Compositions and methods for controlling Leptinotarsa
First Claim
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1. A method for controlling a Leptinotarsa species infestation of a plant comprising:
- (a) contacting said Leptinotarsa species with a double-stranded RNA (dsRNA) comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(b) providing in the diet of said Leptinotarsa species a dsRNA comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(c) causing mortality or stunting in larvae of said Leptinotarsa species by providing in the diet of said larvae at least one dsRNA comprising at least one silencing element comprising a strand comprising 22 contiguous nucleotides that are complementary to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(d) topically applying to said plant a composition comprising at least one dsRNA comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one polynucleotide is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide;
or(e) expressing in said plant at least one dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one polynucleotide is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide;
or(f) providing to said plant at least one dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(g) contacting said Leptinotarsa species with a dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said dsRNA is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide.
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Abstract
Disclosed herein are methods of controlling insect pests, in particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with improved resistance to infestation by Leptinotarsa spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of Leptinotarsa spp.
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Citations
19 Claims
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1. A method for controlling a Leptinotarsa species infestation of a plant comprising:
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(a) contacting said Leptinotarsa species with a double-stranded RNA (dsRNA) comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(b) providing in the diet of said Leptinotarsa species a dsRNA comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(c) causing mortality or stunting in larvae of said Leptinotarsa species by providing in the diet of said larvae at least one dsRNA comprising at least one silencing element comprising a strand comprising 22 contiguous nucleotides that are complementary to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(d) topically applying to said plant a composition comprising at least one dsRNA comprising a strand comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one polynucleotide is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide;
or(e) expressing in said plant at least one dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one polynucleotide is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide;
or(f) providing to said plant at least one dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(g) contacting said Leptinotarsa species with a dsRNA comprising at least one segment that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said dsRNA is provided in a manner that permits a Leptinotarsa species feeding on said plant to ingest said at least one polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. An insecticidal composition for controlling a Leptinotarsa species, comprising:
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(a) at least one double-stranded RNA (dsRNA) consisting of two strands, wherein a first strand comprises at least 22 contiguous nucleotides that are complementary to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one dsRNA causes mortality or stunting of growth in said Leptinotarsa species when ingested or contacted by said Leptinotarsa species;
or(b) at least one dsRNA consisting of at least one silencing element that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one dsRNA causes mortality or stunting of growth in said Leptinotarsa species when ingested or contacted by said Leptinotarsa species;
or(c) at least one dsRNA consisting of two strands, wherein a first strand comprises a sequence that is identical or complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730, wherein said at least one dsRNA causes mortality or stunting of growth in said Leptinotarsa species when ingested or contacted by said Leptinotarsa species;
or(d) a dsRNA molecule that causes mortality or stunting of growth in a Leptinotarsa species when ingested or contacted by said Leptinotarsa species, wherein said dsRNA molecule consists of two strands, wherein a first strand comprises at least 22 contiguous nucleotides that are complementary to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(e) an insecticidal double-stranded RNA molecule that causes mortality or stunting of growth in a Leptinotarsa species when ingested or contacted by said Leptinotarsa species, wherein at least one strand of said insecticidal double-stranded RNA molecule comprises 22 contiguous nucleotides that are complementary to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(f) at least one double-stranded RNA consisting of two strands, wherein the sequence of a first strand is selected from the group consisting of SEQ ID NO;
989, 988, 1103 1105, and 1116-1119, wherein said at least one polynucleotide causes mortality or stunting of growth in said Leptinotarsa species when ingested or contacted by said Leptinotarsa species. - View Dependent Claims (12, 13, 14)
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15. A recombinant DNA construct comprising a heterologous promoter operably linked to:
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(a) DNA comprising a nucleotide sequence that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(b) DNA comprising 22 or more contiguous nucleotides having 100% identity to a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(c) DNA encoding at least one silencing element that is complementary to at least 22 contiguous nucleotides of a sequence corresponding to positions 227 to 758 of SEQ ID NO;
730;
or(e) DNA encoding a RNA comprising at least 22 contiguous nucleotides that are complementary to a nucleotide sequence selected from the group consisting of;
SEQ ID NOs;
989, 988, 1103 1105, and 1116-1119, or the complement thereof, or an orthologous nucleotide sequence from a Leptinotarsa species or a Tribolium species, wherein the orthologous nucleotide sequence has at least 98% sequence identity with a nucleotide sequence selected from the group consisting of;
SEQ ID NOs;
989, 988, 1103 1105, and 1116-1119, wherein the percentage sequence identity is calculated over the same length;
or(f) DNA encoding a RNA comprising at least one double-stranded RNA region, at least one strand of which comprises at least 22 contiguous nucleotides that are complementary to a nucleotide sequence selected from the group consisting of;
SEQ ID NOs;
989, 988, 1103 1105, and 1116-1119, or the complement thereof, or an orthologous nucleotide sequence from a Leptinotarsa species or a Tribolium species, wherein the orthologous nucleotide sequence has at least 98% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs;
989, 988, 1103 1105, and 1116-1119, wherein the percentage sequence identity is calculated over the same length;
or(g) DNA encoding RNA comprising a nucleotide sequence selected from the group consisting of;
SEQ ID NOs;
989, 988, 1103 1105, and 1116-1119, or the complement thereof. - View Dependent Claims (16, 17, 18, 19)
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Specification