Method of controlling the fertility of a plant

• US 6,147,282 A
• Filed: 01/20/1999
• Issued: 11/14/2000
• Est. Priority Date: 03/03/1995
• Status: Expired due to Term

• Current Assignee: SYNGENTA INVESTMENT CORPORATION
• Original Assignee: Novartis Finance Corporation (Novartis Ag)
• Inventors: Goff, Stephen A., Privalle, Laura S., Crossland, Lyle D.
• Primary Examiner(s): Fox, David T.
• Application Number:
  US09/234,190
• Time in Patent Office: 664 Days
• Field of Search: 800/271, 800/274, 800/278, 800/286, 800/287, 800/288, 800/303, 435/69.7, 435/199, 435/468, 536/23.4, 536/23.5, 536/24.1, 514/2, 514/8, 514/21
• US Class Current: 800/303
• 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 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 and a first DNA binding domain;

a second receptor expression cassette which encodes a second receptor polypeptide comprising a second ligand binding domain and a second DNA binding domain, wherein said first and second receptor polypeptides are members of the Class II steroid and thyroid hormone superfamily of nuclear receptors; and

a target expression cassette comprising a 5'"'"' regulatory region operably linked to a nucleotide sequence encoding a target polypeptide that renders fertilization ineffective or restores effective fertilization, wherein said 5'"'"' regulatory region comprises one or more response elements complementary to said first or second DNA binding domain;

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.

2. The method of claim 1 wherein said first receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for said first receptor polypeptide, said second receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for said second receptor polypeptide, or both said first and said second receptor expression cassettes comprise an anther-specific promoter operably linked to the coding sequence for said first and second receptor polypeptides.

3. The method of claim 1 wherein said first receptor expression cassette comprises a pistil-specific promoter operably linked to the coding sequence for said first receptor polypeptide, said second receptor expression cassette comprises a Pistil-specific promoter operably linked to the coding sequence for said second receptor polypeptide, or both said first and said second receptor expression cassettes comprise a pistil-specific promoter operably linked to the coding sequence for said first and second receptor polypeptides.

4. The method of claim 1 wherein said target polypeptide renders fertilization ineffective.

5. The method of claim 4 wherein said target polypeptide is the ribonuclease barnase.

6. The method of claim 1 wherein said target polypeptide restores effective fertilization.

7. The method of claim 6 wherein said target polypeptide is the ribonuclease inhibitor barstar.

8. The method of claim 1 wherein said first receptor polypeptide is Ecdysone Receptor.

9. The method of claim 8 wherein said first receptor polypeptide further comprises a heterologous transactivation domain.

10. The method of claim 9 wherein said heterologous transactivation domain is the transactivation domain from the C1 regulatory gene of maize.

11. The method of claim 8 wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide.

12. The method of claim 11 wherein said first DNA binding domain is the DNA binding domain from the GAL4 protein of yeast.

13. The method of claim 1 wherein said second receptor polypeptide is USP.

14. The method of claim 13 wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

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

16. The method of claim 12 wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

17. The method of claim 1 wherein said chemical ligand is an insect hormone, an insect hormone antagonist or an insect hormone agonist.

18. The method of claim 17 wherein said chemical ligand is fenoxycarb;

CGA 59,205;

RH 5992;

or RH 5849.

19. The method of claim 1 wherein said 5'"'"' regulatory region of said target expression cassette comprises between 1 and 11 response element copies.

20. The method of claim 1 wherein said first receptor polypeptide further comprises a heterologous transactivation domain.

21. The method of claim 1 wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

22. The method of claim 1 wherein said first receptor polypeptide further comprises a heterologous transactivation domain and wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

23. The method of claim 1 wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide.

24. The method of claim 1 wherein said second DNA binding domain is heterologous with respect to said first receptor polypeptide.

25. The method of claim 1 wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide and wherein said second DNA binding domain is heterologous with respect to said first receptor polypeptide.

26. The method of claim 8 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain of Ecdysone Receptor.

27. The method of claim 26 wherein said second receptor polypeptide is USP.

28. The method of claim 27 wherein said second receptor polypeptide further comprises a heterologous transactivation domain from the VP16 protein of herpes simplex.

29. The method of claim 27 wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

30. The method of claim 12 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain from the GAL4 protein of yeast.

31. The method of claim 30 wherein said second receptor polypeptide is USP and wherein said second receptor polypeptide further comprises a heterologous transactivation domain from the VP16 protein of herpes simplex.

32. The method of claim 30 wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize, wherein said second receptor polypeptide is USP, and wherein said second receptor polypeptide further comprises a heterologous transactivation domain from the VP16 protein of herpes simplex.

33. 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 and a first DNA binding domain;

a second receptor expression cassette which encodes a second receptor polypeptide comprising a second ligand binding domain and a second DNA binding domain, wherein said first and second receptor polypeptides are members of the Class II steroid and thyroid hormone superfamily of nuclear receptors; and

a target expression cassette comprising a 5'"'"' regulatory region operably linked to a nucleotide sequence encoding the anti-sense version of a coding sequence critical to effective fertilization, wherein said 5'"'"' regulatory region comprises one or more response elements complementary to said first or second DNA binding domain;

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 anti-sense sequence, wherein said anti-sense sequence renders fertilization ineffective.