Fusion proteins and methods for stimulating plant growth, protecting plants from pathogens, and immobilizing Bacillus spores on plant roots

Patent metadata

Field	Value
Publication number	US-9573980-B2
Application number	US-201414213525-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Feb 21, 2017
Grant date	Feb 21, 2017

Abstract

The present invention is generally directed to fusion proteins containing a targeting sequence that targets the fusion protein to the exosporium of a Bacillus cereus family member. The invention also relates to recombinant Bacillus cereus family members expressing such fusion proteins, formulations containing the recombinant Bacillus cereus family members expressing the fusion proteins. Methods for stimulating plant growth and for protecting plants from pathogens by applying the recombinant Bacillus cereus family members or the formulations to plants or a plant growth medium are also described. The invention also relates to methods for immobilizing spores of a recombinant Bacillus cereus family member expressing a fusion protein on plant roots.

First claim

What is claimed is:

1. A method for stimulating plant growth comprising:
applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant seed, wherein applying the recombinant Bacillus cereus family member to the plant seed comprises: (a) applying the recombinant Bacillus cereus family member to the plant seed at the time of planting; or (b) coating the plant seed with a seed coating formulation comprising the recombinant Bacillus cereus family member and an agriculturally acceptable carrier; and
contacting the seed with a plant growth medium;
wherein the fusion protein comprises at least one plant growth stimulating protein or peptide and a targeting sequence or exosporium protein that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member, and wherein plants grown from plant seeds to which the recombinant Bacillus cereus family member has been applied exhibit increased growth as compared to plants grown from plant seeds to which the recombinant Bacillus cereus family member has not been applied, under the same conditions.

2. A method of claim 1 , wherein the fusion protein comprises a targeting sequence comprising an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%.

3. A method of claim 2 , wherein the targeting sequence comprises an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.

4. A method of claim 2 , wherein the targeting sequence comprises an amino sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.

5. A method of claim 2 , wherein the targeting sequence comprises an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%.

6. A method of claim 2 , wherein the targeting sequence consists of:
(a) a 16 amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO. 1, wherein the identity with amino acids 25-35 is at least about 54%;
(b) amino acids 1-35 of SEQ ID NO. 1;
(c) amino acids 20-35 of SEQ ID NO. 1; or
(d) SEQ ID NO. 1.

7. A method of claim 1 , wherein the targeting sequence comprises the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

8. A method of claim 1 , wherein the plant growth stimulating protein comprises an enzyme.

9. A method of claim 8 , wherein the enzyme comprises a cellulase.

10. A method of claim 9 , wherein the cellulase comprises an endocellulase, an exocellulase, or a β-glucosidase.

11. A method of claim 10 , wherein the endocellulase comprises a Bacillus subtilis endoglucanase, a Bacillus thuringiensis endoglucanase, a Bacillus cereus endoglucanase, or a Bacillus clausii endoglucanase.

12. A method of claim 8 , wherein the enzyme comprises a lipase.

13. A method of claim 12 , wherein the lipase comprises a Bacillus subtilis lipase, a Bacillus thuringiensis lipase, a Bacillus cereus lipase, or a Bacillus clausii lipase.

14. A method of claim 8 , wherein the enzyme comprises a lignin oxidase.

15. A method of claim 14 , wherein the lignin oxidase comprises a lignin peroxidase, a laccase, a glyoxal oxidase, a ligninase, or a manganese peroxidase.

16. A method of claim 8 , wherein the enzyme comprises a protease.

17. A method of claim 16 , wherein the protease comprises a subtilisin, an acid protease, an alkaline protease, a proteinase, a peptidase, an endopeptidase, an exopeptidase, a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase, a serine protease, a glutamic protease, an aspartate protease, a cysteine protease, a threonine protease, or a metalloprotease.

18. A method of claim 8 , wherein the enzyme comprises a glycoside hydrolase.

19. A method of claim 8 , wherein the enzyme comprises a phosphatase.

20. A method of claim 19 , wherein the phosphatase comprises a phosphoric monoester hydrolase, a phosphomonoesterase, a phosphoric diester hydrolase, a phosphodiesterase, a triphosphoric monoester hydrolase, a phosphoryl anhydride hydrolase, a pyrophosphatase, a phytase, a trimetaphosphatase, or a triphosphatase.

21. A method of claim 8 , wherein the enzyme comprises a nitrogenase.

22. A method of claim 21 , wherein the nitrogenase comprises a Nif family nitrogenase.

23. A method of claim 8 , wherein the enzyme comprises a nuclease.

24. A method of claim 8 , wherein the plant growth medium is supplemented with a substrate for an enzyme.

25. A method of claim 24 , wherein the substrate comprises tryptophan, an adenosine monophosphate, an adenosine diphosphate, an adenosine triphosphate, indole, a trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, or a combination thereof.

26. A method of claim 1 , comprising coating the plant seed with the seed coating formulation.

27. A method of claim 1 , wherein the recombinant Bacillus cereus family member comprises Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephensis , or a combination thereof.

28. A method of claim 1 , wherein the plant growth stimulating protein or peptide comprises a peptide hormone.

29. A method of claim 28 , wherein the peptide hormone comprises a phytosulfokine, clavata 3 (CLV3), systemin, ZmlGF, or a SCR/SP11.

30. A method of claim 1 , wherein the plant growth stimulating protein or peptide comprises a non-hormone peptide.

31. A method of claim 30 , wherein the non-hormone peptide comprises a RKN 16D10, Hg-Syv46, an eNOD40 peptide, RHPP, or kunitz trypsin inhibitor.

32. A method of claim 1 , wherein the plant growth stimulating protein or peptide is physically attached to the exosporium of the recombinant Bacillus cereus family member.

33. A method of claim 1 , further comprising inactivating the recombinant Bacillus cereus family member prior to application to the plant seed.

34. A method of claim 1 , wherein the fusion protein comprises:
(a) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;
(b) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;
(c) a targeting sequence comprising SEQ ID NO: 1; or
(d) an exosporium protein comprising SEQ ID NO: 2.

35. A method of claim 1 , wherein the fusion protein comprises:
(a) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 3;
(b) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 3;
(c) a targeting sequence comprising SEQ ID NO: 3; or
(d) an exosporium protein comprising SEQ ID NO: 4.

36. A method of claim 1 , wherein the fusion protein comprises:
(a) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 5;
(b) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 5;
(c) a targeting sequence comprising SEQ ID NO: 5; or
(d) an exosporium protein comprising SEQ ID NO: 6.

37. A method of claim 1 , wherein the fusion protein comprises:
(a) a target

Assignees

• Spogen Biotech Inc

Inventors

• Thompson Brian
• Thompson Katie

Classifications

• C12Y301/01003

  Triacylglycerol lipase (3.1.1.3) · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12N9/20

  Triglyceride splitting, e.g. by means of lipase · CPC title

• A01N37/48

  Nitro-carboxylic acids; Derivatives thereof · CPC title