Biological controls of coleopteran pests

Abstract

Disclosed are double stranded RNA molecules that are toxic to coleopteran insects. In particular, dsRNA molecules that capable of interfering with pest IAP genes and that are toxic to the target pest are provided. Further, methods of making and using the interfering RNA, for example in transgenic plants to confer protection from insect damage are disclosed.

What is claimed is: 
     
         1 . A transgenic plant, or part thereof, comprising a nucleic acid molecule that is or is capable of producing a double stranded (dsRNA) molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18; or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90% complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   wherein the dsRNA molecule is toxic to a  Diabrotica  insect.   
     
     
         2 . The transgenic plant, or part thereof, of  claim 1 , wherein the transgenic plant, or part thereof, is a maize plant or part thereof. 
     
     
         3 . A transgenic seed of the transgenic plant of  claim 1 . 
     
     
         4 . A biological sample from the transgenic plant, or part thereof, of  claim 1 . 
     
     
         5 . A commodity product derived from the transgenic plant, or part thereof, of  claim 1 . 
     
     
         6 . The commodity product of  claim 5 , wherein the commodity product is selected from the group consisting of whole or processed seeds, beans, grains, kernels, hulls, meals, grits, flours, sugars, sugars, starches, protein concentrates, protein isolates, waxes, oils, extracts, juices, concentrates, liquids, syrups, feed, silage, fiber, paper or other food or product produced from plants. 
     
     
         7 . A method of controlling a  Diabrotica  insect comprising contacting the  Diabrotica  insect with a nucleic acid molecule that is or is capable of producing an interfering RNA for inhibiting expression of an IAP gene in the  Diabrotica  insect thereby controlling the  Diabrotica  insect. 
     
     
         8 . The method of  claim 7 , wherein the IAP gene comprises a IAP coding sequence
 a) having from at least about 90% identity to at least about 99% identity to SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:17; or   b) comprising SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:17.   
     
     
         9 . The method of  claim 7 , wherein the interfering RNA is complementary to a portion of a mRNA polynucleotide transcribable from the IAP gene. 
     
     
         10 . The method of  claim 9 , wherein the portion of the mRNA polynucleotide comprises from 20 consecutive nucleotides to at least about 400 consecutive nucleotides of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. 
     
     
         11 . The method of  claim 10 , wherein the portion of the mRNA polynucleotide consists essentially of (a) any 19-mer subsequence of SEQ ID NO:3 (wcrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1603 of SEQ ID NO:3; or (b) any 19-mer subsequence of SEQ ID NO:12 (wcrIAP2) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:12; or (c) any 19-mer subsequence of SEQ ID NO:15 (scrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:15; or (d) any 19-mer subsequence of SEQ ID NO:18 (ncrIAP1) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:18. 
     
     
         12 . The method of  claim 10 , wherein the portion of the mRNA polynucleotide consists essentially of the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15 or SEQ ID NO:18. 
     
     
         13 . The method of  claim 9 , wherein the nucleotide sequence of the interfering RNA consists essentially of (a) any 19-mer subsequence of SEQ ID NO:4 (wcrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to 1603 of SEQ ID NO:4; or (b) any 19-mer subsequence of SEQ ID NO:13 (wcrIAP2*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1444 of SEQ ID NO:13; or (c) any 19-mer subsequence of SEQ ID NO:16 (scrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1018 of SEQ ID NO:16; or (d) any 19-mer subsequence of SEQ ID NO:19 (ncrIAP1*) consisting of N to N+18 nucleotides, wherein N is nucleotide 1 to nucleotide 1020 of SEQ ID NO:19. 
     
     
         14 . The method of  claim 9 , wherein the nucleotide sequence of the interfering RNA consists essentially of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:19. 
     
     
         15 . The method of  claim 7  wherein the  Diabrotica  insect is selected from the group consisting of  D. barberi  (northern corn rootworm),  D. virgifera virgifera  (western corn rootworm),  D. undecimpunctata howardi  (southern corn rootworm),  D. balteata  (banded cucumber beetle),  D. undecimpunctata undecimpunctata  (western spotted cucumber beetle),  D. significata  (3-spotted leaf beetle),  D. speciosa  ( chrysanthemum  beetle) and  D. virgifera zeae  (Mexican corn rootworm). 
     
     
         16 . The method of  claim 15 , wherein the contacting comprises
 a) planting a transgenic seed capable of producing a transgenic plant that expresses the nucleic acid molecule, wherein the  Diabrotica  insect feeds on the transgenic plant, or part thereof; or   b) applying a composition comprising the nucleic acid molecule to a seed or plant, or part thereof,   
       wherein the  Diabrotica  insect feeds on the seed, the plant, or a part thereof. 
     
     
         17 . The method of  claim 16 , wherein the transgenic seed and transgenic plant is a corn seed and a corn plant. 
     
     
         18 . The method of  claim 16 , wherein the seed or plant is a corn seed or corn plant. 
     
     
         19 . A method of reducing an adult  Diabrotica  insect population on a transgenic plant expressing a Cry protein, a hybrid Cry protein or modified Cry protein comprising expressing in the transgenic plant a nucleic acid molecule that is or is capable of producing an interfering RNA capable of inhibiting expression of an IAP gene in an adult  Diabrotica  insect thereby reducing the adult  Diabrotica  insect population. 
     
     
         20 . A method of reducing resistance development in a  Diabrotica  insect population to an interfering RNA capable of inhibiting expression of an IAP gene in a  Diabrotica  insect, the method comprising expressing in a transgenic plant fed upon by the  Diabrotica  insect population an interfering RNA that is capable of inhibiting expression of an IAP gene in a larval and adult  Diabrotica  insect, thereby reducing resistance development in the  Diabrotica  insect population compared to a  Diabrotica  insect population exposed to an interfering RNA capable of inhibiting expression of an IAP gene in a larval or adult  Diabrotica  insect. 
     
     
         21 . A method of reducing the level of a target RNA transcribed from a IAP gene in a  Diabrotica  insect comprising contacting the  Diabrotica  insect with a composition comprising a nucleic acid construct that is or is capable of producing a dsRNA molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 18, or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90$ complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   
       wherein the dsRNA molecule reduces the level of the target RNA in a cell of the  Diabrotica  insect. 
     
     
         22 . The method of  claim 21 , wherein production of an IAP protein encoded by the target RNA is reduced. 
     
     
         23 . The method of  claim 22 , wherein the protein comprises an amino acid sequence of any of SEQ ID NOs:20 to 23. 
     
     
         24 . The method of  claim 21 , wherein the dsRNA is from a transgenic organism expressing the dsRNA. 
     
     
         25 . The method of  claim 24 , wherein the transgenic organism is a transgenic plant, a transgenic bacterium, or a transgenic endophyte. 
     
     
         26 . The method of  claim 21 , wherein the dsRNA is lethal to a  Diabrotica  insect. 
     
     
         27 . The method of  claim 26 , wherein the  Diabrotica  insect is selected from the group consisting of  D. barberi  (northern corn rootworm),  D. virgifera virgifera  (western corn rootworm),  D. undecimpunctata howardi  (southern corn rootworm),  D. balteata  (banded cucumber beetle),  D. undecimpunctata undecimpunctata  (western spotted cucumber beetle),  D. significata  (3-spotted leaf beetle),  D. speciosa  ( chrysanthemum  beetle) and  D. virgifera zeae  (Mexican corn rootworm). 
     
     
         28 . A method of conferring  Diabrotica  insect tolerance to a plant, or part thereof, comprising introducing into the plant, or part thereof, a nucleic acid molecule that is or is capable of producing a dsRNA molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 18, or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90% complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   
       thereby conferring tolerance of the plant or part thereof to the  Diabrotica  insect. 
     
     
         29 . A method of reducing root damage to a plant fed upon by a  Diabrotica  insect, comprising introducing into cells of the plant a nucleic acid molecule that is or is capable of producing a dsRNA molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 18, or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90% complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   
       thereby reducing root damage to the plant. 
     
     
         30 . A method of producing a transgenic plant cell having toxicity to a  Diabrotica  insect, comprising introducing into a plant cell a nucleic acid molecule that is or is capable of producing a dsRNA molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 18, or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90% complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   
       thereby producing the transgenic plant cell having toxicity to the  Diabrotica  insect compared to a control plant cell. 
     
     
         31 . A method of producing a transgenic plant having enhanced tolerance to  Diabrotica  insect feeding damage, comprising introducing into a plant a nucleic acid molecule that is or is capable of producing a dsRNA molecule comprising a sense strand and an antisense strand, wherein:
 a) at least 20 consecutive nucleotides of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene wherein the mRNA polynucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 18, or   b) at least 200 consecutive nucleotides of the antisense strand is at least 90% complementary to a portion of a mRNA polynucleotide transcribable from a  Diabrotica  insect IAP gene, wherein the mRNA polynucleotide comprises at least 200 consecutive nucleotides of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: SEQ ID NO: 15, or SEQ ID NO: 18,   
       thereby producing a transgenic plant having enhanced tolerance to  Diabrotica  insect feeding damage compared to a control plant. 
     
     
         32 . The method of  claim 31 , wherein the introducing step is performed by transforming a plant cell and producing the transgenic plant from the transformed plant cell. 
     
     
         33 . The method of  claim 31 , wherein the introducing step is performed by breeding two plants together. 
     
     
         34 . A method of enhancing control of a  Diabrotica  insect population comprising providing the transgenic plant or part thereof of  claim 1  and applying to the plant a chemical pesticide. 
     
     
         35 . The method of  claim 34 , wherein the chemical pesticide is a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride, a nereistoxin, or a combination thereof. 
     
     
         36 . The method of  claim 35 , wherein the chemical pesticide comprises an active ingredient selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chlorethoxyfos, dimethoate, ethoprophos, malathion, methyl-parathion, phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, bensultap, and a combination thereof. 
     
     
         37 . The method of  claim 36 , wherein the active ingredient is delivered in a product selected from the group consisting of Furadan®, Lanate®, Sevin®, Talstar®, Force®, Ammo®, Cymbush®, Delta Gold®, Karate®, Ambush®, Pounce®, Brigade®, Capture®, ProShield®, Warrior®, Dursban®, Fortress®, Mocap®, Thimet®, AAstar®, Rampart®, Counter®, Cygon®, Dicap®, Regent®, Cruiser®, Gaucho®, Prescribe®, Poncho®, Aztec®, and a combination thereof.