Parental rnai suppression of chromatin remodeling genes to control coleopteran pests

Abstract

This disclosure concerns nucleic acid molecules and methods of use thereof for control of hemipteran pests through RNA interference-mediated inhibition of target coding and transcribed non-coding sequences in hemipteran pests. The disclosure also concerns methods for making transgenic plants that express nucleic acid molecules useful for the control of hemipteran pests, and the plant cells and plants obtained thereby.

1 . An isolated nucleic acid molecule comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide comprises:
 SEQ ID NO:5; the complement of SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:5; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:5; a native coding sequence of a hemipteran insect comprising SEQ ID NO:5; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:5;   SEQ ID NO:7; the complement of SEQ ID NO:7; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:7; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:7; a native coding sequence of a hemipteran insect comprising SEQ ID NO:7; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:7;   SEQ ID NO:10; the complement of SEQ ID NO:10; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; a native coding sequence of a hemipteran insect comprising SEQ ID NO:10; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:10;   SEQ ID NO:8; SEQ ID NO:64; the complement of SEQ ID NO:8; the complement of SEQ ID NO:64; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:8; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:8; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:64; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:64; a native coding sequence of a hemipteran insect comprising SEQ ID NO:8; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:8; a native coding sequence of a hemipteran insect comprising SEQ ID NO:64; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:64; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:69; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:69; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:16; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:16;   SEQ ID NO:10; SEQ ID NO:65; the complement of SEQ ID NO:10; the complement of SEQ ID NO:65; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:10; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:65; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:65; a native coding sequence of a hemipteran insect comprising SEQ ID NO:10; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:10; a native coding sequence of a hemipteran insect comprising SEQ ID NO:65; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:65; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:70; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:70; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:17; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:17;   SEQ ID NO:12; SEQ ID NO:66; the complement of SEQ ID NO:12; the complement of SEQ ID NO:66; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:12; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:12; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:66; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:66; a native coding sequence of a hemipteran insect comprising SEQ ID NO:12; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:12; a native coding sequence of a hemipteran insect comprising SEQ ID NO:66; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:66; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:71; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:71; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:18; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:18;   SEQ ID NO:14; SEQ ID NO:67; the complement of SEQ ID NO:14; the complement of SEQ ID NO:67; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:14; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:14; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:67; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:67; a native coding sequence of a hemipteran insect comprising SEQ ID NO:14; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:14; a native coding sequence of a hemipteran insect comprising SEQ ID NO:67; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:67; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:72; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule comprising SEQ ID NO:72; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:19; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:19;   SEQ ID NO:30; the complement of SEQ ID NO:30; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:30; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:30; a native coding sequence of a hemipteran insect comprising SEQ ID NO:30; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:30; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule translated into the polypeptide of SEQ ID NO:31; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule translated into the polypeptide of SEQ ID NO:31; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:30; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:30;   SEQ ID NO:32; the complement of SEQ ID NO:32; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:32; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:32; a native coding sequence of a hemipteran insect comprising SEQ ID NO:32; the complement of a native coding sequence of a hemipteran insect comprising SEQ ID NO:32; a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule translated into the polypeptide of SEQ ID NO:33; the complement of a native coding sequence of a hemipteran insect that is transcribed into a native RNA molecule translated into the polypeptide of SEQ ID NO:33; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:32; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a hemipteran insect comprising SEQ ID NO:32.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the polynucleotide encodes a double-stranded ribonucleic acid (dsRNA) molecule, the polynucleotide further comprising a second nucleotide sequence and a third nucleotide sequence, wherein the third nucleotide sequence is operably linked to the first nucleotide sequence by the second nucleotide sequence, and wherein the third nucleotide sequence is substantially the reverse complement of the first nucleotide sequence. 
     
     
         3 . A double-stranded ribonucleic acid (dsRNA) molecule comprising a polyribonucleotide encoded by the polynucleotide of the nucleic acid molecule of  claim 1 . 
     
     
         4 . The double-stranded ribonucleic acid molecule of  claim 3 , wherein contacting said ribonucleotide molecule with a hemipteran pest kills or inhibits the growth, reproduction, and/or feeding of the pest. 
     
     
         5 . A cell transformed with the polynucleotide of  claim 1 . 
     
     
         6 . A plant transformation vector comprising the polynucleotide of  claim 1 , wherein the heterologous promoter is functional in a plant cell. 
     
     
         7 . The plant of  claim 6 , wherein the at least one polynucleotide is expressed in the plant as a double-stranded ribonucleic acid molecule. 
     
     
         8 . The plant of  claim 6 , wherein the at least one polynucleotide is expressed in the plant as a ribonucleic acid molecule, and the ribonucleic acid molecule inhibits the expression of an endogenous polynucleotide that is specifically complementary to the at least one polynucleotide when a hemipteran pest ingests a part of the plant. 
     
     
         9 . The polynucleotide of  claim 1 , further comprising at least one additional polynucleotide that encodes an RNA molecule that inhibits the expression of an endogenous pest gene. 
     
     
         10 . The polynucleotide of  claim 9 , wherein the additional polynucleotide encodes an iRNA molecule that results in a parental RNAi phenotype. 
     
     
         11 . The polynucleotide of  claim 9 , wherein the additional polynucleotide encodes an iRNA molecule that inhibits the expression of a hunchback or kruppel gene. 
     
     
         12 . The polynucleotide of  claim 9 , wherein the additional polynucleotide encodes an iRNA molecule that results in decreased growth and/or development and/or mortality in a hemipteran pest that contacts the iRNA molecule (lethal RNAi). 
     
     
         13 . A plant transformation vector comprising the polynucleotide of  claim 9 , wherein the additional polynucleotide(s) are each operably linked to a heterologous promoter functional in a plant cell. 
     
     
         14 . A method for controlling a hemipteran pest population, the method comprising:
 introducing into a hemipteran pest, a ribonucleic acid (RNA) molecule that functions upon contact with the hemipteran pest to inhibit a biological function within the hemipteran pest, wherein the RNA is specifically hybridizable with a polynucleotide selected from the group consisting of any of   SEQ ID NOs:43-62 and 68-72,   the complement of any of SEQ ID NOs:43-62 and 68-72,   a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and 68-72,   the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and 68-72,   a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67,   the complement of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, and SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67,   a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, and SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, and   the complement of a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, and SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67   thereby producing a hemipteran pest having a pRNAi phenotype.   
     
     
         15 . A method for controlling a hemipteran pest population, the method comprising:
 providing an agent comprising a first and a second polynucleotide sequence that functions upon contact with the hemipteran pest to inhibit a biological function within the hemipteran pest, wherein the first polynucleotide sequence comprises a region that exhibits from about 90% to about 100% sequence identity to from about 19 to about 30 contiguous nucleotides of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72, and wherein the first polynucleotide sequence is specifically hybridized to the second polynucleotide sequence.   
     
     
         16 . The method according to  claim 15 , wherein the agent is a double-stranded RNA molecule. 
     
     
         17 . The method according to  claim 15 , wherein the hemipteran pest population is reduced relative to a population of the same pest species infesting a host plant of the same host plant species lacking the transformed plant cell. 
     
     
         18 . The method according to  claim 17 , wherein the ribonucleic acid molecule is a double-stranded ribonucleic acid molecule. 
     
     
         19 . The method according to  claim 17 , wherein the hemipteran pest population is reduced relative to a hemipteran pest population infesting a host plant of the same species lacking the transformed plant cell. 
     
     
         20 . A method of controlling hemipteran pest infestation in a plant, the method comprising providing in the diet of a hemipteran pest a ribonucleic acid (RNA) that is specifically hybridizable with a polynucleotide selected from the group consisting of:
 SEQ ID NOs:43-62 and 68-72,   the complement of any of SEQ ID NOs:43-62 and 68-72,   a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and 68-72,   the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:43-62 and 68-72,   a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67,   the complement of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67,   a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, and   the complement of a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.   
     
     
         21 . The method according to  claim 20 , wherein the diet comprises a plant cell transformed to express the polynucleotide. 
     
     
         22 . The method according to  claim 20 , wherein the specifically hybridizable RNA is comprised in a double-stranded RNA molecule. 
     
     
         23 . The method according to  claim 20 , wherein the RNA molecule is a double-stranded RNA molecule. 
     
     
         24 . A method for producing a transgenic plant cell, the method comprising:
 transforming a plant cell with a vector comprising a means for protecting a plant from a hemipteran pest;   culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transformed plant cells;   selecting for transformed plant cells that have integrated the means for protecting a plant from a hemipteran pest into their genomes;   screening the transformed plant cells for expression of a means for inhibiting expression of an essential gene in a hemipteran pest; and   selecting a plant cell that expresses the means for inhibiting expression of an essential gene in a hemipteran pest.   
     
     
         25 . A method for producing a hemipteran pest-resistant transgenic plant, the method comprising:
 providing the transgenic plant cell produced by the method of  claim 24 ; and   regenerating a transgenic plant from the transgenic plant cell, wherein expression of the means for inhibiting expression of an essential gene in a hemipteran pest is sufficient to modulate the expression of a target gene in a hemipteran pest that contacts the transformed plant.