Double stranded RNA compositions for reducing asian citrus psyllid infestation and methods of use

Patent metadata

Field	Value
Publication number	US-10344291-B2
Application number	US-201715414800-A
Country	US
Kind code	B2
Filing date	Jan 25, 2017
Priority date	Jan 26, 2016
Publication date	Jul 9, 2019
Grant date	Jul 9, 2019

Abstract

dsRNA generated from D. citri trehalase gene is effective in reducing fitness and/or survival of D. citri. Thus genetically altered plants expressing the dsRNA and plants to which dsRNA solutions are applied increase D. citri mortality and reduce D. citri infestation. With reduced D. citri population, the spread of microorganisms for which D. citri is a vector is reduced. Such microorganisms include, but are not limited to, C. Liberibacter species, including: CLas, CLam, and CLaf. Thus, applying of the D. citri trehalase dsRNA to a plant reduces disease and/or microorganism transmission by killing D. citri that feed on the treated plant.

First claim

We, the inventors, claim:

1. A dsRNA comprising a sense region consisting a sequence of at least twenty-one contiguous ribonucleotides equivalent to D. citri trehalase cDNA of SEQ ID NO: 26 and an anti-sense region complementary to said sense region, and wherein the sequence of the anti-sense region is at least 95% identical to the complement of said sense region; and wherein D. citri are killed upon ingestion or adsorption of said dsRNA.

2. The dsRNA of claim 1 wherein said dsRNA comprises a sequence selected from the group consisting of between 21 nt and 469 nt of SEQ ID NO: 4 or a sequence at least 99% identical thereof, between 21 nt and 438 nt of SEQ ID NO: 9 or a sequence at least 99% identical thereof, and between 21 nt and 730 nt of SEQ ID NO: 14 or a sequence at least 99% identical thereof.

3. A dsRNA solution comprising an agriculturally acceptable carrier and said dsRNA of claim 1 .

4. The dsRNA solution of claim 3 , wherein said agriculturally acceptable carrier is selected from the group consisting of water, surfactant, liposome, lipid, protein, peptide, nanotube, chitin, inactivated microorganism, and a combination thereof.

5. The dsRNA solution of claim 3 , wherein said dsRNA solution further comprises a compound that prevent dsRNA degradation, a translaminar chemical, a mineral, a clay, a fertilizer, a sugar, or a combination thereof.

6. A method of reducing Diaphorina citri infestation on a treated plant compared to the D. citri infestation on an untreated plant, said method comprising administering a dsRNA solution having insecticidal activity against said D. citri to an untreated plant in an amount effective to kill said D. citri to generate a treated plant, and allowing said D. citri to ingest or absorb said dsRNA solution, wherein said dsRNA solution comprises an agriculturally acceptable carrier and said dsRNA of claim 1 , and wherein said dsRNA kills said D. citri that ingest or absorb said dsRNA solution and thereby reduces said D. citri infestation on said treated plant compared to said D. citri infestation on said untreated plant.

7. The method of claim 6 , wherein said dsRNA comprises a sequence selected from the group consisting of between 21 nt and 469 nt of SEQ ID NO: 4 or a sequence at least 99% identical thereof, and between 21 nt and 438 nt of SEQ ID NO: 9 or a sequence at least 99% identical thereof, between 21 nt and 730 nt of SEQ ID NO: 14 or a sequence at least 99% identical thereof.

8. The method of claim 6 , wherein said agriculturally acceptable carrier is selected from the group consisting of water, surfactant, liposome, lipid, protein, peptide, nanotube, chitin, inactivated microorganism, and a combination thereof.

9. The method of claim 6 , wherein said dsRNA solution further comprises a compound that prevent dsRNA degradation, a translaminar chemical, a mineral, a clay, a fertilizer, a sugar, or a combination thereof.

10. The method of claim 6 , wherein said administering step comprises spraying said dsRNA solution onto said untreated plant to generate said treated plant.

11. The method of claim 6 , wherein said administering step comprises applying said dsRNA solution to soil surrounding said untreated plant to allow for roots of said untreated plant to absorb said dsRNA and generating said treated plant.

12. The method of claim 6 , wherein said administering step comprising applying said dsRNA solution to one or more roots of said untreated plant to generate said treated plant.

13. A method for reducing fitness or survival of D. citri comprising introducing said dsRNA of claim 1 into a wild-type plant upon which said D. citri feeds, thereby producing an altered plant containing said dsRNA, and allowing D. citri to feed on said altered plant containing said dsRNA, wherein said dsRNA reduces said fitness or survival of said D. citri that ingest said dsRNA.

14. The method of claim 13 , wherein said dsRNA comprises a sequence selected from the group consisting of between 21 nt and 469 nt of SEQ ID NO: 4 or a sequence at least 99% identical thereof, between 21 nt and 438 nt of SEQ ID NO: 9 or a sequence at least 99% identical thereof, and between 21 nt and 730 nt of SEQ ID NO: 14 or a sequence at least 99% identical thereof.

15. The method of claim 14 , wherein said introducing said dsRNA into said wild-type plant comprises spraying a dsRNA solution comprising said dsRNA onto said wild-type plant.

16. The method of claim 14 , wherein said introducing said dsRNA into said wild-type plant comprises applying a dsRNA solution comprising said dsRNA to roots of said wild-type plant.

17. The method of claim 14 , wherein said introducing said dsRNA into said wild-type plant comprises applying a dsRNA solution comprising said dsRNA to soil around said wild-type plant whereby roots of said wild-type plant absorb said dsRNA.

18. The method of claim 13 , wherein said introducing said dsRNA into a wild-type plant comprises transforming a wild-type plant cell with an expression vector encoding said dsRNA to produce an altered plant cell; selecting said altered plant cell that produces said dsRNA; and inducing said altered plant cell that produces said dsRNA to grow into said altered plant that produces said dsRNA; wherein said expression vector comprises at least one heterologous promoter operably linked a polynucleotide encoding said dsRNA comprising a sense region and an anti-sense region, wherein said dsRNA comprises a sequence selected from the group consisting of between 21 nt and 469 nt of SEQ ID NO: 4 or a sequence at least 99% identical thereof, between 21 nt and 438 nt of SEQ ID NO: 9 or a sequence at least 99% identical thereof, between 21 nt and 730 nt of SEQ ID NO: 14 or a sequence at least 99% identical thereof, and between 21 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26 or a sequence at least 95% identical thereof.

19. The method of claim 18 wherein said expression vector comprises one promoter which controls transcription of said sense region and said anti-sense region.

20. The method of claim 18 , wherein said expression vector comprises a first promoter which controls transcription of said sense region and a second promoter which controls transcription of said anti-sense region.

21. A method of reducing transmission by D. citri of a disease-causing microorganism from a treated plant to an untreated plant, said method comprising applying a dsRNA solution to a wild-type plant to produce said treated plant, and allowing D. citri to feed on said treated plant, wherein said dsRNA solution comprises an agriculturally acceptable carrier and said dsRNA of claim 1 , and wherein said dsRNA kills said D. citri that ingests or absorbs said dsRNA, wherein said dead D. citri are unable to transmit said microorganism to an untreated plant.

22. The method of claim 21 wherein said dsRNA comprises a sequence selected from the group consisting of between 21 nt and 469 nt of SEQ ID NO: 4 or a sequence at least 99% identical thereof, between 21 nt and 438 nt of SEQ ID NO: 9 or a sequence at least 99% identical thereof, between 21 nt and 730 nt of SEQ ID NO: 14 or a sequence at least 99% identical thereof, and between 21 nt and 1908 nt of the RNA equivalent of SEQ ID NO: 26 or a sequence at least 95% identical thereof.

23. The method of claim 21 , wherein said applying said dsRNA solution to said wild-type plant comprises spraying said dsRNA solution onto said wild-type plant.

24. The method

Assignees

• Us Agriculture
• Embrapa Pqeb Parque Estacao Biologica
• Empresa Brasileira De Pesquisa Agropecuaria

Inventors

• Hunter Wayne B
• Gonzalez Maria T
• Andrade Eduardo C

Classifications

• C12N15/8218 Primary

  Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS] · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12N15/8286

  for insect resistance · CPC title

• C12N15/8281

  for bacterial resistance · CPC title

• A01N57/16

  containing heterocyclic radicals · CPC title