CONTROL OF PESTS IN PLANTS

• US 20120240288A1
• Filed: 09/15/2010
• Published: 09/20/2012
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Temasek Life Sciences Laboratory Limited
• Original Assignee: Temasek Life Sciences Laboratory Limited
• Inventors: Ye, Jian, Qu, Jing, Chua, Nam-Hai, Gao, Shi-Qiang
• Granted Patent: US 10,017,774 B2
• Time in Patent Office: Days
• US Class Current: 800/279
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8285 for nematode resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention relates to the field of controlling pests, such as insects, using a virus to express pest genes in hosts. More specifically, the present invention relates to a method for rapidly screening for pest genes which can lead to mortality of the pest when the pest has ingested host tissues expressing virus-linked pest gene sequences. The present invention also relates to a method for controlling pests by viral expression of target pest sequences to modify endogenous expression of pest genes in cells or tissues of the pest.

Claims

1. A method of screening pest genes to identify pest genes which can lead to mortality of the pest when expression of the pest gene is silenced in the pest, the method comprising:

(a) inserting a nucleic acid comprising a sequence of a first desired gene into a virus-induced gene silencing (VIGS) vector of a virus that can infect a desired host to produce a modified VIGS vector, wherein the first desired gene is a pest gene to be screened;

(b) inoculating the host with the modified VIGS vector to produce infected host;

(c) growing the infected host under conditions in which the modified VIGS vector is replicated to produce RNA which accumulates in tissue of the host;

(d) feeding the host tissue with RNA to pests; and

(e) determining whether the RNA is toxic to the pest,

wherein pest toxicity identifies the pest gene as a pest gene that leads to mortality of the pest when the pest gene expression is silenced in the pest.

2. The method of claim 1, wherein the virus is a DNA virus or an RNA virus.

4. The method of claim 2, wherein the RNA virus is an RNA virus containing two RNA molecules and one of the RNA molecules is the VIGS vector.

5. The method of claim 4, wherein the RNA virus is tobacco rattle virus (TRV).

6. The method of claim 4, wherein the host is inoculated with the modified VIGS vector and a vector containing the other RNA molecule.

7. The method of claim 1, wherein the host is a plant and the host tissue is a plant leaf.

8. The method of claim 1, wherein the nucleic acid further comprises a sequence of a second desired gene, wherein the second desired gene is a plant host resistance gene.

9. The method of claim 8, wherein the host resistance gene is selected from the group consisting of RNase III Dicer-like 4 (DCL4) gene, RNase III Dicer-like 2 (DCL2) gene, RNase III Dicer-like 3 (DCL3) gene, ARGONAUTE1 (AGO1), ARGONAUTE71 (AGO7), RNA-dependent RNA polymerase 1 (RDR1), RNA-dependent RNA polymerase 6 (RDR6), Suppressor of gene silencing 1 (SGS1), Suppressor of gene silencing 3 (SGS3), and Silencing defective 3 (SDE3).

10. The method of claim 1, wherein the nucleic acid further comprises a sequence of a second desired gene, wherein the second desired gene is a pest small RNA biosynthesis gene.

11. The method of claim 10, wherein the pest small RNA biosynthesis gene is selected from the group consisting of Dicer-1 (DCR1) gene, Pasha gene, Loquacious gene (Loqs), ARGONAUTE1 gene (AGO1), ARGONAUTE2 gene (AGO2), ARGONAUTE3 gene (AGO3), Piwi gene, Stellate gene and Aubergine gene (Aub).

12. The method of claim 1, wherein the RNA produced is dsRNA.

13. The method of claim 12, wherein the dsRNA is an siRNA or modified to form an siRNA.

14. The method of claim 13, wherein the nucleic acid is inserted into the vector as a hairpin structure.

15. The method of claim 1, wherein the RNA produced is ssRNA.

16. The method of claim 15, wherein the nucleic acid is inserted into the vector in a sense orientation.

17. The method of claim 15, wherein the nucleic acid is inserted into the vector in an antisense orientation.

18. The method of claim 1, wherein the sequence is selected from the group consisting of (a) a part of a 5′

UTR of the first desired gene, (b) a part of a 3′

UTR of the first desired gene, (c) a part of a coding sequence of the first desired gene, (d) a part of a 5′

UTR of the first desired gene and a part of a coding sequence of the first desired gene and (e) a part of a coding sequence of the first desired gene and part of a 3′

UTR of the first desired gene.

3. (canceled)

19. A method of controlling pests by viral expression of target pest sequences in a host to modify endogenous expression of pest genes in cells or tissues of the pest, the method comprising:

(a) inserting a nucleic acid comprising a sequence of a first desired gene to be silenced into a virus-induced gene silencing (VIGS) vector of a virus that can infect a desired host to produce a modified VIGS vector, wherein the first desired gene is a pest gene;

(b) inoculating the host with the modified VIGS vector to produce infected host; and

(c) growing the infected host under conditions in which the modified VIGS vector is replicated to produce RNA which accumulates in tissue of the host,

wherein the RNA causes gene silencing in the pest upon ingestion of the RNA produced in the host, whereby pests are controlled.

20. The method of claim 19, wherein the virus is a DNA virus or an RNA virus.

22. The method of claim 20, wherein the RNA virus is an RNA virus containing two RNA molecules and one of the RNA molecules is the VIGS vector.

23. The method of claim 22, wherein the RNA virus is tobacco rattle virus (TRV).

24. The method of claim 22, wherein the host is inoculated with the modified VIGS vector and a vector containing the other RNA molecule.

25. The method of claim 19, wherein the host is a plant and the host tissue is a plant leaf.

26. The method of claim 19, wherein the nucleic acid further comprises a sequence of a second desired gene, wherein the second desired gene is a plant host resistance gene.

27. The method of claim 26, wherein the host resistance gene is selected from the group consisting of RNase III Dicer-like 4 (DCL4) gene, RNase III Dicer-like 2 (DCL2) gene, RNase III Dicer-like 3 (DCL3) gene, ARGONAUTE1 (AGO1), ARGONAUTE71 (AGO7), RNA-dependent RNA polymerase 1 (RDR1), RNA-dependent RNA polymerase 6 (RDR6), Suppressor of gene silencing 1 (SGS1), Suppressor of gene silencing 3 (SGS3), and Silencing defective 3 (SDE3).

28. The method of claim 19, wherein the nucleic acid further comprises a sequence of a second desired gene, wherein the second desired gene is a pest small RNA biosynthesis gene.

29. The method of claim 28, wherein the pest small RNA biosynthesis gene is selected from the group consisting of Dicer-1 (DCR1) gene, Pasha gene, Loquacious gene (Loqs), ARGONAUTE1 gene (AGO1), ARGONAUTE2 gene (AGO2), ARGONAUTE3 gene (AGO3), Piwi gene, Stellate gene and Aubergine gene (Aub).

30. The method of claim 19, wherein the RNA produced is dsRNA.

31. The method of claim 30, wherein the ds RNA is an siRNA or modified to form an siRNA.

32. The method of claim 31, wherein the nucleic acid is inserted into the vector as a hairpin structure.

33. The method of claim 19, wherein the RNA produced is ssRNA.

34. The method of claim 33, wherein the nucleic acid is inserted into the vector in a sense orientation.

35. The method of claim 33, wherein the sequence is nucleic acid into the vector in an antisense orientation.

36. The method of claim 19, wherein the pest gene is selected from the group consisting of gossypol-induced cytochrome P450 (CYP6AE14), H⁺-ATPase (V-ATPase), glutathione-S-transferase (GST), Dicer-1 (DCR1), alpha-tubulin and chitin.

37. The method of claim 19, wherein the sequence is selected from the group consisting of (a) a part of a 5′

UTR of the first desired gene, (b) a part of a 3′

UTR of the first desired gene, (c) a part of a coding sequence of the first desired gene, (d) a part of a 5′

UTR of the first desired gene and a part of a coding sequence of the first desired gene and (e) a part of a coding sequence of the first desired gene and part of a 3′

UTR of the first desired gene.

21. (canceled)