Compositions and methods for controlling insect pests

Abstract

Disclosed herein are methods of controlling insect pests which infest crop plants, in particular Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), Euschistus heros (neotropical brown stink bug), and Plutella xylostella (diamondback moth), and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and plants with improved resistance to infestation by these insects. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of these insect pests.

What is claimed is: 
     
         1 . A method for controlling an insect infestation of a plant comprising contacting with a dsRNA an insect that infests a plant, wherein said dsRNA comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of said insect, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. 
     
     
         2 . The method of  claim 1 , wherein said insect is selected from the group consisting of  Spodoptera  spp.,  Lygus  spp.,  Euschistus  spp., and  Plutella  spp. 
     
     
         3 . The method of  claim 1 , wherein:
 (a) said insect is  Spodoptera frugiperdo  (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.   
     
     
         4 . The method of  claim 1 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:17-19;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:23-25; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.   
     
     
         5 . The method of  claim 1 , wherein said at least one segment is multiple segments. 
     
     
         6 . The method of  claim 1 , wherein said dsRNA is (a) blunt-ended, or (b) has an overhang at at least one terminus. 
     
     
         7 . The method of  claim 1 , wherein said dsRNA is (a) chemically synthesized, or (b) produced by expression in a microorganism, expression in a plant cell, or by microbial fermentation. 
     
     
         8 . The method of  claim 1 , wherein said dsRNA is chemically modified. 
     
     
         9 . The method of  claim 1 , wherein said contacting comprises application of a composition comprising said dsRNA to a surface of said insect or to a surface of said plant infested by said insect. 
     
     
         10 . The method of  claim 9 , wherein said composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment. 
     
     
         11 . The method of  claim 1 , wherein said contacting comprises providing said dsRNA in a composition that further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. 
     
     
         12 . The method of  claim 1 , wherein said contacting comprises providing said dsRNA in a composition that further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein. 
     
     
         13 . The method of  claim 1 , wherein said contacting comprises providing said dsRNA in a composition that is ingested by said insect. 
     
     
         14 . The method of  claim 13 , wherein said composition that is ingested further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. 
     
     
         15 . The method of  claim 13 , wherein said composition that is ingested further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus insecticidal  protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein. 
     
     
         16 . A method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one recombinant RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of said recombinant RNA by said insect results in mortality or stunting in said insect. 
     
     
         17 . The method of  claim 16 , wherein said recombinant RNA comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarily with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         18 . The method of  claim 16 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.   
     
     
         19 . The method of  claim 16 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said silencing clement comprises a sequence selected from the group consisting of SEQ ID NOs:17-19;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:23-25; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.   
     
     
         20 . The method of  claim 16 , wherein said recombinant RNA is dsRNA. 
     
     
         21 . The method of  claim 16 , wherein said recombinant RNA is (a) blunt-ended dsRNA, or (b) dsRNA with an overhang at at least one terminus. 
     
     
         22 . The method of  claim 16 , wherein said recombinant RNA is provided in a microbial or plant cell or in a microbial fermentation product. 
     
     
         23 . The method of  claim 16 , wherein said recombinant RNA is chemically synthesized. 
     
     
         24 . The method of  claim 16 , wherein said insect is in a larval or nymph stage. 
     
     
         25 . The method of  claim 16 , wherein said insect is an adult. 
     
     
         26 . The method of  claim 20 , wherein said dsRNA comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         27 . An insecticidal composition comprising an insecticidally effective amount of a recombinant RNA molecule, wherein said recombinant RNA molecule comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of an insect that infests a plant, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ III NOs:1-12 and 43-44. 
     
     
         28 . The insecticidal composition of  claim 27 , wherein said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         29 . The insecticidal composition of  claim 27 , wherein said recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         30 . The insecticidal composition of  claim 29 , wherein said dsRNA is at least 50 base pairs in length. 
     
     
         31 . The insecticidal composition of  claim 27 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.   
     
     
         32 . The insecticidal composition of  claim 27 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:17-19;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46;   (c) said insect is  Euschistus heros  (neotropical brown stink hug) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:23-25; or   (d) said insect is  Plutella xylostella  (diamondback moth) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.   
     
     
         33 . The insecticidal composition of  claim 27 , wherein:
 (a) said insect is  Spodoptera frugiperda  (fall armyworm) and said recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:17-19;   (b) said insect is  Lygus hesperus  (western tarnished plant bug) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46;   (c) said insect is  Euschistus heros  (neotropical brown stink bug) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:23-25; or   (d) said insect is  Plutella xylostella  (diamondback moth) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.   
     
     
         34 . The insecticidal composition of  claim 27 , further comprising one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. 
     
     
         35 . The insecticidal composition of  claim 27 , further comprising at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus insecticidal  protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein. 
     
     
         36 . The insecticidal composition of  claim 27 , wherein said composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. 
     
     
         37 . A plant treated with the insecticidal composition of  claim 27 , or seed of said plant, wherein said plant exhibits improved resistance to said insect. 
     
     
         38 . A method of providing a plant having improved resistance to an insect, comprising expressing in said plant a recombinant DNA construct comprising DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of said RNA by said insect results in mortality or stunting in said insect. 
     
     
         39 . The method of  claim 38 , wherein said silencing element has a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         40 . The method of  claim 38 , wherein said recombinant DNA construct further comprises a heterologous promoter operably linked to said DNA encoding RNA that includes at least one silencing clement, wherein said heterologous promoter is functional in a plant cell. 
     
     
         41 . The method of  claim 38 , wherein said expressing is by means of transgenic expression or transient expression. 
     
     
         42 . The method of  claim 38 , further comprising expression in said plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus insecticidal  protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein. 
     
     
         43 . The plant having improved resistance to an insect, provided by the method of  claim 38 . 
     
     
         44 . Fruit, seed, or propagatable parts of the plant of  claim 43 . 
     
     
         45 . A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. 
     
     
         46 . The recombinant DNA construct of  claim 45 , wherein said heterologous promoter is (a) functional for expression of said RNA transcript in a bacterium. 
     
     
         47 . The recombinant DNA construct of  claim 46 , wherein said bacterium is selected from the group consisting of  Escherichia coli, Bacillus  species,  Pseudomonas  species,  Xenorhabdus  species, or  Photorhabdus  species. 
     
     
         48 . The recombinant DNA construct of  claim 45 , wherein said heterologous promoter is functional in a plant cell. 
     
     
         49 . A recombinant vector comprising the recombinant DNA construct of  claim 45 . 
     
     
         50 . The recombinant vector of  claim 49 , wherein said recombinant vector is a recombinant plant virus vector or a recombinant baculovirus vector. 
     
     
         51 . A plant chromosome or plastid comprising the recombinant DNA construct of  claim 45 . 
     
     
         52 . A transgenic plant cell having in its genome the recombinant DNA construct of  claim 45   
     
     
         53 . A transgenic plant comprising the transgenic plant cell of  claim 52 . 
     
     
         54 . A commodity product produced from the transgenic plant of  claim 53 . 
     
     
         55 . A transgenic progeny seed or propagatable plant part of the transgenic plant of  claim 53 .