Mutated allene oxygenase 2 (AOS2) gene

Abstract

The invention name of the present invention is mutated allene oxygenase 2 (AOS2) gene. The present invention provides compositions and methods related to gene and / or protein mutations in plants. In certain embodiments, the present disclosure relates to mutations in the allene oxygenase 2 gene (i.e., AOS2). In some embodiments, the present disclosure relates to pathogen-resistant plants.

Description

[0001] The present application is a divisional application of the Chinese application with the application date of March 14, 2014, the application number of 201480024216.X (PCT / US2014 / 029434), and the invention name of “Mutated Allene Oxygenase 2 (AOS2) Gene”.

[0002] This application claims priority to U.S. Provisional Patent Application No. 61 / 785,059, filed March 14, 2013, which is hereby incorporated by reference. Field of the Invention

[0003]

[0014] The present disclosure relates, in part, to gene and / or protein mutations in plants. Background of the Invention

[0004] The following discussion of the background of the invention is provided merely to assist the reader in understanding the present invention and is not admitted to describe or constitute prior art to the present invention.

[0005] Phytophthora infestans (Pi) is an organism belonging to the phylum Oomycota that causes a devastating disease of potatoes (Solanum tuberosum), also known as late blight. The genus Phytophthora also causes diseases in other plant species such as tomatoes, soybeans, peppers, and tobacco. Pi has been managed using chemicals such as methyl bromide and metalaxyl.

[0006] An association between the potato allene oxide synthase (StAOS2) gene and resistance to late blight has been reported. Pajerowska-Mukhtar et al., Planta 228:293 (2008) disclosed "[n]natural variation of potato allene oxide synthase 2 causes differential levels of jasmonates and pathogen resistance in Arabidopsis." Pajerowska-Mukhtar et al., Genetics 181: 1115 (2009) disclosed that “[a]major association was found at the StAOS2 locus encoding alleneoxide synthase 2, a key enzyme in the biosynthesis of jasmonates...” and “[t]wo SNPs at the StAOS2 locus were associated with the largest effect on resistance. StAOS2_snp691 and StAOS2_snp692....” SUMMARY OF THE INVENTION

[0007] The present disclosure relates, 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 producing pathogen-resistant plants. In some aspects and embodiments, the present disclosure may also relate to compositions and methods for producing transgenic or non-transgenic plants with normal or altered maturity levels. In some aspects and embodiments, the present disclosure may also relate to compositions and methods for producing transgenic or non-transgenic plants with elevated jasmonic acid levels. The present disclosure also relates, at least in part, to compositions and methods related to mutations in one or more allene oxygenase 2 (AOS2) genes / alleles.

[0008] In one aspect, plants or plant cells comprising a mutated AOS2 gene are provided. In certain embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. In certain embodiments, plants having plant cells comprising a mutated AOS2 gene can be resistant to pathogens; for example, to plant pathogens such as Phytophthora infestans (Pi). In certain embodiments, plants having plant cells comprising a mutated AOS2 gene can have an altered maturity grade. In certain embodiments, plants having plant cells comprising a mutated AOS2 gene can have elevated jasmonic acid levels.

[0009] In conjunction with any of the various aspects, embodiments, compositions and methods disclosed herein, the plant or plant cell can be a plant or plant cell of any species of dicotyledonous, monocotyledonous or gymnosperm, including any woody plant species that grows as a tree or shrub, any herbaceous plant species, or any species that produces edible fruit, seeds or vegetables, or any species that produces colorful or fragrant flowers. For example, the plant or plant cell can be selected from a plant species selected from potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oats, rice, rapeseed, fruit, vegetable, tobacco, eggplant (aubergine), barley, boxthane, sorghum, tomato, green tomato, tamarillo, mango, peach, apple, pear, strawberry, banana, melon, wolfberry, garden black fruit tree (garden black fruit tree) The plant or plant cell may be selected from the group consisting of: peas, huckleberry, ground cherry, carrot, lettuce, onion, beans, sugarcane, peas, beans, poplar, grape, citrus, alfalfa, rye, oats, lawn and pasture grasses, cucurbits, flax, brassica, cucumber, pumpkin, zucchini, watermelon, cantaloupe, morning glory, impatiens, pepper, sweet pepper, bell pepper, red pepper, green and red pepper, pimento tree, habanero, capsicum, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut-producing plants, as long as they have not been specifically mentioned. The plant or plant cell may be a plant or plant cell of a species selected from the group consisting of Arabidopsis thaliana, potato, Solanum phureja, rice, Amaranthus tuberculatus, and Zea mays. In various embodiments, the plant as disclosed herein can be a plant of any species of the Solanaceae family.

[0010] In conjunction with any of the various aspects, embodiments, compositions, and methods disclosed herein, the plant or plant cell can be any commercial variety of potato. For example, the plant or plant cell can be selected from potato varieties selected from Anya, Arran Victory, Atlantic, Belle de Fontenay, BF-15, Bintje, Cabritas, Camota, Chelina, Chiloé, Cielo, Clavera Blanca, Désirée, Fianna, Fingerling, Flava, Fontana, Golden Wonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, Maris Piper, Nicola, PinkEye, Pink Fir Apple, Primura, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Shepody, Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs's Pink / Meru, Golof, Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro, Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka, B53 (Roslin Eburu), Kiraya, Kenya Akiba, 9, Original, Gituma, Mukorino, Amin, Pimpernel, Anett, B, Gituru, Feldeslohn, C, Kigeni, Romano, Kenya Ruaka, Purplu, Njae, Suzanna, Cardinal, Kathama, Kinare-Mwene, Kibururu, Karoa-Igura, Muturu, Faraja, Kiamucove, Michiri, Rugano, Njine Giathireko, Meru Mix, Blue Baranja, Patrones, Robijn, Roslin Chania, Urgentia, Mirka and Roslin Sasamua.

[0011] As used herein, the term "AOS2 gene" refers to a DNA sequence capable of producing an allene oxygenase 2 (AOS2) polypeptide sharing homology and / or amino acid identity with the amino acid sequence of SEQ ID NO: 1, and / or encoding a protein exhibiting AOS2 activity. In certain embodiments, the AOS2 gene has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a specific AOS2 gene (e.g., a potato AOS2 gene, e.g., StAOS2). In certain embodiments, the AOS2 gene is 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50.

[0012] As used herein, the term "pathogen resistance" refers to a plant trait that reduces pathogen growth upon infection with a pathogenic isolate.

[0013] As used herein, the term "pathogen tolerance" refers to the ability of a plant to mitigate the effects of infection on plant health. In some embodiments, pathogen-resistant plants may have necrotic lesions that are limited and / or do not spread indefinitely. In some embodiments of pathogen-tolerant plants, minimal or no necrosis is observed, but water-soaked lesions may be present. In some embodiments, pathogen-tolerant plants may survive infection with minimal damage or a minimal reduction in the harvest yield of marketable material.

[0014] As used herein, the term "mutation" refers to a single nucleotide change in a nucleic acid sequence and / or a single amino acid change in a polypeptide at least relative to a normal sequence or a wild-type sequence or a reference sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, a mutation refers to a single nucleotide change in a nucleic acid sequence and / or a single amino acid change in a polypeptide at least relative to a nucleotide or amino acid sequence of an AOS2 protein that does not confer an acceptable level of pathogen resistance and / or tolerance. In certain embodiments, a mutation may include a substitution, deletion, inversion, or insertion. In some embodiments, a substitution, deletion, insertion, or inversion may include changes in more than one nucleotide. In certain embodiments, a substitution, deletion, insertion, or inversion may include changes in 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, substitution, deletion, insertion or inversion can include changes in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid positions. The term "nucleic acid" or "nucleic acid sequence" refers to an oligonucleotide, nucleotide or polynucleotide that can be single-stranded or double-stranded and represents a sense strand or antisense strand, and a fragment or portion thereof. Nucleic acid can include DNA or RNA and can be of natural or synthetic origin. For example, nucleic acid can include mRNA or cDNA or genomic DNA. Nucleic acid can include amplified (for example, using polymerase chain reaction) nucleic acid. The convention "NTwt###NTmut" is used to indicate such a mutation: the mutation causes the wild-type nucleotide NTwt at position ### in the nucleic acid to be replaced by the mutant NTmut. The single-letter code for nucleotides is as described in Table 1, Section 2422, Manual of Patent Examining Procedures, U.S. Patent Office. In this regard, the nucleotide designations "R" mean a purine, such as guanine or adenine; "Y" means a pyrimidine, such as cytosine or thymine (or uracil in the case of RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0015] As used herein, the term "mutated AOS2 gene" refers to an allene oxygenase 2 (AOS2) gene having one or more mutations at certain positions of the nucleotide relative to a reference AOS2 nucleic acid sequence (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and / or 50). In certain embodiments, the mutated AOS2 gene has one or more mutations relative to the corresponding wild-type AOS2 sequence. In some embodiments, the mutated AOS2 gene has one or more mutations relative to the corresponding AOS2 sequence encoding an AOS2 protein that does not confer an acceptable level of pathogen resistance and / or tolerance. In some embodiments, the mutated AOS2 gene has one or more mutations at the homologous position of its paralog relative to, for example, SEQ ID NO: 2. In some embodiments, the AOS2 gene is modified by at least one mutation. In certain embodiments, the AOS2 gene is modified by at least two mutations. In certain embodiments, the AOS2 gene is modified by at least three mutations. In some embodiments, the mutated AOS2 gene encodes two or more mutated AOS2 proteins, such as described herein. In some embodiments, the mutated AOS2 gene is a mutated potato AOS2 gene / allele; for example, StAOS2. In some embodiments, the mutated AOS2 gene is a mutated Desiree AOS2 gene / allele. In some embodiments, the mutated AOS2 gene is a mutated Bintje AOS2 gene / allele. In some embodiments, the mutated AOS2 gene is a mutated FontanaAOS2 gene / allele. In some embodiments, the mutated AOS2 gene is a mutated Innovator AOS2 gene / allele.

[0016] In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 691 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 692 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 678 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a T at a position corresponding to position 681 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 727 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 744 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 774 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 879 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 900 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 954 of SEQ ID NO:2.

[0017] As used herein, the term "AOS2 protein" refers to a protein that has homology and / or amino acid identity with the AOS2 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, 41, 43, 45, 47 and / or 49 and / or exhibits AOS2 activity. In certain embodiments, the AOS2 protein is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a specific AOS2 protein (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49), such as a potato AOS2 protein. In some embodiments, the mutated AOS2 protein is a mutated Desiree AOS2 protein. In some embodiments, the mutated AOS2 protein is a mutated Bintje AOS2 protein. In some embodiments, the mutated AOS2 protein is a mutated Fontana AOS2 protein. In some embodiments, the mutated AOS2 protein is a mutated Innovator AOS2 protein. In certain embodiments, the AOS2 protein is selected fromFigure 1 、 3 , 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and / or 49 has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence in , 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and / or 49.

[0018] As used herein, the term "mutated AOS2 protein" refers to an AOS2 protein having one or more mutations at certain amino acid positions relative to a reference AOS2 amino acid sequence or at certain homologous positions of its paralogs. In some embodiments, the mutated AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence (e.g., a reference AOS2 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, 41, 43, 45, 47 or 49, or a portion thereof). In certain embodiments, the mutated AOS2 protein has one or more mutations relative to the corresponding AOS2 wild-type protein. In certain embodiments, the mutant AOS2 protein has one or more mutations at positions corresponding to positions selected from 6, 12, 30, 37, 46, 48, 51, 76, 113, 145, 187, 197, 200, 227, 231, 256, 264, 270, 282, 289, 292, 309, 320, 328, 337, 338, 357, 381, 394, 407, 423, 430, 439, 467, 480, 494, and 495 of SEQ ID NO: 1. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 6. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an R at amino acid position 12. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a P at amino acid position 12. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an A at position 30. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an I at position 37. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 46. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 46. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an I at amino acid position 48.In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 48. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a T at amino acid position 48. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an M at amino acid position 51. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an N at amino acid position 76. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a D at amino acid position 76. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a D at amino acid position 113. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a G at amino acid position 113. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 145. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 187. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a D at amino acid position 197. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an E at amino acid position 197. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a K at amino acid position 200. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an A at amino acid position 227. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an I at amino acid position 231.In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a G at amino acid position 231. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a T at amino acid position 231. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 256. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 256. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an A at amino acid position 264. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 270. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an S at amino acid position 282. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 282. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 289. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an N at amino acid position 289. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an S at amino acid position 289. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 292. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an I at amino acid position 309. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 309.In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 320. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an M at amino acid position 320. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an M at amino acid position 328. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an V at amino acid position 328. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 328. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a D at amino acid position 337. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an E at amino acid position 337. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 338. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 338. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an M at amino acid position 357. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an I at amino acid position 357. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 381. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a P at amino acid position 381. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a T at amino acid position 394.In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a C at amino acid position 407. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a G at amino acid position 407. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an F at amino acid position 423. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an L at amino acid position 430. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an E at position 439. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has an S at amino acid position 467. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a G at amino acid position 467. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a V at amino acid position 480. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a D at amino acid position 494. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a G at amino acid position 494. In certain embodiments, the mutant AOS2 protein has one or more mutations relative to a reference AOS2 amino acid sequence, wherein the reference AOS2 amino acid sequence has a T at amino acid position 495. In another embodiment, the mutated AOS2 protein can be composed of any combination of amino acid mutations at any position in the protein relative to a reference sequence (e.g., EQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, and / or 49). In some embodiments, the mutated AOS2 protein has one or more mutations relative to a corresponding AOS2 protein that confers less than acceptable pathogen resistance and / or tolerance (e.g., resistance to Phytophthora infestans). In some embodiments, the AOS2 protein is modified by one or more mutations.In some embodiments, the AOS2 protein is modified by at least one mutation. In certain embodiments, the AOS2 protein is modified by at least two mutations. In certain embodiments, the AOS2 protein is modified by at least three mutations. In certain embodiments, the AOS2 protein is modified by at least four mutations. In certain embodiments, the AOS2 protein is modified by at least five mutations. In certain embodiments, the AOS2 protein is modified by at least six mutations. In certain embodiments, the AOS2 protein is modified by at least seven mutations. In certain embodiments, the AOS2 protein is modified by at least eight mutations. In certain embodiments, the AOS2 protein is modified by at least nine mutations. In certain embodiments, the AOS2 protein is modified by at least ten mutations. In certain embodiments, the AOS2 protein is modified by at least eleven mutations. In certain embodiments, the AOS2 protein is modified by at least twelve mutations. In some embodiments, the mutated AOS2 protein is one or more potato AOS2 proteins. In some embodiments, the term mutated AOS2 protein refers to an AOS2 protein that confers enhanced resistance and / or tolerance to one or more pathogens compared to a reference protein.

[0019] As used herein, the term "less than an acceptable level of pathogen resistance and / or tolerance" means that the susceptibility of a plant or crop to a pathogen impairs or destroys the commercial profitability of the plant or crop. In certain embodiments, less than an acceptable level of pathogen resistance and / or tolerance reduces the profitability of the plant or crop by at least 10%, or at least 25%, or at least 50%, or at least 75%, or at least 100%, compared to similar plants or crops that are resistant and / or tolerant to the pathogen. In contrast, the profitability of crops or plants with "acceptable levels of resistance and / or tolerance" is not substantially impaired or destroyed by pathogen exposure. In certain embodiments, the profitability of a plant or crop is reduced by less than 20%, or less than 15%, or less than 10% when exposed to a pathogen. The profitability of a plant or crop with "greater than an acceptable level of resistance and / or tolerance" to a pathogen is reduced by less than 10%, or less than 5%, or less than 2% when exposed to a pathogen.

[0020] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, a mutation refers to a single nucleotide change in an AOS2 gene or a single amino acid change in a polypeptide relative to at least the amino acid sequence of an AOS2 gene / protein that confers pathogen resistance and / or tolerance. In some embodiments, a mutation refers to a single nucleotide change in an AOS2 gene or a single amino acid change in a polypeptide relative to at least the amino acid sequence of an AOS2 protein that does not confer an acceptable level of pathogen resistance and / or tolerance. In certain embodiments, a mutation may comprise a substitution, deletion, inversion, or insertion at one or more positions in a gene and / or protein. In some embodiments, the substitutions, deletions, insertions, or inversions can include changes at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 amino acid positions.

[0021] In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the one or more mutations in the mutant AOS2 protein include 6, 12, 30, 37, 46, 48, 51, 76, 113, 145, 187, 197, 200, 227, 231, 256, 264, 270, 282, 289, 292, 309, 320, 328, 337, 338, 357, 381, 382, ​​389, 392, 41, 43, 45, 47 and / or 49. or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more, twenty-six or more, twenty-seven or more, twenty-eight or more, twenty-nine or more, thirty or more, thirty or more, thirty-two or more, thirty-three or more, thirty-four or more, thirty-five or more, thirty-six or more, or thirty-seven or more mutations at positions 394, 407, 423, 430, 439, 467, 480, 494, and 495.

[0022] In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the one or more mutations in the mutant AOS2 protein include S6, P12, R12, , V30, T37, F46, L46, I48, T48, I51, D76, D113, G113, Y145, F187, D197, E197, T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, N289, S289, A292, I309, L309, L320, M320, L328, V328, D337, E337, L338, V338, I357, M357, L381, P381, K394, C407, G407, I423, F430, Δ439 (where Δ indicates a deletion), G467, One or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more mutations at positions S467, T480, D494, G494 and K495.

[0023] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 gene comprises a G at a position corresponding to position 231 of SEQ ID NO: 1 and a V at a position corresponding to position 328 of SEQ ID NO: 1. In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the one or more mutations in the mutated AOS2 protein include one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, seven or more mutations, eight or more mutations, nine or more mutations, or ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more mutations selected from the group consisting of: F6S, R12P, P12R, A30V, I37T, L46F, F46L, V48T, V48I, T48I, I48T, M51I, D76N, N76D, G113D, D113G, F145Y, L187F, D197E, E197D, K200T, A227T, I231T, I231G, G231T, T231G, F256V, V256F, A264T, L270F, F282S, S282F, V289N, V289S, S289N, N289S, V292A, L309I, I309L, M320L, L320M, M328L, M328V, L328V, V328L, E337D, D337E, V338L, L338V, I357M, M357I, P381L, L381P, T394K, G407C, C407G, F423I, L430F, E439Δ, G467S, S467G, V480T, G494D, D494G and T495K. In some embodiments, the one or more mutations in the mutant AOS2 protein include one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, seven or more mutations, eight or more mutations, nine or more mutations, or ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more mutations selected from the group consisting of mutations corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49, phenylalanine at position 6 to serine, arginine at the position corresponding to position 12 to proline, proline at the position corresponding to position 12 to arginine, alanine at the position corresponding to position 30 to valine, isoleucine at the position corresponding to position 37 to threonine, phenylalanine at the position corresponding to position 46 to leucine, leucine at the position corresponding to position 46 to phenylalanine, valine at the position corresponding to position 48 to threonine, valine at the position corresponding to position 48 to isoleucine, isoleucine at the position corresponding to position 48 to threonine, threonine at the position corresponding to position 48 to isoleucine, methionine at the position corresponding to position 51 to isoleucine, asparagine at the position corresponding to position 76 to aspartic acid, , aspartic acid at the position corresponding to position 113 to asparagine, aspartic acid at the position corresponding to position 113 to glycine, glycine at the position corresponding to position 113 to aspartic acid, phenylalanine at the position corresponding to position 145 to tyrosine, leucine at the position corresponding to position 187 to phenylalanine, aspartic acid at the position corresponding to position 197 to glutamic acid, glutamic acid at the position corresponding to position 197 to aspartic acid, lysine at the position corresponding to position 200 to threonine, alanine at the position corresponding to position 227 to threonine, isoleucine at the position corresponding to position 231 to threonine, isoleucine at the position corresponding to position 231 to glycine, and 31 to threonine, threonine to glycine at the position corresponding to position 231, valine to phenylalanine at the position corresponding to position 256, phenylalanine to valine at the position corresponding to position 256, alanine to threonine at the position corresponding to position 264, leucine to phenylalanine at the position corresponding to position 270, serine to phenylalanine at the position corresponding to position 282, phenylalanine to serine at the position corresponding to position 282, valine to asparagine at the position corresponding to position 289, valine to serine at the position corresponding to position 289, serine to asparagine at the position corresponding to position 289, and leucine to phenylalanine at the position corresponding to position 270., asparagine at the position corresponding to position 292 to serine, valine at the position corresponding to position 292 to alanine, isoleucine at the position corresponding to position 309 to leucine, leucine at the position corresponding to position 309 to isoleucine, leucine at the position corresponding to position 320 to methionine, methionine at the position corresponding to position 320 to leucine, methionine at the position corresponding to position 328 to leucine, methionine at the position corresponding to position 328 to valine, valine at the position corresponding to position 328 to leucine, leucine at the position corresponding to position 328 to valine, aspartic acid at the position corresponding to position 337 to glutamic acid, and glutamic acid at the position corresponding to position 337. to aspartic acid, leucine at the position corresponding to position 338 to valine, valine at the position corresponding to position 338 to leucine, methionine at the position corresponding to position 357 to isoleucine, isoleucine at the position corresponding to position 357 to methionine, leucine at the position corresponding to position 381 to proline, proline at the position corresponding to position 381 to leucine, threonine at the position corresponding to position 394 to lysine, cysteine ​​at the position corresponding to position 407 to glycine, glycine to cysteine ​​at the position corresponding to position 407, phenylalanine at the position corresponding to position 423 to isoleucine, and threonine at the position corresponding to position 430 to lysine. , 47, or 49. The amino acid sequence of the amino acid sequence of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, or 49 is deleted from the sequence of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, or 49. The amino acid sequence of the amino acid sequence of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, or 49 is deleted from the sequence of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, or 49.

[0024] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises SEQ ID NO: 1. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises SEQ ID NO:3.

[0025] In another aspect, a method for producing a plant cell is provided. In some embodiments, the plant cell has a mutated AOS2 gene. In certain embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. In certain embodiments, the plant cell can be part of a pathogen-resistant plant. The method can include introducing a gene repair oligonucleobase (GRON) into the plant cell; for example, using a GRON with a targeted mutation to produce a nucleotide change at a homologous position in the AOS2 gene. In certain embodiments, the plant cell produced by the method can include an AOS2 gene capable of expressing a mutated AOS2 protein. The method can further include identifying a plant cell or a plant containing a plant cell that comprises (1) a mutated AOS2 gene, and / or (2) normal or altered growth, and / or AOS2 catalytic activity, enhanced AOS2 enzyme stability, signaling ability, and / or (3) greater pathogen resistance and / or tolerance compared to a corresponding wild-type plant cell. Pathogen-resistant plants having plant cells such as those described herein can be identified in the presence of pathogens. In some embodiments, the plant cell is transgenic. In some embodiments, the plant cells are non-transgenic. Plants comprising plant cells such as those described herein can be non-transgenic pathogen-resistant / pathogen-tolerant plants; for example, the plants and / or plant cells can have a mutated AOS2 gene that confers resistance and / or tolerance to at least one pathogen. In some embodiments, plants comprising plant cells such as those described herein can be produced asexually; for example, from one or more plant cells or from plant tissue composed of one or more plant cells; for example, from tubers or small pieces of potato tubers containing at least one or two eyes (dormant buds), commonly known as seed potatoes. In certain embodiments, plants comprising plant cells such as those described herein can be produced sexually, thereby producing true genetic seeds.

[0026] On the other hand, a method for producing pathogen-resistant and / or tolerant plants is provided. The method may include introducing a gene repair oligonucleobase (GRON) into a plant cell; for example, using a GRON with a targeted mutation to produce a nucleotide change at a homologous position in the AOS2 gene. The method may produce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene may express a mutated AOS2 protein. The method may further include identifying plants having normal or altered growth, AOS2 protein catalytic activity, AOS2 enzyme stability, and / or signaling capacity compared to corresponding wild-type plant cells. The method may further include regenerating pathogen-resistant plants from plant cells with mutated AOS2 genes. The plants may be identified in the presence of pathogens. In some embodiments, the plants are transgenic. In some embodiments, the plants are non-transgenic. The plants may, in some embodiments, be non-transgenic pathogen-resistant plants; for example, the plants may include a mutated AOS2 gene that produces improved resistance and / or tolerance to at least one pathogen. In some embodiments, the plants may include a mutated AOS2 gene that produces plants with an altered maturity grade. In certain embodiments, the plant may comprise a mutated AOS2 gene that results in a plant having an altered late maturity rating.

[0027] On the other hand, a method for producing a plant with an early, medium, medium-early or late maturity rating is provided. The method may include introducing a gene repair oligonucleobase (GRON) into a plant cell; for example, using a GRON with a targeted mutation to produce a nucleotide change at a homologous position in the AOS2 gene. The method may produce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene may express a mutated AOS2 protein. The method may further include identifying a plant cell with normal growth and / or catalytic activity compared to a corresponding wild-type plant cell. The method may further include regenerating a pathogen-resistant plant from a plant cell with a mutated AOS2 gene. In some embodiments, the plant is non-transgenic. The plant may be a non-transgenic plant with a medium-early maturity rating. The plant may be a non-transgenic pathogen-resistant plant in some embodiments; for example, the plant may contain a mutated AOS2 gene that produces resistance and / or tolerance to at least one pathogen. In some embodiments, the plant is transgenic. The plant may be a non-transgenic plant with a medium-early maturity rating. The plant can, in some embodiments, be a transgenic pathogen-resistant plant; for example, the plant can comprise a mutated AOS2 gene that confers resistance and / or tolerance to at least one pathogen.

[0028] On the other hand, a method for increasing jasmonic acid levels in plants is provided. The method may include introducing a gene repair oligonucleobase (GRON) into a plant cell; for example, using a GRON with a targeted mutation to produce a nucleotide change at a homologous position in the AOS2 gene. The method may produce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene may express a mutated AOS2 protein. The method may further include identifying plants having normal or altered growth, AOS2 protein catalytic activity, AOS2 enzyme stability, and / or signaling capacity compared to corresponding wild-type plant cells. The method may further include regenerating plants with elevated jasmonic acid levels from plant cells with the mutated AOS2 gene. The plants may be identified in the presence of pathogens. In some embodiments, the plants are non-transgenic. The plants may, in some embodiments, be non-transgenic pathogen-resistant plants; for example, the plants may contain a mutated AOS2 gene that confers resistance and / or tolerance to at least one pathogen. In some embodiments, the plants may contain a mutated AOS2 gene that produces plants with elevated jasmonic acid levels. In some embodiments, the plants are transgenic. In some embodiments, the plant can be a transgenic pathogen-resistant plant; for example, the plant can comprise a mutated AOS2 gene that confers resistance and / or tolerance to at least one pathogen. In some embodiments, the plant can comprise a mutated AOS2 gene that results in a plant with elevated jasmonic acid levels.

[0029] On the other hand, a method for increasing pathogen resistance and / or tolerance in plants is provided. The method may include introducing a gene repair oligonucleobase (GRON) into a plant cell; for example, using a GRON with a targeted mutation to produce a nucleotide change at a homologous position in the AOS2 gene. The method may produce a plant cell with a mutated AOS2 gene. The mutated AOS2 gene may express a mutated AOS2 protein. The method may further include identifying a plant having normal or altered growth and / or AOS2 protein catalytic activity and / or AOS2 protein stability compared to a corresponding wild-type plant cell. The method may further include regenerating a pathogen-resistant plant from a plant cell with a mutated AOS2 gene. The plant may be identified in the presence of a pathogen. In some embodiments, the plant is non-transgenic. The plant may be a non-transgenic pathogen-resistant plant in some embodiments; for example, the plant may include a mutated AOS2 gene that produces resistance and / or tolerance to at least one pathogen. In some embodiments, the plant may include a mutated AOS2 gene that produces a plant with a medium-early maturity rating. In certain embodiments, the plant may comprise a mutated AOS2 gene that results in a plant with a late maturity rating. In some embodiments, the plant is transgenic. The plant may, in some embodiments, be a transgenic pathogen-resistant plant; for example, the plant may comprise a mutated AOS2 gene that confers resistance and / or tolerance to at least one pathogen. In some embodiments, the plant may comprise a mutated AOS2 gene that results in a plant with a medium-early maturity rating. In certain embodiments, the plant may comprise a mutated AOS2 gene that results in a plant with a late maturity rating.

[0030] In another aspect, plants or plant cells comprising a mutated AOS2 gene are provided. In certain embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. In certain embodiments, the plant or plant cell can be of the Desiree potato variety. In certain embodiments, the plant or plant cell can be of the Bintje potato variety. In certain embodiments, the plant or plant cell can be of the Fontana potato variety. In certain embodiments, the plant or plant cell can be of the Innovator potato variety. In certain embodiments, plants having plant cells comprising a mutated AOS2 gene can be resistant and / or tolerant to pathogens. In certain embodiments, the plant or plant cell is non-transgenic. In certain embodiments, the plant or plant cell is transgenic.

[0031] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the compositions and methods may relate to plants or plant cells having multiple AOS2 genes in two or more sets of chromosomes, each gene having two alleles. For example, a tetraploid plant may contain one, two, three, or four mutant AOS2 alleles. In some embodiments, the multiple AOS2 genes may contain the same mutation or different mutations. In some embodiments, the multiple AOS2 genes may include any combination or arrangement of mutations (e.g., AOS2 mutations as disclosed herein).

[0032] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the plant or plant cell may comprise a mutation in the AOS2 gene / allele / locus on one or more chromosomes. The plant or plant cell may include plants having various multiples of chromosomes, for example, at least one set of chromosomes, at least two sets of chromosomes, at least three sets of chromosomes, at least four sets of chromosomes, at least five sets of chromosomes, at least six sets of chromosomes, at least seven sets of chromosomes, at least eight sets of chromosomes, at least nine sets of chromosomes, at least ten sets of chromosomes, at least eleven sets of chromosomes, and at least twelve sets of chromosomes. In some embodiments, the plant or plant cell includes a plant having four sets of chromosomes.

[0033] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene comprises at least one mutation that confers pathogen resistance and / or tolerance or at least one mutation that confers a late maturity rating. In some embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is the same mutation as the at least one mutation that confers a late maturity rating. In certain embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is different from the at least one mutation that confers a late maturity rating.

[0034] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene comprises at least one mutation that confers pathogen resistance and / or tolerance and at least one mutation that confers a mid-to-early maturity classification. In some embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is the same mutation as the at least one mutation that confers a mid-to-early maturity classification. In certain embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is different from the at least one mutation that confers a mid-to-early maturity classification.

[0035] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene comprises at least one mutation that confers pathogen resistance and / or tolerance and at least one mutation that confers an early maturity rating. In some embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is the same mutation as the at least one mutation that confers an early maturity rating. In certain embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is different from the at least one mutation that confers an early maturity rating.

[0036] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene comprises at least one mutation that confers pathogen resistance and / or tolerance and at least one mutation that confers a medium-ripe rating. In some embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is the same mutation as the at least one mutation that confers a medium-ripe rating. In certain embodiments, the at least one mutation that confers pathogen resistance and / or tolerance is different from the at least one mutation that confers a medium-ripe rating.

[0037] In another aspect, seeds comprising a mutated AOS2 gene are provided. In some embodiments, the seeds have a mutated AOS2 gene. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. In some embodiments, the mutated AOS2 protein is resistant and / or tolerant to pathogens. In some embodiments, the seeds are resistant and / or tolerant to pathogens. In some embodiments, the seeds may comprise a mutated AOS2 gene that produces plants that are resistant and / or tolerant to pathogens. In some embodiments, the seeds are non-transgenic. In some embodiments, the seeds are transgenic. In some embodiments, the seeds may comprise a mutated AOS2 gene that produces plants with a medium-early maturity rating. In some embodiments, the seeds may comprise a mutated AOS2 gene that produces plants with a late maturity rating.

[0038] On the other hand, a vegetative plant material is provided that can produce new plants, including but not limited to tubers or small pieces thereof containing at least a single eye, in vitro grown shoots, rooted shoots, or protoplast-derived callus having at least one mutated AOS2 allele. In some embodiments, such vegetative propagation materials have a mutated AOS2 gene. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein. In some embodiments, the mutated AOS2 protein is resistant and / or resistant to pathogens. In some embodiments, the vegetative material is resistant and / or resistant to pathogens. In some embodiments, the vegetative material may comprise a mutated AOS2 gene that produces plants that are resistant and / or resistant to pathogens. In some embodiments, the vegetative material is non-transgenic. In some embodiments, the vegetative material is transgenic. In some embodiments, the vegetative material may comprise a mutated AOS2 gene that produces plants with a medium-early maturity rating. In some embodiments, the vegetative material may comprise a mutated AOS2 gene that produces plants with a late maturity rating.

[0039] In another aspect, a method for increasing pathogen resistance and / or tolerance in a plant is provided by: (a) crossing a first plant with a second plant, wherein the first plant comprises a mutated AOS2 gene, wherein the gene encodes a mutated AOS2 protein; (b) screening the population resulting from the cross for increased pathogen resistance and / or tolerance; (c) selecting members of the cross with increased pathogen resistance and / or tolerance; and (d) producing seeds from the cross. In some embodiments, hybrid seeds are produced by any of the above methods. In some embodiments, plants are grown from seeds produced by any of the above methods. In some embodiments, the plants and / or seeds are non-transgenic. In some embodiments, the plants and / or seeds are transgenic. In some embodiments, the first and second plants are potato plants. In some embodiments, the plants and / or seeds have an early, early-medium, medium, or late maturity classification.

[0040] In another aspect, isolated nucleic acids of a mutant AOS2 gene are provided. In some embodiments, the isolated nucleic acid encodes a mutant AOS2 protein. In certain embodiments, the isolated nucleic acid encodes a mutant AOS2 protein that is resistant and / or tolerant to pathogens. In some embodiments, the isolated nucleic acid encodes a mutant AOS2 protein that produces plants with an early, medium, early-to-medium, or late maturity rating.

[0041] In another aspect, an expression vector comprising an isolated nucleic acid of a mutated AOS2 gene is provided. In some embodiments, the expression vector comprises an isolated nucleic acid encoding an AOS2 protein.

[0042] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, the methods and compositions disclosed herein include one or more mutated AOS2 genes encoding one or more AOS2 proteins. In some embodiments, the methods and compositions include a mutated chloroplast-targeted AOS2 gene. In some embodiments, the methods and compositions include a mutated AOS2 gene. In some embodiments, the methods and compositions include a mutated potato AOS2 gene; for example, StAOS2. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2-1. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2-6. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2-12. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2-7. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2-8. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB1. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB2. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB3. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB4. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB5. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB6. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB7. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB8. In some embodiments, the methods and compositions include a mutated AOS2 gene allele StAOS2 CB9. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2CB10. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2CB11. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2CB12. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2CB13. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2CB14.In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB15. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB16. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB17. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB18. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB19. In some embodiments, the methods and compositions include a mutant AOS2 gene allele StAOS2 CB20.

[0043] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, a plant or plant cell comprising a mutated AOS2 gene has at least one gene / allele with an A at position 691. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least two genes / alleles with an A at position 691. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least three genes / alleles with an A at position 691. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least four genes / alleles with an A at position 691. In some embodiments, the plant or plant cell is a potato. In some embodiments, the plant or plant cell is a Desiree potato. In some embodiments, the plant or plant cell is a Bintje potato. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

[0044] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, a plant or plant cell comprising a mutated AOS2 gene has at least one gene / allele with a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least two genes / alleles with a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least three genes / alleles with a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least four genes / alleles with a C at position 692. In some embodiments, the plant or plant cell is a potato. In some embodiments, the plant or plant cell is a Desiree potato. In some embodiments, the plant or plant cell is a Bintje potato. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

[0045] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, a plant or plant cell comprising a mutated AOS2 gene has at least one gene / allele with an A at position 691 and a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least two genes / alleles with an A at position 691 and a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least three genes / alleles with an A at position 691 and a C at position 692. In some embodiments, a plant or plant cell comprising a mutated AOS2 gene has at least four genes / alleles with an A at position 691 and a C at position 692. In some embodiments, the plant or plant cell is a potato. In some embodiments, the plant or plant cell is a Desiree potato. In some embodiments, the plant or plant cell is a Bintje potato. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0046] In combination with any of the various aspects, embodiments, compositions, and methods described herein, the plant or plant cell comprises a mutant AOS2 gene with an A at position 691. In some embodiments, the plant or plant cell is polyploid. In some embodiments, at least one mutant AOS2 gene / allele of the polyploid plant has an A at position 691. In some embodiments, at least two mutant AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least three mutant AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least four mutant AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least five mutant AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least six mutant AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least seven of the mutated AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least eight of the mutated AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least nine of the mutated AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, at least ten of the mutated AOS2 genes / alleles of the polyploid plant have an A at position 691. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0047] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, the potato or potato cell comprises a mutated AOS2 gene having an A at position 691. In some embodiments, at least one mutated AOS2 gene / allele of the potato or potato cell has an A at position 691. In some embodiments, at least two mutated AOS2 genes / alleles of the potato or potato cell have an A at position 691. In some embodiments, at least three mutated AOS2 genes / alleles of the potato or potato cell have an A at position 691. In some embodiments, at least four mutated AOS2 genes / alleles of the potato or potato cell have an A at position 691. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0048] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, the Desiree potato or Desiree potato cell comprises a mutated AOS2 gene having an A at position 691. In some embodiments, at least one mutated AOS2 gene / allele of the Desiree potato or Desiree potato cell has an A at position 691. In some embodiments, at least two mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have an A at position 691. In some embodiments, at least three mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have an A at position 691. In some embodiments, at least four mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have an A at position 691. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0049] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, the Bintje potato or Bintje potato cell comprises a mutated AOS2 gene having an A at position 691. In some embodiments, at least one mutated AOS2 gene / allele of the Bintje potato or Bintje potato cell has an A at position 691. In some embodiments, at least two mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have an A at position 691. In some embodiments, at least three mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have an A at position 691. In some embodiments, at least four mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have an A at position 691. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0050] In combination with any of the various aspects, embodiments, compositions, and methods described herein, the plant or plant cell comprises a mutant AOS2 gene having an A at position 692. In some embodiments, the plant or plant cell is polyploid. In some embodiments, at least one mutant AOS2 gene / allele of the polyploid plant has a C at position 692. In some embodiments, at least two mutant AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least three mutant AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least four mutant AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least five mutant AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least six mutant AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least seven of the mutated AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least eight of the mutated AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least nine of the mutated AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, at least ten of the mutated AOS2 genes / alleles of the polyploid plant have a C at position 692. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0051] In conjunction with any of the various aspects, embodiments, compositions, and methods described herein, the potato or potato cell comprises a mutated AOS2 gene having a C at position 692. In some embodiments, at least one mutated AOS2 gene / allele of the potato or potato cell has a C at position 692. In some embodiments, at least two mutated AOS2 genes / alleles of the potato or potato cell have a C at position 692. In some embodiments, at least three mutated AOS2 genes / alleles of the potato or potato cell have a C at position 692. In some embodiments, at least four mutated AOS2 genes / alleles of the potato or potato cell have a C at position 692. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0052] In conjunction with any of the various aspects, embodiments, compositions, and methods disclosed herein, the Desiree potato or Desiree potato cell comprises a mutated AOS2 gene having a C at position 692. In some embodiments, at least one mutated AOS2 gene / allele of the Desiree potato or Desiree potato cell has a C at position 692. In some embodiments, at least two mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have a C at position 692. In some embodiments, at least three mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have a C at position 692. In some embodiments, at least four mutated AOS2 genes / alleles of the Desiree potato or Desiree potato cell have a C at position 692. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0053] In conjunction with any of the various aspects, embodiments, compositions, and methods disclosed herein, the Bintje potato or Bintje potato cell comprises a mutated AOS2 gene having a C at position 692. In some embodiments, at least one mutated AOS2 gene / allele of the Bintje potato or Bintje potato cell has a C at position 692. In some embodiments, at least two mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have a C at position 692. In some embodiments, at least three mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have a C at position 692. In some embodiments, at least four mutated AOS2 genes / alleles of the Bintje potato or Bintje potato cell have a C at position 692. In some embodiments, the one or more genes / alleles are not transgenic. In some embodiments, the AOS2 gene is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0054] In combination with any of the various aspects, embodiments, compositions, and methods disclosed herein, the plant or plant cell is tetraploid. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAAA / CCCC at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAAG / CCCG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAGG / CCCG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAAG / CCGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAGG / CCGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAAG / CCCG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAGG / CCCG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CGGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2.In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAGG / CGGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AGGG / CGGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AAGG / GGGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that produces a genotype of AGGG / GGGG at the nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid plant or plant cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of GGGG / GGGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the plant or plant cell is non-transgenic. In some embodiments, the plant or plant cell is transgenic.

[0055] In conjunction with any of the various aspects, embodiments, compositions, and methods disclosed herein, the plant or plant cell is a potato plant or plant cell. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in an AAAA / CCCC genotype at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in an AAAG / CCCG genotype at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in an AAGG / CCCG genotype at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the tetraploid potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CCCG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / CCCG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / CCGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAAG / CGGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO: 2.In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / CGGG at a nucleotide position corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AGGG / CGGG at a nucleotide position corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AAGG / GGGG at a nucleotide position corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of AGGG / GGGG at a nucleotide position corresponding to 691 / 692 of SEQ ID NO: 2. In some embodiments, the potato plant or potato cell comprises a mutation in one or more AOS2 genes / alleles that results in a genotype of GGGG / GGGG at nucleotide positions corresponding to 691 / 692 of SEQ ID NO:2. In certain embodiments, the potato is a Desiree potato. In certain embodiments, the potato is a Bintje potato. In some embodiments, the plant or plant cell is non-transgenic. In some embodiments, the plant or plant cell is transgenic.

[0056] In conjunction with any of the various aspects, embodiments, compositions and methods disclosed herein, the plant or plant cell is a Solanum tuberosum potato plant or plant cell.

[0057] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, a plant having a plant cell comprising a mutated AOS2 gene may have an early, medium, medium-early, or late maturity classification. In certain embodiments, the plant or plant cell is non-transgenic. In certain embodiments, the plant or plant cell is transgenic. In certain embodiments, the plant or plant cell comprises a mutation in the coding sequence of the AOS2 gene. In certain embodiments, the plant or plant cell comprises a mutation in the non-coding sequence of the AOS2 gene. In certain embodiments, the plant or plant cell comprises a mutation upstream of the coding sequence of the AOS2 gene.

[0058] As used herein, the term "gene" refers to a DNA sequence that contains the control and coding sequences necessary to produce RNA, which may have a non-coding function (e.g., ribosomal RNA or transfer RNA) or may contain a polypeptide or polypeptide precursor. The RNA or polypeptide may be encoded by the full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or function is retained. The term "gene" also refers to and encompasses the corresponding alleles of a plant cultivar or plant line.

[0059] An allele is one of several alternative forms of a gene or nucleotide sequence that is in a specific variation at a given position in a nucleic acid sample. An allele can be represented by one or more base changes (e.g., SNPs) at a given locus. For example, at each autosomal locus, a diploid individual has two alleles, one maternally inherited and the other paternally inherited.

[0060] As used herein, the term "pathogen" refers to a disease-causing agent that causes disease in its host. In certain embodiments, the pathogen is Phytophthora infestans.

[0061] As used herein, the term "coding sequence" refers to a sequence of a nucleic acid, or its complement, or a portion thereof, that can be transcribed and / or translated to produce mRNA for a polypeptide or fragment thereof and / or to produce a polypeptide or fragment thereof. A coding sequence comprises exons within genomic DNA or immature primary RNA transcripts that are linked together by the cell's biochemical machinery to provide mature mRNA. The antisense strand is the complement of such nucleic acid, and the coding sequence can be deduced therefrom.

[0062] As used herein, the term "non-coding sequence" refers to a sequence of a nucleic acid or its complement or portion thereof that is not transcribed into an amino acid in vivo, or in which tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include intron sequences in genomic DNA or immature primary RNA transcripts, as well as gene-associated sequences such as promoters, enhancers, silencers, and the like.

[0063] Nucleobase is a kind of base, and it is purine, pyrimidine or its derivative or analogue in certain preferred embodiments.Nucleoside is the nucleobase containing pentofuranosyl moiety, for example optionally substituted nucleoside or 2 '-deoxynucleoside.Nucleoside can be connected by one of several linking parts, and described linking part can contain phosphorus or can be phosphorus-free.The nucleoside connected by unsubstituted phosphodiester bond is referred to as nucleotide.Term " nucleobase " as used herein comprises the subunit of peptide nucleobase, peptide nucleic acid and morpholino nucleobase and nucleoside and nucleotide.

[0064] Oligonucleobase is a polymer comprising a nucleobase; preferably at least a portion thereof can hybridize with a DNA having a complementary sequence by Watson-Crick base pairing. The oligonucleobase chain can have a single 5' end and a 3' end, which are the final nucleobases of the polymer. Specific oligonucleobase chains can comprise all types of nucleobases. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains, which can be complementary and hybridize by Watson-Crick base pairing. Ribotype nucleobases include pentofuranosyl groups containing nucleobases, wherein the 2' carbon is a methylene group substituted by a hydroxyl group, an alkoxy group or a halogen. Deoxyribotype nucleobases are nucleobases other than ribotype nucleobases and include all nucleobases that do not contain pentofuranosyl moieties.

[0065] In certain embodiments, an oligonucleobase strand can include an oligonucleobase strand and a segment or region of an oligonucleobase strand. An oligonucleobase strand can have a 3' end and a 5' end, and when an oligonucleobase strand is coextensive with a chain, the 3' and 5' ends of the strand are also the 3' and 5' ends of the chain.

[0066] As used herein, the term "gene repair oligonucleobase" or "GRON" refers to oligonucleobases, which include mixed duplex oligonucleotides, molecules without nucleotides, single-stranded oligodeoxynucleotides and other gene repair molecules.

[0067] As used herein, the term "isolated" when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) refers to a nucleic acid that is separated from the substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany such nucleic acid. For example, any nucleic acid produced by synthesis (e.g., by continuous base condensation) is considered isolated. Similarly, nucleic acids that are recombinantly expressed, cloned, produced by primer extension reactions (e.g., PCR), or otherwise excised from the genome are also considered isolated.

[0068] As used herein, the term "amino acid sequence" refers to a polypeptide or protein sequence. The convention "AAwt###AAmut" is used to indicate a mutation that results in the wild-type amino acid AAwt at position ### in the polypeptide being replaced by the mutant AAmut.

[0069] As used herein, the term "complement" refers to the complementary sequence of a nucleic acid according to the standard Watson / Crick pairing rules. The complement sequence can also be an RNA sequence that is complementary to a DNA sequence or its complement sequence, and can also be a cDNA.

[0070] As used herein, the term "substantially complementary" refers to two sequences that hybridize under near stringent hybridization conditions. It will be understood by those skilled in the art that substantially complementary sequences need not hybridize along their entire length.

[0071] As used herein, the term "codon" refers to a sequence of three adjacent nucleotides (RNA or DNA) that constitute the genetic code, which determines the addition of a specific amino acid to a polypeptide chain during protein synthesis or signals to terminate protein synthesis. The term "codon" is also used to refer to the corresponding (and complementary) sequence of three nucleotides in the messenger RNA transcribed from the original DNA.

[0072] As used herein, the term "wild-type" refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a naturally occurring source. A wild-type gene is the gene most commonly observed in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. "Wild-type" can also refer to the sequence at one or more specific nucleotide positions, or the sequence at one or more specific codon positions, or the sequence at one or more specific amino acid positions.

[0073] As used herein, the term "mutant" or "modified" refers to a nucleic acid or protein that exhibits an alteration in sequence and or functional properties (i.e., an altered property) when compared to the wild-type gene or gene product. "Mutant" or "modified" also refers to a sequence at one or more specific nucleotide positions, or a sequence at one or more specific codon positions, or a sequence at one or more specific amino acid positions that exhibits an alteration in sequence and or functional properties (i.e., an altered property) when compared to the wild-type gene or gene product.

[0074] As used herein, the term "homology" refers to sequence similarity between proteins and DNA. The term "homology" or "homologous" refers to the degree of identity. There can be partial homology or complete homology. A partially homologous sequence is one that has less than 100% sequence identity when compared to another sequence.

[0075] As used herein, the term "heterozygous" refers to having different alleles at one or more genetic loci in a homologous chromosome segment. As used herein, "heterozygous" can also refer to a sample, cell, cell mass or organism in which different alleles at one or more genetic loci are detected. The sample of heterozygosity can also be measured via methods known in the art such as nucleic acid sequencing. For example, if the sequencing electrophoretogram shows two peaks at a single locus and the two peak sizes are roughly the same, the sample can be characterized as heterozygous. Alternatively, if a peak is smaller than another peak but is at least about 25% of the larger peak size, the sample can be characterized as heterozygous. In some embodiments, less peak is at least about 15% of the larger peak. In certain embodiments, less peak is at least about 10% of the larger peak. In certain embodiments, less peak is at least about 5% of the larger peak. In certain embodiments, the minimum amount of less peak is detected.

[0076] As used herein, "homozygous" refers to having identical alleles at one or more genetic loci in homologous chromosome segments. "Homozygous" can also refer to a sample, cell, cell population, or organism in which identical alleles at one or more genetic loci are detected. Homozygous samples can be measured via methods known in the art such as nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a specific locus, the sample can be referred to as "homozygous" with respect to the locus.

[0077] The term "hemizygous" refers to a gene or gene segment that occurs only once in the genotype of a cell or organism due to the absence of a second allele. As used herein, "hemizygous" may also refer to a sample, cell, cell population, or organism in which an allele at one or more genetic loci can be detected only once in the genotype.

[0078] As used herein, the term "joint state" refers to a sample, cell population or organism that is heterozygous, homozygous or hemizygous when measured by test methods known in the art and described herein. The term "joint state of a nucleic acid" means determining whether the source of the nucleic acid is heterozygous, homozygous or hemizygous. "Joint state" can refer to the difference in a single nucleotide in a sequence. In some methods, the joining state of a sample with respect to a single mutation can be divided into homozygous wild type, heterozygous (i.e., one wild type allele and one mutant allele), homozygous mutant or hemizygous (i.e., a single copy of a wild type or mutant allele).

[0079] As used herein, the term "about" means plus or minus 10% of a quantitative term. For example, "about 3%" would encompass 2.7-3.3% and "about 10%" would encompass 9-11%. Furthermore, when "about" is used herein in conjunction with a quantitative term, it is understood that in addition to values ​​of plus or minus 10%, the exact value of the quantitative term is also contemplated and described. For example, the term "about 3%" explicitly encompasses, describes, and includes the exact value of 3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The amino acid sequence of potato AOS2 protein, allele 1 (SEQ ID NO: 1) is shown, and the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, AOS2-1 allele (StAOS2-1) is shown (GB accession number ABD15173).

[0081] Figure 2 The nucleic acid sequence of the potato AOS2 gene, allele 1 (SEQ ID NO: 2) is shown, and the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, AOS2-1 allele (StAOS2-1) is shown (GB Accession No. DQ369736).

[0082] Figure 3 The amino acid sequence of potato AOS2 protein, allele 6 (SEQ ID NO: 3) is shown, and the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, AOS2-6 allele (StAOS2-6) is shown (GB accession number ABD15174).

[0083] Figure 4 The nucleic acid sequence of the potato AOS2 gene, allele 6 (SEQ ID NO: 4) is shown, and the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, AOS2-6 allele (StAOS2-6) is shown (GB Accession No. DQ369737).

[0084] Figure 5 The amino acid sequence of potato AOS2 protein, allele 7 (SEQ ID NO: 5) is shown, and the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, AOS2-7 allele (StAOS2-7) is shown (GB accession number ABD15175).

[0085] Figure 6The nucleic acid sequence of the potato AOS2 gene, allele 7 (SEQ ID NO: 6) is shown, and the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, AOS2-7 allele (StAOS2-7) is shown (GB Accession No. DQ369738).

[0086] Figure 7 The amino acid sequence of potato AOS2 protein, allele 8 (SEQ ID NO: 7) is shown, and the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, AOS2-8 allele (StAOS2-8) is shown (GB accession number ABD15176).

[0087] Figure 8 The nucleic acid sequence of the potato AOS2 gene, allele 8 (SEQ ID NO: 8) is shown, and the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, AOS2-8 allele (StAOS2-8) is shown (GB Accession No. DQ369739).

[0088] Figure 9 The amino acid sequence of potato AOS2 protein, allele 12 (SEQ ID NO: 9) is shown, and the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, AOS2-12 allele (StAOS2-12) is shown (GB accession number ABD15172).

[0089] Figure 10 The nucleic acid sequence of the potato AOS2 gene, allele 12 (SEQ ID NO: 10) is shown, and the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, AOS2-12 allele (StAOS2-12) is shown (GB Accession No. DQ369735).

[0090] Figure 11 The amino acid sequence of potato AOS2 protein, allele CB1 (SEQ ID NO: 11) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB1.

[0091] Figure 12 The nucleic acid sequence of the potato AOS2 gene, allele CB1 (SEQ ID NO: 12) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB1.

[0092] Figure 13The amino acid sequence of potato AOS2 protein, allele CB2 (SEQ ID NO: 13) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB2.

[0093] Figure 14 The nucleic acid sequence of the potato AOS2 gene, allele CB2 (SEQ ID NO: 14) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB2.

[0094] Figure 15 The amino acid sequence of potato AOS2 protein, allele CB3 (SEQ ID NO: 15) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB3.

[0095] Figure 16 The nucleic acid sequence of the potato AOS2 gene, allele CB3 (SEQ ID NO: 16) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB3.

[0096] Figure 17 The amino acid sequence of potato AOS2 protein, allele CB4 (SEQ ID NO: 17) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB4.

[0097] Figure 18 The nucleic acid sequence of the potato AOS2 gene, allele CB4 (SEQ ID NO: 18) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB4.

[0098] Figure 19 The amino acid sequence of potato AOS2 protein, allele CB5 (SEQ ID NO: 19) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB5.

[0099] Figure 20 The nucleic acid sequence of the potato AOS2 gene, allele CB5 (SEQ ID NO: 20) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB5.

[0100] Figure 21The amino acid sequence of potato AOS2 protein, allele CB6 (SEQ ID NO: 21) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB6.

[0101] Figure 22 The nucleic acid sequence of the potato AOS2 gene, allele CB6 (SEQ ID NO: 22) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB6.

[0102] Figure 23 The amino acid sequence of potato AOS2 protein, allele CB7 (SEQ ID NO: 23) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB7.

[0103] Figure 24 The nucleic acid sequence of the potato AOS2 gene, allele CB7 (SEQ ID NO: 24) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB7.

[0104] Figure 25 The amino acid sequence of potato AOS2 protein, allele CB8 (SEQ ID NO: 25) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB8.

[0105] Figure 26 The nucleic acid sequence of the potato AOS2 gene, allele CB8 (SEQ ID NO: 26) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB8.

[0106] Figure 27 The amino acid sequence of potato AOS2 protein, allele CB9 (SEQ ID NO: 27) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB9.

[0107] Figure 28 The nucleic acid sequence of the potato AOS2 gene, allele CB9 (SEQ ID NO: 28) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB9.

[0108] Figure 29The amino acid sequence of potato AOS2 protein, allele CB10 (SEQ ID NO: 29) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB10.

[0109] Figure 30 The nucleic acid sequence of the potato AOS2 gene, allele CB10 (SEQ ID NO: 30) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB10.

[0110] Figure 31 The amino acid sequence of potato AOS2 protein, allele CB11 (SEQ ID NO: 31) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB11.

[0111] Figure 32 The nucleic acid sequence of the potato AOS2 gene, allele CB11 (SEQ ID NO: 32) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB11.

[0112] Figure 33 The amino acid sequence of potato AOS2 protein, allele CB12 (SEQ ID NO: 33) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB12.

[0113] Figure 34 The nucleic acid sequence of the potato AOS2 gene, allele CB12 (SEQ ID NO: 34) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB12.

[0114] Figure 35 The amino acid sequence of potato AOS2 protein, allele CB13 (SEQ ID NO: 35) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB13.

[0115] Figure 36 The nucleic acid sequence of the potato AOS2 gene, allele CB13 (SEQ ID NO: 36) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB13.

[0116] Figure 37The amino acid sequence of potato AOS2 protein, allele CB14 (SEQ ID NO: 37) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB14.

[0117] Figure 38 The nucleic acid sequence of the potato AOS2 gene, allele CB14 (SEQ ID NO: 38) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB14.

[0118] Figure 39 The amino acid sequence of potato AOS2 protein, allele CB15 (SEQ ID NO: 39) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB15.

[0119] Figure 40 The nucleic acid sequence of the potato AOS2 gene, allele CB15 (SEQ ID NO: 40) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB15.

[0120] Figure 41 The amino acid sequence of potato AOS2 protein, allele CB16 (SEQ ID NO: 41) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB16.

[0121] Figure 42 The nucleic acid sequence of the potato AOS2 gene, allele CB16 (SEQ ID NO: 42) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB16.

[0122] Figure 43 The amino acid sequence of potato AOS2 protein, allele CB17 (SEQ ID NO: 43) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB17.

[0123] Figure 44 The nucleic acid sequence of the potato AOS2 gene, allele CB17 (SEQ ID NO: 44) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB17.

[0124] Figure 45The amino acid sequence of potato AOS2 protein, allele CB18 (SEQ ID NO: 45) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB18.

[0125] Figure 46 The nucleic acid sequence of the potato AOS2 gene, allele CB18 (SEQ ID NO: 46) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB18.

[0126] Figure 47 The amino acid sequence of potato AOS2 protein, allele CB19 (SEQ ID NO: 47) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB19.

[0127] Figure 48 The nucleic acid sequence of the potato AOS2 gene, allele CB19 (SEQ ID NO: 48) shows the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB19.

[0128] Figure 49 The amino acid sequence of potato AOS2 protein, allele CB20 (SEQ ID NO: 49) is shown, showing the amino acid sequence of potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB20.

[0129] Figure 50 The nucleic acid sequence of the potato AOS2 gene, allele CB20 (SEQ ID NO: 50) is shown, showing the nucleic acid sequence of the potato allene oxidase synthase 2 (AOS2) gene, StAOS2_CB20. DETAILED DESCRIPTION

[0130] Allene oxidase synthase protein

[0131] Allene oxygenase 2 (AOS2) proteins belong to the cytochrome P450 superfamily and are included in the CYP74 group, which is specialized in the metabolism of hydroperoxides. These proteins play a role in the plant oxylipin biosynthetic pathway, which is important for the production of substances that play an important role in various plant stress and developmental processes (including pathogen / insect attack) and plant fertility. Hughes et al., Chembiochem 10:1122 (2009). These enzymes are encoded by three different genes, AOS1, 2, and 3, which catalyze the production of C6 aldehydes, jasmonic acid (JA), and C9 aldehydes, respectively. AOS1 and AOS2 are enzymes located in the chloroplasts, while the expression of AOS3 is reportedly restricted to underground organs in potatoes. Stumpe et al., Plant J 47:883 (2006). All three enzymes are unusual cytochrome P450 proteins that do not bind molecular oxygen but instead use oxygenated fatty acid hydroperoxide substrates as oxygen donors. Schaller and Stintzi, Phytochemistry 70:1532 (2009). The AOS2 protein catalyzes the crucial step in the formation of jasmonic acid (JA) in plants. The important role of JA in the induction of plant defenses in response to plant wounding and pathogen attack is well known.

[0132] Allene oxidase synthase 2 (AOS2) alleles and SNPs associated with pathogen resistance and / or tolerance.

[0133] The AOS2 gene product is called allene oxygenase 2 and catalyzes the conversion of hydroperoxide to allene oxide, which is a key step in jasmonic acid (JA) biosynthesis. Jasmonic acid and its derivatives, collectively known as jasmonic acid compounds, are key signaling molecules involved in the induction of plant defense responses in response to pathogen attack or injury. Failure to produce JA or loss of sensitivity to it leads to increased disease susceptibility in plants, such as the Arabidopsis coi1 mutant (Feys et al., Plant Cell. 6(5):751-759(1994)). In potatoes, JA is applied to inhibit sporangium germination and mycelial growth of Phytophthora infestans (Pi). The potato AOS2 (StAOS2) gene is mapped to a quantitative resistance locus (QRL) on potato chromosome XI with the R3a resistance gene, which plays a role in species-specific disease resistance to Pi. Pajerowska et al., Planta 228:293(2008). Furthermore, silencing the AOS2 gene in potato resulted in a significant reduction in jasmonic acid levels in wounded plants and increased lesion expansion upon infection with Pi (Pajerowska-Mukhtar et al., 2008, Planta 228:293 (2008). StAOS2 complemented an Arabidopsis AOS2 knockout line lacking JA, and the complemented plant line exhibited enhanced resistance to Arabidopsis bacterial pathogens compared to the gene-deficient line (Pajerowska-Mukhtar et al., 2008).

[0134] The sequences of five AOS2 alleles derived from diploid potatoes used for the previous breeding population are known. Pajerowska et al., (2008); Pajerowska-Mukhtar et al., Genetics 181:1115 (2009). These five different alleles are divided into three categories: "resistance" (StAOS2-1, StAOS2-6), "neutrality" (StAOS2-12) and "susceptibility" (StAOS2-7, StAOS2-8). In the above-mentioned published research, two populations of the F1 progeny of the heterozygous parental system were divided into quantitative resistance, quantitative neutrality and quantitative susceptibility according to late blight development. Subsequently, these classifications were associated with the specific alleles of the StAOS2 gene listed above. Complementation analysis of Arabidopsis JA-deficient mutants with StAOS2 alleles resulted in the recovery of JA production (and 12-oxo-phytodienoate (OPDA) reductase, an intermediate in JA biosynthesis). In addition, complementation of the "resistant" allele resulted in a 10-fold increase in JA production compared to the level of JA produced by the "susceptible" allele. The "neutral" allele had intermediate levels of JA and OPDA. In addition, pathogen assays using Erwinia carotovora ssp. carotovora on these complemented Arabidopsis lines confirmed the JA production profile by showing 10-fold greater bacterial growth in plants complemented by the "susceptible" allele compared to the "resistant" allele.

[0135] Comparison of the amino acid sequences of these five different alleles revealed multiple amino acid differences along the otherwise highly conserved sequences of the AOS2 gene alleles. Twenty-five amino acid changes and one InDel (insertion / deletion polymorphism) are present in these five alleles, while five amino acids (N76D, V289S, V292A, M328L, and T495K) and InDels are numbered as "resistance" allele-specific based on the susceptibility allele StAOS2-7 (SEQ ID NO:5). No amino acid changes are specific for the "susceptibility" allele. Three substitutions (Y145F, T231I / G, and K394T) are present in the neutral allele. Pajerowska et al., Planta 228:293 (2008).

[0136] The amino acid changes T495K and N76D are in close proximity to the active site. It has been suggested that the F256V polymorphism between StAOS2-1 and StAOS2-6 can explain the slightly poor performance of StAOS2-6 based on the position of StAOS2-6 relative to the substrate binding pocket. In addition, the Y145F of the neutral allele can contribute to its centered activity spectrum because this residue is adjacent to the active site. Pajerowska-Mukhtar et al., Planta 228:293 (2008).

[0137] Evaluation of field resistance to Pi in potato cultivars revealed that the AOS2 gene is an important locus that controls the resistance phenotype of certain cultivars. Pajerowska-Mukhtar et al., Genetics 181:1115 (2009). Two SNPs, StAOS2_SNP691 (A) and StAOS2_SNP692 (C), were associated with field resistance (rAUDPC values ​​of 0.15, indicating very low disease establishment). In this study, the genotype with the highest resistance to late blight had a homozygous AAAA / CCCC genotype, and a positive correlation was observed between the degree of deviation from this and the severity of late blight development. It has been reported that these two SNPs are also associated with plant maturity (PM). In general, there is a positive correlation between potato maturity classification (early and late maturing cultivars) and Pi resistance. Wastie RL, Adv Plant Pathology 7:193 (1999). However, those individuals homozygous for the A and C alleles fall into the moderately early maturing category, separating them from the highly desirable late maturing phenotype. Pajerowska-Mukhtar et al., Genetics 181: 1115 (2009).

[0138] Potato is a very heterozygous tetraploid, which makes it difficult to transmit the required proterties between cultivars with expression in progeny. In addition, some species of Solanum (Solanum) with natural resistance to insect pests and diseases (such as several species present in Peru and Central America) are diploid and difficult for tetraploid potato breeding. The homologous tetraploid genome and asexual propagation used for potato breeding bring challenges in developing new cultivars with required proterties. The resistance proterties shown in diploid species, for example, Solanum bulbocastanum, cannot be used for breeding because the endosperm balance number of this species is 1, while the endosperm balance number of potato is 4.

[0139] Using RTDS in Potato TM Compared with traditional breeding, transgenic genetic engineering has some advantages. TMThis allows manipulation of the endogenous AOS2 gene, eliminating the need for backcrossing required to remove undesirable traits in traditional breeding. TM This allows the introduction of mutations in genes that confer resistance and / or tolerance to traits exhibited by other species that do not have a ploidy compatible with potato. TM Compared with transgenic genetic engineering, RTDS has advantages. TM Ability to manipulate endogenous genes.

[0140] Rapid Trait Development System (RTDS) TM )

[0141] In any of the various aspects and embodiments of the compositions and methods disclosed herein, the rapid trait development system (RTDS) developed by Cibus can be used, for example. TM ) techniques for gene and protein mutation. Plants containing any of the mutations disclosed herein, in combination or individually, can form the basis for new pathogen-resistant and / or pathogen-tolerant products. Also provided are seeds / vegetative materials produced from mutant plants, wherein the AOS2 gene is homozygous or heterozygous for the mutation. The mutations disclosed herein can be combined with any other known mutations or with mutations discovered in the future.

[0142] In some embodiments, RTDS TM Based on: By utilizing the cell's own gene repair system to change the targeted gene, the gene sequence is specifically modified in situ without inserting foreign DNA and gene expression control sequences. This procedure can achieve precise changes in the genetic sequence while the rest of the genome sequence remains unchanged. Compared with traditional genetically modified GMOs, no foreign genetic material is integrated and no foreign genetic material remains in the plant. In many embodiments, the RTDS TM The changes introduced into the genetic sequence are not inserted randomly. Because the affected genes remain in their original locations, there are no random, uncontrolled, or adverse expression patterns.

[0143] RTDS TM The process is realized using chemically synthesized oligonucleotides (gene repair oligonucleotides (GRONs)) that can be composed of DNA and modified RNA bases and other chemical moieties, and is designed to hybridize at the targeted gene position to produce one or more mismatched base pairs. The mismatched base pairs act as a signal to attract the cell's own natural gene repair system to the site and correct (replace, insert or delete) one or more nucleotides specified in the gene. Once the correction process is complete, the GRON molecule degrades and continues to express the modified or repaired gene under the normal endogenous control mechanism of the gene.

[0144] Gene Repair Oligonucleobases ("GRONs")

[0145] The methods and compositions disclosed herein can be implemented or prepared using, for example, "gene repair oligonucleobases" having the conformations and chemical structures described in detail below. The "gene repair oligonucleobases" contemplated herein have also been described in the published scientific and patent literature using other names, including "recombination-inducing oligonucleobases," "RNA / DNA chimeric oligonucleotides," "chimeric oligonucleotides," "mixed duplex oligonucleotides" (MDONs), "RNA DNA oligonucleotides (RDOs)," "gene targeting oligonucleotides," "genoplasts," "single-stranded modified oligonucleotides," "single-stranded oligodeoxynucleotide mutation vectors" (SSOMVs), "duplex mutation vectors," and "heteroduplex mutation vectors."

[0146] Oligonucleobases having the conformation and chemical structure described in U.S. Patent No. 5,565,350 to Kmiec (Kmiec I) and U.S. Patent No. 5,731,181 to Kmiec (Kmiec II), which are hereby incorporated by reference, are suitable for use as the "gene repair oligonucleobases" of the present disclosure. The gene repair oligonucleobases in Kmiec I and / or Kmiec II contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides ("RNA segment") that base-pair with the DNA-type nucleotides of the other strand.

[0147] Kmiec II discloses that non-nucleotides containing purine and pyrimidine bases can replace nucleotides.Other gene repair molecules that can be used for the present invention are 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 in International Patent No. PCT / US00 / 23457; And described in International Patent Publication Nos. WO 98 / 49350, WO 99 / 07865, WO 99 / 58723, WO 99 / 58702 and WO 99 / 40789, which are each hereby incorporated by reference in their entirety.

[0148] In one embodiment, the gene repair oligonucleotide is a mixed duplex oligonucleotide (MDON), wherein the RNA type nucleotide of the mixed duplex oligonucleotide is resistant to RNA enzymes by replacing 2'-hydroxyl with fluorine, chlorine, bromine functional groups or by placing a substituent on 2'-O. Suitable substituents include the substituents taught by Kmiec II. Alternative substituents include the substituents taught by U.S. Patent No. 5,334,711 (Sproat) and the substituents taught by patent disclosures EP 629 387 and EP 679 657 (both Martin applications), which are hereby incorporated by reference. As used herein, the 2'-fluoro, chlorine or bromine derivatives of ribonucleotides or the ribonucleotides in which 2'-OH is replaced by the substituents described in Martin applications or Sproa are referred to as "2'-substituted ribonucleotides". As used herein, the term "RNA-type nucleotide" means a 2'-hydroxyl or 2'-substituted nucleotide that is linked to the other nucleotides of a mixed duplex oligonucleotide via an unsubstituted phosphodiester bond or any non-natural bond as taught by Kmiec I or Kmiec II. As used herein, the term "deoxyribonucleotide" means a nucleotide with a 2'-H bond that can be linked to the other nucleotides of a gene repair oligonucleotide via an unsubstituted phosphodiester bond or any non-natural bond as taught by Kmiec I or Kmiec II.

[0149] In a specific embodiment of the present invention, the gene repair oligonucleotide is a mixed duplex oligonucleotide (MDON) connected only by an unsubstituted phosphodiester bond. In an alternative embodiment, the connection is carried out by a non-phosphodiester bond, a phosphodiester derivative, and a non-phosphorus-based bond as taught by Kmiec II. In another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2'-substituted nucleotide. The particularly preferred embodiment of the 2'-substituted ribonucleotide is a 2'-fluoro, 2'-methoxy, 2'-propoxy, 2'-allyloxy, 2'-hydroxyethoxy, 2'-methoxyethoxy, 2'-fluoropropoxy and 2'-trifluoropropoxy-substituted ribonucleotide. The more preferred embodiment of the 2'-substituted ribonucleotide is a 2'-fluoro, 2'-methoxy, 2'-methoxyethoxy and 2'-allyloxy-substituted nucleotide. In another embodiment, the mixed duplex oligonucleotide is connected by an unsubstituted phosphodiester bond.

[0150] Although it is more convenient to synthesize mixed duplex oligonucleotides (MDONs) with only a single type of 2'-substituted RNA-type nucleotides, the method of the present invention can be implemented with mixed duplex oligonucleotides with two or more types of RNA-type nucleotides. The function of the RNA segment can be unaffected by the interruption caused by the introduction of deoxynucleotides between two RNA-type trinucleotides, and therefore, the term RNA segment encompasses terms such as "interrupted RNA segment". Uninterrupted RNA segments are referred to as continuous RNA segments. In an alternative embodiment, RNA segments can contain alternating RNase-resistant nucleotides and unsubstituted 2'-OH nucleotides. The mixed duplex oligonucleotide preferably has less than 100 nucleotides and more preferably less than 85 nucleotides, but more than 50 nucleotides. The first chain and the second chain undergo Watson-Crick base pairing. In one embodiment, the chains of the mixed duplex oligonucleotide are covalently bonded by a linker such as a single-stranded hexanucleotide, pentanucleotide or tetranucleotide so that the first chain and the second chain are segments of a single oligonucleotide chain with a single 3' end and a single 5' end. The 3' and 5' ends can be protected by adding a "hairpin cap" to allow the nucleotides at the 3' and 5' ends to undergo Watson-Crick pairing with adjacent nucleotides. Alternatively, a second hairpin cap can be placed at the junction between the first and second strands, away from the 3' and 5' ends, to stabilize Watson-Crick pairing between the first and second strands.

[0151] The first chain and the second chain contain two regions, and these two regions are homologous to the two fragments of the target gene, that is, they have the same sequence as the target gene. The homologous region contains the nucleotides of the RNA segment and can contain one or more DNA-type nucleotides of the connecting DNA segment and can also contain DNA-type nucleotides that are not in the intervening DNA segment. The two homologous regions are separated by a region (called a "heterologous region") having a sequence different from the target gene sequence, and each region is adjacent to the "heterologous region". The heterologous region can contain one, two, or three or more mismatched nucleotides. The mismatched nucleotides can be continuous or can be separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen nucleotides that are homologous to the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide can be different from the sequence of the target gene only by deleting one, two, three or five or fewer nucleotides from the mixed duplex oligonucleotide. In this case, the length and position of the heterologous region is considered the length of the deletion, even if no nucleotides in the mixed duplex oligonucleotide are within the heterologous region. The distance between the target gene segments complementary to the two heterologous regions is the same as the length of the heterologous region in which the substitution or substitutions are intended to be made. When the heterologous region contains an insertion, the homologous region is therefore separated in the mixed duplex oligonucleotide further in the gene than its complementary homologous segment, while the opposite applies when the heterologous region encodes a deletion.

[0152] The RNA segments of the mixed duplex oligonucleotide are each part of a homologous region (i.e., a region identical in sequence to the target gene fragment), and these segments together preferably contain at least 13 RNA-type nucleotides and preferably 16 to 25 RNA-type nucleotides or even more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, the RNA segments of the homologous regions are separated and adjacent to the intervening DNA segments, i.e., are "connected" by the intervening DNA segments. In one embodiment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. The intervening DNA segment containing the heterologous region of the mixed duplex oligonucleotide is called a "mutator segment."

[0153] In another embodiment of the present disclosure, the gene repair oligonucleobase (GRON) is a single-stranded oligodeoxynucleotide mutagenesis vector (SSOMV), such as disclosed in International Patent Application PCT / US2000 / 23457, U.S. Patent Nos. 6,271,360, 6,479,292, and 7,060,500, which are incorporated by reference in their entirety. The sequence of SSOMVs is based on the same principles as the mutagenesis vectors described in U.S. Patents 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, as well as International Publication Nos. WO 98 / 49350, WO 99 / 07865, WO 99 / 58723, WO 99 / 58702, and WO 99 / 40789. The sequence of SSOMVs contains two regions that are homologous to the target sequence, separated by a region called a mutator region containing the desired genetic alteration. The mutator region can have a sequence that is the same length as the sequence separating the homologous region in the target sequence, but has a different sequence. Such a mutator region can induce substitutions. Alternatively, the homologous regions in the SSOMV can be contiguous with one another, while regions in the target gene with identical sequences are separated by one, two, or more nucleotides. Such SSOMVs cause deletion of nucleotides from the target gene that are not present in the SSOMV. Finally, sequences of the target gene identical to the homologous regions can be adjacent in the target gene but separated by one, two, or more nucleotides in the sequence of the SSOMV. Such SSOMVs cause insertions into the target gene sequence.

[0154] The nucleotides of the SSOMV are deoxyribonucleotides connected by unmodified phosphodiester bonds, except that the 3' and / or 5' end internucleotide linkage or the two 3' and / or 5' end internucleotide linkages can be phosphorothioate or phosphoamidate. As used herein, an internucleotide linkage is a linkage between the nucleotides of the SSOMV and does not include the linkage between the 3' terminal nucleotide or the 5' terminal nucleotide and the end-blocking substituent. In a specific embodiment, the length of the SSOMV is between 21 and 55 deoxynucleotides, and the length of the homology region is a total length of at least 20 deoxynucleotides, and at least two homology regions should each have a length of at least 8 deoxynucleotides.

[0155] SSOMVs can be designed to complement either the coding or noncoding strand of the target gene. When the desired mutation is a single base substitution, it is preferred that both the mutator nucleotide and the targeting nucleotide are pyrimidines. For consistency in achieving the desired functional outcome, it is preferred that both the mutator nucleotide and the targeting nucleotide in the complementary strand are pyrimidines. Particularly preferred are SSOMVs that encode transversion mutations, i.e., mismatches between a C or T mutator nucleotide and a C or T nucleotide, respectively, in the complementary strand.

[0156] In addition to oligodeoxynucleotides, SSOMVs may also contain a 5' capping substituent that is linked to the 5' carbon via a linker. The chemical nature of the linker (except for its length) is not critical; the linker length should preferably be at least 6 atoms long and the linker should be flexible. A variety of non-toxic substituents can be used, such as biotin, cholesterol or other steroids, or non-intercalating cationic fluorescent dyes. A particularly preferred reagent for preparing SSOMVs is Cy3 TM and Cy5 TM Cy3 is a commercially available reagent that is a blocked phosphoramidite that, upon incorporation into oligonucleotides, yields 3,3,3',3'-tetramethyl N,N'-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. TM is particularly preferred. When the indocarbocyanine is substituted with an N-oxyalkyl group, it can be conveniently linked to the 5' end of the oligodeoxynucleotide as a phosphodiester with a 5' terminal phosphate. The chemical nature of the dye linker between the dye and the oligodeoxynucleotide is not critical and is selected for ease of synthesis. When commercially available Cy3 TM When the aminophosphite is used, the resulting 5' modification consists of a capping substituent and a linker group, which are N-hydroxypropyl, N'-phosphatidylpropyl 3,3,3',3'-tetramethylindoline monocarbocyanine.

[0157] In a preferred embodiment, the indole carbocyanine dye is tetrasubstituted at the 3 and 3' positions of the indole ring. Without being limited by theory, these substitutions prevent the dye from becoming an intercalating dye. The identity of the substituents at these positions is not critical. The SSOMV may additionally have a 3'-blocking substituent. The chemical nature of the 3'-blocking substituent is also not critical.

[0158] The mutations described herein can also be obtained by mutagenesis (random, somatic, or directed) and other DNA editing or recombination techniques, including but not limited to gene targeting using site-specific homologous recombination by zinc finger nucleases, meganucleases, or other nucleases.

[0159] Delivering gene repair oligonucleobases into plant cells

[0160] Any commonly known method for transforming plant cells can be used to deliver the gene repair oligonucleobase. Illustrative methods are described below.

[0161] Microcarriers and microfibers

[0162] The use of metal microcarriers (microspheres) to introduce large DNA fragments into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the art (hereinafter referred to as biolistic delivery). US Patent Nos. 4,945,050, 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for their delivery.

[0163] Specific conditions for using microcarriers in the methods of the present invention are described in International Publication No. WO 99 / 07865. In an illustrative technique, ice-cold microcarriers (60 mg / mL), mixed duplex oligonucleotide (60 mg / mL), 2.5 M CaCl2, and 0.1 M spermidine are added sequentially; the mixture is gently agitated, for example by vortexing, for 10 minutes and then allowed to stand at room temperature for 10 minutes. The microcarriers are then diluted in 5 volumes of ethanol, centrifuged, and resuspended in 100% ethanol. Good results are achieved with an adhesion solution concentration of 8-10 μg / μL microcarriers, 14-17 μg / mL mixed duplex oligonucleotide, 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 oligonucleotide, 1.3 M CaCl2, and 21 mM spermidine.

[0164] For the practice of the present invention, microfibers can also be used to pass through the cell wall and cell membrane to introduce gene repair oligonucleotides into plant cells. U.S. Patent No. 5,302,523 to Coffee et al. describes the use of 30X 0.5μm and 10X 0.3μm silicon carbide fibers to promote the transformation of Black Mexican Sweet corn suspension cultures. Any mechanical technology that can apply microfibers to introduce DNA for plant cell transformation can be used to deliver gene repair oligonucleotides for transmutation.

[0165] An illustrative technique for microfiber delivery of gene repair oligonucleotides 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 suspension culture is allowed to settle and equal volumes of concentrated cells and sterile fiber / nucleotide suspension are vortexed for 10 minutes before plating. Depending on the specific trait, selective medium is applied immediately or delayed for up to approximately 120 hours.

[0166] Protoplast electroporation

[0167] In an alternative embodiment, the gene repair oligonucleotide can be delivered to the plant cell by electroporation of protoplasts derived from plant parts or plant cell suspensions. According to techniques well known to those skilled in the art, protoplasts are formed by enzymatic treatment of plant parts, particularly leaves. 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. Illustrative conditions for electroporation are 3X10 5 protoplasts, and the concentration of gene repair oligonucleobases was between 0.6-4 μg / mL.

[0168] PEG-mediated DNA uptake into protoplasts

[0169] In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the membrane-modifying agent polyethylene glycol according to techniques well known to those skilled in the art (see, e.g., Gharti-Chhetri et al., Physiol. Plant. 85:345-351 (1992); Datta et al., Plant Molec. Biol. 20:619-629 (1992)).

[0170] Microinjection

[0171] In an alternative embodiment, the gene repair oligonucleobase can be delivered by microcapillary injection into plant cells or protoplasts (see, eg, Miki B. et al., Meth. Cell Science 12: 139-144 (1989); Schnorf M, et al., Transgen. Res. 1: 23-30 (1991)).

[0172] genetically modified

[0173] In any of the various aspects and embodiments of the compositions and methods disclosed herein, mutations in genes and proteins can be achieved using, for example, transgenic technology. In some embodiments, the compositions and methods include plants or plant cells transformed with a nucleic acid construct comprising a promoter operably linked to an AOS2 nucleotide sequence disclosed herein. The methods disclosed herein may include introducing an AOS2 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 sequence encoding an AOS2 protein that is resistant and / or resistant to pathogens as disclosed herein, particularly in the presence of a pathogen. Figure 2 and Figure 4 The nucleotide sequence shown in , and fragments and variants thereof. The method further relates to the use of a promoter capable of driving gene expression in plant cells. In one embodiment, such promoter is a constitutive promoter or a tissue-preferred promoter. The plants produced by these methods can have increased or stable AOS2 activity and / or elevated jasmonic acid and / or 12-oxo-phytodienoic acid (OPDA) levels, thereby resulting in enhanced resistance and / or tolerance to pathogens compared to untransformed plants. Therefore, the method can be used to enhance or improve the resistance and / or tolerance of plants to at least one pathogen.

[0174] In one embodiment, the method for producing a pathogen-resistant and / or tolerant plant comprises 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 regenerating a transformed plant from the transformed plant cell. The nucleotide sequence is selected from those encoding the pathogen-resistant and / or tolerant AOS2 disclosed herein, particularly wherein Figure 2 and Figure 4 The nucleotide sequence shown in , and fragments and variants thereof. The pathogen-resistant and / or tolerant plants produced by this method include enhanced resistance and / or tolerance to at least one pathogen (eg, Phytophthora infestans) compared to untransformed plants.

[0175] The disclosed nucleic acid molecules can be used in nucleic acid constructs to transform plants, for example crop plants such as potatoes. In one embodiment, nucleic acid constructs containing the disclosed nucleic acid molecules can be used to generate transgenic plants to provide resistance and / or tolerance to pathogens such as Phytophthora infestans. The nucleic acid constructs can be used in expression cassettes, expression vectors, transformation vectors, plasmids, and the like. Transgenic plants obtained after transformation with such constructs exhibit increased resistance and / or tolerance to pathogens such as Phytophthora infestans.

[0176] Construct

[0177] The nucleic acid molecules disclosed herein (eg, mutant AOS2 genes) can be used to generate recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the invention can be used to prepare nucleic acid constructs, eg, expression cassettes for expression in plants of interest.

[0178] The expression cassette may comprise a regulatory sequence operably linked to the AOS2 nucleic acid sequence disclosed herein. The cassette may further comprise at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene or genes may be provided on multiple expression cassettes.

[0179] The nucleic acid construct may be provided with a plurality of restriction sites for inserting the AOS2 nucleic acid sequence so that it is under the transcriptional regulation of the regulatory region. The nucleic acid construct may further comprise a nucleic acid molecule encoding a selectable marker gene.

[0180] Any promoter can be used in the production of nucleic acid constructs. The promoter can be native or similar, or exogenous or heterologous to the plant host and / or to the AOS2 nucleic acid sequence disclosed herein. In addition, the promoter can be a natural sequence, or a synthetic sequence. When a promoter is "exogenous" or "heterologous" to the plant host, it means that the promoter is not present in the natural plant into which the promoter is introduced. When a promoter is "exogenous" or "heterologous" to the AOS2 nucleic acid sequence disclosed herein, it means that the promoter is not a native or naturally occurring promoter for the operably connected AOS2 nucleic acid sequence disclosed herein. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous thereto.

[0181] In some embodiments, a heterologous promoter is used to express the AOS2 nucleic acid sequence disclosed herein, but native promoter sequences can be used in the preparation of the construct. Such constructs will alter the expression level of the AOS2 protein in the plant or plant cell, thereby changing the phenotype of the plant or plant cell.

[0182] Any promoter can be used in the preparation of the construct to control expression of the AOS2 coding sequence, such as a constitutive promoter, a tissue-preferred promoter, an inducible promoter, or other promoters used for expression in plants. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43 838 and U.S. Patent 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); ALS promoter (U.S. Patent No. 5,659,026), etc. 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.

[0183] Tissue-preferred promoters can be used to direct AOS2 expression in specific plant tissues. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include 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.

[0184] The nucleic acid construct can also include a transcriptional termination region. When using a transcriptional termination region, any termination region can be used to prepare a nucleic acid construct. For example, the termination region can be natural for the transcription initiation region, can be natural for the AOS2 sequence of interest that is operably connected, can be natural for a plant host, or can be derived from another source (that is, exogenous or heterologous for the promoter, the AOS2 nucleic acid molecule of interest, the plant host, or any combination thereof). Examples of the termination region that can be used for constructs of the present invention include those derived from the Ti plasmid of Agrobacterium tumefaciens (A.tumefaciens), such as octopine synthase and nopaline synthase termination regions. See also 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.

[0185] In some embodiments, nucleic acids can be optimized to enhance expression in transformed plants. That is, nucleic acids encoding mutant AOS2 proteins can be synthesized using plant-preferred codons to improve expression. For host-preferred codon usage, see, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11. Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498.

[0186] In addition, other sequence modifications can be made to the nucleic acid sequences disclosed herein. For example, additional sequence modifications can enhance gene expression in a cellular host. These include eliminating sequences encoding spurious polyadenylation signals, exon / intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can also be adjusted to the average level of the target cellular host, as calculated by reference to known genes expressed in the host cell. Furthermore, the sequence can be modified to avoid predicted hairpin secondary mRNA structures.

[0187] Other nucleic acid sequences can also be used to prepare constructs of the present invention, for example, to enhance expression of the AOS2 coding sequence. Such nucleic acid sequences include intron 1 of the maize Adh1 gene (Callis et al. (1987) Genes and Development 1: 1183-1200), and leader sequences (W-sequences) from tobacco mosaic virus (TMV), maize chlorotic mottle virus, and alfalfa mosaic virus (Gallie et al. (1987) Nucleic Acids Res. 15: 8693-8711 and Skuzeski et al. (1990) Plant Mol. Biol. 15: 65-79). It has been shown that the first intron from the maize shrunken-1 locus increases gene expression in chimeric gene constructs. U.S. Patent Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al., Plant Physiol. 106:929-939 (1994) have also demonstrated that introns are useful for regulating gene expression on a tissue-specific basis. To further enhance or optimize AOS2 gene expression, the plant expression vectors disclosed herein may also include a DNA sequence containing a matrix attachment region (MAR). Plant cells transformed with such modified expression systems may then exhibit overexpression or constitutive expression of the nucleotide sequences of the present invention.

[0188] The expression construct disclosed herein can also include a nucleic acid sequence capable of directing the expression of the AOS2 sequence to the chloroplast. Such nucleic acid sequences include chloroplast targeting sequences encoding a chloroplast transit peptide to direct the gene product of interest to the chloroplasts of plant cells. Such transit peptides are known in the art. With respect to chloroplast targeting sequences, "operably connected" means that the nucleic acid sequence encoding the transit peptide (i.e., the chloroplast targeting sequence) is connected to the AOS2 nucleic acid molecule of the present invention so that the two sequences are continuous and in the same reading frame. See, for example, 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. Although the AOS2 proteins disclosed herein may include a native chloroplast transit peptide, any chloroplast transit peptide known in the art can be fused to the amino acid sequence of a mature AOS2 protein of the present invention by operably linking a chloroplast targeting sequence to the 5'-terminus of the nucleotide sequence encoding the mature AOS2 protein of the present invention.

[0189] Chloroplast targeting sequences are known in the art and include 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(1):100-101); 1):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.

[0190] In another embodiment, a nucleic acid construct can be prepared to direct the expression of the mutant AOS2 coding sequence from plant cell chloroplasts. Methods for transforming chloroplasts are 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. This method relies on particle gun delivery of DNA containing a selective marker and targeting the DNA to the plastid genome by homologous recombination. In addition, plastid transformation can be accomplished by transactivating a silent plastid-carried transgene through tissue-preferred expression of nuclear-encoded and plastid-mediated RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0191] Nucleic acids of interest that are targeted to the chloroplast can be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this way, nucleic acids of interest can be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, incorporated herein by reference.

[0192] The nucleic acid construct can be used to transform plant cells and regenerate transgenic plants containing the mutant AOS2 coding sequence. A variety of plant transformation vectors and methods are available for transforming plants. See, for example, 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. 203-212; Cousins ​​et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, PP et al. (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 et al. (1993) Plant Sci.91:139-148; Franklin, CI et al. (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 et al. (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 CellRep.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 et al. (1994) Plant Physiol. 104:3748. The construct can also be transformed into plant cells using homologous recombination.

[0193] The disclosed constructs comprising the AOS2 nucleic acid sequences disclosed herein can be used in a variety of methods to generate transgenic host cells, such as bacteria, yeast, and to transform plant cells and, in some cases, regenerate transgenic plants. For example, a method for generating transgenic crop plants containing an AOS2 mutant protein disclosed herein, wherein expression of one or more nucleic acids in the plant results in pathogen resistance and / or tolerance compared to wild-type plants or to known AOS2 mutant plants, the method comprising: (a) introducing into a plant cell an expression vector comprising a nucleic acid encoding the mutant AOS2 protein, and (b) generating a pathogen-resistant and / or tolerant transgenic plant from the plant cell.

[0194] AOS2 mutation

[0195] The compositions and methods may at least partially relate to mutations in the AOS2 gene, such as mutations that render the plant resistant or tolerant to pathogens. The compositions and methods, in certain embodiments, also relate to the use of gene repair oligonucleotides to carry out the desired mutations in the gene encoding the AOS2 protein in the chromosome or episomal sequence of the plant. The mutated protein may, in some embodiments, substantially retain the catalytic activity of the wild-type protein, allowing the plant to have enhanced resistance or tolerance to pathogens, and thus, in some embodiments, allow the plant, its organs, tissues or cells to grow or develop substantially normally or altered compared to wild-type plants, regardless of the presence or absence of pathogens. The compositions and methods also relate to non-transgenic plant cells in which the AOS2 gene has mutated, non-transgenic plants regenerated therefrom, and plants produced, for example, by hybridizing the regenerated non-transgenic plant with a plant having a mutation in a different AOS2 gene or in the same AOS2 gene. The compositions and methods also relate to transgenic plant cells in which the AOS2 gene has mutated, transgenic plants regenerated therefrom, and plants produced, for example, by hybridizing the regenerated transgenic plant with a plant having a mutation in a different AOS2 gene or in the same AOS2 gene.

[0196] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutant AOS2 protein has one or more mutations at a position corresponding to a position selected from the group consisting of 6, 12, 30, 37, 46, 48, 51, 76, 113, 145, 187, 197, 200, 227, 231, 256, 264, 270, 282, 289, 292, 309, 320, 328, 337, 338, 357, 381, 394, 407, 423, 430, 439, 467, 480, 494, and 495 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein has one or more mutations at a position corresponding to position 6 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein has one or more mutations at a position corresponding to position 12 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 30 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 37 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 46 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 48 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 51 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 76 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 113 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 145 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 187 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein has one or more mutations at a position corresponding to position 197 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein has one or more mutations at a position corresponding to position 200 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein has one or more mutations at a position corresponding to position 227 of SEQ ID NO: 5.In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 231 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 256 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 264 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 270 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 282 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 289 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 292 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 309 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 320 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 328 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 337 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 338 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 357 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 381 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 394 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 407 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 423 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 430 of SEQ ID NO: 5.In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 439 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 467 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 480 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 494 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein has one or more mutations at a position corresponding to position 495 of SEQ ID NO: 5.

[0197] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein has an F at amino acid position 6 of SEQ ID NO: 7, a P at amino acid position 12 of SEQ ID NO: 5, an R at amino acid position 12 of SEQ ID NO: 11, an A at amino acid position 30 of SEQ ID NO: 5, an I at amino acid position 37 of SEQ ID NO: 5, an L at amino acid position 46 of SEQ ID NO: 5, an F at amino acid position 46 of SEQ ID NO: 3, a T at amino acid position 48 of SEQ ID NO: 5, an I at amino acid position 48 of SEQ ID NO: 27, a V at amino acid position 48 of SEQ ID NO: 7, an M at amino acid position 51 of SEQ ID NO: 5, a D at amino acid position 76 of SEQ ID NO: 5, an N at amino acid position 76 of SEQ ID NO: 5, a G at amino acid position 113 of SEQ ID NO: 5, an F at amino acid position 46 of SEQ ID NO: 3, an T at amino acid position 48 of SEQ ID NO: 5, an I at amino acid position 48 of SEQ ID NO: 27, a V at amino acid position 48 of SEQ ID NO: 7, an M at amino acid position 51 of SEQ ID NO: 5, an D at amino acid position 76 of SEQ ID NO: 5, an N at amino acid position 76 of SEQ ID NO: 5, an G at amino acid position 113 of SEQ ID NO: 5, an F at amino acid position 46 of SEQ ID NO: 3, an F at amino acid position 48 of SEQ ID NO: 5 NO:49 has a D at amino acid position 113, an F at amino acid position 145 of SEQ ID NO:9, an L at amino acid position 187 of SEQ ID NO:5, an E at amino acid position 197 of SEQ ID NO:5, a D at amino acid position 197 of SEQ ID NO:3, a K at amino acid position 200 of SEQ ID NO:7, an A at amino acid position 227 of SEQ ID NO:5, a T at amino acid position 231 of SEQ ID NO:5, an I at amino acid position 231 of SEQ ID NO:7, a G at amino acid position 231 of SEQ ID NO:9, an F at amino acid position 256 of SEQ ID NO:5, a V at amino acid position 256 of SEQ ID NO:3, an A at amino acid position 264 of SEQ ID NO:7, an L at amino acid position 270 of SEQ ID NO:7, an F at amino acid position 282 of SEQ ID NO:5, and a V at amino acid position 283 of SEQ ID NO:7. SEQ ID NO: 41 has an S at amino acid position 282, a V at amino acid position 289 of SEQ ID NO: 5, an S at amino acid position 289 of SEQ ID NO: 11, an N at amino acid position 289 of SEQ ID NO: 13, a V at amino acid position 292 of SEQ ID NO: 5, an L at amino acid position 309 of SEQ ID NO: 5, an I at amino acid position 309 of SEQ ID NO: 19, an M at amino acid position 320 of SEQ ID NO: 5, an N at amino acid position 289 of SEQ ID NO: 13, an L at amino acid position 309 of SEQ ID NO: 5, an I at amino acid position 320 of SEQ ID NO: 5, an M at amino acid position 321 of SEQ ID NO: 5, an N at amino acid position 289 of SEQ ID NO: 13, an N at amino acid position 292 of SEQ ID NO: 5, an L at amino acid position 309 of SEQ ID NO: 19, an M at amino acid position 320 of SEQ ID NO: 5, an N at amino acid position 289 of SEQ ID NO: 13has an L at amino acid position 320 of SEQ ID NO:23, an M at amino acid position 328 of SEQ ID NO:5, an L at amino acid position 328 of SEQ ID NO:19, a V at amino acid position 328 of SEQ ID NO:27, an E at amino acid position 337 of SEQ ID NO:5, a D at amino acid position 337 of SEQ ID NO:13, a V at amino acid position 338 of SEQ ID NO:5, an L at amino acid position 338 of SEQ ID NO:13, an I at amino acid position 357 of SEQ ID NO:5, an M at amino acid position 357 of SEQ ID NO:3, a P at amino acid position 381 of SEQ ID NO:5, an L at amino acid position 381 of SEQ ID NO:35, a T at amino acid position 394 of SEQ ID NO:9, a G at amino acid position 407 of SEQ ID NO:5, a C at amino acid position 407 of SEQ ID NO:13, and an L at amino acid position 358 of SEQ ID NO:13. In some embodiments, the AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 423 of SEQ ID NO: 7, an L at amino acid position 430 of SEQ ID NO: 5, a deletion of amino acid E at position 439 of SEQ ID NO: 5, a G at amino acid position 467 of SEQ ID NO: 5, an S at amino acid position 467 of SEQ ID NO: 39, a V at amino acid position 480 of SEQ ID NO: 5, a G at amino acid position 494 of SEQ ID NO: 5, a D at amino acid position 494 of SEQ ID NO: 21, and / or a T at amino acid position 495 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 6 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a P at amino acid position 12 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an R at amino acid position 12 of SEQ ID NO: 11. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an A at amino acid position 30 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 37 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 37 of SEQ ID NO: 5.In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 46 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 46 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a T at amino acid position 48 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 48 of SEQ ID NO: 27. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 48 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 51 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an N at amino acid position 76 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 76 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a G at position 113 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a D at position 113 of SEQ ID NO:49. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 145 of SEQ ID NO:9. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 187 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an E at amino acid position 197 of SEQ ID NO:5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 197 of SEQ ID NO: 3. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a K at amino acid position 200 of SEQ ID NO: 7. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a K at amino acid position 200 of SEQ ID NO: 7.NO:5 comprises one or more mutations. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having a T at amino acid position 231 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having an I at amino acid position 231 of SEQ ID NO:7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having a G at amino acid position 231 of SEQ ID NO:9. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having an F at amino acid position 256 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having a V at amino acid position 256 of SEQ ID NO:3. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to the AOS2 amino acid sequence having an A at amino acid position 264 of SEQ ID NO:7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 270 of SEQ ID NO:7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 282 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an S at amino acid position 282 of SEQ ID NO:41. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 289 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an S at amino acid position 289 of SEQ ID NO: 11. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an N at amino acid position 289 of SEQ ID NO:13. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 292 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 309 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 309 of SEQ ID NO: 5.In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 320 of SEQ ID NO: 5.

[0198] In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 320 of SEQ ID NO:23. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 320 of SEQ ID NO:23. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 328 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 328 of SEQ ID NO:19. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 328 of SEQ ID NO:27. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an E at amino acid position 337 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 337 of SEQ ID NO:13. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 338 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 338 of SEQ ID NO:13. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 357 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 357 of SEQ ID NO:3. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a P at amino acid position 381 of SEQ ID NO:5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 381 of SEQ ID NO:35. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a T at amino acid position 394 of SEQ ID NO: 9. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 407 of SEQ ID NO: 5.In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a C at amino acid position 407 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 423 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 430 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an amino acid E deleted at position 439 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 467 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having an S at amino acid position 467 of SEQ ID NO: 39. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 480 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 494 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 494 of SEQ ID NO: 21. In some embodiments, the mutant AOS2 protein comprises one or more mutations relative to an AOS2 amino acid sequence having and / or a T at amino acid position 495 of SEQ ID NO: 5.

[0199] In combination with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene encodes a mutated AOS2 protein. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 691 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 692 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 678 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a T at a position corresponding to position 681 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 727 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 744 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 774 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 879 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes an A at a position corresponding to position 900 of SEQ ID NO: 2. In some embodiments, the mutated AOS2 gene includes a C at a position corresponding to position 954 of SEQ ID NO: 2.

[0200] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 gene can encode a mutated AOS2 protein. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein having an F at amino acid position 6 of SEQ ID NO:7, a P at amino acid position 12 of SEQ ID NO:5, an R at amino acid position 12 of SEQ ID NO:11, an A at amino acid position 30 of SEQ ID NO:5, an I at amino acid position 37 of SEQ ID NO:5, an L at amino acid position 46 of SEQ ID NO:5, an F at amino acid position 46 of SEQ ID NO:3, a T at amino acid position 48 of SEQ ID NO:5, an I at amino acid position 48 of SEQ ID NO:27, a V at amino acid position 48 of SEQ ID NO:7, an M at amino acid position 51 of SEQ ID NO:5, a D at amino acid position 76 of SEQ ID NO:5, an N at amino acid position 76 of SEQ ID NO:5, a G at amino acid position 113 of SEQ ID NO:5, an F at amino acid position 46 of SEQ ID NO:3, an I at amino acid position 48 of SEQ ID NO:5, an I at amino acid position 48 of SEQ ID NO:27, a V at amino acid position 48 of SEQ ID NO:7, an M at amino acid position 51 of SEQ ID NO:5, a D at amino acid position 76 of SEQ ID NO:5, an N at amino acid position 76 of SEQ ID NO:5, an NO:49 has a D at amino acid position 113, an F at amino acid position 145 of SEQ ID NO:9, an L at amino acid position 187 of SEQ ID NO:5, an E at amino acid position 197 of SEQ ID NO:5, a D at amino acid position 197 of SEQ ID NO:3, a K at amino acid position 200 of SEQ ID NO:7, an A at amino acid position 227 of SEQ ID NO:5, a T at amino acid position 231 of SEQ ID NO:5, an I at amino acid position 231 of SEQ ID NO:7, a G at amino acid position 231 of SEQ ID NO:9, an F at amino acid position 256 of SEQ ID NO:5, a V at amino acid position 256 of SEQ ID NO:3, an A at amino acid position 264 of SEQ ID NO:7, an L at amino acid position 270 of SEQ ID NO:7, an F at amino acid position 282 of SEQ ID NO:5, a SEQ ID NO: 41 has an S at amino acid position 282, a V at amino acid position 289 of SEQ ID NO: 5, an S at amino acid position 289 of SEQ ID NO: 11, an N at amino acid position 289 of SEQ ID NO: 13, a V at amino acid position 292 of SEQ ID NO: 5, an L at amino acid position 309 of SEQ ID NO: 5, an N at amino acid position 293 of SEQ ID NO: 5, an L at amino acid position 307 of SEQ ID NO: 5, an N at amino acid position 310 of SEQ ID NO: 5, an N at amino acid position 329 of SEQ ID NO: 5, an N at amino acid position 330 of SEQ ID NO: 5, an N at amino acid position 341 of SEQ ID NO: 5, an N at amino acid position 359 of SEQ ID NO: 5, an N at amino acid position 360 of SEQ ID NO: 5, an N at amino acid position 371 of SEQ ID NO: 5, an N at amino acid position 389 of SEQ ID NO: 5, an N at amino acid position 390 of SEQ ID NO: 5, an N at amino acid position 309 of SEQ ID NO: 5, an N at amino acid position 311 of SEQ ID NO: 5, an N at amino acid position 332 of SEQ ID NO: 5, an N at amino acid position 343 of SEQ ID NO: 537 of SEQ ID NO:5, an M at amino acid position 357 of SEQ ID NO:5, an L at amino acid position 35 ...3, an M at amino acid position 357 of SEQ ID NO:5, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:5, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID NO:3, an L at amino acid position 357 of SEQ ID NO:3, an M at amino acid position 357 of SEQ ID The AOS2 amino acid sequence having a T at amino acid position 394 of SEQ ID NO: 9, a G at amino acid position 407 of SEQ ID NO: 5, a C at amino acid position 407 of SEQ ID NO: 13, an F at amino acid position 423 of SEQ ID NO: 7, an L at amino acid position 430 of SEQ ID NO: 5, a deletion of amino acid E at position 439 of SEQ ID NO: 5, a G at amino acid position 467 of SEQ ID NO: 5, an S at amino acid position 467 of SEQ ID NO: 39, a V at amino acid position 480 of SEQ ID NO: 5, a G at amino acid position 494 of SEQ ID NO: 5, a D at amino acid position 494 of SEQ ID NO: 21 and / or a T at amino acid position 495 of SEQ ID NO: 5 comprises one or more mutations. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 6 of SEQ ID NO:7. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a P at amino acid position 12 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an R at amino acid position 12 of SEQ ID NO:11. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an R at amino acid position 12 of SEQ ID NO:11.NO:5 comprises one or more mutations. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 37 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 46 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 46 of SEQ ID NO:3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a T at amino acid position 48 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 48 of SEQ ID NO:27. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 48 of SEQ ID NO:7. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 51 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 76 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an N at amino acid position 76 of SEQ ID NO:5. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 113 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 113 of SEQ ID NO: 49. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 145 of SEQ ID NO: 9.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 187 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 187 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an E at amino acid position 197 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 197 of SEQ ID NO:3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a K at amino acid position 200 of SEQ ID NO:7. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an A at amino acid position 227 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a T at amino acid position 231 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 231 of SEQ ID NO:7. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 231 of SEQ ID NO:9. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 256 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 256 of SEQ ID NO:3. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having an A at amino acid position 264 of SEQ ID NO: 7. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having an A at amino acid position 264 of SEQ ID NO: 7.NO:7 comprises one or more mutations. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having an F at amino acid position 282 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having an S at amino acid position 282 of SEQ ID NO:41. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having a V at amino acid position 289 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to the AOS2 amino acid sequence having an S at amino acid position 289 of SEQ ID NO:11. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an N at amino acid position 289 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 292 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 309 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 309 of SEQ ID NO: 19. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 320 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 320 of SEQ ID NO: 23. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 328 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 328 of SEQ ID NO: 5.NO:19 comprises one or more mutations. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 328 of SEQ ID NO:27. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an E at amino acid position 337 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 337 of SEQ ID NO:13. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 338 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 338 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an I at amino acid position 357 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an M at amino acid position 357 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a P at amino acid position 381 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 381 of SEQ ID NO: 35. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a T at amino acid position 394 of SEQ ID NO: 9. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 407 of SEQ ID NO: 5. In some embodiments, the mutant AOS2 gene encodes a mutant AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 407 of SEQ ID NO: 5.NO:13 comprises one or more mutations. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an F at amino acid position 423 of SEQ ID NO:7. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an L at amino acid position 430 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an amino acid E deleted at position 439 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 467 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an S at amino acid position 467 of SEQ ID NO:39. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a V at amino acid position 480 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a G at amino acid position 494 of SEQ ID NO:5. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having a D at amino acid position 494 of SEQ ID NO:21. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising one or more mutations relative to an AOS2 amino acid sequence having an AOS2 A and / or AOS2 A at amino acid position 495 of SEQ ID NO:5.

[0201] In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises S6, P12, R12, V30, T37, F46, L46, I48, T48, I51, D76, N76, D113, G113, Y145, F187, D197, E197, T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, N289, S289, A292, I309, L309, L320, M320, L328, V328, D337, or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, or twenty-five or more mutations at positions E337, L338, V338, I357, M357, L381, P381, K394, C407, G407, I423, F430, Δ439 (where Δ indicates a deletion), G467, S467, T480, D494, G494, and K495.In some embodiments, the mutated AOS2 protein comprises two or more mutations, wherein at least one mutation is in a region corresponding to S6, P12, R12, V30, T37, F46, L46, I48, T48, I51, D76, D113, G113, Y145, F187, D197, E197, T200, T227, G231, T231, F256, V256, T264, F270, F282, S282, At the amino acid positions of N289, S289, A292, 1309, L309, L320, M320, L328, V328, D337, E337, L338, V338, 1357, M357, L381, P381, K394, C407, G407, 1423, F430, Δ439 (where Δ indicates a deletion), G467, S467, T480, D494, G494, and K495. In some embodiments, the mutated AOS2 gene comprises three or more mutations, wherein at least one mutation is in a region corresponding to S6, P12, R12, V30, T37, F46, L46, I48, T48, I51, D76, D113, G113, Y145, F187, D197, E197, T200, T227, G231, T231, F256, V256, T264, at amino acid positions F270, F282, S282, N289, S289, A292, 1309, L309, L320, M320, L328, V328, D337, E337, L338, V338, 1357, M357, L381, P381, K394, C407, G407, 1423, F430, Δ439 (where Δ indicates a deletion), G467, S467, T480, D494, G494, and K495.

[0202] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position F6 of SEQ ID NO: 7 or 9. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position R12 of SEQ ID NO: 1, 3, 7, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position P12 of SEQ ID NO: 11. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises a mutation at amino acid position A30 corresponding to a position of SEQ ID NO: 5. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position V30 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at amino acid position 137 corresponding to a position of SEQ ID NO: 5. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at amino acid position F46 corresponding to a position of SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position L46 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position I48 of SEQ ID NO: 27, 47 or 49. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position V48 of SEQ ID NO: 7.In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position T48 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, 39, 41, 43 or 45. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position M51 of SEQ ID NO: 5. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position N76 of SEQ ID NO: 5, 7, 9, 19, 21, 23, 25, 29, 31 or 43. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position G113 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position D113 of SEQ ID NO: 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position F145 of SEQ ID NO: 9. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position L187 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position E197 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position D197 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position K200 of SEQ ID NO: 7 or 9. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position A227 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position I231 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position G231 of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 29, 43, or 45.In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position T231 of SEQ ID NO: 1, 3, 5, 23, 25, 27, 31, 33, 35, 37, 39, 41, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position F256 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position V256 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position A264 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position L270 of SEQ ID NO: 7. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position F282 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47 or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position S282 of SEQ ID NO: 41. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position N289 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position V289 of SEQ ID NO: 5, 7 or 9. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position S289 of SEQ ID NO: 1, 3, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position V292 of SEQ ID NO: 5, 7, 9, or 13. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position L309 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 41, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position 1309 of SEQ ID NO: 19, 21, 23, 25, or 43.In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position M320 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position L320 of SEQ ID NO: 23. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position V328 of SEQ ID NO: 27, 33, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position M328 of SEQ ID NO: 5, 7, 9, 13, or 15. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position L328 of SEQ ID NO: 1, 3, 11, 17, 19, 21, 23, 25, 29, 31, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position E337 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position D337 of SEQ ID NO: 13 or 15. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position V338 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position L338 of SEQ ID NO: 13 or 15. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position I357 of SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position M357 of SEQ ID NO:3.In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position P381 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position L381 of SEQ ID NO: 35. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position T394 of SEQ ID NO: 9. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position C407 of SEQ ID NO: 13 or 15. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position G407 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position F423 of SEQ ID NO: 7, 25, 27, 33, 47 or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position L430 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position S467 of SEQ ID NO: 39. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position G467 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position V480 of SEQ ID NO: 5. In some embodiments, the mutated AOS2 protein comprises a mutation at an amino acid position corresponding to position D494 of SEQ ID NO: 21, 23, 31, or 43. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position G494 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 25, 27, 29, 33, 35, 37, 39, 41, 45, 47, or 49. In some embodiments, the mutant AOS2 protein comprises a mutation at an amino acid position corresponding to position T495 of SEQ ID NO: 5, 7, or 9.In some embodiments, the mutated AOS2 protein comprises a deletion at an amino acid position corresponding to position E439 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47, or 49.

[0203] In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid serine at a position corresponding to position 6 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid proline at a position corresponding to position 12 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid arginine at a position corresponding to position 12 of SEQ ID NO: 11. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid valine at a position corresponding to position 30 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid threonine at a position corresponding to position 37 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 46 of SEQ ID NO: 1. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 46 of SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid isoleucine at a position corresponding to position 48 of SEQ ID NO: 27, SEQ ID NO: 47 or SEQ ID NO: 49. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 48 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid isoleucine at a position corresponding to position 51 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid aspartic acid at a position corresponding to position 76 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid asparagine at a position corresponding to position 76 of SEQ ID NO: 1 or SEQ ID NO: 3.In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid glycine at a position corresponding to position 113 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid aspartic acid at a position corresponding to position 113 of SEQ ID NO: 49. In some embodiments, the mutated AOS2 protein comprises the amino acid tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 187 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 protein comprises the amino acid glutamic acid at a position corresponding to position 197 of SEQ ID NO: 1. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid aspartic acid at a position corresponding to position 197 of SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 200 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 227 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 231 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid glycine at a position corresponding to position 231 of SEQ ID NO: 9. In some embodiments, the mutant AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 256 of SEQ ID NO: 1. In some embodiments, the mutant AOS2 protein comprises the amino acid valine at a position corresponding to position 256 of SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 264 of SEQ ID NO: 1 or SEQ ID NO: 3.In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 270 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 282 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 protein comprises the amino acid serine at a position corresponding to position 282 of SEQ ID NO: 41. In some embodiments, the mutated AOS2 protein comprises the amino acid serine at a position corresponding to position 289 of SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the mutated AOS2 protein comprises the amino acid asparagine at a position corresponding to position 289 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 protein comprises the amino acid alanine at a position corresponding to position 292 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 309 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid isoleucine at a position corresponding to position 309 of SEQ ID NO: 19. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid methionine at a position corresponding to position 320 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 320 of SEQ ID NO: 23. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 328 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid valine at a position corresponding to position 328 of SEQ ID NO: 27. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid glutamic acid at a position corresponding to position 337 of SEQ ID NO: 1 or SEQ ID NO: 3.In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid aspartic acid at a position corresponding to position 337 of SEQ ID NO: 13 or SEQ ID NO: 15. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid valine at a position corresponding to position 338 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 338 of SEQ ID NO: 13 or SEQ ID NO: 15. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid isoleucine at a position corresponding to position 357 of SEQ ID NO: 1. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid methionine at a position corresponding to position 357 of SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid proline at a position corresponding to position 381 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid leucine at a position corresponding to position 381 of SEQ ID NO: 35. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid lysine at a position corresponding to position 394 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid glycine at a position corresponding to position 407 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid cysteine ​​at a position corresponding to position 407 of SEQ ID NO: 13 or SEQ ID NO: 15. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid isoleucine at a position corresponding to position 423 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid phenylalanine at a position corresponding to position 430 of SEQ ID NO: 1 or SEQ ID NO: 3.In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises a deletion of the amino acid glutamic acid at a position corresponding to position 439 of SEQ ID NO: 5. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid glycine at a position corresponding to position 466 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid serine at a position corresponding to position 467 of SEQ ID NO: 39. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid threonine at a position corresponding to position 479 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 protein comprises the amino acid glycine at a position corresponding to position 493 of SEQ ID NO: 1 or SEQ ID NO: 3. In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutant AOS2 protein comprises the amino acid aspartic acid at a position corresponding to position 494 of SEQ ID NO: 21. In some embodiments, the mutant AOS2 protein comprises the amino acid lysine at a position corresponding to position 494 of SEQ ID NO: 1 or SEQ ID NO: 3.

[0204] In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the mutant AOS2 gene encodes a mutant AOS2 protein having one or more mutations, two or more mutations, three or more mutations, four or more mutations, five or more mutations, six or more mutations, seven or more, eight or more, nine or more, or ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty or more, twenty-two or more, twenty-three or more, twenty-four or more, twenty-five or more mutations selected from the group consisting of: 45, 47 or 49, phenylalanine to serine at position 6, arginine to proline at the position corresponding to position 12, proline to arginine at the position corresponding to position 12, alanine to valine at the position corresponding to position 30, isoleucine to threonine at the position corresponding to position 37, phenylalanine to leucine at the position corresponding to position 46, leucine to phenylalanine at the position corresponding to position 46, valine to threonine at the position corresponding to position 48, valine to isoleucine at the position corresponding to position 48, isoleucine to threonine at the position corresponding to position 48, threonine to isoleucine at the position corresponding to position 48, methionine to isoleucine at the position corresponding to position 51, asparagine to aspartic acid at the position corresponding to position 76, 76 to asparagine, aspartic acid at the position corresponding to position 113 to glycine, glycine at the position corresponding to position 113 to aspartic acid, phenylalanine at the position corresponding to position 145 to tyrosine, leucine at the position corresponding to position 187 to phenylalanine, aspartic acid at the position corresponding to position 197 to glutamic acid, glutamic acid at the position corresponding to position 197 to aspartic acid, lysine at the position corresponding to position 200 to threonine, alanine at the position corresponding to position 227 to threonine, isoleucine at the position corresponding to position 231 to threonine, isoleucine at the position corresponding to position 231 to glycine, glycine at the position corresponding to position 231 to threonine, threonine at the position corresponding to position 231 to glycine, and threonine at the position corresponding to position 231 to glycine.256 to phenylalanine, phenylalanine at the position corresponding to position 256 to valine, alanine at the position corresponding to position 264 to threonine, leucine at the position corresponding to position 270 to phenylalanine, serine at the position corresponding to position 282 to phenylalanine, phenylalanine at the position corresponding to position 282 to serine, valine at the position corresponding to position 289 to asparagine, valine at the position corresponding to position 289 to serine, serine at the position corresponding to position 289 to asparagine, asparagine at the position corresponding to position 289 to serine, valine at the position corresponding to position 292 to alanine, isoleucine at the position corresponding to position 309 to leucine, leucine at the position corresponding to position 309 to isoleucine, leucine at the position corresponding to position 320 to methionine, methionine at the position corresponding to position 320 to leucine, to leucine at the position corresponding to position 328, to methionine at the position corresponding to position 328, to valine at the position corresponding to position 328, to leucine at the position corresponding to position 328, to leucine at the position corresponding to position 328, to valine at the position corresponding to position 328, to leucine at the position corresponding to position 328, to aspartic acid at the position corresponding to position 337, to glutamic acid at the position corresponding to position 337, to glutamic acid at the position corresponding to position 337, to leucine at the position corresponding to position 338, to valine at the position corresponding to position 338, to leucine at the position corresponding to position 357, to isoleucine at the position corresponding to position 357, to methionine at the position corresponding to position 381, to leucine at the position corresponding to position 382, ​​to proline at the position corresponding to position 383, to glutamic acid at the position corresponding to position 387, to aspartic acid at the position corresponding to position 388 1 to leucine, threonine to lysine at the position corresponding to position 394, cysteine ​​to glycine at the position corresponding to position 407, glycine to cysteine ​​at the position corresponding to position 407, phenylalanine to isoleucine at the position corresponding to position 423, leucine to phenylalanine at the position corresponding to position 430, serine to glycine at the position corresponding to position 467, glycine to serine at the position corresponding to position 467, valine to threonine at the position corresponding to position 480, aspartic acid to glycine at the position corresponding to position 494, glycine to aspartic acid at the position corresponding to position 494, threonine to lysine at the position corresponding to position 495, and glutamic acid at the position corresponding to position 439 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 39, 41, 43, 45, 47 or 49.

[0205] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to serine at a position corresponding to position 6 of SEQ ID NO: 1, 3, 5, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from arginine to proline at a position corresponding to position 12 of SEQ ID NO: 1, 3, 5, 7, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from proline to arginine at a position corresponding to position 12 of SEQ ID NO: 11. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from alanine to valine at a position corresponding to position 30 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to threonine at a position corresponding to position 37 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to leucine at a position corresponding to position 46 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to phenylalanine at a position corresponding to position 46 of SEQ ID NO:3.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to threonine at a position corresponding to position 48 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to threonine at a position corresponding to position 48 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from threonine to isoleucine at a position corresponding to position 48 of SEQ ID NO: 27, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to isoleucine at a position corresponding to position 48 of SEQ ID NO: 27, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from methionine to isoleucine at a position corresponding to position 51 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from asparagine to aspartic acid at a position corresponding to position 76 of SEQ ID NO: 1, 3, 11, 13, 15, 17, 27, 33, 35, 37, 39, 41, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from asparagine to aspartic acid at a position corresponding to position 76 of SEQ ID NO: 1, 3, 11, 13, 15, 17, 27, 33, 35, 37, 39, 41, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from aspartic acid to glycine at a position corresponding to position 113 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glycine to aspartic acid at a position corresponding to position 113 of SEQ ID NO: 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to phenylalanine at a position corresponding to position 187 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glutamic acid to aspartic acid at a position corresponding to position 197 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from aspartic acid to glutamic acid at a position corresponding to position 197 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from lysine to threonine at a position corresponding to position 200 of SEQ ID NO: 1, 3, 5, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from alanine to threonine at a position corresponding to position 227 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to threonine at a position corresponding to position 231 of SEQ ID NO: 1, 3, 5, 23, 25, 27, 31, 33, 35, 37, 39, 41, 47, or 49.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to glycine at a position corresponding to position 231 of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 29, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from threonine to glycine at a position corresponding to position 231 of SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 29 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glycine to threonine at a position corresponding to position 231 of SEQ ID NO: 1, 3, 5, 23, 25, 27, 31, 33, 35, 37, 39, 41, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to valine at a position corresponding to position 256 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to phenylalanine at a position corresponding to position 256 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from alanine to threonine at a position corresponding to position 264 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to phenylalanine at a position corresponding to position 270 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to serine at a position corresponding to position 282 of SEQ ID NO:41.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from serine to phenylalanine at a position corresponding to position 282 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to asparagine at a position corresponding to position 289 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to serine at a position corresponding to position 289 of SEQ ID NO: 1, 3, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from asparagine to serine at a position corresponding to position 289 of SEQ ID NO: 1, 3, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from serine to asparagine at a position corresponding to position 289 of SEQ ID NO: 13. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to alanine at a position corresponding to position 292 of SEQ ID NO: 1, 3, 11, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to isoleucine at a position corresponding to position 309 of SEQ ID NO: 19, 21, 23, 25, or 43. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to leucine at a position corresponding to position 309 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 27, 29, 31, 33, 35, 37, 39, 41, 45, 47 or 49.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to methionine at a position corresponding to position 320 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from methionine to leucine at a position corresponding to position 320 of SEQ ID NO: 23. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from methionine to leucine at a position corresponding to position 328 of SEQ ID NO: 27, 33, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from methionine to leucine at a position corresponding to position 328 of SEQ ID NO: 1, 3, 11, 17, 19, 21, 23, 25, 29, 31, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to valine at a position corresponding to position 328 of SEQ ID NO: 27, 33, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to leucine at a position corresponding to position 328 of SEQ ID NO: 1, 3, 11, 17, 19, 21, 23, 25, 29, 31, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from aspartic acid to glutamic acid at a position corresponding to position 337 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glutamic acid to aspartic acid at a position corresponding to position 337 of SEQ ID NO: 13 or 15.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to valine at a position corresponding to position 338 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to leucine at a position corresponding to position 338 of SEQ ID NO: 13 or 15. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from methionine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from isoleucine to methionine at a position corresponding to position 357 of SEQ ID NO: 3. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to proline at a position corresponding to position 381 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from proline to leucine at a position corresponding to position 381 of SEQ ID NO: 35. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from threonine to lysine at a position corresponding to position 394 of SEQ ID NO: 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from cysteine ​​to glycine at a position corresponding to position 407 of SEQ ID NO: 1, 3, 5, 7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glycine to cysteine ​​at a position corresponding to position 407 of SEQ ID NO: 13 or 15. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from phenylalanine to isoleucine at a position corresponding to position 423 of SEQ ID NO: 1, 3, 5, 9, 11, 13, 15, 17 19, 21, 23, 29, 31, 35, 37, 39, 41, 43, or 45. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from leucine to phenylalanine at a position corresponding to position 430 of SEQ ID NO: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from serine to glycine at a position corresponding to position 467 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 41, 43, 45, 47, or 49, or position 466 of SEQ ID NO: 1, 3, 29, 31, 35, or 37. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glycine to serine at a position corresponding to position 467 of SEQ ID NO: 39. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from valine to threonine at a position corresponding to position 480 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, or position 479 of SEQ ID NO: 1, 3, 29, 31, 35, or 37. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from aspartic acid to glycine at a position corresponding to position 494 of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 25, 27, 29, 33, 35, 37, 39, 41, 45, 47 or 49, or position 493 of SEQ ID NO: 1, 3, 29, 35 or 37.In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from glycine to aspartic acid at a position corresponding to position 494 of SEQ ID NO: 21, 23, or 43, or position 493 of SEQ ID NO: 31. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation from threonine to lysine at a position corresponding to position 495 of SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, or position 494 of SEQ ID NO: 1, 3, 29, 31, 35, or 37. In some embodiments, the mutated AOS2 gene encodes a mutated AOS2 protein comprising an amino acid mutation at a position corresponding to position 439 of SEQ ID NO: 1, 3, 29, 31, 35, or 37, in which glutamic acid is deleted.

[0206] In combination with any of the aspects, embodiments, compositions and methods disclosed herein, the mutated AOS2 gene comprises at least one mutation, at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, at least ten mutations, at least eleven mutations, at least twelve mutations, at least thirteen mutations, at least fourteen mutations, at least fifteen mutations, at least sixteen mutations, at least seventeen mutations, at least eighteen mutations, at least nineteen mutations, at least twenty mutations, at least twenty-one mutations, at least twenty-two mutations, at least twenty-three mutations, at least twenty-four mutations, at least twenty-five mutations, at least twenty-six mutations, at least twenty-seven mutations, at least twenty-eight mutations, at least twenty-nine mutations, at least thirty mutations, at least thirty-one mutations, at least thirty-two mutations, at least thirty-three mutations, at least thirty-four mutations, at least thirty-five mutations, at least thirty-six mutations, or at least thirty-seven mutations.

[0207] Paralogs

[0208] The subject mutations in the AOS2 gene are generally described using selected potato AOS2 genes and proteins, with amino acid positions referenced to positions in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49, and nucleic acid positions referenced to positions in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. The compositions and methods also encompass mutant AOS2 genes and proteins (paralogs) from other potato cultivars, as well as other plant species. However, due to the variation in the AOS2 genes of different species, the number of amino acid residues to be changed in one species may be different from another species. Nevertheless, similar positions can still be easily identified by sequence homology by those skilled in the art. Therefore, similar positions in paralogs can be identified and mutated.

[0209] pathogens

[0210] The compositions and methods provided herein include AOS2 genes and AOS2 proteins that confer resistance and / or tolerance to pathogens. In some embodiments, the pathogen is a Phytophthora pathogen. In a specific embodiment, the pathogen is a Phytophthora infestans. In a specific embodiment, the pathogen is a virus, bacterium, nematode, fungus, etc. Viral pathogens include any plant virus, such as tobacco or cucumber mosaic virus, potato virus Y, ringspot virus, necrosis virus, corn dwarf mosaic virus, etc. Fungal, oomycete and viral pathogens of major crops include, but are not limited to, Phytophthora, Fusarium ssp, Alternaria, Pythium spp, soybean mosaic virus, tobacco ringspot virus, tobacco streak virus, tomato spotted wilt virus, Sclerotinia, Peronospora, Cladosporium, Erysiphe, Aspergillus, Puccinia spp, Botrytis spp, Blumeria spp, and Trichoderma.Bacterial plant pathogens include any bacterial species that infects plants and include, but are not limited to, Xanthomonas (e.g., Xanthomonas axonopodis pv. aurantifolii, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. vesicatoria), Pseudomonas (Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. syringae), Pseudomonas spp. syringae), Erwinia (e.g., Erwinia carotovora subsp. atroseptica), Ralstonia (e.g., Ralstonia solanacearum), Clavibacter michiganensis, and Xylella fastidiosa.

[0211] Also provided are transgenic or non-transgenic plants or plant cells having one or more mutations in the AOS2 gene, such as those disclosed herein. In certain embodiments, the plants or plant cells having one or more mutations in the AOS2 gene have increased resistance and / or tolerance to pathogens. In certain embodiments, the plants or plant cells having one or more mutations in the AOS2 gene may exhibit substantially normal growth or development of the plant, its organs, tissues, or cells compared to corresponding wild-type plants or cells. In particular, aspects and embodiments are provided of non-transgenic plants having mutations in the AOS2 gene, such as those disclosed herein, which plants, in certain embodiments, have increased resistance and / or tolerance to Phytophthora infestans.

[0212] Further provided are methods for producing plants having a mutated AOS2 gene (e.g., having one or more mutations as described herein); preferably, the plant substantially maintains the catalytic activity of the wild-type protein, regardless of the presence or absence of the relevant pathogen. In certain embodiments, the method comprises introducing into a plant cell a gene repair oligonucleobase having one or more targeted mutations in the AOS2 gene (such as disclosed herein) and identifying cells, seeds, or plants having a mutated AOS2 gene.

[0213] plant species

[0214] In conjunction with any of the various aspects, embodiments, compositions and methods disclosed herein, the plant or plant cell can be a plant or plant cell of any species of dicotyledonous, monocotyledonous or gymnosperm, including any woody plant species that grows as a tree or shrub, any herbaceous plant species, or any species that produces edible fruit, seeds or vegetables, or any species that produces colorful or fragrant flowers. For example, the plant or plant cell can be selected from a plant species selected from potato, sunflower, sugar beet, corn, cotton, soybean, wheat, rye, oats, rice, rapeseed, fruit, vegetables, tobacco, eggplant (aubergine), barley, boxthane, sorghum, tomato, green tomato, tamarillo, mango, peach, apple, pear, strawberry, banana, melon, wolfberry, horticultural black fruit tree, ground cherry, carrot, lettuce, onion, bean, sugarcane, pea, silkworm Beans, poplars, grapes, citrus, alfalfa, rye, oats, lawn and forage grasses, cucurbits, flax, Brassica, cucumbers, pumpkins, zucchini, watermelons, cantaloupes, morning glories, impatiens, peppers, sweet peppers, bell peppers, red peppers, green and red peppers, pimento trees, habanero peppers, capsicum, eggplant, marigolds, lotus flowers, cabbage, daisies, carnations, tulips, irises, lilies, and nut-producing plants, as long as they have not been specifically mentioned. The plant or plant cell can be a plant or plant cell of a species selected from Arabidopsis thaliana, potato, Solanum phureja, Oryza sativa, Amaranthus tuberculatus, and Zea mays. In various embodiments, the plant as disclosed herein can be a plant of any species of the Solanaceae family.

[0215] In some embodiments, the plant or plant cell can be tomato. In some embodiments, the plant or plant cell can be eggplant. In some embodiments, the plant or plant cell can be pepper. In some embodiments, the plant or plant cell can be soybean. In some embodiments, the plant or plant cell can be tobacco.

[0216] In conjunction with any of the aspects, embodiments, compositions, and methods disclosed herein, the plant can be any commercial variety of potato. For example, the plant or plant cell can be selected from a potato variety selected from Anya, Arran Victory, Atlantic, Belle de Fontenay, BF-15, Bintje, Cabritas, Camota, Chelina, Chiloé, Cielo, Clavera Blanca, Désirée, Fianna, Fingerling, Fontana, Flava, Golden Wonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, Maris Piper, Nicola, Pink Eye, Pink FirApple, Primura, Red Norland, Red Pontiac, Rooster, Russet Burbank, RussetNorkotah, Shepody, Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs'sPink / Meru, Golof, Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro, America, Roslin Bvumbwe, Njine, Roslin Gucha, Arka, B53 (Roslin Eburu), Kiraya, Kenya Akiba, 9, Original, Gituma, Mukorino, Amin, Pimpernel, Anett, B, Gituru, Feldeslohn, C, Kigeni, Romano, Kenya Ruaka,Purplu,Njae,Suzanna, Cardinal, Kathama, Kinare-Mwene, Kibururu, Karoa-Igura, Muturu, Faraja, Kiamucove, Michiri, Rugano, NjineGiathireko, Meru Mix, Blue Baranja, Patrones, Robijn, Roslin Chania, Urgentia, Mirka and Roslin Sasamua.

[0217] In various embodiments, the plants or plant cells disclosed herein can be any commercial variety of potato. In some embodiments, the plant or plant cell can be of the potato variety Anya. In some embodiments, the plant or plant cell can be of the potato variety Arran Victory. In some embodiments, the plant or plant cell can be of the potato variety Atlantic. In some embodiments, the plant or plant cell can be of the potato variety Belle de Fontenay. In some embodiments, the plant or plant cell can be of the potato variety BF-15. In some embodiments, the plant or plant cell can be of the potato variety Bintje. In some embodiments, the plant or plant cell can be of the potato variety Cabritas. In some embodiments, the plant or plant cell can be of the potato variety Camota. In some embodiments, the plant or plant cell can be of the potato variety Chelina. In some embodiments, the plant or plant cell can be of the potato variety Chiloé, Cielo. In some embodiments, the plant or plant cell can be of the potato variety Clavera Blanca. In some embodiments, the plant or plant cell may be of the potato variety Désirée. In some embodiments, the plant or plant cell may be of the potato variety Fianna. In some embodiments, the plant or plant cell may be of the potato variety Fingerling. In some embodiments, the plant or plant cell may be of the potato variety Flava. In some embodiments, the plant or plant cell may be of the potato variety Fontana. In some embodiments, the plant or plant cell may be of the potato variety Golden Wonder. In some embodiments, the plant or plant cell may be of the potato variety Innovator. In some embodiments, the plant or plant cell may be of the potato variety Jersey Royal. In some embodiments, the plant or plant cell may be of the potato variety Kerr's Pink. In some embodiments, the plant or plant cell may be of the potato variety Kestrel. In some embodiments, the plant or plant cell may be of the potato variety King Edward. In some embodiments, the plant or plant cell may be of the potato variety Kipfler. In some embodiments, the plant or plant cell can be of the potato variety Lady Balfour. In some embodiments, the plant or plant cell can be of the potato variety Maris Piper. In some embodiments, the plant or plant cell can be of the potato variety Nicola.In some embodiments, the plant or plant cell may be a potato variety. In some embodiments, the plant or plant cell may be of the potato variety Pink Eye. In some embodiments, the plant or plant cell may be of the potato variety PinkFir Apple. In some embodiments, the plant or plant cell may be of the potato variety Primura. In some embodiments, the plant or plant cell may be of the potato variety Red Norland. In some embodiments, the plant or plant cell may be of the potato variety Red Pontiac. In some embodiments, the plant or plant cell may be of the potato variety Rooster. In some embodiments, the plant or plant cell may be of the potato variety Russet Burbank. In some embodiments, the plant or plant cell may be of the potato variety Russet Norkotah. In some embodiments, the plant or plant cell may be of the potato variety Shepody. In some embodiments, the plant or plant cell may be of the potato variety Spunta. In some embodiments, the plant or plant cell may be of the potato variety Vivaldi. In some embodiments, the plant or plant cell may be of the potato variety Yukon Gold. In some embodiments, the plant or plant cell may be of the potato variety Nyayo. In some embodiments, the plant or plant cell may be of the potato variety Mukori. In some embodiments, the plant or plant cell may be of the potato variety Roslin Tana. In some embodiments, the plant or plant cell may be of the potato variety Kerrs's Pink / Meru. In some embodiments, the plant or plant cell may be of the potato variety Golof. In some embodiments, the plant or plant cell may be of the potato variety Kinongo. In some embodiments, the plant or plant cell may be of the potato variety Ngure. In some embodiments, the plant or plant cell may be of the potato variety KenyaBaraka. In some embodiments, the plant or plant cell may be of the potato variety Maritta. In some embodiments, the plant or plant cell may be of the potato variety Kihoro. In some embodiments, the plant or plant cell may be of the potato variety Americar. In some embodiments, the plant or plant cell may be of the potato variety Roslin Bvumbwe. In some embodiments, the plant or plant cell can be of the potato variety Njine. In some embodiments, the plant or plant cell can be of the potato variety Roslin Gucha. In some embodiments, the plant or plant cell can be of the potato variety Arka.In some embodiments, the plant or plant cell may be of the potato variety B53 (Roslin Eburu). In some embodiments, the plant or plant cell may be of the potato variety Kiraya. In some embodiments, the plant or plant cell may be of the potato variety Kenya Akiba. In some embodiments, the plant or plant cell may be of the potato variety 9. In some embodiments, the plant or plant cell may be of the potato variety Original. In some embodiments, the plant or plant cell may be of the potato variety Gituma. In some embodiments, the plant or plant cell may be of the potato variety Mukorino. In some embodiments, the plant or plant cell may be of the potato variety Amin. In some embodiments, the plant or plant cell may be of the potato variety Pimpernel. In some embodiments, the plant or plant cell may be of the potato variety Anett. In some embodiments, the plant or plant cell may be of the potato variety B. In some embodiments, the plant or plant cell may be of the potato variety Gituru. In some embodiments, the plant or plant cell may be of the potato variety Feldeslohn. In some embodiments, the plant or plant cell may be of the potato variety C. In some embodiments, the plant or plant cell may be of the potato variety Kigeni. In some embodiments, the plant or plant cell may be of the potato variety Romano. In some embodiments, the plant or plant cell may be of the potato variety Kenya Ruaka. In some embodiments, the plant or plant cell may be of the potato variety Purplu. In some embodiments, the plant or plant cell may be of the potato variety Njae. In some embodiments, the plant or plant cell may be of the potato variety Suzanna. In some embodiments, the plant or plant cell may be of the potato variety Cardinal. In some embodiments, the plant or plant cell may be of the potato variety Kathama. In some embodiments, the plant or plant cell may be of the potato variety Kinare-Mwene. In some embodiments, the plant or plant cell may be of the potato variety Kibururu. In some embodiments, the plant or plant cell can be of the potato variety Karoa-Igura. In some embodiments, the plant or plant cell can be of the potato variety Muturu. In some embodiments, the plant or plant cell can be of the potato variety Faraja. In some embodiments, the plant or plant cell can be of the potato variety Kiamucove.In some embodiments, the plant or plant cell may be of the potato variety Michiri. In some embodiments, the plant or plant cell may be of the potato variety Rugano. In some embodiments, the plant or plant cell may be of the potato variety Njine Giathireko. In some embodiments, the plant or plant cell may be of the potato variety Meru Mix. In some embodiments, the plant or plant cell may be of the potato variety Blue Baranja. In some embodiments, the plant or plant cell may be of the potato variety Patrones. In some embodiments, the plant or plant cell may be of the potato variety Robijn. In some embodiments, the plant or plant cell may be of the potato variety Roslin Chania. In some embodiments, the plant or plant cell may be of the potato variety Urgentia. In some embodiments, the plant or plant cell may be of the potato variety Mirka. In some embodiments, the plant or plant cell may be of the potato variety Roslin Sasamua.

[0218] Gene repair oligonucleobases can be introduced into plant cells using any method commonly used in the art, including but not limited to microcarriers (biolistic delivery), microfibers, polyethylene glycol (PEG)-mediated uptake, electroporation, and microinjection.

[0219] Also provided are methods and compositions involving culturing cells mutated according to the methods disclosed herein to obtain plants that produce seeds (hereinafter "fertile plants"), and producing seeds and additional plants from such fertile plants.

[0220] Also provided are methods and compositions involving culturing cells mutated according to the methods disclosed herein to obtain plants that produce substantially normal tubers at a substantially normal yield, such that substantially normal plants are produced from tubers or small pieces containing at least one or two eyes (dormant buds), commonly referred to as seed potatoes.

[0221] Also provided are mutations in the AOS2 gene that confer resistance and / or tolerance to relevant pathogens on the plant or wherein the mutated AOS2 gene has substantially the same or altered enzymatic activity compared to wild-type AOS2.

[0222] Selection of pathogen-resistant plants and application of pathogens

[0223] Plants and plant cells can be tested for resistance and / or tolerance to pathogens using methods generally known in the art, for example, by growing the plant or plant cells in the presence of the pathogen and measuring the growth rate compared to the growth rate in the absence of the pathogen. Pathogen challenges for selecting resistant and / or tolerant plants can be achieved by applying pathogen sporangia or zoospores. The resistance level of plants subjected to these challenges can be assessed using various methods, such as determining the rate of increase in pathogen DNA from infected plant material, the rate of progression of lesion size, and the like.

[0224] As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as: the growth rate or cell division rate of the plant, plant organ, plant tissue or plant cell is at least 35%, at least 50%, at least 60% or at least 75% of the growth rate or cell division rate of the corresponding plant, plant organ, plant tissue or plant cell expressing wild-type AOS2 protein.

[0225] As used herein, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as one or more developmental events occurring in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing a wild-type AOS2 protein.

[0226] In certain embodiments, plant organs provided herein include but are not limited to leaves, stems, roots, leaf buds, flower buds, meristems, embryos, cotyledons, endosperms, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or parts, slices, disks etc. taken from them. Plant tissues include but are not limited to callus, ground tissue, vascular tissue, storage tissue, meristems, 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 and aggregates of various sizes thereof, and protoplasts.

[0227] When a plant is treated with a relevant pathogen and provides a dose / response curve that shifts to the right compared to a dose / response curve provided by a non-tolerant similar plant that receives a similar treatment, the plant is essentially "resistant" to the pathogen. Such a dose / response curve has a "dose" plotted on the x-axis and a "kill percentage," "pathogenic effect," etc. plotted on the y-axis. Tolerant plants will require more pathogens than non-tolerant similar plants to produce a given pathogenic effect. When treated with pathogens at typical concentrations and rates at which pathogens are exposed in the field, essentially "resistant" pathogen plants rarely, if ever, exhibit necrosis, cracking, chlorosis, or other pathological changes. Pathogen-resistant plants are also resistant to pathogens.

[0228] Polymerase chain reaction method for detection and quantification of pathogens in plants

[0229] Established methods and those known to those skilled in the art can be used to determine host resistance to pathogens. Generally, a variety of methods are typically used for a variety of pathogens, but the following methods are generally applicable to fungal and bacterial pathogens.

[0230] Pathogen resistance and / or tolerance can be determined by monitoring the presence and amount of pathogen-specific nucleic acids in plants. For example, plant leaflets are inoculated on both sides of the midrib with 10 μL droplets of a sporangium suspension (30-40 sporangia / μL). Oberhagemann, P, et al. Mol. Breed. Vol. 5, pp. 399-415 (1999). Disease symptoms can be scored 7 days after infection. DNA is extracted from infected plant material. Pathogen growth is monitored using P. infestans-ribosomal DNA-specific primers as described (exemplary forward primer sequence: 5'-GAAAGGCATAGAAGGTAGA-3' and exemplary reverse primer sequence: 5'-TAACCGACCAAGTAGTAAA-3'). The intensity of the P. infestans amplicon is calibrated against the potato tubulin DNA band. Band intensities are quantified and converted to arbitrary units relative to absolute values ​​obtained from control plants. Judelson, HS, et al. Phytopathology, Vol. 90, pp. 1112-1119 (2000).

[0231] The pathogen resistance level of potato plants of interest can be assessed by challenging the plants with Phytophthora infestans or other pathogens of interest. For Phytophthora infestans, leaves from 6-8 week old plants are removed and placed abaxially up on 4% water agar plates. The leaves are inoculated with a Pasteur pipette onto the abaxial side of the leaf with a drop of a sporangium suspension (40,000-100,000 sporangia / mL). The plates are placed in an incubator at 18°C ​​with a 12-hour photoperiod.

[0232] Disease development will be scored 6 days after inoculation and, when necessary, according to published methods, such as those described in Vleeshouwers et al. (2000) Physiol and Mol Plant Pathology, Vol. 57, pp. 35-42; Vleeshouwers et al. (1999) Europ J of Plant Pathology, Vol. 105, pp. 241-250; Oberhagemann et al. (1999) Molecular Breeding, Vol. 5, pp. 399-415.

[0233] For fungal infection assays, infection level assessments will be performed according to published methods for each fungal-host interaction. References include Rogers et al. (1994) Plant Cell, Vol. 6, pp. 935–945; Valent et al. (1991) Genetics, Vol. 127, pp. 87–101; Thomas et al. (1997) Plant Cell, Vol. 9, pp. 2209–2224.

[0234] Typically, for sporulating fungi, inoculation is performed using an inoculum containing spores of the fungus of interest at the desired concentration. This inoculum will be sprayed onto the plant at a specific developmental stage (e.g., before the fourth leaf emerges / 6-8 weeks of age, etc.). The inoculated plants will be incubated for 24 hours after inoculation under high humidity conditions and then transferred to the desired growth conditions, which are under a diurnal cycle suitable for host plant growth. Infection density will typically be assessed 3-4 days after infection and scored according to established methods for host-pathogen systems. Typically, lesions that do not form sporulation will be assessed as a "resistant" response, while lesions that form sporulation will be considered a "susceptible" response. The severity of infection is assessed for the latter based on the size and appearance of the lesions.

[0235] For the assessment of disease severity associated with bacterial pathogens, published methods for each bacterial species will be used as described in the following literature: Elibox, W, et al. (2008) Phytopathology, Vol. 98, pp. 421-426; Chaudhry et al. (2006) Pakistan J of Botany, Vol. 38, No. 1, pp. 193-203; Zhao et al. (2005) J of Bacteriology, Vol. 187, pp. 8088. Typically, for bacterial pathogens, a predetermined density (e.g., 5x10 4 A bacterial suspension containing 200 (600) colony-forming units (CFU) will be infiltrated into the leaves of host plants at a specific developmental stage (e.g., 3-week-old plants). The inoculated plants will be maintained under high humidity for 3-4 days, and then the severity of the infection will be assessed by collecting two to three leaf discs, crushing them, and plating the resulting supernatant onto bacterial growth medium to count the bacterial CFUs produced by the infected plant material.

[0236] The severity of infection in transformed plants will be assessed by evaluating the colony forming units produced by infected tissue of transformed plants compared to the colony forming units produced by infected tissue of wild-type plants.

[0237] Those skilled in the art will readily appreciate that the present invention is well able to carry out the objects of the invention and obtain the ends and advantages mentioned as well as those inherent therein.The examples provided herein represent preferred embodiments and are exemplary and are not intended to limit the scope of the invention.

[0238] Example

[0239] Below are examples, which illustrate procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0240] Example 1: Using RTDS TM Technology improves plant pathogen resistance

[0241] Evaluate the genotype of the cultivar of interest at the AOS2 locus

[0242] Using techniques known to those skilled in the art, the potato cultivars of interest were subjected to genotyping as follows: genomic DNA of the plant cultivars of interest was extracted by known methods and subjected to polymerase chain reaction (PCR)-mediated gene amplification to isolate all AOS2 alleles present in the genomic DNA sample. The PCR primers used for amplification were as follows: forward primer 5'-CACCTTTGTATCACTAACATTACCCATCC-3' (SEQ ID NO: 51) and reverse primer 5'-GCATGTGTTGCTTGTTCTTATAATTTCAG-3' (SEQ ID NO: 52). The amplified fragments were cloned into the TOPO 2.1 vector (Invitrogen Corporation, Carlsbad, CA) and sequenced at 12 clones per amplification. The resulting sequences were aligned with the reference sequence (SEQ ID NO 2) and polymorphic sites were determined using the Vector NTI software analysis package (Invitrogen Corporation, Carlsbad, CA). The nucleic acid sequences were also translated into protein coding sequences using Vector NTI sequence analysis software, and the resulting sequences were compared to a reference protein sequence (SEQ ID NO 1) to identify polymorphic amino acids. All detected amino acid polymorphisms collected to date and their positions in the protein sequence are provided in Table 1. Amino acid positions are named according to their amino acid positions in the reference protein sequence given by SEQ ID NO 1.

[0243] Characterization of the biochemical activity of AOS2 alleles (in vitro)

[0244] The nucleic acid of the identified AOS2 allele was PCR amplified using primers as described earlier, but Xma I and Pst I sites were added to the forward and reverse primers, respectively, to introduce Xma I and Pst I sites at the 5' and 3' ends of the allele, respectively, to facilitate cloning of the amplified product into the pQE30 vector for heterologous expression in Escherichia coli (E. coli) (M15 strain, Qiagen Inc, Valencia, CA). The PCR amplified fragment was digested with Xma I and Pst I restriction enzymes and then cloned into a similarly digested pQE30b vector (which underwent site-directed mutagenesis to insert nucleotides 5' of the Xma I site so that any gene fragment cloned into the Xma I site was in frame with the coding sequence of the pQE30 vector) and clones were selected by transformation into the E. coli strain XL-1 Blue. The resulting expression plasmid was extracted from XL-1 Blue cells and subjected to colony PCR and sequencing to confirm any frameshifted clones and the absence of any fragments. Confirmed clones were transformed into M15 cells (Qiagen Inc, Valencia, CA) and used for protein expression analysis.

[0245] For protein expression analysis, 500 μL of a 5 mL overnight culture of the strain of interest (e.g., a vector-only strain with a plasmid but no AOS2 gene and a strain of interest with a single allele of the AOS2 gene) was inoculated into 10 mL LB medium supplemented with carbenicillin (100 μg / mL) and kanamycin (25 μg / mL). The culture was incubated at 37 ° C with 250 rpm stirring until the absorbance at 600 nm (A600) reached the desired OD unit (e.g., 0.6-0.8 OD units). The cells were then induced for protein expression with 1 mM IPTG and incubated for the desired period of time (e.g., 3-7 days) at the desired temperature (e.g., 12 ° C) with 100 rpm stirring. Protein expression was monitored by SDS PAGE gel electrophoresis, and the expression of type I cytochrome P450 proteins was monitored by spectral analysis. AOS2 protein purification was performed using commercially available Ni NTA binding columns according to the manufacturer's instructions (Thermo Scientific, Rockford, IL).

[0246] Biochemical assays used to characterize the catalytic activity of AOS2 protein

[0247] Purified proteins expressed in Escherichia coli were used to determine the catalytic activity of proteins encoded by identified alleles of the AOS2 gene. The assays were performed according to a published protocol (Schreier and Lorenz (1982) Z. Naturforsch, Vol. 37, p. 165). Generally, 13S-hydroperoxy-9Z,11E-octadecatrienoic acid (13-HPODE) and 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (HPOTrE) served as substrates for the enzyme assays, and a reference sample without enzyme was used as a negative control. To assess the catalytic activity of different proteins encoded by different AOS2 alleles, known amounts of purified protein, standardized by spectroscopic analysis or other means, were assayed with 3–13 μM substrate solutions in 0.1 M phosphate buffer (pH 6.0). The rate of decrease in absorbance at A234 was monitored over time, and the resulting kinetic data were used to calculate the specific activity of each protein of interest. Enzymes with the highest specific activities were considered those of interest, and the amino acid sequences of such enzymes were compared to those with lower specific activities to identify specific amino acid positions that confer superior catalytic activity to the AOS2 protein.

[0248] To evaluate the effect of the G231T mutation, nucleotides (nt) 691 / 692 of the StAOS2 alleles StAOS2_CB17 and StAOS2_CB18 of Bintje were converted from G / G to A / C using site-directed mutagenesis (SDM), resulting in a G231T shift in the corresponding AOS2 proteins. The amino acid (aa) polymorphisms found throughout these AOS2 proteins are given in Table 2. Those clones were subjected to biochemical assays as described above, and the specific activities of those proteins, as well as those altered at the 231 aa position, are given in Table 3.

[0249] Table 2: Genotypic differences between StAOS2 alleles of Bintje at amino acid positions 48, 76, 231, 328, 423 and 494 assayed for biochemical activity.

[0250] 48 76 231 328 423 494 StAOS2_CB17 T N G L I D StAOS2_CB17_G231T T N T L I D StAOS2_CB18 T D G L I G StAOS2_CB18_G231T T D T L I G

[0251] Table 3: Specific activities of protein-encoding StAOS2 alleles, namely StAOS2_CB18 and StAOS2_CB17 and their derivatives. Genotypes at positions 691 / 692 nt as G / G and A / C, respectively, correspond to G and T at 231 aa in the encoded protein.

[0252]

[0253]

[0254] As shown in Table 3, when the specific activities of isogenic proteins that differ only at the 231aa position are compared with each other, the conversion of the genotype at the 691 / 692nt position of the StAOS2 gene allele from G / G to A / C results in an increase in the specific activity of the encoded protein.

[0255] Further evaluation of the effect of the amino acid profile at positions 231 and 328 of the protein encoded by StAOS2_CB18 showed that the combination of amino acid compositions at these two positions increased the specific activity of the AOS2 protein. The data are provided in Table 4.

[0256] Table 4: Specific activities of the protein encoded by the StAOS2_CB18 allele and its derivatives differing at residues 231 and 328 aa.

[0257]

[0258] The AOS2 protein encoded by the StAOS2_CB18 allele has an amino acid (aa) profile at position 328aa changed from L to V (StAOS2_CB1_L328V), which increases activity when combined with G at position 231aa, but decreases activity when combined with T at position 231aa (StAOS2_CB18_G231_L328V). The data indicate that the G231T transition leads to a decrease in the specific activity of the AOS2 protein when combined with the L328V mutation and suggest that the interaction between the aa profiles at these two positions affects the activity of the AOS2 protein.

[0259] In vitro activity assays were also used to test the effect of the D76N mutation in StAOS2_CB19. StAOS2_CB19 was subjected to SDM to generate the StAOS2_CB19_D76N allele, in which the 76th residue in the AOS2 protein was converted from aspartic acid (D) to asparagine (N). The specific activity differences were evaluated using the above-mentioned method. The data collected from these independent experiments showed that the D76N mutation caused an approximately 30% decrease in enzyme activity.

[0260] Those alleles with superior catalytic activity were selected for in planta assays.

[0261] Characterization of the biochemical activity of AOS2 alleles (in vivo)

[0262] To evaluate the hypothesis that AOS2 proteins with excellent in vitro biochemical activity would also have excellent in planta biochemical activity, AOS2 alleles exhibiting superior specific activity were cloned into plant binary vectors under either the constitutive or Arabidopsis AOS2 promoter. These constructs were transformed into the Arabidopsis AOS2 gene-disrupted plant line CS6149 (TAIR, http: / / www.arabidopsis.org / ) using Agrobacterium tumefaciens-mediated transformation methods using established methods (Bent et al. (2000) Plant Physiol, Vol. 124, p. 1540). Transformants were identified by appropriate selection (depending on the presence of a selectable marker in the binary vector—i.e., kanamycin for the nptII gene—as a selectable marker), molecular methods, and the ability of the introduced AOS2 gene to complement the abnormal pollination / silique development AOS2-deficient phenotype caused by male sterility due to the absence of a functional AOS2 gene. JA and / or OPDA levels were assessed in AOS2 gene-complemented plant lines under basal and induced conditions using established methods (Chebab et al. (2008), PLoS ONE, Vol. 3: p. e1904; Schmelz et al. (2003) Plant Physiol, Vol. 133: p. 295; Engelberth et al. (2003) Anal Biochem, Vol. 312, p. 242). Alternatively, the complemented lines were used in plant disease assays with Arabidopsis pathogens (such as Erwinia carotovora or ssp. carotovora or Hyaloperonospora arabidopsidis and / or other fungi) to test the hypothesis that higher JA levels or AOS2 catalytic activity lead to enhanced resistance and / or tolerance to pathogens.

[0263] To evaluate the effect of aa polymorphisms in the AOS2 protein on jasmonic acid (JA) accumulation in plants, two alleles of the Bintje potato cultivar, StAOS2_CB18_G231T, driven by the Arabidopsis thaliana AtAOS2 promoter, were used to complement the null mutant phenotype of Arabidopsis thaliana aos2 mutant plants. The resulting transgenic plants were introduced into the T3 generation to obtain homozygotes, and the resulting plants were subjected to JA quantification studies according to the methods described (Chebab et al. (2008), PLoS ONE, Vol. 3: p. e1904; Schmelz et al. (2003) Plant Physiol., Vol. 133: p. 295; Engelberth et al. (2003) Anal Biochem., Vol. 312, p. 242). The results are shown in Table 5.

[0264] Table 5: JA accumulation profiles of Arabidopsis transgenic lines with StAOS2_CB19 or StAOS2_Cb18_G231T alleles. The average JA amounts shown represent the JA levels present in Arabidopsis leaf tissue at basal expression levels (expressed in ng per gram fresh weight). The results shown are the averages of two replicates including multiple leaves.

[0265] StAOS2 allele Plant strains Average JA Standard error StAOS2_CB19 1001-13-6 32.34 4.25 1001-14-6 29.94 5.69 1001-14-7 59.91 1.96 1001-19-4 107.21 22.59 1001-4-6 16.04 1.63 1001-9-1 20.48 7.47 StAOS2_CB18_G231T 1003-10-8 20.90 6.26 1003-16-6 84.20 24.58 1003-17-1 97.60 18.37 1003-17-2 81.08 1.81 1003-4-4 95.50 3.61 1003-7-9 67.66 30.93 aos2 0.16 0.02 Co1-0 74.17 7.53

[0266] The experiments indicated that, in general, Arabidopsis transgenics with StAOS2_CB18_G231T, which carries a T and L profile at positions 231 and 328 aa, respectively, accumulated higher levels of JA (44.32 ng JA / g fresh weight, average) than those with StAOS2_CB19, which carries a T and V aa profile at said positions (74.45 ng JA / g fresh weight, average). This data is consistent with that shown in Table 3, provided that the interaction between positions 231 and 328 of the AOS2 protein plays an important role in regulating AOS2 protein activity. This data also validates the in vitro data collected in plants described herein, indicating that positions 231 and 328 of the AOS2 protein play a role in regulating JA levels in plants.

[0267] To evaluate the effect of StAOS2 genotype and the subsequent aa profile of AOS2 protein on disease tolerance, Arabidopsis transgenic plants with StAOS2 alleles StAOS2_CB18_G231T and StAOS2_CB19 of the Bintje potato cultivar were inoculated with Erwinia carotovora ssp.carotovora (Ecc) at 5x104 cfu / ml according to established methods (Kariola et al., (2003) Arabidopsis, 16: MPMI, 179-187). At multiple time points after inoculation, leaf samples were retrieved and bacterial titers were quantified. Bacterial growth was significantly lower in Arabidopsis transgenic plants with StAOS2_CB18_G231T than in those with the StAOS2_CB19 allele.

[0268] Evaluation of the 231aa gene panel for resistance to Phytophthora infestans in potato

[0269] In order to associate functional differences with genotype differences in StAOS2 gene alleles and test the following hypothesis: StAOS2 gene alleles with A / C at 691 / 692nt give enhanced tolerance when compared with those containing G / G at these positions, two variants of StAOS2 alleles, StAOS2_CB18 and StAOS2_CB18_G231T, with G / G and A / C at 691 / 692nt positions, were overexpressed in potato under the 35S promoter. Standard detached leaf assay was used to test the tolerance of some resulting strains to Phytophthora infestans. In brief, for each allele tested, leaves were removed from approximately six independent 4-8 week-old transgenic potato plants grown in soil and inoculated with 300 spores at 4 positions on the abaxial surface of the leaves. Leaves were kept in the dark for 24 hours after inoculation and then incubated for 8 days at 18°C ​​with 12 hours of darkness and light. The experiment was repeated using independent detached leaf assays, with identical results. While leaves from plants overexpressing StAOS2_CB18 developed lesions similar to those in wild-type Bintje potato plants and blank vector control transgenics, leaves from transgenic plants harboring StAOS2_CB18_G231T showed significantly reduced or no lesion development. This suggests that overexpression of the StAOS2 gene allele with an A / C genotype at 691 / 692nt results in enhanced tolerance to P. infestans in potato plants.

[0270] Similarly, these two gene constructs are also expressed in potato plants under the natural promoter of StAOS2 gene. Potato cultivar Bintje is a genetically modified parental line, and Bintje_pJIHoon is a vector-controlled transgenic strain. Standard detached leaf assay (described herein) is also used to make the plant strain of gained accepted phytophthora infestans infection. Similar to the result obtained by transgenic overexpression plant, although those leaves deriving from the plant of overexpression StAOS2_CB18 have produced lesions similar to the blank vector control transgenic, the leaves deriving from the plant with StAOS2_CB1_G231T demonstrate that the lesions significantly reduced produce or do not have lesions.

[0271] RTDS TM mediated AOS2 allele conversion

[0272] To access the RTDS TMThe AOS2 allele of interest is transformed by delivering AOS2 GRONs to plant protoplasts carrying specific changes at the target nucleic acid residue of interest (i.e., via PEG-mediated nucleic acid uptake, electroporation, etc.). For example, to obtain the desired A / C conversion at positions 691 / 692 of the AOS2 gene, respectively, GRONs carry the same sequence upstream and downstream of the 691 / 692 positions of the target AOS2 allele, but with AC at positions 691 / 692. GRON-treated cells are developed into callus using established methods.

[0273] Selection of plants / calli with desired genotypic changes

[0274] Those plants / calli with the desired change are selected by pathogen challenge selection (in the potato late blight disease system, the pathogen challenge would consist of the application of Phytophthora sporangia or zoospores). Alternatively, plants / calli with the desired change are selected based on non-selection methods, such as sequencing of the callus / plant material, e.g., primer-mediated specific amplification of the desired target to identify those with the desired change.

[0275] Multi-round RTDS TM Evaluation and application of

[0276] Once plant material with the desired change in the AOS2 gene is identified, genotyping of the AOS2 locus is repeated to fully evaluate the nature of the AOS2 allelic diversity. If "susceptible" or "intermediate" alleles are still present, those plants / calli are again subjected to RTDS manipulation to produce the desired change in the allele. If necessary, RTDS and selection are iteratively repeated as necessary for several rounds until the desired genotype at one or more AOS2 loci is obtained.

[0277] Final evaluation of cultivars with desired changes.

[0278] In case calli with targeted changes are identified, those are regenerated into plants. Such plants are evaluated using pathogen assays, JA / OPDA level assessments, protein expression analysis. For these efforts, wild-type plants are used as controls to assess the degree of expected changes, such as higher pathogen resistance, higher JA / OPDA levels in plants containing desired transformations.

[0279] Example 2: Identification of novel mutations in the AOS2 gene that enhance AOS2 activity and in planta functional assays.

[0280] Generation of new alleles of the StAOS2 gene

[0281] In order to find those amino acids that are not observed in nature and that can enhance the catalytic activity or stability of the AOS2 protein or those amino acids that are not detected by such genotyping analysis (see above), random mutagenesis efforts using error-prone PCR or site-directed mutagenesis (SDM) or more direct efforts with targeted mutagenesis of specific target residues of the AOS2 protein were made. For site-directed mutagenesis, the target site can be composed of the following sites: sites in the AOS2 gene identified by the above-mentioned genotyping efforts and in Table 1 (e.g., N76D and T495K); other sites, such as those predicted to be near the enzyme active site that may have an effect on substrate binding or catalytic activity or other sites that may affect catalytic activity at a certain distance.

[0282] For such efforts, plasmid DNA containing a construct of a reference gene (such as that given by SEQ ID NO: 2) is used and subjected to mutagenesis using established methods (Diversify random PCR mutagenesis kit, Clonetech, Mountain view, CA); Error prone refs; QuikChange XL site-directed mutagenesis kit, Stratagene, San Diego, CA). Mutated clones are selected and subjected to sequence analysis to identify mutations, and those of interest are selected for heterologous protein expression using the pQE30 expression system (see below) from Qiagen Inc, Valencia, CA.

[0283] Alternatively, a library of such mutagenesis constructs is cloned into a binary vector and transformed into plant protoplasts, and the transformants are developed into callus and regenerated into plants. By established methods (see, for example, Chebab et al., (2008), PLoS ONE, Vol. 3: p. e1904; Schmelz (2003) Plant Physiol, Vol. 133: p. 295; Engelberth et al. (2003) Anal Biochem, Vol. 312, p. 242), the resulting callus is subjected to JA / OPDA level quantification, and the tolerance of these strains is assessed using pathogens of interest (e.g., Phytophthora infestans).

[0284] Example 3: Identification of novel mutations in the AOS2 gene that enhance AOS2 activity and complementation analysis in Arabidopsis

[0285] AOS2 gene variants collected through the above genotyping analysis or mutagenesis procedures were transformed into Arabidopsis aos2 mutant line CS6149 (TAIR, http: / / www.arabidopsis.org / ), and the AOS2 alleles of interest were selected by JA / OPDA levels or by pathogen assays as described above.

[0286] Although the present invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use the invention, various alternatives, modifications, and improvements will be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments and are exemplary and not intended to limit the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. Such modifications are within the spirit of the invention and are defined by the scope of the claims.

[0287] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the spirit and scope of the invention.

[0288] All patents and publications mentioned in this specification are indicative of the levels of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0289] The invention exemplarily described herein may suitably be practiced in the absence of any element or elements, any limitation or limitations, not specifically disclosed herein. Thus, for example, in various instances herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions that have been adopted are used as descriptive and not restrictive terms, and in the use of such terms and expressions, it is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications may be made within the scope of the invention protected by the claims. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention, the scope of which is defined by the appended claims.

[0290] Other embodiments are set forth in the claims. Sequence Listing <110> Hibos USA, Inc. Hibos Europe Ltd. <120> Mutated allene oxygenase 2 (AOS2) gene <130> CIBUS-019-PCT <140> PCT / US2014 / 029434 <141> 2014-03-14 <150> 61 / 785,059 <151> 2013-03-14 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 509 <212> PRT <213> potato <400> 1 Met Ala Leu Thr Ser Ser Phe Ser Leu Pro Leu Pro Ser Leu His Gln 1 5 10 15 Gln Phe Pro Ser Lys Tyr Ser Thr Phe Arg Pro Ile Ile Val Ser Leu 20 25 30 Ser Glu Lys Pro Thr Ile Val Val Thr Gln Pro Thr Lys Leu Pro Thr 35 40 45 Arg Thr Ile Pro Gly Asp Tyr Gly Leu Pro Gly Ile Gly Pro Trp Lys 50 55 60 Asp Arg Leu Asp Tyr Phe Tyr Asn Gln Gly Lys Asp Glu Phe Phe Glu 65 70 75 80 Ser Arg Val Val Lys Tyr Lys Ser Thr Ile Phe Arg Thr Asn Met Pro 85 90 95 Pro Gly Pro Phe Ile Ser Ser Asn Pro Lys Val Ile Val Leu Leu Asp 100 105 110 Gly Lys Ser Phe Pro Val Leu Phe Asp Val Ser Lys Val Glu Lys Lys 115 120 125 Asp Leu Phe Thr Gly Thr Tyr Met Pro Ser Thr Glu Leu Thr Gly Gly 130 135 140 Tyr Arg Val Leu Ser Tyr Leu Asp Pro Ser Glu Pro Asn His Glu Lys 145 150 155 160 Leu Lys Lys Leu Met Phe Phe Leu Leu Ser Ser Arg Arg Asp His Val 165 170 175 Ile Pro Lys Phe His Glu Thr Tyr Thr Glu Phe Phe Glu Thr Leu Asp 180 185 190 Lys Glu Met Ala Glu Lys Gly Thr Ala Gly Leu Asn Ser Gly Asn Asp 195 200 205 Gln Ala Ala Phe Asn Phe Leu Ala Arg Ser Leu Phe Gly Val Asn Pro 210 215 220 Val Glu Thr Lys Leu Gly Thr Asp Gly Pro Thr Leu Ile Gly Lys Trp 225 230 235 240 Val Leu Leu Gln Leu His Pro Val Leu Thr Leu Gly Leu Pro Lys Phe 245 250 255 Leu Asp Asp Leu Ile Leu His Thr Phe Arg Leu Pro Phe Leu Val 260 265 270 Lys Lys Asp Tyr Gln Arg Leu Tyr Asp Phe Phe Tyr Thr Asn Ser Ala 275 280 285 Ser Leu Phe Ala Glu Ala Glu Lys Leu Gly Ile Ser Glu Glu Ala 290,295,300 Cys Phe Asn Ser Phe Gly Gly Met 305 310 315 320 Lys Ile Phe Phe Pro Asn Met Leu Lys Ser Ile Ala Lys Ala Gly Val 325 330 335 Glu Val His Thr Arg Leu Ala Asn Glu Ile Arg Ser Glu Val Lys Ser 340 345 350 Ala Gly Gly Lys Ile Thr Met Ser Ala Met Glu Lys Met Pro Leu Met 355 360 365 Lys Ser Val Val Tyr Glu Ala Leu Arg Val Asp Pro Pro Val Ala Ser 370 375 380 Gln Tyr Gly Arg Ala Lys Gln Asp Leu Lys With Glu Ser His Asp Ala 385 390 395 400 Val Phe Glu Val Lys Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe 405 410 415 Ala Thr Lys Asp Pro Lys Ile Phe Asp Arg Pro Glu Glu Phe Val Ala 420 425 430 Asp Arg Phe Val Gly Glu Gly Glu Lys Leu Leu Lys Tyr Val Leu Trp 435 440 445 Ser Asn Gly Pro Glu Thr Glu Ser Pro Thr Val Gly Asn Lys Gln Cys 450 455 460 Ala Gly Lys Asp Phe Val Val Met Val Ser Arg Leu Phe Val Thr Glu 465 470 475 480 Phe Phe Leu Arg Tyr Asp Thr Phe Asn Val Asp Val Gly Lys Ser Ala 485 490 495 Leu Gly Ala Ser Ile Thr Ile Thr Ser Leu Lys Lys Ala 500 505 <210> 2 <211> 1530 <212> DNA <213> Potato <400> 2 atggcattaa cttcatcttt ttctcttcct cttccttctc ttcaccaaca atttccatca 60 aaatactcca catttcgtcc tattattgtt tctttatcgg aaaagccaac aatcgtggta 120 acccaaccta caaaattacc tactaggaca atacctggcg actatgggtt gccgggtatt 180 ggtccatgga aagataggct tgattacttt tacaatcaag ggaaagacga attttcgaa 240 tcaagagtag tgaaatacaa atcaactata ttcagaacga acatgccacc gggaccattc 300 atttttcta acccgaaggt tattgttttg ctcgacggca agagtttccc agtccttttc 360 gatgtttcga aagtcgaaaa aaaggacctc ttcaccggaa cttacatgcc gtcgactgaa 420 ctcaccggtg gttaccgtgt tctttcttat cttgacccat ctgaaccaaa ccatgaaaaa 480 ttgaaaaaat tgatgttctt ccttctttct tctcgtcgtg atcacgttat acccaaattc 540 catgaaactt atacagagtt ttttgaaacc ctagataagg aaatggcgga aaaaggtaca 600 gctggtttaa actccggcaa tgatcaagct gcgtttaatt tcttagctag atcgttgttc 660 ggagttaacc cagttgaaac taaactcgga actgatggtc cgacattgat cggaaaatgg 720 gttttgcttc agcttcatcc tgtactcact ctcggtcttc cgaagtttct agacgactta 780 atcctccata ctttccggtt acctccgttt ctggtgaaga aagattacca gagactttac 840 gatttctttt acaccaactc cgccagttta ttcgccgaag ctgaaaaact cggcatttca 900 aaagaagaag cttgtcataa tcttctcttc gctacttgct tcaattcctt cggcgggatg 960 aagattttct tcccgaatat gctgaaatcg atagcgaaag caggggtgga ggtccatacc 1020 cgtttagcaa acgagatccg atcggaagta aaatccgctg gcgggaagat cacgatgtcg 1080 gcgatggaga aaatgccgtt aatgaaatca gtagtttatg aagctttgcg agttgatcct 1140 ccggtagctt cacaatacgg aagagccaaa caggacctta agatcgaatc acacgacgcc 1200 gttttcgagg tgaaaaaagg tgaaatgcta ttcgggtacc aaccatttgc aacgaaggat 1260 ccgaaaattt ttgaccggcc ggaagagttc gtcgccgatc ggttcgtcgg agaaggagaa 1320 aagttattga aatatgtatt atggtctaat ggaccggaaa cggaaagtcc aacagtgggg 1380 aataaacagt gtgctggcaa agattttgta gtgatggttt cgaggttatt cgtaacggag 1440 ttttttctcc gttacgatac attcaacgtc gacgttggta agtcggcgtt gggggcttca 1500 attactataa cttctttgaa aaaagcttag 1530 <210> 3 <211> 509 <212> PRT <213> Potato <400> 3 Met Ala Leu Thr Ser Ser Phe Ser Leu Pro Leu Pro Ser Leu His Gln 1 5 10 15 Gln Phe Pro Ser Lys Tyr Ser Thr Phe Arg Pro Ile Ile Val Ser Leu 20 25 30 Ser Glu Lys Pro Thr Ile Val Val Thr Gln Pro Thr Lys Phe Pro Thr 35 40 45 Arg Thr Ile Pro Gly Asp Tyr Gly Leu Pro Gly Ile Gly Pro Trp Lys 50 55 60 Asp Arg Leu Asp Tyr Phe Tyr Asn Gln Gly Lys Asp Glu Phe Phe Glu 65 70 75 80 Ser Arg Val Val Lys Tyr Lys Ser Thr Ile Phe Arg Thr Asn Met Pro 85 90 95 Pro Gly Pro Phe Ile Ser Ser Asn Pro Lys Val Ile Val Leu Leu Asp 100 105 110 Gly Lys Ser Phe Pro Val Leu Phe Asp Val Ser Lys Val Glu Lys Lys 115 120 125 Asp Leu Phe Thr Gly Thr Tyr Met Pro Ser Thr Glu Leu Thr Gly Gly 130 135 140 Tyr Arg Val Leu Ser Tyr Leu Asp Pro Ser Glu Pro Asn His Glu Lys 145 150 155 160 Leu Lys Lys Leu Met Phe Phe Leu Leu Ser Ser Arg Arg Asp His Val 165 170 175 Ile Pro Lys Phe His Glu Thr Tyr Thr Glu Phe Phe Glu Thr Leu Asp 180 185 190 Lys Glu Met Ala Asp Lys Gly Thr Ala Gly Leu Asn Ser Gly Asn Asp 195 200 205 Gln Ala Ala Phe Asn Phe Leu Ala Arg Ser Leu Phe Gly Val Asn Pro 210 215 220 Val Glu Thr Lys Leu Gly Thr Asp Gly Pro Thr Leu Ile Gly Lys Trp 225 230 235 240 Val Leu Leu Gln Leu His Pro Val Leu Thr Leu Gly Leu Pro Lys Val 245 250 255 Leu Asp Asp Leu Ile Leu His Thr Phe Arg Leu Pro Pro Phe Leu Val 260 265 270 Lys Lys Asp Tyr Gln Arg Leu Tyr Asp Phe Phe Tyr Thr Asn Ser Ala 275 280 285 Ser Leu Phe Ala Glu Ala Glu Lys Leu Gly Ile Ser Lys Glu Glu Ala 290 295 300 Cys His Asn Leu Leu Phe Ala Thr Cys Phe Asn Ser Phe Gly Gly Met 305 310 315 320 Lys Ile Phe Phe Pro Asn Met Leu Lys Ser Ile Ala Lys Ala Gly Val 325 330 335 Glu Val His Thr Arg Leu Ala Asn Glu Ile Arg Ser Glu Val Lys Ser 340 345 350 Ala Gly Gly Lys Met Thr Met Ser Ala Met Glu Lys Met Pro Leu Met 355 360 365 Lys Ser Val Val Tyr Glu Ala Leu Arg Val Asp Pro Pro Val Ala Ser 370 375 380 Gln Tyr Gly Arg Ala Lys Gln Asp Leu Lys Ile Glu Ser His Asp Ala 385 390 395 400 Val Phe Glu Val Lys Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe 405 410 415 Ala Thr Lys Asp Pro Lys Ile Phe Asp Arg Pro Glu Glu Phe Val Ala 420 425 430 Asp Arg Phe Val Gly Glu Gly Glu Lys Leu Leu Lys Tyr Val Leu Trp 435 440 445 Ser Asn Gly Pro Glu Thr Glu Ser Pro Thr Val Gly Asn Lys Gln Cys 450 455 460 Ala Gly Lys Asp Phe Val Val Met Val Ser Arg Leu Phe Val Thr Glu 465 470 475 480 Phe Phe Leu Arg Tyr Asp Thr Phe Asn Val Asp Val Gly Lys Ser Ala 485 490 495 Leu Gly Ala Ser Ile Thr Ile Thr Ser Leu Lys Lys Ala 500 505 <210> 4 <211> 1530 <212> DNA <213> Potato <400> 4 atggcattaa cttcatcttt ttctcttcct cttccttctc ttcaccaaca atttccatca 60 aaatactcca catttcgtcc tattattgtt tctttatcgg aaaagccaac aatcgtggta 120 acccaaccta caaaatttcc tactaggaca atacctggcg actatgggtt gccgggtatt 180 ggtccatgga aagataggct tgattacttt tacaatcaag ggaaagacga atttttcgaa 240 tcaagagtag tgaaatacaa atcaactata ttcagaacga acatgccacc gggaccattc 300 atttcttcta acccgaaggt tattgttttg ctcgacggca agagtttccc agtccttttc 360 gatgtttcga aagtcgaaaa aaaggacctc ttcaccggaa cttacatgcc gtcgactgaa 420 ctcaccggtg gttaccgtgt tctttcttat cttgacccat ctgaaccaaa ccatgaaaaa 480 ttgaaaaaat tgatgttctt ccttctttct tctcgtcgtg atcacgttat acccaaattc 540 catgaaactt atacagagtt ttttgaaacc ctagataagg aaatggcgga taaaggtaca 600 gctggtttaa actccggcaa tgatcaagct gcgtttaatt tcttagctag atcgttgttc 660 ggagttaacc cagttgaaac taaactcgga actgatggtc cgacattgat cggaaaatgg 720 gttttgcttc agcttcatcc tgtactcact ctcggtcttc cgaaagttct agacgactta 780 atcctccata ctttccggtt acctccgttt ctggtgaaga aagattacca gagactttac 840 gatttctttt acaccaactc cgccagttta ttcgccgaag ctgaaaaact cggcatttca 900 aaagaagaag cttgtcataa tcttctcttc gctacttgct tcaattcctt cggcgggatg 960 aagattttct tcccgaatat gctgaaatcg atagcgaaag caggagtgga ggtccatacc 1020 cgtttagcaa acgagatccg atcggaagta aaatccgctg gcgggaagat gacgatgtcg 1080 gcgatggaga aaatgccgtt aatgaaatca gtagtttatg aagcgttgcg agttgatcct 1140 ccggtagctt cacaatacgg aagagccaaa caggacctta agatcgaatc acacgacgcc 1200 gttttcgagg tgaaaaaagg tgaaatgcta ttcgggtacc aaccatttgc aacgaaggat 1260 ccgaaaattt ttgaccggcc ggaagagttc gtcgccgatc ggttcgtcgg agaaggagaa 1320 aagttattga aatatgtatt atggtctaat ggaccggaaa cggaaagtcc aacagtgggg 1380 aataaacagt gtgctggcaa agattttgta gtgatggttt cgaggttatt cgtaacggag 1440 ttttttctcc gttacgatac attcaacgtc gacgttggta agtcggcgtt gggggcttca 1500 attactataa cttctttgaa aaaagcttag 1530 <210> 5 <211> 510 <212> PRT ​​​​​​​​​​​​​​​​​​​​Asp Arg Leu Asp Tyr Phe Tyr Asn Gln Gly Lys Asn Glu Phe Phe Glu 65 70 75 80 Ser Arg Val Val Lys Tyr Lys Ser Thr Ile Phe Arg Thr Asn Met Pro 85 90 95 Pro Gly Pro Phe Ile Ser Ser Asn Pro Lys Val Ile Val Leu Leu Asp 100 105 110 Gly Lys Ser Phe Pro Val Leu Phe Asp Val Ser Lys Val Glu Lys Lys 115 120 125 Asp Leu Phe Thr Gly Thr Tyr Met Pro Ser Thr Glu Leu Thr Gly Gly 130 135 140 Tyr Arg Val Leu Ser Tyr Leu Asp Pro Ser Glu Pro Asn His Glu Lys 145 150 155 160 Leu Lys Lys Leu Met Phe Phe Leu Leu Ser Ser Arg Arg Asp His Val 165 170 175 Ile Pro Lys Phe His Glu Thr Tyr Thr Glu Leu Phe Glu Thr Leu Asp 180 185 190 Lys Glu Met Ala Glu Lys Gly Thr Ala Gly Leu Asn Ser Gly Asn Asp 195 200 205 Gln Ala Ala Phe Asn Phe Leu Ala Arg Ser Leu Phe Gly Val Asn Pro 210 215 220 Val Glu Ala Lys Leu Gly Thr Asp Gly Pro Thr Leu Ile Gly Lys Trp 225 230 235 240 Val Leu Leu Gln Leu His Pro Val Leu Thr Leu Gly Leu Pro Lys Phe 245 250 255 Leu Asp Asp Leu Ile Leu His Thr Phe Arg Leu Pro Pro Phe Leu Val 260 265 270 Lys Lys Asp Tyr Gln Arg Leu Tyr Asp Phe Phe Tyr Thr Asn Ser Ala 275 280 285 Asn Leu Phe Val Glu Ala Glu Lys Leu Gly Ile Ser Lys Glu Glu Ala 290 295 300 Cys His Asn Leu Leu Phe Ala Thr Cys Phe Asn Ser Phe Gly Gly Met 305 310 315 320 Lys Ile Phe Phe Pro Asn Met Met Lys Ser Ile Ala Lys Ala Gly Val 325 330 335 Glu Val His Thr Arg Leu Ala Asn Glu Ile Arg Ser Glu Val Lys Ser 340 345 350 Ala Gly Gly Lys Ile Thr Met Ser Ala Met Glu Lys Met Pro Leu Met 355 360 365 Lys Ser Val Val Tyr Glu Ala Leu Arg Val Asp Pro Pro Val Ala Ser 370 375 380 Gln Tyr Gly Arg Ala Lys Gln Asp Leu Lys Ile Glu Ser His Asp Ala 385 390 395 400 Val Phe Glu Val Lys Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe 405 410 415 Ala Thr Lys Asp Pro Lys Ile Phe Asp Arg Pro Glu Glu Leu Val Ala 420 425 430 Asp Arg Phe Val Gly Glu Glu Gly Glu Lys Leu Leu Lys Tyr Val Leu 435 440 445 Trp Ser Asn Gly Pro Glu Thr Glu Ser Pro Thr Val Gly Asn Lys Gln 450 455 460 Cys Ala Gly Lys Asp Phe Val Val Met Val Ser Arg Leu Phe Val Val 465 470 475 480 Glu Phe Phe Leu Arg Tyr Asp Thr Phe Asn Val Asp Val Gly Thr Ser 485 490 495 Ala Leu Gly Ala Ser Ile Thr Ile Thr Ser Leu Lys Lys Ala 500 505 510 <210> 6 <211> 1533 <212> DNA <213> Potato <400> 6 atggcattaa cttcatcttt ttctcttcct cttccttctc ttcaccaaca atttccatca 60 aaatactcca catttcgtcc tattattgct tcgttatccg aaaaaccaat aatcgtggta 120 acccaaccta caaaattacc taccaggaca atgcccggcg actatgggtt accgggtatt 180 ggtccatgga aagataggct tgattacttt tacaatcaag gcaaaaaacga atttttcgaa 240 tcaagagtag tgaaatacaa atcaactata ttcagaacga acatgccacc gggaccattc 300 atttttcta acccgaaggt tattgttttg ctcgacggca agagtttccc agtccttttc 360 gatgtttcga aagtcgaaaa aaaggacctc ttcactggaa cttacatgcc gtcgactgaa 420 ctcaccggtg gttaccgtgt tctttcttat cttgacccat ctgaaccaaa ccatgaaaaa 480 ttgaaaaaat tgatgttctt ccttctttct tctcgtcgtg atcacgttat acccaaattc 540 catgaaactt atacagagtt gtttgaaacc ctagataagg aaatggcgga aaaaggtaca 600 gctggtttaa actccggcaa tgatcaagct gcgtttaatt tcttagctag atcgttgttc 660 ggagttaacc cagttgaagc taaactcgga actgatggtc cgacattgat cggaaaatgg 720 gttttgcttc agcttcatcc tgtgcttact ctcggtcttc cgaagtttct agacgactta 780 atcctccata ctttccggtt acctccgttt ctggtgaaaa aagattacca gagactttac 840 gatttctttt acaccaattc cgccaattta ttcgtcgaag ctgaaaaact cggcatttct 900 aaagaagaag cttgtcataa tcttctcttc gctacttgct tcaattcctt cggcgggatg 960 aagattttct tcccgaatat gatgaaatcg atagcgaaag caggggtgga ggtccatacc 1020 cgtttagcaa acgagatccg atcggaagta aaatccgccg gcgggaagat cacgatgtcg 1080 gcgatggaga aaatgccgtt aatgaaatca gtagtatatg aagctttacg agttgatcct 1140 ccggtagctt cacaatacgg aagagccaaa caggacctta agatcgaatc acacgacgcc 1200 gttttcgagg tgaaaaaagg tgaaatgcta ttcgggtacc aaccatttgc aacgaaggat 1260 ccgaaaattt ttgaccgacc ggaagagctc gtcgccgatc ggttcgtcgg agaagaagga 1320 gaaaagttat tgaaatatgt attatggtct aatggaccgg aaacggaaag tccgacagtg 1380 gggaataaac agtgtgctgg aaaagatttt gtagtgatgg tttcgaggtt attcgtagtg 1440 gagttttttc tccgttacga tacattcaac gtcgacgttg gtacgtcggc gttgggggct 1500 tcaattacta taacttcttt gaaaaaagct tag 1533 <210> 7 <211> 510 <212> PRT <213> Potato <400> 7 Met Ala Leu Thr Ser Phe Phe Ser Leu Pro Leu Pro Ser Leu His Gln 1 5 10 15 Gln Phe Pro Ser Lys Tyr Ser Thr Phe Arg Pro Ile Ile Val Ser Leu 20 25 30 Ser Glu Lys Pro Thr Ile Val Val Thr Gln Pro Thr Lys Leu Pro Val 35 40 45 Arg Thr Ile Pro Gly Asp Tyr Gly Leu Pro Gly Ile Gly Pro Trp Lys 50 55 60 Asp Arg Leu Asp Tyr Phe Tyr Asn Gln Gly Lys Asn Glu Phe Phe Glu 65 70 75 80 Ser Arg Val Val Lys Tyr Lys Ser Thr Ile Phe Arg Thr Asn Met Pro 85 90 95 Pro Gly Pro Phe Ile Ser Ser Asn Pro Lys Val Ile Val Leu Leu Asp 100 105 110 Gly Lys Ser Phe Pro Val Leu Phe Asp Val Ser Lys Val Glu Lys Lys 115 120 125 Asp Leu Phe Thr Gly Thr Tyr Met Pro Ser Thr Glu Leu Thr Gly Gly 130 135 140 Tyr Arg Val Leu Ser Tyr Leu Asp Pro Ser Glu Pro Asn His Glu Lys 145 150 155 160 Leu Lys Lys Leu Met Phe Phe Leu Leu Ser Ser Arg Arg Asp His Val 165 170 175 Ile Pro Lys Phe His Glu Thr Tyr Thr Glu Phe Phe Glu Thr Leu Asp 180 185 190 Lys Glu Met Ala Glu Lys Gly Lys Ala Gly Leu Asn Ser Gly Asn Asp 195 200 205 Gln Ala Ala Phe Asn Phe Leu Ala Arg Ser Leu Phe Gly Val Asn Pro 210 215 220 Val Glu Thr Lys Leu Gly Ile Asp Gly Pro Thr Leu Ile Gly Lys Trp 225 230 235 240 Val Leu Leu Gln Leu His Pro Val Leu Thr Leu Gly Leu Pro Lys Phe 245 250 255 Leu Asp Asp Leu Ile Leu His Ala Phe Arg Leu Pro Pro Leu Leu Val 260 265 270 Lys Lys Asp Tyr Gln Arg Leu Tyr Asp Phe Phe Tyr Thr Asn Ser Ala 275 280 285 Asn Leu Phe Val Glu Ala Glu Lys Leu Gly Ile Ser Lys Glu Glu Ala 290 295 300 Cys His Asn Leu Leu Phe Ala Thr Cys Phe Asn Ser Phe Gly Gly Met 305 310 315 320 Lys Ile Phe Phe Pro Asn Met Met Lys Ser Ile Ala Lys Ala Gly Val 325 330 335 Glu Val His Thr Arg Leu Ala Asn Glu Ile Arg Ser Glu Val Lys Ser 340 345 350 Ala Gly Gly Lys Ile Thr Met Ser Ala Met Glu Lys Met Pro Leu Met 355 360 365 Lys Ser Val Val Tyr Glu Ala Leu Arg Val Asp Pro Pro Val Ala Ser 370 375 380 Gln Tyr Gly Arg Ala Lys Gln Asp Leu Lys Ile Glu Ser His Asp Ala 385 390 395 400 Val Phe Glu Val Lys Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe 405 410 415 Ala Thr Lys Asp Pro Lys Phe Phe Asp Arg Pro Glu Glu Phe Val Ala 420 425 430 Asp Arg Phe Val Gly Glu Glu Gly Glu Lys Leu Leu Lys Tyr Val Leu 435 440 445 Trp Ser Asn Gly Pro Glu Thr Glu Ser Pro Thr Val Gly Asn Lys Gln 450 455 460 Cys Ala Gly Lys Asp Phe Val Val Met Val Ser Arg Leu Phe Val Thr 465 470 475 480 Glu Phe Phe Leu Arg Tyr Asp Thr Phe Asn Val Asp Val Gly Thr Ser 485 490 495 Ala Leu Gly Ala Ser Ile Thr Ile Thr Ser Leu Lys Lys Ala 500 505 510 <210> 8 <211> 1533 <212> DN...

Claims

1. A method for producing a non-transgenic potato plant cell having a mutated AOS2 gene, comprising: A gene repair oligonucleobase (GRON) having a targeted mutation in a potato allene oxygenase (AOS2) gene is introduced into a potato plant cell to produce a potato plant cell having a potato AOS2 gene, wherein the potato AOS2 gene expresses a mutated potato AOS2 protein having a mutation based on SEQ ID NO:43, wherein for the mutated potato AOS2 protein, the amino acid at the position corresponding to residue 48 of SEQ ID NO:43 is threonine, the amino acid at the position corresponding to residue 76 of SEQ ID NO:43 is asparagine, the amino acid at the position corresponding to residue 231 of SEQ ID NO:43 is threonine, the amino acid at the position corresponding to residue 328 of SEQ ID NO:43 is leucine, the amino acid at the position corresponding to residue 423 of SEQ ID NO:43 is isoleucine, and the amino acid at the position corresponding to residue 494 of SEQ ID NO:43 is aspartic acid, and wherein the mutated potato AOS2 protein is 50% amino acid residues relative to SEQ ID NO:

43. The potato AOS2 protein of NO:43 exhibited increased AOS2 catalytic activity.

2. The method according to claim 1, further comprising: (a) identifying a potato plant cell having normal or above normal growth and AOS2 catalytic activity in the presence of a pathogen compared to a corresponding wild type potato plant cell; as well as (b) regenerating a non-transgenic potato plant having a mutated AOS2 gene from said potato plant cell.

3. The method of claim 2, wherein the pathogen is one or more species selected from bacteria, fungi, viruses and mycoplasma species.

4. The method of claim 2, wherein the pathogen species is one or more selected from the group consisting of: species of the infesting Phytophthora spp. Fusarium, species of Botrytis, species of Alternaria, species of Pythium, species of Downy Mildew, species of Cladosporium, species of Powdery Mildew, species of Aspergillus, species of Rust, species of Broomella and / or species of Trichoderma, Xanthomonas, Pseudomonas, Erwinia and Ralstonia.

5. The method of claim 2, wherein the pathogen species is one or more selected from the group consisting of: Xanthomonas campestris pv. citrus, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. blister, Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. bean, Pseudomonas syringae pv. syringae, Erwinia carotovora subsp. blackleg, and Ralstonia solanacearum.

6. The method of claim 2, wherein the pathogen species is Phytophthora canker, Xylella fastidiosa, soybean mosaic virus, tobacco ringspot virus, tobacco streak virus, and tomato spotted wilt virus.

7. The method of claim 2, wherein the pathogen is Phytophthora infestans.

8. The method of any one of claims 1-7, wherein the mutation generates an A at the position corresponding to 691 of SEQ ID NO:

2.

9. The method of any one of claims 1-7, wherein the mutation generates A at at least two positions corresponding to 691 of SEQ ID NO:

2.

10. The method of any one of claims 1-7, wherein the mutation generates A at at least three positions corresponding to 691 of SEQ ID NO:

2.

11. The method of any one of claims 1-7, wherein the mutation generates A at at least four positions corresponding to 691 of SEQ ID NO:

2.

12. The method of any one of claims 1-7, wherein the mutation generates a C at the position corresponding to 692 of SEQ ID NO:

2.

13. The method of any one of claims 1-7, wherein the mutation generates a C at at least two positions corresponding to 692 of SEQ ID NO:

2.

14. The method of any one of claims 1-7, wherein the mutation generates a C at at least three positions corresponding to 692 of SEQ ID NO:

2.

15. The method of any one of claims 1-7, wherein the mutation generates C at at least four positions corresponding to 692 of SEQ ID NO:

2.

16. The method according to any one of claims 1 to 7, wherein the non-transgenic potato plant cell is tetraploid; and wherein the mutation confers a homozygous genotype of AAAA at the nucleotide position corresponding to position 691 of SEQ ID NO:2 and a homozygous genotype of CCCC at the position corresponding to position 692 of SEQ ID NO:

2.

17. The method according to any one of claims 1 to 7, wherein the non-transgenic potato plant cells are cells of a potato variety selected from the group consisting of Anya, Arran Victory, Atlantic, Belle de Fontenay, BF-15, Bintje, Cabritas, Camota, Chelina, Chiloé, Cielo, Clavera Blanca, Désirée, Fianna, Fingerling, Flava, Fontana, Golden Wonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, Maris Piper, Nicola, PinkEye, Pink Fir Apple, Primura, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Shepody, Spunta, Vivaldi, Yukon Gold, Nyayo, Mukori, Roslin Tana, Kerrs's Pink, Golof, Kinongo, Ngure, Kenya Baraka, Maritta, Kihoro, Americar, Roslin Bvumbwe, Njine, Roslin Gucha, Arka, Anett, Pimpernel, B53 (Roslin Eburu), Patrones, Robijn, Roslin Chania, Urgentia, Feldeslohn, Kenya Akiba, Mirka and Roslin Sasamua.

18. The method according to any one of claims 1-7, wherein said non-transgenic potato plant cells are propagated asexually, wherein said non-transgenic potato plant cells are produced from tubers.

19. The method of any one of claims 1-7, wherein the non-transgenic potato plant cell has a medium early maturity classification.

20. Use of a mutant AOS2 gene in producing a potato plant or plant cell comprising the mutant AOS2 gene, wherein the gene encodes a mutant protein, and wherein the mutant protein is a mutant potato AOS2 protein having a mutation based on SEQ ID NO: 43, wherein for the mutant potato AOS2 protein, the amino acid at the position corresponding to residue 48 of SEQ ID NO: 43 is threonine, the amino acid at the position corresponding to residue 76 of SEQ ID NO: 43 is asparagine, the amino acid at the position corresponding to residue 231 of SEQ ID NO: 43 is threonine, the amino acid at the position corresponding to residue 328 of SEQ ID NO: 43 is leucine, the amino acid at the position corresponding to residue 423 of SEQ ID NO: 43 is isoleucine and the amino acid at the position corresponding to residue 494 of SEQ ID NO: 43 is aspartic acid, and wherein the mutant potato AOS2 protein has a mutation based on SEQ ID NO: 43, wherein ... The potato AOS2 protein of NO:43 exhibits increased AOS2 catalytic activity, and wherein the potato plant or plant cell is resistant to pathogens, wherein the potato plant or plant cell is resistant to Phytophthora infestans.

21. The use according to claim 20, wherein the mutation produces an A at the position corresponding to 691 of SEQ ID NO:

2.

22. The use according to claim 20, wherein the mutation produces A at at least two positions corresponding to 691 of SEQ ID NO:

2.

23. The use according to claim 20, wherein the mutation produces A at at least three positions corresponding to 691 of SEQ ID NO:

2.

24. The use according to claim 20, wherein the mutation produces A at at least four positions corresponding to 691 of SEQ ID NO:

2.

25. The use according to any one of claims 20-24, wherein the mutation generates a C at the position corresponding to 692 of SEQ ID NO:

2.

26. The use according to any one of claims 20-24, wherein the mutation generates a C at at least two positions corresponding to 692 of SEQ ID NO:

2.

27. The use according to any one of claims 20-24, wherein the mutation generates C at at least three positions corresponding to 692 of SEQ ID NO:

2.

28. The use according to any one of claims 20-24, wherein the mutation generates C at at least four positions corresponding to 692 of SEQ ID NO:

2.

29. The use according to any one of claims 20 to 24, wherein the potato plant or plant cell is tetraploid; and wherein the mutation confers a homozygous genotype of AAAA at the nucleotide position corresponding to position 691 of SEQ ID NO:2 or SEQ ID NO:4 and a homozygous genotype of CCCC at the position corresponding to position 692 of SEQ ID NO:

2.

30. Use according to any one of claims 20 to 24, said potato plant cell being a Désirée potato plant cell or a Bintje potato plant cell.

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