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

• US 5,880,333 A
• Filed: 01/21/1998
• Issued: 03/09/1999
• 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., Crossland, Lyle D., Privalle, Laura S.
• Primary Examiner(s): Fox, David T.
• Application Number:
  US09/010,050
• Time in Patent Office: 412 Days
• Field of Search: 435/69.1, 435/69.7, 435/468, 800/278, 800/288, 800/298, 800/320.1, 800/320.3, 536/23.4, 536/23.5, 536/24.1, 514/2, 514/8, 514/21
• US Class Current: 800/288
• 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 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 wherein said first and second receptor polypeptides function together as a heterodimer; and

a target expression cassette comprising a 5'"'"' regulatory region operably linked to a nucleotide sequence encoding a target polypeptide, 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.

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

3. The method of claim 2 wherein said first receptor polypeptide further comprises a heterologous transactivation domain.

4. The method of claim 3 wherein said heterologous transactivation domain is the transactivation domain from the C1 regulatory gene of maize.

5. The method of claim 2 wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide.

6. The method of claim 5 wherein said first DNA binding domain is the DNA binding domain from the GAL4 protein of yeast.

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

8. The method of claim 7 wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

9. The method of claim 8 wherein said heterologous transactivation domain is the transactivation domain from the VP16 protein of herpes simplex.

10. The method of claim 6 wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulator gene of maize.

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

12. The method of claim 11 wherein said chemical ligand is fenoxycarb;

CGA 59,205;

RH 5992;

or RH 5849.

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

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

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

23. 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.

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

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

26. 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.

27. The method of claim 2 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain of Ecdysone Receptor.

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

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

30. The method of claim 28 wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

31. The method of claim 6 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain from the GAL4 protein of yeast.

32. The method of claim 31 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. The method of claim 31 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.

14. A transgenic plant comprising:

a) a first receptor expression cassette which encodes a first receptor polypeptide comprising a first DNA binding domain;

b) a second receptor expression cassette encoding a second receptor polypeptide comprising 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 wherein said first and second receptor polypeptides function together as a heterodimer; and

c) a target expression cassette comprising a 5'"'"' regulatory region operably linked to a nucleotide sequence encoding a target polypeptide, wherein said 5'"'"' regulatory region comprises one or more response elements complementary to said first or second DNA binding domain.

15. The transgenic plant of claim 14 wherein said plant is maize.

16. The transgenic plant of claim 14 wherein said plant is wheat.

34. The transgenic plant of claim 14 wherein said first receptor polypeptide is Ecdysone Receptor.

35. The transgenic plant of claim 14 wherein said second receptor polypeptide is USP.

36. The transgenic plant of claim 14 wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide.

37. The transgenic plant of claim 36 wherein said first receptor polypeptide is Ecdysone Receptor and wherein said heterologous DNA binding domain is the DNA binding domain from the GAL4 protein of yeast.

38. The transgenic plant of claim 14 wherein said second receptor polypeptide further comprises a heterologous transactivation domain.

39. The transgenic plant of claim 38 wherein said second receptor polypeptide is USP and wherein said heterologous transactivation domain is from the VP16 protein of herpes simplex.

40. The transgenic plant of claim 14 wherein said first receptor polypeptide further comprises a heterologous transactivation domain.

41. The transgenic plant of claim 40 wherein said first receptor polypeptide is Ecdysone Receptor and wherein said heterologous transactivation domain is from the C1 regulatory gene of maize.

42. The transgenic plant of claim 14 wherein said first receptor polypeptide is Ecdysone Receptor, wherein said first DNA binding domain is heterologous with respect to said first receptor polypeptide, wherein said first DNA binding domain is the DNA binding domain from the GAL4 protein of yeast, and wherein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

43. The transgenic plant of claim 34 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain of Ecdysone Receptor.

44. The transgenic plant of claim 43 wherein said second receptor polypeptide is USP.

45. The transgenic plant of claim 44 wherein said second receptor polypeptide further comprises a heterologous transactivation domain from the VP16 protein of herpes simplex.

46. The transgenic plant of claim 44 herein said first receptor polypeptide further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

47. The transgenic plant of claim 37 wherein said 5'"'"' regulatory region comprises one or more response elements complementary to the DNA binding domain from the GAL4 protein of yeast.

48. The transgenic plant of claim 47 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.

49. The transgenic plant of claim 47 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.

17. 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 an Ecdysone receptor comprising a heterologous transactivation domain from the C1 regulatory gene of maize; and

c) a 3'"'"' terminating sequence.

18. 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 an Ecdysone receptor comprising a heterologous DNA binding domain from the GAL4 protein of yeast; and

c) a 3'"'"' terminating sequence.

19. The receptor expression cassette of claim 18 wherein said receptor further comprises a heterologous transactivation domain from the C1 regulatory gene of maize.

20. 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 USP receptor polypeptide comprising a heterologous transactivation domain from the VP16 protein of herpes simplex; and

c) a 3'"'"' terminating sequence.