Novel insect inhibitory proteins

Abstract

A pesticidal protein class exhibiting toxic activity against Coleopteran, Lepidopteran, and Hemipteran pest species is disclosed, and includes, but is not limited to, TIC5290. DNA constructs are provided which contain a recombinant nucleic acid sequence encoding the TIC5290 pesticidal protein. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran, Coleopteran and Hemipteran infestation are provided which contain recombinant nucleic acid sequences encoding the TIC5290 pesticidal protein of the present invention. Methods for detecting the presence of the recombinant nucleic acid sequences or the protein of the present invention in a biological sample, and methods of controlling Coleopteran, Lepidopteran, and Hemipteran species pests using the TIC5290 pesticidal protein are also provided.

What is claimed is: 
     
         1 . A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a pesticidal protein or pesticidal fragment thereof, wherein:
 a. said pesticidal protein comprises the amino acid sequence of SEQ ID NO:2; or   b. said pesticidal protein comprises an amino acid sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:2; or   c. said polynucleotide segment hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.   
     
     
         2 . The recombinant nucleic acid molecule of  claim 1 , wherein:
 a. the recombinant nucleic acid molecule comprises a sequence that functions to express the pesticidal protein in a plant; or   b. the recombinant nucleic acid molecule is expressed in a plant cell to produce a pesticidally effective amount of the pesticidal protein; or   c. the recombinant nucleic acid molecule is in operable linkage with a vector, and said vector is selected from the group consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial or yeast artificial chromosome.   
     
     
         3 . The recombinant nucleic acid molecule of  claim 1 , defined as present within a host cell, wherein said host cell is selected from the group consisting of a bacterial and a plant cell. 
     
     
         4 . The recombinant nucleic acid molecule of  claim 3 , wherein the bacterial host cell is from a genus of bacteria selected from the group consisting of:  Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea , and  Erwinia.    
     
     
         5 . The recombinant nucleic acid molecule of  claim 4 , wherein the  Bacillus  species is  Bacillus cereus  or  Bacillus thuringiensis , said  Brevibacillus  is  Brevibacillus laterosperus , or said  Escherichia  is  Escherichia coli.    
     
     
         6 . The recombinant nucleic acid molecule of  claim 3 , wherein said plant cell is a dicotyledonous or a monocotyledonous plant cell. 
     
     
         7 . The recombinant nucleic acid molecule of  claim 6 , wherein said plant host cell is selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage,  Brassica , carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell. 
     
     
         8 . The recombinant nucleic acid molecule of  claim 1 , wherein said pesticidal protein exhibits activity against a Coleopteran insect. 
     
     
         9 . The recombinant nucleic acid molecule of  claim 8 , wherein said insect is Western Corn Rootworm, Southern Corn Rootworm, Northern Corn Rootworm, Mexican Corn Rootworm, Brazilian Corn Rootworm, or Brazilian Corn Rootworm complex consisting of  Diabrotica viridula  and  Diabrotica speciosa.    
     
     
         10 . The recombinant nucleic acid molecule of  claim 1 , wherein said pesticidal protein exhibits activity against an insect species of the order of Lepidoptera. 
     
     
         11 . The recombinant nucleic acid molecule of  claim 10 , wherein said insect is Velvet bean caterpillar, Sugarcane borer, Lesser cornstalk borer, Corn earworm, Tobacco budworm, Soybean looper, Black armyworm, Southern armyworm, Fall armyworm, Beet armyworm, Old World bollworm, Oriental leaf worm, Pink bollworm, Black cutworm, Southwestern Corn Borer, or European corn borer. 
     
     
         12 . The recombinant nucleic acid molecule of  claim 1 , wherein said pesticidal protein exhibits activity against a Hemipteran insect. 
     
     
         13 . The recombinant nucleic acid molecule of  claim 12 , wherein said insect is a Western tarnished plant bug, a Tarnished plant bug, or a Cotton fleahopper. 
     
     
         14 . A plant or part thereof comprising the recombinant nucleic acid molecule of  claim 1 . 
     
     
         15 . The plant or part thereof of  claim 14 , wherein said plant is a monocot plant or a dicot plant. 
     
     
         16 . The plant or part thereof of  claim 14 , wherein the plant is selected from the group consisting of an alfalfa, banana, barley, bean, broccoli, cabbage,  Brassica , carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat. 
     
     
         17 . A seed of the plant of  claim 14 , wherein said seed comprises said recombinant nucleic acid molecule. 
     
     
         18 . An insect inhibitory composition comprising the recombinant nucleic acid molecule of  claim 1 . 
     
     
         19 . The insect inhibitory composition of  claim 18 , further comprising a nucleotide sequence encoding at least one other pesticidal agent that is different from said pesticidal protein. 
     
     
         20 . The insect inhibitory composition of  claim 19 , wherein said at least one other pesticidal agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, and an ancillary protein. 
     
     
         21 . The insect inhibitory composition of  claim 19 , wherein said at least one other pesticidal agent exhibits activity against one or more pest species of the orders Lepidoptera, Coleoptera, or Hemiptera. 
     
     
         22 . The insect inhibitory composition of  claim 21 , wherein said at least one other pesticidal protein is selected from the group consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-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 variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657 and a DIG-11protein. 
     
     
         23 . The insect inhibitory composition of  claim 18 , defined as comprising a plant cell that expresses said recombinant nucleic acid molecule. 
     
     
         24 . A commodity product produced from the plant or part thereof of  claim 14 , wherein the commodity product comprises a detectable amount of said recombinant nucleic acid molecule or a pesticidal protein encoded thereby. 
     
     
         25 . The commodity product of  claim 24 , selected from the group consisting of commodity corn bagged by a grain handler, corn flakes, corn cakes, corn flour, corn meal, corn syrup, corn oil, corn silage, corn starch, corn cereal, and the like, whole or processed cotton seed, cotton oil, lint, seeds and plant parts processed for feed or food, fiber, paper, biomasses, and fuel products such as fuel derived from cotton oil or pellets derived from cotton gin waste, whole or processed soybean seed, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk, soybean cheese, soybean wine, animal feed comprising soybean, paper comprising soybean, cream comprising soybean, soybean biomass, and fuel products produced using soybean plants and soybean plant parts. 
     
     
         26 . A method of producing seed comprising:
 a. planting at least a first seed according to  claim 17 ;   b. growing a plant from the seed; and   c. harvesting seed from the plant, wherein said harvested seed comprises said recombinant nucleic acid molecule.   
     
     
         27 . A plant resistant to insect infestation, wherein the cells of said plant comprise the recombinant nucleic acid molecule of  claim 1 . 
     
     
         28 . A method for controlling a Coleopteran or Lepidopteran or Hemipteran species pest or pest infestation, said method comprising:
 a. contacting the pest with an insecticidally effective amount of a pesticidal protein as set forth in SEQ ID NO:2; or   b. contacting the pest with an insecticidally effective amount of one or more pesticidal proteins comprising an amino acid sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:2.   
     
     
         29 . A method of detecting the presence of the recombinant nucleic acid molecule of  claim 1  in a sample comprising plant genomic DNA, comprising:
 a. contacting the sample with a nucleic acid probe that hybridizes under stringent hybridization conditions with genomic DNA from a plant comprising the DNA molecule of  claim 1 , and does not hybridize under such hybridization conditions with genomic DNA from an otherwise isogenic plant that does not comprise the recombinant nucleic acid molecule of  claim 1 , wherein the probe is homologous or complementary to SEQ ID NO:1, SEQ ID NO:3, or a sequence that encodes a pesticidal protein comprising an amino acid sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:2; 
 b. subjecting the sample and probe to stringent hybridization conditions; and 
 c. detecting hybridization of the probe with DNA of the sample. 
 
     
     
         30 . A method of detecting the presence of a pesticidal protein or fragment thereof in a sample comprising protein, wherein said pesticidal protein comprises the amino acid sequence of SEQ ID NO:2; or said pesticidal protein comprises an amino acid sequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99% or about 100% amino acid sequence identity to SEQ ID NO:2; comprising:
 a. contacting the sample with an immunoreactive antibody; and   b. detecting the presence of the protein.   
     
     
         31 . The method of  claim 30 , wherein the step of detecting comprises an ELISA, or a Western blot.