Control of pests in plants

• US 10,017,774 B2
• Filed: 09/15/2010
• Issued: 07/10/2018
• 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, Chua, Nam-Hai, Qu, Jing, Gao, Shi-Qiang
• Primary Examiner(s): Baum, Stuart F
• Application Number:
  US13/497,729
• Publication Number:
  US 20120240288A1
• Time in Patent Office: 2,855 Days
• Field of Search: None
• 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 a cotton insect pest gene to determine whether the cotton insect pest gene can lead to mortality of the cotton insect pest when expression of the cotton insect pest gene is silenced in the cotton insect pest, the method comprising:

(a) inserting a first nucleic acid comprising a target sequence of a cotton insect pest gene to be silenced and a second nucleic acid comprising a target sequence of a cotton insect pest Dicer-1 gene to be silenced into a vector of a virus-induced gene silencing (VIGS) system wherein the vector comprises a tobacco rattle virus (TRV) RNA2 sequence to produce a modified vector comprising a modified TRV RNA2 sequence, wherein the cotton insect pest gene is selected from the group consisting of a gene in a metabolic pathway, a gene in an energy metabolism pathway, a gene encoding a detoxification protein, a gene involved in organ or tissue differentiation, a gene involved in development regulation, a gene involved in molting processing and a gene encoding a cytoskeleton protein, wherein the target sequence in the first nucleic acid is 200 to 1200 nucleotides in length and wherein the target sequence of the cotton insect pest Dicer-1 gene is part of the coding sequence set forth in SEQ ID NO;

20;

(b) preparing a mixed culture of Agrobacterium comprising Agrobacterium containing a vector comprising a TRV RNA1 sequence and Agrobacterium containing the modified vector comprising the modified TRV RNA2 sequence;

(c) inoculating the cotton plant host with the mixed culture of Agrobacterium to produce an infected cotton plant host;

(d) growing the infected cotton plant host under conditions in which the modified vector comprising the modified TRV RNA2 sequence is replicated and systemically spreads in the infected cotton plant host to produce RNA of the cotton insect pest gene which accumulates in tissue of the infected cotton plant host and to produce RNA of the cotton insect pest Dicer-1 gene which accumulates in tissue of the infected cotton host plant;

(e) feeding the infected cotton plant host tissue with the accumulated RNA of the cotton insect pest gene and RNA of the cotton insect pest Dicer-1 gene to cotton insect pests;

(f) feeding infected cotton plant host tissue with accumulated RNA of the cotton insect pest gene to be silenced to cotton insect pests;

(g) feeding infected cotton plant host tissue with accumulated RNA of the cotton insect pest Dicer-1 gene to cotton insect pests;

(h) comparing mortality rate and/or mortality speed of the cotton insect pests of step (e) to mortality rate and/or mortality speed of the cotton insect pests of step (f) and step (g) and

(i) selecting the cotton insect pest gene as a cotton insect pest gene that leads to mortality of the cotton insect pest

if the mortality rate and/or mortality speed of the cotton insect pests of step (e) is greater than the mortality rate and/or mortality speed of the cotton insect pests of step (f) and step (g), and the difference between the mortality rates and/or mortality speed from the cotton insect pests of step (e) and step (f) is statistically significant as measured by having a p-value of less than 0.01.

2. The method of claim 1, wherein the infected cotton plant host tissue is a new, systemically infected cotton plant leaf.

3. The method of claim 1, wherein the RNA of the cotton insect pest gene and/or the RNA of the cotton insect pest Dicer-1 gene that is produced is dsRNA.

4. The method of claim 3, wherein the dsRNA is an siRNA or modified to form an siRNA.

5. The method of claim 4, wherein the first nucleic acid and/or the second nucleic acid is inserted into the vector as a hairpin structure.

6. The method of claim 1, wherein the RNA of the cotton insect pest gene and/or the RNA of the cotton insect pest Dicer-1 gene that is produced is ssRNA.

7. The method of claim 6, wherein the first nucleic acid and second nucleic acid are independently inserted into the vector in a sense orientation or an antisense orientation.

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

UTR of the gene, (b) a part of a 3′

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

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

UTR of the gene.

18. The method of claim 1, wherein the cotton insect pest gene is selected from the group consisting of a gene with the sequence of SEQ ID NO:

5 (CYP6AE14 gene), SEQ ID NO;

8 (VATP-A gene), SEQ ID NO;

29 (DCR1), SEQ ID NO;

35 (chitinase), SEQ ID NO;

36 (GST gene), and SEQ ID NO;

37 (tubulin).

19. The method of claim 1, wherein the target sequence in the first nucleic acid is 300 to 1000 nucleotides in length.

21. The method of claim 1, wherein the cotton insect pest is cotton bollworm.

9. A method of increasing larval mortality in larvae of cotton insect pests by viral expression of target cotton insect pest sequences in a cotton plant host to modify endogenous expression of the cotton insect pest genes in cells or tissues of the cotton insect pest, the method comprising:

(a) inserting a first nucleic acid comprising a target sequence of a cotton insect pest gene to be silenced and a second nucleic acid comprising a target sequence of a cotton insect pest Dicer-1 gene to be silenced into a vector of a virus-induced gene silencing (VIGS) system wherein the vector comprises a tobacco rattle virus (TRV) RNA2 sequence to produce a modified vector comprising a modified TRV RNA2 sequence, wherein the cotton insect pest gene is selected from the group consisting of a gene in a metabolic pathway, a gene in an energy metabolism pathway, a gene encoding a detoxification protein, a gene involved in organ or tissue differentiation, a gene involved in development regulation, a gene involved in molting processing and a gene encoding a cytoskeleton protein, wherein the target sequence in the first nucleic acid is 200 to 1200 nucleotides in length and wherein the target sequence of the cotton insect pest Dicer-1 gene is part of the coding sequence set forth in SEQ ID NO;

20;

(b) preparing a mixed culture of Agrobacterium comprising Agrobacterium containing a vector comprising a TRV RNA1 sequence and Agrobacterium containing the modified vector comprising the modified TRV RNA2 sequence;

(c) inoculating cotton plant host with the mixed culture of Agrobacterium to produce an infected cotton plant host; and

(d) growing the infected cotton plant host under conditions in which the modified vector comprising the modified TRV RNA2 sequence is replicated and systemically spreads in the infected cotton plant host to produce RNA of the cotton insect pest gene which accumulates in tissue of the infected cotton plant host and to produce RNA of the cotton insect pest Dicer-1 gene which accumulates in tissue of the infected cotton host plant;

wherein the target sequence of the first nucleic acid has been shown to silence the cotton insect pest gene;

wherein the accumulated RNA of the cotton insect pest gene and the accumulated RNA of the cotton insect pest Dicer-1 gene causes gene silencing in the cotton insect pest upon ingestion of the RNA produced in the infected cotton plant host.

10. The method of claim 9, wherein the infected cotton plant host tissue is a new, systemically infected cotton plant leaf.

11. The method of claim 9, wherein the RNA of the cotton insect pest gene and/or the RNA of the cotton insect pest Dicer-1 gene that is produced is dsRNA.

12. The method of claim 11, wherein the dsRNA is an siRNA or modified to form an siRNA.

13. The method of claim 12, wherein the first nucleic acid and/or the second nucleic acid is inserted into the vector as a hairpin structure.

14. The method of claim 9, wherein the RNA of the cotton insect pest gene and/or the RNA of the cotton pest Dicer-1 gene that is produced is ssRNA.

15. The method of claim 14, wherein the first nucleic acid and second nucleic acid are independently inserted into the vector in a sense orientation or an antisense orientation.

16. The method of claim 9, wherein the cotton insect pest gene is selected from the group consisting of a gene with the sequence of SEQ ID NO:

5 (CYP6AE14 gene), SEQ ID NO;

8 (VATP-A gene), SEQ ID NO;

29 (DCR1), SEQ ID NO;

35 (chitinase), SEQ ID NO;

36 (GST gene), and SEQ ID NO;

37 (tubulin).

17. The method of claim 9, wherein each target sequence is independently selected from the group consisting of (a) a part of a 5′

UTR of the gene, (b) a part of a 3′

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

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

UTR of the gene.

20. The method of claim 9, wherein the target sequence in the first nucleic acid is 300 to 1000 nucleotides in length.

22. The method of claim 9, wherein the cotton insect pest is cotton bollworm.