COMPOSITIONS AND METHODS TO CONTROL INSECT PESTS
Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a Coleopteran plant pest or a Diabrotica plant pest, decrease the expression of a target sequence in the pest. Plants, plant parts, 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 polynucleotide comprising:
(a) the nucleotide sequence comprising any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof;
(b) the nucleotide sequence comprising at least 90% sequence identity to any one of nucleotides SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof; wherein said polynucleotide encodes a silencing element having insecticidal activity against a Coleoptera plant pest; or
(c) the nucleotide sequence comprising at least 19 consecutive nucleotides of any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof;
wherein said polynucleotide encodes a silencing element having insecticidal activity against a Coleoptera plant pest.
2 . The expression cassette of claim 1 , wherein said coleopteran plant pest is a Diabrotica 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, 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 any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements 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 any one SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof; or,
(b) the nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof,
wherein said silencing element, when ingested by a Coleoptera plant pest, reduces the level of a target sequence in said Coleoptera plant pest and thereby controls the Coleoptera plant pest.
10 . The plant cell of claim 9 , wherein the Coleoptera plant pest is a Diabrotica plant pest.
11 . The plant cell of claim 9 , wherein said silencing element comprises
(a) a polynucleotide comprising the sequence set forth in any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof; or
b) a polynucleotide comprising at least 130 consecutive nucleotides of the sequence set forth in any one SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof.
12 . The plant cell of claim 9 , wherein said plant cell comprises the expression cassette of claim 8 .
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, 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 any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements 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 . The plant cell of claim 9 , wherein said plant cell is from a dicot.
20 . The plant cell of claim 19 , wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis , or cotton.
21 . A plant or plant part comprising a plant cell of claim 9 .
22 . A transgenic seed from the plant of claim 21 .
23 . A method for controlling a Coleoptera plant pest comprising feeding to a Coleoptera plant pest a composition comprising a silencing element, wherein said silencing element, when ingested by said Coleoptera plant pest, reduces the level of a target Coleoptera plant pest sequence and thereby controls the Coleoptera plant pest, wherein said target Coleoptera plant pest sequence comprise a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof.
24 . The method of claim 23 , wherein said Coleoptera plant pest comprises a Diabrotica plant pest.
25 . The method of claim 23 , wherein said silencing element comprises
a) a fragment of at least 19 consecutive nucleotides of any one SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof; or
b) a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof.
26 . The method of claim 23 , wherein said Diabrotica plant pest comprises D. virgifera virgifera, D. virgifera zeae, D. speciosa, D. barberi, D. virgifera zeae , or D. undecimpunctata howardi.
27 . The method of claim 23 , wherein said composition comprises a plant or plant part having stably incorporated into its genome a polynucleotide comprising said silencing element.
28 . The method of claim 27 , wherein said silencing element comprises
(a) a polynucleotide comprising the sense or antisense sequence of the sequence set forth in any one SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements 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 any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729; or
(c) a polynucleotide comprising the sense or antisense sequence of a sequence having at least 130 contiguous nucleotides of any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof.
29 . The method of claim 27 , wherein said silencing element expresses a double stranded RNA.
30 . The method of claim 27 , wherein said silencing element comprises a hairpin RNA.
31 . The method of claim 30 , 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 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 19 nucleotides having at least 85% complementarity to the first segment.
32 . The method of claim 27 , wherein said silencing element is operably linked to a heterologous promoter.
33 . The method of claim 27 , 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.
34 . The method of claim 27 , wherein said plant is a monocot.
35 . The method of claim 33 , wherein said monocot is maize, barley, millet, wheat or rice.
36 . The method of claim 27 , wherein said plant is a dicot.
37 . The method of claim 36 , wherein said plant is soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis , or cotton.
38 . An isolated polynucleotide comprising a nucleotide sequence comprising:
(a) a nucleotide sequence comprising any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof;
(b) a nucleotide sequence comprising at least 90% sequence identity to any one of nucleotides SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof; or
(c) the nucleotide sequence comprising at least 19 consecutive nucleotides of any one of SEQ ID NOs: 724, 725, 726, 727, 728, 729, or active variants and fragments thereof, and complements thereof;
wherein said polynucleotide encodes a silencing element having insecticidal activity against a Coleoptera plant pest.
39 . The isolated polynucleotide of claim 38 , wherein said Coleoptera plant pest is a Diabrotica plant pest.