Juvenile hormone or one of its agonists as a chemical ligand to control gene expression in plants by receptor mediated transactivation

• US 6,362,394 B1
• Filed: 08/17/1999
• Issued: 03/26/2002
• Est. Priority Date: 10/10/1995
• Status: Expired due to Fees

• Current Assignee: Novartis Ag
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Crossland, Lyle Dean, Goff, Stephen Arthur
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): Davis, Katharine F
• Application Number:
  US09/376,107
• Time in Patent Office: 952 Days
• Field of Search: 800/278, 800/288, 800/298, 800/320.1, 800/320.3, 435/69.1, 435/69.7, 435/468, 435/320.1, 435/375, 536/23.1, 536/24.1
• US Class Current: 800/278
• CPC Class Codes: C07K 14/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8238 chemically inducible, e.g. tetracycline
• CPC Class Codes: C12N 15/8287 for fertility modification, e.g. apomixis

Abstract

The present invention is drawn to a method of controlling gene expression in plants. Specifically, the method comprises transforming a plant with a USP receptor expression cassette which encodes a USP receptor and at least one target expression cassette which encodes a target polypeptide. Contacting said transformed plant with juvenile hormone or one of its agonists activates expression of the target polypeptide in the presence of said USP receptor polypeptide. Optionally, additional “secondary” receptor expression cassettes may be used, wherein the secondary receptor expression cassette encodes a receptor polypeptide distinct from USP. The method is useful for controlling various traits of agronomic importance, such as plant fertility. Also disclosed is a method of identifying previously unknown ligands for USP. Substances to be tested are identified by placing them in contact with plant cells transformed with a USP receptor expression cassette and a target expression cassette. The target expression cassette encodes a reporter polypeptide whose expression can be determined quantitatively or qualitatively, whereby the test substance is identified as a ligand for USP.

Claims

1. A method of controlling gene expression in a transgenic plant cell transformed with an Ultraspiracle (USP) receptor expression cassette that encodes a USP receptor polypeptide and a target expression cassette that encodes a target polypeptide, comprising:

a) expressing the receptor polypeptide in said transgenic plant cell; and

b) contacting said transgenic plant cell with juvenile hormone or one of its agonists, wherein the receptor polypeptide controls expression of the target polypeptide in the presence of juvenile hormone or one of its agonists.

2. The method according to claim 1 wherein expression of the target polypeptide is increased or activated in the presence of juvenile hormone or one of its agonists.

3. The method according to claim 1 wherein expression of the target polypeptide is decreased or inhibited in the presence of juvenile hormone or one of its agonists.

4. The method according to claim 1 wherein the USP receptor polypeptide comprises a heterologous transactivation domain.

5. The method according to claim 1 wherein said agonist is selected from the group consisting of fenoxycarb, diofenolan, kinoprene, methoprene, hyrdoprene, diofenolan, methoprene acid, triflumuron, hexamflumuron, teflubenzuron, flufenoxuron, flucycloxuron, lufenuron, diflubenzuron and chlorfluzuron.

6. The method according to claim 1 wherein said target expression cassette comprises a 5′

regulatory region comprising between 1 and 11 copies of a response element.

7. The method according to claim 1 wherein said transgenic plant cell additionally comprises a secondary receptor expression cassette encoding a secondary receptor polypeptide distinct from the USP receptor polypeptide.

8. The method according to claim 1 wherein said transgenic plant cell is a maize cell.

9. The method according to claim 1 wherein said transgenic plant cell is a wheat cell.

10. The method according to claim 1 wherein said USP receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for the USP receptor polypeptide.

11. The method according to claim 1 wherein said USP receptor expression cassette comprises a pistil-specific promoter operably linked to the coding sequence for the USP receptor polypeptide.

12. The method according to claim 1 wherein said target polypeptide is the ribonuclease barnase.

13. The method according to claim 1 wherein said target polypeptide is the ribonuclease inhibitor barstar.

14. The method according to claim 4 wherein said heterologous transactivation domain is the transactivation domain from the VP16 protein of herpes simplex.

15. The method according to claim 7 wherein said secondary receptor polypeptide is selected from the group consisting of Ecdysone Receptor (EcR), Drosophila Hormone Receptor 38 (DHR38), and Retinoic X Receptor (RXR).

16. A method of controlling gene expression in a transgenic plant cell transformed with a USP receptor expression cassette that encodes a USP receptor polypeptide and a target expression cassette that encodes an anti-sense sequence comprising:

a) expressing the receptor polypeptide in said transgenic plant cell; and

b) contacting said transgenic plant cell with juvenile hormone or one of its agonists, wherein the receptor polypeptide controls expression of the antisense sequence in the presence of juvenile hormone or one of its agonists.

17. A method of controlling gene expression, comprising the steps of:

a) providing a transgenic plant or plant cell transformed with chimeric DNA comprising;

i) a USP receptor expression cassette that encodes a USP receptor polypeptide, and

ii) a target expression cassette that encodes a target polypeptide;

b) expressing said receptor polypeptide in said transgenic plant or plant cell; and

c) contacting said transgenic plant or plant cell with juvenile hormone or one of its agonists, wherein the receptor polypeptide controls expression of the target polypeptide in the presence of juvenile hormone or one of its agonists.

18. The method according to claim 17 wherein said transgenic plant or plant cell additionally comprises a secondary receptor expression cassette encoding a secondary receptor polypeptide distinct from the USP receptor polypeptide.

19. The method according to claim 17 wherein expression of said target polypeptide is increased or activated in the presence of juvenile hormone or one of its agonists.

20. The method according to claim 17 wherein expression of said target polypeptide is decreased or inhibited in the presence of juvenile hormone or one of its agonists.

21. The method according to claim 17 wherein said USP receptor polypeptide comprises a heterologous transactivation domain.

22. The method according to claim 17 wherein the agonist is selected from the group consisting of fenoxycarb, diofenolan, kinoprene, methoprene, hyrdoprene, diofenolan, methoprene acid, triflumuron, hexamflumuron, teflubenzuron, flufenoxuron, flucycloxuron, lufenuron, diflubenzuron, and chlorfluzuron.

23. The method according to claim 17 wherein said target expression cassette comprises a 5′

regulatory region comprising between 1 and 11 copies of a response element.

24. The method according to claim 17 wherein said transgenic plant or plant cell is maize.

25. The method according to claim 17 wherein said transgenic plant or plant cell is wheat.

26. The method according to claim 17 wherein said USP receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for the USP receptor polypeptide.

27. The method according to claim 17 wherein said USP receptor expression cassette comprises a pistil-specific promoter operably linked to the coding sequence for the USP receptor polypeptide.

28. The method according to claim 17 wherein said target polypeptide is the ribonuclease barnase.

29. The method according to claim 17 wherein said target polypeptide is the ribonuclease inhibitor barstar.

30. The method according to claim 18 wherein said secondary receptor polypeptide is selected from the group consisting of EcR, DHR38, and RXR.

31. The method according to claim 21 wherein said heterologous transactivation domain is the transactivation domain from the VP16 protein of herpes simplex.

32. A method of controlling gene expression, comprising the steps of:

a) providing a transgenic plant or plant cell transformed with chimeric DNA comprising;

i) a USP receptor expression cassette that encodes a USP receptor polypeptide, and

ii) a target expression cassette that encodes an anti-sense sequence;

b) expressing said receptor polypeptide in said transgenic plant or plant cell; and

c) contacting said transgenic plant or plant cell with juvenile hormone or one of its agonists, wherein the receptor polypeptide controls expression of the anti-sense sequence in the presence of juvenile hormone or one of its agonists.