Insecticidal Compositions and Methods for Making Insect-Resistant Transgenic Plants

• US 20090068159A1
• Filed: 08/30/2006
• Published: 03/12/2009
• Est. Priority Date: 08/31/2005
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Chay, Catherine A., Anderson, Heather, Roberts, James K., Zhang, Bei, Baum, James A.
• Granted Patent: US 9,121,035 B2
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: A01N 63/23 B. thuringiensis
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/325 Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention provides isolated polynucleotide sequences encoding ET37, TIC810 and TIC812 proteins from Bacillus thuringiensis, and nucleotide sequences for use in expressing TIC809, ET37, TIC810 and TIC812, and fusions of various insecticidally effective combinations of these proteins such as TIC 127, in plants. Methods of making and using the polynucleotide sequences and the proteins in the development of transgenic plant cells and transgenic plants exhibiting improved insect resistance against (1) Coleopteran insects including Western Corn Rootworm (Dibrotica virgifera), Southern Corn Rootworm (Dibrotica undecempunctata), Northern Corn Rootworm (Diabrotica barbed), Mexican Corn Rootworm (Diabrotica virgifera zeae), Brazilian Corn Rootworm (Diabrotica balteata) and Brazilian Corn Rootworm complex (Diabrotica viridula and Diabrotica speciosa), and against Hemipteran insects such as Lygus bugs, are disclosed.

Claims

1. An isolated and purified nucleotide sequence encoding an insecticidal protein, said protein being selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, and SEQ ID NO;

47.

2. The isolated and purified nucleotide sequence of claim 1 selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, and SEQ ID NO;

46.

3. An isolated and purified insecticidal protein selected from the group consisting of an ET29, an ET37, a TIC810, a TIC812, a TIC809, and a TIC127.

4. A plant or plant cell transformed with a nucleotide sequence encoding an insecticidal protein, said insecticidal protein being selected from the group consisting of an ET29, an ET37, a TIC810, a TIC812, a TIC809, and a TIC127.

5. Progeny or seed from the plant of claim 4, wherein said progeny or seed comprises said nucleotide sequence.

6. A vector comprising a nucleotide sequence encoding a protein selected from the group consisting of an ET29, ET37, TIC810, TIC812, TIC809, and TIC127.

7. A method for enhancing the accumulation of a first insecticidal protein in a host cell, said method comprising contemporaneously expressing said first protein selected from the group consisting of ET29, TIC809, and ET37 with a second insecticidal protein selected from the group consisting of TIC810 and TIC812, wherein said contemporaneous expression enhances the accumulation of the first protein compared to the accumulation of said first protein expressed in the absence of said second protein.

8. A composition exhibiting insecticidal activity comprising a first protein selected from the group consisting of ET29, ET37, and TIC809, and a second protein selected from the group consisting of TIC810 and TIC812.

9. The composition of claim 8 wherein said first protein is TIC809 and said second protein is TIC810.

10. The composition of claim 8 wherein the ratio of said first protein to said second protein is from about 1:

1 to about 2;

1.

11. The composition of claim 8 provided in the diet of a Coleopteran or a Hemipteran plant pest in an agriculturally acceptable formulation.

12. The composition of claim 11 wherein said formulation consists of both proteins expressed in a plant cell.

13. The composition of claim 11 wherein said Coleopteran pest is a corn rootworm and said Hemipteran pest is a Lygus bug.

14. A commodity product comprising a detectable amount of a first protein selected from the group consisting of ET29, ET37 and TIC809 and a detectable amount of a second protein selected from the group consisting of TIC810 and TIC812.

16. The commodity product of any of claims 14-15 selected from the group consisting of cotton seed, cotton seed oil, cotton seed meal, soybean seed, soybean oil, soybean meal, soybean flour, distillers'"'"' dry goods solids, corn seed, corn flour, corn meal, corn syrup, corn oil, corn starch, animal feed, and cereals manufactured in whole or in part to contain soybean and/or corn by-products.

15. A commodity product comprising a detectable amount of (1) a first nucleotide sequence encoding a first protein selected from the group consisting of ET29, ET37 and TIC809 and second nucleotide sequence encoding a second protein selected from the group consisting of TIC810 and TIC812;

(2) a first protein selected from the group consisting of ET29, ET37, and TIC809 and a second protein selected from the group consisting of TIC810 and TIC812;

(3) both (1) and (2);

or (4) any combination of (1) and (2).

17. A method for making a plant cell resistant to an insect pest comprising transforming said plant cell to express an insecticidally effective amount of a toxin composition, said toxin composition comprising a first protein selected from the group consisting of ET29, ET37 and TIC809 and a second protein selected from the group consisting of TIC810 and TIC812.

18. The method of claim 17 wherein said plant cell is regenerated into a fertile transgenic plant.

19. The method of claim 17 wherein said plant cell is selected from the group consisting of a monocot plant cell and a dicot plant cell.

20. The method of claim 19, wherein (1) said monocot plant cell is selected from the group consisting of corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass (fescue, timothy, brome, orchard, St. Augustine, Bermuda, bentgrass), and barley plant cell, and (2) said dicot plant cell is selected from the group consisting of alfalfa, apple, apricot, asparagus, bean, berry, blackberry, blueberry, canola, carrot, cauliflower, celery, cherry, chickpea, citrus tree, cotton, cowpea, cranberry, cucumber, cucurbit, egg plant, fruit tree, grape, lemon, lettuce, linseed, melon, mustard, nut bearing tree, okra, orange, pea, peach, peanut, pear, plum, potato, soybeans, squash, strawberry, sugar beet, sunflower, sweet potato, tobacco, tomato, turnip, and vegetable plant cells.

21. The method of claim 18, wherein progeny plants and seeds are produced from said transgenic plant, and wherein said progeny plants and seed comprise said first and second protein.

22. A host cell comprising a nucleotide sequence encoding a cyt protein, said protein being selected from the group consisting of a TIC809, a ET37, a TIC810, a TIC812, and a TIC127.

23. The host cell of claim 22 selected from the group consisting of a plant cell, a fungal cell and a bacterial cell.

24. The host cell of claim 23 wherein said plant cell is selected from the group consisting of a monocot plant cell and a dicot plant cell.

25. The host cell of claim 24 wherein (1) said monocot plant cell is selected from the group consisting of corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass (fescue, timothy, brome, orchard, St. Augustine, Bermuda, bentgrass), and barley plant cell, and (2) said dicot plant cell is selected from the group consisting of alfalfa, apple, apricot, asparagus, bean, berry, blackberry, blueberry, canola, carrot, cauliflower, celery, cherry, chickpea, citrus tree, cotton, cowpea, cranberry, cucumber, cucurbit, egg plant, fruit tree, grape, lemon, lettuce, linseed, melon, mustard, nut bearing tree, okra, orange, pea, peach, peanut, pear, plum, potato, soybeans, squash, strawberry, sugar beet, sunflower, sweet potato, tobacco, tomato, turnip, and vegetable plant cells.

26. An expression cassette for use in expressing an insecticidal protein in a host cell, wherein said expression cassette comprises in operable linkage a promoter sequence functional in said host cell and a nucleotide sequence encoding said protein, said protein being selected from the group consisting of TIC809, TIC810, TIC812, ET37, and TIC127.

27. The expression cassette of claim 26 wherein said host cell is selected from the group consisting of a bacterial cell, a fungal cell, a mammalian cell, and a plant cell.

28. The expression cassette of claim 27 wherein

(a) said bacterial cell is selected from the group consisting of a Bacillus species cell, a Enterobacteriacae species cell, a Pseudomonas species cell, a Clostridium species cell, and a Rhizobium species cell, and a Agrobacterium species cell;

and

(b) said plant cell is selected from the group of plants consisting of a dicotyledonous plant and a monocotyledonous plant, said dicotyledonous plant being further selected from the group consisting of alfalfa, apple, apricot, asparagus, bean, berry, blackberry, blueberry, canola, carrot, cauliflower, celery, cherry, chickpea, citrus tree, cotton, cowpea, cranberry, cucumber, cucurbit, egg plant, fruit tree, grape, lemon, lettuce, linseed, melon, mustard, nut bearing tree, okra, orange, pea, peach, peanut, pear, plum, potato, soybeans, squash, strawberry, sugar beet, sunflower, sweet potato, tobacco, tomato, turnip, and vegetable, and said monocotyledonous plant being further selected from the group consisting of corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass (fescue, timothy, brome, orchard, St. Augustine, Bermuda, bentgrass), and barley.

29. The expression cassette of claim 26 wherein said host cell is a plant cell and said expression cassette further comprises in operable linkage a sequence selected from the group consisting of an expression enhancer sequence, an untranslated leader sequence, an intron sequence, a chloroplast targeting peptide encoding sequence, and a transcription termination and polyadenylation sequence.

30. A vector comprising the expression cassette of any of claims 26 through claim 29.

31. A transgenic plant or plant cell resistant to insect infestation comprising a nucleotide sequence encoding a first cyt toxin selected from the group consisting of ET29, TIC809, and ET37 and a second cyt toxin selected from the group consisting of TIC810 and TIC812.

32. The transgenic plant or plant cell of claim 31 wherein said transgenic plant is selected from the group consisting of a dicot plant or dicot plant cell and a monocot plant or monocot plant cell, said dicot plant or dicot plant cell being further selected from the group consisting of alfalfa, apple, apricot, asparagus, bean, berry, blackberry, blueberry, canola, carrot, cauliflower, celery, cherry, chickpea, citrus tree, cotton, cowpea, cranberry, cucumber, cucurbit, egg plant, fruit tree, grape, lemon, lettuce, linseed, melon, mustard, nut bearing tree, okra, orange, pea, peach, peanut, pear, plum, potato, soybeans, squash, strawberry, sugar beet, sunflower, sweet potato, tobacco, tomato, turnip, and vegetable, and said monocot plant being further selected from the group consisting of corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass (fescue, timothy, brome, orchard, St. Augustine, Bermuda, bentgrass), and barley.

33. The transgenic plant or plant cell of claim 32 resistant to (1) coleopteran insect infestation, wherein said coleopteran insect is selected from the group consisting of a Chrysomelidae, Cucujidae, Scarabaeidae, Trogositidae, Tenebrionidae, Curculionidae, Elateridae and Brichidae, and (2) hemiptera insect infestation, wherein said hemiptera are selected from the sub-orders Heteroptera and Homoptera.

34. The transgenic plant or plant cell of claim 33, wherein said Chrysomelidea is a Diabrotica species selected from the group consisting of D. virgifera (western corn rootworm), D. undecempunctata (southern corn rootworm), D. barberi (northern corn rootworm), D. virgifera zeae (Mexican corn rootworm), D. balteata (brazilian corn rootworm) and Brazilian Corn Rootworm complex (BCR) further comprising D. viridula and D. speciosa.

35. Progeny or seed of the transgenic plant or plant cell of claim 33, wherein said progeny or seed comprise said nucleotide sequence.

36. A method for controlling coleopteran or hemipteran insect infestation of a plant by providing in the diet of the insect one or more plant cells transformed with a nucleic acid sequence comprising in operable linkage a plant functional promoter and a nucleotide sequence encoding a fusion protein, wherein said fusion protein comprises a first amino acid sequence selected from the group consisting of a SEQ ID NO:

2, SEQ ID NO;

8, and SEQ ID NO;

14, and a second amino acid sequence selected from the group consisting of SEQ ID NO;

4 and SEQ ID NO;

6.

37. A method for protecting a crop in a field from insect infestation comprising growing a transgenic crop plant comprising an insecticidally effective amount of a first protein and a second protein, said first protein being selected from the group consisting of an ET37, an ET29, and a TIC809 and said second protein being selected from the group consisting of TIC810 and TIC812, and providing the proteins together in the diet of the insect to inhibit the insect from surviving on said transgenic crop plant.

38. The method of claim 37 wherein said insect is selected from the group consisting of a coleopteran insect and a Hemipteran insect.

39. The method of claim 37, wherein said transgenic crop plant further comprises an additional insecticidal agent toxic to the same insect as the first and second proteins, wherein said additional insecticidal agent is selected from the group consisting of a Bacillus toxin, a Xenorhabdus toxin, a Photorlabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect.

40. The method of claim 37-39 wherein the yield of said crop is improved compared to the yield of an isogenic crop lacking said insecticidal proteins.