Compositions and methods for insecticidal control of stinkbugs

Abstract

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a Pentatomidae plant pest, decrease the expression of a target sequence in the pest. The present invention provides various target polynucleotides set forth in any one of SEQ ID NOS: 6-12, 18-40 or active variants and fragments thereof, wherein a decrease in expression of one or more the sequences in the target pest controls the pest (i.e., has insecticidal activity). Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

What is claimed is: 
     
         1 . An expression cassette, comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence comprising any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof;   (b) a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof, wherein said polynucleotide encodes a silencing element having insecticidal activity against a Pentatomidae plant pest;   (c) a nucleotide sequence comprising at least 19 consecutive nucleotides of any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof, wherein said polynucleotide encodes a silencing element having insecticidal activity against a Pentatomidae plant pest; and,   (d) a nucleotide sequence that hybridizes under stringent conditions to the full length complement of the nucleotide sequence of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C., wherein said polynucleotide encodes a silencing element having insecticidal activity against a Pentatomidae plant pest.   
     
     
         2 . The expression cassette of  claim 1 , wherein said Pentatomidae plant pest is a  N. viridula  plant pest. 
     
     
         3 . The expression cassette of  claim 1 , wherein said polynucleotide is operably linked to a heterologous promoter. 
     
     
         4 . The expression cassette of  claim 1 , wherein said polynucleotide is expressed as a double stranded RNA. 
     
     
         5 . The expression cassette of  claim 1 , wherein said polynucleotide comprise a silencing element which is expressed as a hairpin RNA. 
     
     
         6 . The expression cassette of  claim 5 , wherein the silencing element comprises, a first segment, a second segment, and a third segment, wherein
 a) said first segment comprises at least about 19 nucleotides having at least 90% sequence complementarity to a target sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof;   b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,   c) said third segment comprises at least about 19 nucleotides having at least 85% complementarity to the first segment.   
     
     
         7 . The expression cassette of  claim 1 , wherein said polynucleotide is flanked by a first operably linked convergent promoter at one terminus of the polynucleotide and a second operably linked convergent promoter at the opposing terminus of the polynucleotide, wherein the first and the second convergent promoters are capable of driving expression of the polynucleotide. 
     
     
         8 . A host cell comprising a heterologous expression cassette of  claim 1 . 
     
     
         9 . A plant cell having stably incorporated into its genome a heterologous polynucleotide comprising a silencing element, wherein said silencing element comprises
 a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof; or,   b) the nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof,   wherein said silencing element, when ingested by a Pentatomidae plant pest, reduces the level of a target sequence in said Pentatomidae plant pest and thereby controls the Pentatomidae plant pest.   
     
     
         10 . The plant cell of  claim 9 , wherein the Pentatomidae plant pest is a  N. viridula  plant pest. 
     
     
         11 . The plant cell of  claim 9 , wherein said silencing element comprises
 a) a polynucleotide comprising the sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof;   b) a polynucleotide comprising at least 75 consecutive nucleotides of the sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof.   
     
     
         12 . The plant cell of  claim 9 , wherein said plant cell comprises the expression cassette of  claim 7 . 
     
     
         13 . The plant cell of  claim 9 , wherein said silencing element expresses a double stranded RNA. 
     
     
         14 . The plant cell of  claim 9 , wherein said silencing element expresses a hairpin RNA. 
     
     
         15 . The plant cell of  claim 14 , wherein said polynucleotide comprising the silencing element comprises, a first segment, a second segment, and a third segment, wherein
 a) said first segment comprises at least about 19 nucleotides having at least 90% sequence complementarity to a target sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof;   b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,   c) said third segment comprises at least about 19 nucleotides having at least 85% complementarity to the first segment.   
     
     
         16 . The plant cell of  claim 9 , wherein said silencing element is operably linked to a heterologous promoter. 
     
     
         17 . The plant cell of  claim 9 , wherein said plant cell is from a monocot. 
     
     
         18 . The plant cell of  claim 17 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         19 - 24 . (canceled) 
     
     
         25 . The plant cell of  claim 9 , wherein said plant cell is from a dicot. 
     
     
         26 . The plant cell of  claim 25 , wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis,  or cotton. 
     
     
         27 - 32 . (canceled) 
     
     
         33 . A plant or plant part comprising a plant cell of  claim 9 . 
     
     
         34 . A transgenic seed from the plant of  claim 33 . 
     
     
         35 . A method for controlling a Pentatomidae plant pest comprising feeding to a Pentatomidae plant pest a composition comprising a silencing element, wherein said silencing element, when ingested by said Pentatomidae plant pest, reduces the level of a target Pentatomidae plant pest sequence and thereby controls the Pentatomidae plant pest, wherein said target Pentatomidae plant pest sequence comprise a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof. 
     
     
         36 . The method of  claim 35 , wherein said Pentatomidae plant pest comprises a  N. viridula  plant pest. 
     
     
         37 . The method of  claim 35 , wherein said silencing element comprises
 a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof or,   b) a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof.   
     
     
         38 . The method of  claim 35 , wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide comprising said silencing element. 
     
     
         39 . The method of  claim 38 , wherein said silencing element comprises
 a) a polynucleotide comprising the sense or antisense sequence of the sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof;   b) a polynucleotide comprising the sense or antisense sequence of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof;   c) a polynucleotide comprising the sense or antisense sequence of a sequence having at least 75 contiguous nucleotides of SEQ ID NOS: 6-12, 18-40, a fragment or variant thereof, or a complement thereof.   
     
     
         40 . The method of  claim 39 , wherein said silencing element expresses a double stranded RNA. 
     
     
         41 . The method of  claim 39 , wherein said silencing element comprises a hairpin RNA. 
     
     
         42 . The method of  claim 41 , wherein said polynucleotide comprising the silencing element comprises, a first segment, a second segment, and a third segment, wherein
 a) said first segment comprises at least about 20 nucleotides having at least 90% sequence complementarity to the target polynucleotide;   b) said second segment comprises a loop of sufficient length to allow the silencing element to be transcribed as a hairpin RNA; and,   c) said third segment comprises at least about 20 nucleotides having at least 85% complementarity to the first segment.   
     
     
         43 . The method of  claim 35 , wherein said silencing element is operably linked to a heterologous promoter. 
     
     
         44 . The method of  claim 35 , wherein said silencing element is flanked by a first operably linked convergent promoter at one terminus of the silencing element and a second operably linked convergent promoter at the opposing terminus of the polynucleotide, wherein the first and the second convergent promoters are capable of driving expression of the silencing element. 
     
     
         45 . The method of  claim 35 , wherein said plant is a monocot. 
     
     
         46 . The method of  claim 45 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         47 . The method of  claim 38 , wherein said plant is a monocot. 
     
     
         48 - 54 . (canceled) 
     
     
         55 . The method of any one of  claims 35 - 37 , wherein said plant is a dicot. 
     
     
         56 . The method of  claim 45 , wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis,  or cotton. 
     
     
         57 - 66 . (canceled)