HPPD variants and methods of use
Compositions and methods for conferring herbicide tolerance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include polynucleotides encoding herbicide tolerance polypeptides, vectors comprising those polynucleotides, and host cells comprising the vectors. The nucleotide sequences of the invention can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Compositions also include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated polynucleotides encoding HPPD inhibitor tolerance polypeptides are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.
1. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a 4-hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide of an HPPD enzyme, wherein the amino acid sequence of said HPPD polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises mutations selected from the group consisting of:
(i) the amino acid substitutions of:
(a) an aspartic acid at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1;
(b) an alanine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1;
(c) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1;
(d) an aspartic acid at the amino acid position corresponding to amino acid position 336 of SEQ ID NO:1;
(e) a serine at the amino acid position corresponding to amino acid position 337 of SEQ ID NO:1;
(f) a tyrosine at the amino acid position corresponding to amino acid position 339 of SEQ ID NO:1;
(g) an arginine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1;
(h) a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and
(i) a methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; and
(ii) the amino acid substitutions of:
(a) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1;
(b) an aspartic acid at the amino acid position corresponding to amino acid position 336 of SEQ ID NO:1;
(c) a serine at the amino acid position corresponding to amino acid position 337 of SEQ ID NO:1;
(d) a tyrosine at the amino acid position corresponding to amino acid position 339 of SEQ ID NO:1; and
(e) an arginine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1;
wherein an HPPD enzyme comprising said recombinant HPPD polypeptide retains HPPD activity in the presence of 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (Cmpd. 1) that is greater than that of an HPPD enzyme comprising the evo41 polypeptide (SEQ ID NO:6).
2. The recombinant nucleic acid molecule of claim 1 wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 10 and nucleotide sequences having at least 90% sequence identity to SEQ ID NO:10.
3. The recombinant nucleic acid molecule of claim 1 , wherein said nucleotide sequence encodes an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.
4. The recombinant nucleic acid molecule of claim 1 , wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.
5. The recombinant nucleic acid molecule of claim 1 , further comprising a promoter capable of directing expression of the nucleotide sequence in a plant cell.
6. The recombinant nucleic acid molecule of claim 1 , wherein said HPPD inhibitor herbicide is selected from the group consisting of an N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide, an N-(1,2,5-oxadiazol-3-yl)benzamide, tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione, isoxaflutole, pyrasulfotole, and mesotrione.
7. An expression cassette comprising the nucleic acid molecule of claim 1 .
8. The expression cassette of claim 7 , further comprising a nucleic acid molecule encoding a heterologous polypeptide.
9. A host cell that contains the recombinant nucleic acid molecule of claim 1 .
10. The host cell of claim 9 that is a bacterial host cell.
11. The host cell of claim 9 that is a plant cell.
12. A transgenic plant comprising the recombinant nucleic acid molecule of claim 1 .
13. The plant of claim 12 , wherein said plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.
14. A transgenic seed comprising the recombinant nucleic acid molecule of claim 1 .
15. A recombinant HPPD polypeptide of an HPPD enzyme, wherein said HPPD enzyme is tolerant to an HPPD inhibitor herbicide, and wherein the amino acid sequence of said HPPD polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises mutations selected from the group consisting of:
(i) the amino acid substitutions of:
(a) an aspartic acid at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1;
(b) an alanine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1;
(c) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1;
(d) an aspartic acid at the amino acid position corresponding to amino acid position 336 of SEQ ID NO:1;
(e) a serine at the amino acid position corresponding to amino acid position 337 of SEQ ID NO:1;
(f) a tyrosine at the amino acid position corresponding to amino acid position 339 of SEQ ID NO:1;
(g) an arginine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1;
(h) a glutamine at the amino acid position corresponding to amino acid position 344 of SEQ ID NO:1; and
(i) a methionine at the amino acid position corresponding to amino acid position 345 of SEQ ID NO:1; and
(iii) the amino acid substitutions of:
(a) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1;
(b) an aspartic acid at the amino acid position corresponding to amino acid position 336 of SEQ ID NO:1;
(c) a serine at the amino acid position corresponding to amino acid position 337 of SEQ ID NO:1;
(d) a tyrosine at the amino acid position corresponding to amino acid position 339 of SEQ ID NO:1; and
(e) an arginine at the amino acid position corresponding to amino acid position 340 of SEQ ID NO:1.
16. The recombinant polypeptide of claim 15 , wherein said amino acid sequence has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.
17. The recombinant polypeptide of claim 15 , wherein said amino acid sequence is set forth in SEQ ID NO:4.
18. The recombinant polypeptide of claim 15 , wherein said HPPD inhibitor herbicide is selected from the group consisting of an N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide, an N-(1,2,5-oxadiazol-3-yl)benzamide, tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione, isoxaflutole, pyrasulfotole, and mesotrione.
19. The recombinant polypeptide of claim 15 further comprising a heterologous polypeptide.
20. A method for conferring tolerance to an HPPD inhibitor herbicide in a plant, said method comprising transforming said plant with a DNA construct, said construct comprising a promoter that drives expression, in a plant cell, of the nucleotide sequence of claim 1 .
21. The plant of claim 12 , having stably incorporated into its genome a DNA construct, said construct comprising a promoter capable of driving expression of the nucleotide sequence encoding said HPPD polypeptide.
22. A plant cell, a plant tissue, or a plant seed of a plant of claim 21 .
23. The plant of claim 21 , wherein said plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.
24. A commodity product comprising a detectable amount of the HPPD polypeptide of claim 15 , or of a nucleic acid encoding said polypeptide.
25. Transgenic seed of the plant of claim 21 .
26. A method of controlling weeds in a field comprising planting the plant of claim 21 or a seed thereof in a field and applying to said field an effective concentration of an HPPD inhibitor herbicide.
27. The method of claim 26 , wherein said HPPD inhibitor herbicide is selected from the group consisting of an N-(tetrazol-4-yl)- or N-(triazol-3-yl)arylcarboxamide, an N-(1,2,5-oxadiazol-3-yl)benzamide, tembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione, isoxaflutole, pyrasulfotole, and mesotrione.
28. The transgenic plant of claim 21 , wherein said plant further comprises a polynucleotide that confers tolerance to glyphosate and a polynucleotide that confers tolerance to glufosinate.