Plants resistant to insect pests

• US 9,845,479 B2
• Filed: 05/02/2012
• Issued: 12/19/2017
• Est. Priority Date: 04/20/2011
• Status: Expired due to Fees

• Current Assignee: deVGen NV (China National Chemical Corporation)
• Original Assignee: deVGen NV (China National Chemical Corporation)
• Inventors: Beghyn, Myriam, Bogaert, Thierry Andre Olivier Eddy, Feldmann, Pascale, Raemaekers, Romaan
• Primary Examiner(s): Kovalenko, Mykola V
• Application Number:
  US13/462,610
• Publication Number:
  US 20120297501A1
• Time in Patent Office: 2,057 Days
• Field of Search: None
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: A01N 63/60 Isolated nucleic acids
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention relates to genetic control of infestation by insect pest species, particularly prevention and/or control of pest infestation of plants, using interfering ribonucleic acid (RNA) molecules. The invention provides transgenic plants that (i) express or are capable of expressing interfering RNAs of the invention and (ii) are resistant to infestation by insect pest species.

Claims

1. A transgenic plant, reproductive or propagation material from a transgenic plant, or a cultured plant cell, which comprises a nucleic acid molecule that produces an interfering ribonucleic acid (RNA), wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91 complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

2. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 1, wherein the interfering RNA comprises at least two silencing elements, wherein each silencing element comprises or consists of a sequence of nucleotides which is at least partially complementary to said troponin I target nucleotide sequence within the target gene.

3. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 2, wherein each of the silencing elements comprises or consists of a different sequence of nucleotides which is complementary to a different target nucleotide sequence within said troponin I target gene.

4. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 1, wherein the transgenic plant, material therefrom or plant cell additionally comprises a heterologous gene.

5. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 4, wherein the heterologous gene encodes a protein toxic to a plant pest species.

6. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 5, wherein the protein is selected from the group consisting of a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporus insecticidal protein, and a Bacillus sphaericus insecticidal protein.

7. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant of claim 6, wherein said Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, and a Cry 22.

8. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 4, wherein the heterologous gene encodes a protein conferring herbicide tolerance.

9. The transgenic plant, reproductive or propagation material from a transgenic plant, or cultured transgenic plant cell of claim 8, wherein the protein is selected from a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a dicamba monooxygenase or a phosphinothricin acetyl transferase.

10. Seed produced from the transgenic plant of claim 1, wherein the seed is transgenic and comprises the nucleic acid molecule that produces an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

12. A method for generating a transgenic plant of claim 1, comprising transforming a plant cell with a DNA construct comprising a polynucleotide sequence encoding an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

11. A method for generating a transgenic plant resistant to infestation by Lygus hesperus comprising transforming a plant cell with a DNA construct comprising a polynucleotide sequence encoding an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest, and regenerating a transformed plant from said plant cell.

13. The method of claim 11, wherein the method further comprises the steps of generating progeny from the transformed plant and testing the progeny for resistance to a Lygus hesperus insect.

14. A method for generating a transgenic plant resistant to infestation by Lygus hesperus comprising:

(i) crossing a transgenic plant obtained by the method of claim 11 with a second plant, wherein the second plant is not resistant to infestation by Lygus hesperus,

(ii) selecting progeny resistant to said pest.

15. The method of claim 14 further comprising the step of backcrossing the progeny plant that is resistant to infestation by Lygus hesperus with the second plant, wherein the second plant is not resistant to infestation by Lygus hesperus, and further selecting for progeny resistant to said pest.

16. A method of generating a transgenic plant resistant to infestation by Lygus hesperus comprising:

(i) sexually crossing a transgenic plant obtained by the method of claim 11 with a second plant that lacks the resistance to Lygus hesperus thereby producing a plurality of progeny plants;

(ii) selecting a first progeny plant that is resistant to Lygus hesperus;

(iii) selfing said first progeny plant, thereby producing a plurality of second progeny plants;

(iv) repeated selfing of said progeny for 1, 2, 3, 4, or 5 more generations; and

(v) selecting from any of the progeny plants a plant that is resistant to infestation by Lygus hesperus.

17. The method of claim 14, further comprising identifying the insect-resistant progeny by detecting the presence of a polynucleotide encoding an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

18. The method of claim 14, wherein the second plant comprises a heterologous gene.

19. The method of claim 18, wherein the heterologous gene encodes a protein toxic to a plant pest species.

20. The method of claim 19, wherein the protein is selected from the group consisting of a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporus insecticidal protein, and a Bacillus sphaericus insecticidal protein.

21. The method of claim 20, wherein the Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, and a Cry 22.

22. The method of claim 18, wherein the heterologous gene encodes a protein conferring herbicide tolerance.

23. The method according to claim 11 or claim 20, wherein the plant resistant to infestation by Lygus hesperus is selected from the group consisting of cotton, potato, rice, canola, sunflower, sorghum, pearl millet, corn, strawberry, soy, alfalfa, tomato, eggplant, pepper and tobacco.

24. A transgenic plant produced by the method of claim 11.

25. Seed produced by the plant of claim 24, wherein the seed is transgenic and comprises a DNA construct comprising a polynucleotide sequence encoding an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

26. A method for preventing and/or controlling infestation by Lygus hesperus in a field of crop plants, said method comprising expressing in said plants an effective amount of an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

27. A host cell comprising a polynucleotide encoding an interfering RNA, wherein the interfering RNA comprises at least one silencing element, wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 70 contiguous nucleotides that is at least 91% complementary to a fragment of SEQ ID NO:

1 or SEQ ID NO;

2, or a sequence of at least 27 contiguous nucleotides that is complementary to SEQ ID NO;

1 or SEQ ID NO;

2, wherein the interfering RNA down regulates the troponin I gene in a Lygus hesperus pest.

28. The host cell of claim 27 wherein the host cell is a prokaryotic or a eukaryotic cell.

29. The host cell of claim 28 wherein the host cell is a bacterial cell or a plant cell.