Compositions and methods for insecticidal control of stinkbugs
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.
1. An expression cassette, comprising a heterologous promoter operably linked to a polynucleotide encoding a double stranded RNA, wherein the double stranded RNA comprises the sense and antisense sequence of a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence comprising any one of SEQ ID NOS: 9, or 30-34;
(b) a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NOS: 9, or 30-34; and
(c) a nucleotide sequence comprising at least 23 consecutive nucleotides of any one of SEQ ID NOS: 9, or 30-34;
wherein said double stranded RNA has 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 double stranded RNA is expressed as a hairpin RNA.
4. The expression cassette of claim 3 , wherein the double stranded RNA comprises, a first segment, a second segment, and a third segment, wherein
a) said first segment comprises a sense or antisense nucleic acid sequence of at least 23 nucleotides of a target sequence set forth in SEQ ID NOS: 9, or 30-34;
b) said second segment comprises a loop of sufficient length to allow the double stranded RNA to be transcribed as a hairpin RNA; and,
c) said third segment comprises a sense or antisense nucleic acid sequence of at least 23 nucleotides having at least 95% complementarity to the first segment.
5. 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 double stranded RNA.
6. A host cell comprising a heterologous expression cassette of claim 1 .
7. A plant cell having stably incorporated into its genome a heterologous polynucleotide encoding a double stranded RNA, wherein the double stranded RNA comprises the sense and antisense sequence of a nucleotide sequence selected from the group consisting of:
a) at least 23 consecutive nucleotides of SEQ ID NOS: 9, or 30-34; or,
b) the nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NOS: 9, or 30-34,
wherein said polynucleotide encodes a double stranded RNA that controls the Pentatomidae plant pest.
8. The plant cell of claim 7 , wherein the Pentatomidae plant pest is a N. viridula plant pest.
9. The plant cell of claim 7 , wherein said double stranded RNA comprises
a) a polynucleotide comprising a sense or antisense sequence of a nucleotide sequence as set forth in SEQ ID NOS: 9, or 30-34;
b) a polynucleotide comprising a sense or antisense sequence of at least 75 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NOS: 9, or 30-34.
10. The plant cell of claim 7 , wherein said double stranded RNA expresses a hairpin RNA.
11. The plant cell of claim 10 , wherein said polynucleotide comprising the double stranded RNA comprises a first segment, a second segment, and a third segment, wherein
a) said first segment comprises a sense or antisense nucleic acid sequence of at least 23 nucleotides complementarity to a target sequence set forth in SEQ ID NOS: 9, or 30-34;
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 a sense or antisense nucleic acid sequence of at least 23 nucleotides having at least 95% complementarity to the first segment.
12. The plant cell of claim 7 , wherein said polynucleotide is operably linked to a heterologous promoter.
13. The plant cell of claim 7 , wherein said plant cell is from a monocot.
14. The plant cell of claim 13 , wherein said monocot is maize, barley, millet, wheat or rice.
15. The plant cell of claim 7 , wherein said plant cell is from a dicot.
16. The plant cell of claim 15 , wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis , or cotton.
17. A plant or plant part comprising a plant cell of claim 7 .
18. A transgenic seed from the plant of claim 17 .
19. A method for controlling a Pentatomidae plant pest comprising feeding to a Pentatomidae plant pest a composition comprising a double stranded RNA, wherein said double stranded RNA, when ingested by said Pentatomidae plant pest, reduces the level of a target Pentatomidae plant pest sequence and thereby controls the Pentatomidae plant pest, and wherein said double stranded RNA comprises
a) the sense and antisense sequence of at least 23 consecutive nucleotides of SEQ ID NOS: 9, or 30-34; or,
b) the sense and antisense sequence of a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NOS: 9, or 30-34.
20. The method of claim 19 , wherein said Pentatomidae plant pest comprises a N. viridula plant pest.
21. The method of claim 19 , wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide encoding said double stranded RNA.
22. The method of claim 21 , wherein said double stranded RNA comprises
a) the sense or antisense sequence of the sequence set forth in SEQ ID NOS: 9, or 30-34;
b) the sense or antisense sequence of a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NOS: 9, or 30-34;
c) the sense or antisense sequence of a sequence having at least 75 contiguous nucleotides of SEQ ID NOS: 9, or 30-34.
23. The method of claim 22 , wherein said double stranded RNA comprises a hairpin RNA.
24. The method of claim 23 , wherein said polynucleotide encoding the double stranded RNA comprises a first segment, a second segment, and a third segment, wherein
a) said first segment comprises a sense or antisense nucleic acid sequence of at least 23 nucleotides 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 a sense or antisense nucleic acid sequence of at least 23 nucleotides having at least 95% complementarity to the first segment
wherein said double stranded RNA has insecticidal activity against a Pentatomidae plant pest.
25. The method of claim 21 , wherein said polynucleotide is operably linked to a heterologous promoter.
26. The method of claim 21 , 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 double stranded RNA.
27. The method of claim 19 , wherein said plant is a monocot.
28. The method of claim 27 , wherein said monocot is maize, barley, millet, wheat or rice.
29. The method of claim 19 , wherein said plant is a dicot.
30. The method of claim 27 , wherein said dicot is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis , or cotton.