Insecticidal compositions and methods for making insect-resistant transgenic plants
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.
1. A method for enhancing the accumulation of a first insecticidal protein in a recombinant host cell, said method comprising:
contemporaneously expressing said first insecticidal protein, said first insecticidal protein selected from the group consisting of: ET37 as set forth in SEQ ID NO:2 or SEQ ID NO:18, ET29 as set forth in SEQ ID NO:8, and TIC809 as set forth in SEQ ID NO:14, with a second insecticidal protein, said second insecticidal protein selected from the group consisting of: TIC810 as set forth in SEQ ID NO:4 or SEQ ID NO:16, and TIC812 as set forth in SEQ ID NO:6 or SEQ ID NO:20, said first insecticidal protein and said second insecticidal protein being under the control of one or more heterologous promoters in said recombinant host cell,
wherein said contemporaneous expression of said first insecticidal protein and said second insecticidal protein enhances the accumulation of the first protein compared to the accumulation of said first insecticidal protein expressed in a recombinant host cell in the absence of said second insecticidal protein.
2. A composition exhibiting insecticidal activity, said composition comprising TIC809 as set forth in SEQ ID NO:14 and TIC810 as set forth in SEQ ID NO:4 or SEQ ID NO:16.
3. A method for making a plant cell resistant to an insect pest, the method 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 as set forth in SEQ ID NO:8, ET37 as set forth in SEQ ID NO:2 or SEQ ID NO:18, and TIC809 as set forth in SEQ ID NO:14, and a second protein selected from the group consisting of: TIC810 as set forth in SEQ ID NO:4 or SEQ ID NO:16 and TIC812 as set forth in SEQ ID NO:6 or SEQ ID NO:20.
4. The method of claim 3 , wherein said plant cell is selected from the group consisting of a monocot plant cell and a dicot plant cell.
5. The method of claim 4 , wherein:
(1) said monocot plant cell is selected from the group consisting of: corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass, 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.
6. A transgenic plant or plant cell resistant to insect infestation, said plant or plant cell comprising a pesticidally effective amount of an insecticidal composition, said insecticidal composition comprising a first polynucleotide encoding a first protein and a second polynucleotide encoding a second protein, wherein said first protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, and SEQ ID NO:18, and said second protein comprises an amino acid sequence exhibiting at least 99% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:16.
7. The transgenic plant or plant cell of claim 6 , wherein said transgenic plant or plant cell 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 plant or plant cell, and said monocot plant being further selected from the group consisting of: corn, wheat, oat, rice, sorghum, milo, buckwheat, rye, grass, and barley plant or plant cell.
8. The transgenic plant or plant cell of claim 6 , wherein said transgenic crop plant further comprises an additional insecticidal agent toxic to the insect infestation, wherein said additional insecticidal agent is selected from the group consisting of: a Bacillus toxin, a Xenorhabdus toxin, a Photorhabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect.
9. An isolated and purified polynucleotide encoding an insecticidal protein, said protein comprising the amino acid sequence of SEQ ID NO:47.
10. An expression cassette for use in expressing an insecticidal protein in a recombinant host cell, wherein said expression cassette comprises, in operable linkage, a heterologous promoter sequence functional in said host cell and a nucleotide sequence encoding said insecticidal protein, wherein said insecticidal protein comprises the amino acid sequence as set forth in SEQ ID NO:4.
11. The expression cassette of claim 10 , wherein said recombinant host cell is selected from the group consisting of: a bacterial cell, a fungal cell, a mammalian cell, and a plant cell.
12. The expression cassette of claim 11 , 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, and barley.
13. The expression cassette of claim 10 , 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.
14. A vector comprising the expression cassette of any one of claims 10 - 13 .
15. The composition of claim 3 , wherein said composition comprises a fusion of said TIC809 and said TIC810 as set forth in SEQ ID NO:47.
16. A commodity product comprising a detectable amount of:
a polynucleotide as set forth in SEQ ID NO:46;
(2) a fusion of TIC809 and TIC810 as set forth in SEQ ID NO:47; or
(3) both SEQ ID NO:46 and SEQ ID NO:47.
17. The method of claim 3 ,
wherein said first protein is TIC809 and said second protein is TIC810, and
wherein said toxin composition comprises a fusion of said first protein and said second protein as set forth in SEQ ID NO:47.
18. The transgenic plant or plant cell of claim 6 ,
wherein said first protein is TIC809 and said second protein is TIC810, and
wherein said insecticidal composition comprises a fusion of said first protein and said second protein as set forth in SEQ ID NO:47.
19. A method for controlling Coleopteran or Hemipteran insect infestation of a plant, said method comprising providing in the diet of the insect the transgenic plant or plant cell of claim 6 .
20. The method of claim 19 , wherein said Coleopteran insect is a corn rootworm or said Hemipteran insect is a Lygus bug.
21. A method for protecting a crop in a field from insect infestation, said method comprising:
growing a transgenic crop plant in the field, wherein the transgenic crop plant comprises an insecticidally effective amount of a first protein and a second protein, wherein said first protein is selected from the group consisting of: ET37 as set forth in SEQ ID NO:2 or SEQ ID NO:18, ET29 as set forth in SEQ ID NO:8, and TIC809 as set forth in SEQ ID NO:14, and said second protein is selected from the group consisting of: TIC810 as set forth in SEQ ID NO:4 or SEQ ID NO:16 and TIC812 as set forth in SEQ ID NO:6 or SEQ ID NO:20, said first protein and said second protein expressed contemporaneously in said transgenic crop plant under the control of one or more heterologous promoters, thereby preventing an insect from surviving on said transgenic crop plant.
22. The method of claim 21 , wherein said insect is selected from the group consisting of: a Coleopteran insect and a Hemipteran insect.
23. The method of claim 22 , wherein said Coleopteran insect is a corn rootworm and said Hemipteran insect is a Lygus bug.
24. The method of claim 21 , wherein said transgenic crop plant further comprises an additional insecticidal agent toxic to the same insect as said first protein and said second protein, wherein said additional insecticidal agent is selected from the group consisting of: a Bacillus toxin, a Xenorhabdus toxin, a Photorhabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect.