Control of gene expression in plants by receptor mediated transactivation in the presence of a chemical ligand

• US 20010022004A1
• Filed: 03/21/2001
• Published: 09/13/2001
• Est. Priority Date: 03/03/1995
• Status: Active Grant

• Current Assignee: SYNGENTA INVESTMENT CORPORATION
• Original Assignee: Novartis Finance Corporation (Novartis Ag)
• Inventors: Goff, Stephen A., Crossland, Lyle D., Privalle, Laura S.
• Granted Patent: US 6,939,711 B2
• Time in Patent Office: Days
• 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 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8217 Gene switch
• CPC Class Codes: C12N 15/8237 Externally regulated expression systems
• CPC Class Codes: C12N 15/8289 Male sterility
• CPC Class Codes: C12N 15/829 Female sterility

Abstract

The present invention is drawn to a method of controlling gene expression in plants. Specifically, the method comprises obtaining a transgenic plant comprising at least two receptor expression cassettes and at least one target expression cassette. The first receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a first receptor polypeptide, and a 3′ termination region. The second receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a second receptor polypeptide, and a 3′ termination region. The target expression cassette comprises a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence which encodes a target polypeptide, and a 3′ termination region, wherein the 5′ regulatory region of said target expression cassette is activated by said first and second receptor polypeptides in the presence of one or more chemical ligands which are complementary to the ligand binding domain of said receptor polypeptides, whereby expression of said target polypeptide is accomplished. The method is useful for controlling various traits of agronomic importance, such as plant fertility.

Claims

1. A method of controlling gene expression in a plant comprising:

a) transforming said plant with a first receptor expression cassette which encodes a first receptor polypeptide comprising a first ligand binding domain, a second receptor expression cassette which encodes a second receptor polypeptide comprising a second ligand binding domain, wherein said first and second receptor polypeptides are mutually distinct, and a target expression cassette encoding a target polypeptide;

b) expressing said first and second receptor polypeptides in said transformed plant; and

c) contacting said transformed plant with one or more chemical ligands which are complementary to the ligand binding domain of said first or second receptor polypeptides whereby said receptor polypeptides in the presence of said chemical ligand activate the expression of said target polypeptide.

2. The method of

claim 1

wherein said first and second receptor polypeptides are members of the Class II steroid and thyroid hormone superfamily of nuclear receptors.

3. The method of

claim 2

wherein said first receptor polypeptide is Ecdysone Receptor.

4. The method of

claim 3

wherein said first receptor polypeptide further comprises a heterologous transactivation domain.

5. The method of

claim 4

wherein said heterologous transactivation domain is the transactivation domain from the C1 regulatory gene of maize.

6. The method of

claim 3

wherein said first receptor polypeptide further comprises a heterologous DNA binding domain.

7. The method of

claim 6

wherein said DNA binding domain is the DNA binding domain from the GAL4 protein of yeast.

8. The method of

claim 2

wherein said second receptor polypeptide is USP.

9. The method of

claim 3

wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

10. The method of

claim 9

wherein said heterologous kansactivation domain is the transactivation domain from the VP1 6 protein of herpes simplex.

11. The method of

claim 1

wherein said first or second receptor polypeptide has been mutated in the ligand binding domain.

12. The method of

claim 1

wherein said chemical ligand is an insect hormone, an insect hormone antagonist or an insect hormone agonist.

13. The method of

claim 12

wherein said chemical ligand is fenoxycarb, CGA 59,205, MIMIC®

or RH 5849.

14. The method of

claim 1

wherein said target expression cassette comprises a 5′

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

15. A method of controlling the fertility of a plant comprising:

a) transforming said plant with a first receptor expression cassette which encodes a first receptor polypeptide comprising a first ligand binding domain, a second receptor expression cassette which encodes a second receptor polypeptide comprising a second ligand binding domain, wherein said first and second receptor polypeptides are mutually distinct, and a target expression cassette encoding a target polypeptide;

b) expressing said first and second receptor polypeptides in said transformed plant;

and

c) contacting said transformed plant with one or more chemical ligands which are complementary to the ligand binding domain of said first or second receptor polypeptides whereby said receptor polypeptides in the presence of said chemical ligand activate the expression of said target polypeptide, wherein said target polypeptide renders fertilization ineffective or restores effective fertilization.

16. The method of

claim 15

wherein said receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for said receptor polypeptide.

17. The method of

claim 15

wherein said receptor expression cassette comprises a pistil-specific promoter operably linked to the coding sequence for said receptor polypeptide.

18. The method of

claim 15

wherein said target polypeptide renders fertilization ineffective.

19. The method of

claim 18

wherein said target polypeptide is the ribonuclease barnase.

20. The method of

claim 15

wherein said target expression cassette encodes the anti-sense version of a coding sequence critical to effective fertilization, thereby rendering fertilization ineffective.

21. The method of

claim 15

wherein said target polypeptide restores effective fertilization.

22. The method of

claim 21

wherein said target polypeptide is the ribonuclease inhibitor barstar.

23. A transgenic plant comprising:

a) a first receptor expression cassette which encodes a first receptor polypeptide;

b) a second receptor expression cassette encoding a second receptor polypeptide; and

c) a target expression cassette encoding a target polypeptide.

24. The plant of

claim 23

wherein said plant is maize.

25. The plant of

claim 23

wherein said plant is wheat.

26. A receptor expression cassette comprising:

a) a 5′

regulatory region capable of promoting expression in a plant cell;

b) an operably linked coding sequence encoding a receptor polypeptide comprising a ligand binding domain; and

c) a 3′

terminating sequence.

27. The receptor expression cassette of

claim 26

wherein said receptor polypeptide is a member of the Class II steroid and thyroid hormone superfamily of nuclear receptors.

28. The receptor expression cassette of

claim 27

wherein said receptor polypeptide is Ecdysone Receptor.

29. The receptor expression cassette of

claim 28

wherein said receptor polypeptide further comprises a heterologous transactivation domain.

30. The receptor expression cassette of

claim 29

wherein said heterologous transactivation domain is the transactivation domain from the C1 regulatory gene of maize.

31. The receptor expression cassette of

claim 28

wherein said receptor polypeptide further comprises a heterologous DNA binding domain.

32. The receptor expression cassette of

claim 31

wherein said heterologous DNA binding domain is the DNA binding domain from the GAL4 of yeast.

33. The receptor expression cassette of

claim 26

wherein said receptor polypeptide is USP.

34. The receptor expression cassette of

claim 33

wherein said receptor polypeptide further comprises a heterologous transactivation domain.

35. The receptor expression cassette of

claim 34

wherein said heterologous transactivation domain is the transactivation domain from the VP16 protein of herpes simplex.

36. The receptor expression cassette of

claim 26

wherein said receptor polypeptide has been mutated in the ligand binding domain.