Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants

Patent metadata

Field	Value
Publication number	US-9850289-B2
Application number	US-201414775892-A
Country	US
Kind code	B2
Filing date	Mar 17, 2014
Priority date	Mar 15, 2013
Publication date	Dec 26, 2017
Grant date	Dec 26, 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 and formulations containing the recombinant Bacillus cereus family members expressing the fusion proteins. Methods for stimulating plant growth, for protecting plants from pathogens, and for enhancing stress resistance in a plant 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 plants.

First claim

What is claimed is:

1. A method for stimulating plant growth comprising introducing a recombinant Bacillus cereus family member expressing a fusion protein into a plant growth medium or applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant, a plant seed, or an area surrounding a plant or a plant seed, wherein the fusion protein comprises:
a targeting sequence, exosporium protein, or exosporium protein fragment selected from the group consisting of:
(a) 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%;
(b) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;
(c) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;
(d) a targeting sequence comprising SEQ ID NO: 1:
(e) a targeting sequence comprising SEQ ID NO: 60;
(f) a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1;
(g) a targeting sequence comprising amino acids 22-33 of SEQ ID NO: 1;
(h) a targeting sequence comprising amino acids 20-31 of SEQ ID NO: 1; and

an enzyme, wherein the enzyme is phospholipase;
and wherein the recombinant Bacillus cereus family member comprises a plant-growth promoting strain of bacteria, the plant-growth promoting strain of bacteria comprising Bacillus mycoides BT155 (NRRL No. B-50921), Bacillus mycoides EE118 (NRRL No. B-50918), Bacillus mycoides EE141 (NRRL No. B-50916), Bacillus mycoides BT46-3 (NRRL No. B-50922), Bacillus cereus family member EE128 (NRRL No. B-50917), Bacillus thuringiensis BT013A (NRRL No. B-50924), or Bacillus cereus family member EE349 (NRRL No. B-50928).

2. The method of claim 1 , wherein the targeting sequence, exosporium protein, or exosporium protein fragment further comprises a methionine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein fragment.

3. The method of claim 1 , wherein the targeting sequence comprises SEQ ID NO: 60.

4. The method of claim 1 , wherein the enzyme is physically attached to the exosporium of the recombinant Bacillus cereus family member.

5. The method for stimulating plant growth of claim 1 , wherein plants grown in the presence of the recombinant Bacillus cereus family member exhibit increased growth as compared to the growth of plants grown in the absence of the recombinant Bacillus cereus family member under the same conditions.

6. The method of claim 1 , wherein the plant-growth promoting strain of bacteria comprises Bacillus thuringiensis BT013A (NRRL No. B-50924).

7. The method of claim 1 , wherein the plant-growth promoting strain of bacteria comprises Bacillus cereus family member EE349 (NRRL No. B-50928).

8. The method of claim 1 , further comprising introducing at least one agrochemical into the plant growth medium or applying at least one agrochemical to the plant, plant seed, or area surrounding the plant.

9. The method of claim 8 , wherein the agrochemical comprises a fertilizer, a micronutrient fertilizer material, an insecticide, a herbicide, a fungicide, a molluscicide, an algicide, a plant growth amendment, a bacterial inoculant, a fungal inoculant, or a combination thereof.

10. The method of claim 9 , wherein the agrochemical comprises the fertilizer.

11. The method of claim 9 , wherein the fertilizer comprises ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrate, magnesian limestone, magnesia, urea, urea-formaldehyde, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K 2 SO 4 -2MgSO 4 , kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, superphosphate, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, humic acid, or a combination thereof.

12. The A method of claim 1 , 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%.

13. The A method of claim 1 , 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%.

14. The A method of claim 1 , 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%.

15. The A method of claim 1 , wherein the targeting sequence consists of:
(a) an amino acid sequence consisting of 16 amino acids and 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: one;
(c) amino acids 20-35 of SEQ ID NO: 1;
(d) SEQ ID NO: 1; or
(e) SEQ ID NO: 60.

16. The method of claim 1 , wherein targeting sequence, the exosporium protein, or the exosporium protein fragment comprises the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.

17. The method of claim 1 , wherein the fusion protein further comprises an amino acid linker between the targeting sequence, the exosporium protein, or the exosporium protein fragment and the enzyme.

18. The method of claim 17 , wherein the linker comprises a polyalanine linker, a polyglycine linker, or a linker comprising a mixture of both alanine and glycine residues.

19. The method of claim 5 , wherein:
plants grown in the plant growth medium comprising the recombinant Bacillus cereus family member exhibit increased growth as compared to the growth of plants in the identical plant growth medium that does not contain the recombinant Bacillus cereus family member;
plants to which the recombinant Bacillus cereus family member has been applied exhibit increased growth as compared to the growth of plants to which the recombinant Bacillus cereus family member has not been applied, under the same conditions;
plants grown from plant seeds to which the recombinant Bacillus cereus family member has been applied exhibit increased growth as compared to the growth of plants grown from plant seeds to which the recombinant Bacillus cereus family member has not been applied, under the same conditions; or
plants grown in the area to which the recombinant Bacillus cereus family member has been applied exhibit increased growth as compared to the growth of plants in an area to which the recombinant Bacillus cereus family member has not been applied, under the same conditions.

20. The method of claim 6 , wherein the fusion protein comprises the targeting sequence of SEQ ID NO: 60.

21. The method of claim 1 , further comprising inactivating the rec

Assignees

• Spogen Biotech Inc

Inventors

• Thompson Brian
• Thompson Katie

Classifications

• C12Y301/01003

  Triacylglycerol lipase (3.1.1.3) · CPC title

• C12N9/48

  acting on peptide bonds (3.4) · CPC title

• C12Y118/06001

  Nitrogenase (1.18.6.1) · CPC title

• C12Y304/00

  Hydrolases acting on peptide bonds, i.e. peptidases (3.4) · CPC title

• C12Y302/01004

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