fragment within the encoding sequence can be replaced or deleted, or an additional DNA fragment can be inserted into the encoding sequence to engineer a new toxin-encoding sequence. The new encoding sequence can encode a toxin protein having new properties such as increased activity or spectrum against insect pests and provide activity against one or more insect pest species, where resistance to the original insect toxin protein has developed or may develop. Whole plants expressing the new toxin protein can be generated from plant cells containing the gene-edited toxin-encoding sequence by methods known in the art.

[0068] For each of TIC11207, TIC11207PL, TIC11304 and TIC11304PL, fragments and amino acid sequence variants that retain or exhibit improved insect inhibitory activity are contemplated. The variants can be truncated forms in which one or more amino acids are deleted from the N-terminus, C-terminus, middle or a combination thereof of the protein, where the variant retains or exhibits improved insect inhibitory activity compared to the naturally occurring sequence. These fragments can be naturally occurring or synthetic (artificial) variants or derived protein variants of TIC11207, TIC11207PL, TIC11304 or TIC11304PL, but should retain at least the insect inhibitory activity of TIC11207, TIC11207PL, TIC11304 or TIC11304PL.

[0069] A variety of computer-based algorithms known in the art can be used to identify and compare proteins similar to the TIC11207 and TIC11304 proteins. The amino acid sequence identities reported in this application are the result of Clustal W alignments using these default parameters: weight matrix: blosum, gap open penalty: 10.0, gap extension penalty: 0.05, hydrophilic gaps: on, hydrophilic residues: GPSNDQERK, residue-specific gap penalties: on (Thompson et al. (1994) Nucleic Acids Research, 22:4673-4680). The percentage of amino acid identity is further calculated by multiplying 100% by the product of (amino acid identity of the subject protein / length). Other alignment algorithms are also available in the art and provide results similar to those obtained using Clustal W alignments and are considered herein.

[0070] Proteins expected to exhibit insect inhibitory activity against hemipteran or lepidopteran insect species are used in the query (e.g., in a Clustal W alignment) and are related to TIC11207 and TIC11304, and the proteins of the present invention as shown in SEQ ID NO:2 or 12 are identified as hits in such alignments, where the query protein exhibits at least 80% to about 100% amino acid identity along the length of the query protein, which is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any fraction within this range.

[0071] The amino acid sequence identity matches closest to TIC11207 and TIC11304 have been disclosed in U.S. Patent Application Publication US20180066277 as the insect toxin protein APG01463 (SEQ ID NO:42), which has 80% and 78.1% identity with TIC11207 and TIC11304, respectively. Figure 1 A multiple alignment of the Clustal W analysis of TIC11207, TIC11304, and APG01463 is shown. APG01463 exhibits activity against Spodoptera frugiperda but does not exhibit activity against Nezara viridula or Aphis glycines. Table 1 below shows the multiple alignment of TIC11207, TIC11304, and APG01463 using Clustal W, where the numbers in parentheses are the number of identical amino acids.

[0072] Table 1. Percent identity of TIC11207, TIC11304, and APG01463.

[0073] toxin TIC11207 TIC11304 APG01463 TIC11207 - 96.6(313) 79(256) TIC11304 96.6(313) - 77.2(250) APG01463 80(256) 78.1(250) -

[0074] In addition to percent identity, TIC11207 and TIC11304 can also be related by primary structure (conserved amino acid motifs), length, and other characteristics. The properties of the TIC11207 and TIC11304 insecticidal proteins are reported in Table 2 below.

[0075] Table 2. Selected properties of the TIC11207 and TIC11304 insecticidal proteins.

[0076]

[0077] As further described in the examples of the present application, the synthetic nucleic acid coding sequences encoding TIC11207 and TIC11304 are designed for use in plants and are encoded by SEQ ID NO: 3, 5, 6, 7, 8, 9, 10, 13, and 15. Given the redundancy of the genetic code, generating any number of other sequences for encoding the toxin protein is within the skill in the art. However, it should be understood that sequences generated for expression in planta should avoid problems known in the art that impede or limit the efficient expression of the coding sequence, particularly as described in U.S. Patent No. 5,500,365.

[0078] Expression cassettes and vectors containing recombinant nucleic acid sequence molecules can be constructed and introduced into plants, particularly into plant cells such as maize, soybean, or cotton plants, according to transformation methods and techniques known in the art. For example, Agrobacterium-mediated transformation is described in U.S. Patent Application Publication 2009 / 0138985A1 (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (maize), 2008 / 0282432 (cotton), 2008 / 0256667 (cotton), 2003 / 0110531 (wheat), 2001 / 0042257A1 (beet), U.S. Patent No. 5,750,871 (rapeseed), 7,026,528 (wheat), and 6,365,807 (rice) as well as Arencibia et al. (1998) Transgenic Res. 7:213 - 222 (sugarcane), all of which are incorporated herein by reference in their entirety. The transformed cells can be regenerated into transformed plants expressing TIC2199, and insecticidal activity can be demonstrated by bioassays performed using plant leaf discs obtained from the transformed plants in the presence of lepidopteran pest larvae. Plants can be derived from plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0079] As an alternative to traditional transformation methods, a DNA sequence (such as a transgene, one or more expression cassettes, etc.) can be inserted or integrated into a specific locus or site within the genome of a plant or plant cell via site-directed integration. Thus, one or more recombinant DNA constructs and one or more molecules of the present disclosure can contain a donor template sequence that includes at least one transgene, expression cassette, or other DNA sequence for insertion into the genome of a plant or plant cell. This donor template for site-directed integration can also include one or two homology arms flanking the insertion sequence (i.e., the sequence, transgene, cassette, etc. to be inserted into the plant genome). One or more recombinant DNA constructs of the present disclosure can also contain one or more expression cassettes that encode site-specific nucleases and / or any one or more associated proteins for site-directed integration. These one or more nuclease expression cassettes can be present in the same molecule or vector as the donor template (cis), or in a separate molecule or vector (trans). Several methods for site-directed integration are known in the art and involve different proteins (or complexes of proteins and / or guide RNAs) that cleave genomic DNA to create a double-strand break (DSB) or nick at a desired genomic site or locus. Briefly, as understood in the art, during the process of repairing the DSB or nick introduced by the nuclease, the donor template DNA can be integrated into the genome at the site of the DSB or nick. The presence of one or more homology arms in the donor template can facilitate the adoption of the insertion sequence and its targeting to the plant genome during the repair process by homologous recombination, although the insertion event can occur by non-homologous end joining (NHEJ). Examples of site-specific nucleases that can be used include zinc finger nucleases, engineered or native 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 contain sequences encoding one or more guide RNAs to direct the nuclease to a desired site within the plant genome.

[0080] Recombinant nucleic acid molecule compositions encoding the TIC11207 and TIC11304 proteins expressed in bacteria and plants can be expressed using recombinant DNA constructs, wherein a polynucleotide molecule having an ORF encoding said protein is operably linked to genetic expression elements such as a promoter and any other regulatory elements required for expression in the system for which the construct is intended. Non-limiting examples include a plant functional promoter operably linked to the TIC11207 or TIC11304 protein coding sequence for expressing said protein in plants or a Bt functional promoter operably linked to the TIC11207 or TIC11304 protein coding sequence for expressing said protein in Bt bacteria or other Bacillus species. Other elements can be operably linked to the TIC11207 and TIC11304 protein coding sequences, including but not limited to enhancers, introns, untranslated leader sequences, protein immobilization tags (HIS-tag) encoded, 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 heterologous promoters operably linked to polynucleotides (such as SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 18) encoding the TIC11207 or TIC11304 proteins having the amino acid sequences shown in SEQ ID NO: 2, 4, 12, 14, 17, and 19. SEQ ID NO: 16 and 18 encode TIC11207_His (SEQ ID NO: 17) and TIC11304-His (SEQ ID NO: 19), respectively, which contain a histidine tag at the carboxyl terminus, thereby allowing expression in Escherichia coli (E. coli) for isolation and purification. The heterologous promoter can also be operably linked to a synthetic coding sequence encoding plastid-targeted TIC11207 or TIC11304. 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).

[0081] Recombinant DNA constructs containing the TIC11207 or TIC11304 protein coding sequence can also contain a DNA region encoding one or more insect inhibitors, which can be configured to be co-expressed or co-expressed with the DNA sequence encoding the TIC11207 or TIC11304 protein, an insect inhibitory dsRNA molecule, or an accessory protein. Accessory proteins include but are not limited to cofactors, enzymes, binding partners, or other agents that contribute to the effectiveness of the insect inhibitor, such as by assisting in its expression, affecting its stability in plants, optimizing the free energy of oligomerization reactions, enhancing its toxicity, and increasing its activity spectrum. Accessory proteins can promote, for example, the uptake of one or more insect inhibitors or enhance the toxic effects of toxic agents.

[0082] Recombinant DNA constructs can be assembled such that all proteins or dsRNA molecules are expressed from one promoter, or each protein or dsRNA molecule is under the control of a separate promoter, or some combination thereof. The proteins of the invention can be expressed by a multi-gene expression system, in which TIC11207 or TIC11304 is expressed from a common nucleotide segment that also contains other open reading frames and promoters, depending on the type of expression system selected. For example, a bacterial multi-gene expression system can utilize a single promoter to drive the expression of multiple linked / tandem open reading frames within 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.

[0083] A recombinant polynucleotide or recombinant DNA construct comprising the TIC2199 protein coding sequence can be delivered to a host cell by a vector, such as a plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage, or viral vector. Such vectors can be used to achieve stable or transient expression of the TIC11207 or TIC11304 protein coding sequence in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that contains the TIC11207 or TIC11304 protein coding sequence and is introduced into a host cell is referred to herein as a "transgene".

[0084] The present disclosure provides transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain recombinant polynucleotides expressing TIC11207 or TIC11304, or related family toxin protein-encoding sequences. The terms "bacterial cell" or "bacterium" can include, but are not limited to, Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, Brevibacillus, Klebsiella, Erwinia, or Rhizobium cells. The terms "plant cell" or "plant" can include, but are not limited to, dicotyledonous or monocotyledonous plants. The terms "plant cell" or "plant" can also include, but are not limited to, alfalfa, banana, barley, bean, broccoli, cabbage, Brassica plants (e.g., canola), carrot, cassava, castor bean, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, pasture, pea, peanut, pepper, pigeon pea, pine, potato, poplar, round orange squash, 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 feed corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cells or plants. In certain embodiments, transgenic plants and transgenic plant parts regenerated from the transgenic plant cells are provided. In certain embodiments, the transgenic plants can be obtained from transgenic seeds by cutting, breaking, grinding, or otherwise separating the part from the plant. In certain embodiments, the plant part can be a seed, boll, leaf, flower, stem, root, or any part thereof, or a non-regenerable part of the transgenic plant part. As used herein, a "non-regenerable" part of a transgenic plant part is a part that cannot be induced to form a whole plant, or a part that cannot be induced to form a whole plant capable of sexual and / or asexual reproduction. In certain embodiments, the non-regenerable part of the plant part is a part of a transgenic seed, boll, leaf, flower, stem, or root.

[0085] Methods are provided for producing transgenic plants that contain an insect lepidopteran-inhibiting or hemipteran-inhibiting amount of a TIC11207 or TIC11304 protein. Such plants can be produced by introducing a recombinant polynucleotide encoding any of the proteins provided herein into a plant cell and selecting plants derived from the plant cell that express an insect lepidopteran-inhibiting amount of the protein. The plants can be derived from the plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art.

[0086] The present disclosure also provides processed plant products, wherein the processed products comprise a detectable amount of TIC11207 or TIC11304, an insect inhibitory segment or fragment thereof, or any distinguishable portion thereof. In certain embodiments, the processed products are selected from the group consisting of: plant parts, plant biomass, oils, meals, sugars, animal feeds, flours, flakes, bran, lint, hulls, processed seeds, and seeds. In certain embodiments, the processed products are non-renewable. The plant products can include commodities or other commercial products derived from transgenic plants or transgenic plant parts, wherein the commodities or other products can be commercially traced by detecting a nucleotide segment or expressed RNA or protein encoding or comprising a distinguishable portion of TIC11207 or TIC11304.

[0087] Plants expressing the TIC11207 or TIC11304 proteins can be hybridized by breeding with transgenic events expressing other toxin proteins and / or expressing other transgenic traits (such as herbicide tolerance genes, genes conferring yield or stress tolerance traits, etc.), or such traits can be combined in a single stacked vector such that all traits are linked.

[0088] As further described in the Examples, the TIC11207 or TIC11304 protein coding sequences and sequences having a significant percentage identity to TIC11207 or TIC11304 can be identified using methods known to those of ordinary skill in the art, such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, the proteins TIC11207 and TIC11304 can be used to generate antibodies that specifically bind to related proteins and can be used to screen for and find other closely related protein members.

[0089] In addition, nucleotide sequences encoding the TIC11207 or TIC11304 toxin proteins can be used as probes and primers for screening using thermal cycling or isothermal amplification and hybridization methods to identify other members of the class. For example, oligonucleotides derived from the sequences shown in SEQ ID NOs: 3, 5, 6, 7, 8, 9, 10, 13, and 15 can be used to determine the presence of the TIC11207 or TIC11304 transgenes in deoxyribonucleic acid samples derived from commercial products. Given the sensitivity of certain nucleic acid detection methods using oligonucleotides, it is expected that oligonucleotides derived from the sequences shown in SEQ ID NOs: 3, 5, 6, 7, 8, 9, 10, 13, and 15 can be used to detect the TIC11207 or TIC11304 transgenes in commercial products of mixed origin, where only a small fraction of the commercial product is derived from transgenic plants containing any transgenes. It is further recognized that such oligonucleotides can be used to introduce nucleotide sequence variations in each of SEQ ID NOs: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 18. Such "mutagenic" oligonucleotides can be used to identify TIC11207 and TIC11304 amino acid sequence variants that exhibit a range of insect inhibitory activities or differential expression in transgenic plant host cells.

[0090] Nucleotide sequence homologs, such as insecticidal proteins encoded by nucleotide sequences that hybridize under stringent hybridization conditions to each or any of the sequences disclosed in this application, are also embodiments of the invention. The invention also provides a method for detecting a first nucleotide sequence that hybridizes to a second nucleotide sequence, where the first nucleotide sequence (or its reverse complement) encodes an insecticidal protein or an insecticidal fragment thereof and hybridizes to the second nucleotide sequence. In such cases, the second nucleotide sequence can be any nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 18 under stringent hybridization conditions. The nucleotide coding sequences hybridize to each other under appropriate hybridization conditions, such as stringent hybridization conditions, and the proteins encoded by these nucleotide sequences cross-react with antisera raised against any one of the other proteins. Stringent hybridization conditions as defined herein include at least hybridization at 42 °C, followed by washing twice at room temperature with 2X SSC, 0.1% SDS for five minutes each time, and then washing twice at 65 °C in 0.5X SSC, 0.1% SDS for thirty minutes each time. Washing at even higher temperatures constitutes even more stringent conditions, for example, hybridization at 68 °C, followed by washing at 68 °C in 2x SSC containing 0.1% SDS.

[0091] Those skilled in the art will recognize that due to the redundancy of the genetic code, many other sequences are capable of encoding such related proteins, and those sequences, insofar as their function of expressing insecticidal proteins in Bacillus strains or plant cells is concerned, are embodiments of the present invention, and of course it is recognized that many such redundant coding sequences do not hybridize with the native Bacillus sequences encoding the TIC11207 and TIC11304 variants under these conditions. This application contemplates the use of these and other identification methods known to those of ordinary skill in the art to identify TIC11207 and TIC11304 variant protein coding sequences and sequences having a significant percentage identity with the TIC11207 and TIC11304 variant protein coding sequences.

[0092] The present disclosure also contemplates the use of molecular methods known in the art to engineer and clone commercially useful proteins, which include chimeras of proteins from insecticidal proteins; for example, chimeras can be assembled from segments of the TIC11207 and TIC11304 proteins to obtain additional useful embodiments, which include the assembly of segments of the TIC11207 and TIC11304 proteins with segments of different proteins that are different from the TIC11207 and TIC11304 proteins and related proteins. The TIC11207 and TIC11304 proteins can be aligned with each other and with other Arthrobacter, Bacillus, Paenibacillus, or other insecticidal proteins (regardless of whether these proteins are closely or distantly phylogenetically related), and segments of each such protein can be identified that can be used for substitution between the aligned proteins, thereby resulting in the construction of chimeric proteins. Pest bioassay analysis can be performed on such chimeric proteins, and it can be characterized whether there is increased biological activity or an expanded target pest spectrum compared to the parental proteins from which each such segment in the chimera is derived. The insecticidal activity of the polypeptide can be further engineered to obtain activity against specific pests or a broader spectrum of pests by exchanging domains or segments with other proteins or by using directed evolution methods known in the art.

[0093] The present application discloses methods for controlling insect, particularly Hemiptera, infestations of crop plants using the TIC11207 or TIC11304 proteins. Such methods can include growing plants that contain an insect or Hemiptera inhibitory amount of the TIC11207 or TIC11304 toxin protein. In certain embodiments, such methods can further include any one or more of the following: (i) applying any composition that contains or encodes the TIC11207 or TIC11304 toxin protein to the plant or the seeds from which the plant is produced; and (ii) transforming the plant or the plant cells from which the plant is produced with a polynucleotide that encodes the TIC11207 or TIC11304 toxin protein. Generally, the TIC11207 or TIC11304 toxin protein can be provided in a composition, provided in a microorganism, or provided in a transgenic plant to confer insect inhibitory activity against Hemiptera insects.

[0094] In certain embodiments, the recombinant nucleic acid molecule of the TIC11207 or TIC11304 toxin protein is the insecticidal active ingredient of an insect inhibitory composition prepared by culturing a recombinant Bacillus or any other recombinant bacterial cell that has been transformed to express the TIC11207 or TIC11304 toxin protein under conditions suitable for the expression of the TIC11207 or TIC11304 toxin protein. Such a composition can be prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting, or concentrating a culture of such recombinant cells that express / produce the recombinant polypeptide. Such a process can yield 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, the composition containing the recombinant polypeptide can contain bacterial cells, bacterial spores, and parasporal inclusions and can be formulated for various uses, including as an agricultural insect inhibitory spray product or as an insect inhibitory preparation in a dietary bioassay.

[0095] In one embodiment, to reduce the likelihood of resistance development, an insect inhibitory composition comprising the TIC11207 or TIC11304 protein may further comprise at least one additional polypeptide that exhibits insect inhibitory activity against the same hemipteran or lepidopteran insect species but is different from the TIC11207 or TIC11304 toxin protein. Possible additional polypeptides for such compositions include insect inhibitory proteins and insect inhibitory dsRNA molecules. An example of using such ribonucleotide sequences to control insect pests is described by Baum et al. (U.S. Patent Publication 2006 / 0021087A1). Such additional polypeptides for controlling hemipteran pests may be selected from the group consisting of insect inhibitory proteins such as, but not limited to, TIC1415 (U.S. Patent Publication 2013-0097735A1), TIC807 (U.S. Patent No. 8609936), TIC834 (U.S. Patent Publication 2013-0269060A1), AXMI-036 (U.S. Patent Publication 2010-0137216A1), and AXMI-171 (U.S. Patent Publication 2013-0055469A1). In addition, polypeptides for controlling coleopteran pests may be selected from the group consisting of insect inhibitory proteins such as, but not limited to, Cry3Bb (U.S. Patent No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, AXMI134 (U.S. Patent Publication 2013-0167264A1), AXMI-184 (U.S. Patent Publication 2010-0004176A1), AXMI-205 (U.S. Patent Publication 2014-0298538A1), AXMI-207 (U.S. Patent Publication 2013-0303440A1), AXMI-218, AXMI-220 (U.S. Patent Publication 2014-0245491A1), AXMI-221z, AXMI-223z (U.S. Patent Publication 2014-0196175A1), AXMI-279 (U.S. Patent Publication 2014-0223599A1), AXMI-R1 and its variants (U.S. Patent Publication 2010-0197592A1), TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication 2010-0319092A1), eHIPs (U.S. Patent Application Publication No. 2010 / 0017914A1), IP3 and its variants (U.S. Patent Publication 2012-0210462A1), IPD102Aa and its homologs (International Patent Publication WO2020076958 A1), IPD110Aa and its homologs (U.S. Patent Application Publication No. 2021-0355174A1) and -Hexatoxin-Hv1a (US Patent Application Publication US2014-0366227A1).

[0096] In other embodiments, such compositions / formulations may further comprise at least one additional polypeptide that exhibits insect inhibitory activity against insects not inhibited by the other insect inhibitory proteins of the present invention to broaden the obtained insect inhibitory spectrum. For example, for controlling Lepidoptera pests, combinations of the insect inhibitory proteins of the present invention can be used in combination with Lepidoptera-active proteins such as, but not limited to, Cry1A (U.S. Patent No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. Patent Publication No. 10 / 525,318), Cry1C (U.S. Patent No. 6,033,874), Cry1D, Cry1Da and variants thereof, Cry1E, Cry1F and Cry1A / F chimeras (U.S. Patent Nos. 7,070,982; 6,962,705; and 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras such as, but not limited to, TIC836, TIC860, TIC867, TIC869 and TIC1100 (International Application Publication WO2016 / 061391 (A2)), TIC2160 (International Application Publication WO2016 / 061392 (A2)), Cry2A, Cry2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, TIC4029, TIC4064, TIC13085, TIC13087, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 (U.S. Patent Publication 2013-0117884A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication 2013-0310543A1), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication 2013-0104259A1), AXMI-134 (U.S. Patent Publication 2013-0167264A1), AXMI-150 (U.S. Patent Publication 2010-0160231A1), AXMI-184 (U.S. Patent Publication 2010-0004176A1), AXMI-196, AXMI-204,AXMI-207, AXMI-209 (U.S. Patent Publication 2011-0030096A1), AXMI-218, AXMI-220 (U.S. Patent Publication 2014-0245491A1), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175A1), AXMI-238 (U.S. Patent Publication 2014-0033363A1), AXMI-270 (U.S. Patent Publication 2014-0223598A1), AXMI-345 (U.S. Patent Publication 2014-0373195A1), AXMI-335 (International Application Publication WO2013 / 134523(A2)), DIG-3 (U.S. Patent Publication 2013-0219570A1), DIG-5 (U.S. Patent Publication 2010-0317569A1), DIG-11 (U.S. Patent Publication 2010-0319093A1), AfIP-1A and its derivatives (U.S. Patent Publication 2014-0033361A1), AfIP-1B and its derivatives (U.S. Patent Publication 2014-0033361A1), PIP-1A PIP-1B (U.S. Patent Publication 2014-0007292A1), PSEEN3174 (U.S. Patent Publication 2014-0007292A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292A1), DIG-657 (International Application Publication WO2015 / 195594A2), Pput_1064 (U.S. Patent Publication 2014-0007292A1), GS-135 and its derivatives (U.S. Patent Publication 2012-0233726A1), GS153 and its derivatives (U.S. Patent Publication 2012-0192310A1), GS154 and its derivatives (U.S. Patent Publication 2012-0192310A1), GS155 and its derivatives (U.S. Patent Publication 2012-0192310A1), SEQ ID NO:2 or 4 and their derivatives as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 or 4 and their derivatives as described in U.S. Patent Publication 2012-0047606A1, SEQ ID NO:2 or 4 and their derivatives as described in U.S. Patent Publication 2011-0154536A1, SEQ ID NO:2 or 4 and their derivatives as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 or 4 and 4 and their derivatives as described in U.S. Patent Publication 2010-0192256A1,SEQ ID NO:2 or 4 and their derivatives as described in US Patent Publication 2010-0077507A1, SEQ ID NO:2 or 4 and their derivatives as described in US Patent Publication 2010-0077508A1, SEQ ID NO:2 or 4 and their derivatives as described in US Patent Publication 2009-0313721A1, SEQ ID NO:2 or 4 and their derivatives as described in US Patent Publication 2010-0269221A1, SEQ ID NO:2 or 4 and their derivatives as described in US Patent No. 7,772,465 (B2), CF161_0085 and its derivatives as described in WO2014 / 008054A2, lepidopteran toxin proteins and their derivatives as described in US Patent Publications US2008-0172762A1, US2011-0055968A1 and US2012-0117690A1; SEQ ID NO:2 or 4 and their derivatives as described in US7510878 (B2), SEQ ID NO:2 or 4 and their derivatives as described in US Patent No. 7812129 (B1); IPD072Aa and its homologs (US Patent Application Publication No. 2016-0366891A1) and IPD103 and its homologs (International Application Publication WO2018005411 A1), PIP-50 and PIP-65 and their homologs (US Patent Application Publication No. 2017-0166921A1), PIP-83 and its homologs (US Patent Application Publication No. 2016-0347799A1); etc.

[0097] Additional polypeptides for controlling coleopteran, lepidopteran, and hemipteran insect pests that can be combined with insect inhibitory proteins of the TIC11207 and TIC11304 classes can be found on the Bacillus thuringiensis toxin nomenclature website maintained by Neil Crickmore (World Wide Web URL: btnomenclature.info). Broadly speaking, any insect inhibitory protein known to those of ordinary skill in the art can be combined with proteins of the TIC11207 and TIC11304 families in planta (by breeding or molecular stacking) or in a composition or formulation as a biopesticide or a combination of biopesticides.

[0098] The possibility of insects developing resistance to certain insecticides has been documented in the art. One insect resistance management strategy is to use transgenic crops that express two different insect inhibitors that act by different modes of action. Thus, any insect that is resistant to either insect inhibitor can be controlled by the other insect inhibitor. Another insect resistance management strategy involves using plants that are not protected against the target lepidopteran pest species to provide a refuge for such unprotected plants. A specific example is described in U.S. Patent No. 6,551,962, which is incorporated herein by reference in its entirety.

[0099] Other embodiments for use with the proteins in seed treatment, spray formulations, drip formulations, or wipe formulations (such as topically applied pesticidal chemicals that are designed to control pests also controlled by the proteins disclosed herein) can be applied directly to the soil (soil drench), to growing plants expressing the proteins disclosed herein, or formulated 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 adhesives and tackifiers known in the art. Such formulations can contain insecticides that are synergistic with the disclosed proteins in their mode of action such that the formulated insecticides act by different modes of action to control the same or similar pests that can be controlled by the disclosed proteins, or such insecticides are used to control pests over a broader host range or plant pest species that are not effectively controlled by the TIC11207 or TIC11304 insecticidal proteins.

[0100] The above compositions / formulations can also contain agriculturally acceptable carriers such as baits, powders, dusts, pellets, granules, sprays, emulsions, colloidal suspensions, aqueous solutions, Bacillus spore / crystal formulations, seed treatments, recombinant plant cells / plant tissues / seeds / plants transformed to express one or more of the said proteins, or bacteria transformed to express one or more of the said proteins. Depending on the inherent insect inhibitory or insecticidal inhibitory level of the recombinant polypeptide and the level of the formulation applied to the plant or diet assay, the compositions / formulations can contain different amounts by weight of the recombinant polypeptide, for example, 0.0001% to 0.001% to 0.01% to 1% to 99% by weight of the recombinant polypeptide.

[0101] In view of the foregoing, those skilled in the art will understand that changes can be made in the specific aspects that have been disclosed and still obtain similar or like results without departing from the spirit and scope of the invention. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting. It is understood that the entire disclosure of each reference cited herein is incorporated into the disclosure of this application.

[0102] Examples

[0103] Example 1

[0104] Discovery, cloning and expression of TIC11207 and TIC11304

[0105] The insecticidal protein TIC11207 was identified by sequence analysis of the genome of the Arthrobacter nitroguajacolicus strain MDI-0030264. DNA was isolated from MDI-0030264 and sequenced. The assembled sequences were then subjected to bioinformatics analysis. The TIC11207 protein was identified as an ETX_MTX2 type protein by pfam analysis. A homolog of TIC11207, TIC11304, was identified by sequence analysis of the genome of the Arthrobacter nitroguajacolicus species MDI-0030376. A search of patents and public sequences revealed the homolog protein APG01463, which appears in US Patent Application Publication US20180066277 (SEQ ID NO:42), has approximately 80% identity with TIC11207, and has approximately 78% identity with TIC11307. Figure 1 An alignment between TIC11207, TIC11304 and APG01463 is shown, where identical amino acids are indicated by asterisks below the alignment.

[0106] Polymerase chain reaction (PCR) primers were designed to amplify full-length copies of the coding regions of TIC11207 and TIC11304 from total genomic DNA isolated from the Arthrobacter nitroguajacolicus strains MDI-0030264 and MDI-0030376. The PCR amplicons were cloned into two plasmid constructs using methods known in the art: one was cloned into an Escherichia coli (Ec) expression vector operably linked to an Ec-expressible promoter and a histidine tag for protein purification; and the other was cloned into a Bt expression vector operably linked to a Bt-expressible promoter. The coding sequences of the histidine-tagged TIC11207-his and TIC11304-his proteins (SEQ ID NO:17 and 19) are presented as SEQ ID NO:16 and 18, respectively. Preparations of TIC11207 and TIC11304 derived from both Ec and Bt were used in bioassays.

[0107] Example 2

[0108] In insect bioassays, TIC11207 demonstrated hemipteran activity, while TIC11304 demonstrated hemipteran and lepidopteran activity

[0109] The insecticidal proteins TIC11207 and TIC11304 were expressed in recombinant Ec and Bt using the vectors described in Example 1, and the toxicity of the resulting proteins expressed in these systems against different species of hemipterans, lepidopterans and coleopterans was determined.

[0110] The toxicities of TIC11207 were determined against the lepidopteran insect species Spodoptera frugiperda (FAW), and the hemipteran insect species Nezara viridula (SGSB), Euschistus heros (NBSB), and Lygus hesperus (WTP). The toxicities of TIC11304 were determined against the lepidopteran insect species Spodoptera frugiperda (FAW), Spodoptera eridania (SAW), and Pseudoplusia includens (SBL); the hemipteran insect species Nezara viridula, Euschistus heros, and Lygus hesperus; and the coleopteran species Diabrotica virgifera (WCR). The bioassay results are presented in Table 3 below, where "+" indicates activity, "-" indicates inactivity, and "NT" indicates not tested.

[0111] Table 3. Activities of TIC11207 and TIC11304 against lepidopteran, coleopteran, and hemipteran insect species.

[0112]

[0113] From the data presented in Table 3, it can be seen that TIC11207 exhibited activity against WTP, SGSB, and NBSB. TIC11304 exhibited activity against SAW, SBL, WTP, SGSB, and NBSB.

[0114] Example 3

[0115] Artificial coding sequences for TIC11207PL and TIC11304PL designed for expression in plants.

[0116] The artificial coding sequences SEQ ID NO:3, 5, 6, 7, 8, 9, 10, 13, and 15 encoding TIC11207PL and TIC11304PL were designed for expression in plant cells. The artificial (or synthetic) sequences were synthesized according to the methods generally described in U.S. Patent 5,500,365 to avoid certain harmful problem sequences, such as ATTTA-rich and A / T-rich plant polyadenylation sequences, while retaining the amino acid sequences of the native Bacillus proteins. The amino acid sequences of TIC11207PL and TIC11304PL contain additional alanine residues inserted after the start methionine of TIC11207 and TIC11304 for enhanced expression.

[0117] Using techniques known in the art, artificial coding sequences encoding TIC11207PL and TIC11304PL were cloned into a plant transformation vector, downstream of and functionally linked to a plant promoter, thereby driving the expression of the coding sequences when in plant cells. The transformation vector for transforming soybean plants comprises: a right border sequence from Agrobacterium tumefaciens; a first transgene cassette for selecting transformed plant cells using spectinomycin selection; a second transgene cassette for expressing TIC11207PL or TIC11304PL, comprising a constitutive promoter, operably 5'-linked to a leader sequence, operably 5'-linked to an intron, operably 5'-linked to an artificial coding sequence encoding TIC11207PL or TIC11304PL, said artificial coding sequence in turn being operably 5'-linked to a 3' UTR; and a left border sequence from Agrobacterium tumefaciens.

[0118] Example 4

[0119] TIC11207PL exhibits activity against the Neotropical brown stink bug (NBSB, Euschistus heros) when expressed in stably transformed soybean plants.

[0120] A binary plant transformation vector containing a transgene cassette designed to express TIC11207PL using the coding sequence of SEQ ID NO:3 was cloned using methods known in the art. The resulting vector was used to stably transform soybean plants. The transformed soybean plants were infested with the Neotropical brown stink bug (NBSB, Euschistus heros), and their activity against NBSB was determined.

[0121] Stably transformed R0 soybean plants expressing TIC11207PL with at least one R5.5 pod were infested with NBSB eggs by adhering two egg masses (each egg mass containing approximately 19 eggs in agar on rayon fabric) to intact soybean trifoliate leaves. The plants were then covered with fine mesh bags to prevent escape of nymphs after hatching. Approximately 80% of the eggs hatched per plant. On day 15, the bags containing the soybean plants were collected by cutting the stem below the bag closure with hand pruners. Approximately 92% of the bugs were recovered by this method. The number of live bugs and the instar of each nymph were recorded for each R0 event. Untransformed control plants were also used in this assay for comparison with plants expressing TIC11207PL. The average percentage survival of NBSB is presented in Table 4 below and shown graphically in Figure 2 as follows.

[0122] Table 4. Average percentage survival of the Neotropical brown stink bug (NBSB, Euschistus heros) in stably transformed R0 plants expressing TIC11207PL.

[0123] toxin average value standard error control 80.77 1.71 TIC11207PL 54.08 8.84

[0124] As can be seen in Table 4 and Figure 2 it can be seen that, compared to the control plants, soybean plants expressing TIC11207PL exhibit activity against NBSB. The following Table 5 and Figure 3 show individual events for calculating the mean survival percentages of control and TIC11207PL.

[0125] Table 5. Survival percentages of Neotropical brown stink bug (NBSB, Euschistus heros) in R0 plants stably transformed to express TIC11207PL.

[0126]

[0127] As can be seen in the above Table 5 and Figure 3 it can be seen that 5 R0 soybean plants expressing TIC11207PL exhibit NBSB survival percentages of less than 50%.

[0128] Soybean plants expressing TIC11207PL exhibit resistance to the Neotropical brown stink bug (Euschistus heros).

[0129] All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in accordance with the disclosure herein. While the compositions of the present invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those skilled in the art that changes, alterations, modifications, and substitutions may be applied to the compositions described herein without departing from the true spirit, nature, and scope of the present invention. More specifically, it is apparent that certain agents related chemically and physiologically can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0130] All publications and patent documents disclosed in this specification are incorporated herein by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding an insecticidal protein or a fragment thereof, wherein optionally: a. the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2, 4, 12, 14, 17, or 19; b. the insecticidal protein comprises an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 12, 14, 17, or 19; or c. the polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, or 18.

2. The recombinant nucleic acid molecule of claim 1, wherein: a. the recombinant nucleic acid molecule is expressed in a plant cell to produce an insecticidally effective amount of the insecticidal protein or insecticidal fragment; or b. the recombinant nucleic acid molecule is operably linked to a vector, and the vector is selected from the group consisting of plasmids, phagemids, bacmids, cosmids, and bacterial or yeast artificial chromosomes.

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 bacterial cells and plant cells.

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, Arthrobacter, 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 claim 2, wherein the 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 plants, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, loblolly pine, millet, melon, nut, oat, olive, onion, ornamental plant, palm, pasture, pea, peanut, pepper, pigeon pea, pine, potato, poplar, round orange squash, radiata pine, radish, rapeseed, rice, rootstock, rye, safflower, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, corn, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon, and wheat plant cells.

8. The recombinant nucleic acid molecule of claim 1, wherein the insecticidal protein exhibits activity against hemipteran insect species.

9. The recombinant nucleic acid molecule according to claim 8, wherein the insect species is Nezara viridula (Southern green stink bug), Thyanta perditor (Neotropical brown stink bug), and Lygus hesperus (Western tarnished plant bug).

10. The recombinant nucleic acid molecule according to claim 1, wherein the insecticidal protein exhibits activity against lepidopteran insect species.

11. The recombinant nucleic acid molecule according to claim 10, wherein the insect species is Mythimna separata (Southern armyworm) and Autographa nigrisigna (Soybean looper).

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

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

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

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

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

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

18. The insect-inhibiting composition according to claim 17, wherein the at least one other insecticide is selected from the group consisting of insect-inhibiting proteins, insect-inhibiting dsRNA molecules, chemical molecules, and accessory proteins, and wherein the at least one other insecticide is toxic to the same pest as the insecticidal protein or its insecticidal fragment.

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

20. The insect-inhibiting composition according to claim 17, wherein the at least one other insecticidal protein is selected from the group consisting of: Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1D variants, 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, TIC4029, TIC4064, TIC13085, TIC13087, VIP3A, VIP3B, VIP3Ab, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113 and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and its variants, IP3 and its variants, DIG-3, DIG-5, DIG-10, DIG-657, DIG-11 protein, IPD102Aa and its homologs, IPD110Aa and its homologs, TIC868, Cry1Da1_7, BCW003, TIC1100, TIC867, TIC867_23, TIC6757, TIC7641, IPD072Aa and its homologs, TIC5290, TIC3668, TIC3669, TIC3670, IPD103 and its homologs, PIP-50 and PIP-65 and their homologs, PIP-83 and its homologs, fern proteins toxic to hemipteran species, and Cry1B.

34.

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

22. A commercial product produced from the plant according to claim 11, or a part thereof, wherein the commercial product contains a detectable amount of the recombinant nucleic acid molecule, the insecticidal protein, or its insecticidal fragment.

23. The commercial product according to claim 22, which is selected from the group consisting of: commercial corn bagged by a grain processor, corn flakes, tortillas, corn flour, cornmeal, corn syrup, corn oil, corn silage, corn starch, corn cereal, etc., and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut, fruit, melon and vegetable commercial products, including, where applicable, fruit juice, concentrate, jam, jelly, marmalade and other edible forms of such commercial products containing a detectable amount of such polynucleotides and / or polypeptides of the present application, whole or processed cottonseed, cotton oil, lint, seeds and plant parts processed for feed or food, fibers, 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, soy protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soy cheese, soy wine, animal feed containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass and fuel products produced using soybean plants and soybean plant parts.

24. A method for producing progeny seeds comprising the recombinant nucleic acid molecule according to claim 1, 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 seeds from the plant, wherein the harvested seeds comprise the recombinant nucleic acid molecule.

25. A plant resistant to insect infestation, wherein the cells of the plant comprise the recombinant nucleic acid molecule according to claim 1.

26. A method for controlling pests or pest infestations of Lepidoptera or Hemiptera species, the method comprising: a. contacting the pest with an insecticidally effective amount of an insecticidal protein as shown in SEQ ID NO: 2, 4, 12, 14, 17 or 19; or b. contacting the pest with an insecticidally effective amount of one or more insecticidal proteins, the one or more insecticidal proteins comprising an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQ ID NO: 2, 4, 12, 14, 17 or 19.

27. A method for detecting the presence of the recombinant nucleic acid molecule according to claim 1 in a sample comprising plant genomic DNA, which comprises: a. contacting the sample with a nucleic acid probe that hybridizes under stringent hybridization conditions to the genomic DNA of a plant comprising the recombinant nucleic acid molecule according to claim 1 and does not hybridize under such hybridization conditions to the genomic DNA of other isogenic plants that do not comprise the recombinant nucleic acid molecule according to claim 1, wherein the probe is homologous or complementary to SEQ ID NO: 3, 5, 6, 7, 8, 9, 10, 13 or 15; b. placing the sample and the probe under stringent hybridization conditions; and c. detecting the hybridization of the nucleic acid probe to the recombinant nucleic acid molecule.

28. A method for detecting the presence of an insecticidal protein or a fragment thereof in a sample containing protein, wherein the insecticidal protein comprises the amino acid sequence of SEQ ID NO: 2, 4, 12, 14, 17 or 19; or the insecticidal protein comprises an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity with SEQ ID NO: 2, 4, 12, 14, 17 or 19; the method comprises: a. contacting the sample with an immunoreactive antibody; and b. detecting the presence of the insecticidal protein or a fragment thereof.

29. The method according to claim 28, wherein the detecting step comprises ELISA or Western blotting.

30. An insecticidally effective amount of a protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, 4, 12, 14, 17 or 19.

31. A method for controlling hemipteran or lepidopteran pest species or pest infestations 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, 4, 12 or 14; or b. growing a crop plant expressing an insecticidally effective amount of one or more insecticidal proteins, the one or more insecticidal proteins comprising an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or about 100% amino acid sequence identity with SEQ ID NO: 2, 4, 12 or 14; and optionally c. releasing a transgenic hemipteran or 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.

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

33. The method according to claim 32, wherein the dicotyledonous crop plant is soybean, cotton or rapeseed.

34. The method according to claim 33, wherein the dicotyledonous crop plant is soybean.

35. The method according to claim 32, wherein the monocotyledonous crop plant is corn, wheat, sorghum, rice, rye or millet.

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