RU2781075C2 - New proteins with inhibitory effect relatively to insects - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a recombinant nucleic acid molecule for giving resistance to an insect pest of Lepidoptera order. A transgenic plant, a part of the plant, and a seed containing the specified recombinant nucleic acid molecule; a composition, and a feed, food, cosmetic, and industrial product containing the specified recombinant nucleic acid molecule are also disclosed. A method for control of a pest or pest damage, where the pest is an insect pest of Lepidoptera order, using the specified nucleic acid; and a method for detection of the presence of the specified recombinant nucleic acid molecule are disclosed.

EFFECT: invention allows for effective control of insect pests of Lepidoptera order.

29 cl, 11 tbl, 6 ex

Description

[01] This application claims priority over U.S. Provisional Application No. 62/406082, filed October 10, 2016, which is incorporated herein by reference in its entirety.

INCLUDING A SEQUENCE LIST

[02] A file named "MONS426WO_ST25.txt" containing the Sequence Listing in machine-readable format was created on October 8, 2017. This file is 77030 bytes in size (as measured by MS-Windows®), simultaneously filed electronically (using the EFS-Web filing system with the US Patent Office) and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[03] The invention generally relates to the field of insect-inhibiting proteins. A new class of proteins exhibiting inhibitory activity against agricultural pests of cultivated plants and seeds has been disclosed. In particular, the disclosed class of proteins has insecticidal activity against agricultural pests of cultivated plants and seeds, especially Lepidopteran insect pest species. Plants, plant parts, and seeds are provided that contain a recombinant polynucleotide construct encoding one or more of the disclosed toxic proteins.

BACKGROUND OF THE INVENTION

[04] Increasing the yield of important crops, including but not limited to corn, soybeans, sugar cane, rice, wheat, vegetables and cotton, is becoming increasingly important. In addition to the growing need for agricultural products to feed, clothe and provide energy for a growing population, climate-related effects and pressure from a growing population to use land for purposes other than agriculture are predicted to reduce the amount of arable land available for agriculture. These factors lead to gloomy forecasts for food security, in particular due to the lack of major improvements in plant biotechnology and farming practices. In light of such pressures, environmentally sustainable improvements in technology, farming practices, and pest management are vital tools for expanding crop production on the limited amount of arable land available for farming.

[05] Insects, especially those of the orders Lepidoptera and Coleoptera , are considered the main cause of damage to field crops, resulting in reduced yields in infested areas. Lepidoptera pest species that adversely affect agriculture include, but are not limited to: tomato cutworm ( Spodoptera exigua ), cotton cutworm (Helicoverpa zea), American cotton cutworm (Alabama argillacea), corn moth ( Ostrinia nubilalis ) , Fall Armyworm ( Spodoptera frugiperda ), Cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera eridania , Diatraea grandiosella , Earias vittella , Diatraea saccharalis , Heliothis and Virescenalis .

[06] Historically, heavy use of synthetic chemical insecticides has been relied upon as a pest control agent. Concerns about the environment and human health, in addition to emerging sustainability issues, have spurred the research and development of biological pesticides. These studies have led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

[07] The biological control paradigm changed when the potential of entomopathogenic bacteria, especially bacteria belonging to the genus Bacillus , was discovered and realized as a biological pest control agent. Strains of the bacterium Bacillus thuringiensis (Bt) have been used as a source of pesticidal proteins because Bt strains have been found to exhibit high toxicity to specific insects. Bt strains are known to produce delta-endotoxins that localize to parasporal crystalline inclusion bodies at the onset of sporulation and during the stationary phase of growth (eg, Cry proteins) and are also known to produce a secreted insecticidal protein. When ingested by a susceptible insect, delta-endotoxins, like secreted toxins, exert their effect on the surface of the epithelium of the small intestine, destroying the cell membrane, which leads to cell destruction and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt , including other Bacillus and various additional bacterial species such as Brevibacillus laterosporus, Lysinibacillus sphaericus (" Ls " formerly known as Bacillus sphaericus ), and Paenibacillus popilliae .

[08] Crystalline and secreted soluble insecticidal toxins are very specific to their target organisms and have gained worldwide acceptance as an alternative to chemical insecticides. For example, insecticidal toxin proteins have been used in various agricultural practices to protect agriculturally important plants from insect attack, to reduce the need for chemical pesticides, and to increase yields. Insecticidal toxin proteins are used to control crop pests by mechanical methods such as spraying to disperse microbial preparations containing various bacterial strains on plant surfaces, and by genetic transformation methods to obtain transgenic plants and seeds expressing the insecticidal protein. -toxin.

[09] The use of transgenic plants expressing insecticidal toxin proteins has been universally adopted. For example, in 2012, 26.1 million ha were planted with transgenic crops expressing Bt toxins (James, C., Global Status of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44). The global use of transgenic insect-protected crops and the limited amount of insecticidal toxin proteins used in these crops has created selection pressure on existing insect alleles that confer resistance to currently used insecticidal proteins.

[010] The development of resistance in target pests to insecticidal toxin proteins creates a continuing need to discover and develop new forms of insecticidal toxin proteins that can be used to control increased insect resistance to transgenic crops expressing insecticidal toxin proteins. Newer white toxins with improved potency, which show control over a wider range of susceptible insect species, will reduce the number of surviving insects that can develop resistance alleles. In addition, the use in the same plant of two or more transgenic insecticidal toxin proteins that are toxic to the same pest and exhibit different modes of action reduces the likelihood of resistance in any individual target insect species.

[011] Thus, the inventors disclose herein a new family of protein toxins from Bacillus thuringiensis , together with similar toxic proteins, protein variants, and illustrative recombinant proteins, that exhibit insecticidal activity against target Lepidoptera species, in particular against the tomato armyworm ( Spodoptera exigua ), Helicoverpa zea , American cotton bollworm (Alabama argillacea ), Corn moth ( Ostrinia nubilalis ), Fall armyworm ( Spodoptera frugiperda ), Cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera eridania , Diatraea grandiosella , Earias vittella , Diatraea saccharalis , Heliothis virescens , and Anticarsia gemmatalis .

SUMMARY OF THE INVENTION

[012] Disclosed herein is a new group of pesticidal proteins with insect inhibitory activity (toxic proteins), referred to herein as TIC4472, TIC1425, and TIC2613, belonging to the TIC4472 protein toxin class, which have been shown to exhibit inhibitory activity against one or more crop pests. The TIC4472 protein and proteins in the TIC4472 class of protein toxins can be used alone or in combination with other insecticidal proteins and toxic agents in formulations and in planta , thereby offering alternatives to the insecticidal proteins and chemical insecticides currently used in agricultural systems.

[013] In one embodiment, this application discloses a recombinant nucleic acid molecule containing a heterologous promoter fragment operably linked to a polynucleotide segment encoding a pesticidal protein or fragment thereof, wherein (a) said pesticidal protein contains the amino acid sequence of SEQ ID NO:4 , SEQ ID NO:2, or SEQ ID NO:6; or (b) said pesticidal protein contains an amino acid sequence having: (i) at least 93% or 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (ii) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10; or (c) said polynucleotide segment hybridizes to a polynucleotide having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9; or (d) said polynucleotide segment encoding the pesticidal protein or fragment thereof contains a polynucleotide sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% sequence identity with the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9; or (e) said recombinant nucleic acid molecule is operably linked to a vector, and said vector is selected from the group consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial or yeast artificial chromosome. The recombinant nucleic acid molecule may contain a sequence that functions to express the pesticidal protein in the plant; or expressed in a plant cell to produce a pesticidally effective amount of the pesticidal protein.

[014] In another embodiment of this application, proposed host cells containing a recombinant nucleic acid molecule according to this application, and the host cell is selected from the group consisting of bacterial and plant cells. Considered bacterial host cells include Agrobacterium, Rhizobium , Bacillus , Brevibacillus , Escherichia , Pseudomonas , Klebsiella , Pantoec , and Erwinia . In some embodiments, said Bacillus species is Bacillus cereus or Bacillus thuringiensis , said Brevibacillus is Brevibacillus laterosperous , or Escherichia is Escherichia coli . Contemplated plant host cells include a dicotyledonous plant cell and a monocotyledonous plant cell. The considered plant cells additionally include the plant cell of alfalfa, banana, barley, bean, broccoli, cabbage, ornamental cabbage, carrot, cassava, castor bean, cauliflower, celery, chickpea, bok choy, citrus, coconut, coffee, corn, clover , cotton ( Gossypium sp.), cucurbits, cucumber, Menzies pseudo-hemlock, eggplant, eucalyptus, flax, garlic, grapes, hops, leek, lettuce, frankincense pine, millet, melon, walnut, oats, olive tree, onion onions, ornamental plants, palm, pasture grasses, peas, peanuts, peppers, pigeon peas, pine, potatoes, poplars, pumpkins, radiata pine, radish, rapeseed, rice, rhizomes, rye, wild saffron, shrubs, sorghum, southern pine, soybeans, spinach, cucurbits, strawberries, sugar beet, sugar cane, sunflower, sweet corn, amberwood, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon and wheat.

[015] In another embodiment, the pesticidal protein exhibits activity against Lepidoptera insects, including the tomato cutworm ( Spodoptera exigua ), Helicoverpa zea , American cotton cutworm (Alabama argillacea), corn moth ( Ostrinia nubilalis ), corn fall armyworm ( Spodoptera frugiperda ), cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera eridania , Diatraea grandiosella , Earias vittella , Diatraea saccharalis , Heliothis virescens , and Anticarsia gemmatalis .

[016] This application also contemplates plants containing a recombinant nucleic acid molecule containing a heterologous promoter fragment operably linked to a polynucleotide segment encoding a pesticidal protein or fragment thereof, wherein: (a) said pesticidal protein contains the amino acid sequence of SEQ ID NO:4 , SEQ ID NO:2, or SEQ ID NO:6; or (b) said pesticidal protein contains an amino acid sequence having: (i) at least 93% or 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (ii) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10; or (c) said polynucleotide segment hybridizes under stringent hybridization conditions to a complementary nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:9; or (d) said plant exhibits a certain amount of said pesticidal protein. In some embodiments, the pesticidal protein comprises SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. In one embodiment, the plant is either a dicot plant or a monocot plant. In another embodiment, the plant is further selected from the group consisting of: alfalfa, banana, barley, bean, broccoli, cabbage, kale, carrot, cassava, castor bean, cauliflower, celery, chickpea, bok choy, citrus, coconut palm, coffee, corn, clover, cotton (Gossypium sp.), cucurbits, cucumber, Menzies pseudo-hemzies, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, lettuce, frankincense pine, millet, melon, walnut, oats , olive tree, onion, ornamental plants, palm, pasture grasses, peas, peanuts, peppers, pigeon peas, pine, potatoes, poplars, pumpkins, pine radish, radish, rapeseed, rice, rhizomes, rye, wild saffron, shrubs, sorghum, southern pine, soybean, spinach, cucurbits, strawberry, sugar beet, sugar cane, sunflower, sweet corn, amberwood, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon and wheat.

[017] In additional embodiments, seeds containing recombinant nucleic acid molecules are disclosed.

[018] In another embodiment, an insect-inhibiting composition is contemplated comprising the recombinant nucleic acid molecules disclosed herein. The insect-inhibiting composition may further comprise a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. In some embodiments, the at least one other pesticidal agent is selected from the group consisting of an insect-inhibiting protein, an insect-inhibiting dsRNA molecule, and an accessory protein. It is also contemplated that at least one other pesticidal agent in the insect-inhibiting composition is active against one or more pest species of the orders Lepidoptera , Coleoptera , or Hemiptera . At least one other pesticidal agent in the insect control composition in one embodiment is selected from the group consisting of Cry1A protein, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, chimeras Cry1A/F, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1 variants , ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100 , VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-, 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 -223z, AXMI-224z and AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants, IP3 and its variants, DIG-3, DIG-5, DIG-10, DIG-657 and DIG-11.

[019] Commercial products containing a detectable amount of the recombinant nucleic acid molecules disclosed in this application are also contemplated. Such marketable products include market corn packaged in a grain processor, corn flakes, corn pancakes, fine corn meal, corn meal, corn syrup, corn oil, corn silage, corn starch, breakfast corn, and the like, as well as similar soy, rice, wheat, sorghum, pigeon peas, peanuts, fruits, melons, and vegetable commercial products, including, where applicable, juices, concentrates, jams, jellies, marmalades and other edible forms of such commercial products containing a detectable amount of such polynucleotides and/or polypeptides according to this application, whole or processed cotton seeds, cottonseed oil, fluff, seeds and plant parts processed for feed or food, fiber, paper, biomass and fuel products such as fuel derived from cottonseed oil or pellets from cotton gin waste, whole or processed soybean seeds, soybean oil, soy protein, soybean meal, soy flour, soy flakes, soy bran, soy milk, soy cheese, soy wine, soy-containing animal feed, soy-containing paper, soy-containing cream, soy biomass, and fuel products produced using soy plants and plant parts soybeans.

[020] This application also discusses methods for obtaining seeds containing recombinant nucleic acid molecules disclosed in this application. The method includes planting at least one seed containing the recombinant nucleic acid molecules disclosed in this application; growing a plant from a seed; and harvesting plant seeds, wherein the harvested seeds contain the recombinant nucleic acid molecules of this application.

[021] In another exemplary embodiment, a plant is provided that is resistant to insect attack, wherein said plant cells comprise: (a) a recombinant nucleic acid molecule encoding an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO:4, SEQ ID NO :2, or SEQ ID NO:6; or (b) an insecticidally effective amount of a protein comprising an amino acid sequence having: (i) at least 93%, or 95%, or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (ii) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10.

[022] This application also discloses methods for controlling lepidoptera pest species and controlling plant damage by lepidopteran pest species, in particular, cultivated plants. The method includes, in one embodiment, (a) contacting the pest with an insecticidally effective amount of the pesticidal proteins set forth in SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (b) contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having: (i) at least 93% or 95% or about 100% amino acid sequence identity with SEQ ID NO: 4, SEQ ID NO:2, or SEQ ID NO:6; or (ii) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10.

[023] Additionally, this document provides a method for detecting the presence of a recombinant nucleic acid molecule containing a polynucleotide segment encoding a pesticidal protein or a fragment thereof, wherein: (a) said pesticidal protein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (b) said pesticidal protein contains an amino acid sequence having: (i) at least 93% or 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (ii) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10; or (c) said polynucleotide segment hybridizes to a polynucleotide having the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. In one embodiment of the invention, the method includes contacting a nucleic acid sample with a nucleotide probe that hybridizes under stringent hybridization conditions to genomic DNA from a plant containing a polynucleotide segment encoding a pesticidal protein or fragment thereof as provided herein, and not hybridizes under such hybridization conditions with genomic DNA from another isogenic plant that does not contain a segment, while the probe is homologous or complementary to SEQ ID NO:3 or SEQ ID NO:9, or a sequence that encodes a pesticidal protein containing an amino acid sequence having: (a) at least 93% or 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2, or SEQ ID NO:6; or (b) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10. The method may further include (a) subjecting the sample and probe to stringent hybridization conditions; and (b) detecting hybridization of the probe with sample DNA.

[024] The invention also provides methods for detecting the presence of a pesticidal protein or a fragment thereof in a sample containing a protein, said pesticidal protein comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO:10; or said pesticidal protein contains an amino acid sequence having: (a) at least 93% or 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2 , or SEQ ID NO:6; or (b) at least 73% or 75% or 80% or 85% or 90% or 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10. In one embodiment, the method includes: (a) contacting the sample with an immunoreactive antibody; and (b) detecting the presence of the protein. In some embodiments, the detection step includes an ELISA or a Western blot.

BRIEF DESCRIPTION OF SEQUENCES

[025] SEQ ID NO:1 is a nucleic acid sequence encoding the pesticide protein TIC4472 derived from the species Bacillus thuringiensis EG10742.

[026] SEQ ID NO:2 is the amino acid sequence of the pesticide protein TIC4472.

[027] SEQ ID NO:3 is a synthetic coding sequence encoding the pesticidal TIC4472PL protein for plant cell expression, with an additional alanine codon inserted immediately after the methionine start codon.

[028] SEQ ID NO:4 is the amino acid sequence of TIC4472PL encoded by the synthetic coding sequence for plant cell expression (SEQ ID NO:3), with an additional alanine amino acid inserted immediately after the start methionine.

[029] SEQ ID NO:5 is a nucleic acid sequence encoding the pesticide protein TIC1425 derived from the species Bacillus thuringiensis EG10731.

[030] SEQ ID NO:6 is the amino acid sequence of the TIC1425 pesticide protein.

[031] SEQ ID NO:7 is a nucleic acid sequence encoding the pesticide protein TIC2613 derived from the species Bacillus thuringiensis EG5408.

[032] SEQ ID NO:8 is the amino acid sequence of the pesticide protein TIC2613.

[033] SEQ ID NO:9 is a synthetic coding sequence encoding the pesticidal TIC2613PL protein for plant cell expression, with an additional alanine codon inserted immediately after the methionine start codon.

[034] SEQ ID NO:10 is the amino acid sequence of TIC2613PL encoded by the synthetic coding sequence for plant cell expression (SEQ ID NO:9), with an additional alanine amino acid inserted immediately after the starter methionine.

DETAILED DESCRIPTION OF THE INVENTION

[035] A problem in the field of 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 that can be expressed in plants without causing undesirable agronomic consequences, and provide an alternative mode of action compared to the current toxins that are commercially used in plants.

[036] Novel pesticidal proteins, exemplified by TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL, are disclosed herein and address each of these problems, in particular with respect to a wide range of lepidopteran insect pests, and more specifically with respect to the fall armyworm. ( Spodoptera exigua ), Helicoverpa zea , American cotton bollworm (Alabama argillacea), Corn moth ( Ostrinia nubilalis ), Fall armyworm ( Spodoptera frugiperda ), Cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera eridania , Diatraea grandiosella , Earias vittella , Diatraea saccharalis , Heliothis virescens , and Anticarsia gemmatalis .

[037] Reference in this application to TIC4472, "TIC4472 protein", "TIC4472 protein toxin", "TIC4472 toxic protein", "TIC4472 pesticide protein", "TIC4472 related toxins", "TIC4472 related toxic proteins", TIC4472PL, "TIC4472PL protein", "TIC4472PL protein toxin", "TIC4472PL toxic protein", "TIC4472PL pesticide protein", "TIC4472PL related toxins", "TIC4472PL related toxic proteins", TIC1425, "TIC1425 protein", "TIC1425 protein toxin" , "TIC1425 toxic protein", "TIC1425 pesticidal protein", "TIC1425 related toxins", "TIC1425 toxic related proteins", TIC2613, "TIC2613 protein", "TIC2613 protein toxin", "TIC2613 toxic protein", "Pesticide protein TIC2613", "TIC2613 related toxins", "TIC2613 related toxic proteins", TIC2613PL, "TIC2613PL protein", "TIC2613PL protein toxin", "TIC2613PL toxic protein", "TIC2613PL pesticidal protein", "TIC2613PL related toxins", "toxic proteins related to TIC2613PL", etc. ., refer to any novel pesticidal protein or insect inhibitory protein that contains, consists of, is essentially a homolog similar to, or is derived from any pesticidal protein or insect inhibitory protein sequence TIC4472 (SEQ ID NO:2), TIC4472PL (SEQ ID NO:4), TIC1425 (SEQ ID NO:6), TIC2613 (SEQ ID NO:8), or TIC2613PL (SEQ ID NO:10) and its pesticidal or insect-inhibiting segment, or combinations thereof that provide activity against Lepidoptera pests, including any protein exhibiting pesticidal or insect-inhibiting activity, if alignment of such protein with TIC4472, TIC4472PL, or TIC1425 results in an amino acid sequence identity of any percentage from about 93% to about 100% percent; or if alignment of such a protein with TIC2613 or TIC2613PL results in an amino acid sequence identity of any percentage from about 73% to about 100%. The TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins include both plastid-targeted and non-plastid-targeted forms of the proteins.

[038] The term "segment" or "fragment" is used in this application to describe continuous amino acid or nucleotide sequences that are shorter than the full amino acid or nucleotide sequence describing the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein. This application also discloses a segment or fragment exhibiting insect-inhibiting activity if the alignment of such segment or fragment with the corresponding part of the TIC4472 protein shown in SEQ ID NO:2, the TIC4472PL protein shown in SEQ ID NO:4, the TIC1425 protein shown in SEQ ID NO:6, results in an amino acid sequence identity of any percentage from about 93 to about 100 percent between the segment or fragment and the corresponding portion of the TIC4472, TIC4472PL, or TIC1425 protein; or if alignment of such a segment or fragment with the corresponding portion of TIC2613 as set forth in SEQ ID NO:8 or the TIC2613PL protein as set forth in SEQ ID NO:10 results in an amino acid sequence identity of any percentage from about 73 to about 100 percent between a segment, or fragment, and the corresponding part of the TIC2613 or TIC2613PL protein.

[039] In yet further specific embodiments, a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein fragment can be determined to exhibit the pesticidal activity of the original protein molecule from which it is derived. A fragment of a nucleic acid sequence encoding a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein can be defined as encoding a protein that exhibits pesticidal activity possessed by the protein molecule encoded by the original nucleotide sequence from which it is derived. The fragment or variant described herein may further comprise the domain identified herein that is responsible for the pesticidal activity of the protein.

[040] In specific embodiments, provided are TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein fragments containing at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1150, or at least about 1175 consecutive amino acids, or more, of a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein having pesticidal activity as described herein. In some embodiments, the present invention provides fragments of any of SEQ ID NOs: 2, 4, 6, 8, or 10 having full-length sequence activity. Methods for obtaining such fragments from the original molecule are well known in the art.

[041] Reference in this application to the terms "active" or "activity", "pesticidal activity" or "pesticidal" or "insecticidal activity", "insect-inhibiting" or "insecticidal" refers to the effectiveness of a toxic agent, such as a protein toxin, with regard to inhibition (inhibition of growth, nutrition, fertility or viability), suppression (inhibition of growth, nutrition, fertility or viability), control (control of pest infestation, control of pest feeding activities on a specific crop containing an effective amount of the protein TIC4472, TIC4472PL, TIC1425, TIC2613 or TIC2613PL) or destruction (causing pathology, mortality, or reduced fertility) of the pest. These terms are intended to include the result of exposing a pest to a pesticidally effective amount of a toxic protein when the effect of the toxic protein on the pest results in injury, mortality, reduced fertility, or stunted growth. The terms also include repelling a pest from a plant, plant tissue, plant part, seed, plant cell, or a particular geographic location where the plant may grow, by delivering a pesticidally effective amount of a toxic protein to or on the plant. In general, pesticidal activity generally refers to the ability of a toxic protein to be effective in inhibiting growth, development, viability, feeding behavior, mating behavior, fertility, or any measurable reduction in side effects caused by insect ingestion of that protein, protein fragment, segment protein or polynucleotide in a particular target pest, including but not limited to insects of the order Lepidoptera. The toxic protein may be produced by the plant or may be applied to the plant or the environment where the plant is located. The terms "biological activity", "effective", "effective" or variations thereof are also terms used interchangeably in this application to describe the effect of the proteins of this invention on harmful target insects.

[042] A pesticidally effective amount of a toxic agent, when delivered to the diet of a target pest, exhibits pesticidal activity when the toxic agent comes into contact with the pest. The toxic agent may be a pesticidal protein, or one or more chemical agents known in the art. The pesticidal or insecticidal chemical agents and the pesticidal or insecticidal proteinaceous agents may be used alone or in combination with each other. Chemical agents include, but are not limited to, dsRNA molecules targeting specific genes for suppression in the target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal or insecticidal proteinaceous agents include the proteinaceous toxins provided herein, as well as other proteinaceous toxicants, including those that target Lepidoptera, as well as proteinaceous toxins which are used to control other plant pests, such as Cry proteins and Cyt available in the art for use in the control of Coleopteran , Hemipteran and Homopteran species.

[043] Reference to a pest, in particular a crop pest, is intended to mean crop pests, in particular those Lepidoptera insect pests that are controlled by the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein toxin class. However, a pest reference may also include plant pests from Coleopteran , Hemipteran , and Homopteran , as well as nematodes and fungi, when the toxic agents targeting these pests are co-localized or present with the TIC4472, TIC4472PL, or TIC1425 protein, or a protein that is 93 to about 100% identical to TIC4472, TIC4472PL, or TIC1425; or a TIC2613 or TIC2613PL protein or a protein that is 73% to about 100% identical to TIC2613 or TIC2613PL.

[044] The TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins are similar in general function and exhibit insecticidal activity against Lepidoptera insect pests, including adult insects, pupae, larvae, and hatched larvae.

[045] Insects of the order Lepidoptera (Lepidoptera) include, but are not limited to: insects from the family Noctuidae (armyworms, cutworms, loopers, heliothines), e.g. corn fallowworm ( Spodoptera frugiperda ), tomato cutworm ( Spodoptera exigua ), Spodoptera exempta , Spodoptera eridania , Mamestra configurata , Upsilon cutworm ( Agrotis ipsilon ), Trichoplusia ni , Pseudoplusia includens , Anticarsia gemmatalis , Hypena scabra , Heliothis virescens , Agrotis subterranea , Pseudaletia unipuncta , Agrotis orthogonia ; grinders, case-bearers and other insects from the family of moths ( Pyralidae ), such as corn moth ( Ostrinia nubilalis ), Amyelois transitella , Crambus caliginosellus , Herpetogramma licarsisalis , Homoeosoma electellum , Elasmopalpus lignosellus ; insects from the leafworm family ( Tortricidae ), e.g. apple codling moth ( Cydia pomonella ), Endopiza viteana , eastern codling moth Grapholita molesta , Suleima helianthana ; and many other economically important Lepidoptera, such as the cabbage moth ( Plutella xylostella ), Pectinophora gossypiella , and the gypsy moth ( Lymantria dispar). Other insect pests of the Lepidoptera order include, for example, Alabama argillacea , Archips argyrospila , Archips rosana and other Archips species, ( Chilo suppressalis, yellow rice moth), Cnaphalocrocis medinalis , Crambus caliginosellus , Crambus teterrellus , Diatraea grandiosella saccharalis , Earias insulana , Earias vittella , cotton bollworm ( Helicoverpa armigera ), Helicoverpa zea , Heliothis virescens , Herpetogramma licarsisalis , Striacosta albicosta , bunch leafworm ( Lobesia botrana ), Phyllocnistis citrella , cabbage ( Pieris brassicae ), turnip ( Pierisptrapera exigua , Spodoptera litura , and the tomato miner moth ( Tuta absoluta ).

[046] Reference in this application to "an isolated DNA molecule", or an equivalent term or phrase, is intended to mean that a DNA molecule is a molecule that is present alone or in combination with other compounds, but not in its natural environment. For example, nucleic acid elements, such as a coding sequence, an intron sequence, an untranslated leader sequence, a promoter sequence, a transcription termination sequence, and the like, that are naturally found in the DNA of an organism's genome are not considered "isolated" as long as the element is found in the organism's genome and at the location in the genome where it is naturally found. However, each of these elements, and portions of such elements, will be "isolated" within the scope of this disclosure if the element is not in the organism's genome, nor the location in the genome where it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein, or any naturally occurring insecticidal variant of such a protein, will be an isolated nucleotide sequence, as long as the nucleotide sequence is not in the DNA of a bacterium in which the sequence encoding the protein is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e. a DNA nucleotide sequence inserted into the genome of plant or bacterial cells, or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar structure, used for cell transformation, in the genome of a plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commercial products derived from a plant or bacterium.

[047] As further described herein, an open reading frame (ORF) encoding TIC4747 (SEQ ID NO:1) was found in DNA obtained from Bacillus thuringiensis strain EG10742. The coding sequence has been cloned and expressed in microbial host cells to generate recombinant proteins used in bioassays. An open reading frame (ORF) encoding TIC1425 (SEQ ID NO:5) was found in DNA obtained from Bacillus thuringiensis strain EG10731. An open reading frame (ORF) encoding TIC2613 (SEQ ID NO:7) was found in DNA obtained from Bacillus thuringiensis strain EG5408. Bioassay using TIC4472 proteins derived from microbial host cells demonstrated activity against Lepidoptera species - Tomato cutworm ( Spodoptera exigua ), Helicoverpa zea , American cotton cutworm (Alabama argillacea), Corn moth ( Ostrinia nubilalis ), Corn fall armyworm ( Spodoptera frugiperda) ), cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera eridania , Diatraea grandiosella , Earias vittella , Diatraea saccharalis , Heliothis virescens , and Anticarsia gemmatalis . In addition, activity against the yellow fever mosquito ( Aedes aegypti ) was also observed. Bioassay using TIC1425 proteins derived from microbial host cells demonstrated activity against the Lepidoptera species of American cotton bollworm (Alabama argillacea), corn moth ( Ostrinia nubilalis ), fall armyworm ( Spodoptera frugiperda ), Diatraea saccharalis , and Diatraea grandiosella . Bioassay using TIC2613 proteins derived from microbial host cells demonstrated activity against Lepidoptera species Helicoverpa zea , American cotton bollworm Alabama argillacea , Corn moth ( Ostrinia nubilalis ), Fall armyworm ( Spodoptera frugiperda ), Chrysodeixis includens , Diatraea grandiosella , and Heliothis virescens .

[048] For expression in plant cells, the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins can be expressed to localize in the cytosol, or can be targeted to various plant cell organelles. For example, targeting a protein to a chloroplast can result in increased levels of expressed protein in a transgenic plant while preventing off-target phenotypes. Targeting may also lead to an increase in the effectiveness of pest resistance in the transgenic transformant. A targeting peptide or transit peptide is a short (3-70 amino acids long) peptide chain that directs protein transport to a specific region of the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome, and plasma membrane. Some targeting peptides are cleaved from the protein by signal peptidases after protein transport. For chloroplast targeting, proteins contain transit peptides that contain about 40-50 amino acids. For a description of the use of chloroplast transit peptides, see US Pat. Examples of such isolated chloroplast proteins include, but are not limited to: ribulose-1,5,-bisphosphate carboxylase small subunit (SSU)-associated proteins, ferredoxin, ferredoxin oxidoreductase, light-trapping complex protein I and protein II, thioredoxin F, enolpyruvil shikimate phosphate synthase (EPSPS), and the transit peptides described in US Pat. No. 7,193,133. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to the chloroplast using heterologous CTP fusion proteins, and that CTP is sufficient to target the protein to the chloroplast . It has been shown that insertion of a suitable chloroplast transit peptide such as Arabidopsis thaliana EPSPS CTP (CTP2) (see Klee et al., Mol. Gen. Genet. 210 :437-442, 1987) or Petunia hybrida EPSPS CTP (CTP4) ( see della - Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986) targeted heterologous EPSPS protein sequences to chloroplasts in transgenic plants (see US Pat. Nos. 5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP 508909; and EP 924299). To target the toxic TIC6757 or TIC6757PL protein to the chloroplast, the sequence encoding the chloroplast transit peptide is placed 5' in operable relationship and in reading frame with a synthetic coding sequence encoding the toxic TIC6757 or TIC6757PL protein that has been designed for optimal expression in plant cells.

[049] It is contemplated that additional toxic protein sequences related to TIC4472, TIC1425, or TIC2613 can be created using the amino acid sequence of TIC4472, TIC1425, or TIC2613 to create new proteins with new properties. Toxic TIC4472, TIC1425, or TIC2613 proteins can be aligned to fold differences, at the amino acid sequence level, into new amino acid sequence variants, and make corresponding changes in the recombinant nucleotide sequence encoding the variants.

[050] This disclosure further contemplates that improved variants of the TIC4472 protein toxin class can be engineered in planta using various gene editing techniques known in the art. Such technologies used for genome editing include, but are not limited to: ZFN (zinc finger nuclease), meganucleases, TALEN (transcriptional activator-like effector nucleases) and CRISPR (regularly arranged short palindromic repeats)/Cas ( CRISPR-associated) systems. These genome editing methods can be used to change a toxic protein coding sequence transformed in a plant cell to a different toxin coding sequence. In particular, using these methods, one or more codons in the toxin-coding sequence are changed to construct a new protein amino acid sequence. Alternatively, a fragment within the coding sequence is replaced or deleted, or additional DNA fragments are inserted into the coding sequence to construct a new toxin-coding sequence. The novel coding sequence can encode a toxic protein with novel properties such as increased activity or spectrum against insect pests, as well as activity against insect pest species that develop resistance to the original insect toxic protein. A plant cell containing an edited toxin-coding gene sequence can be used by methods known in the art to produce whole plants expressing the new toxic protein.

[051] It is also contemplated that fragments of TIC4472, TIC1425, or TIC2613, or protein variants thereof, may be truncated forms in which one or more amino acids are removed from the N-terminus, C-terminus, middle of the protein, or combinations thereof, wherein the fragments and variants retain insect-inhibiting activity. Such fragments may occur naturally or be synthetic variants of TIC4472, TIC1425 or TIC2613, or derived protein variants, but must retain insect inhibitory activity of at least TIC4472, TIC1425 or TIC2613. The fragment or variant described herein may further comprise the domain identified herein that is responsible for the pesticidal activity of the protein.

[052] Proteins that resemble TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins can be identified and compared to each other using various computer algorithms known in the art (see Tables 1 and 2). The amino acid sequence identities reported in this application are the result of Clustal W alignment using the following default parameters: Weight matrix: blosum, Gap opening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps: On, Hydrophilic residues: GPSNDQERK, Penalties for specific residue gaps: On. (Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percent amino acid identity is additionally calculated by the product multiplied by 100% (amino acid identity/length of the protein in question). Other alignment algorithms are also available in the art and provide similar results to those obtained using Clustal W alignment and are discussed in this document.

[053] A protein exhibiting insect-inhibiting activity against Lepidoptera insect species is assumed to be TIC4472, TIC4472PL, or TIC1425 if the protein used as a query, for example, in the Clustal W alignment, and the proteins of the present invention shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 are identified as matches in such an alignment in which the query protein shows at least 93% to about 100% amino acid identity along the length of the query protein, which is about 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any percentage in this range. It is also assumed that a protein exhibiting insect inhibitory activity against Lepidoptera species of insects, refers to TIC2613 or TIC2613PL, if the protein used as a query, for example, in the alignment of Clustal W, and the proteins according to this invention, presented in SEQ ID NO: 8 or SEQ ID NO:10 are identified as matches in such an alignment in which the query protein shows at least 73% to about 100% amino acid identity along the length of the query protein, which is about 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any percentage within this range.

[054] The exemplary proteins TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL were aligned to each other using the Clustal W algorithm. As reported in Table 1, a pairwise matrix of percent amino acid sequence identity was generated for each of the full length proteins.

Table 1 Demonstration of a pairwise matrix of exemplary proteins TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL.

Toxin TIC4472 (SEQ ID NO:2) TIC4472PL (SEQ ID NO:4) TIC1425 (SEQ ID NO:6) TIC2613 (SEQ ID NO:8) TIC2613PL (SEQ ID NO:10) TIC4472 (SEQ ID NO:2) - 99.9 (1186) 99.9 (1186) 68.1 (808) 68 (807) TIC4472PL (SEQ ID NO:4) 99.8 (1186) - 99.7 (1185) 67.9 (807) 68.1 (809) TIC1425 (SEQ ID NO:6) 99.9 (1186) 99.8 (1185) - 68.2 (809) 68.1 (808) TIC2613 (SEQ ID NO:8) 68.6 (808) 68.5 (807) 68.7 (809) - 99.9 (1177) TIC2613PL (SEQ ID NO:10) 68.4 (807) 68.6 (809) 68.5 (808) 99.8 (1177) -

Table Description: The Clustal W alignment between (X) and (Y) is described in a pairwise matrix. The percent amino acid identity between all pairs is calculated and is shown as the first number in each cell. The second number (in brackets) in each cell indicates the number of identical amino acids for the pair.

[055] In addition to percent identity, association between TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL and related proteins can also be identified by primary structure (conserved amino acid motifs), length (about 1187 amino acids), and other characteristics. Characteristics of protein toxins TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL are shown in Table 2.

Table 2 Selected characteristics of the TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins.

Protein Molecular weight (in Daltons) Length in amino acids isoelectric point Charge at pH 7.0 Number of strongly basic (-) amino acids Amount of strongly acidic amino acids Number of hydrophobic amino acids Number of polar amino acids TIC4472 134446.86 1187 4.7545 -36 137 162 611 576 TIC4472PL 134517.94 1188 4.7545 -36 137 162 612 576 TIC1425 134460.88 1187 4.7545 -36 137 162 611 576 TIC2613 134636.07 1178 4.6653 -42 132 162 588 590 TIC2613PL 134707.15 1179 4.6653 -42 132 162 589 590

[056] As further described in the Examples of this application, synthetic nucleic acid molecule sequences encoding the TIC4472 variant, TIC4472PL and the TIC2613 variant, TIC2613PL, were constructed for use in plants. An exemplary sequence of a recombinant nucleic acid molecule that has been developed for use in plants encoding the TIC4472PL protein is shown as SEQ ID NO:3. An exemplary sequence of a recombinant nucleic acid molecule that has been developed for use in plants encoding the TIC2613PL protein is shown as SEQ ID NO:9. Proteins TIC4472PL and TIC2613PL contain an additional amino acid alanine immediately after the starting methionine, relative to proteins TIC4472 and TIC2613, respectively. An additional alanine residue inserted into the TIC4472 and TIC2613 amino acid sequences is thought to improve in planta protein expression. Similarly, synthetic nucleic acid molecule sequences encode TIC1425 variants and can be developed for use in plants.

[057] A leaf disc assay using _R0 cotton leaf tissue expressing the TIC4472PL protein showed high activity against Chrysodeixis includens and Heliothis virescens , and low activity against Helicoverpa zea and fall armyworm ( Spodoptera frugiperda ). A leaf disc assay using soybean R ₀ leaf tissue expressing the TIC4472PL protein showed activity against Spodoptera eridania and Chrysodeixis includens .

[058] Leaf samples of R ₀ soybean plants expressing TIC4472PL and TIC2613PL proteins showed activity against Spodoptera eridania and Chrysodeixis includens .

[059] Expression cassettes and vectors containing the sequence of the recombinant nucleic acid molecule can be constructed and introduced into cells of corn, soybean, cotton or other plants in accordance with the methods and methods of transformation known in the art. For example, Agrobacterium -mediated transformation is described in US Patent Application Publications 2009/0138985A1 (soybean), 2008/0280361A1 (soybean), 2009/0142837A1 (corn), 2008/0282432 (cotton), 2008/0256667 (cotton), 2003/011051 (wheat), 2001/0042257 A1 (sugar beet), US Pat . (1998) Transgenic Res. 7:213-222 (sugarcane), each of which is incorporated herein by reference in its entirety. Transformed cells can be regenerated into transformed plants that express TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins and exhibit pesticidal activity through bioassays conducted in the presence of lepidopteran pest larvae using plant leaf disks derived from transformed plants. Plants can be obtained from plant cells by regeneration, seed, pollen or meristem transformation methods. Methods for transforming plants are known in the art.

[060] As an alternative to traditional transformation methods, a DNA sequence such as a transgene, expression cassette(s), etc., can be inserted or integrated at a specific site or locus in the genome of a plant or plant cell by site-directed insertion. The recombinant DNA construct(s) and molecule(s) of the invention may thus comprise a donor template sequence containing at least one transgene, expression cassette or other DNA sequence for insertion into the plant genome or plant cell. Such a site-directed integration donor template may further comprise one or two homologous arms flanking the insertion sequence (ie, the sequence, transgene, cassette, etc. that is inserted into the plant genome). The recombinant DNA construct(s) of this invention may further comprise an expression cassette(s) encoding a site-specific nuclease and/or any related protein(s) for site-directed insertion. Such nuclease-expressing cassettes may be present in the same molecule or vector as the donor template ( in cis ) or in a separate molecule or vector ( in trans ). Several site-directed insertion methods are known in the art using various proteins (or protein complexes and/or guide RNA) that cut genomic DNA to produce a double-strand break (DSB) or a single-strand break at the desired genomic site or locus. Briefly, as is understood in the art, during the repair of an LSB or single-strand break introduced by a nuclease enzyme, the donor template DNA can be integrated into the genome at the site of the LSB or single-strand break. The presence of the homologous arm(s) in the donor template may facilitate entry and targeting of the insert sequence in the plant genome during the repair process via homologous recombination, although the insertion event may occur via 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-directed endonucleases (eg, Cas9 or Cpf1). For methods using site-specific RNA-directed nucleases (eg, Cas9 or Cpf1), the recombinant DNA construct(s) will also contain a sequence encoding one or more guide RNAs to target the nuclease to the desired site in the plant genome.

[061] As used herein, the term "recombinant DNA molecule" is a DNA molecule containing a combination of DNA molecules that would not naturally occur together without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that consists of at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence that differs from naturally occurring DNA sequences, or a DNA molecule that that has been incorporated into the DNA of the host cell by genetic transformation or gene editing. Similarly, a "recombinant protein molecule" is a protein molecule containing a combination of amino acids that would not naturally occur together without human intervention. For example, a recombinant protein molecule may be a protein molecule that consists of at least two amino acid molecules that are heterologous to each other, a protein molecule that contains an amino acid sequence that differs from naturally occurring amino acid sequences, or a protein molecule that expressed in the host cell as a result of genetic transformation of the host cell or due to gene editing of the host cell's genome.

[062] Compositions of recombinant nucleic acid molecules that encode TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL are contemplated. For example, the TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins can be expressed using recombinant DNA constructs in which a polynucleotide molecule with an ORF encoding a protein is operably linked to genetic expression elements such as a promoter and any other regulatory element necessary for expression. in the system for which the design is intended. Non-limiting examples include a plant-functional promoter operably linked to a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein-coding sequence for protein expression in plants, or a Bt -functional promoter operably linked to a TIC4472, TIC4472PL, protein-coding sequence, TIC1425, TIC2613 or TIC2613PL, for protein expression in Bt bacteria or other Bacillus species. Other elements may be operably linked to the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein coding sequence, including but not limited to: enhancers, introns, untranslated leader sequences, encoded protein immobilization tags (HIS tag), translocation peptides (ie, plastid transit peptides, signal peptides), polypeptide sequences for post-translational modifying enzymes, ribosome binding sites, and RNAi target sites. Illustrative recombinant polynucleotide molecules provided herein include, but are not limited to: a heterologous promoter operably linked to a polynucleotide such as SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9 which encodes the corresponding polypeptides or proteins having the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO :ten. The heterologous promoter can also be operably linked with synthetic DNA sequences encoding plastid-targeted TIC4472PL or TIC2613PL; or non-targeted TIC4472PL or TIC2613PL. Codons of a recombinant nucleic acid molecule encoding the proteins disclosed herein may be replaced with synonymous codons (known in the art as silent substitution).

[063] A recombinant DNA construct containing sequences encoding TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins may additionally contain a DNA region that encodes one or more insect inhibitory agents that can be arranged for simultaneous expression or co-expression with a DNA sequence encoding a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein, a protein other than the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein, an insect-inhibiting dsRNA molecule, or an accessory protein. Accessory proteins include, but are not limited to: cofactors, enzymes, binding partners, or other agents that act to contribute to the effectiveness of the insect-inhibiting agent, for example, aiding its expression, affecting its stability in plants, optimizing free energy for oligomerization, increasing its toxicity and increasing its spectrum of activity. The accessory protein may facilitate uptake of, for example, one or more insect inhibitory agents or enhance the toxic effect of a toxic agent.

[064] A recombinant DNA construct can be assembled such that all proteins or dsRNA molecules are expressed from a single promoter, or each protein or dsRNA molecule is under the control of a single promoter or some combination thereof. The proteins of this invention can be expressed from a multigene expression system in which one or more TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins are expressed from a common nucleotide segment that also contains other open reading frames and promoters, depending on the type of system chosen. expression. For example, a bacterial multigene expression system may use a single promoter to drive the expression of multiple-linked/tandem open reading frames within a single operon (ie, polycistronic expression). In another example, a plant multigene expression system may use multiple-unlinked or linked expression cassettes, with each cassette expressing a different protein or different agent, such as one or more dsRNA molecules.

[065] Recombinant polynucleotides or recombinant DNA constructs containing a sequence encoding a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein can be delivered to host cells using vectors, e.g., plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage or viral vector. Such vectors can be used to achieve stable or transient expression of a TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein coding sequence in a host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that contains a sequence encoding a TIC4472, TIC4472PL, TIC1425, TIC2613 or TIC2613PL protein and which is introduced into a host cell is referred to in this application as a "transgene".

[066] Provided herein are transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain a recombinant polynucleotide that expresses any one or more TIC4472 or a sequence encoding a toxic related family protein. The term "bacterial cell" or "bacteria" may include, but is not limited to: Agrobacterium cell, Bacillus cell, Escherichia cell, Salmonella cell, Pseudomonas cell, Brevibacillus cell, Klebsiella cell, Erwinia cell, Rhizobium cell. The term "plant cell" or "plant" may include, but is not limited to, dicotyledonous or monocotyledonous plants. The term "plant cell" or "plant" may also include, but is not limited to: a plant cell or plant of alfalfa, banana, barley, bean, broccoli, cabbage, ornamental cabbage, carrot, cassava, castor bean, cauliflower, celery , chickpea, bok choy, citrus, coconut, coffee, corn, clover, cotton, cucurbits, cucumber, menzies, eggplant, eucalyptus, flax, garlic, grapes, hops, leek, lettuce, frankincense pine, millet , melon, walnut, oats, olive tree, onion, ornamental plants, palm, pasture grasses, peas, peanuts, peppers, pigeon peas, pines, potatoes, poplars, pumpkins, radiant pine, radishes, rapeseed, rice, rhizomes, rye , wild saffron, bush, sorghum, southern pine, soybeans, spinach, cucurbits, strawberries, sugar beet, sugar cane, sunflower, sugar corn, amber tree, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon and n shenitsy. In some embodiments, transgenic plants and transgenic plant parts regenerated from a transgenic plant cell are provided. In some embodiments, transgenic plants can be obtained from transgenic seed by cutting, breaking off, crushing, or otherwise separating a portion from the plant. In some embodiments, the plant part may be a seed, pod, leaf, flower, stem, root, or any part thereof or a non-regenerated part of the transgenic plant part. As used herein, a "non-regenerating" portion of a transgenic plant part is a portion that cannot be stimulated to form a whole plant, or that cannot be stimulated to form a whole plant capable of sexual and/or asexual reproduction. In some embodiments, the non-regenerated portion of the plant part is a portion of a transgenic seed, pod, leaf, flower, stem, or root.

[067] Methods are provided for producing transgenic plants that contain an amount of Lepidoptera inhibitory protein TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL. Such plants can be obtained by introducing a recombinant polynucleotide that encodes any of the proteins provided herein into a plant cell and selecting a plant derived from said plant cell that expresses an amount of Lepidoptera inhibitory proteins. Plants can be obtained from plant cells by regeneration, seed, pollen or meristem transformation methods. Methods for transforming plants are known in the art.

[068] Also disclosed herein are processed plant products, wherein the processed product contains a detectable amount of TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein, an insect-inhibiting segment or fragment thereof, or any distinctive portion thereof. In certain embodiments, the processed product is selected from the group consisting of plant parts, plant biomass, oil, flour, sugar, animal feed, flour, flakes, bran, fluff, husks, treated seeds, and seeds. In certain embodiments, the processed product is non-recyclable. A plant product may include commercial or other commercial products derived from a transgenic plant or part of a transgenic plant, and commercial or other products can be traced through trade links, by detecting nucleotide segments or expressed RNA or proteins that encode or contain distinctive parts of the TIC4472 protein. , TIC4472PL, TIC1425, TIC2613 or TIC2613PL.

[069] Plants expressing TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins can be crossed by selection with transgenic transformants expressing other toxic proteins and/or expressing other transgenic traits such as herbicide resistance genes, genes that confer yield traits, or stress tolerance, etc., or such traits can be combined into one vector so that all traits are linked.

[070] Reference in this application to an "isolated" DNA molecule or amino acid molecule, or an equivalent term or phrase, is intended to mean that the DNA molecule or amino acid molecule is present alone or in combination with other compounds, but not in its natural environment. For example, a DNA molecule or amino acid molecule will be "isolated" within the scope of this disclosure if the element is not found in the genome of the organism or at the locus of the genome in which it occurs naturally. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e. a DNA nucleotide sequence inserted into the genome of plant or bacterial cells, or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present in a plasmid or similar structure, used for cell transformation, in the genome of a plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commercial products derived from a plant or bacterium.

[071] As further described in the Examples, sequences encoding TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins and sequences having a significant percent identity with TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL can be identified using methods known to those skilled in the art. in the art, for example, polymerase chain reaction (PCR), thermal amplification and hybridization. For example, the TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL proteins can be used to generate antibodies that specifically bind to related proteins and can be used to screen and search for other members of the protein class that are related.

[072] In addition, the nucleotide sequences encoding the toxic proteins TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL can be used as probes and primers for screening to identify other members of the class using thermal cycling, or isothermal amplification and hybridization methods. For example, oligonucleotides derived from the sequence shown in SEQ ID NO:3 can be used to determine the presence or absence of the TIC4472PL transgene in a deoxyribonucleic acid sample obtained from a commercial product. Oligonucleotides derived from the sequence shown in SEQ ID NO:7 can be used to determine the presence or absence of the TIC2613PL transgene in a deoxyribonucleic acid sample obtained from a commercial product. Given the sensitivity of some nucleic acid detection methods that use oligonucleotides, it is expected that oligonucleotides derived from the sequences shown in SEQ ID NO:3 or SEQ ID NO:9 can be used to detect the TIC4472PL or TIC2613PL transgene in commercial products obtained from pooled sources where only part of the commercial product is derived from a transgenic plant containing any of the transgenes. It is further recognized that such oligonucleotides can be used to introduce variations in the nucleotide sequence of each SEQ ID NO:3 and SEQ ID NO:9. Such "mutation" oligonucleotides are useful for identifying TIC4472PL and TIC2613PL amino acid sequence variants exhibiting a range of insect inhibitory activity or diverse expression in transgenic plant host cells.

[073] Nucleotide sequence homologues, for example, insecticidal proteins encoded by nucleotide sequences that hybridize to each or any of the sequences disclosed herein under stringent hybridization conditions, are also an embodiment of this invention. The invention also relates to a method for detecting a first nucleotide sequence that hybridizes to a second nucleotide sequence, wherein the first nucleotide sequence (or its reverse complementary sequence) encodes a pesticidal protein or a pesticidal fragment thereof, and hybridizes to the second nucleotide sequence. In such a case, the second nucleotide sequence may be any of the nucleotide sequences shown as SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 under stringent hybridization conditions. Nucleotide coding sequences hybridize with each other under appropriate hybridization conditions, such as stringent hybridization conditions, and the proteins encoded by these nucleotide sequences cross-react with antisera designed against any of such proteins. Stringent hybridization conditions, as defined herein, include at least hybridization at 42°C followed by two washes for five minutes, each at room temperature, with 2X SSC, 0.1% SDS, followed by two washes for thirty minutes each at 65° C. in 0.5X SSC, 0.1% SDS. Washes at even higher temperatures set even more stringent conditions, eg hybridization conditions at 68°C followed by a wash at 68°C in 2xSSC containing 0.1% SDS.

[074] One skilled in the art will appreciate that, due to the redundancy of the genetic code, many other sequences are capable of encoding such related proteins, and such sequences to the extent that they function to express pesticidal proteins either in Bacillus strains or in a plant cell , are embodiments of this invention, recognizing, of course, that many such redundant coding sequences will not hybridize under given conditions to native Bacillus sequences encoding TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL. This application discusses the use of these and other identification methods known to those skilled in the art to identify sequences encoding the TIC4472, TIC1425 and TIC2613 proteins and sequences that have a significant percentage of identity with sequences encoding the TIC4472, TIC1425 and TIC2613 proteins.

[075] This disclosure also provides for the use of molecular methods known in the art to construct and clone commercially useful proteins, including protein chimeras from pesticidal proteins; for example, chimeras can be assembled from TIC4472, TIC4472PL, TIC1425, TIC2613, or TIC2613PL protein segments to provide additional useful embodiments, including combining TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL protein segments with segments of different proteins other than TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL, and related proteins. The TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins can be aligned with each other, and with others from Bacillus thuringiensis or other pesticidal proteins (regardless of whether they are closely or distantly related phylogenetically), and segments of each such can be identified. protein, which is convenient for exchange between aligned proteins, resulting in the construction of chimeric proteins. Such chimeric proteins can be subjected to a bioassay with a pest, and characterized for the presence or absence of increased biological activity or broadening of the spectrum of target pests, compared with the original proteins from which each such segment of the chimera was derived. The pesticidal activity of polypeptides can be further engineered for activity against a particular pest, or a broader range of pests, by replacing domains or segments with other proteins, or using directed evolution techniques known in the art.

[076] Also disclosed in this application are methods for controlling insects, in particular Lepidoptera infestation of cultivated plants, using the TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL proteins. Such methods may include growing a plant containing an insect or lepidoptera inhibitory amount of the toxic protein TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL. In certain embodiments, such methods may further include any one or more of: (i) applying any composition containing or encoding the toxic TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL protein to a plant or seed that gives rise to a plant ; and (ii) transforming a plant or plant cell that produces a plant with a polynucleotide encoding the toxic protein TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL. In general, it is generally contemplated that the toxic protein TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL can be provided in a composition, provided in a microorganism, or provided in a transgenic plant, to confer insect-inhibiting activity against Lepidoptera.

[077] In some embodiments, the recombinant nucleic acid molecule of the toxic TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL toxic proteins is an insecticidally active ingredient of an insect-inhibiting composition obtained by culturing recombinant Bacillus or any other recombinant bacterial cell transformed to express the toxic TIC4472 protein, TIC4472PL, TIC1425, TIC2613 and TIC2613PL, under conditions suitable for the expression of the toxic protein TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL. Such a composition can be obtained by drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of a culture of such recombinant cells expressing/producing said recombinant polypeptide. Such a process may result in the production of a Bacillus or other entomopathogenic bacterial cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet. By providing recombinant polypeptides thus produced, a composition that contains the recombinant polypeptides can contain bacterial cells, bacterial spores, and parasporal inclusion bodies, and can be formulated for a variety of uses, including as insect-inhibiting agricultural aerosols or as formulations that inhibit insects in food bioassays.

[078] In one embodiment, to reduce the likelihood of developing resistance, an insect inhibitory composition comprising TIC4472, TIC4472PL, TIC1425, TIC2613, and TIC2613PL may further comprise at least one additional polypeptide that exhibits insect inhibitory activity against those same Lepidoptera species of insects, but which differs from the toxic protein TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL. Possible additional polypeptides for such a composition include an insect inhibitory protein and an insect inhibitory dsRNA molecule. One example of the use of such ribonucleotide sequences for insect pest control is described in Baum et al. (US Patent Publication 2006/0021087 A1). Such additional Lepidoptera pest control polypeptide may be selected from the group consisting of an insect inhibitory protein such as, but not limited to, Cry1A (US Pat. No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B US No. 10/525318), Cry1C (US patent No. 6033874), Cry1D, Cry1Da and its derivatives, Cry1E, Cry1F, and Cry1A/F chimeras (US patent No. 7070982; 6962705; and 6713063), 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. 7064249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI- 001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 (US Patent Publication 2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI- 112 AXMI-117, AXMI-100 (US Patent Publication 2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (US Patent Publication 2013-0104259 A1), AXMI-134 (US Patent Publication 2013-0167264 A1) , AXMI-150 (US Patent Publication 2010-0160231 A1), AXMI-184 (US Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207, axmi209 (US Patent Publication 2011-0030096 A1), AXMI-218, AXMI-220 (US Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (US Patent Publication 2014-0196175 A1), AXMI-238 ( US Patent Publication 2014-0033363 A1), AXMI-270 (US Patent Publication 2014-0223598 A1), AXMI-345 (US Patent Publication 2014-0373195 A1), AXMI-335 (International Application Publication WO2013/134523(A2)), DIG-3 (US Patent Publication 2013-0219570 A1), DIG-5 US Patent Publication 2010-0317569 A1), DIG-11 (US Patent Publication 2010-0319093 A1), AfIP-1A and its derivatives (US Patent Publication 2014- 0033361 A1), AfIP-1B and its derivatives (US Patent Publication 2 014-0033361 A1), PIP-1APIP-1B (US Patent Publication 2014-0007292 A1), PSEEN3174 (US Patent Publication 2014-0007292 A1), AECGF-592740 (US Patent Publication 2014-0007292 A1), Pput_1063 (US Patent Publication 2014-0007292 A1), DIG-657 (International Application Publication WO2015/195594(A2)), Pput_1064 (US Patent Publication 2014-0007292 A1), GS-135 and its derivatives (US Patent Publication 2012-0233726 A1), GS153 and its derivatives (US Patent Publication 2012-0192310 A1), GS154 and its derivatives (US Patent Publication 2012-0192310 A1), GS155 and its derivatives (US Patent Publication 2012-0192310 A1), SEQ ID NO:2 and its derivatives, as described in US Patent Publication 2012-0167259 A1, SEQ ID NO:2 and derivatives thereof, as described in US Patent Publication 2012-0047606 A1, SEQ ID NO:2 and derivatives thereof, as described in US Patent Publication 2011-0154536 A1, SEQ ID NO:2 and derivatives thereof as described in US Patent Publication 2011-0112013 A1, SEQ ID NO:2 and 4 and derivatives thereof as described in 2010-0192256 A1, SEQ ID NO:2 and derivatives thereof as described in US Patent Publication 2010-0077507 A1, SEQ ID NO:2 and derivatives thereof as described in US Patent Publication 2010-0077508 A1, SEQ ID NO:2 and its derivatives as described in US Patent Publication 2009-0313721 A1, SEQ ID NO:2 or 4, and their derivatives as described in US Patent Publication 2010-0269221 A1, SEQ ID NO:2 and its derivatives, as described in US Pat. -0117690 A1; SEQ ID NO:2 and its derivatives, as described in US7510878(B2), SEQ ID NO:2 and its derivatives, as described in US patent No. 7812129(B1); etc.

[079] In other embodiments, such a composition/formulation may further comprise at least one additional polypeptide that exhibits insect-inhibiting activity against insects that are not inhibited by another insect-inhibiting protein of the present invention to broaden the resulting inhibition spectrum. insects. For example, to control arthropod pests, combinations of insect inhibitory proteins of the present invention can be used with anti-arthropod proteins such as TIC1415 (U.S. Patent Publication 2013-0097735 A1), TIC807 (U.S. Patent No. 2013-0269060 A1), AXMI-036 (US Patent Publication 2010-0137216 A1), and AXMI-171 (US Patent Publication 2013-0055469 A1). Additionally, the hemipteran pest control polypeptide can be selected from the group consisting of an insect inhibitory protein such as, but not limited to: Cry3Bb (US Pat. No. 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34/ 35, 5307, AXMI134 (US Patent Publication 2013-0167264 A1) AXMI-184 (US Patent Publication 2010-0004176 A1), AXMI-205 (US Patent Publication 2014-0298538 A1), axmi207 (US Patent Publication 2013-0303440 A1) , AXMI-218, AXMI-220 (U.S. Patent Pub 20140245491A1), AXMI-221z, AXMI-223z (U.S. Patent Pub 2014-0196175 A1), AXMI-279 (U.S. Patent Pub 2014-0223599 A1), AXMI-R1 and its variants (US Patent Publication 2010-0197592 A1, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (US Patent Publication 2010-0319092 A1), eHIPs (US Patent Application Publication No. 2010/0017914 ), IP3 and variants thereof (US Patent Publication 2012-0210462 A1), and ϖ-Hexatoxin-Hv1a (US Patent Application Publication US2014-0366227 A1).

[080] Additional polypeptides for controlling insect pests from Coleoptera, Lepidoptera, Arthrostomes can be found on the Bacillus thuringiensis toxin classification website maintained by Neil Crickmore (on the World Wide Web at btnomenclature.info).

[081] Insect resistance to certain insecticides has been documented in the art. One strategy for managing insect resistance is to use transgenic crops that express two different insect inhibitory agents that act in different ways. Therefore, any insect that is resistant to one of the insect inhibitory agents can be controlled by the other insect inhibitory agent. Another insect resistance management strategy involves the use of plants that are not protected against the target Lepidoptera pest species, providing a protected site for such exposed plants. One specific example is described in US Pat. No. 6,551,962, which is incorporated by reference in its entirety.

[082] Other embodiments, such as topical pesticidal chemical agents that are designed to control pests that are also controlled by the proteins disclosed herein, should be used with the proteins in seed treatment, spray, water, or wettings can be applied directly to the soil (ground irrigation), used to grow plants expressing the proteins disclosed herein, or prepared for application to seeds containing one or more transgenes encoding one or more of the disclosed proteins. Such formulations for use in seed treatment can be applied with various adhesives and tackifiers known in the art. Such formulations may contain pesticides that are synergistic in mode of action with the disclosed proteins, provided that the pesticide formulation acts differently to control the same or similar pests that can be controlled by the disclosed proteins, or provided the pesticides act to control pests within a wider range of hosts or plant pest species that are not effectively controlled by the pesticidal proteins TIC4472, TIC4472PL, TIC1425, TIC2613 and TIC2613PL.

[083] The above composition/formulation may further comprise an agriculturally acceptable carrier, such as feed, powder, dust, pellets, granules, spray, emulsion, colloidal suspension, aqueous solution, Bacillus spore/crystal preparation, seed fertilizer, a recombinant plant cell/plant tissue/seed/plant transformed to express one or more proteins, or a bacterium transformed to express one or more proteins. Depending on the level of insect inhibition or insecticidal inhibition inherent in the recombinant polypeptide and the amount of drug applied to the plant or nutritional analysis, the composition/formulation may contain various mass amounts of the recombinant polypeptide, for example, from 0.0001% to 0.001% to 0 01% to 1% to 99% by weight of the recombinant polypeptide.

[084] In view of the foregoing, those skilled in the art will appreciate that changes can be made to the specific aspects that are disclosed and still achieve a similar or similar result without deviating from the spirit and scope of the invention. Thus, the specific structural and functional details disclosed herein should not be interpreted as limiting. It should be understood that the full disclosure of each source cited in this document is included in the disclosure of this application.

EXAMPLES

Example 1

Detection, cloning and expression of TIC4472, TIC1425 and TIC2613

[085] The sequences encoding three new Bacillus thuringiensis pesticidal proteins have been identified, cloned, validated and tested in insect bioassays. Pesticide proteins TIC4472, TIC1425, and TIC2613 isolated from Bacillus thuringiensis strains EG10742, EG10731, and EG5408, respectively, represent new Cry1Ca-like proteins.

[086] Polymerase chain reaction (PCR) primers were designed to amplify a complete copy of the coding region for TIC4472, TIC1425, and TIC2613 from total genomic DNA isolated from Bacillus thuringiensis strains EG10742, EG10731, and EG5408, respectively. The PCR amplicons also contained translation initiation and translation termination codons for each coding sequence.

[087] Each of the amplicons was cloned using methods known in the art into Bt ( Bacillus thuringiensis ) expression vectors operably linked to an expressed Bt promoter.

Example 2

TIC4472, TIC1425 and TIC2613 show activity against Lepidoptera in an insect bioassay

[088] The biological activity of the pesticide proteins TIC4472, TIC1425 and TIC2613 was evaluated by protein production in a Bt -expressing host. The Bt strain expressing TIC4472, TIC1425, and TIC2613 was grown for twenty-four (24) hours, and then either a crystal spore preparation or a solubilized protein preparation was added to insect food. Mortality and stunting were assessed by comparing the growth and development of insects on a diet with a culture from a Bt strain expressing TIC4472, TIC1425 and TIC2613 with insects on a diet with an untreated control culture.

[089] TIC4472 preparations were analyzed against Lepidoptera species - Helicoverpa zea (two colonies (CEW and CEWUC)), American cotton cutworm ( CLW, Alabama argillacea), Corn moth (ECB, Ostrinia nubilalis ), Corn fall armyworm (FAW, Spodoptera frugiperda ), Chrysodeixis includens (SBL), Spodoptera eridania (SAW), Diatraea grandiosella (SWCB), Diatraea saccharalis (SCB), Cry2Ab resistant colony Diatraea saccharalis (SCB2R), Heliothis virescens (TBW), and Anticarsia gemmatalis (VBW); Coleoptera species - Colorado potato beetle (CPB, Leptinotarsa decemlineata ) and Diabrotica virgifera virgifera (WCB); arthroprobosmic species - Lygus lineolaris (TPB), Lygus hesperus (WTP); and the Diptera species, the yellow fever mosquito ( Aedes aegypti ). TIC1425 preparations were analyzed against species of Lepidoptera - ipsilon cutworm (BCW, Agrotis ipsilon ), American cotton cutworm (CLW, Alabama argillacea), corn moth (ECB, Ostrinia nubilalis ), fall armyworm (FAW, Spodoptera frugiperda ), Agrotis ipsilon ( BCW), Diatraea grandiosella (SWCB), and Diatraea saccharalis (SCB); and beetle species - Diabrotica virgifera virgifera (WCB) and Diabrotica undecimpunctata howardi. TIC2613 preparations were analyzed against Lepidoptera species - Helicoverpa zea (two colonies (CEW and CEWUC)), American cotton bollworm (CLW, Alabama argillacea), Corn moth (ECB, Ostrinia nubilalis ), Fall armyworm (FAW, Spodoptera frugiperda ), Chrysodeixis includens (SBL), Spodoptera eridania (SAW), epsilon cutworms (BCW, Agrotis ipsilon ), Diatraea grandiosella (SWCB), Heliothis virescens (TBW), and Anticarsia gemmatalis (VBW); beetle species - Colorado potato beetle (CPB, Leptinotarsa decemlineata ), Diabrotica virgifera virgifera (WCB) and Diabrotica undecimpunctata howardi ; and species of arthropods - Lygus lineolaris (TPB), Lygus hesperus (WTP).

[090] The bioassay activity observed for each protein grown in the Bt host is shown in Tables 3 and 4 below, with "+" indicating activity, "NT" indicating that the toxin was not assayed against that particular insect pest, "S" stands for stunting, and "M" stands for mortality. TIC4472, TIC1425, and TIC2613 preparations showed no activity against beetles and arthropods analyzed for each protein. TIC4472 has also shown activity against the yellow fever mosquito ( Aedes aegypti ). All three toxins showed resistance to numerous Lepidoptera insect pests, as shown in Tables 3 and 4.

Table 3. Bioassay of activity of TIC4472, TIC1425 and TIC2613 against insect pests.

CEW CEWUC CLW ECB FAW SBL SAW Toxin S M S M S M S M S M S M S M TIC4472 + + + + + + + + + + + + + TIC1425 NT NT + + + + NT NT TIC2613 + + + + + + + + + +

Table 4. Bioassay of activity of TIC4472, TIC1425 and TIC2613 against insect pests.

BCW SCB SCB2R SWCB TBW VBC YFM Toxin S M S M S M S M S M S M S M TIC4472 NT NT + + + + + + + + + + + TIC1425 + NT NT + NT NT NT NT NT NT TIC2613 + + NT NT + + + + NT NT

[091] As can be seen from Tables 3 and 4 above, insecticide TIC4472 demonstrated activity against all Lepidoptera pests analyzed (CEW, CEWUC, CLW, ECB, FAW, SBL, SAW, SCB, SCB2R, SWCB, TBW, and VBC); as well as YFM. The insecticide TIC1425 has shown activity against CLW, ECB, FAW, SCB and SWCB. The insecticide TIC2613 has shown activity against CEW, CLW, ECB, FAW, SBL, SAW, SCB, SWCB, TBW and VBC. No activity was observed for TIC1425 and TIC2613 when assayed against BCW.

[092] In a separate set of experiments, TIC4472 protein preparations were analyzed, using an overlapping food assay, against lepidopteran insect pests - Spodoptera exigua (BAW), Pectinophora gossypiella (PBW), Cry1Ac resistant Pectinophora gossypiella (PBW_Cry1Ac ^r ), cotton bollworm ( OWB, Helicoverpa armigera ), Spodoptera litura (OLW), and Earias vittella (SBW, Earias vittella ). Table 5 shows the activity observed against each of these Lepidoptera pests analyzed in an overlapping food bioassay, where "+" denotes activity.

Table 5. Bioassay of TIC4472 activity against Lepidoptera insect pests.

Lepidoptera insect pests BAW PBW PBW_Cry1Ac ^r OWB OLW SBW + + + + + +

[093] As can be seen from Table 5 above, TIC4472 showed activity against all lepidopteran insect pests analyzed in the overlapping diet bioassay, including a Cry1Ac resistant Pectinophora gossypiella colony.

[094] As shown in Tables 3-5, TIC4472, TIC1425 and TIC2613 show activity against a wide range of Lepidoptera insect pests.

Example 3

Development of synthetic coding sequences encoding TIC4472PL and TIC2613PL for expression in plant cells

[095] Synthetic coding sequences were designed for use in expression of the encoded protein in plants, cloned into binary plant transformation vectors, and used to transform plant cells. Synthetic sequences were synthesized according to methods generally described in US Pat. No. 5,500,365, avoiding certain problematic sequences such as plant sequences rich in ATTTA and A/T. The synthetic coding sequences encode the TIC4472PL and TIC2613PL proteins, which contain an additional alanine residue immediately following the start methionine relative to the TIC4472 and TIC2613 proteins, respectively. An additional alanine residue is included in the synthetic coding sequence to improve the expression of the insect-toxic protein.

[096] The synthetic coding sequence encoding TIC4472PL (SEQ ID NO:4) is presented herein as SEQ ID NO:3. The synthetic coding sequence encoding TIC2613PL (SEQ ID NO:10) is shown as SEQ ID NO:9. The synthetic coding sequences were used in binary plant transformation vectors to generate transgenic plants expressing the TIC4472PL and TIC2613PL proteins and analyzed for activity against Lepidoptera pests.

Example 4

Analysis of TIC4472PL Activity Against Lepidoptera Pests in Stably Transformed Cotton Plants

[097] Binary plant transformation vectors containing transgenic cassettes for expressing the untargeted pesticidal TIC4472PL protein were cloned using methods known in the art. The resulting vectors were used for stable transformation of cotton plants. Tissues were harvested from transformants and used in insect bioassays against various Lepidoptera pests.

[098] Synthetic coding sequences were designed for use in plant expression of the encoded protein, cloned into a binary plant transformation vector, and used to transform cotton plant cells. The resulting plant transformation vectors contained the first transgenic cassette for the expression of the pesticide protein TIC4472PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to a synthetic coding sequence encoding the untargeted TIC4472PL protein, which, in turn, was operably linked at 5' to 3'UTR; and a second transgenic cassette for selecting transformed plant cells using selectinomycin.

[099] Three binary plant transformation vectors were constructed as described above and contained different constitutive promoters. Two constructs, Construct 1 and Construct 3, also contained an intron sequence that was operably linked 3' to the leader sequence and 5' to the synthetic coding sequence encoding the untargeted TIC4472PL protein. Construct 2 was without introns. Cotton plants were transformed with three different binary transformation vectors using the Agrobacterium -mediated transformation method. Transformed cells were induced to form plants using methods known in the art. Bioassays using plant leaf discs were performed similarly to those described in US Pat. No. 8,344,207. One freshly hatched larvae, up to one day old, was placed on each leaf disc sample and allowed to feed for about four days. An untransformed cotton plant was used to obtain tissue, which was used as a negative control. _R0 transformants with multiple transformations with one copy of each binary vector insert were evaluated against Helicoverpa zea (CBW), fall armyworm (FAW, Spodoptera frugiperda ), Chrysodeixis includens (SBL), and Heliothis virescens (TBW). The transformant was scored with a Leaf Damage Rating (LDR) score ranging from one (1) to four (4). The LDR scores are based on the percentage of damage observed for each leaf disk. Table 6 below shows the LDR scores and the corresponding percentage damage range associated with each score. For an LDR score of one (1), the penetrance of the feature is also assessed. High penetrance (as denoted by "(H)") is defined as more than fifty (50) percent of the evaluated transformants for each construct having leaf damage less than or equal to ten (10) percent. Low penetrance (as denoted by "(L)") is defined as no more than fifty (50) percent of the evaluated transformants for each construct having leaf damage less than or equal to ten (10) percent. Penetrance does not apply to LDR scores greater than one (1).

Table 6. Leaf Damage Rating (LDR) scores for cotton, and corresponding damage percentage and penetrance.

LDR score Leaf damage rating (LDR) range Penetrance one LDR < 10% High => 50% transformants one Low=< 50% transformants 2 10% < LDR < 17.5% n/a 3 17.5% < LDR < 35% n/a four LDR > 35% n/a

[0100] Leaf damage scores for transformed R ₀ cotton plants expressing TIC4472PL are shown in Table 7 below. The number of transformants showing an LDR score out of the total number of transformants analyzed is shown in brackets, followed by the penetrance for those LDR scores equal to one (1).

Table 7. Leaf Damage Rating (LDR) Scores, Number of Transformants Showing LDR and Penetrance for Transformed R Plants ₀ cotton expressing TIC4472PL.

Design CBW FAW SBL TBW Design 1 3 (1/25) 2 (2/25) 1 (25/25)H 1 (24/25)H Construction 2 3 (3/25) 2 (1/25) 1 (24/25)H 1 (16/25)H Design 3 4 (23/23) 1 (1/23) L 1 (21/23) H 1 (6/23) L

[0101] Transformed _R0 cotton plants expressing TIC4472PL were highly effective (defined as having less than or exactly ten percent leaf damage) against SBL and TBW, as shown in Table 7. Activity against CBW and FAW was also observed in several transformants.

[0102] Cotton R ₁ transformants were selected from the transformed R ₀ cotton plants evaluated above and used in the leaf disc assay against FAW, SBL and TBW. Table 8 shows the leaf damage scores for transformed R ₁ cotton plants expressing TIC4472PL.

Table 8. Leaf Damage Rating (LDR) Scores, Number of Transformants Showing LDR and Penetrance for Transformed R Plants _one cotton expressing TIC4472PL.

Design FAW SBL TBW Design 1 1 (3/4) H 1 (4/4)H 1 (4/4) H Construction 2 1 (1/4) L 1 (4/4) H 1 (1/4) L

[0103] As can be seen from Table 8 above, the selected transformants showed high efficiency against FAW, SBL and TBW. Penetrance was high for construct 1 transformants for all three insect pests. With regard to the transformants transformed with Construct 2, the level of penetrance was high for SBL.

[0104] The above demonstrates that transformed cotton plants expressing the TIC4472PL protein exhibit resistance to fall armyworm (FAW, Spodoptera frugiperda ), Chrysodeixis includens (SBL), and Heliothis virescens (TBW).

Example 5

Analysis of the Lepidoptera Pest Activity of TIC4472PL and TIC2613PL in Stably Transformed Soybean Plants

[0105] Binary plant transformation vectors containing a transgenic cassette designed to express plastid-targeted pesticidal protein TIC4472PL or non-targeted TIC2613PL were cloned using methods known in the art. The resulting vector was used for stable transformation of soybean plants. Tissues were harvested from transformants and used in insect bioassays against various Lepidoptera pests.

[0106] The synthetic coding sequences designed for expression in plants described in Example 3 were cloned into plant transformation binary vector constructs and used to transform soybean plant cells. Three binary vector constructs were constructed using methods known in the art to express plastid-targeted and untargeted TIC4472PL. Construct 4 contained the first transgenic cassette for the expression of untargeted TIC4472PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to a synthetic coding sequence encoding the untargeted TIC4472PL protein, which in turn was operably linked 5' from 3'UTR and; a second transgenic cassette for selection of transformed plant cells using selectinomycin. Construct 5 contained the first transgenic cassette for the expression of targeted TIC4472PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to an intron operably linked 5' to a synthetic coding sequence encoding plastid-targeted protein TIC4472PL, which in turn was operably linked at 5' to 3' UTR and; a second transgenic cassette for selection of transformed plant cells using selectinomycin. Construct 6 contained the first transgenic cassette for the expression of untargeted TIC4472PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to an intron operably linked 5' to a synthetic coding sequence encoding the untargeted TIC4472PL protein, which in turn was operably linked at 5' to 3' UTR and; a second transgenic cassette for selection of transformed plant cells using selectinomycin.

[0107] Two binary vector constructs were constructed using methods known in the art to express untargeted TIC2613PL. Construct 7 contained the first transgene cassette for the expression of untargeted TIC2613PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to a synthetic coding sequence encoding the untargeted TIC2613PL protein, which in turn was operably linked 5' from 3'UTR and; a second transgenic cassette for selection of transformed plant cells using selectinomycin. Construct 8 contained the first transgenic cassette for the expression of untargeted TIC2613PL, which contained a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to an intron operably linked 5' to a synthetic coding sequence encoding the untargeted TIC2613PL protein, which in turn was operably linked at 5' to 3' UTR and; a second transgenic cassette for selection of transformed plant cells using selectinomycin.

[0108] The resulting binary transformation vector constructs were used to transform soybean cells using an Agrobacterium -mediated transformation method. Transformed soybean cells were induced to form plants using methods known in the art. Bioassays using plant leaf disks were performed similarly to those described in US Pat. No. 8,344,207. An untransformed soybean plant was used to obtain tissue, which was used as a negative control. Soybean multiple R ₀ transformants obtained with binary vectors were evaluated against Spodoptera eridania (SAW), Chrysodeixis includens (SBL), Helicoverpa zea (SPW). Leaf Damage Rating (LDR) scores were similar to those for cotton, but differed in the percentage range of damage used to determine the score. The leaf damage rating scores and their respective percentage leaf damage rating ranges are shown in Table 9 below.

Table 9. Soybean Leaf Damage Rating (LDR) scores and corresponding damage percentage range.

LDR score Leaf Damage Rating (LDR) Range one LDR ≤ 20% 2 20% < LDR ≤ 35% 3 35% < LDR ≤ 70% four LDR > 70%

[0109] Leaf injury scores for transformed R ₀ cotton plants expressing TIC4472PL and TIC2613PL are shown in Table 10 below. The number of transformants showing an LDR score out of the total number of transformants evaluated is shown in brackets.

Table 10. Leaf Damage Rating (LDR) Scores and Number of Transformants Showing LDR for Transformed Plants R ₀ soybeans expressing TIC4472PL and TIC2613PL.

Toxin Design SAW SBL SPW TIC4472PL Design 4 1 (23/29) 1 (29/29) 3 (8/29) TIC4472PL Design 5 1 (20/29) 1 (28/29) 1 (1/29) TIC4472PL Design 6 1 (10/29) 1 (15/15) 3 (3/15) TIC2613PL Design 7 1 (22/30) 1 (24/30) 3 (1/30) TIC2613PL Design 8 1 (14/25) 1 (22/25) 2 (2/25)

[0110] As can be seen from Table 10 above, the expression of both TIC4472PL and TIC2613PL showed high efficacy against SAW and SBL. The activity was lower for both proteins against SPW.

[0111] Soybean R ₁ transformants were selected from transformed soybean R ₀ plants expressing TIC4472PL and TIC2613PL evaluated above and used in leaf disc assays against SAW, SBL, and SPW. Soybean transformants R ₁ expressing TIC4472PL were also evaluated against Anticarsia gemmatalis (VBW). Table 11 shows the leaf damage scores for transformed soybean R ₁ plants expressing TIC4472PL and TIC2613PL.

Table 11. Leaf Damage Rating (LDR) scores and number of transformants showing LDR for transformed plants R _one soybeans expressing TIC4472PL and TIC2613PL.

Toxin Design SAW SBL SPW VBC TIC4472PL Design 5 1 (3/8) 1 (5/8) 3 (2/8) TIC4472PL Design 6 1 (1/9) 1 (6/9) 3 (1/9) 1 (3/9) TIC2613PL Design 8 2 (2/10) 1 (9/10) 4 (4/10)

[0112] As can be seen from Table 11, a number of soybean R ₁ transformants transformed with TIC4472PL showed high efficacy against SAW and SBL. In addition, several soybean R ₁ transformants transformed using Construct 6 showed high efficacy against VBC. Soybean R ₁ transformants transformed with TIC2613PL showed high efficacy against SBL.

[0113] The foregoing demonstrates that transformed soybean plants expressing TIC4472PL or TIC2613PL show resistance to Lepidoptera, specifically Spodoptera eridania and Chrysodeixis includens , and Anticarsia gemmatalis.

Example 6

Analysis of Lepidoptera Pest Activity of TIC4472PL and TIC2613PL in Stably Transformed Corn Plants

[0114] Binary plant transformation vectors containing transgenic cassettes designed to express the plastid-targeted and non-targeted pesticide protein TIC4472PL or TIC2613PL were cloned using methods known in the art. The resulting vectors are used to stably transform maize plants. Tissues are harvested from transformants and used in insect bioassays against various Lepidoptera pests.

[0115] The synthetic coding sequence encoding the TIC4472PL protein (SEQ ID NO:3) or TIC2613 (SEQ ID NO:9) is cloned into binary vectors for transformation. For a plastid-targeted protein, the synthetic sequence encoding the pesticide protein TIC4472PL or TIC2613PL is operably linked in frame to the sequence encoding the chloroplast-targeting signal peptide. The resulting vectors for plant transformation contain the first transgenic cassette for the expression of the pesticide protein TIC4472PL or TIC2613PL, which contains a constitutive promoter operably linked 5' to a leader sequence operably linked 5' to an intron operably linked 5' to a synthetic coding sequence encoding plastid-targeted and untargeted protein TIC4472PL or TIC2613PL, which in turn is operably linked 5' to 3'UTR; and a second transgenic cassette for selecting transformed plant cells using selectinomycin.

[0116] Maize plants are transformed with the binary transformation vectors described above using the Agrobacterium -mediated transformation method. Transformed cells are induced to form plants by methods known in the art. Bioassays using plant leaf discs were performed similarly to those described in US Pat. No. 8,344,207. An untransformed corn plant was used to obtain tissue to be used as a negative control. Transformants with multiple transformations of each binary vector are evaluated against Spodoptera exigua (BAW), epsilon cutworm (BCW, Agrotis ipsilon ), Helicoverpa zea (CEW), American cotton cutworm (CLW, Alabama argillacea), corn moth (ECB, Ostrinia nubilalis ), fall armyworm (FAW, Spodoptera frugiperda ), cotton bollworm (OWB, Helicoverpa armigera ), Spodoptera litura (OLW), Pectinophora gossypiella (PBW), Chrysodeixis includens (SBL), Earias vittella (SBW), Diatraea grandiosella (SWCB), Diatraea saccharalis (SCB), Heliothis virescens (TBW), and Anticarsia gemmatalis (VBW), as well as other Lepidoptera pests.

[0117] Insect death and stunting caused by ingestion of provided leaf discs expressing TIC4472PL or TIC2613PL is noted and compared to leaf discs derived from non-transformed corn plants.

[0118] All compositions disclosed and claimed herein can be prepared and used without undue experimentation in view of this disclosure. Although the compositions of this invention have been described in terms of the above illustrative embodiments, it should be apparent to those skilled in the art that variations, changes, modifications, and edits may be made to the composition described herein without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such analogous substituents and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined in the appended claims.

[0119] All publications and published patent documents cited in the description are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated for inclusion by reference.

--->

SEQUENCE LIST

<110> Monsanto Technology LLC

Bowen, David J

Chay, Catherine A

Howe, Arlene R

Kesanapalli, Uma

<120> NOVEL INSECT INHIBITORY PROTEINS

<130> MONS:426WO

<140> US 62/406082

<141> 2016-10-10

<160> 10

<170>PatentIn version 3.5

<210> 1

<211> 3564

<212> DNA

<213> Bacillus thuringienses

<220>

<221> misc_feature

<222> (1)..(3564)

<223> Nucleic acid sequence encoding a TIC4472 pesticidal protein

obtained from Bacillus thuringiensis species EG10742.

<400> 1

atgaataata atattgaaaa ccaatgcgta ccttacaatt gtttaagtaa tcctgaagaa 60

gtaattttgg atggagaacg gatatcaact ggtaattcat caattgatat ttctctgtca 120

cttgttcaac ttctggtatc taactttgta ccaggcggag gatttttagt agggttaata 180

gattttgtat ggggaatagt aggcccttct ccatgggatg catttctagt gcaaattgaa 240

caattaattc agcaaagaat agaagcatat gctagggctg cagcaatttc taatttagaa 300

ggaataggaa acaatttcaa tatatatgtg gaagcatttc aagaatggga agaagatcct 360

aataatccag caaccaggaa tagagtagtt gatcgctttc gtatacttga tgggctactt 420

gaaagggaca ttccttcgtt tcgaatttct ggatttgaag tccccctttt atccgtttat 480

gctcaagcgg ccaatctgca tctagctata ttaagagatt ctgtaatttt tggagaaaga 540

tggggattga caacaacaaa tgtcaatgaa aactataata gacaaatcag gcatattgat 600

gaatatgctg atcactgtgc aaatacgtat aatcggggat taaataattt accgaaatct 660

acgtatcaag attggataac atataatcga ttacggagag aattaacatt gactgtatta 720

gatatcgctg ctttctttcc aaactatgac aataggcggt atccaattca gccagttggt 780

caactaacaa gggaagttta tacggaccca ttaattactt ttaatcccca gttacagtct 840

gtagctcaat tacctacttt taacgttatg gaaagcaacg caattagaaa tcctcatttg 900

tttgatatat tgaataatct tacaattttt acggattggt ttagtgttgg acgcaacttt 960

tattggggag gacatcgagt aacttctaac tatataggag gaggcaacat aacatctcct 1020

atatatggaa gagaggcgaa ccaggagcct ccaagatctt ttacttttaa tggacctgtt 1080

tttaggactt tatcaaatcc tactttacga ttattacagc aaccttggcc agcaccacca 1140

tttaatttac gtggtgttga aggagtagaa ttttctacac ctacaaaatag ctttacgtat 1200

cgaggaagag gtacagttga ttctttaacc gaattaccgc ctgaggataa tagtgtgcta 1260

cctcgcgagg gatatagtca tcgtttatgt catgcaactt ttgttcaaag atctggaaca 1320

ccatttttaa caacgggtgt agtattttct tggacgcatc gcagtgcaac tcttacaaat 1380

acaattgatc cagacaaaat tactcagata cctttagtga aaggatttag agtttggagt 1440

ggcgcctctg tcgttacagg accaggtttt acaggagggg atatccttcg aagaactaac 1500

tttggggatt ttgtatctat gcaagttaat attaattcac caataacaca aagataccgt 1560

1620

1680

1740

agagctaatc cagatataat tcgtataaat gaacaaccac tattcggtgc aggctctatt 1800

agtagtggtg aactttatat agataaaatt gaaattattc tagcagatgc aacatttgaa 1860

gcagaatatg atttggaaag agcacagaag gcggtgaatg cgctgtttac ttctacaaac 1920

1980

gagtgtttat cggatgaatt ttgtctggat gaaaaacgag aattgtccga gaaagtcaaa 2040

catgcgaagc gactcagtga tgagcgaaat ttactccagg atcgaaattt cacatccatt 2100

2160

gacgtattca aagagaatta cgtcacacta ctgggtacct ttgatgagtg ctatccaacg 2220

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga 2280

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacac 2340

gaaacagtaa atgtgccagg tacaggttcc ttatggccgc tttcagtcga aagtccaatc 2400

ggaaagtgtg gagaaccgaa tcgatgcaca ccacaccttg aatggaatcc taatctagat 2460

tgttcctgca gagacgggaa aacatgtgca catcattctc atcatttctc cttggacatt 2520

gatgttggat gtacagactt aaatgaagat ctaggtgtat gggtgatatt caagattaag 2580

acgcaagatg gtcatgcaag actaggaaat ctagagtttc tcgaagagaa accattagta 2640

ggagaagcgt tagctcgtgt gaaaagagcg gagaaaaaat ggagagacaa acgcgaaaaa 2700

ttgcaattag aaacaaatat cgtttacaaa gaggcaaaag aagctgtgga tgctttattt 2760

gtaaactctc aatatgatag attacaagtg gatacgaaca ttgccatgat tcatgcggca 2820

2880

gtcaatgcgg ctattttcga agaattagaa gggtgtgttt tcactgcatt ctccctatat 2940

gatgcgagaa atgtcattaa aaatggcgat tttaataatg gcttatcatg ctggaacgtg 3000

aaagggcatg tagaagaaca aaacgaccat cgttcggtcc ttgttgtccc ggaatgggaa 3060

cacaagaggt tcgtgtctgt ccaggtcgtg gctatatcct tcgtgttaca 3120

gcatacaaag agggatatgg agaaggctgt gtaaccattc atgggatcga gaacaataca 3180

gacgaactga agtttagcaa ctgcgtagaa gaggaagtct atccaaacaa cacggtaacg 3240

tgtaatgatt atcctgcaaa tcaagaagaa tacgggggtg cgtacacttc tcgtaatcgt 3300

ggatatgacg aaacttatgg aagcaattct tccgtatcag ctgattatgc gtcagtttat 3360

gaagaaaaag cgtatacaga tggacgaaga gacaatccat gtgaatttaa cagagggtat 3420

ggggattata cgccactacc agctggctat gtaacaaaag aattagaata cttcccagaa 3480

3540

gaattactcc ttatggagga atag 3564

<210> 2

<211> 1187

<212> PRT

<213> Bacillus thuringienses

<220>

<221> MISC_FEATURE

<222> (1)..(1187)

<223> The amino acid sequence of the TIC4472 pesticidal protein.

<400> 2

Met Asn Asn Asn Ile Glu Asn Gln Cys Val Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Ile Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn

20 25 30

Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Leu Leu Val Ser Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp

50 55 60

Gly Ile Val Gly Pro Ser Pro Trp Asp Ala Phe Leu Val Gln Ile Glu

65 70 75 80

Gln Leu Ile Gln Gln Arg Ile Glu Ala Tyr Ala Arg Ala Ala Ala Ile

85 90 95

Ser Asn Leu Glu Gly Ile Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala

100 105 110

Phe Gln Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Asn Arg

115 120 125

Val Val Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile

130 135 140

Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Glu Asn Tyr

180 185 190

Asn Arg Gln Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn

195 200 205

Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp

210 215 220

Trp Ile Thr Tyr Asn Arg Leu Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile

245 250 255

Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile

260 265 270

Thr Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn

275 280 285

Val Met Glu Ser Asn Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu

290 295 300

Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe

305 310 315 320

Tyr Trp Gly Gly His Arg Val Thr Ser Asn Tyr Ile Gly Gly Gly Asn

325 330 335

Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg

340 345 350

Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr

355 360 365

Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg

370 375 380

Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr

385 390 395 400

Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp

405 410 415

Asn Ser Val Leu Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala

420 425 430

Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val

435 440 445

Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro

450 455 460

Asp Lys Ile Thr Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Ser

465 470 475 480

Gly Ala Ser Val Val Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu

485 490 495

Arg Arg Thr Asn Phe Gly Asp Phe Val Ser Met Gln Val Asn Ile Asn

500 505 510

Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser

515 520 525

Arg Asp Ala Arg Leu Thr Val Ala Thr Gly Ala Ala Asn Thr Gly Val

530 535 540

Gly Gly Gln Ile Ser Val Asp Met Ala Leu Gln Lys Thr Met Glu Ile

545 550 555 560

Gly Glu Ser Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn

565 570 575

Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Arg Ile Asn Glu Gln

580 585 590

Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp

595 600 605

Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Tyr Asp

610 615 620

Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn

625 630 635 640

Gln Arg Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val

645 650 655

Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys

660 665 670

Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu

675 680 685

Arg Asn Leu Leu Gln Asp Arg Asn Phe Thr Ser Ile Asn Gly Gln Leu

690 695 700

Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp

705 710 715 720

Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp Glu

725 730 735

Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys

740 745 750

Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp

755 760 765

Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn

770 775 780

Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile

785 790 795 800

Gly Lys Cys Gly Glu Pro Asn Arg Cys Thr Pro His Leu Glu Trp Asn

805 810 815

Pro Asn Leu Asp Cys Ser Cys Arg Asp Gly Lys Thr Cys Ala His His

820 825 830

Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn

835 840 845

Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly

850 855 860

His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val

865 870 875 880

Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp

885 890 895

Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala

900 905 910

Lys Glu Ala Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu

915 920 925

Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val

930 935 940

His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly

945 950 955 960

Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Cys Val Phe Thr Ala

965 970 975

Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn

980 985 990

Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Glu Glu Gln Asn

995 1000 1005

Asp His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val

1010 1015 1020

Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg

1025 1030 1035

Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile

1040 1045 1050

His Gly Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys

1055 1060 1065

Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp

1070 1075 1080

Tyr Pro Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg

1085 1090 1095

Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Ser

1100 1105 1110

Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ala Tyr Thr Asp Gly

1115 1120 1125

Arg Arg Asp Asn Pro Cys Glu Phe Asn Arg Gly Tyr Gly Asp Tyr

1130 1135 1140

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe

1145 1150 1155

Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly

1160 1165 1170

Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1175 1180 1185

<210> 3

<211> 3567

<212> DNA

<213> Artificial

<220>

<223> A synthetic coding sequence encoding a TIC4472PL pesticidal

protein designed for expression in a plant cell where an

additional alanine codon is inserted immediately following the

initiating methionine codon.

<400> 3

atggctaata acaacatcga gaaccagtgc gtgccctaca actgcctttc gaacccggag 60

gaagtgatcc tggacggcga aaggatctcg accgggaata gcagcatcga catctcgctt 120

tcgctcgtgc agcttctagt cagtaacttc gttccgggcg gagggtttct cgtgggcctt 180

attgacttcg tttggggcat cgtgggccca tctccttggg acgcattcct cgtgcagatc 240

gagcaactga tccagcagcg tatcgaggcg tacgctaggg ctgccgctat ctccaacctg 300

gagggcatcg gcaacaactt caacatctac gttgaagcct tccaagaatg ggaggaagat 360

cctaataacc cagctacgcg gaacagagtg gtggatcgct ttagaatcct cgacggcctc 420

ctggaaaggg acatcccgag cttccgtatt tccggcttcg aggtgccgct gctgagcgtg 480

tacgcgcaag cggccaatct gcacctggcg attctccggg actctgtgat cttcggcgag 540

cggtggggcc tcaccaccac taacgtgaac gagaactaca accgccagat ccgccacatc 600

660 gacgagtacg cggaccactg cgccaacaca

agcacttacc aagattggat cacctacaac aggctccgcc gggaactcac tctcacagtc 720

ctcgacatcg ctgccttctt cccgaactac gacaaccgcc gctacccgat tcagccagtc 780

ggccagctca cccgtgaggt gtacaccgat ccactgataa ctttcaatcc gcagctccag 840

tctgtcgcac agttgcccac cttcaacgtc atggaaagca acgccatcag gaacccacac 900

ttgttcgaca tccttaacaa cctgactatc ttcaccgact ggttcagcgt cggacggaac 960

ttctactggg gcggacaccg cgtcacctca aactacatcg gcggcggcaa cattacttcg 1020

cccatctacg gccgggaggc gaatcaggag ccgccacgca gctttacatt caacggtcct 1080

gtgtttcgca cgttatcgaa cccgacactc aggctgctcc agcagccctg gcctgcgccg 1140

ccgtttaatt tgcgcggcgt cgaaggcgtc gagttcagta cgccgaccaa cagcttcacc 1200

tatcgcggac gcgggactgt tgactccctg acagagctgc cgccggagga caactcggtt 1260

ctgccgcgtg agggctacag ccataggctt tgtcacgcga cctttgtgca gcgatccggg 1320

acaccgttcc ttacaaccgg cgtggtgttc tcctggacac accgcagtgc aactctgacg 1380

1440

tctggtgcct ctgtagtcac tgggcctggc tttacgggtg gcgacatcct ccgtcgcacg 1500

aactttggcg acttcgtgtc catgcaagtg aacattaaca gccctattac gcaacgctac 1560

cggctgaggt tcagatacgc ttcctcgcgg gacgcccgtc ttacggtggc gacgggcgca 1620

gcgaacactg gagttggcgg ccaaatctcc gtggacatgg ctttgcagaa gactatggag 1680

atcggtgagt ctctcacatc tcgcacgttc cgctacacgg atttctccaa ccctttctcc 1740

ttccgcgcca atccggacat cattcggatc aacgaacagc cgctcttcgg cgcgggctcc 1800

atctcatccg gtgagcttta cattgataag attgagataa ttctggccga cgcgaccttt 1860

gaggcagagt atgatctgga gcgcgcacag aaggccgtga acgcgctgtt tacgtccacg 1920

aaccagcgcg ggctcaagac agacgtcaca gactaccaca tcgaccaagt ctccaacctt 1980

gtcgagtgtc tctccgacga gttctgcctg gacgagaagc gggagcttag tgagaaggtg 2040

aagcacgcaa agcgcctgtc tgacgagcgg aaccttctac aagaccgtaa cttcacctcc 2100

attaacgggc agctagaccg tggctggcgc gggtccaccg acatcactat ccaaggtggc 2160

gacgacgtct tcaaggagaa ctacgtgacg ctgctcggca cctttgacga gtgctacccg 2220

acatacctct atcagaagat tgacgagtct aagctcaagg cttacacccg ttacgagctg 2280

cgtggctaca tcgaggactc ccaggatctg gaaatctatc tcatcagata caacgcgaag 2340

cacgagacag tcaacgtacc tgggacaggc tctctctggc ctctgtctgt ggagagtccc 2400

atcggcaaat gtggcgagcc gaacagatgt actccgcacc tggagtggaa tcccaacttg 2460

gactgtagtt gccgcgacgg caagacctgc gcgcaccact cccaccactt ctccctggac 2520

attgacgtcg gctgcacgga tctcaacgag gatctgggcg tttgggtcat cttcaagata 2580

aagacccagg acggacacgc cagactggga aacctagagt tccttgagga gaagccgctg 2640

gtcggcgaag cactggcgcg ggtcaagagg gccgagaaga agtggcggga caaacggggag 2700

aaacttcaac tcgaaacgaa catcgtttac aaggaggcaa aggaggccgt ggacgcactg 2760

ttcgtgaact cgcagtacga ccgcctccaa gtggacacca acatcgccat gatccatgca 2820

gcggacaagc gcgtgcaccg aatcagggaa gcgtacttgc ccgagttgtc cgttatccct 2880

ggcgtgaacg ctgccatctt cgaggaactg gagggctgcg tgttcaccgc attctccctg 2940

tacgacgcac gaaacgtcat caagaatggc gacttcaaca acggcctgag ctgctggaac 3000

gtgaagggcc acgtggagga gcagaacgac caccgctccg tgttagtggt cccggaatgg 3060

3120

accgcctaca aggagggcta tggcgaggggc tgcgtgacga tacacggtat cgagaacaac 3180

accgatgagc ttaagttctc gaactgcgtg gaggaggagg tgtacccgaa taacacagtg 3240

acgtgcaatg actacccggc caaccaggag gagtacggcg gtgcctacac gagccgaaac 3300

3360

tacgaggaga aggcgtacac ggacgggcgg cgcgacaacc cttgcgagtt caatagaggc 3420

tatggcgact acacgccgct gcccgccggt tatgtgacga aggagttgga atacttccca 3480

gagacggaca aggtgtggat cgagattggc gagaccgagg gcacgttcat tgtggacagc 3540

gttgagctgc tactgatgga ggagtga 3567

<210> 4

<211> 1188

<212> PRT

<213> Artificial

<220>

<223> The amino acid sequence of TIC4472PL encoded by a synthetic

coding sequence designed for expression in a plant cell (SEQ ID

NO:3), and although an additional alanine amino acid is inserted

immediately following the initiating methionine.

<400> 4

Met Ala Asn Asn Asn Ile Glu Asn Gln Cys Val Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Glu Val Ile Leu Asp Gly Glu Arg Ile Ser Thr Gly

20 25 30

Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Leu Leu Val Ser

35 40 45

Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val

50 55 60

Trp Gly Ile Val Gly Pro Ser Pro Trp Asp Ala Phe Leu Val Gln Ile

65 70 75 80

Glu Gln Leu Ile Gln Gln Arg Ile Glu Ala Tyr Ala Arg Ala Ala Ala

85 90 95

Ile Ser Asn Leu Glu Gly Ile Gly Asn Asn Phe Asn Ile Tyr Val Glu

100 105 110

Ala Phe Gln Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Asn

115 120 125

Arg Val Val Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp

130 135 140

Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val

145 150 155 160

Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val

165 170 175

Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Glu Asn

180 185 190

Tyr Asn Arg Gln Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala

195 200 205

Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln

210 215 220

Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Glu Leu Thr Leu Thr Val

225 230 235 240

Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro

245 250 255

Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu

260 265 270

Ile Thr Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe

275 280 285

Asn Val Met Glu Ser Asn Ala Ile Arg Asn Pro His Leu Phe Asp Ile

290 295 300

Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn

305 310 315 320

Phe Tyr Trp Gly Gly His Arg Val Thr Ser Asn Tyr Ile Gly Gly Gly

325 330 335

Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro

340 345 350

Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro

355 360 365

Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu

370 375 380

Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr

385 390 395 400

Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu

405 410 415

Asp Asn Ser Val Leu Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His

420 425 430

Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val

435 440 445

Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp

450 455 460

Pro Asp Lys Ile Thr Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp

465 470 475 480

Ser Gly Ala Ser Val Val Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile

485 490 495

Leu Arg Arg Thr Asn Phe Gly Asp Phe Val Ser Met Gln Val Asn Ile

500 505 510

Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser

515 520 525

Ser Arg Asp Ala Arg Leu Thr Val Ala Thr Gly Ala Ala Asn Thr Gly

530 535 540

Val Gly Gly Gln Ile Ser Val Asp Met Ala Leu Gln Lys Thr Met Glu

545 550 555 560

Ile Gly Glu Ser Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser

565 570 575

Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Arg Ile Asn Glu

580 585 590

Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile

595 600 605

Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Tyr

610 615 620

Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr

625 630 635 640

Asn Gln Arg Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln

645 650 655

Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu

660 665 670

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp

675 680 685

Glu Arg Asn Leu Leu Gln Asp Arg Asn Phe Thr Ser Ile Asn Gly Gln

690 695 700

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly

705 710 715 720

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp

725 730 735

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu

740 745 750

Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln

755 760 765

Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val

770 775 780

Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Glu Ser Pro

785 790 795 800

Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Thr Pro His Leu Glu Trp

805 810 815

Asn Pro Asn Leu Asp Cys Ser Cys Arg Asp Gly Lys Thr Cys Ala His

820 825 830

His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu

835 840 845

Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp

850 855 860

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu

865 870 875 880

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg

885 890 895

Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu

900 905 910

Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg

915 920 925

Leu Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg

930 935 940

Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro

945 950 955 960

Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Cys Val Phe Thr

965 970 975

Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe

980 985 990

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Glu Glu Gln

995 1000 1005

Asn Asp His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu

1010 1015 1020

Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu

1025 1030 1035

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr

1040 1045 1050

Ile His Gly Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn

1055 1060 1065

Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn

1070 1075 1080

Asp Tyr Pro Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser

1085 1090 1095

Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val

1100 1105 1110

Ser Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ala Tyr Thr Asp

1115 1120 1125

Gly Arg Arg Asp Asn Pro Cys Glu Phe Asn Arg Gly Tyr Gly Asp

1130 1135 1140

Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr

1145 1150 1155

Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu

1160 1165 1170

Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1175 1180 1185

<210> 5

<211> 3564

<212> DNA

<213> Bacillus thuringienses

<220>

<221> misc_feature

<222> (1)..(3564)

<223> Nucleic acid sequence encoding a TIC1425 pesticidal protein

obtained from Bacillus thuringiensis species EG10731.

<400> 5

atgaataata atattgaaaa ccaatgcgta ccttacaatt gtttaagtaa tcctgaagaa 60

gtaattttgg atggagaacg gatatcaact ggtaattcat caattgatat ttctctgtca 120

cttgttcaac ttctggtatc taactttgta ccaggcggag gatttttagt agggttaata 180

gattttgtat ggggaatagt aggcccttct ccatgggatg catttctagt gcaaattgaa 240

caattaattc agcaaagaat agaagcatat gctagggctg cagcaatttc taatttagaa 300

ggaataggaa acaatttcaa tatatatgtg gaagcatttc aagaatggga agaagatcct 360

aataatccag caaccaggaa tagagtagtt gatcgctttc gtatacttga tgggctactt 420

gaaagggaca ttccttcgtt tcgaatttct ggatttgaag tccccctttt atccgtttat 480

gctcaagcgg ccaatctgca tctagctata ttaagagatt ctgtaatttt tggagaaaga 540

tggggattga caacaacaaa tgtcaatgaa aactataata gacaaatcag gcatattgat 600

gaatatgctg atcactgtgc aaatacgtat aatcggggat taaataattt accgaaatct 660

acgtatcaag attggataac atataatcga ttacggagag aattaacatt gactgtatta 720

gatatcgctg ctttctttcc aaactatgac aataggcggt atccaattca gccagttggt 780

caactaacaa gggaagttta tacggaccca ttaattactt ttaatcccca gttacagtct 840

gtagctcaat tacctacttt taacgttatg gaaagcaacg caattagaaa tcctcatttg 900

tttgatatat tgaataatct tacaattttt acggattggt ttagtgttgg acgcaacttt 960

tattggggag gacatcgagt aacttctaac tatataggag gaggcaacat aacatctcct 1020

atatatggaa gagaggcgaa ccaggagcct ccaagatctt ttacttttaa tggacctgtt 1080

tttaggactt tatcaaatcc tactttacga ttattacagc aaccttggcc agcaccacca 1140

tttaatttac gtggtgttga aggagtagaa ttttctacac ctacaaaatag ctttacgtat 1200

cgaggaagag gtacagttga ttctttaacc gaattaccgc ctgaggataa tagtgtgcta 1260

cctcgcgagg gatatagtca tcgtttatgt catgcaactt ttgttcaaag atctggaaca 1320

ccatttttaa caacgggtgt agtattttct tggacgcatc gcagtgcaac tcttacaaat 1380

acaattgatc cagacaaaat tactcagata cctttagtga aaggatttag agtttggagt 1440

ggcgcctctg tcgttacagg accaggtttt acaggagggg atatccttcg aagaactaac 1500

tttggggatt ttgtatctat gcaagttaat attaattcac caataacaca aagataccgt 1560

1620

1680

1740

agagctaatc cagatataat tcgtataaat gaacaaccac tattcggtgc aggctctatt 1800

agtagtggtg aactttatat agataaaatt gaaattattc tagcagatgc aacatttgaa 1860

gcagaatatg atttggaaag agcacagaag gcggtgaatg cgctgtttac ttctacaaac 1920

1980

gagtgtttat cggatgaatt ttgtctggat gaaaaacgag aattgtccga gaaagtcaaa 2040

catgcgaagc gactcagtga tgagcgaaat ttactccagg atcgaaattt cacatccatt 2100

2160

gacgtattca aagagaatta cgtcacacta ctgggtacct ttgatgagtg ctatccaacg 2220

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga 2280

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacac 2340

gaaacagtaa atgtgccagg tacaggttcc ttatggccgc tttcagtcga aagtccaatc 2400

ggaaagtgtg gagaaccgaa tcgatgcaca ccacaccttg aatggaatcc taatctagat 2460

tgttcctgca gagacgggaa aacatgtgca catcattctc atcatttctc cttggacatt 2520

gatgttggat gtacagactt aaatgaagat ctaggtgtat gggtgatatt caagattaag 2580

acgcaagatg gtcatgcaag actaggaaat ctagagtttc tcgaagagaa accattagta 2640

ggagaagcgt tagctcgtgt gaaaagagcg gagaaaaaat ggagagacaa acgcgaaaaa 2700

ttgcaattag aaacaaatat cgtttacaaa gaggcaaaag aagctgtgga tgctttattt 2760

gtaaactctc aatatgatag attacaagtg gatacgaaca ttgccatgat tcatgcggca 2820

2880

gtcaatgcgg ctattttcga agaattagaa gggtgtattt tcactgcatt ctccctatat 2940

gatgcgagaa atgtcattaa aaatggcgat tttaataatg gcttatcatg ctggaacgtg 3000

aaagggcatg tagaagaaca aaacgaccat cgttcggtcc ttgttgtccc ggaatgggaa 3060

cacaagaggt tcgtgtctgt ccaggtcgtg gctatatcct tcgtgttaca 3120

gcatacaaag agggatatgg agaaggctgt gtaaccattc atgggatcga gaacaataca 3180

gacgaactga agtttagcaa ctgcgtagaa gaggaagtct atccaaacaa cacggtaacg 3240

tgtaatgatt atcctgcaaa tcaagaagaa tacgggggtg cgtacacttc tcgtaatcgt 3300

ggatatgacg aaacttatgg aagcaattct tccgtatcag ctgattatgc gtcagtttat 3360

gaagaaaaag cgtatacaga tggacgaaga gacaatccat gtgaatttaa cagagggtat 3420

ggggattata cgccactacc agctggctat gtaacaaaag aattagaata cttcccagaa 3480

3540

gaattactcc ttatggagga ataa 3564

<210> 6

<211> 1187

<212> PRT

<213> Bacillus thuringienses

<220>

<221> MISC_FEATURE

<222> (1)..(1187)

<223> The amino acid sequence of the TIC1425 pesticidal protein.

<400> 6

Met Asn Asn Asn Ile Glu Asn Gln Cys Val Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Ile Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn

20 25 30

Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Leu Leu Val Ser Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp

50 55 60

Gly Ile Val Gly Pro Ser Pro Trp Asp Ala Phe Leu Val Gln Ile Glu

65 70 75 80

Gln Leu Ile Gln Gln Arg Ile Glu Ala Tyr Ala Arg Ala Ala Ala Ile

85 90 95

Ser Asn Leu Glu Gly Ile Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala

100 105 110

Phe Gln Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Asn Arg

115 120 125

Val Val Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile

130 135 140

Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Glu Asn Tyr

180 185 190

Asn Arg Gln Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn

195 200 205

Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp

210 215 220

Trp Ile Thr Tyr Asn Arg Leu Arg Arg Glu Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile

245 250 255

Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile

260 265 270

Thr Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn

275 280 285

Val Met Glu Ser Asn Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu

290 295 300

Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe

305 310 315 320

Tyr Trp Gly Gly His Arg Val Thr Ser Asn Tyr Ile Gly Gly Gly Asn

325 330 335

Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg

340 345 350

Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr

355 360 365

Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg

370 375 380

Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr

385 390 395 400

Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp

405 410 415

Asn Ser Val Leu Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala

420 425 430

Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val

435 440 445

Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro

450 455 460

Asp Lys Ile Thr Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Ser

465 470 475 480

Gly Ala Ser Val Val Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu

485 490 495

Arg Arg Thr Asn Phe Gly Asp Phe Val Ser Met Gln Val Asn Ile Asn

500 505 510

Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser

515 520 525

Arg Asp Ala Arg Leu Thr Val Ala Thr Gly Ala Ala Asn Thr Gly Val

530 535 540

Gly Gly Gln Ile Ser Val Asp Met Ala Leu Gln Lys Thr Met Glu Ile

545 550 555 560

Gly Glu Ser Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn

565 570 575

Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Arg Ile Asn Glu Gln

580 585 590

Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp

595 600 605

Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Tyr Asp

610 615 620

Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn

625 630 635 640

Gln Arg Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val

645 650 655

Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys

660 665 670

Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu

675 680 685

Arg Asn Leu Leu Gln Asp Arg Asn Phe Thr Ser Ile Asn Gly Gln Leu

690 695 700

Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp

705 710 715 720

Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp Glu

725 730 735

Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys

740 745 750

Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp

755 760 765

Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn

770 775 780

Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile

785 790 795 800

Gly Lys Cys Gly Glu Pro Asn Arg Cys Thr Pro His Leu Glu Trp Asn

805 810 815

Pro Asn Leu Asp Cys Ser Cys Arg Asp Gly Lys Thr Cys Ala His His

820 825 830

Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn

835 840 845

Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly

850 855 860

His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val

865 870 875 880

Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp

885 890 895

Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala

900 905 910

Lys Glu Ala Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu

915 920 925

Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val

930 935 940

His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly

945 950 955 960

Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Cys Ile Phe Thr Ala

965 970 975

Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn

980 985 990

Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Glu Glu Gln Asn

995 1000 1005

Asp His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val

1010 1015 1020

Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg

1025 1030 1035

Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile

1040 1045 1050

His Gly Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys

1055 1060 1065

Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp

1070 1075 1080

Tyr Pro Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg

1085 1090 1095

Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Ser

1100 1105 1110

Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ala Tyr Thr Asp Gly

1115 1120 1125

Arg Arg Asp Asn Pro Cys Glu Phe Asn Arg Gly Tyr Gly Asp Tyr

1130 1135 1140

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe

1145 1150 1155

Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly

1160 1165 1170

Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1175 1180 1185

<210> 7

<211> 3537

<212> DNA

<213> Bacillus thuringienses

<220>

<221> misc_feature

<222> (1)..(3537)

<223> Nucleic acid sequence encoding a TIC2613 pesticidal protein

obtained from Bacillus thuringiensis species EG5408.

<400> 7

atggataaca atatcaagaa ccaatgcatt ccttacaatt gtttaaatga tcctgaggta 60

gaaatattag gtgaagaggg gataactact agtaatgaaa atctcgaatt tttcttatcg 120

ctaacgaaat ttgtcttgaa taggtttgtc cctggtggag catatgtagc tggcctattt 180

gatgtattct ggggatggtt aaaaccttct gattggtctg caatccttga acaaattgaa 240

gaattaatta accaaaaaat tgagacgttt gctagaaatc aagcaattag tagattggaa 300

gggttaagca acctttatga aatttacgca gatactttta aagaatggga aaaagatccg 360

actaatccag cattaagaga agaaatgcgt acacaattta atgacatgaa cagctctttt 420

gtaacagcta tgcctctttt ttcagttcaa aattttgaag ttcctctttt agcagtatac 480

gctcaagctg caaatttaca tctatcagtt ttaagggatg tctcagtttt tggtcaaaat 540

tggggatttg attcagccac tgtcaatagt cgttataatg atttagtaag aaatattcgt 600

660

acgtaccaag attggttaaa ttatcatcgc tttagaagag aattaacaat aactgcattg 720

gatatcatta ccatattccc acactacgat aataaaatgt atccaattca accccatttt 780

caattaacaa gagagattta tacggatcca ctaattaatt tcaatccggc gttacagtct 840

gtagcacaat tacctctatt taatgagatg gaaaatagta caattagaag ccctcattta 900

gttgattttt taaataggct tacaatttat acagattggt atagtctcgg aagacactat 960

tattggggag gacatcaaat agtctctaga caaacaggat caacttccac tattacattc 1020

1080

gtctttagaa cactgtcaaa tcctacttta acacgtttaa tgcaaccttg gccagcccca 1140

gcatttcagt tgcgtcgtct tgagggagtt gaatttcaaa caactacagg taattttacg 1200

1260

ccagcgcgtg aaggatatag tcatcgttta tgtcatgcaa catttatcag aaaatctggg 1320

1380

aatataattt atcctgataa aatcactcaa gtaccattgg tgaaagcatc taaccttcat 1440

tctagtgctt tcgttttaaa aggaccagga tttacaggag gggacatact tggaagaact 1500

1560

cgcgtacgaa ttcggtatgc ctctactaca aacttacaat ttcatacgac aattaacggc 1620

agggccgtaa atcaggctaa tttcccagca actatgaata gagtagaaga cttagaatat 1680

aattccttta gaacgataag tttcggtact ccttttaact ttttagatgc tcaaagtacc 1740

ttcaggttag gtgtatggag cttttcttca ggtaccgttt taatagatag aattgaagtt 1800

gtaccaatgg aagtaacatt tgaagcagaa tctgatttag aaagagcaca aaaggcggtg 1860

aacgctctgt ttacttctat aaatcaaaaa ggactaaaaa cagatgtaac agattatcac 1920

attgatcaag tatccaattt ggtcgaatgt ctatccgatg aattttgtct agatgaaaag 1980

agagaactat ttgagaaagt caaatatgcg aaacgactca gtgacgaacg gaatttactt 2040

gcagatccaa atttcacatc tattaatggg caactagatc gtggatggag aggaagtacg 2100

gatattacca ttcaaggggg cgatgacgta ttcaaagaaa actacgtcac actatcaggt 2160

acccttgatg agtgttatcc aacctattta tatcaaaaaa tagacgaatc gaaattaaaa 2220

gcgtataccc gttacgagtt acgaggatat atcgaagata gtcaagattt agaagtatac 2280

ttgattcgtt acaataccaa acatgaaaca ttgaatgtac caggtacagg gggcctatgg 2340

ccgcttgcag tagaaagttc aatcggaggg tgtggcgaac caaaccgatg cgcaccacaa 2400

atggaatggg atccaaatct agaatgttct tgtagcgacg aggagaaatg tgcgcatcat 2460

tcccatcatt tctctctcga tattgatgtt ggatgtactg atttaaatga aaatctaggt 2520

2580

tttctcgaag agaaaccatt aataggggaa gcgttagctc gtgttaagcg agtggagaaa 2640

aaatggaaag acaaacgtga aaaattagaa tttgaaacga atatagtcta caacgaggca 2700

aaagaagctg tggatgcact attcgtaaat tcacaatatg atagattgca agctgataca 2760

aatatcgcaa tgattcatgc ggcggataac aaagttcata aaattcgcga ggcgtacctc 2820

2880

atttttacag cattctccct ttacgatgca agaaatgtga taaaaaatgg ggatttcaat 2940

aatggattat cttgttggaa tgtgaagggc caagtagatg tagaccaaaa tgaccatcgt 3000

3060

gatcgtggat acattcttcg tgttacggcg tacaaagaag gatatggaga aggctgcgta 3120

3180

gaagtatctc taaataatgc ggtgacatgt gatgagtata ctacaaatca agaagtagga 3240

gggtatgcgg atgtacgtca atccaataat cgtggatcta atgaggccta tgtaaatcct 3300

acttccacat caactgatta tgcatcgctc tacgaggaag agtcgtatac gaatgaacag 3360

acatataatt cttgtgaatc taacagaggg tatggtaatc aaatgccatt accgtctggc 3420

3480

gaaacagaag gaacattcat cgtagacagt gtggaattac tccttatgga ggaataa 3537

<210> 8

<211> 1178

<212> PRT

<213> Bacillus thuringienses

<220>

<221> MISC_FEATURE

<222> (1)..(1178)

<223> The amino acid sequence of the TIC2613 pesticidal protein.

<400> 8

Met Asp Asn Asn Ile Lys Asn Gln Cys Ile Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asp Pro Glu Val Glu Ile Leu Gly Glu Glu Gly Ile Thr Thr Ser Asn

20 25 30

Glu Asn Leu Glu Phe Phe Leu Ser Leu Thr Lys Phe Val Leu Asn Arg

35 40 45

Phe Val Pro Gly Gly Ala Tyr Val Ala Gly Leu Phe Asp Val Phe Trp

50 55 60

Gly Trp Leu Lys Pro Ser Asp Trp Ser Ala Ile Leu Glu Gln Ile Glu

65 70 75 80

Glu Leu Ile Asn Gln Lys Ile Glu Thr Phe Ala Arg Asn Gln Ala Ile

85 90 95

Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Glu Ile Tyr Ala Asp Thr

100 105 110

Phe Lys Glu Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu

115 120 125

Met Arg Thr Gln Phe Asn Asp Met Asn Ser Ser Phe Val Thr Ala Met

130 135 140

Pro Leu Phe Ser Val Gln Asn Phe Glu Val Pro Leu Leu Ala Val Tyr

145 150 155 160

Ala Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val

165 170 175

Phe Gly Gln Asn Trp Gly Phe Asp Ser Ala Thr Val Asn Ser Arg Tyr

180 185 190

Asn Asp Leu Val Arg Asn Ile Arg Thr Tyr Thr Asn Tyr Val Val Arg

195 200 205

Trp Tyr Asn Thr Gly Leu Ala Arg Leu Arg Gly Thr Thr Tyr Gln Asp

210 215 220

Trp Leu Asn Tyr His Arg Phe Arg Arg Glu Leu Thr Ile Thr Ala Leu

225 230 235 240

Asp Ile Ile Thr Ile Phe Pro His Tyr Asp Asn Lys Met Tyr Pro Ile

245 250 255

Gln Pro His Phe Gln Leu Thr Arg Glu Ile Tyr Thr Asp Pro Leu Ile

260 265 270

Asn Phe Asn Pro Ala Leu Gln Ser Val Ala Gln Leu Pro Leu Phe Asn

275 280 285

Glu Met Glu Asn Ser Thr Ile Arg Ser Pro His Leu Val Asp Phe Leu

290 295 300

Asn Arg Leu Thr Ile Tyr Thr Asp Trp Tyr Ser Leu Gly Arg His Tyr

305 310 315 320

Tyr Trp Gly Gly His Gln Ile Val Ser Arg Gln Thr Gly Ser Thr Ser

325 330 335

Thr Ile Thr Phe Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Ala Pro

340 345 350

Arg Thr Tyr Asn Phe Ser Gln Pro Val Phe Arg Thr Leu Ser Asn Pro

355 360 365

Thr Leu Thr Arg Leu Met Gln Pro Trp Pro Ala Pro Ala Phe Gln Leu

370 375 380

Arg Arg Leu Glu Gly Val Glu Phe Gln Thr Thr Thr Gly Asn Phe Thr

385 390 395 400

Tyr Arg Gly Arg Gly Thr Val Asp Ser Phe Asp Glu Leu Pro Pro Asp

405 410 415

Asp Thr Ser Val Pro Ala Arg Glu Gly Tyr Ser His Arg Leu Cys His

420 425 430

Ala Thr Phe Ile Arg Lys Ser Gly Thr Pro Tyr Leu Thr Thr Gly Val

435 440 445

Thr Leu Ser Trp Thr His Asn Ser Asn Thr Pro Thr Asn Ile Ile Tyr

450 455 460

Pro Asp Lys Ile Thr Gln Val Pro Leu Val Lys Ala Ser Asn Leu His

465 470 475 480

Ser Ser Ala Phe Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile

485 490 495

Leu Gly Arg Thr Ser Val Gly Asn Ile Ala Asp Ile Gln Met Asn Ile

500 505 510

Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser

515 520 525

Thr Thr Asn Leu Gln Phe His Thr Thr Ile Asn Gly Arg Ala Val Asn

530 535 540

Gln Ala Asn Phe Pro Ala Thr Met Asn Arg Val Glu Asp Leu Glu Tyr

545 550 555 560

Asn Ser Phe Arg Thr Ile Ser Phe Gly Thr Pro Phe Asn Phe Leu Asp

565 570 575

Ala Gln Ser Thr Phe Arg Leu Gly Val Trp Ser Phe Ser Ser Gly Thr

580 585 590

Val Leu Ile Asp Arg Ile Glu Val Val Pro Met Glu Val Thr Phe Glu

595 600 605

Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe

610 615 620

Thr Ser Ile Asn Gln Lys Gly Leu Lys Thr Asp Val Thr Asp Tyr His

625 630 635 640

Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys

645 650 655

Leu Asp Glu Lys Arg Glu Leu Phe Glu Lys Val Lys Tyr Ala Lys Arg

660 665 670

Leu Ser Asp Glu Arg Asn Leu Leu Ala Asp Pro Asn Phe Thr Ser Ile

675 680 685

Asn Gly Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile

690 695 700

Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly

705 710 715 720

Thr Leu Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu

725 730 735

Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu

740 745 750

Asp Ser Gln Asp Leu Glu Val Tyr Leu Ile Arg Tyr Asn Thr Lys His

755 760 765

Glu Thr Leu Asn Val Pro Gly Thr Gly Gly Leu Trp Pro Leu Ala Val

770 775 780

Glu Ser Ser Ile Gly Gly Cys Gly Glu Pro Asn Arg Cys Ala Pro Gln

785 790 795 800

Met Glu Trp Asp Pro Asn Leu Glu Cys Ser Cys Ser Asp Glu Glu Lys

805 810 815

Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys

820 825 830

Thr Asp Leu Asn Glu Asn Leu Gly Ile Trp Val Ile Phe Lys Ile Lys

835 840 845

Thr Gln Asn Gly Tyr Ala Lys Leu Gly Asn Leu Glu Phe Leu Glu Glu

850 855 860

Lys Pro Leu Ile Gly Glu Ala Leu Ala Arg Val Lys Arg Val Glu Lys

865 870 875 880

Lys Trp Lys Asp Lys Arg Glu Lys Leu Glu Phe Glu Thr Asn Ile Val

885 890 895

Tyr Asn Glu Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asn Ser Gln

900 905 910

Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala

915 920 925

Asp Asn Lys Val His Lys Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser

930 935 940

Val Ile Pro Gly Val Asn Ala Thr Val Phe Glu Glu Leu Glu Glu Arg

945 950 955 960

Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn

965 970 975

Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly Gln Val

980 985 990

Asp Val Asp Gln Asn Asp His Arg Ser Val Leu Val Ile Pro Gly Trp

995 1000 1005

Glu Ser Glu Val Ser Gln Glu Val His Val Cys Pro Asp Arg Gly

1010 1015 1020

Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly

1025 1030 1035

Cys Val Thr Ile His Glu Ile Asp Asn His Thr Asp Glu Leu Lys

1040 1045 1050

Phe Lys Asn Cys Phe Glu Glu Glu Val Ser Leu Asn Asn Ala Val

1055 1060 1065

Thr Cys Asp Glu Tyr Thr Thr Asn Gln Glu Val Gly Gly Tyr Ala

1070 1075 1080

Asp Val Arg Gln Ser Asn Asn Arg Gly Ser Asn Glu Ala Tyr Val

1085 1090 1095

Asn Pro Thr Ser Thr Ser Thr Asp Tyr Ala Ser Leu Tyr Glu Glu

1100 1105 1110

Glu Ser Tyr Thr Asn Glu Gln Thr Tyr Asn Ser Cys Glu Ser Asn

1115 1120 1125

Arg Gly Tyr Gly Asn Gln Met Pro Leu Pro Ser Gly Tyr Val Thr

1130 1135 1140

Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu

1145 1150 1155

Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu

1160 1165 1170

Leu Leu Met Glu Glu

1175

<210> 9

<211> 3540

<212> DNA

<213> Artificial

<220>

<223> A synthetic coding sequence encoding a TIC2613PL pesticidal

protein designed for expression in a plant cell where an

additional alanine codon is inserted immediately following the

initiating methionine codon.

<400> 9

atggctgaca acaacatcaa gaaccagtgc atcccgtaca actgcctcaa cgacccggag 60

gtcgagatcc tcggcgagga gggcataacg acgagcaacg agaaccttga gttcttcctc 120

agcctcacga agttcgtcct gaaccgcttc gtgccggggcg gagcctacgt ggctggcctg 180

ttcgatgtgt tctggggatg gctcaagcca agcgactggt ccgcgattct ggagcagatc 240

gaggaactca tcaaccagaa gatcgagaca ttcgcccgca accaggccat cagccgcctg 300

gagggcctct cgaacctcta cgaaatctac gccgatacgt tcaaggagtg ggagaaggat 360

ccgacgaacc cggccttgcg cgaggagatg aggacgcaat tcaacgacat gaactccagc 420

ttcgtcaccg ccatgccgct gttctccgtc cagaacttcg aggtgccctt gctcgccgtg 480

tacgcgcaag ctgcgaactt acatcttagc gtcctccgcg acgtcagcgt cttcggccag 540

aactggggat tcgattccgc gacggtgaac tcacggtaca atgatctcgt gcggaacatc 600

660

actacctacc aggactggct caactaccac cggttccgcc gcgaactcac aatcacagcg 720

ctggacatca ttaccatctt cccgcactac gacaacaaga tgtacccaat ccagcctcac 780

ttccagctta cccgtgagat ctacacggac ccgctcatca acttcaatcc cgcactgcaa 840

tcagtagccc aattgccact cttcaacgag atggagaact cgacaatccg aagccctcac 900

ctcgtggact tcctcaaccg cctgaccatc tacacggatt ggtactctct tggtcggcac 960

tactattggg gcgggcacca aatcgtgtcc aggcagaccg gctctacctc taccataacc 1020

ttcccgatct atggccggga ggccaaccag gaggctccga ggacttacaa cttcagtcag 1080

ccagtgttcc gcacactctc caacccgact ctcactcgtt tgatgcagcc ctggcccgct 1140

cccgcgtttc agctcagaag attggagggc gtggagttcc aaacaacgac gggcaacttc 1200

acctaccgtg gccgtgggac ggtggacagt ttcgacgagt tgcctccgga cgacaccagc 1260

gtgcctgcaa gggaaggcta ctcgcacagg ctgtgccacg cgacgttcat ccgcaagtct 1320

gggacaccct acctgacaac cggcgtcact ctctcctgga cccacaacag caacacaccc 1380

accaacataa tctaccctga caagataaca caagtgccgc tggtgaaggc ttcgaacctc 1440

cattcctccg ccttcgtcct caagggtccg ggcttcaccg gcggcgacat cctgggtcgc 1500

acgtcggtcg gcaacatcgc ggacattcag atgaacatta ccgcacctct gtcccagcgc 1560

tacagagtgc gtatccgcta cgcgagtacg accaacctcc aattccacac tacgatcaat 1620

gggagggcgg ttaatcaggc caacttcccg gccacgatga accgggtcga agacctggag 1680

tacaactcgt ttcggaccat ctctttcggc acgccgttca acttcctaga cgcccagtca 1740

acctttcggc tgggagtttg gagcttcagc agcggcacag tcctcatcga ccgaatagag 1800

gtggttccga tggaggtcac gttcgaggcg gagtcggacc tggagcgagc gcagaaggct 1860

gtaaatgcgt tgttcacgag cattaaccag aagggcctca agaccgatgt cacagactac 1920

cacatcgacc aagtgtcgaa cctggtggag tgtctgtcgg atgagttctg tcttgacgag 1980

aagcgggagc tgttcgagaa ggtgaagtat gctaagcggc tgagcgacga gcggaacttg 2040

ttggctgacc cgaacttcac cagcatcaac ggacagctcg accgtgggtg gcgaggttcc 2100

accgacatca cgatacaggg cggagacgat gtgttcaagg agaactatgt gaccctctca 2160

ggaacactgg atgagtgcta cccgacctat ctctaccaga agatcgacga gagcaagctc 2220

aaggcttaca cgcgctacga actccgtggc tacatcgaag actcccagga tcttgaggtg 2280

tacctcatac gctacaacac aaagcacgag acgctcaacg ttcctggcac cggtggtctt 2340

tggcccttgg ccgtggagag tagcatcggc gggtgcggtg agccaaaccg atgcgcgcca 2400

cagatggaat gggatccgaa cctagagtgc tcctgctcag acgaggagaa gtgcgcccac 2460

cactcccacc acttctcgct cgacattgac gttggctgca cggatctcaa cgagaaccta 2520

ggaatctggg tgatcttcaa gattaagacc cagaacggct acgccaagct cgggaatctg 2580

gagtttcttg aggagaagcc gctgatcggc gaggccctcg cgcgcgtgaa gcgagtcgag 2640

aagaagtgga aagacaagcg ggagaagcta gagtttgaaa cgaacattgt ttacaacgag 2700

gcaaaggaag ccgtggacgc tctgttcgta aacagtcagt acgaccgtct ccaggccgac 2760

acgaacatcg caatgataca cgcggcggat aacaaggtgc acaagattcg ggaggcttac 2820

ctgcccgagc tgtcggtcat cccaggcgta aacgctaccg tgttcgagga gctggaggaa 2880

cggatcttca ccgcgttctc cctctatgac gcaaggaacg tcatcaagaa cggcgacttc 2940

aacaacggcc tgagctgctg gaacgtgaag ggccaagtgg acgtcgatca gaacgatcac 3000

cgctccgttc tggtcattcc agggtgggag tccgaggtga gccaagaggt ccatgtgtgc 3060

ccggaccgtg gctacatcct tcgggtgacc gcgtacaagg agggctacgg cgaaggctgc 3120

3180 gtgaccatac acgagatcga caaccacacc

gaggaggtgt cactgaacaa cgccgtgacc tgcgacgagt acacgaccaa tcaggaggtc 3240

ggcggctacg ccgacgtccg ccagtcgaac aatcgaggca gcaacgaggc gtacgtgaac 3300

ccaacctcca cctcgacgga ctacgccagc ctctacgagg aggagtccta cacaaacgag 3360

cagacctaca actcgtgcga gagcaaccga ggttacggga accagatgcc gctaccgtcc 3420

gggtacgtga cgaaggagct ggagtatttc ccagagaccg acaaggtgtg gatcgagatc 3480

ggcgagacag agggcacgtt catcgtggac agcgtcgagc tgctgttgat ggaggagtga 3540

<210> 10

<211> 1179

<212> PRT

<213> Artificial

<220>

<223> The amino acid sequence of TIC2613PL encoded by a synthetic

coding sequence designed for expression in a plant cell (SEQ ID

NO:9), and where an additional alanine amino acid is inserted

immediately following the initiating methionine.

<400> 10

Met Ala Asp Asn Asn Ile Lys Asn Gln Cys Ile Pro Tyr Asn Cys Leu

1 5 10 15

Asn Asp Pro Glu Val Glu Ile Leu Gly Glu Glu Gly Ile Thr Ser

20 25 30

Asn Glu Asn Leu Glu Phe Phe Leu Ser Leu Thr Lys Phe Val Leu Asn

35 40 45

Arg Phe Val Pro Gly Gly Ala Tyr Val Ala Gly Leu Phe Asp Val Phe

50 55 60

Trp Gly Trp Leu Lys Pro Ser Asp Trp Ser Ala Ile Leu Glu Gln Ile

65 70 75 80

Glu Glu Leu Ile Asn Gln Lys Ile Glu Thr Phe Ala Arg Asn Gln Ala

85 90 95

Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Glu Ile Tyr Ala Asp

100 105 110

Thr Phe Lys Glu Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg Thr Gln Phe Asn Asp Met Asn Ser Ser Phe Val Thr Ala

130 135 140

Met Pro Leu Phe Ser Val Gln Asn Phe Glu Val Pro Leu Leu Ala Val

145 150 155 160

Tyr Ala Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser

165 170 175

Val Phe Gly Gln Asn Trp Gly Phe Asp Ser Ala Thr Val Asn Ser Arg

180 185 190

Tyr Asn Asp Leu Val Arg Asn Ile Arg Thr Tyr Thr Asn Tyr Val Val

195 200 205

Arg Trp Tyr Asn Thr Gly Leu Ala Arg Leu Arg Gly Thr Thr Tyr Gln

210 215 220

Asp Trp Leu Asn Tyr His Arg Phe Arg Arg Glu Leu Thr Ile Thr Ala

225 230 235 240

Leu Asp Ile Ile Thr Ile Phe Pro His Tyr Asp Asn Lys Met Tyr Pro

245 250 255

Ile Gln Pro His Phe Gln Leu Thr Arg Glu Ile Tyr Thr Asp Pro Leu

260 265 270

Ile Asn Phe Asn Pro Ala Leu Gln Ser Val Ala Gln Leu Pro Leu Phe

275 280 285

Asn Glu Met Glu Asn Ser Thr Ile Arg Ser Pro His Leu Val Asp Phe

290 295 300

Leu Asn Arg Leu Thr Ile Tyr Thr Asp Trp Tyr Ser Leu Gly Arg His

305 310 315 320

Tyr Tyr Trp Gly Gly His Gln Ile Val Ser Arg Gln Thr Gly Ser Thr

325 330 335

Ser Thr Ile Thr Phe Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Ala

340 345 350

Pro Arg Thr Tyr Asn Phe Ser Gln Pro Val Phe Arg Thr Leu Ser Asn

355 360 365

Pro Thr Leu Thr Arg Leu Met Gln Pro Trp Pro Ala Pro Ala Phe Gln

370 375 380

Leu Arg Arg Leu Glu Gly Val Glu Phe Gln Thr Thr Thr Gly Asn Phe

385 390 395 400

Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Phe Asp Glu Leu Pro Pro

405 410 415

Asp Asp Thr Ser Val Pro Ala Arg Glu Gly Tyr Ser His Arg Leu Cys

420 425 430

His Ala Thr Phe Ile Arg Lys Ser Gly Thr Pro Tyr Leu Thr Thr Gly

435 440 445

Val Thr Leu Ser Trp Thr His Asn Ser Asn Thr Pro Thr Asn Ile Ile

450 455 460

Tyr Pro Asp Lys Ile Thr Gln Val Pro Leu Val Lys Ala Ser Asn Leu

465 470 475 480

His Ser Ser Ala Phe Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Asp

485 490 495

Ile Leu Gly Arg Thr Ser Val Gly Asn Ile Ala Asp Ile Gln Met Asn

500 505 510

Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala

515 520 525

Ser Thr Thr Asn Leu Gln Phe His Thr Thr Ile Asn Gly Arg Ala Val

530 535 540

Asn Gln Ala Asn Phe Pro Ala Thr Met Asn Arg Val Glu Asp Leu Glu

545 550 555 560

Tyr Asn Ser Phe Arg Thr Ile Ser Phe Gly Thr Pro Phe Asn Phe Leu

565 570 575

Asp Ala Gln Ser Thr Phe Arg Leu Gly Val Trp Ser Phe Ser Ser Gly

580 585 590

Thr Val Leu Ile Asp Arg Ile Glu Val Val Pro Met Glu Val Thr Phe

595 600 605

Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu

610 615 620

Phe Thr Ser Ile Asn Gln Lys Gly Leu Lys Thr Asp Val Thr Asp Tyr

625 630 635 640

His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe

645 650 655

Cys Leu Asp Glu Lys Arg Glu Leu Phe Glu Lys Val Lys Tyr Ala Lys

660 665 670

Arg Leu Ser Asp Glu Arg Asn Leu Leu Ala Asp Pro Asn Phe Thr Ser

675 680 685

Ile Asn Gly Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr

690 695 700

Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser

705 710 715 720

Gly Thr Leu Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp

725 730 735

Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile

740 745 750

Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu Ile Arg Tyr Asn Thr Lys

755 760 765

His Glu Thr Leu Asn Val Pro Gly Thr Gly Gly Leu Trp Pro Leu Ala

770 775 780

Val Glu Ser Ser Ile Gly Gly Cys Gly Glu Pro Asn Arg Cys Ala Pro

785 790 795 800

Gln Met Glu Trp Asp Pro Asn Leu Glu Cys Ser Cys Ser Asp Glu Glu

805 810 815

Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly

820 825 830

Cys Thr Asp Leu Asn Glu Asn Leu Gly Ile Trp Val Ile Phe Lys Ile

835 840 845

Lys Thr Gln Asn Gly Tyr Ala Lys Leu Gly Asn Leu Glu Phe Leu Glu

850 855 860

Glu Lys Pro Leu Ile Gly Glu Ala Leu Ala Arg Val Lys Arg Val Glu

865 870 875 880

Lys Lys Trp Lys Asp Lys Arg Glu Lys Leu Glu Phe Glu Thr Asn Ile

885 890 895

Val Tyr Asn Glu Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asn Ser

900 905 910

Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala

915 920 925

Ala Asp Asn Lys Val His Lys Ile Arg Glu Ala Tyr Leu Pro Glu Leu

930 935 940

Ser Val Ile Pro Gly Val Asn Ala Thr Val Phe Glu Glu Leu Glu Glu

945 950 955 960

Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys

965 970 975

Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly Gln

980 985 990

Val Asp Val Asp Gln Asn Asp His Arg Ser Val Leu Val Ile Pro Gly

995 1000 1005

Trp Glu Ser Glu Val Ser Gln Glu Val His Val Cys Pro Asp Arg

1010 1015 1020

Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu

1025 1030 1035

Gly Cys Val Thr Ile His Glu Ile Asp Asn His Thr Asp Glu Leu

1040 1045 1050

Lys Phe Lys Asn Cys Phe Glu Glu Glu Val Ser Leu Asn Asn Ala

1055 1060 1065

Val Thr Cys Asp Glu Tyr Thr Thr Asn Gln Glu Val Gly Gly Tyr

1070 1075 1080

Ala Asp Val Arg Gln Ser Asn Asn Arg Gly Ser Asn Glu Ala Tyr

1085 1090 1095

Val Asn Pro Thr Ser Thr Ser Thr Asp Tyr Ala Ser Leu Tyr Glu

1100 1105 1110

Glu Glu Ser Tyr Thr Asn Glu Gln Thr Tyr Asn Ser Cys Glu Ser

1115 1120 1125

Asn Arg Gly Tyr Gly Asn Gln Met Pro Leu Pro Ser Gly Tyr Val

1130 1135 1140

Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile

1145 1150 1155

Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu

1160 1165 1170

Leu Leu Leu Met Glu Glu

1175

<---

Claims (51)

1. A recombinant nucleic acid molecule for imparting resistance to an insect pest from the Lepidoptera order , containing a heterologous promoter operably linked to a polynucleotide segment encoding a pesticidal protein or a pesticidal fragment, wherein: a. the specified pesticidal protein contains the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; or b. said pesticidal protein contains an amino acid sequence having: i. at least 95%, or 98%, or 99%, or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2 or SEQ ID NO:6 and exhibits pesticidal activity; or ii. at least 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10 and exhibits pesticidal activity; or c. said polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. 2. Recombinant nucleic acid molecule according to claim 1, characterized in that a. said recombinant nucleic acid molecule contains a sequence that functions to express the pesticidal protein in the plant; or b. said recombinant nucleic acid molecule is expressed in a plant cell to produce a pesticidally effective amount of a pesticidal protein; or c. said recombinant nucleic acid molecule is operably linked to a vector, and said vector is selected from the group consisting of: plasmids, phagemids, bacmids, cosmids, and a bacterial or yeast artificial chromosome. 3. A recombinant nucleic acid molecule according to claim 1, defined as being present in a host cell, said host cell being selected from the group consisting of bacterial and plant cells. 4. Recombinant nucleic acid molecule according to claim 3, characterized in that the bacterial host cell belongs to a genus of bacteria selected from the group consisting of: Agrobacterium, Rhizobium , Bacillus , Brevibacillus , Escherichia , Pseudomonas , Klebsiella , Pantoea and Erwinia . 5. The recombinant nucleic acid molecule according to claim 3, wherein said Bacillus species is Bacillus cereus or Bacillus thuringiensis , said Brevibacillus is Brevibacillus laterosperous , and said Escherichia is Escherichia coli . 6. Recombinant nucleic acid according to claim 3, characterized in that said plant cell is a cell of a dicotyledonous or monocotyledonous plant. 7. Recombinant nucleic acid according to claim 6, characterized in that said plant cell host is selected from the group consisting of a plant cell of alfalfa, banana, barley, bean, broccoli, cabbage, ornamental cabbage, carrot, cassava, castor bean, cauliflower , celery, chickpea, bok choy, citrus, coconut, coffee, corn, clover, cotton, cucurbits, cucumber, menzies, eggplant, eucalyptus, flax, garlic, grapes, hops, leek, lettuce, frankincense pine , millet, melon, walnut, oats, olive tree, onion, ornamental plants, palm, pasture grasses, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, radiata pine, radish, rapeseed, rice, rhizomes , rye, wild saffron, bush, sorghum, southern pine, soybean, spinach, cucurbits, strawberry, sugar beet, sugar cane, sunflower, sugar corn, amber tree, sweet potato, switchgrass, tea, tobacco, tomato, triticale, etc. thorn grass, watermelon and wheat. 8. A recombinant nucleic acid molecule according to claim 1, characterized in that said protein exhibits activity against an insect pest from the order Lepidoptera. 9. Recombinant nucleic acid molecule according to claim 8, characterized in that said insect is selected from the group consisting of: tomato cutworm ( Spodoptera exigua ), cotton cutworm ( Helicoverpa zea), American cotton cutworm (Alabama argillacea), corn moth ( Ostrinia nubilalis ), fall armyworm ( Spodoptera frugiperda ), cotton bollworm ( Helicoverpa armigera ), Spodoptera litura , Pectinophora gossypiella , Cry1Ac resistant Pectinophora gossypiella , Chrysodeixis includens , Spodoptera materidania , Diatraea grandiosella , Earias Antivittella , Diatraea , Herescaralis viscaralis . 10. A transgenic plant resistant to an insect pest from the Lepidoptera order , containing a recombinant nucleic acid molecule according to claim 1. 11. A transgenic plant according to claim 10, characterized in that said plant is a monocot plant or a dicot plant. 12. Transgenic plant according to claim 10, characterized in that said plant is selected from the group consisting of: alfalfa, banana, barley, beans, broccoli, cabbage, ornamental cabbage, carrots, cassava, castor beans, cauliflower, celery, chickpeas, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, cucurbits, cucumber, Menzies pseudo-hemlock, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, lettuce, frankincense pine, millet, melon, walnut, oat, olive tree, onion, ornamental plants, palm, pasture grasses, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, radish pine, radish, rapeseed, rice, rhizomes, rye, wild saffron shrubs, sorghum, southern pine, soybean, spinach, cucurbits, strawberry, sugar beet, sugar cane, sunflower, sweet corn, amberwood, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon and wheat. 13. The seed of a transgenic plant according to claim 10, characterized in that said seed is intended to produce a plant with resistance to an insect pest from the order Lepidoptera , and where said seed contains said recombinant nucleic acid molecule. 14. An insect-inhibiting composition for conferring resistance to an insect pest from the order Lepidoptera , comprising an agriculturally acceptable carrier and a recombinant nucleic acid molecule according to claim 1. 15. An insect inhibitory composition according to claim 14, further comprising a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. 16. An insect inhibitory composition according to claim 15, wherein said at least one other pesticidal agent is selected from the group consisting of a protein exhibiting an insect inhibitory effect, a dsRNA molecule exhibiting an inhibitory effect action against insects, and an auxiliary protein. 17. An insect-inhibiting composition according to claim 16, wherein said at least one other pesticidal agent is active against one or more pest species from the orders Lepidoptera , Coleoptera or Hemiptera . 18. An insect inhibitory composition according to claim 17, characterized in that said at least one other pesticidal protein is selected from the group consisting of proteins: Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3 variants, Cry3A, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9 variants , Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC100, TIC201, TIC201 , TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-, 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 their variants, IP3 and its variants, DIG-3, DIG-5, DIG-10, DIG-657 and DIG-11. 19. An insect-inhibiting composition for conferring resistance to an insect of the order Lepidoptera according to claim 14, wherein the agriculturally acceptable carrier is a plant cell that expresses said recombinant nucleic acid molecule. 20. A food product for commercial use obtained from a transgenic plant according to claim 10, characterized in that said food product contains a processed plant product and a detectable amount of said recombinant nucleic acid molecule or said encoded pesticidal protein. 21. A food product for commercial use obtained from a transgenic plant according to claim 10, characterized in that said food product contains a processed plant product and a detectable amount of said recombinant nucleic acid molecule or said encoded pesticidal protein. 22. A cosmetic product for commercial use obtained from a transgenic plant according to claim 10, characterized in that said cosmetic product contains a processed plant product and a detectable amount of said recombinant nucleic acid molecule or said encoded pesticidal protein. 23. An industrial product for commercial use obtained from a transgenic plant according to claim 10, characterized in that said industrial product contains a processed plant product and a detectable amount of said recombinant nucleic acid molecule or said encoded pesticidal protein. 24. A transgenic plant resistant to Lepidoptera insect pests, wherein the cells of said plant contain a recombinant nucleic acid molecule according to claim 1. 25. A method for controlling a pest or pest infestation, where the pest is an insect pest from the order Lepidoptera , where the method includes: a. contacting the pest with an insecticidally effective amount of the pesticidal protein set forth in SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10; or b. bringing the pest into contact with an insecticidally effective amount of one or more pesticidal proteins containing an amino acid sequence having: i. at least 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2 or SEQ ID NO:6 and exhibits pesticidal activity; or ii. at least 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10 and exhibits pesticidal activity. 26. A method for detecting the presence of a recombinant nucleic acid molecule to confer resistance to an insect pest from the Lepidoptera order according to claim 1 in a sample containing plant genomic DNA, including: a. bringing the sample into contact with a nucleotide probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant containing the DNA molecule according to claim 1, and does not hybridize under such hybridization conditions with genomic DNA from another isogenic plant that does not contain the recombinant nucleic acid molecule 1, wherein said probe is homologous or complementary to SEQ ID NO:3 or SEQ ID NO:9 or a sequence that encodes a pesticide protein containing an amino acid sequence having: i. at least 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2 or SEQ ID NO:6 and exhibits pesticidal activity; or ii. at least 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10 and exhibits pesticidal activity; b. subjecting the sample and probe to stringent hybridization conditions; and c. detection of probe hybridization with sample DNA. 27. A method for detecting the presence of a pesticidal protein or a fragment thereof for conferring resistance to an insect pest from the order Lepidoptera in a sample containing said pesticidal protein, said pesticidal protein comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO :6, SEQ ID NO:8 or SEQ ID NO:10 or said pesticidal protein contains an amino acid sequence having: a. at least 95% or 98% or 99% or about 100% amino acid sequence identity with SEQ ID NO:4, SEQ ID NO:2 or SEQ ID NO:6 and exhibits pesticidal activity; or b. at least 95% or about 100% amino acid sequence identity with SEQ ID NO:8 or SEQ ID NO:10 and exhibits pesticidal activity; including: c. contacting the sample with an immunoreactive antibody; and d. detecting the presence of a protein. 28. The method of claim 25, wherein said detection step comprises ELISA or Western blotting. 29. Part of a transgenic plant with resistance to an insect pest from the Lepidoptera order , containing a recombinant nucleic acid molecule according to claim 1.