Plant Hormone Biosensors

• US 20140090111A1
• Filed: 09/05/2013
• Published: 03/27/2014
• Est. Priority Date: 09/05/2012
• Status: Abandoned Application

• Current Assignee: Carnegie Institution of Washington
• Original Assignee: Carnegie Institution of Washington
• Inventors: Frommer, Wolf B., Jones, Alexander M.
• Application Number:
  US14/019,062
• Publication Number:
  US 20140090111A1
• Time in Patent Office: Days
• US Class Current: 800/298
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/8212 Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
• CPC Class Codes: C12N 15/8293 Abscisic acid [ABA]
• CPC Class Codes: G01N 2333/415 from plants
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The invention provides fusion proteins comprising at least two fluorescent proteins, with the fluorescent proteins emitting different wavelengths of light from one another, at least one plant hormone binding domain that changes three-dimensional conformation upon specifically binding to a plant hormone, and two linker peptides, with the first linker linking the first fluorescent protein to the N-terminus of the plant hormone binding domain and the second linker linking the second fluorescent protein to the C-terminus of the plant hormone binding domain. The invention also provides for methods of using the fusion proteins of the present invention and nucleic acids encoding the fusion proteins.

Claims

1. A fusion protein comprising

a) at least a first and second fluorescent protein, wherein the first and second fluorescent proteins emit wavelengths of light that are different from one another

b) at least one plant hormone binding domain comprising an N-terminus and a C-terminus, wherein the binding domain changes three-dimensional conformation upon specifically binding to a plant hormone, and

c) at least a first and second fluorescent protein linker peptide, wherein the first fluorescent protein linker peptide links the first fluorescent protein to the N-terminus of the at least one plant hormone binding domain and the second fluorescent protein linker peptide links the second fluorescent protein to the C-terminus of the at least one plant hormone binding domain.

2. The fusion protein of claim 1, wherein the at least one plant hormone binding domain comprises at least two subdomains that are linked together with a subdomain linker peptide.

3. The fusion protein of claim 2, wherein the first and second fluorescent protein linker peptides are the same.

4. The fusion protein of claim 3, wherein the subdomain linker peptide is different from the first and second fluorescent protein linker peptides.

5. The fusion protein of claim 4, wherein the subdomain linker peptide comprises the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

6. The fusion protein of claim 5, wherein the first and second fluorescent protein linker peptides comprise an amino acid sequence of SEQ ID NO:

3 or SEQ ID NO;

4.

7. The fusion protein of claim 6, wherein the first and second fluorescent proteins are selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), citrine, cerulean, VENUS and teal fluorescent protein (TFP).

8. The fusion protein of claim 7, wherein the plant hormone binding domain specifically binds to abscisic acid (ABA) or a derivative thereof, an auxin (IAA), a gibberellin (GA) and jasmonic acid (JA) or a derivative thereof.

9. The fusion protein of claim 8, wherein the plant hormone binding domain specifically binds GA and the plant hormone binding domain comprises two subdomains with the first subdomain selected from the group consisting of RGA1 and RGA2, and the second subdomain selected from the group consisting of GID1A, GID1B and GID1C.

10. The fusion protein of claim 8, wherein the plant hormone binding domain specifically binds IAA and the plant hormone binding domain comprises two subdomains with the first subdomain being TIR1, and the second subdomain selected from the group consisting of IAA1, IAA3, IAA7, IAA12, IAA17, IAA28, IAA18 and IAA9.

11. The fusion protein of claim 8, wherein the plant hormone binding domain specifically binds JA or a derivative thereof and the plant hormone binding domain comprises two subdomains with the first subdomain being COI1, and the second subdomain selected from the group consisting of JAZ1, JAZ3, JAZ6 and JAZ9.

12. The fusion protein of claim 8, wherein the plant hormone binding domain specifically binds ABA or a derivative thereof and the plant hormone binding domain comprises two subdomains with the first subdomain selected from the group consisting of ABI1 and HAB1, and the second subdomain selected from the group consisting of PYR1, PYL1, PYL4, PYL5, PYL8 and PYL9.

13. A nucleic acid that encodes the fusion protein of claim 1.

14. A vector comprising the nucleic acid of claim 13.

15. A host cell comprising the vector of claim 14.

16. A plant comprising the host cell of claim 15.

18. A method of detecting a plant hormone in a sample, the method comprising contacting the fusion protein of claim 1 with the sample and determining the amount of fluorescence resonance energy transfer (FRET) between the first and second fluorescent proteins that occurs after the plant hormone binds to the plant hormone binding moiety of the fusion protein.

19. The method of claim 18, wherein the plant hormone is selected from the group consisting of abscisic acid (ABA) or a derivative thereof, an auxin (IAA), a gibberellin (GA) and jasmonic acid (JA) or a derivative thereof.

20. The method of claim 19, wherein the sample is in a plant or tissue thereof.

17. A method of producing a fusion protein, the method comprising culturing a host cell in conditions that promote protein expression and recovering the fusion protein from the culture, wherein the host cell comprises a vector encoding a fusion protein, wherein the fusion protein comprises

a) at least a first and second fluorescent protein, wherein the first and second fluorescent proteins emit wavelengths of light that are different from one another

b) at least one plant hormone binding domain comprising an N-terminus and a C-terminus, wherein the binding domain changes three-dimensional conformation upon specifically binding to a plant hormone, and

c) at least a first and second fluorescent protein linker peptide, wherein the first fluorescent protein linker peptide links the first fluorescent protein to the N-terminus of the at least one plant hormone binding domain and the second fluorescent protein linker peptide links the second fluorescent protein to the C-terminus of the at least one plant hormone binding domain.