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 or a N. viridula, Acrosternum hilare, Piezodorus guildini , and/or Halymorpha halys plant pest, decrease the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant. The present invention provides various target polynucleotides set forth in any one of SEQ ID NOS: 1-292 or 302-304 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). Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is Pentatomidae . 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.

That which is claimed: 
     
         1 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) the nucleotide sequence comprising any one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 or a complement thereof;   (b) the nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 or a complement thereof, wherein said polynucleotide encodes a silencing element having insecticidal activity against a  Pentatomidae  plant pest;   (c) the nucleotide sequence comprising at least 19 consecutive nucleotides of any one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof, wherein said polynucleotide encodes a silencing element having insecticidal activity against a  Pentatomidae  plant pest; and,   (d) the 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 isolated polynucleotide of  claim 1 , wherein said  Pentatomidae  plant pest is a  N. viridula  plant pest. 
     
     
         3 . An expression cassette comprising a heterologous polynucleotide of  claim 1  operably linked to a seed-preferred promoter. 
     
     
         4 . The expression cassette of  claim 3 , wherein said polynucleotide is expressed as a double stranded RNA. 
     
     
         5 . The expression cassette of  claim 3 , 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, in the following order, 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: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263;   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 6 , wherein said target sequence comprises the sequences set forth any one of SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 337, 338, 339, 340, 341, 342, 343 or 344 or a sequence having at least 90% sequence identity to SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 337, 338, 339, 340, 341, 342, 343 or 344. 
     
     
         8 . The expression cassette of  claim 6 , wherein said expression cassette comprises any one of SEQ ID NOS: 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328. 
     
     
         9 . The expression cassette of  claim 3 , 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. 
     
     
         10 . A host cell comprising the heterologous expression cassette of  claim 3 . 
     
     
         11 . A plant cell having stably incorporated into its genome a heterologous polynucleotide comprising a silencing element operably linked to a seed-preferred promoter, wherein said silencing element, when ingested by a  Pentatomidae  plant pest, reduces the level of expression of any one of the target sequences set forth in SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 in said  Pentatomidae  plant pest and thereby controls the  Pentatomidae  plant pest. 
     
     
         12 . The plant cell of  claim 11 , wherein said silencing element comprises
 a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof; or,   b) the nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 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.   
     
     
         13 . The plant cell of  claim 12 , wherein the  Pentatomidae  plant pest is a  N. viridula  plant pest. 
     
     
         14 . The plant cell of  claim 12 , wherein said silencing element comprises the sequences set forth in any one of SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 305, 306, 307, 308, 309, 310, 311, 312, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343 or 344 or a complement thereof. 
     
     
         15 . The plant cell of  claim 11 , wherein said plant cell comprises the expression cassette of  claim 9 . 
     
     
         16 . The plant cell of  claim 11 , wherein said silencing element expresses a double stranded RNA. 
     
     
         17 . The plant cell of  claim 11 , wherein said silencing element expresses a hairpin RNA. 
     
     
         18 . The plant cell of  claim 17 , wherein said polynucleotide comprising the silencing element comprises, in the following order, 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: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328;   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.   
     
     
         19 . The plant cell of  claim 11 , wherein said plant cell is from a monocot. 
     
     
         20 . The plant cell of  claim 19 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         21 . The plant cell of  claim 11 , wherein said plant cell is from a dicot. 
     
     
         22 . The plant cell of  claim 21 , wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis , or cotton. 
     
     
         23 . A plant or plant part comprising a plant cell of  claim 11 . 
     
     
         24 . A transgenic seed from the plant of  claim 23 , wherein said transgenic seed comprises said heterologous polynucleotide comprising said silencing element. 
     
     
         25 . A method of 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 expression of any one of the target  Pentatomidae  plant pest sequences set forth in SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 and thereby controls the  Pentatomidae  plant pest. 
     
     
         26 . The method of  claim 25 , wherein said silencing element comprises
 a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof; or,   b) the nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 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.   
     
     
         27 . The method of  claim 26 , wherein said  Pentatomidae  plant pest comprises a  N. viridula  plant pest. 
     
     
         28 . The method of  claim 26 , wherein said silencing element comprises the sequence set forth in any one of SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 305, 306, 307, 308, 309, 310, 311, 312, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343 or 344 or a complement thereof. 
     
     
         29 . The method of  claim 25 , wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide comprising said silencing element, wherein said silencing element is operably linked to a seed-preferred promoter. 
     
     
         30 . The method of  claim 25 , wherein said silencing element comprises
 a) a polynucleotide comprising the sense or antisense sequence of the sequence set forth in SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18, 263, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311 or 312 or a complement thereof; or,   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:   284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18, 263, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311 or 312 ora complement thereof.   
     
     
         31 . The method of  claim 25 , wherein said silencing element expresses a double stranded RNA. 
     
     
         32 . The method of  claim 25 , wherein said silencing element comprises a hairpin RNA. 
     
     
         33 . The method of  claim 32 , wherein said polynucleotide comprising the silencing element comprises, in the following order, 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.   
     
     
         34 . The method of  claim 29 , 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. 
     
     
         35 . The method of  claim 29 , wherein said plant is a monocot. 
     
     
         36 . The method of  claim 35 , wherein said monocot is maize, barley, millet, wheat or rice. 
     
     
         37 . The method of  claim 29 , wherein said plant is a dicot. 
     
     
         38 . The method of  claim 37 , wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco,  Arabidopsis , or cotton.