Mutated protoporphyrinogen ix oxidase (PPX) genes

Abstract

To provide a plant or plant cell that can have resistance to a PPX-inhibiting herbicide, including a mutated protoporphyrinogen IX oxidase (PPX) gene.SOLUTION: The present invention relates to a plant or plant cell including a PPX gene having a targeted mutation introduced artificially into a specific amino acid, and a method for producing the plant cell, including a gene repair oligonucleobase (GRON) having a targeted mutation in a PPX gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates, at least in part, to gene and / or protein mutations in plants. [Background technology]

[0002] The following description is provided solely to aid in the understanding of the present invention and is not admitted to describe or constitute prior art.

[0003] Examples of specific mutations in plant PPX genes have been reported. For example, U.S. Patent No. 5,767,373 discloses "a eukaryotic DNA sequence encoding a herbicide-resistant native protoporphyrinogen oxidase (protox) or a modified version of said enzyme"; U.S. Patent No. 6,282,837 discloses "a method for suppressing weeds using a eukaryotic DNA sequence encoding a herbicide-resistant native protoporphyrinogen oxidase (protox) or a modified version of said enzyme and a plant having modified protox activity that confers herbicide resistance"; U.S. Patent No. 6,308,458 discloses "a method for suppressing the growth of undesirable vegetation, comprising applying an effective amount of a protox-inhibiting herbicide to a population of transgenic plants or plant seeds transformed with a DNA sequence encoding a modified protox enzyme that is resistant to a protox-inhibiting herbicide, or to a locus in which said transgenic plants or plant seeds are cultivated"; U.S. Patent No. No. 6,905,852 discloses "a protoporphyrinogen oxidase resistant to photobleaching herbicides and their derivatives, comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 [PPX protein] or a mutated peptide derived therefrom by deletion, addition, substitution, etc. of one or more amino acids in said amino acid sequence, and having activity substantially corresponding to that of protoporphyrinogen oxidase"; U.S. Pat. No. ( ) discloses "a method for conferring resistance to protoporphyrinogen-inhibiting herbicides in crop plants.US Patent Application Publication No. 20020086395 discloses "a method for evaluating the ability of a compound to inhibit protoporphyrinogen oxidase activity, comprising: (1) culturing a transformant expressing a protoporphyrinogen oxidase gene present in a DNA fragment in a medium substantially free of protoheme compounds in each comparative system in the presence and absence of a test compound to measure the growth rate of the transformant under each condition (wherein the transformant arises from a host cell deficient in growth ability based on protoporphyrinogen oxidase activity transformed by a DNA fragment to which a promoter operable in the host cell and a protoporphyrinogen oxidase gene are operably linked); and (2) determining the ability of the compound to inhibit protoporphyrinogen oxidase activity by comparing the growth rates, etc.; Patzoldt WL et al., PNAS USA 103:12329-34 (2006) discloses "a 3-bp deletion corresponding to the G210 codon" in PPX; Li X et al., Plant Physiology 133:736-47 (2003) discloses "Isolation of a plant protoporphyrinogen (PPO) gene and isolation of a herbicide-resistant mutation." The terms PPO and PPX are used synonymously herein. Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure relates, at least in part, to methods and compositions related to gene and protein mutations in plants. In some aspects and embodiments, the present disclosure may also relate to compositions and methods for creating herbicide-resistant plants. The methods and compositions of the present disclosure relate, at least in part, to mutations in the protoporphyrinogen IX oxidase (PPX) gene. [Means for solving the problem]

[0005] In one aspect, a plant or plant cell is provided that comprises a mutated PPX gene.In certain embodiments, the mutated PPX gene encodes a mutated PPX protein.In certain embodiments, the plant that has a plant cell that comprises a mutated PPX gene can have herbicide resistance, for example, resistance to herbicides that inhibit PPX.In certain embodiments, the plant or plant cell is non-transgenic.In certain embodiments, the plant or plant cell is transgenic.The present disclosure also provides a recombinant vector that comprises this mutated PPX gene, as well as a transgenic plant that comprises this mutated PPX gene.

[0006] As used herein, the term "PPX gene" refers to a DNA sequence capable of producing a PPX polypeptide that shares homology and / or amino acid identity with the amino acid sequence SEQ ID NO:1 and / or encodes a protein exhibiting PPX activity. In certain embodiments, the PPX gene has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a particular PPX gene, e.g., StmPPX1 or StmPPX2; or, e.g., a plastidial Russet Burbank PPX gene, e.g., StcPPX1, e.g., a mitochondrial Russet Burbank PPX gene. In certain embodiments, the PPX gene has 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from the sequences of Figures 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 43, and 45. In some embodiments, the PPX gene is a mitochondrial PPX gene, e.g., StmPPX1 or StmPPX2. In some embodiments, the PPX gene is a plastidial PPX gene, e.g., StcPPX. In some embodiments, the PPX gene is an allele of a mitochondrial PPX gene, e.g., StmPPX2.1 or StmPPX2.2. In some embodiments, the PPX gene is an allele of a plastidial PPX gene, e.g., StcPPX1 or StcPPX1.1. In some plants, such as water hemp, the protein product of a single PPX gene is both a mitochondrial and a plastidial product, as disclosed by Patzoldt WL et al., PNAS USA 103:1, 2329-34 (2006).

[0007] As used herein, the term "mutation" refers to at least one nucleotide change in a nucleic acid sequence and / or one amino acid change in a polypeptide compared to a normal or wild-type sequence or reference sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a mutation refers to at least one nucleotide change in a nucleic acid sequence and / or one amino acid change in a polypeptide compared to the nucleotide or amino acid sequence of a PPX protein that is not herbicide-resistant. In certain embodiments, a mutation can include a substitution, deletion, inversion, or insertion. In some embodiments, a substitution, deletion, insertion, or inversion can include a change at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, a substitution, deletion, insertion, or inversion can include a mutation at 1, 2, 3, 4, 5, 6, 7, or 8 amino acid positions. The term "nucleic acid" or "nucleic acid sequence" refers to oligonucleotides, nucleotides, or polynucleotides, and fragments or portions thereof, which may be single-stranded or double-stranded, and may represent either the sense or antisense strand. Nucleic acids include DNA or RNA and can be naturally occurring or synthetic. For example, nucleic acids can include mRNA or cDNA. Nucleic acids can include nucleic acids that have been amplified (e.g., using the polymerase chain reaction). The term "NTwt###NTmut" is used to represent a mutation that results in the wild-type nucleotide NTwt at position ### in the nucleic acid being replaced by the mutant NTmut. Single-alphabet codes for nucleotides follow the description in U.S. Manual of Patent Examining Procedures, Section 2422, Table 1. In this regard, the nucleotide designation "R" refers to a purine, such as guanine or adenine; "Y" refers to a pyrimidine, such as cytosine or thymine (or uracil in the case of RNA); "M" refers to adenine or cytosine; "K" refers to guanine or thymine; and "W" refers to adenine or thymine.

[0008] As used herein, the term "mutated PPX gene" refers to a PPX gene having one or more mutations at a nucleotide position compared to a reference PPX nucleic acid sequence. In certain embodiments, the mutated PPX gene has one or more mutations compared to the corresponding wild-type PPX sequence. As used herein, the term "wild-type" may also be used to designate the standard allele at a locus, i.e., the allele with the highest frequency in a particular population. In some cases, the wild-type allele may be represented by a particular amino acid or nucleic acid sequence. For example, a wild-type potato plastid PPX protein may be represented by SEQ ID NO: 7. For example, a wild-type potato mitochondrial PPX protein may be represented by SEQ ID NO: 9. In some embodiments, the mutated PPX gene has one or more mutations compared to a reference PPX nucleic acid sequence, e.g., at homologous positions in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 43, or 45, or paralogs thereof. In some embodiments, the mutated PPX gene is modified by at least one mutation. In other embodiments, the mutated PPX gene is modified by at least two mutations. In other embodiments, the mutated PPX gene is modified by at least three mutations. In some embodiments, the mutated PPX gene encodes a mutated PPX protein. In some embodiments, the mutated PPX gene comprises mutations in two or more nucleic acid sequences selected from Tables 2, 3a, and 3b. In some embodiments, the mutated PPX gene encodes one or more mutated mitochondrial PPX proteins. In other embodiments, the mutated PPX gene encodes one or more mutated plastidial PPX proteins. In some embodiments, the mutated PPX gene is a mutated mitochondrial PPX gene, such as a mutated StmPPX1. In some embodiments, the mutated PPX gene is a mutated mitochondrial PPX gene, such as a mutated StmPPX2.In some embodiments, the mutated PPX gene is a mutated plastidial PPX gene, e.g., a mutated StcPPX1. In some embodiments, the mutated PPX gene is a mutated mitochondrial PPX gene allele, e.g., a mutated StmPPX2.1 or a mutated StmPPX2.2. In some embodiments, the mutated PPX gene is a mutated plastidial PPX gene allele, e.g., a mutated StcPPX1 or a mutated StcPPX1.1. In some embodiments, there is at least one mutation in the plastidial PPX gene and at least one mutation in the mitochondrial PPX gene. In some embodiments, one or more mutations in the PPX gene lead to herbicide resistance, e.g., resistance to herbicides that inhibit PPX. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that has increased resistance to one or more herbicides compared to a reference PPX protein.

[0009] In some embodiments, the mutations in the mutated PPX gene encode a protein having a combination of two or more mutations. In certain embodiments, at least one mutation is present in a plastidic PPX gene and at least one mutation is present in a mitochondrial PPX gene. In certain embodiments, the combination is selected from Tables 4a and 4b. In some embodiments, the mutations in the mutated PPX gene encode a protein having a combination of three or more mutations, for example, a combination selected from Tables 4a and 4b. In some embodiments, the at least one mutation in the plastidic PPX gene and the at least one mutation in the mitochondrial PPX gene are present at the same corresponding position. In other embodiments, the at least one mutation in the plastidic PPX gene and the at least one mutation in the mitochondrial PPX gene are present at different corresponding positions.

[0010] As used herein, the term "PPX protein" refers to a protein having homology and / or amino acid identity to and / or exhibiting PPX activity with a PPX protein of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, or 44. In certain embodiments, the PPX protein has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a particular PPX protein, e.g., a mitochondrial Russet Burbank PPX protein or a plastidial Russet Burbank PPX protein. In certain embodiments, the PPX protein has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from the sequences of Figure 1, Figure 3, Figure 5, Figure 7, Figure 9, Figure 11, Figure 13, Figure 15, Figure 17, Figure 19, Figure 21, Figure 23, Figure 25, Figure 27, Figure 29, Figure 31, Figure 33, Figure 35, Figure 37, Figure 39, Figure 40, Figure 42, or Figure 44.

[0011] As used herein, the term "mutated PPX protein" refers to a PPX protein having one or more mutations at amino acid positions relative to a reference PPX amino acid sequence or homologous positions of a paralog thereof. In some embodiments, the mutated PPX protein has one or more mutations relative to a reference PPX amino acid sequence having SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, or 44, or a portion thereof. In certain embodiments, the mutated PPX protein has one or more mutations compared to the corresponding wild-type protein. In some embodiments, the mutated PPX protein has one or more mutations compared to a corresponding protein that is not herbicide-resistant. In some embodiments, the PPX protein is modified by at least one mutation. In other embodiments, the PPX protein is modified by at least two mutations. In other embodiments, the PPX protein is modified by at least three mutations. In some embodiments, one or more mitochondrial PPX proteins are mutated. In other embodiments, one or more plastidial PPX proteins are mutated. In yet another embodiment, one or more mitochondrial PPX proteins and one or more plastidial PPX proteins are mutated. In some embodiments, the term "mutated PPX protein" refers to a PPX protein that has increased resistance to one or more herbicides compared to a reference protein.

[0012] In some embodiments, the mutated PPX protein is selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 340, 341, 342, 343, 344, 345, 346, 347, 348, and comprising a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of: 2, 305, 311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508, and 525. In some embodiments, the mutated PPX protein contains a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO:9.In some embodiments, the plant or plant cell is a plant that contains a gene selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, S332, S343, A354, L357, K359, The gene may include a mutated protoporphyrinogen IX oxidase (PPX) gene encoding a protein containing a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525. In some embodiments, the plant or plant cell is a plant that contains a gene selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, X195, P214, I215, K229, K230, C271, D274, F283, A292, S296, The gene may include a mutated protoporphyrinogen IX oxidase (PPX) gene encoding a protein containing a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470 and T500.In some embodiments, the PPX protein is a paralogue of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato plastid PPX protein), and the PPX protein can have an N at position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; a K at position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; an S at position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S; and / or an S at position corresponding to position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S. In some embodiments, the mutated PPX protein can be a paralogue of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato plastid PPX protein), and the PPX protein can have an N at position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; a K at position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; an S at position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S; and / or an S at position corresponding to position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S. G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E 212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, and two or more mutations, at least one mutation at an amino acid position corresponding to a position selected from the group consisting of S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525.In some embodiments, the mutated PPX protein comprises D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, X195, P214, I215, K229, K230, C271, D27 of SEQ ID NO: 9. 4, two or more mutations, at least one mutation at an amino acid position corresponding to a position selected from the group consisting of F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470, and T500. In some embodiments, the PPX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato plastid PPX protein), and the PPX protein has two or more mutations and further has one or more of the following: (1) an N at a position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; (2) a K at a position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; (3) an S at a position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S; and / or (4) an S at a position corresponding to position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S.In some embodiments, the mutated PPX protein comprises any of the following: G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, S332, S343, A354, L357, K359, and comprising three or more, at least one mutation at an amino acid position corresponding to a position selected from the group consisting of L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525. In some embodiments, the mutated PPX protein comprises D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, X195, P214, I215, K229, K230, C271, D192, C193, H194, L195, P214, I215, K229, K230, C271, D195, P214, I215, K230, C271, D196, P214, I215, K229, K230, C271, D197, P214, I215, K230, C271, D198, P214, I215, K229, K230, C271, D199, P215, I216, K230, C272, D199, P215, I216, K230, C271, D199, P215, I215, K229, K230, C271, D199, P215, I215 ... and three or more, at least one mutation at an amino acid position corresponding to a position selected from the group consisting of 274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470, and T500.In some embodiments, the PX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato PPX protein), and the PPX protein has three or more mutations and further has one or more of the following: (1) an N at a position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; (2) a K at a position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; (3) an S at a position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S; and / or (4) an S at position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S.

[0013] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX protein comprises one or more amino acid mutations selected from Table 1, Table 2, Table 3a, Table 3b, Table 4a, Table 4b, Tables 8a-f, Tables 9a-d and Table 10. In some embodiments, the mutated PPX protein comprises two or more amino acids selected from Table 1, Table 2, Table 3a, Table 3b, Table 4a, Table 4b, Tables 8a-f, Tables 9a-d and Table 10. In some embodiments, the mutated PPX protein comprises three or more amino acid mutations selected from Table 1, Table 2, Table 3a, Table 3b, Table 4a, Table 4b, Tables 8a-f, Tables 9a-d and Table 10. In some embodiments, the mutated PPX protein comprises one or more nucleic acid sequence mutations selected from Table 2, Table 3a and Table 3b. In some embodiments, the one or more mutations in the mutated PPX protein are glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1; asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1; glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO:1; glycine to asparagine at a position corresponding to position 130 of SEQ ID NO:1; aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO:1; proline to arginine at a position corresponding to position 143 of SEQ ID NO:1; arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1; Arginine to leucine at a position corresponding to position 144 of SEQ ID NO:1; arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1; phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1; phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1; leucine to valine at a position corresponding to position 147 of SEQ ID NO:1; phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO:1; alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1; proline to arginine at a position corresponding to position 185 of SEQ ID NO:1;Proline to histidine at a position corresponding to position 185 of SEQ ID NO:1; Proline to tyrosine at a position corresponding to position 185 of SEQ ID NO:1; Glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO:1; Glutamic acid to lysine at a position corresponding to position 192 of SEQ ID NO:1; Serine to threonine at a position corresponding to position 193 of SEQ ID NO:1; Arginine to leucine at a position corresponding to position 199 of SEQ ID NO:1; Valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO:1; Tyrosine to serine at a position corresponding to position 219 of SEQ ID NO:1; Alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1; Alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1; Alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1; Alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 alanine to valine at position 226 of SEQ ID NO:1; leucine to methionine at position 228 of SEQ ID NO:1; lysine to glutamine at position 229 of SEQ ID NO:1; alanine to phenylalanine at position 230 of SEQ ID NO:1; serine to glycine at position 244 of SEQ ID NO:1; serine to threonine at position 244 of SEQ ID NO:1; arginine to histidine at position 256 of SEQ ID NO:1; arginine to serine at position 256 of SEQ ID NO:1; lysine to glutamic acid at position 270 of SEQ ID NO:1; lysine to glutamine at position 270 of SEQ ID NO:1; proline to arginine at position 271 of SEQ ID NO:1; glutamine to phenylalanine at position 272 of SEQ ID NO:1;Serine to leucine at a position corresponding to position 305 of SEQ ID NO:1, glutamic acid to arginine at a position corresponding to position 311 of SEQ ID NO:1, threonine to glycine at a position corresponding to position 316 of SEQ ID NO:1, threonine to glycine at a position corresponding to position 318 of SEQ ID NO:1, serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1, leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1, lysine to arginine at a position corresponding to position 359 of SEQ ID NO:1, lysine to threonine at a position corresponding to position 359 of SEQ ID NO:1, leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO:1, leucine to lysine at a position corresponding to position 360 of SEQ ID NO:1, alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO:1, leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1 leucine to serine at a position corresponding to position 393 of SEQ ID NO:1, leucine to valine at a position corresponding to position 403 of SEQ ID NO:1, leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1; leucine to serine at a position corresponding to position 403 of SEQ ID NO:1; leucine to serine at a position corresponding to position 424 of SEQ ID NO:1; tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1;Serine to leucine at a position corresponding to position 430 of SEQ ID NO:1, lysine to serine at a position corresponding to position 438 of SEQ ID NO:1, glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO:1, valine to isoleucine at a position corresponding to position 444 of SEQ ID NO:1, leucine to valine at a position corresponding to position 455 of SEQ ID NO:1, lysine to valine at a position corresponding to position 457 of SEQ ID NO:1, valine to serine at a position corresponding to position 470 of SEQ ID NO:1, valine to tyrosine at a position corresponding to position 470 of SEQ ID NO:1, phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1, phenylalanine to serine at a position corresponding to position 483 of SEQ ID NO:1 phenylalanine to glycine, aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO:1, isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO:1, aspartic acid to glycine at a position corresponding to position 487 of SEQ ID NO:1, lysine to asparagine at a position corresponding to position 490 of SEQ ID NO:1, leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO:1, valine to threonine at a position corresponding to position 508 of SEQ ID NO:1, and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the PPX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato PPX protein), wherein the PPX protein has an N at a position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; a K at a position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; or an S at a position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S;and / or has an S at a position corresponding to position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S. In such embodiments, the one or more mutations in the mutated PPX protein are an asparagine to lysine at a position corresponding to position 52 of SEQ ID NO:1; asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1; arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1; arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1; phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1; phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1; alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1; proline to arginine at a position corresponding to position 185 of SEQ ID NO:1; proline to histidine at a position corresponding to position 185 of SEQ ID NO:1; alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1; alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1; alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1; Alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1; alanine to valine at a position corresponding to position 220 of SEQ ID NO:1; leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1; methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1; serine to glycine at a position corresponding to position 244 of SEQ ID NO:1; serine to threonine at a position corresponding to position 244 of SEQ ID NO:1; lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1;Serine to leucine at a position corresponding to position 305 of SEQ ID NO:1, serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1, leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1, serine to arginine at a position corresponding to position 359 of SEQ ID NO:1, serine to threonine at a position corresponding to position 359 of SEQ ID NO:1, leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1, leucine to serine at a position corresponding to position 393 of SEQ ID NO:1, leucine to valine, leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1; leucine to serine at a position corresponding to position 403 of SEQ ID NO:1; leucine to serine at a position corresponding to position 424 of SEQ ID NO:1; tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1; tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1; SEQ ID NO: Tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:1; Tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1; Tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1; Tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1; Phenylanine to serine at a position corresponding to position 478 of SEQ ID NO:1; Isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1; aspartic acid to asparagine in SEQ ID NO:9, glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO:9; glycine to cysteine ​​at a position corresponding to position 74 of SEQ ID NO:9; glycine to asparagine at a position corresponding to position 84 of SEQ ID NO:9; leucine to histidine at a position corresponding to position 93 of SEQ ID NO:9, lysine to arginine at a position corresponding to position 97 of SEQ ID NO:9; arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, Arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, alanine to valine at a position corresponding to position 101 of SEQ ID NO:9, serine to asparagine at a position corresponding to position 119 of SEQ ID NO:9, phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9; asparagine to tyrosine, asparagine to arginine at a position corresponding to position 139 of SEQ ID NO:9, asparagine to histidine at a position corresponding to position 139 of SEQ ID NO:9, glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO:9, glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO:9, serine to threonine at a position corresponding to position 151 of SEQ ID NO:9, glutamine to leucine at a position corresponding to position 157 of SEQ ID NO:9;Valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO:9; Valine to alanine at a position corresponding to position 164 of SEQ ID NO:9; Aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO:9, Cysteine ​​to serine at a position corresponding to position 177 of SEQ ID NO:9; Histidine to glutamine at a position corresponding to position 187 of SEQ ID NO:9; Leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, Asparagine to lysine at a position corresponding to position 195 of SEQ ID NO:9; Proline to serine at a position corresponding to position 214 of SEQ ID NO:9; Proline to histidine at a position corresponding to position 214 of SEQ ID NO:9; isoleucine to serine at a position corresponding to position 215 of SEQ ID NO:9; isoleucine to histidine at a position corresponding to position 215 of SEQ ID NO:9; lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO:9; lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9; lysine to arginine at a position corresponding to position 230 of SEQ ID NO:9; cysteine ​​to arginine at a position corresponding to position 271 of SEQ ID NO:9; aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO:9; phenylalanine to glycine at a position corresponding to position 283 of SEQ ID NO:9, Alanine to glycine at a position corresponding to position 292 of SEQ ID NO:9; serine to leucine at a position corresponding to position 296 of SEQ ID NO:9; cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9; asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO:9; asparagine to lysine at a position corresponding to position 324 of SEQ ID NO:9; aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO:9; serine to leucine at a position corresponding to position 396 of SEQ ID NO:9; alanine to serine at a position corresponding to position 404 of SEQ ID NO:9;Arginine to lysine at a position corresponding to position 406 of SEQ ID NO:9, lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO:9, leucine to valine at a position corresponding to position 421 of SEQ ID NO:9, alanine to valine at a position corresponding to position 423 of SEQ ID NO:9, cysteine ​​to serine at a position corresponding to position 434 of SEQ ID NO:9; cysteine ​​to tyrosine at a position corresponding to position 434 of SEQ ID NO:9; aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO:9; serine to alanine at a position corresponding to position 448 of SEQ ID NO:9, the amino acid sequence of SEQ ID NO:9 includes one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of: valine to glutamic acid at a position corresponding to position 449 of SEQ ID NO:9, aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO:9, aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO:9; tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO:9, lysine to threonine at a position corresponding to position 470 of SEQ ID NO:9, and threonine to serine at a position corresponding to position 500 of SEQ ID NO:9;

[0014] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, in certain embodiments, the mutated PPX protein may comprise a combination of multiple mutations, for example, a combination of mutations selected from Tables 4a and 4b. In some embodiments, the mutated PPX protein comprises a combination of two or more mutations; for example, a combination of mutations selected from Tables 4a and 4b. In some embodiments, the mutated PPX protein comprises a combination of three or more mutations; for example, a combination selected from Tables 4a and 4b. In some embodiments, the combination of mutations in the mutated PPX gene encodes a protein having a mutation at a position corresponding to Y426 of SEQ ID NO: 1 and mutations at one or more amino acid positions corresponding to positions selected from the group consisting of N85, R144, F145, A180, A220, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations in the mutated PPX gene encodes a protein having a mutation at a position corresponding to L393 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of R144, F145, A220, S224, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to L403 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of F145, A220, and L226 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to R144 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of G52, N85, A220, S244, L226, M228, K272, S332, L393, L424, Y426, and 1525 of SEQ ID NO: 1.In some embodiments, the combination of mutations encodes a protein having a mutation at one or more amino acid positions corresponding to N85 of SEQ ID NO: 1 and a position selected from the group consisting of R144, F145, A180, A220, L226, M228, and Q272 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to L424 of SEQ ID NO: 1 and a mutation at an amino acid position corresponding to a position selected from the group consisting of R144, F145, A220, L226, and L393 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to I525 of SEQ ID NO: 1 and a mutation at an amino acid position corresponding to positions N85, F144, F145, A180, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to R144 of SEQ ID NO:1 and a mutation at a position corresponding to position A220 of SEQ ID NO:1. In some embodiments, the PPX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato PPX protein), and the PPX protein has an N at a position corresponding to position 52 of SEQ ID NO:1, where the N is substituted with an amino acid other than N; a K at a position corresponding to position 272 of SEQ ID NO:1, where the K is substituted with an amino acid other than K; an S at a position corresponding to position 359 of SEQ ID NO:1, where the S is substituted with an amino acid other than S; or an S at a position corresponding to position 525 of SEQ ID NO:1, where the S is substituted with an amino acid other than S. In such embodiments, the mutated PPX protein comprises two or more combinations; for example, a combination selected from Table 4a and Table 4b. In such embodiments, the mutated PPX protein comprises a combination of three or more mutations; for example, a combination selected from Table 4a and Table 4b.In some embodiments, the combination of mutations in the mutated PPX gene encodes a protein having a mutation at a position corresponding to Y426 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of N85, R144, F145, A180, A220, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations in the mutated PPX gene encodes a protein having a mutation at a position corresponding to L393 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of R144, F145, A220, S244, and S224 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to L403 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of F145, A220, and L226 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to R144 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of N52, N85, A220, S244, L226, M228, K272, S332, L393, L424, Y426, and S525 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to N85 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of R144, F145, A180, A220, L226, M228, and K272 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to L424 of SEQ ID NO:1 and a mutation at an amino acid position corresponding to a position selected from the group consisting of R144, F145, A220, L226 and L393 of SEQ ID NO:1.In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to S525 of SEQ ID NO: 1, and mutations at amino acid positions corresponding to positions N85, F144, F145, A180, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to 98 of SEQ ID NO: 9, and mutations at amino acid positions corresponding to positions selected from the group consisting of 74, 93, 97, 98, 119, 121, 124, 139, 150, 151, 164, 188, 214, 229, 230, 271, 274, 292, 307, 324, 396, 410, 423, 434, 447, 448, 451, 465, 470, and 500 of SEQ ID NO: 9. In certain embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to 98 of SEQ ID NO:9 and a mutation at an amino acid position corresponding to a position selected from the group consisting of 271, 274, 292, 307, 324, 330, 396, 404, 406, 410, 423, 434, 447, 448, 454, 465, 470 and 500 of SEQ ID NO:9. In certain embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to 98 of SEQ ID NO:9 and a mutation at an amino acid position corresponding to a position selected from the group consisting of 307 and 423 of SEQ ID NO:9. In certain embodiments, the combination of mutations encodes a protein having a mutation at a position corresponding to 98 of SEQ ID NO:9 and a mutation at an amino acid position corresponding to a position selected from the group consisting of 124, 188, 214 and 229 of SEQ ID NO:9.

[0015] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the PPX protein may be a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein may be a potato PPX protein), and the PPX protein may have one or more corresponding PPX amino acids to SEQ ID NO: 9. In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the one or more mutations in the mutated PPX gene may be at positions 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, The gene encoding the mutated PPX protein may encode a mutated PPX protein having one or more mutations, two or more mutations, three or more mutations selected from the group consisting of mutated PPX proteins which may contain one or more mutations at amino acid positions corresponding to one or more positions selected from the group consisting of 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500.

[0016] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the plant cell may have a mutated PPX gene. In certain embodiments, the mutated PPX gene encodes a mutated PPX protein. In certain embodiments, the plant cell may be part of a herbicide-tolerant plant. The method may include introducing a gene repair oligonucleobase (GRON) into the plant cell; for example, using a GRON in combination with a targeted mutation in the PPX gene. In certain embodiments, the plant cell produced by the method may contain a PPX gene capable of expressing a mutated PPX protein. The method may further include identifying a plant cell or a plant containing the plant cell that contains (1) a mutated PPX gene and / or (2) normal growth and / or catalytic activity compared to corresponding wild-type plant cells. Herbicide-tolerant plants having plant cells as described herein may be identified in the presence of a herbicide that inhibits PPX. In some embodiments, the plant cell is non-transgenic. In some embodiments, the plant cell is transgenic. The plant comprising the plant cell as described herein can be a non-transgenic or transgenic herbicide-tolerant plant; for example, the plant and / or plant cell can have a mutated PPX gene, which leads to the acquisition of resistance to at least one herbicide.In some embodiments, the plant comprising the plant cell as described herein can be produced by asexual reproduction; for example, it can be produced from one or more plant cells or from plant tissues that consist of one or more plant cells; for example, it can be produced from tubers.In other embodiments, the plant comprising the plant cell as described herein can be produced by sexual reproduction.

[0017] In another aspect, a method for producing herbicide-resistant plants is provided.This method can include introducing gene repair oligonucleobase (GRON) into plant cells; for example, using GRON designed for targeted mutation in PPX gene.Mutated PPX gene can express mutated PPX protein.This method can further include identifying plants that have normal growth and / or catalytic activity compared with corresponding wild-type plant cells.The plant can be identified in the presence of herbicide that inhibits PPX.In some embodiments, the plant is non-transgenic.In some embodiments, the plant is a non-transgenic herbicide-resistant plant; for example, the plant can comprise a mutated PPX gene that leads to the acquisition of resistance to at least one herbicide.

[0018] In another aspect, seeds comprising mutated PPX gene are provided.In some embodiments, the seeds have mutated PPX gene.In some embodiments, the mutated PPX encodes mutated PPX protein.In some embodiments, the mutated PPX protein can be resistant to herbicides, for example, herbicides that inhibit PPX.In some embodiments, the plants that are grown from the seeds are resistant to at least one herbicide, for example, herbicides that inhibit PPX.

[0019] In another aspect, a method for increasing herbicide resistance in a plant is provided by: (a) crossing a first plant with a second plant, wherein the first plant contains a mutated PPX gene, the gene encoding a mutated PPX protein; (b) screening the population resulting from the cross for increased herbicide resistance, e.g., increased resistance to herbicides that inhibit PPX; (c) selecting members resulting from the cross with increased herbicide resistance; and / or (d) producing seeds resulting from the cross. In some embodiments, the hybrid seeds are produced by any of the methods described herein. In some embodiments, plants are grown from seeds produced by any of the methods described herein. In some embodiments, the plants or seeds are non-transgenic. In some embodiments, the plants and / or seeds are transgenic.

[0020] In another aspect, a method for suppressing weeds in a field containing plants is provided by applying an effective amount of at least one herbicide to the field containing the weeds and the plants. In some embodiments of the method, the at least one herbicide is a herbicide that inhibits PPX. In some embodiments of the method, one or more of the plants in the field contain a mutated PPX gene, for example, a mutated PPX gene as described herein. In some embodiments of the method, one or more of the plants in the field include non-transgenic plants or transgenic plants having a mutated PPX gene as described herein. In some embodiments, the mutated PPX gene encodes a mutated PPX protein. In some embodiments, one or more of the plants in the field are herbicide-resistant, for example, resistant to a herbicide that inhibits PPX.

[0021] In another aspect, provided is an isolated nucleic acid that encodes PPX protein or a part thereof.In some embodiments, the isolated nucleic acid comprises one or more PPX gene mutations as described herein.In some embodiments, the isolated nucleic acid encodes a mutated PPX protein as disclosed herein.In certain embodiments, the isolated nucleic acid encodes herbicide-resistant PPX protein; for example, the PPX protein is resistant to herbicides that inhibit PPX.

[0022] In another aspect, an expression vector comprising an isolated nucleic acid of a mutated PPX gene is provided. In some embodiments, the expression vector comprises an isolated nucleic acid encoding a PPX protein. In some embodiments, the isolated nucleic acid encodes a protein having a mutation selected from those shown in Table 1, Table 2, Table 3a, Table 3b, Table 4a, Table 4b, Tables 8a-8f, Tables 9a-9d, and Table 10. In certain embodiments, the isolated nucleic acid encodes a protein having two or more mutations. In some embodiments, the two or more mutations are selected from those shown in Table 1, Table 2, Table 3a, Table 3b, Table 4a, Table 4b, Tables 8a-8f, Tables 9a-9d, and Table 10. In certain embodiments, the isolated nucleic acid encodes a PPX protein that is herbicide-resistant, e.g., resistant to herbicides that inhibit PPX.

[0023] As used herein, the term "herbicide" refers to any chemical or substance capable of killing a plant or arresting or reducing the growth and / or vigor of a plant. In some embodiments, herbicide resistance is the genetic ability of a plant to survive and reproduce after being treated with a concentration of herbicide that would normally kill or severely injure an unmodified wild-type plant. In some embodiments, in conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the herbicide is a herbicide that inhibits PPX. In some embodiments, the herbicide that inhibits PPX is an herbicide from a chemical family selected from the group of chemical families listed in Table 5. In some embodiments, the herbicide that inhibits PPX is an herbicide from a chemical family selected from the group of chemical families consisting of N-phenylphthalimides, triazolinones, and pyrimidinediones. In some embodiments, the herbicide that inhibits PPX is selected from the group of herbicides listed in Table 5. In some embodiments, the PPX-inhibiting herbicide is selected from the group of herbicides consisting of flumioxazin, sulfentrazone, and saflufenacil. In other embodiments, the PPX-inhibiting herbicide is a flumioxazin herbicide. In other embodiments, the PPX-inhibiting herbicide is a sulfentrazone herbicide. In other embodiments, the PPX-inhibiting herbicide is a saflufenacil herbicide.

[0024] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the plant or plant cell is derived from a plant crop selected from the group consisting of potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oats, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soybean spp, sugarcane, pea, field bean, poplar, grape, citrus, alfalfa, rye, oats, turf and grasses, flax, oilseed rape, cucumber, morning glory, impatiens, pepper, eggplant, marigold, water lily, cabbage, daisy, carnation, petunia, tulip, iris, lily, and, unless specifically mentioned already, nut-bearing plants. In some embodiments, the plant or plant cell belongs to a species selected from Table 6. In some embodiments, the plant or plant cell belongs to a species selected from the group consisting of Arabidopsis, haliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Amaranthus tuberculatus, Zea mays, Brassica napus, and Glycine max. In some embodiments, the plant or plant cell is a Russet Burbank potato cultivar. In some embodiments, the mutated PPX gene encodes a Russet Burbank PPX protein. In some embodiments, the mutated PPX gene encodes an Arabidopsis thaliana PPX protein. In some embodiments, the mutated PPX gene encodes a Solanum tuberosum PPX protein. In some embodiments, the mutated PPX gene encodes a Solanum phureja PPX protein. In some embodiments, the mutated PPX gene encodes a Zea mays PPX protein. In some embodiments, the mutated PPX gene encodes an Oryza sativ PPX protein.In some embodiments, the mutated PPX gene encodes an Amaranthus tuberculatus PPX protein. In some embodiments, the mutated PPX gene encodes a Sorghum bicolor PPX protein. In some embodiments, the mutated PPX gene encodes a Ricinus communis PPX protein. In some embodiments, the mutated PPX gene encodes a Brassica napus PPX protein. In some embodiments, the mutated PPX gene encodes a Glycine max PPX protein. In some embodiments, the mutated PPX gene At4G01690 encodes an Arabidopsis thalian PPX protein. In some embodiments, the mutated PPX gene AT5g14220 encodes an Arabidopsis thalian PPX protein.

[0025] Aspects, embodiments, methods, and / or compositions disclosed herein may include one or more mutated PPX genes. In some embodiments, the methods and compositions require one or more mutated PPX genes encoding one or more mitochondrial PPX proteins. In other embodiments, the methods and compositions include one or more mutated PPX genes encoding one or more plastidic PPX proteins. In some embodiments, the methods and compositions include one or more mutated PPX genes encoding one or more plastidic PPX proteins and one or more mutated PPX genes encoding a mitochondrial PPX protein. In some embodiments, the methods and compositions include a mutated mitochondrial PPX gene, StmPPX1. In some embodiments, the methods and compositions include a mutated mitochondrial PPX gene, StmPPX2. In some embodiments, the plant includes a mutated plastidic PPX gene, StcPPX1. In some embodiments, the methods and compositions include a mutated mitochondrial PPX gene allele, StcPPX2.1. In some embodiments, the methods and compositions include a mutated mitochondrial PPX gene allele, StcPPX2.2. In some embodiments, the methods and compositions comprise a mutated plastid PPX gene allele, StcPPX1. In some embodiments, the methods and compositions comprise a mutated plastid PPX gene allele, StcPPX1.1.

[0026] As used herein, the term "gene" refers to a DNA sequence that contains control and coding sequences necessary for the production of RNA, which may have a non-coding function (e.g., ribosomal or transfer RNA) or may encode a polypeptide or polypeptide precursor. The RNA or polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or function is maintained.

[0027] As used herein, the term "coding sequence" refers to a nucleic acid or its complement, or a portion thereof, sequence that can be transcribed and / or translated to produce mRNA and / or a polypeptide or fragment thereof. A coding sequence includes exons in genomic DNA or immature primary RNA transcripts, which are linked together by the cell's biochemical machinery to give mature mRNA. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0028] As used herein, the term "non-coding sequence" refers to a sequence or portion of a nucleic acid or its complement that is not transcribed into amino acids in vivo, or a sequence that does not interact with or attempt to interact with tRNA to position an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts and gene-associated sequences such as promoters, enhancers, silencers, etc.

[0029] Nucleobase is a base, and in certain preferred embodiments, it is purine, pyrimidine, or their derivatives or analogs.Nucleoside is an acid base containing a pentose-furanosyl moiety, such as an optionally substituted riboside or 2'-deoxyriboside.This moiety can be any group that increases DNA binding and / or reduces nuclease degradation compared with nucleosides that do not have this moiety.Nucleosides can be linked by one of several linking moieties, which may or may not contain phosphorus.Nucleosides that are linked by unsubstituted phosphodiester bonds are called nucleotides.

[0030] Oligonucleobase is a polymer containing nucleobases, preferably at least a portion of which can hybridize to DNA with a complementary sequence through Watson-Crick base pairing. An oligonucleobase chain can have a single 5' and 3' end, which is the final nucleobase in the polymer. A specific oligonucleobase chain can contain all types of nucleobases. An oligonucleobase compound is a compound that contains one or more oligonucleobase chains that are complementary and can hybridize through Watson-Crick base pairing. Ribo-type nucleobases are pentose-furanosyl-containing nucleobases, and the 2' carbon is methylene substituted with hydroxyl, alkyloxy, or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases, including all nucleobases that do not contain pentose-furanosyl moieties.

[0031] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, an oligonucleobase strand can include both an oligonucleobase chain and an oligonucleobase segment or region. An oligonucleobase strand can have a 5'-end and a 3'-end, and when an oligonucleobase strand is coextensive with a chain, the 5'-end and 3'-end of the oligonucleobase strand are also the 5'-end and 3'-end of the chain.

[0032] As used herein, the term "gene repair oligonucleobase" or "GRON" refers to oligonucleobases, including mixed duplex oligonucleotides, non-nucleotide-containing molecules, single-stranded oligodeoxynucleotides and other gene repair molecules.

[0033] As used herein, the term "transgenic" refers to an organism or cell that has DNA originating from another organism inserted into its genome. For example, in some embodiments, a transgenic organism or cell includes inserted DNA that includes a heterologous promoter and / or coding region.

[0034] As used herein, the term "non-transgenic" refers to an organism or cell that does not have DNA from another organism inserted into its genome, although a non-transgenic plant or cell may have one or more artificially introduced targeted mutations.

[0035] As used herein, the term "isolated," when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or mixed polymers), refers to a nucleic acid that is separated from a substantial portion of the genome in which it naturally occurs and / or that is substantially separated from other cellular components that naturally accompany such nucleic acid. For example, a nucleic acid that is synthetically produced (e.g., by sequential base condensation) is considered isolated. Similarly, a nucleic acid that is recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or excised from a genome by other methods is also considered isolated.

[0036] As used herein, the term "amino acid sequence" refers to the sequence of a polypeptide or protein. The notation "AAwt###AAmut" is used to represent a mutation in which the wild-type amino acid AAwt is replaced with the mutant AAmut at position ### in a polypeptide.

[0037] As used herein, the term "complement" refers to the complementary sequence of a nucleic acid that follows the standard Watson-Crick base pairing rules. A complementary sequence may be an RNA sequence that is complementary to a DNA sequence or its complement, or may be cDNA.

[0038] As used herein, the term "substantially complementary" refers to two sequences that hybridize under stringent hybridization conditions. One of skill in the art will understand that substantially complementary sequences need not hybridize over their entire length.

[0039] As used herein, the term "codon" refers to a sequence of three adjacent nucleotides (either RNA or DNA) that constitutes the genetic code that determines the insertion of a particular amino acid into a polypeptide chain during protein synthesis or a signal to stop protein synthesis. The term "codon" is also used to refer to the corresponding (and complementary) sequence of three nucleotides in the messenger RNA into which the original DNA is transcribed.

[0040] As used herein, the term "homology" refers to sequence similarity between proteins and DNA. The terms "homology" or "homology" refer to the degree of identity. There may be partial or complete homology. A partially homologous sequence is one that has less than 100% sequence identity when compared to another sequence.

[0041] As used herein, the quantitative term "about" refers to plus or minus 10%. For example, "about 3%" includes 2.7 to 3.3%, and "about 10%" includes 9 to 11%. Furthermore, when "about" is used in conjunction with a quantitative term herein, the exact value of the quantitative term is contemplated and described in addition to the value plus or minus 10%. For example, the term "about 3%" explicitly contemplates, describes, and includes exactly 3%. [Brief explanation of the drawings]

[0042] [Figure 1] 1 is the amino acid sequence of the PPX protein from Arabidopsis thaliana chloroplasts (plastids) (SEQ ID NO: 1). [Figure 2] This is the nucleic acid sequence of Arabidopsis thaliana chloroplast (plastid) PX cDNA (SEQ ID NO: 2). [Figure 3] 1 is the amino acid sequence of the Arabidopsis thaliana mitochondrial PPX protein (SEQ ID NO: 3). [Figure 4]1 is the nucleic acid sequence of Arabidopsis thaliana mitochondrial PPX cDNA (SEQ ID NO: 4). [Figure 5] Amino acid sequence of Amaranthus tuberculatus mitochondrial PPX protein (SEQ ID NO: 5). [Figure 6] Amino acid sequence of Amaranthus tuberculatus mitochondrial PPX cDNA (SEQ ID NO: 6). [Figure 7] 1 is the amino acid sequence of the Solanum tuberosum plastid PPX protein StcPPX (SEQ ID NO: 7). [Figure 8] 1 is the nucleic acid sequence of the PPX cDNA from Solanum tuberosum plastid (SEQ ID NO: 8). [Figure 9] 1 is the amino acid sequence of the Solanum tuberosum mitochondrial PPX protein (SEQ ID NO: 9). [Figure 10] 1 is the nucleic acid sequence of Solanum tuberosum mitochondrial PPX cDNA (SEQ ID NO: 10). [Figure 11] Amino acid sequence of the PPX protein from Zea mays plastid (SEQ ID NO: 11). [Figure 12] Nucleic acid sequence of the Zea mays plastid PPX cDNA (SEQ ID NO: 12). [Figure 13] Amino acid sequence of Zea mays mitochondrial PPX protein (SEQ ID NO: 13). [Figure 14] Nucleic acid sequence of Zea mays mitochondrial PPX cDNA (SEQ ID NO: 14). [Figure 15] 1 is the amino acid sequence of the PPX protein from Oryza sativa plastid (SEQ ID NO: 15). [Figure 16] 1 is the nucleic acid sequence of the Oryza sativa plastid PPX cDNA (SEQ ID NO: 16). [Figure 17]1 is the amino acid sequence of the Oryza sativa mitochondrial PPX protein cDNA (SEQ ID NO: 17). [Figure 18] 1 is the nucleic acid sequence of Oryza sativa mitochondrial PPX cDNA (SEQ ID NO: 18). [Figure 19] Amino acid sequence of the PPX protein from Sorghum bicolor plastid (SEQ ID NO: 19). [Figure 20] 1 is the nucleic acid sequence of the PPX cDNA from Sorghum bicolor plastid (SEQ ID NO: 20). [Figure 21] Amino acid sequence of Sorghum bicolor mitochondrial PPX protein (SEQ ID NO: 21). [Figure 22] 1 is the nucleic acid sequence of the Sorghum bicolor mitochondrial PPX cDNA (SEQ ID NO: 22). [Figure 23] Amino acid sequence of the PPX protein from Ricinus communis plastid (SEQ ID NO: 23). [Figure 24] Nucleic acid sequence of the PPX cDNA from Ricinus communis plastid (SEQ ID NO: 24). [Figure 25] Amino acid sequence of Ricinus communis mitochondrial PPX protein (SEQ ID NO: 25). [Figure 26] Nucleic acid sequence of Ricinus communis mitochondrial PPX cDNA (SEQ ID NO: 26). [Figure 27] 1 is the amino acid sequence of the Solanum Tuberosum mitochondrial PPX protein StmPPX1 (SEQ ID NO: 27). [Figure 28] Nucleic acid sequence of Solanum Tuberosum mitochondrial PPX cDNA StmPPX1 (SEQ ID NO: 28). [Figure 29]1 is the amino acid sequence of the Solanum Tuberosum mitochondrial PPX protein StmPPX2.1 (SEQ ID NO: 29). [Figure 30] Nucleic acid sequence of Solanum Tuberosum mitochondrial PPX cDNA StmPPX2.1 (SEQ ID NO: 30). [Figure 31] 1 is the amino acid sequence of the Solanum Tuberosum mitochondrial PPX protein StmPPX2.2 (SEQ ID NO: 31). [Figure 32] Nucleic acid sequence of Solanum Tuberosum mitochondrial PPX cDNAStmPPX2.2 (SEQ ID NO: 32). [Figure 33] 1 is the amino acid sequence of the PPX protein BncPPX1 from Brassica napus plastid (SEQ ID NO: 33). [Figure 34]

[0047] Figure 1 is the nucleic acid sequence of Brassica napus PPX cDNA BncPPX1 (SEQ ID NO: 34). [Figure 35] 1 is the amino acid sequence of the Brassica napus plastid PPX protein BncPPX2 (SEQ ID NO: 35). [Figure 36]

[0041] Figure 1 is the nucleic acid sequence of Brassica napus PPX cDNA BncPPX2 (SEQ ID NO: 36). [Figure 37] Partial amino acid sequence of the PPX protein BncPPX3 from Brassica napus plastid (SEQ ID NO: 37). [Figure 38] Partial nucleic acid sequence of Brassica napus PPX cDNA BncPPX3 (SEQ ID NO: 38). [Figure 39] 1 is the amino acid sequence of the Glycine max plastid PPX protein GmcPPX1-1 (SEQ ID NO: 39). [Figure 40] 1 is the amino acid sequence of the Glycine max plastid PPX protein GmcPPX1-2 (SEQ ID NO: 40). [Figure 41]

[0047] Figure 1 is the nucleic acid sequence of the Glycine max plastid PPX protein GmcPPX1 (SEQ ID NO: 41). [Figure 42] 1 is the amino acid sequence of the Glycine max plastid PPX protein GmcPPX2 (SEQ ID NO: 42). [Figure 43]

[0047] Figure 1 is the nucleic acid sequence of the Glycine max plastid PPX protein GmcPPX2 (SEQ ID NO: 43). [Figure 44] 1 is the amino acid sequence of the Glycine max mitochondrial PPX protein GmcPPX (SEQ ID NO: 44). [Figure 45]

[0047] Figure 1 is the nucleic acid sequence of the Glycine max mitochondrial PPX protein GmcPPX (SEQ ID NO: 45). [Figure 46] Alignment of PPX proteins from various plant species. [Figure 47] 1 is a table of homologous amino acid positions in plant PPX amino acid sequences of various species. [Figure 48] 1 is a table of homologous amino acid positions in the PPX amino acid sequences of various plant species. DETAILED DESCRIPTION OF THE INVENTION

[0043] Rapid Trait Development System (RTDS (registered trademark)) In any of the various aspects and embodiments of the compositions and methods disclosed herein, mutations in genes and proteins can be created, for example, using the Rapid Trait Development System (RTDS®) ​​technology developed by Cibus. Plants containing any of the mutations disclosed herein, in combination or alone, can form the basis of novel herbicide-tolerant products. Also provided are seeds produced from mutated plants that are either homozygous or heterozygous for mutations in the PPX gene. The mutations disclosed herein can be combined with any other known mutations or with mutations discovered in the future.

[0044] As used herein, the term "heterozygous" refers to having different alleles at one or more loci in homologous chromosomal segments. As used herein, "heterozygous" can also refer to a sample, cell, cell population, or organism in which different alleles can be detected at one or more loci. Heterozygous samples can be determined by methods known in the art, such as nucleic acid sequencing. For example, if the sequencing electropherogram shows two peaks at a single locus, both peaks being approximately the same size, the sample can be characterized as heterozygous. Alternatively, if one peak is smaller than the other but at least about 25% the size of the larger peak, the sample can be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the smaller peak is at least about 10% of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

[0045] The term "hemizygous" refers to a gene or gene segment that is present only once in the genotype of a cell or organism due to the absence of a second allele. As used herein, "hemizygous" can also refer to a sample, cell, cell population, or organism in which an allele can be detected only once in the genotype at one or more loci.

[0046] In some embodiments, RTDS relies on utilizing a cell's own gene repair system to specifically modify gene sequences in situ, altering targeted genes without inserting foreign DNA and / or gene expression control sequences. This method allows for precise changes to be made to gene sequences while leaving the rest of the genome unchanged. In contrast to traditional transgenic GMOs, there is no integration of foreign genetic material, and no foreign genetic material remains in the plant. In many embodiments, the genetic sequence changes introduced by RTDS are not inserted randomly. Because the affected genes remain in their natural locations, random, uncontrolled, or deleterious expression patterns do not occur.

[0047] The RTDS that causes this change is a chemically synthesized oligonucleotide (e.g., using gene repair oligonucleobases (GRONs)) that can be composed of both DNA and modified RNA bases, as well as other chemical moieties, designed to hybridize at the targeted gene location and generate a mismatched base pair(s). This mismatched base pair acts as a signal to attract the cell's own natural gene repair system to the site, which corrects (substitutes, inserts, or deletes) the specified nucleotide(s) in the gene. Once the correction process is complete, the RTDS molecule is degraded, and the now altered or repaired gene is expressed under the gene's normal endogenous regulatory mechanisms.

[0048] Gene Repair Oligonucleobases ("GRON") Methods and compositions disclosed herein can be carried out or manufactured using " gene repair oligonucleobase ", for example, with the conformation and chemistry described in detail below.The " gene repair oligonucleobase " contemplated herein is described in open scientific literature and patent literature under other names, such as " recombinogenic oligonucleobase "; " RNA / DNA chimeric oligonucleotide "; " chimeric oligonucleotide "; " mixed duplex oligonucleotide " (MDONs) "; " RNA DNA oligonucleotide (RDOs) "; " gene targeting oligonucleotide "; " genoplast "; " single-stranded modified oligonucleotide "; " single-stranded oligodeoxynucleotide mutation vector " (SSOMVs) "; " duplex mutation vector "; and " heteroduplex mutation vector ".

[0049] The oligonucleobase of conformation and chemistry described in United States Patent No. 5,565,350 (Kmiec I) and United States Patent No. 5,731,181 (Kmiec II) of Kmiec, which are incorporated herein by reference, is suitable for use as " gene repair oligonucleobase " of the present disclosure.The gene repair oligonucleobase of Kmiec I and / or Kmiec II comprises two complementary strands, one of which comprises at least one segment of RNA type nucleotide (" RNA segment ") that base pairs with the DNA type nucleotide of the other strand.

[0050] Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be used instead of nucleotide.Additional gene repair molecules that can be used in the present disclosure are described in United States Patent No. 5,756,325;5,871,984;5,760,012;5,888,983;5,795,972;5,780,296;5,945,339;6,004,804;and 6,010,907, and International Application No. PCT / US00 / 23457;and International Publication No. WO98 / 49350;WO99 / 07865;WO99 / 58723;WO99 / 58702;and WO99 / 40789, each of which is incorporated herein by reference in its entirety.

[0051] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, gene repair oligonucleobase is a mixed duplex oligonucleotide (MDON), wherein the RNA-type nucleotide of the mixed duplex oligonucleotide is made RNase resistant by replacing 2'-hydroxyl with fluoro, chloro or bromo functional group, or by placing a substituent on 2'-O.Suitable substituents include the substituents taught by Kmiec II.Alternative substituents include, but are not limited to, the substituents taught by U.S. Patent No. 5,334,711 (Sproat) and the substituents taught by Patent Publications EP 629,387 and EP 679657 (collectively referred to as Martin Application), which are incorporated herein by reference.As used herein, 2'-fluoro, chloro or bromo derivatives of ribonucleotide, or ribonucleotides with 2'-OH substituted with the substituents described in Martin Application or Sproat, are referred to as "2'-substituted ribonucleotides". As used herein, the term "RNA-type nucleotide" refers to a 2'-hydroxyl or 2'-substituted nucleotide that is linked to other nucleotides of mixed duplex oligonucleotides by unsubstituted phosphodiester bonds or any non-natural bonds as taught by Kmiec I or Kmiec II.As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has 2'-H, which can be linked to other nucleotides of gene repair oligonucleobases by unsubstituted phosphodiester bonds or any non-natural bonds as taught by Kmiec I or Kmiec II.

[0052] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, gene repair oligonucleobase can be a mixed duplex oligonucleotide (MDON) that is exclusively linked by unsubstituted phosphodiester bonds.In alternative embodiments, linkage is by substituted phosphodiester, phosphodiester derivatives and non-phosphorus-based bonds as taught by Kmiec II.In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2'-substituted nucleotide.Particularly preferred embodiments of 2'-substituted ribonucleotides include, but are not limited to, 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, 2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'-trifluoropropyloxy substituted ribonucleotides.More preferred embodiments of 2'-substituted ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy and 2'-allyloxy substituted nucleotides. In another embodiment, the mixed duplex oligonucleotides are linked by unsubstituted phosphodiester bonds.

[0053] Although mixed duplex oligonucleotides (MDONs) containing only one type of 2'-substituted RNA-type nucleotide are more conveniently synthesized, the methods of the present invention can also be practiced using mixed duplex oligonucleotides containing two or more types of RNA-type nucleotides. The function of the RNA segment is not affected by interruptions caused by the introduction of deoxynucleotides between two RNA-type trinucleotides. Therefore, the term "RNA segment" encompasses terms such as "interrupted RNA segment." Uninterrupted RNA segments are referred to as continuous RNA segments. In alternative embodiments, the RNA segment can contain alternating RNase-resistant and unsubstituted 2'-OH nucleotides. The mixed duplex oligonucleotide preferably has fewer than 100 nucleotides, more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands form Watson-Crick base pairs. In one embodiment, the strands of the mixed duplex oligonucleotide are covalently linked by a linker, e.g., a single-stranded hexa-, penta-, or tetranucleotide, such that the first and second strands are segments of a single oligonucleotide strand with a single 3' end and a single 5' end. The 3' and 5' ends can be protected by adding a "hairpin cap," which allows the 3' and 5' terminal nucleotides to form Watson-Crick pairs with adjacent nucleotides. Additionally, a second hairpin cap can be placed at the junction between the first and second strands, away from the 3' and 5' ends, thereby stabilizing the Watson-Crick pairing between the first and second strands.

[0054] The first and second strands contain two regions homologous to the two fragments of the target gene, i.e., they have the same sequence as the target gene. The homologous regions contain nucleotides from the RNA segment and may contain one or more DNA-type nucleotides from the linked DNA segment, or may contain DNA-type nucleotides not present in the intervening DNA segment. The two regions of homology are separated by a region, called a "non-homologous region," whose sequence differs from that of the target gene and are adjacent to each other. The non-homologous region may contain one, two, or three mismatched nucleotides. The mismatched nucleotides may be contiguous or separated by one or two nucleotides homologous to the target gene. Alternatively, the non-homologous region may contain an insertion of one, two, three, five, or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from that of the target gene only by the deletion of one, two, three, five, or fewer nucleotides from the mixed duplex oligonucleotide. In this case, the length and position of the non-homologous region are considered to be the length of the deletion, even if the nucleotide of the mixed duplex oligonucleotide is not in the non-homologous region.When substitution (single or multiple) is intended, the distance between the fragments of the target gene that are complementary to the two homologous regions is the same as the length of the non-homologous region.If the non-homologous region contains an insertion, this makes the homologous region in the mixed duplex oligonucleotide more distant than its complementary homologous fragments are in the gene, and the opposite is true if the non-homologous region codes for a deletion.

[0055] Each RNA segment of a mixed duplex oligonucleotide is part of a homologous region, i.e., a region identical in sequence to a fragment of a target gene. Together, these segments preferably contain at least 13 RNA-type nucleotides, preferably 16 to 25 RNA-type nucleotides, or even more preferably 18 to 22 RNA-type nucleotides, or most preferably 20 nucleotides. In one embodiment, the RNA segments of the homologous region are separated and adjacent, i.e., "connected," by an intervening DNA segment. In one embodiment, each nucleotide of the non-homologous region is a nucleotide of the intervening DNA segment. The intervening DNA segment comprising the non-homologous region of a mixed duplex oligonucleotide is referred to as the "mutagenesis segment."

[0056] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the gene repair oligonucleobase (GRON) is a single-stranded oligodeoxynucleotide mutagenesis vector (SSOMV), as disclosed, for example, by International Patent Application No. PCT / US00 / 23457, U.S. Patent Nos. 6,271,360, 6,479,292 and 7,060,500, the entireties of which are incorporated herein by reference. The sequence of SSOMV is based on the same principle as the mutagenesis vectors described in U.S. Patent Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and International Publication Nos. WO 98 / 49350; WO 99 / 07865; WO 99 / 58723; WO 99 / 58702; and WO 99 / 40789. The sequence of SSOMV contains two regions of homology with the target sequence, separated by a region containing the desired genetic alteration, referred to as the mutagenesis region. The mutagenized region can have a sequence that is the same length as the sequence separating the homologous regions in the target sequence, but has a different sequence. Such a mutagenized region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous with each other, but regions with the same sequence in the target gene are separated by one, two, or more nucleotides. Such an SSOMV causes the deletion of nucleotides from the target gene that are not present in the SSOMV. Finally, sequences identical to the homologous regions of the target gene can be adjacent in the target gene, but separated by one, two, or more nucleotides in the SSOMV sequence. Such an SSOMV causes an insertion into the sequence of the target gene.

[0057] The nucleotides of the SSOMV are deoxyribonucleotides linked by unmodified phosphodiester bonds, except for the 3'- and / or 5'-terminal internucleotide linkages, or the two 3'- and / or 5'-terminal internucleotide linkages may be phosphorothioates or phosphoramidites. As used herein, internucleotide linkages refer to linkages between the nucleotides of the SSOMV and do not include the linkage between the 3'- or 5'-terminal nucleotide and a blocking substituent. In certain embodiments, the length of the SSOMV is 21 to 55 deoxynucleotides; therefore, the length of the homology regions should be at least 20 deoxynucleotides in total, with at least two homology regions each having a length of at least 8 deoxynucleotides.

[0058] SSOMV can be designed to be complementary to either the coding strand or non-coding strand of target gene.When desired mutation is a single base substitution, it is preferred that both the mutated nucleotide and the target nucleotide are pyrimidine.To the extent consistent with achieving desired functional results, it is preferred that both the mutated nucleotide and the target nucleotide in the complementary strand are pyrimidine.Particularly preferred is SSOMV that encodes transversion mutation, that is, C or T mutated nucleotide is mismatched with C or T nucleotide in the complementary strand, respectively.

[0059] In addition to the oligodeoxynucleotide, the SSOMV can contain a 5'-blocking substituent attached to the 5'-terminal carbon via a linker. The chemistry of the linker is not critical except for its length, which should preferably be at least six atoms long and flexible. Various non-toxic substituents can be used, such as biotin, cholesterol, other steroids, or non-intercalating cationic fluorescent dyes. Particularly preferred reagents for preparing SSOMVs are those commercially available from Glen Research, Sterling, Virginia (now GE Healthcare) as Cy3™ and Cy5™, which are blocked phosphoramidites that, upon incorporation into an oligonucleotide, yield 3,3,3',3'-tetramethyl-N,N'-isopropyl-substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is particularly preferred. When the indocarbocyanine is N-oxyalkyl-substituted, it can be conveniently attached to the 5' end of the oligodeoxynucleotide as a phosphodiester with a 5'-terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When commercially available Cy3 phosphoramidites are used as directed, the resulting 5' modification, together with the blocking substituent and linker, consists of N-hydroxypropyl, N'-phosphatidylpropyl 3,3,3',3'-tetramethylindomonocarbocyanine.

[0060] In a preferred embodiment, the indocarbocyanine dye is tetrasubstituted at the 3 and 3' positions of the indole ring. Without being bound by theory, because of these substitutions, the dye is not an intercalating dye. The identity of the substituents at these positions is not critical. The SSOMV can further have a 3'-blocking substituent. Again, the chemistry of the 3'-blocking substituent is not critical.

[0061] The mutations described herein can also be obtained by mutagenesis (random, somatic, or directed) or other DNA editing or recombination techniques, such as, but not limited to, gene targeting using site-specific homologous recombination with zinc finger nucleases.

[0062] Delivery of gene repair oligonucleobases into plant cells Any known method used for the transformation of plant cells can be used to deliver the gene repair oligonucleobases. Exemplary methods are described below.

[0063] Microcarriers and Microfibers The use of metal microcarriers (microspheres) to introduce large fragments of DNA into plant cells with cellulose cell walls by projectile penetration is well known to those skilled in the art (hereinafter referred to as biolistic delivery). U.S. Patent Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for their projection.

[0064] Specific conditions for using microcarriers in the methods disclosed herein are described in International Publication WO 99 / 07865. In an exemplary procedure, ice-cold microcarriers (60 mg / mL), mixed duplex oligonucleotides (60 mg / mL), 2.5 M CaCl2, and 0.1 M spermidine are added in this order, and the mixture is gently stirred, for example, by vortexing for 10 minutes, then left at room temperature for 10 minutes. The microcarriers are then diluted with 5 volumes of ethanol, centrifuged, and resuspended in 100% ethanol. Good results can be obtained when the adhesion solution contains 8-10 μg / μL of microcarriers, 14-17 μg / mL of mixed duplex oligonucleotides, 1.1-1.4 M CaCl2, and 18-22 mM spermidine. Optimal results were observed with 8 μg / μL microcarriers, 16.5 μg / mL mixed duplex oligonucleotides, 1.3 M CaCl 2 and 21 mM spermidine.

[0065] In carrying out the present invention, gene repair oligonucleic acid bases can also be introduced into plant cells using microfibers to penetrate cell wall and cell membrane.Coffee et al., U.S. Patent No. 5,302,523, describes the use of 30x0.5 μm and 10x0.3 μm silicon carbide fibers to facilitate the transformation of corn (Black Mexican Sweet) suspension culture.Any mechanical method that can be used to introduce DNA for the transformation of plant cells using microfibers can be used to deliver gene repair oligonucleic acid bases for transmutation.

[0066] An exemplary procedure for microfiber delivery of gene repair oligonucleobases is as follows: Sterile microfibers (2 μg) are suspended in 150 μL of plant culture medium containing approximately 10 μg of mixed duplex oligonucleotides. The suspended culture is allowed to settle, and the collected cells are vortexed for 10 minutes in equal volumes with the sterile fiber / nucleotide suspension and plated. Selection media is applied immediately or delayed for up to approximately 120 hours, as appropriate for specific characteristics.

[0067] Electroporation of protoplasts In alternative embodiments, gene repair oligonucleobases can be delivered to plant cells by electroporation of protoplasts derived from plant parts.Protoplasts are formed by treating plant parts, particularly leaves, with enzymes according to methods well known to those skilled in the art.See, for example, Gallois et al., 1996, Methods in Molecular Biology 55:89-107, Humana Press, Totowa, NJ; Kipp et al., 1999, Methods in Molecular Biology 133:213-221, Humana Press, Totowa, NJ.Protoplasts do not need to be cultured in growth medium before electroporation.An example of electroporation conditions is 3x10 in a total volume of 0.3mL. 5 protoplasts, and the concentration of gene repair oligonucleobases is 0.6-4 μg / mL.

[0068] Protoplast PEG-mediated DNA uptake In an alternative embodiment, nucleic acids are taken up into plant protoplasts in the presence of the membrane-altering agent polyethylene glycol, according to techniques well known to those skilled in the art (see, e.g., Gharti-Chhetri et al., 1992; Datta et al., 1992).

[0069] Microinjection In an alternative embodiment, gene repair oligonucleobases can be delivered by injection into plant cells or protoplasts using a microcapillary (see, e.g., Miki et al., 1989; Schnorf et al., 1991).

[0070] transgenic In any of the various aspects and embodiments of the compositions and methods disclosed herein, mutations in genes and proteins can be generated, for example, using transgenic techniques. In some embodiments, the compositions and methods include plants or plant cells harboring a transformed nucleic acid construct comprising a promoter operably linked to a PPX nucleotide disclosed herein. The methods disclosed herein can also include introducing a PPX nucleic acid construct disclosed herein into at least one plant cell and regenerating a transformed plant therefrom. The nucleic acid construct comprises at least one nucleotide encoding a herbicide-resistant PPX protein as disclosed herein, particularly the nucleotide sequences and fragments set forth in Figures 2, 4, 6, 8, 10, and 12, and variants thereof. The methods can also involve the use of a promoter capable of driving gene expression in plant cells. In one embodiment, such a promoter is a constitutive promoter or a tissue-preferred promoter. Plants produced by these methods can have increased PPX activity and / or particularly herbicide-resistant PPX activity compared to untransformed plants. Thus, these methods are useful in enhancing or increasing the resistance of a plant to at least one herbicide that increases the activity of the PPX enzyme, particularly in the presence of an herbicide that inhibits PPX.

[0071] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, a method for producing a herbicide-tolerant plant can include transforming a plant cell with a nucleic acid construct comprising a nucleotide sequence operably linked to a promoter that drives expression in the plant cell, and further regenerating a transformed plant from the transformed plant cell. The nucleotide sequence is selected from nucleotide sequences encoding the herbicide-tolerant PPXs disclosed herein, particularly the nucleotide sequences and fragments and variants thereof set forth in Figures 2, 4, 6, 8, 10, and 12. The herbicide-tolerant plants produced by this method comprise enhanced resistance to at least one herbicide, particularly a herbicide that interferes with the activity of the PPX enzyme, such as a herbicide that inhibits PPX, compared to an untransformed plant.

[0072] The disclosed nucleic acid molecules can be used in nucleic acid constructs for the purpose of transforming plants, e.g., crop plants such as Solanum tuberosum. In one embodiment, such nucleic acid constructs containing the disclosed nucleic acid molecules can be used to generate transgenic plants for the purpose of creating resistance to herbicides, such as herbicides known to inhibit PPX activity, such as PPX-inhibiting herbicides. The nucleic acid constructs can be used in expression cassettes, expression vectors, transformation vectors, plasmids, and the like. Following transformation with such constructs, the resulting transgenic plants exhibit increased resistance to PPX-inhibiting herbicides, such as flumioxazin and sulfentrazone herbicides.

[0073] construct The nucleic acid molecules (e.g., mutated PPX genes) disclosed herein can be used to generate recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the present disclosure can be used to prepare nucleic acid constructs, e.g., expression cassettes, for expression in plants of interest.

[0074] The expression cassette may include a regulatory sequence operably linked to the PPX nucleic acid sequence disclosed herein. The cassette may further include at least one additional gene that is co-transformed into the organism. Alternatively, the gene(s) may be provided in multiple expression cassettes.

[0075] The nucleic acid construct may be provided with multiple restriction enzyme recognition sites for insertion of the PPX nucleic acid sequence under the transcriptional control of the regulatory region. The nucleic acid construct may further include a nucleic acid molecule encoding a selectable marker gene.

[0076] Any promoter can be used to generate the nucleic acid construct. The promoter may be native, analogous, foreign, or heterologous to the plant host and / or the PPX nucleic acid sequence disclosed herein. Furthermore, the promoter may be a natural sequence or a synthetic sequence. When a promoter is "foreign" or "heterologous" to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. When a promoter is "foreign" or "heterologous" to the PPX nucleic acid sequence disclosed herein, it is intended that the promoter is not a native or naturally occurring promoter to the operably linked PPX nucleic acid sequence disclosed herein. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region heterologous to the coding sequence.

[0077] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, the PPX nucleic acid sequences disclosed herein may be expressed using a heterologous promoter, but the native promoter sequence can also be used to prepare constructs. Such constructs will alter the expression level of the PPX protein in plants or plant cells, thereby altering the phenotype of the plant or plant cell.

[0078] Any promoter can be used to prepare constructs for controlling expression of the PPX coding sequence, including, for example, a constitutive promoter, a tissue-preferred promoter, an inducible promoter, or other promoter that allows for expression in plants. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171), ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); the ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0079] Tissue-preferred promoters can be used to direct PPX expression in specific plant tissues, including, but not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters are described in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

[0080] The nucleic acid construct may include a transcription termination region. When a transcription termination region is used, any transcription termination region may be used in preparing the nucleic acid construct. For example, the termination region may be the same native as the transcription initiation region, the same native as the operably linked PPX sequence of interest, the same native as the plant host, or derived from another source (i.e., foreign or heterologous to the promoter, the PPX nucleic acid molecule, the plant host, or any combination thereof). Examples of termination regions that can be used in the constructs of the present disclosure include those obtained from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions (Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639).

[0081] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, nucleic acids can be optimized for enhanced expression in transformed plants. That is, nucleic acids encoding mutant PPX proteins can be synthesized using plant-preferred codons for improved expression. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a review of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

[0082] In addition, other sequence modifications can be made to the nucleic acid sequences disclosed herein. For example, additional sequence modifications are known to enhance gene expression in cellular hosts. These include removing sequences encoding spurious polyadenylation signals, exon / intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GG content of the sequence can also be adjusted to an average level for the target cellular host, calculated by comparing known genes expressed in the host cell. Furthermore, the sequence can be modified to avoid predicted hairpin secondary structures in mRNA.

[0083] Other nucleic acid sequences can also be used in preparing the constructs of the present disclosure, for example, to enhance expression of the PPX coding sequence. Such nucleic acid sequences include the maize intron AdhI, intron 1 gene (Callis et al. (1987) Genes and Development 1:1183-1200), and the leader sequences (W sequences) from tobacco mosaic virus (TMV), maize chlorotic mottle virus, and alfalfa mosaic virus (Gallie et al. (1987) Nucleic Acid Res. 15:8693-8711 and Skuzeski et al. (1990) Plant Mol. Biol. 15:65-79, 1990). The first intron from the maize shrunken-1 locus has been shown to increase gene expression in chimeric gene constructs. U.S. Patent Nos. 5,424,412 and 5,593,874 disclose the use of certain introns in gene expression constructs, and Gallie et al. ((1994) Plant Physiol. 106:929-939) also demonstrated the usefulness of introns for tissue-specific control of gene expression. To further enhance or optimize PPX gene expression, the plant expression vectors disclosed herein can also contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems may then exhibit overexpression or constitutive expression of the nucleotide sequences of the present disclosure.

[0084] The expression constructs disclosed herein can also include a nucleic acid sequence capable of directing expression of a PPX sequence in chloroplasts. Such nucleic acid sequences also include a chloroplast targeting sequence encoding a chloroplast transit peptide for directing a gene product of interest to the plant cell chloroplast. Such transit peptides are known in the art. With respect to a chloroplast targeting sequence, "operably linked" means that the nucleic acid sequence encoding the transit peptide (i.e., the chloroplast targeting sequence) is linked to a PPX nucleic acid molecule disclosed herein such that the two sequences are contiguous and in the same reading frame. See, e.g., Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481. The PPX proteins disclosed herein include native chloroplast transit peptides, although any chloroplast transit peptide known in the art can be fused to the amino acid sequence of a mature PPX protein by operably linking a chloroplast targeting sequence to the 5' end of the nucleotide sequence encoding the mature PPX protein.

[0085] Chloroplast targeting sequences are known in the art and include those found in the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem. 272(33) ):20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and light-harvesting chlorophyll a / b-binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0086] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, a nucleic acid construct can be prepared to induce expression of a mutant PPX coding sequence from plant cell chloroplasts. Methods for chloroplast transformation are also known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting the DNA to the plastid genome by homologous recombination. In addition, plastid transformation can be achieved by transactivation of a silent transgene carried in the plastid through tissue-preferential expression of a nuclear-encoded, plastid-directed RNA polymerase. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0087] The nucleic acid of interest that is targeted to chloroplast can be optimized for expression in chloroplast, taking into account the difference in codon usage between plant nucleus and this organelle.In this way, the nucleic acid of interest can be synthesized using chloroplast-preferred codons.See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0088] The nucleic acid constructs can be used to transform plant cells and regenerate transgenic plants containing the mutated PPX coding sequence. Many plant transformation vectors and methods for transforming plants are available. See, e.g., U.S. Patent No. 6,753,458; An, G. et al. (1986) Plant Physiol., 81:301-305; Fry, J. et al. (1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl Genet. 76:767-774; Hinchee et al. (1990) Stadler. Genet. Symp. 203212.203-212; Cousins ​​et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, PP and Slightom, JL (1992) Gene 118:255-260; Christou et al. (1992) Trends. Biotechnol. 10:239-246; D'Halluin et al. (1992) Bio / Technol. 10:309-314; Dhir et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant;29P:119-124; Davies et al. (1993) Plant Cell Rep. 12:180-183; Dong, JA and McHughen, A. (1993) Plant Sci. 91:139-148; Franklin, CI and Trieu, TN (1993) Plant. Physiol. 102:167; Golovkin et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano et al. (1994) Plant Cell Rep. 13; Ayeres NM and Park, WD (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo et al. (1994) Plant. J.5:583-592; Becker et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol. Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5:17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman et al. (1994) Bio-Technology 12:919-923; Ritala et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, YC and Lemaux, PG (1994) Plant Physiol. 104:3748. Constructs can also be transformed into plant cells using homologous recombination. .

[0089] Constructs containing or maintaining the PPX nucleic acid sequences disclosed herein can be used in a variety of ways to produce transgenic host cells, such as bacteria and yeast, and to transform plant cells and, in some cases, regenerate transgenic plants. For example, a method for producing a transgenic crop plant containing a PPX mutant protein disclosed herein includes (a) introducing into a plant cell an expression vector containing a nucleic acid encoding the mutant PPX protein, where expression of the nucleic acid in the plant leads to herbicide resistance compared to a wild-type plant or a known PPX mutant plant, and (b) generating a herbicide-resistant transgenic plant from the plant cell.

[0090] PPX mutations The compositions and methods may relate at least in part to mutations in PPX genes, such as mutations that confer resistance or tolerance to herbicides belonging to the PPX inhibitor family of herbicides in plants.In certain embodiments, the compositions and methods also relate to the use of gene repair oligonucleobases to generate desired mutations in chromosomes or episodic sequences in genes encoding PPX proteins.In some embodiments, the mutated proteins can substantially maintain the catalytic activity of wild-type proteins, leading to increased plant resistance or tolerance to herbicides belonging to the PPX inhibitor family, and in some embodiments, allowing plants, their organs, tissues or cells to grow or develop substantially normally compared to wild-type plants, regardless of the presence or absence of herbicides.The compositions and methods also relate to non-transgenic or transgenic plant cells in which mutations have occurred in PPX genes, non-transgenic or transgenic plants regenerated therefrom, and plants resulting from crossing the regenerated non-transgenic or transgenic plants with, for example, a plant having a mutation in a different PPX gene. These mutations can also be applied to target resistance to these inhibitors in plant and mammalian systems, including crop plants, algae, bacteria, and fungi.

[0091] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, at least one mutation in the mutated PPX protein may be any of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 and 525. In some embodiments, the mutated PPX protein comprises a mutation at one or more amino acid positions corresponding to positions selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470, and 50 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 85 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 111 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 130 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 139 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 143 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 147 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 165 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 192 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 193 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 199 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 206 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 219 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 229 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 230 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 244 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 256 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 270 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 271 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 305 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 311 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 316 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 318 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 332 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to 357 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 360 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 366 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 438 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 440 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 444 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 455 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 457 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 470 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 478 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 483 of SEQ ID NO:1 from phenylalanine to glycine. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 484 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 485 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 487 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 490 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 503 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 508 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 58 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 64 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 74 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 84 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 93 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 97 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 101 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 119 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 121 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 124 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 139 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 150 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 151 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 157 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 164 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 170 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 177 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 187 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 188 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 195 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 215 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 230 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 271 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 274 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 278 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 283 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 292 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 296 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 330 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 396 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 404 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 406 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 410 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 421 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 434 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 447 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 448 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 449 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 451 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 454 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 465 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position 470 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 500 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises two or more mutations, at least one of which mutations is at 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 3 and wherein the amino acid sequence is at an amino acid position corresponding to a position selected from the group consisting of: 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508, and 525.In some embodiments, the mutated PPX protein comprises two or more mutations, at least one of which mutations is at an amino acid position corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises three or more mutations, at least one of which mutations is selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, and at an amino acid position corresponding to a position selected from the group consisting of 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508 and 525. In some embodiments, the mutated PPX protein comprises three or more mutations, at least one of which mutations is at an amino acid position corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO:9.

[0092] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, at least one mutation in the mutated PPX protein may be selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, The amino acid sequence can be present at an amino acid position corresponding to a position selected from the group consisting of T318, S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525. In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, at least one mutation in the mutated PPX protein may be selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, X195, D196, D197, D200, D210, D211, D212, D213, D214, D215, D220, D225, D230, D231, D240, D241, D252, D253, D260, D261, D270, D272, D273, D274, D280, D285, D290, D295, D300, D310, D311, D312, D313, D400, D410, D420, D430, D440, D450, D460, D470, D480, D500, D510, D520, D600, D610, D620, D630, D640, D740, D740, D84, D93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, X195, D196, D207, D210, D221, D232, D243, D254 The PPX protein may be present at an amino acid position corresponding to a position selected from the group consisting of P214, I215, K229, K230, C271, D274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470, and T500. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position G52 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to N85 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to E111 of SEQ ID NO: 1.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to G130 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to D139 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to P143 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to R144 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F145 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to L147 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F165 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L167 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position 1170 in SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to A180 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position P185 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E192 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S193 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position R199 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to V206 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E212 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to Y219 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to A220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position G221 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to L226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position M228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K229 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A230 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K237 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S244 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position R256 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position R257 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K270 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position P271 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position Q272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S305 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E311 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position T316 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position T318 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S332 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S343 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A354 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L357 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K359 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to L360 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A366 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position Y426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S430 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K438 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E440 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position V444 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L455 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K457 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position V470 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F478 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F483 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D484 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position I485 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D487 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K490 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L503 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position V508 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position I525 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D58 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E64 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position G74 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position G84 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L93 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K97 of SEQ ID NO: 9.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K98 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A101 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S119 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F121 of SEQ ID NO: 9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position T124 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position N139 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position E150 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S151 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position Q157 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position V164 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D170 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position C177 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position H187 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L188 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position N195 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position P214 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position I215 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K230 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position C271 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to D274 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F283 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A292 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S296 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position C307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position N324 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D330 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S396 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A404 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position R406 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K410 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L421 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position C434 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D447 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S448 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position V449 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D451 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position D454 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position Y465 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K470 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position T500 of SEQ ID NO:9. In some embodiments, the PPX protein is a paralogue of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato plastid PPX protein), and the PPX protein can have an N at a position corresponding to position 52 of SEQ ID NO:1, where the N is replaced with an amino acid other than N; a K at a position corresponding to position 272 of SEQ ID NO:1, where the K is replaced with an amino acid other than K; an S at a position corresponding to position 359 of SEQ ID NO:1, where the S is replaced with an amino acid other than S; and / or an S at a position corresponding to position 525 of SEQ ID NO:1, where the S is replaced with an amino acid other than S. In such embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position N52 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position N85 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position R144 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position F145 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at the amino acid position corresponding to position A180 of SEQ ID NO: 1.In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position P185 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position A220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position M228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S244 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position K272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S305 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S332 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L357 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S359 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L393 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L403 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position L424 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position Y426 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position F478 of SEQ ID NO: 1. In some embodiments, the mutated PPX protein comprises a mutation at an amino acid position corresponding to position S525 of SEQ ID NO: 1.

[0093] In some embodiments, the mutated PPX protein comprises two or more mutations, at least one of which is at an amino acid position corresponding to a position selected from the group consisting of G52, N85, R144, F145, A180, P185, A220, L226, M228, S244, Q272, S305, S332, L357, K359, L393, L403, L424, Y426, F478 and I525 of SEQ ID NO: 1. In some embodiments, the PPX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato PPX protein), and the PPX protein has two or more mutations and further has one or more of the following: (1) an N at a position corresponding to position 52 of SEQ ID NO: 1, where the N is replaced with an amino acid other than N; (2) a K at a position corresponding to position 272 of SEQ ID NO: 1, where the K is replaced with an amino acid other than K; (3) an S at a position corresponding to position 359 of SEQ ID NO: 1, where the S is replaced with an amino acid other than S; and / or (4) an S at a position corresponding to position 525 of SEQ ID NO: 1, where the S is replaced with an amino acid other than S. In such embodiments, the mutated PPX protein has two or more mutations, at least one of which is at an amino acid position corresponding to a position selected from the group consisting of N52, N85, R144, F145, A180, P185, A220, L226, M228, S244, K272, S305, S332, L357, S359, L393, L403, L424, Y426, F478 and S525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein is present at an amino acid position corresponding to a position selected from the group consisting of G52, N85, R144, F145, A180, P185, A220, L226, M228, S244, Q272, S305, S332, L357, K359, L393, 403, L424, Y426, F478 and I525 of SEQ ID NO: 1.In some embodiments, the PPX protein is a paralog of an Arabidopsis thaliana PPX protein (e.g., the PPX protein can be a potato PPX protein), and the PPX protein has three or more mutations and further has one or more of the following: (1) an N at a position corresponding to position 52 of SEQ ID NO: 1, where the N is replaced with an amino acid other than N; (2) a K at a position corresponding to position 272 of SEQ ID NO: 1, where the K is replaced with an amino acid other than K; (3) an S at a position corresponding to position 359 of SEQ ID NO: 1, where the S is replaced with an amino acid other than S; and / or (4) an S at position 525 of SEQ ID NO: 1, where the S is replaced with an amino acid other than S. In such embodiments, the mutated PPX protein has three or more mutations, at least one of which is at an amino acid position corresponding to a position selected from the group consisting of N52, N85, R144, F145, A180, P185, A220, L226, M228, S244, K272, S305, S332, L357, S359, L393, L403, L424, Y426, F478 and S525 of SEQ ID NO: 1.

[0094] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX protein may comprise one or more mutations selected from the mutations set forth in the table.

[0095] [Table 1]

[0096] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the one or more mutations in the mutated PPX gene may be from glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1, asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1, glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO:1, glycine to asparagine at a position corresponding to position 130 of SEQ ID NO:1, aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO:1, proline to arginine at a position corresponding to position 143 of SEQ ID NO:1, arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1, arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1, arginine to leucine at a position corresponding to position 144 of SEQ ID NO:1, phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1, phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1, Leucine to valine at a position corresponding to position 147 of SEQ ID NO:1, phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO:1, alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1, proline to histidine at a position corresponding to position 185 of SEQ ID NO:1, proline to arginine at a position corresponding to position 185 of SEQ ID NO:1, proline to tyrosine at a position corresponding to position 192 of SEQ ID NO:1 glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO:1; glutamic acid to lysine at a position corresponding to position 193 of SEQ ID NO:1; serine to threonine at a position corresponding to position 199 of SEQ ID NO:1; arginine to leucine at a position corresponding to position 199 of SEQ ID NO:1; valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO:1; tyrosine to serine at a position corresponding to position 219 of SEQ ID NO:1; alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1;Alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1, alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1, alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1, alanine to valine at a position corresponding to position 220 of SEQ ID NO:1, leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1, methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1, and lysine at a position corresponding to position 229 of SEQ ID NO:1 to glutamine, alanine to phenylalanine at a position corresponding to position 230 of SEQ ID NO:1, serine to glycine at a position corresponding to position 244 of SEQ ID NO:1, serine to threonine at a position corresponding to position 244 of SEQ ID NO:1, arginine to histidine at a position corresponding to position 256 of SEQ ID NO:1, arginine to serine at a position corresponding to position 256 of SEQ ID NO:1, lysine to glutamic acid at a position corresponding to position 270 of SEQ ID NO:1, and lysine to glutamic acid at a position corresponding to position 270 of SEQ ID NO:1. lysine to glutamic acid at a position corresponding to position 271 of SEQ ID NO:1, proline to arginine at a position corresponding to position 272 of SEQ ID NO:1, glutamine to phenylalanine at a position corresponding to position 273 of SEQ ID NO:1, serine to leucine at a position corresponding to position 305 of SEQ ID NO:1, glutamic acid to arginine at a position corresponding to position 311 of SEQ ID NO:1, threonine to glycine at a position corresponding to position 316 of SEQ ID NO:1, threonine to glycine at a position corresponding to position 318 of SEQ ID NO:1, Serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1, serine to leucine at a position corresponding to position 332 of SEQ ID NO:1, leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1, lysine to arginine at a position corresponding to position 359 of SEQ ID NO:1, lysine to threonine at a position corresponding to position 359 of SEQ ID NO:1, leucine to lysine at a position corresponding to position 360 of SEQ ID NO:1, leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO:1,Alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO:1, leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1, leucine to serine at a position corresponding to position 393 of SEQ ID NO:1, leucine to valine at a position corresponding to position 393 of SEQ ID NO:1, leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1, leucine to serine at a position corresponding to position 403 of SEQ ID NO:1, leucine to valine at a position corresponding to position 424 of SEQ ID NO:1 to serine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to cysteine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to phenylalanine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to histidine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to isoleucine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to leucine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to arginine ...cysteine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to phenylalanine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to histidine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to isoleucine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 to leucine, tyrosine at a position corresponding to position 426 of SEQ ID NO:1 tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1, lysine to serine at a position corresponding to position 438 of SEQ ID NO:1, glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO:1, valine to isoleucine at a position corresponding to position 444 of SEQ ID NO:1, leucine to valine at a position corresponding to position 455 of SEQ ID NO:1, lysine to valine at a position corresponding to position 457 of SEQ ID NO:1, and glutamic acid to lysine at a position corresponding to position 470 of SEQ ID NO:1. valine to serine at a position corresponding to position 470 of SEQ ID NO:1, valine to tyrosine at a position corresponding to position 478 of SEQ ID NO:1, phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1, phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO:1, aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO:1, isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO:1, and lysine to asparagine at a position corresponding to position 490 of SEQ ID NO:1.encoding a mutated PPX protein having one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO: 1, valine to threonine at a position corresponding to position 508 of SEQ ID NO: 1, and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1;

[0097] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can encode a mutated PPX protein comprising glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glycine to asparagine at a position corresponding to position 130 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to arginine at a position corresponding to position 143 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises arginine to leucine at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising leucine to valine at a position corresponding to position 147 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to histidine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to arginine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to tyrosine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to lysine at a position corresponding to position 192 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to threonine at a position corresponding to position 193 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to leucine at a position corresponding to position 199 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to serine at a position corresponding to position 219 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to valine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to phenylalanine at a position corresponding to position 230 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises arginine to histidine at a position corresponding to position 256 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to serine at a position corresponding to position 256 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to glutamic acid at a position corresponding to position 270 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to glutamic acid at a position corresponding to position 270 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to arginine at a position corresponding to position 271 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to leucine at a position corresponding to position 305 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises glutamic acid to arginine at a position corresponding to position 311 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises threonine to glycine at a position corresponding to position 316 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises threonine to glycine at a position corresponding to position 318 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to leucine at a position corresponding to position 332 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains lysine to arginine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains lysine to threonine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to lysine at a position corresponding to position 360 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises a leucine to serine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises a leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises a leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises a leucine to serine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises a leucine to serine at a position corresponding to position 424 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes lysine to serine at a position corresponding to position 438 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that includes valine to isoleucine at a position corresponding to position 444 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising leucine to valine at a position corresponding to position 455 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to valine at a position corresponding to position 457 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising valine to serine at a position corresponding to position 470 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising valine to tyrosine at a position corresponding to position 470 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains lysine to asparagine at a position corresponding to position 490 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains valine to threonine at a position corresponding to position 508 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.

[0098] [Table 2] *"AA mtn" refers to an amino acid mutation; "NA mtn" refers to a nucleic acid mutation.

[0099] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can comprise a GGG → AAA nucleic acid mutation, which encodes a mutated PPX protein comprising glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AAT → GAT nucleic acid mutation, which encodes a mutated PPX protein comprising asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AGG → TGC or TGT nucleic acid mutation, which encodes a mutated PPX protein comprising arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AGG → CAC or CAT nucleic acid mutation, which encodes a mutated PPX protein comprising arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTT → CTT nucleic acid mutation, which encodes a mutated PPX protein comprising phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTT to TAT nucleic acid mutation, which encodes a mutated PPX protein containing phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GCA to ACA nucleic acid mutation, which encodes a mutated PPX protein containing alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a CCG to CAC or CAT nucleic acid mutation, which encodes a mutated PPX protein containing proline to arginine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a CCG to CGT nucleic acid mutation, which encodes a mutated PPX protein containing proline to histidine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a CCG to CGG nucleic acid mutation, which encodes a mutated PPX protein containing proline to arginine at a position corresponding to position 185 of SEQ ID NO:1.In some embodiments, the mutated PPX gene comprises a GCT→TGT nucleic acid mutation that encodes a mutated PPX protein containing alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GCT→ATT nucleic acid mutation that encodes a mutated PPX protein containing alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GCT→CTT nucleic acid mutation that encodes a mutated PPX protein containing alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GCT→ACT nucleic acid mutation that encodes a mutated PPX protein containing alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GCT→GTT nucleic acid mutation that encodes a mutated PPX protein containing alanine to valine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a GTG to ATG nucleic acid mutation that encodes a mutated PPX protein containing leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an ATG to CTG nucleic acid mutation that encodes a mutated PPX protein containing methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AGC to GGC nucleic acid mutation that encodes a mutated PPX protein containing serine to glycine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AGC to ACC nucleic acid mutation that encodes a mutated PPX protein containing serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a CAG to TTC or TTT nucleic acid mutation that encodes a mutated PPX protein containing glutamine to asparagine at a position corresponding to position 272 of SEQ ID NO:1.In some embodiments, the mutated PPX gene comprises a TCA to TTA nucleic acid mutation, which encodes a mutated PPX protein containing serine to leucine at a position corresponding to position 305 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TCT to TGT nucleic acid mutation, which encodes a mutated PPX protein containing serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a CTC to ATC nucleic acid mutation, which encodes a mutated PPX protein containing leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AAA to AGA nucleic acid mutation, which encodes a mutated PPX protein containing lysine to arginine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises an AAA to ACT nucleic acid mutation, which encodes a mutated PPX protein containing lysine to threonine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTG to ATG nucleic acid mutation that encodes a mutated PPX protein containing leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTG to TCG nucleic acid mutation that encodes a mutated PPX protein containing leucine to serine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTG to GTG nucleic acid mutation that encodes a mutated PPX protein containing leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTA to CGA nucleic acid mutation that encodes a mutated PPX protein containing leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTA to TCA nucleic acid mutation that encodes a mutated PPX protein containing leucine to serine at a position corresponding to position 403 of SEQ ID NO:1.In some embodiments, the mutated PPX gene comprises a TTG to TCG nucleic acid mutation, which encodes a mutated PPX protein comprising leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC to TGC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC to TTC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC to CAC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC to ATC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC → TTA or CTC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC → CGC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC → ACC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TAC → GTC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene comprises a TTT → TCT nucleic acid mutation, which encodes a mutated PPX protein comprising phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1.In some embodiments, the mutated PPX gene comprises an ATT to ACT nucleic acid mutation that encodes a mutated PPX protein containing an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.

[0100] [Table 3] *"AA mtn" refers to an amino acid mutation; "NA mtn" refers to a nucleic acid mutation.

[0101] In some embodiments, in conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the one or more mutations in the mutated PPX gene are asparagine to lysine at a position corresponding to position 52 of SEQ ID NO:1, asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1, arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1, arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1, phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1, phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1, alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1, proline to histidine at a position corresponding to position 185 of SEQ ID NO:1, proline to arginine at a position corresponding to position 185 of SEQ ID NO:1, alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1, alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1, alanine to leucine at a position corresponding to position 145 of SEQ ID NO:1, isoleucine, alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1, alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1, alanine to valine at a position corresponding to position 220 of SEQ ID NO:1, leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1, methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1, serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 serine to threonine at a position corresponding to position 272 of SEQ ID NO:1, lysine to phenylalanine at a position corresponding to position 305 of SEQ ID NO:1, serine to leucine at a position corresponding to position 332 of SEQ ID NO:1, leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1, serine to arginine at a position corresponding to position 359 of SEQ ID NO:1, serine to threonine at a position corresponding to position 359 of SEQ ID NO:1,Leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1, leucine to serine at a position corresponding to position 393 of SEQ ID NO:1, leucine to valine at a position corresponding to position 393 of SEQ ID NO:1, leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1, leucine to serine at a position corresponding to position 403 of SEQ ID NO:1, leucine to serine at a position corresponding to position 424 of SEQ ID NO:1, tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1, The polypeptides may encode mutated PPX proteins having one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1, tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1, phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1, and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.

[0102] In some embodiments, in conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can encode a mutated PPX protein comprising asparagine to lysine at a position corresponding to position 52 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein, including a mutated PPX protein that comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises proline to histidine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises proline to arginine at a position corresponding to position 185 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to leucine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to valine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to glycine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains serine to leucine at a position corresponding to position 305 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to arginine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to threonine at a position corresponding to position 359 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to methionine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to serine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to serine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO:1. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.

[0103] In some embodiments, the mutated PPX gene comprises an AAT to AAA nucleic acid mutation, which encodes a mutated PPX protein containing asparagine to lysine at a position corresponding to position 52 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AAT to GAT nucleic acid mutation, which encodes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CGC to TGC nucleic acid mutation, which encodes a mutated PPX protein containing arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CGC to CAC nucleic acid mutation, which encodes a mutated PPX protein containing arginine to histidine at a position corresponding to position 144 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTT to CTT nucleic acid mutation, which encodes a mutated PPX protein containing phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTT to TAT nucleic acid mutation, which encodes a mutated PPX protein comprising phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a GCC to ACC nucleic acid mutation, which encodes a mutated PPX protein comprising alanine to threonine at a position corresponding to position 180 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CCT to CAT nucleic acid mutation, which encodes a mutated PPX protein comprising proline to arginine at a position corresponding to position 185 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CCT to CGT nucleic acid mutation, which encodes a mutated PPX protein comprising proline to histidine at a position corresponding to position 185 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a GCC to TGC nucleic acid mutation, which encodes a mutated PPX protein comprising alanine to cysteine ​​at a position corresponding to position 220 of SEQ ID NO:7.In some embodiments, the mutated PPX gene comprises a GCC to ATC nucleic acid mutation that encodes a mutated PPX protein comprising alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a GCC to CTC nucleic acid mutation that encodes a mutated PPX protein comprising alanine to leucine at a position corresponding to position 220 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a GCC to ACC nucleic acid mutation that encodes a mutated PPX protein comprising alanine to threonine at a position corresponding to position 220 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a GCC to GTC nucleic acid mutation that encodes a mutated PPX protein comprising alanine to valine at a position corresponding to position 220 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTG to ATG nucleic acid mutation that encodes a mutated PPX protein comprising leucine to methionine at a position corresponding to position 226 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an ATG to CTG nucleic acid mutation that encodes a mutated PPX protein containing methionine to leucine at a position corresponding to position 226 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AGC to GGC nucleic acid mutation that encodes a mutated PPX protein containing serine to glycine at a position corresponding to position 244 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AGC to ACC nucleic acid mutation that encodes a mutated PPX protein containing serine to threonine at a position corresponding to position 244 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AAA to AAT nucleic acid mutation that encodes a mutated PPX protein containing lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TCT to CTT nucleic acid mutation that encodes a mutated PPX protein containing serine to leucine at a position corresponding to position 305 of SEQ ID NO:7.In some embodiments, the mutated PPX gene comprises an AGT to TGT nucleic acid mutation that encodes a mutated PPX protein containing serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CTT to ATT nucleic acid mutation that encodes a mutated PPX protein containing leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AGT to AGA nucleic acid mutation that encodes a mutated PPX protein containing serine to arginine at a position corresponding to position 359 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises an AGT to ACT nucleic acid mutation that encodes a mutated PPX protein containing serine to threonine at a position corresponding to position 359 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTG to ATG nucleic acid mutation that encodes a mutated PPX protein containing leucine to methionine at a position corresponding to position 393 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTG to TCG nucleic acid mutation that encodes a mutated PPX protein comprising leucine to serine at a position corresponding to position 393 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTG to GTG nucleic acid mutation that encodes a mutated PPX protein comprising leucine to valine at a position corresponding to position 393 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CTA to CGA nucleic acid mutation that encodes a mutated PPX protein comprising leucine to arginine at a position corresponding to position 403 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a CTA to TCA nucleic acid mutation that encodes a mutated PPX protein comprising leucine to serine at a position corresponding to position 403 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTG to TCG nucleic acid mutation that encodes a mutated PPX protein comprising leucine to serine at a position corresponding to position 424 of SEQ ID NO:7.In some embodiments, the mutated PPX gene comprises a TAC to TGC nucleic acid mutation that encodes a mutated PPX protein comprising tyrosine to cysteine ​​at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to AAC nucleic acid mutation that encodes a mutated PPX protein comprising tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to CAC nucleic acid mutation that encodes a mutated PPX protein comprising tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to ATC nucleic acid mutation that encodes a mutated PPX protein comprising tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to TTC nucleic acid mutation that encodes a mutated PPX protein comprising tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to CGC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to ACC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TAC to GTC nucleic acid mutation, which encodes a mutated PPX protein comprising tyrosine to valine at a position corresponding to position 426 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TTT to TCT nucleic acid mutation, which encodes a mutated PPX protein comprising phenylalanine to serine at a position corresponding to position 428 of SEQ ID NO:7. In some embodiments, the mutated PPX gene comprises a TCT to ACT nucleic acid mutation, which encodes a mutated PPX protein comprising isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7.

[0104] [Table 4] *"AA mtn" refers to an amino acid mutation; "NA mtn" refers to a nucleic acid mutation.

[0105] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can encode a mutated PPX protein comprising aspartic acid to asparagine at a position corresponding to position 58 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glycine to cysteine ​​at a position corresponding to position 74 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glycine to asparagine at a position corresponding to position 84 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising leucine to histidine at a position corresponding to position 93 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to arginine at a position corresponding to position 97 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to valine at a position corresponding to position 101 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to asparagine at a position corresponding to position 119 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO:9.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to threonine at a position corresponding to position 151 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising glutamine to leucine at a position corresponding to position 157 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises valine to alanine at a position corresponding to position 164 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises cysteine ​​to serine at a position corresponding to position 177 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises histidine to glutamine at a position corresponding to position 187 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to lysine at a position corresponding to position 195 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to histidine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising proline to serine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising isoleucine to histidine at a position corresponding to position 215 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising isoleucine to serine at a position corresponding to position 215 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to arginine at a position corresponding to position 230 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising cysteine ​​to arginine at a position corresponding to position 271 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising aspartic acid to glycine at a position corresponding to position 278 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising phenylalanine to glycine at a position corresponding to position 283 of SEQ ID NO:9.In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to glycine at a position corresponding to position 292 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to leucine at a position corresponding to position 296 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising asparagine to lysine at a position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising serine to leucine at a position corresponding to position 396 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising alanine to serine at a position corresponding to position 404 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising arginine to lysine at a position corresponding to position 406 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising leucine to valine at a position corresponding to position 421 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein comprising cysteine ​​to serine at a position corresponding to position 434 of SEQ ID NO:9.In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises cysteine ​​to tyrosine at a position corresponding to position 434 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises serine to alanine at a position corresponding to position 448 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises valine to glutamic acid at a position corresponding to position 449 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that comprises aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains lysine to threonine at a position corresponding to position 470 of SEQ ID NO:9. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that contains threonine to serine at a position corresponding to position 500 of SEQ ID NO:9.

[0106] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene comprises a GAT to AAT nucleic acid mutation encoding a mutated PPX protein comprising aspartic acid to asparagine at a position corresponding to position 58 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAA to GTA nucleic acid mutation encoding a mutated PPX protein comprising glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GGT to TGT nucleic acid mutation encoding a mutated PPX protein comprising glycine to cysteine ​​at a position corresponding to position 74 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GGA to GAT nucleic acid mutation encoding a mutated PPX protein comprising glycine to asparagine at a position corresponding to position 84 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CGC to TGC nucleic acid mutation encoding a mutated PPX protein comprising leucine to histidine at a position corresponding to position 93 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CGC to CAC nucleic acid mutation, which encodes a mutated PPX protein containing arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CGC to CTC nucleic acid mutation, which encodes a mutated PPX protein containing arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAT to TAT nucleic acid mutation, which encodes a mutated PPX protein containing asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAA to GAT nucleic acid mutation, which encodes a mutated PPX protein containing glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAA to AAA nucleic acid mutation, which encodes a mutated PPX protein containing glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO:9.In some embodiments, the mutated PPX gene comprises an AGT → ACT nucleic acid mutation, which encodes a mutated PPX protein containing serine to threonine at a position corresponding to position 151 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CAG → CTG nucleic acid mutation, which encodes a mutated PPX protein containing glutamine to leucine at a position corresponding to position 157 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GTT → TTT nucleic acid mutation, which encodes a mutated PPX protein containing valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAT → GAA nucleic acid mutation, which encodes a mutated PPX protein containing aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CAC → CAG nucleic acid mutation, which encodes a mutated PPX protein containing histidine to glutamine at a position corresponding to position 187 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CTT to TTT nucleic acid mutation, which encodes a mutated PPX protein containing leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAT to AAA nucleic acid mutation, which encodes a mutated PPX protein containing asparagine to lysine at a position corresponding to position 195 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CCT to CAT nucleic acid mutation, which encodes a mutated PPX protein containing proline to histidine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a CCT to TCT nucleic acid mutation, which encodes a mutated PPX protein containing proline to serine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAG to GAG nucleic acid mutation, which encodes a mutated PPX protein containing lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO:9.In some embodiments, the mutated PPX gene comprises an AAG to CAG nucleic acid mutation, which encodes a mutated PPX protein containing lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAG to AGG nucleic acid mutation, which encodes a mutated PPX protein containing lysine to arginine at a position corresponding to position 230 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAC ​​to GGC nucleic acid mutation, which encodes a mutated PPX protein containing aspartic acid to glycine at a position corresponding to position 283 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TCA to TTA nucleic acid mutation, which encodes a mutated PPX protein containing serine to leucine at a position corresponding to position 296 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TGT to AGT nucleic acid mutation, which encodes a mutated PPX protein containing cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAT to GAT nucleic acid mutation that encodes a mutated PPX protein containing asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAT to AAA nucleic acid mutation that encodes a mutated PPX protein containing asparagine to lysine at a position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAT to GAA nucleic acid mutation that encodes a mutated PPX protein containing aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GCC to TCC nucleic acid mutation that encodes a mutated PPX protein containing alanine to serine at a position corresponding to position 404 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AGG to AAG nucleic acid mutation that encodes a mutated PPX protein containing arginine to lysine at a position corresponding to position 406 of SEQ ID NO:9.In some embodiments, the mutated PPX gene comprises a AAA to ATA nucleic acid mutation, which encodes a mutated PPX protein containing lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a XXX GCT to GTT nucleic acid mutation, which encodes a mutated PPX protein containing alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TGC to AGC nucleic acid mutation, which encodes a mutated PPX protein containing cysteine ​​to serine at a position corresponding to position 434 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TGC to TAC nucleic acid mutation, which encodes a mutated PPX protein containing cysteine ​​to tyrosine at a position corresponding to position 434 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TCA to GCA nucleic acid mutation, which encodes a mutated PPX protein containing serine to alanine at a position corresponding to position 448 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAT to GGT nucleic acid mutation that encodes a mutated PPX protein containing aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a GAC ​​to AAC nucleic acid mutation that encodes a mutated PPX protein containing aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises a TAT to TTT nucleic acid mutation that encodes a mutated PPX protein containing tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an AAG to ACG nucleic acid mutation that encodes a mutated PPX protein containing lysine to threonine at a position corresponding to position 470 of SEQ ID NO:9. In some embodiments, the mutated PPX gene comprises an ACC to AGC nucleic acid mutation that encodes a mutated PPX protein containing threonine to serine at a position corresponding to position 500 of SEQ ID NO:9.

[0107] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can comprise a GGG→AAA mutation, which encodes a mutated PPX protein comprising a glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, the mutated PPX gene comprises an AAT→GAT nucleic acid mutation, which encodes a mutated PPX protein comprising an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1.

[0108] In some embodiments, in conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX gene can include a combination of mutations, such as two or more, three or more, four or more, five or more, or six or more mutations in the PPX gene. In certain embodiments, the combination of mutations is selected from the combinations of mutations shown in Table 4a and Table 4b.

[0109] [Table 5]

[0110] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1 and serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1. In other embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1, arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1, and serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1 and serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1.

[0111] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1 and serine to cysteine ​​at a position corresponding to position 332 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:7 and lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises asparagine to lysine at a position corresponding to position 52 of SEQ ID NO:7, arginine to histidine at a position corresponding to position 144 of SEQ ID NO:7, and serine to threonine at a position corresponding to position 244 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 144 of SEQ ID NO:1 and serine to threonine at a position corresponding to position 244 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and methionine to leucine at a position corresponding to position 228 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises an arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises an alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises a leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 244 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 220 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO:1.In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:1 and leucine to serine at a position corresponding to position 424 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:1 and leucine to arginine at a position corresponding to position 403 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and leucine to valine at a position corresponding to position 393 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 144 of SEQ ID NO:1 and tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:1 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:1. In some embodiments, the mutated PPX protein comprises serine to glycine at a position corresponding to position 244 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises alanine to threonine at a position corresponding to position 180 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7.In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7. In some embodiments, the mutated PPX protein comprises leucine to methionine at a position corresponding to position 226 of SEQ ID NO:7 and isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO:7.

[0112] [Table 6] TIFF2025143393000007.tif104162

[0113] In conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, the mutated PPX protein comprises glycine to cysteine ​​at a position corresponding to position 74 of SEQ ID NO:9 and arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises leucine to histidine at a position corresponding to position 93 of SEQ ID NO:9 and valine to alanine at a position corresponding to position 164 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9 and proline to histidine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises serine to asparagine at a position corresponding to position 119 of SEQ ID NO:9 and asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO:9 and glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises serine to threonine at a position corresponding to position 151 of SEQ ID NO:9, lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO:9, and lysine to arginine at a position corresponding to position 230 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises glutamine to leucine at a position corresponding to position 157 of SEQ ID NO:9, and histidine to glutamine at a position corresponding to position 187 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises cysteine ​​to arginine at a position corresponding to position 271 of SEQ ID NO:9, and aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises serine to leucine at a position corresponding to position 396 of SEQ ID NO:9 and lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises cysteine ​​to serine at a position corresponding to position 434 of SEQ ID NO:9 and threonine to serine at a position corresponding to position 500 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO:9 and alanine to glycine at a position corresponding to position 292 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises serine to alanine at a position corresponding to position 448 of SEQ ID NO:9 and asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO:9 and lysine to threonine at a position corresponding to position 470 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 243 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 243 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 243 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 243 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9 and proline to histidine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to cysteine ​​at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9 and proline to histidine at a position corresponding to position 214 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, and lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and alanine to valine at a position corresponding to position 423 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9.In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, proline to histidine at a position corresponding to position 214 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9. In some embodiments, the mutated PPX protein comprises arginine to histidine at a position corresponding to position 98 of SEQ ID NO:9, threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO:9, lysine to glutamine at a position corresponding to position 229 of SEQ ID NO:9, and cysteine ​​to serine at a position corresponding to position 307 of SEQ ID NO:9.

[0114] Paralog The subject mutations in the PPX gene are generally described herein using the PPX gene and protein of Solanum tuberosum plastid (see, e.g., Figures 8 and 7, respectively), with amino acid positions referring to positions in Arabidopsis thaliana (SEQ ID NO: 1). The compositions and methods also encompass mutated PPX genes and proteins of other species (paralogs). However, due to variations in PPX genes of other species, the number of amino acid residues targeted for alteration in one species may be different in another. However, analogous positions are readily identified by those skilled in the art by sequence homology. For example, Table 6 summarizes homologous amino acid positions in paralogs of PPX coding sequences from various plants, and Figure 33 shows an amino acid sequence alignment of PPX paralogs from various plants. Thus, homologous positions in these and other paralogs can be identified and mutated.

[0115] herbicides The compositions and methods provided herein include PPX genes and PPX proteins that confer resistance to PPX-inhibiting herbicides. In some embodiments, the PPX-inhibiting herbicides include the diphenyl ether, phenylpyrazole, N-phenylphthalimide, thiadiazole, oxadiazole, riazolinone, oxazolidinedione, and pyrimidinedione chemical families. Representative active ingredients of PPX-inhibiting herbicides and their respective chemical families are summarized in Table 5.

[0116] [Table 7]

[0117] In some embodiments, the PPX-inhibiting herbicide is acifluorfen-Na. In some embodiments, the PPX-inhibiting herbicide is bifenox. In some embodiments, the PPX-inhibiting herbicide is clomethoxyfen. In some embodiments, the PPX-inhibiting herbicide is fluoroglycofen-ethyl. In some embodiments, the PPX-inhibiting herbicide is fomesafen. In some embodiments, the PPX-inhibiting herbicide is halosafen. In some embodiments, the PPX-inhibiting herbicide is lactofen. In some embodiments, the PPX-inhibiting herbicide is oxyfluorfen. In some embodiments, the PPX-inhibiting herbicide is fluazolate. In some embodiments, the PPX-inhibiting herbicide is pyraflufen-ethyl. In some embodiments, the PPX-inhibiting herbicide is cinidon-ethyl. In some embodiments, the PPX-inhibiting herbicide is flumioxazin. In some embodiments, the PPX-inhibiting herbicide is flumicloracpentyl. In some embodiments, the PPX-inhibiting herbicide is fluthiacet-methyl. In some embodiments, the PPX-inhibiting herbicide is thidiazimine. In some embodiments, the PPX-inhibiting herbicide is oxadiazon. In some embodiments, the PPX-inhibiting herbicide is oxadiargyl. In some embodiments, the PPX-inhibiting herbicide is azafenidin. In some embodiments, the PPX-inhibiting herbicide is carfentrazone-ethyl. In some embodiments, the PPX-inhibiting herbicide is sulfentrazone. In some embodiments, the PPX-inhibiting herbicide is pentoxazone. In some embodiments, the PPX-inhibiting herbicide is benzfendizone. In some embodiments, the PPX-inhibiting herbicide is butafenacil. In some embodiments, the PPX-inhibiting herbicide is saflufenacil. In some embodiments, the PPX-inhibiting herbicide is pyrazogyl. In some embodiments, the PPX-inhibiting herbicide is profluazole.

[0118] Also provided are transgenic or non-transgenic plants or plant cells having one or more mutations in a PPX gene, such as those disclosed herein. In certain embodiments, plants or plant cells having one or more mutations in a PPX gene have increased resistance or tolerance to members of the PPX-inhibiting herbicide family. In certain embodiments, plants or plant cells having one or more mutations in a PPX gene can exhibit substantially normal growth or development of the plant, its organs, tissues, or cells compared to corresponding wild-type plants or cells. In certain aspects and embodiments, transgenic or non-transgenic plants are provided that have mutations in a PPX gene, such as those disclosed herein, and in certain embodiments, have increased resistance or tolerance to one or more members of the chemical family of PPX-inhibiting herbicides, and can exhibit substantially normal growth or development of the plant, its organs, tissues, or cells compared to corresponding wild-type plants or cells. That is, in the presence of one or more herbicides, such as flumioxazin, sulfentrazone, or saflufenacil, the mutated PPX protein has substantially the same catalytic activity as the wild-type PPX protein.

[0119] Further provided is a method for producing a plant with mutated PPX gene, for example, one or more mutations as described herein; preferably, the plant maintains the catalytic activity of wild-type protein, regardless of the presence or absence of relevant herbicide.In certain embodiments, the method comprises: introducing the gene repair oligonucleotide into plant cell, which has one or more targeted mutations (such as the mutations disclosed herein) in PPX gene, and identifying the cell, seed or plant with mutated PPX gene.

[0120] plant seeds The plants disclosed herein, in conjunction with any of the aspects, embodiments, methods and / or compositions disclosed herein, can belong to any species of dicotyledonous, monocotyledonous or gymnosperm, and include any woody plant species that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruits, seeds or vegetables, or any species that has colorful or fragrant flowers. For example, the plant or plant cell may be selected from the group consisting of potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oats, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soybean spp., sugarcane, pea, field bean, poplar, grape, citrus, alfalfa, rye, oats, turf and grass, flax, rapeseed, cucumber, morning glory, impatiens, pepper, eggplant, marigold, water lily, cabbage, daisy, carnation, petunia, tulip, iris, lily, and, unless specifically mentioned already, nut-bearing plants. The plant or plant cell may also belong to a species selected from Table 6. The plant or plant cell may also belong to a species selected from the group consisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Amaranthus tuberculatus, Sorghum bicolor, Ricinus communis and Zea mays.

[0121] [Table 8] In TIFF2025143393000010.tif223160DQ386118, deletion of G210 leads to resistance to PPX inhibitors. P refers to plastids, M refers to mitochondria, and B refers to both.

[0122] Gene repair oligonucleobases can be introduced into plant cells using any method commonly used in the art, including, but not limited to, microcarriers (gene gun delivery), microfibers, polyethylene glycol (PEG)-mediated uptake, electroporation, and microinjection.

[0123] Also provided are methods and compositions for culturing cells mutated according to the methods disclosed herein to obtain seed-producing plants (hereinafter referred to as "fertile plants"), and for the production of seeds and additional plants from such fertile plants.

[0124] Also provided is a method for selectively suppressing weeds in a field, the field comprising plants and weeds having the disclosed PPX gene modifications, the method comprising spraying the field with a herbicide to which the plants have been rendered resistant.

[0125] Also provided are mutations in the PPX gene that confer resistance or tolerance to the plant against a member of the herbicide family, or alternatively, the mutated PPX gene has substantially the same enzymatic activity as wild-type PPX.

[0126] Selection of herbicide-tolerant plants and herbicide application Plants and plant cells can be tested for resistance or tolerance to herbicides using methods commonly known in the art, such as growing the plant or plant cell in the presence of the herbicide and measuring the growth rate compared to the growth rate in the absence of the herbicide.

[0127] As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or cell division rate in the plant, plant organ, plant tissue or plant cell that is at least 35%, at least 50%, at least 60% or at least 70% of the growth rate or cell division rate in the corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type PPX protein.

[0128] As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as one or more growth events occurring in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in the corresponding plant, plant organ, plant tissue or plant cell expressing a wild-type PPX protein.

[0129] In conjunction with any of the aspects, embodiments, methods, and / or compositions disclosed herein, plant organs provided herein can include, but are not limited to, leaves, stems, roots, shoots, flower buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries, and fruits, or clusters, fragments, or discs extracted therefrom. Plant tissues include, but are not limited to, callus tissue, ground tissue, vascular tissue, storage tissue, meristematic tissue, leaf tissue, stem tissue, root tissue, gall tissue, plant tumor tissue, and reproductive tissue. Plant cells include, but are not limited to, isolated cells with cell walls, aggregates of various sizes thereof, and protoplasts.

[0130] A plant is substantially "tolerant" to a herbicide if, when the herbicide is applied to it and a dose-response curve is generated, it is shifted to the right compared to that obtained from a similar, non-tolerant plant treated in the same way. Such a dose-response curve is plotted with "dose" on the x-axis and "percent kill," "herbicidal effect," etc. on the y-axis. Tolerant plants require more herbicide to achieve a given herbicidal effect than similar, non-tolerant plants. Plants that are substantially "resistant" to a herbicide exhibit little, if any, necrosis, lysis, chlorosis, or other lesions when the herbicide is applied at concentrations and rates typically used in the agrochemical community to control field weeds. Plants that are resistant to a herbicide are also tolerant to the herbicide.

[0131] In some embodiments, "increased resistance to herbicides" or "increased tolerance to herbicides" refers to the level of resistance or tolerance that a plant, seed, or plant part having a mutated PPX gene or protein as disclosed herein has to a plant herbicide above a defined reference concentration. The defined reference level of resistance to herbicides is the level of resistance exhibited by a plant of the same species that does not have the corresponding mutation(s). In some embodiments, the resistance is increased substantially above the defined reference level, for example, by 20% or more, 50% or more, 75% or more, or 100%.

[0132] Example The following are examples to illustrate the practice of the present invention. These examples should not be considered limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise specified.

[0133] Example 1: Cloning and characterization of plastid and mitochondrial PPX genes Plastidial and mitochondrial PPX genes were amplified from both cDNA and genomic DNA from the Russet Burbank cultivar. Plastidial PPX clones were divided into two classes, designated StcPPX1 and StcPPX1.1, which likely represent alleles of a single potato PPX gene. Within the amino acid coding sequence, these clones contained 10 distinct polymorphisms, three of which led to amino acid differences, only two of which were found in the mature protein. One amino acid difference was in the chloroplast transit peptide. In one of the StcPPX1.1 clones, intron 3 was not spliced.

[0134] We obtained a full-length, error-free genomic clone for plastid PPX. Analysis of approximately 5 kb of genomic sequence from five independent clones and the StcPPX cDNA sequencing results suggests that the characterized Russet Burbank species is heterozygous, with very little polymorphism between the two alleles.

[0135] First, five full-length StmPPX genomic DNA clones were cloned and sequenced. These five represented both alleles and contained the same SNPs as those in the cDNA. Genomic DNA fragments from shorter amplicons were cloned and sequenced to test for additional alleles. Cloning of this internal amplicon of mitochondrial PPX revealed the presence of three alleles; six of the 22 clones contained a deletion within one of the introns, while the remaining 16 clones suggested that the two alleles observed in the cDNA clones were evenly distributed. Next, another 12 full-length StmPPX genomic DNA clones were sequenced.

[0136] Completed sequencing of the mitochondrial PPX gene in Russet Burbank potato suggested the existence of two genes, which we named StmPPX1 and StmPPX2. There are two StmPPX2 alleles, and eight SNPs were identified between the two. Between StmPPX1 and StmPPX2.1, there is one insertion, four deletions, and 30 SNPs, and between StmPPX1 and StmPPX2.2, there is one insertion, four deletions, and 29 SNPs. Supplementary details are provided in Table 7.

[0137] Gene sequences of plastidial and mitochondrial potato PPX genes from Russet Burbank (Solanum tuberosum) were compared to our locally installed database based on the scaffold of the recent release of the first complete potato (Solanum phureja) genome using the Basic Local Alignment Search Tool (BLAST). Only one plastidial and one mitochondrial PPX gene were found.

[0138] [Table 9] Example 2: PPX complementation

[0139] StcPPX1, minus its chloroplast transit peptide, was cloned from cDNA into a Cibus proprietary functional screening vector. This vector can be used for both functional screening and GRON QC. The potato PPX gene was used to complement a HemG mutant strain of Escherichia coli, which lacks a functional HemG gene, the bacterial homolog of PPX. Without the complementing gene, E. coli growth requires supplementation of the medium with hematin. Transformation of clones of the plastid PPX gene (pACYStcPPX Col6) and the mitochondrial PPX gene (pACYStmPPX Col6, 12, and 21) revealed that all genes / alleles complemented the HemG mutant E. coli strain, enabling E. coli to grow in the absence of hematin.

[0140] To evaluate mutations conferring resistance to PPX inhibitors, such as Chateau (flumioxazin - Valent / Sumitomo), Naja (diphenyl ether - MAI), or Kixor (saflufenacil - BASF), the PPX-inhibiting herbicides are listed in Table 5. The pure active ingredients of the PPX-inhibiting herbicides Chateau (flumioxazin - Valent / Sumitomo), Spartan (sulfentrazone - FMC), and Kixor / Sharpen (saflufenacil - BASF) were obtained. A wild-type potato plastid PPX clone (pACYStcPPX Col6) was transformed into a hemG mutant E. coli strain to complement it. A series of concentrations of the active ingredient of the PPX-inhibiting herbicide Spartan (sulfentrazone - FMC) was selected to determine the concentration at which the complemented HemG-less strain failed to grow. Because the wild-type construct did not grow at 2.5 mM sulfentrazone, selection for resistance mutants was performed at this concentration. This was further refined and resistance was selected for with 0.75 mM sulfentrazone. The wild-type construct showed limited growth on 10 mM flumioxazin in liquid selection and no growth at all on 0.3 mM saflufenacil in plate-based selection, concentrations used to test for resistant mutants. All potato mitochondrial PPX genes and alleles were tested for natural resistance to sulfentrazone and flumioxazin, but none were resistant.

[0141] Example 3: PPX PCR mutagenesis and selection of mutagenized clones Mutagenesis experiments were first performed on two overlapping fragments (5' and 3') of the potato plastid PPX gene to identify mutations in the potato plastid PPX coding sequence that confer herbicide resistance.

[0142] Liquid Selection Standardization Liquid medium selection conditions were developed for both sulfentrazone and flumioxazin. Concentrations of sulfentrazone and flumioxazin ranging from 0 to 10 mM were tested. The 0 mM sample contained 25 μL of DMSO (2.5%) to replicate the DMSO concentration in the 10 mM herbicide sample. For uniformity, each tube was inoculated with 10 μL of an overnight culture of HemG cells complemented with the wild-type PPX plasmid. Spectrophotometric measurements (OD600) were taken for each sample (1:4 dilution), and each sample was plated onto LB-Chlor-IPTG plates to determine whether OD600 correlated with the number of viable colonies. For sulfentrazone-treated cells, plates were inoculated with a 10% dilution of the overnight culture, and for flumioxazin-treated cells, plates were inoculated with a 1% dilution (see results in Table 8, af, and Table 9, ad).

[0143] Both sulfentrazone and flumioxazin flocculated in the liquid medium, causing it to become opaque even before inoculation with bacteria. As the OD of sulfentrazone indicates, this herbicide eventually went into solution, whereas flumioxazin did not. Although the lack of solubility of flumioxazin distorted the OD readings, flumioxazin, in contrast to the WT gene, showed a steady decrease in colonies, suggesting that the cells were able to absorb flumioxazin from the medium.

[0144] 5'-end mutagenesis The 5' end of the PPX gene was mutagenized using the Stratagene GeneMorph II Random Mutagenesis Kit and cloned into XL-1 Blue to confirm the mutation rate. Results showed that 14 of 16 sequenced colonies were mutated. 90% of the XL-1 Blue plates (approximately 4,000 colonies) were scraped, plasmids prepared, transformed into HemG, and plated on 2.5 mM sulfentrazone. Approximately 200 colonies grew on sulfentrazone plates, and a lawn of colonies grew on 10% LB-Chlor-IPTG plates. Table 9, a through d, describes the nucleotide and amino acid substitutions found in the resistant clones.

[0145] Selection of mutated clones Randomly mutagenized plasmids (5' and 3' ends) were transformed into XL1-Blue E. coli cells. The resulting colonies were pooled, and plasmid DNA was isolated and transformed into HemG (PPX mutant E. coli) cells. For flumioxazin selection, cells were allowed to recover in liquid minimal medium for 1 hour, then herbicide was added and the cells were allowed to recover overnight. The next day, for selection of complemented plasmids, cells were plated at appropriate dilutions onto LB plates containing antibiotic. Colonies from each plate were sequenced. After liquid selection with 10 mM flumioxazin, approximately 200–1200 colonies emerged for the mutagenized plasmids, whereas approximately 30 colonies emerged on wild-type (WT) PPX plates. For sulfentrazone selection, cultures were grown overnight in minimal medium and diluted with sulfentrazone at 0 mM and 0.75 mM concentrations before plating. Colony counts were compared between the two, and mutation tolerance was determined based on the percentage of colonies on the 0.75 mM sulfentrazone plates compared to the 0 mM plates. The number of colonies appearing on a plate served as a method for ranking the mutations.

[0146] 3'-end mutagenesis Mutagenesis was performed on the 3' end of the PPX gene. Clones were transformed into HemG and grown overnight in 2.5 mM, 5 mM, and 10 mM flumioxazin for selection. Selection on 5 mM flumioxazin yielded many more colonies than on 10 mM selection plates. Selection of clones mutagenized with 10 mM flumioxazin yielded four clones. Selection of clones mutagenized with 5 mM flumioxazin yielded 200 colonies. One-third of the 200 colonies obtained for 3' end mutagenesis were screened in 10 mM flumioxazin. All resistant colonies (approximately 130) were sequenced, and the best flumioxazin-resistant mutations were evaluated by colony count on flumioxazin and the 3' end for resistance to sulfentrazone.

[0147] Example 4: Analysis of amino acid substitutions that confer resistance All possible amino acid substitutions at each position that confer resistance to sulfentrazone or flumioxazin were tested for tolerance. The single amino acid substitutions were then combined in all permutations and combinations to evaluate complementation and herbicide tolerance. The results of single and multiple mutation combinations on flumioxazin are shown in Tables 8a, 8b, and 8c, with the last column indicating the number of colonies reported for each mutation on 10 mM flumioxazin. The results of single and multiple mutation combinations on sulfentrazone are shown in Tables 9a and 9b, with the last column indicating the number of colonies reported for each mutation on 0.75 mM sulfentrazone. The results of single and multiple mutation combinations on saflufenacil are shown in Table 10, with the fourth column indicating the number of colonies reported for each mutation on 0.3 mM saflufenacil.

[0148] [Table 10] TIFF2025143393000013.tif163161

[0149]

Table 11

[0150]

Table 12

[0151]

Table 13

[0152]

Table 14

[0153]

Table 15

[0154] Table 16

[0155] Table 17 TIFF2025143393000024.tif169164

[0156] Table 18 TIFF2025143393000026.tif150162

[0157] Table 19 TIFF2025143393000028.tif186163

[0158] [Table 20] TIFF2025143393000030.tif100159

[0159] Example 5 Plant Cell Culture Medium - Herbicide Killing Curve Flumioxazin herbicidal curve A herbicide selection experiment was performed to determine the herbicide concentration required to kill protoplast-induced microcoleus over a defined treatment period. Based on the initial kill curve results, in which a concentration of 1.25 μM was sufficient to kill all cells within one week, a new kill curve was designed using lower concentrations of flumioxazin to determine the concentration at which 99% of the cells were killed (see Table 11). Herbicides were suspended in DMSO, with a final DMSO concentration of 1% in the herbicide treatments. Cell development was assessed microscopically once per week. Except for the control treatment, division was prevented in all flumioxazin treatments after one week, and no microcoleus developed at any of the concentrations tested after one month. A concentration of 0.032 mM flumioxazin was sufficient to prevent microcoleus development from potato protoplasts.

[0160] [Table 21]

[0161] Sulfentrazone herbicidal curve The herbicide curves of sulfentrazone on shoot apex-induced protoplasts and cell suspensions showed that a concentration of 7.8 μM sulfentrazone was sufficient to kill all protoplast-derived cells (see Table 12). Therefore, new herbicide curves were started with lower concentrations of sulfentrazone: 0, 0.5, 0.6, 0.7, and 0.8 μM, as shown in Table 13. The results suggest that GRON-treated protoplasts can be selected at herbicide concentrations from 0.6 μM to 0.7 μM.

[0162] [Table 22]

[0163] [Table 23]

[0164] Example 6: Leaf disc herbicidal curves To establish the concentration of sulfentrazone that inhibits callus formation in leaf disc explants, leaf discs were prepared from 5-week-old in vitro-grown potato seedlings by punching with a sterile punch. The prepared leaf discs were cultured in Petri dishes containing solid Haberlach medium containing various concentrations of sulfentrazone in a final concentration of 1% DMSO. Six leaf discs were cultured per herbicide concentration. The plates were sealed with micropore tape and incubated at room temperature (approximately 23°C). In initial experiments, 7.8 μM sulfentrazone, the lowest concentration tested in the experiment, was sufficient to stop callus formation and bleach all leaf discs within 20 days. Kill curve results using lower concentrations of sulfentrazone showed that a concentration of 3.0 μM sulfentrazone was sufficient to inhibit callus formation on almost all leaf discs after 20 days, but some leaf disc veins grown on 2.0 μM sulfentrazone induced callus after 13 days. Similar leaf disc kill curve experiments were performed with saflufenacil, and 0.5 μM of this herbicide was sufficient to inhibit callus formation on almost all leaf discs after 20 days.

[0165] Example 7: Materials and methods for cell culture and GRON transduction [Table 24]

[0166] Cell Culture Procedure: For example, seeds, tubers, axillary buds, leaves, stems, roots, callus, or shoots derived from microspore-derived embryos are propagated in vitro under sterile conditions. Explants are subcultured, for example, every 3 to 4 weeks, in approximately 100 mL of medium, such as MS medium, in Magenta GA7 culture vessels with vented lids (Phytotechnology Laboratories, Shawnee Mission, Kansas, USA) according to the method described by Murashige and Skoog (A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant 15 (1962) 473-49) or a modified version thereof. Vessels can be sealed with micropore tape (3M). Young leaves, shoot tips, roots, microtubers, or sections of leaves and axillary stems, as well as callus derived from these tissues, can be used for protoplast isolation. Protoplasts can also be isolated from suspension cultures derived from young leaves, shoot tips, roots, microtubers, or segments of leaves and axillary stems, as well as callus derived from these tissues.

[0167] Isolation of protoplasts from shoot tips Approximately 200 shoot tips from 2- to 8-week-old in vitro stems are cultured under a normal day / night regime or, preferably, kept in the dark for 2 days before protoplast isolation. Stems may be cut into small pieces using a scalpel in a Petri dish containing sterile water. After all shoot tips have been cut, the water is replaced with protoplast medium, preferably BN medium (B5 salts and vitamins (Phytotechnology Laboratories)), 20 g / L glucose, 70 g / L mannitol, 5 mg / L α-naphthaleneacetic acid, 600 mg / L additional CaCl2x 2H2O, 250 mg / L casein hydrolysate, 10 mg / L cysteine-HCl, and 5 g / L polyvinylpyrrolidone (MW 10,000). After approximately 1-2 hours, the protoplast medium is replaced with an enzyme solution, e.g., BN medium containing 0.5% (wt / vol) Cellulase YC and 0.75% (wt / vol) Macerozyme R10 (both from Karlan Research Products, Cottonwood, Arizona), 1 g / L bovine serum albumin, and 1 g / L 2-morpholinoethanesulfonic acid. The ratio of shoot apex numbers to enzyme solution volume can be between 10 and 16, with 13 being preferred. The dish containing the shoot apex segments in the enzyme solution is incubated in the dark at 25-30°C, preferably 28°C, in a shaking incubator set at 50 rpm. After overnight incubation, the protoplast suspension is purified using an iodixanol density gradient (Optiprep Application Sheet C18; Purification of Intact Plant Protoplasts; Axis-Shield USA, 10 Commerce Way, Norton, MA 02776). After density gradient centrifugation, the band containing purified protoplasts is removed with approximately 5 mL of W5 medium (Frigerio et al., 1998). The protoplasts are grown to a density of 1 x 10 in BN medium containing 2 mg / L of 2,6-dichlorobenzonitrile (a cellulose synthesis inhibitor). 6 The solution is adjusted to 1 / mL and the protoplasts are cultured in the dark at 30°C for about 16 hours.

[0168] Isolation of protoplasts from cell suspensions Isolation of protoplasts from cell suspension follows the same procedure as described for isolation of protoplasts from shoot tips, with the following exceptions: 1. Use a fast-growing cell suspension, preferably 3 days after the last subculture. Transfer 1.5 mL of the settled cell volume to approximately 15 mL of BN medium, and after 2 hours, replace with enzyme solution. 2. After protoplast purification, immediately perform GRON / PEG treatment.

[0169] Introduction of gene repair oligonucleotides (GRON) The protoplast suspension was mixed with an equal volume of W5 medium, transferred to a 50 mL centrifuge tube, and centrifuged for 10 minutes at the lowest setting (approximately 50 × g) in a clinical centrifuge. The supernatant was removed and replaced with TM medium (Klaus, S. Markerfreie transplastome Tabakpflanzen (Marker-free transplastomic tobacco plants). PhD Dissertation, 2002, Ludwig-Maximilians-Universitat Munchen, p. 109) to achieve a protoplast density of 5 × 10. 6 / mL. 5 A 100 µL aliquot containing 100 protoplasts is placed in a 12 mL round-bottom centrifuge tube. Next, GRONs (such as those listed in Table 4) targeting one or more mutations in one or both mitochondrial and plastid PPX genes are introduced into the protoplasts using PEG treatment. To introduce GRONs into the protoplasts, 12.5 µg of GRON is dissolved in 25 µL of pure water and 125 µL of polyethylene glycol solution (5 g PEG MW 1500, 638 mg mannitol, 207 mg CaNO3 x 4HO, and 8.75 mL of pure water; pH adjusted to approximately 9.0) is added. After incubation on ice for 10–30 min, the protoplast-PEG suspension is washed with W5 medium and resuspended in medium BN. The suspension is kept overnight in the dark at room temperature.

[0170] GRON can be introduced into a plasmid by electroporation, cationic lipids, nanoparticles, polycations such as hexadimethrine bromide (polybrene) or spermidine, or GRON conjugated to various cell penetrating peptides (CPPs), including but not limited to TAT, pVEC, transportan, nona-arginine, BAX inhibitor peptide (VPMLK), or those described in Patel et al., Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives, Pharmaceutical Research, 24 (2007) 1977-1992 or Veldhoen et al., Recent developments in peptide-based nucleic acid delivery, International Journal of Molecular Science (2008) 1276-1320. In another embodiment, GRON is introduced into protoplasts using negatively charged polymers, including but not limited to d...

Claims

1. A plant comprising a mutated protoporphyrinogen IX oxidase (PPX) gene, wherein the gene encodes a protein containing a leucine to arginine substitution at a position corresponding to position 403 of SEQ ID NO: 1, wherein the plant is a non-transgenic plant having an artificially introduced targeted mutation.

2. 2. The plant of claim 1, wherein the gene encodes a protein further comprising a phenylalanine to tyrosine substitution at a position corresponding to position 145 of SEQ ID NO:

1.

3. A plant cell comprising a mutated protoporphyrinogen IX oxidase (PPX) gene, wherein the gene encodes a protein containing a leucine to arginine substitution at a position corresponding to position 403 of SEQ ID NO: 1, wherein the plant cell is a non-transgenic plant cell having an artificially introduced targeted mutation.

4. 4. The plant cell of claim 3, wherein the gene encodes a protein further comprising a phenylalanine to tyrosine substitution at a position corresponding to position 145 of SEQ ID NO:

1.

5. 5. The plant of claim 1 or 2 or the plant cell of claim 3 or 4, characterized in that the plant or the plant from which the plant cell is derived is selected from the group consisting of potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oats, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soybean spp., sugarcane, pea, field bean, poplar, grape, citrus, alfalfa, rye, oats, turf and grasses, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, water lily, cabbage, daisy, carnation, petunia, tulip, iris, lily, and nut-bearing plants.

6. 5. The plant according to claim 1 or 2 or the plant cell according to claim 3 or 4, characterized in that the plant or the plant from which the plant cell is derived is a species selected from the group consisting of potato (Solanum tuberosum), rice (Oryza sativa) and corn (Zea mays) or the Russet Burbank potato cultivar.

7. 5. A plant according to claim 1 or 2 or a plant cell according to claim 3 or 4, characterized in that the plant or the plant from which the plant cell is derived is produced by asexual reproduction or is produced from a tuber.

8. 8. The plant or plant cell of claim 1, wherein the plant or plant from which the plant cell is derived is resistant to one or more of the PPX-inhibiting herbicides selected from the group consisting of acifluorfen-Na, bifenox, clomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimine, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, saflufenacil, pyrazogyl, and profluazole.

9. 8. The plant or plant cell according to claim 1, wherein the plant or the plant from which the plant cell is derived is resistant to one or more herbicides selected from the group consisting of flumioxazin, sulfentrazone, and saflufenacil.

10. The method for producing non-transgenic plant cells with mutated protoporphyrinogen IX oxidase (PPX) gene, comprising: introducing the gene repair oligonucleotide (GRON) of targeted mutation into plant cells, so as to produce the plant cells with PPX gene, expressing PPX protein, comprising leucine to arginine substitution at the position corresponding to the 403 position of SEQ ID NO:

1.

11. 11. The method of claim 10, wherein the PPX protein further comprises a leucine to arginine substitution at a position corresponding to position 403 of SEQ ID NO:

1.

12. and further identifying plant cells that have substantially normal growth and catalytic activity in the presence of the herbicide compared to corresponding wild-type plant cells; and 12. The method of claim 10 or 11 for producing a herbicide-tolerant plant, comprising regenerating a non-transgenic herbicide-tolerant plant having a mutated PPX gene from said plant cell.

13. 13. The method of any one of claims 10 to 12, characterized in that the plant cell is derived from a plant selected from the group consisting of potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oat, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soybean spp., sugarcane, pea, field bean, poplar, grape, citrus, alfalfa, rye, oat, turf and grass, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, water lily, cabbage, daisy, carnation, petunia, tulip, iris, lily, and nut-bearing plants.

14. 13. The method according to any one of claims 10 to 12, characterized in that the plant cells are derived from a species selected from the group consisting of potato (Solanum tuberosum), rice (Oryza sativa), sorghum (Sorghum bicolor), castor bean (Ricinus communis), oilseed rape (Brassica napus), soybean (Glycine max), and maize (Zea mays) or from the Russet Burbank potato cultivar.

15. 15. The method of claim 13 or 14, wherein the plant is resistant to one or more of the following PPX-inhibiting herbicides: acifluorfen-Na, bifenox, clomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimine, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, saflufenacil, pyrazogyl, or profluazole.

16. 15. The method of claim 13 or 14, wherein the plant is resistant to one or more herbicides selected from the group consisting of flumioxazin, sulfentrazone and saflufenacil.

17. 13. The method according to any one of claims 10 to 12, characterized in that the plant cells are derived from plants produced by asexual reproduction or from tubers.

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