Plants having increased tolerance to herbicides

A method of identifying a nucleotide sequence encoding a mut-HPPD which is resistant or tolerant to a bicycloarylcarboxamide, the method comprising:

• a) generating a library of mut-HPPD-encoding nucleic acids,
• b) screening a population of the resulting mut-HPPD-encoding nucleic acids by expressing each of said nucleic acids in a cell or plant and treating said cell or plant with a bicycloarylcarboxamide
• c) comparing the bicycloarylcarboxamide-tolerance levels provided by said population of mut-HPPD encoding nucleic acids with the bicycloarylcarboxamide-tolerance level provided by a control HPPD-encoding nucleic acid,
• d) selecting at least one mut-HPPD-encoding nucleic acid that provides a significantly increased level of tolerance to a bicycloarylcarboxamide as compared to that provided by the control HPPD-encoding nucleic acid.

An isolated nucleic acid encoding a mut-HPPD, wherein the mut-HPPD comprises a polypeptide which differs from an amino acid sequence of a wild-type HPPD at one or more positions corresponding to or at the following positions of SEQ ID NO:2:

• the amino acid corresponding to or at position 236 is other than alanine;
• the amino acid corresponding to or at position 411 is other than glutamic acid;
• the amino acid corresponding to or at position 320 is other than leucine;
• the amino acid corresponding to or at position 403 is other than glycine;
• the amino acid corresponding to or at position 334 is other than leucine;
• the amino acid corresponding to or at position 353 is other than leucine;
• the amino acid corresponding to or at position 321 is other than proline;
• the amino acid corresponding to or at position 212 is other than valine;
• the amino acid corresponding to or at position 407 is other than glycine;
• the amino acid corresponding to or at position 377 is other than phenylalanine;
• the amino acid corresponding to or at position 412 is other than leucine;
• the amino acid corresponding to or at position 278 is other than glutamine;
• the amino acid corresponding to or at position 406 is other than lysine;
• the amino acid corresponding to or at position 404 is other than phenylalanine;
• the amino acid corresponding to or at position 409 is other than phenylalanine;
• the amino acid corresponding to or at position 416 is other than isoleucine;
• the amino acid corresponding to or at position 250 is other than leucine;
• the amino acid corresponding to or at position 267 is other than asparagine;
• the amino acid corresponding to or at position 252 is other than serine;
• the amino acid corresponding to or at position 265 is other than proline;
• the amino acid corresponding to or at position 371 is other than glycine;
• the amino acid corresponding to or at position 375 is other than threonine;
• the amino acid corresponding to or at position 309 is other than arginine;
• the amino acid corresponding to or at position 279 is other than isoleucine;
• the amino acid corresponding to or at position 366 is other than phenylalanine;
• the amino acid corresponding to or at position 238 is other than phenylalanine;
• the amino acid corresponding to or at position 213 is other than valine;
• the amino acid corresponding to or at position 215 is other than asparagine;
• the amino acid corresponding to or at position 410 is other than serine;
• the amino acid corresponding to or at position 254 is other than valine.

An isolated nucleic acid encoding a mut-HPPD, wherein the mut-HPPD comprises a polypeptide which differs from an amino acid sequence of a wild-type HPPD at one or more positions corresponding to the following positions of SEQ ID NO:53:

• the amino acid corresponding to or at position 228 is other than valine;
• the amino acid corresponding to or at position 230 is other than asparagine;
• the amino acid corresponding to or at position 251 is other than alanine;
• the amino acid corresponding to or at position 253 is other than phenylalanine;
• the amino acid corresponding to or at position 265 is other than leucine;
• the amino acid corresponding to or at position 267 is other than serine;
• the amino acid corresponding to or at position 280 is other than proline;
• the amino acid corresponding to or at position 282 is other than asparagine;
• the amino acid corresponding to or at position 291 is other than lysine;
• the amino acid corresponding to or at position 293 is other than glutamine;
• the amino acid corresponding to or at position 294 is other than isoleucine;
• the amino acid corresponding to or at position 324 is other than arginine;
• the amino acid corresponding to or at position 335 is other than methionine;
• the amino acid corresponding to or at position 336 is other than proline;
• the amino acid corresponding to or at position 337 is other than serine;
• the amino acid corresponding to or at position 339 is other than proline;
• the amino acid corresponding to or at position 340 is other than proline;
• the amino acid corresponding to or at position 363 is other than glutamic acid;
• the amino acid corresponding to or at position 368 is other than leucine;
• the amino acid corresponding to or at position 381 is other than phenylalanine;
• the amino acid corresponding to or at position 385 is other than leucine;
• the amino acid corresponding to or at position 386 is other than glycine;
• the amino acid corresponding to or at position 390 is other than threonine;
• the amino acid corresponding to or at position 392 is other than phenylalanine;
• the amino acid corresponding to or at position 393 is other than an isoleucine;
• the amino acid corresponding to or at position 419 is other than phenylalanine;
• the amino acid corresponding to or at position 421 is other than lysine;
• the amino acid corresponding to or at position 422 is other than glycine;
• the amino acid corresponding to or at position 424 is other than phenylalanine;
• the amino acid corresponding to or at position 427 is other than leucine;
• the amino acid corresponding to or at position 431 is other than isoleucine;
• the amino acid corresponding to or at position 425 is other than serine;
• the amino acid corresponding to or at position 269 is other than valine.

A transgenic plant cell transformed by a wild-type or mut-HPPD nucleic acid, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a HPPD-inhibiting herbicide as compared to a wild type variety of the plant cell,

• and wherein the wild-type or mut-HPPD nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
• a) a polynucleotide as shown in SEQ ID NO:1, 51, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 52, 54, 56, 68, 69, or a variant or derivative thereof;
• b) a polynucleotide as shown in SEQ ID NO:47 or 49, or a variant or derivative thereof;
• c) a polynucleotide encoding a polypeptide as shown in SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 53, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 48, 50, or a variant or derivative thereof;
• d) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) through c);
• and e) a polynucleotide complementary to the polynucleotide of any of a) through d).

A plant that expresses a mutagenized or recombinant mut-HPPD comprising SEQ ID NO:2, a homologue, orthologue or paralogue thereof, in which the amino acid sequence differs from a HPPD amino acid sequence of a corresponding wild-type plant at one or more amino acid positions:

• the amino acid corresponding to or at position 236 is other than alanine;
• the amino acid corresponding to or at position 411 is other than glutamic acid;
• the amino acid corresponding to or at position 320 is other than leucine;
• the amino acid corresponding to or at position 403 is other than glycine;
• the amino acid corresponding to or at position 334 is other than leucine;
• the amino acid corresponding to or at position 353 is other than leucine;
• the amino acid corresponding to or at position 321 is other than proline;
• the amino acid corresponding to or at position 212 is other than valine;
• the amino acid corresponding to or at position 407 is other than glycine;
• the amino acid corresponding to or at position 377 is other than phenylalanine;
• the amino acid corresponding to or at position 412 is other than leucine;
• the amino acid corresponding to or at position 278 is other than glutamine;
• the amino acid corresponding to or at position 406 is other than lysine;
• the amino acid corresponding to or at position 404 is other than phenylalanine;
• the amino acid corresponding to or at position 409 is other than phenylalanine;
• the amino acid corresponding to or at position 416 is other than isoleucine;
• the amino acid corresponding to or at position 250 is other than leucine;
• the amino acid corresponding to or at position 267 is other than asparagine;
• the amino acid corresponding to or at position 252 is other than serine;
• the amino acid corresponding to or at position 265 is other than proline;
• the amino acid corresponding to or at position 371 is other than glycine;
• the amino acid corresponding to or at position 375 is other than threonine;
• the amino acid corresponding to or at position 309 is other than arginine;
• the amino acid corresponding to or at position 279 is other than isoleucine;
• the amino acid corresponding to or at position 366 is other than phenylalanine;
• the amino acid corresponding to or at position 238 is other than phenylalanine;
• the amino acid corresponding to or at position 213 is other than valine;
• the amino acid corresponding to or at position 215 is other than asparagine;
• the amino acid corresponding to or at position 410 is other than serine;
• the amino acid corresponding to or at position 254 is other than valine.
• and wherein said mutagenized or recombinant mut-HPPD confers upon the plant increased herbicide tolerance, preferably bicycloarylcarboxamide herbicide tolerance, as compared to the corresponding wild-type variety of the plant when expressed therein.

A plant that expresses a mutagenized or recombinant mut-HPPD comprising SEQ ID NO:53, a homologue, orthologue or paralogue thereof, in which the amino acid sequence differs from a HPPD amino acid sequence of a corresponding wild-type plant at one or more amino acid positions:

• the amino acid corresponding to or at position 228 is other than valine;
• the amino acid corresponding to or at position 230 is other than asparagine;
• the amino acid corresponding to or at position 251 is other than alanine;
• the amino acid corresponding to or at position 253 is other than phenylalanine;
• the amino acid corresponding to or at position 265 is other than leucine;
• the amino acid corresponding to or at position 267 is other than serine;
• the amino acid corresponding to or at position 280 is other than proline;
• the amino acid corresponding to or at position 282 is other than asparagine;
• the amino acid corresponding to or at position 291 is other than lysine;
• the amino acid corresponding to or at position 293 is other than glutamine;
• the amino acid corresponding to or at position 294 is other than isoleucine;
• the amino acid corresponding to or at position 324 is other than arginine;
• the amino acid corresponding to or at position 335 is other than methionine;
• the amino acid corresponding to or at position 336 is other than proline;
• the amino acid corresponding to or at position 337 is other than serine;
• the amino acid corresponding to or at position 339 is other than proline;
• the amino acid corresponding to or at position 340 is other than proline;
• the amino acid corresponding to or at position 363 is other than glutamic acid;
• the amino acid corresponding to or at position 368 is other than leucine;
• the amino acid corresponding to or at position 381 is other than phenylalanine;
• the amino acid corresponding to or at position 385 is other than leucine;
• the amino acid corresponding to or at position 386 is other than glycine;
• the amino acid corresponding to or at position 390 is other than threonine;
• the amino acid corresponding to or at position 392 is other than phenylalanine;
• the amino acid corresponding to or at position 393 is other than an isoleucine;
• the amino acid corresponding to or at position 419 is other than phenylalanine;
• the amino acid corresponding to or at position 421 is other than lysine;
• the amino acid corresponding to or at position 422 is other than glycine;
• the amino acid corresponding to or at position 424 is other than phenylalanine;
• the amino acid corresponding to or at position 427 is other than leucine;
• the amino acid corresponding to or at position 431 is other than isoleucine;
• the amino acid corresponding to or at position 425 is other than serine;
• the amino acid corresponding to or at position 269 is other than valine,
• and wherein said mutagenized or recombinant mut-HPPD confers upon the plant increased herbicide tolerance, preferably bicycloarylcarboxamide herbicide tolerance, as compared to the corresponding wild-type variety of the plant when expressed therein.

A method of identifying or selecting a transformed plant cell, plant tissue, plant or part thereof comprising:

• i) providing a transformed plant cell, plant tissue, plant or part thereof, wherein said transformed plant cell, plant tissue, plant or part thereof comprises a polynucleotide as shown in SEQ ID NO:1, 51, 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 52, 54, 56, 68, 69, or a variant or derivative thereof, wherein the polynucleotide encodes an HPPD polypeptide that is used as a selection marker, and wherein said transformed plant cell, plant tissue, plant or part thereof may comprise a further isolated polynucleotide;
• ii) contacting the transformed plant cell, plant tissue, plant or part thereof with at least one HPPD-inhibiting compound, preferably bicycloarylcarboxamide;
• iii) determining whether the plant cell, plant tissue, plant or part thereof is affected by the inhibiting compound;
• and iv) identifying or selecting the transformed plant cell, plant tissue, plant or part thereof.