For plants resistant to the purslane mosaic virus infection

Abstract

This invention relates to plants containing inactivated genes in their genomes that make the plants resistant to Pepino mosaic virus (PepMV) infection. The invention also relates to the inactivation of genes required for PepMV infection. The invention covers portions of these plants and their progeny that exhibit the inactivation of the genes and thus an improved phenotype in terms of resistance to PepMV infection. Methods for obtaining plants, plant parts, or seeds resistant to PepMV infection are also part of this invention. The invention further relates to genes and linked sequences as markers for selecting plants resistant to PepMV infection. Therefore, this invention belongs to the agricultural field.

Description

Technical Field

[0001] This invention relates to plants containing an inactivated gene in their genome that makes the plant resistant to Pepino mosaic virus (PepMV) infection. The invention also relates to the inactivation of a gene required for PepMV infection. The invention covers portions of these plants and their progeny that contain the inactivated gene and thus exhibit an improved phenotype of resistance to PepMV infection. Methods for obtaining plants, plant parts, or seeds resistant to PepMV infection are also part of this invention. The invention further relates to genes and linked sequences as markers for selecting plants resistant to PepMV infection. Therefore, this invention belongs to the field of agriculture. Background Technology

[0002] Viruses, especially plant viruses, possess small genomes encoding very small protein reservoirs; with these few tools, viruses must complete their life cycle, including replication, transport within the host, resistance to host defenses, and spread within the host, to name just a few of the main functions. To achieve this, viruses hijack and disrupt the plant cellular machinery, interacting with host factors that possess proviral functions (Hyodo and Okuno, 2020, Advances in Virus Research, vol. 107. Academic Press, Cambridge, pp. 37-86); host proviral factors are often referred to as host susceptibility factors because in their absence (or in the presence of isotypes that are nonfunctional to the virus), the host is not susceptible or not fully susceptible. Considering the phenotype of plant-virus interactions, loss of susceptibility is equivalent to plant resistance (Kourelis and van der Hoorn, 2018, Plant Cell 30, 285-299), and therefore it has extremely important benefits in breeding. For years, plant breeders have used recessive resistance genes to construct virus-resistant cultivars; in cases studied in sufficient depth, all recessive viral resistance genes conform to the hypothesis of encoding proviral factors (Nicaise, 2014, Front Plant Sci 5, 1-18). To date, the recessive viral resistance genes characterized within the natural diversity of crop species appear to belong to only one category, encoding eukaryotic translation initiation (eIF) factors of the 4E and 4G families (Truniger and Aranda, 2009, Advances in Virus Research, vol 75, Academic Press, Cambridge, pp 119-159). A different picture emerges when screening mutant sets from model species for loss of susceptibility to viruses; in this case, host factors distinct from eIF4E or 4G have been described (Makiinen, 2020, Ann Appl Biol 176, 122-129). However, their use in breeding virus-resistant crop varieties remains an underexplored possibility.

[0003] PepMV is a single-stranded, positive-sense RNA virus belonging to the genus *Potexvirus* (family Alphaflexiviridae) and is prevalent in tomato crops worldwide; in fact, PepMV is causing very significant economic losses in intensive tomato crops globally. This species is quite diverse, with at least five strains described to date. Given its economic impact, screening has been conducted in registered *Solanum* species to identify sources of resistance to PepMV (Soler et al., 2011, *J. Plant Dis Prot 118*, 149-155), but with limited success; the identified resistances are partial and / or strain-specific, which, along with the genetic distance from the source of resistance to cultivated tomatoes, limits their breeding benefits. Host factors interacting with PepMV factors were also identified; this is the case with the heat shock isotype 70 (Hsc70) that interacts with the PepMV coat protein (CP) (Mathioudakis et al., 2014, Mol Plant-Microbe Interact 27, 135.6-1369). Hsc70 appears to possess proviral function against PepMV, but its silencing induces a severe phenotype in target plants (Mathioudakis et al., 2014, Mol Plant-Microbe Interact 27, 135.6-1369), rendering it unsuitable for breeding. A few other proviral factors have been identified for potato X virus, but their function against PepMV has not been tested, or they have not been envisioned for breeding PepMV-resistant tomato varieties.

[0004] Therefore, there is a need for new mechanisms to reduce plant susceptibility to PepMV infection, which are applicable to several or all PepMV strains and are stable mechanisms to resist viral adaptation and evasion, so that they can be used for breeding.

[0005] Invention Description

[0006] To address the aforementioned lack of available PepMV resistance in plants, the authors screened a batch of tomato mutant plants to identify mutations showing an association with reduced susceptibility to PepMV. This screening resulted in the discovery of mutant plants with reduced viral loads and none of the typical symptoms of PepMV infection. The genomes of these mutants were characterized by segregating population grouping analysis coupled with high-throughput sequencing to determine the source of the plant's resistance to PepMV infection. The source was ultimately determined to be the inactivation of a specific protein-coding gene. Backcrossing with wild-type plants and progeny analysis confirmed that the resistance was recessive (see Examples), as the segregation frequencies almost perfectly matched the expected gene segregation in models where resistance was a single gene and recessive. The mutant plants exhibited no other significant phenotypes besides improved PepMV resistance or reduced susceptibility to PepMV (see Examples).

[0007] Therefore, a first aspect of the invention relates to a plant (hereinafter referred to as the plant of the invention) or a portion thereof, reproductive or propagating plant material (including seeds) (hereinafter referred to as the reproductive or propagating plant material of the invention), or plant cell (hereinafter referred to as the plant cell of the invention), characterized in that it comprises a gene encoding a protein (hereinafter referred to as the gene of the invention), wherein said protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, and said gene has been inactivated. In a preferred embodiment, the plant of the invention or a portion thereof, the reproductive or propagating plant material of the invention, or the plant cell of the invention is not obtained solely by a substantially biological process.

[0008] As stated above, the inventors have identified mutant plants resistant to PepMV infection. The term "plant" as used herein includes the whole plant, any "reproductive or propagational material" of a plant, offspring of a plant, and parts of a plant, including seeds, siliques, fruits, leaves, flowers, seedlings, stems, tubers, roots, isolated plant cells, callus, tissues, and organs. References to plants may also include plant cells, plant protoplasts, plant tissue cultures, plant callus, plant flower clusters, and plant cells that are intact within a plant or part of a plant, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cereals, spikes, ears of grain, husks, stems, roots, root tips, etc. Any offspring, variants, and mutants of any plant described herein are within the scope of this invention. Seeds of any of the said plants are also included. As used herein, the term "part of a plant" includes any part of a plant, including seeds, siliques, fruits, leaves, flowers, seedlings, stems, and / or roots.

[0009] As is known and well understood by those skilled in the art, the present invention can also be practiced in plant cells. As used herein, the term "plant cell" includes plant cells derived from and / or isolated from plant cell tissues or plant cell cultures.

[0010] The invention described herein relates to plants having improved resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection. In another preferred embodiment, the plant or part thereof, or the reproductive or propagating plant material of the invention, or the plant cells of the invention are characterized by having improved resistance to PepMV infection or reduced susceptibility to PepMV infection, or an improved phenotype in terms of resistance to PepMV infection, compared to a wild-type control plant.

[0011] As used herein, the expressions “resistance to PepMV infection” and “reduced susceptibility to PepMV infection” refer to a reduced or absent virus titer in the plants of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cells of the present invention, compared to wild-type control plants, materials, or plant cells. In a preferred embodiment, the reduction in virus titer compared to wild-type plants is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Furthermore, the expressions “resistance to PepMV infection” and “improved phenotype in terms of resistance to PepMV infection” refer to a reduction or absence of symptoms caused by infection. In a preferred embodiment, resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection leads to symptom reduction according to a disease scale test (see examples), resulting in a better score than wild-type plants on such tests, i.e., a score from 2 to 1 or to 0 on a 0-2 scale (where 0 is the absence of symptoms, 1 is sporadic bright yellow spots in new leaves, and 2 is bright yellow mottling affecting all new leaves).

[0012] The inventors of this invention have determined that resistance to PepMV infection, or an improved phenotype in terms of resistance to PepMV infection, is due to the inactivation of the gene of this invention. In a preferred embodiment, the plant or part thereof of this invention, or the reproductive or propagating plant material of this invention, or the plant cell of this invention, is characterized in that the inactivated gene encodes a protein, wherein said protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. In another preferred embodiment, the plant of this invention, or the reproductive or propagating plant material of this invention, or the plant cell of this invention, is characterized in that the inactivated gene encodes a protein, wherein said protein has an amino acid sequence consisting of SEQ ID NO:1.

[0013] SEQ ID NO:1

[0014] MIQSSFSADSPSMAANSTFSPPPAAGDGDFNYDVAWYGNIQYLLNISAIGALTCLLIFIFGKLRSDHRRMPGPTAIVSKLLAAWHATGVEIARHCGADAAQYLLIEGGSSALLLFLALLSLAVMLPLNIYAGKAPMADQFSKTTINHIEKGSPLLWIHFIFVVIVVVLVHYGISEIQERLKITRLRDGYGNPSNSGTNVSAIFSIMVQGVPKTLGFDKTPLVEYFQHKYPGKVYRVVVPMDLCALDDLATELVKVREDISKLVSRIELRGYLNEGEEDEYNNDSVNGRGLLERLCFLWRKAKDTWYHVVDQLGFSDEERLRKLQELRADLEMEMASYKEGRARGAGVAFVVFKDVFTANKAVQDLRNEKRRRYGRFFSVIELQLQRNQWKVERAPLATDIYWNHLGSTKFSLKLRRVLVNTCLLLMLLFCSSPLAVISAIQSAGRIINAEAMDHAQMWLNWVQGSSWLATIIFQFLPNVLIFVSMYIVVPSVLSYLSKFEQHLTVSGEQRAELLKMVCFFLVNLILLRALVESTLEGALLSMGRCYLDGEDCKKIEQYMTASFLTRTCLSSLAFLITSSFLGISFDLLAPIPWIKKKLQKFRKNDMLQLVPERSEEYPLENQDIDSLERPLIHERSSTVIADNNGFLHDASPNEIDFPGQDLSEYPPVSRTSPVPKPKFDFAQYYAFNLTIFALTLIYCSFAPLVVPVGAVYFGYRYLVDKYNFLFVYRVRGFPAGNDGRLMDTVLSIMRFCVDLFLLSMLLFFSVRGDSTKLQAIFTLGLLVVYKLLPSDKDSFQPALLQGIQTIDNIVEGPTDYEVFSQPTFDWDTYNS

[0015] As used herein, the term "inactivated" means that the expression of a target gene or target allele is reduced, decreased, or completely or partially suppressed by at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, to 100% compared to its normal expression in wild-type plants. Additionally, the term "inactivated" is also used herein as a synonym for "silenced" and as a synonym for "knockout," the latter referring to the modification of a gene nucleotide sequence in a manner that produces non-functional messenger RNA or reduced functional or non-functional proteins. In a more preferred embodiment, the plant of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, is characterized in that the inactivated gene encodes a protein, wherein said protein comprises SEQ ID NO:2, preferably consisting of SEQ ID NO:2. SEQ ID NO:2 is generated from a mutation at position 554 in SEQ ID NO:1, wherein the amino acid lysine is replaced by a stop codon. In another preferred embodiment, the plant of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, is characterized in that the inactivated gene encodes a protein, said protein comprising SEQ ID NO:3, preferably consisting of SEQ ID NO:3. SEQ ID NO:3 arises from the deletion of amino acids 11 to 408 of SEQ ID NO:1.

[0016] SEQ ID NO:2

[0017] MIQSSFSADSPSMAANSTFSPPPAAGDGDFNYDVAWYGNIQYLLNISAIGALTCLLIFIFGKLRSDHRRMPGPTAIVSKLLAAWHATGVEIARHCGADAAQYLLIEGGSSALLLFLALLSLAVMLPLNIYAGKAPMADQFSKTTINHIEKGSPLLWIHFIFVVIVVVLVHYGISEIQERLKITRLRDGYGNPSNSGTNVSAIFSIMVQGVPKTLGFDKTPLVEYFQHKYPGKVYRVVVPMDLCALDDLATELVKVREDISKLVSRIELRGYLNEGEEDEYNNDSVNGRGLLERLCFLWRKAKDTWYHVVDQLGFSDEERLRKLQELRADLEMEMASYKEGRARGAGVAFVVFKDVFTANKAVQDLRNEKRRRYGRFFSVIELQLQRNQWKVERAPLATDIYWNHLGSTKFSLKLRRVLVNTCLLLMLLFCSSPLAVISAIQSAGRIINAEAMDHAQMWLNWVQGSSWLATIIFQFLPNVLIFVSMYIVVPSVLSYLSKFEQHLTVSGEQRAELLKMVCFFLVNLILLRALVESTLEGALLSMGRCYLDGEDCK

[0018] SEQ ID NO:3

[0019] MIQSSFSADSQKFSLKLRRVLVNTCLLLMLLFCSPLAVISAIQSAGRIINAEAMDHAQMWLNWVQGSSWLATIIFQFLPNVLIFVSMYIVVPSVLSYLSKFEQHLTV SGEQRAELLKMVCFFLVNLILLRALVESTLEGALLSMGRCYLDGEDCKKIEQYMTASFLTRTCLSSLAFLITSSFLGISFDLLAPIPWIKKKLQKFRKNDMLQLVPERS EEYPLENQDIDSLERPLIHERSSTVIADNNGFLHDASPNEIDFPGQDLSEYPPVSRTSPVPKPKFDFAQYYAFNLTIFALTLIYCSFAPLVVPVGAVYFGYRYLVDKY NFLFVYRVRGFPAGNDGRLMDTVLSIMRFCVDLFLLSMLLFFSVRGDSTKLQAIFTLGLLVVYKLLPSDKDSFQPALLQGIQTIDNIVEGPTDYEVFSQPTFDWDTYNS

[0020] In this invention, inactivation involves the gene of the invention. As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, the gene includes coding sequences and / or regulatory sequences required for its expression. The gene also includes unexpressed DNA segments, which, for example, form recognition sequences for other proteins. The gene can be obtained from a wide variety of sources, including cloning from a target source or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters. The expression "encoding protein" as used herein refers to the fact that the gene of the invention contains an essential nucleotide sequence that allows it to be transcribed into messenger RNA, which is then translated into an amino acid sequence that will fold into a functional protein. In a preferred embodiment, the gene of the invention comprises the genomic DNA sequence SEQ ID NO:4. In another preferred embodiment, the gene of the invention is inactivated and comprises the nucleotide sequence according to SEQ ID NO:5 or SEQ ID NO:6. In yet another preferred embodiment, the gene of the invention is inactivated and consists of the nucleotide sequence SEQ ID NO:5 or SEQ ID NO:6.

[0021] As mentioned above, plants exhibiting resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection refer to plants with lower viral titers or milder infection symptoms compared to wild-type plants. Similarly, the inactivation of the gene of the present invention also includes a reduction in the expression level of the gene, a reduction in the level of mRNA transcribed from the gene, a reduction in the amount of functional protein encoded by the gene, and the complete elimination of the gene and / or functional protein. Where a reduction exists, it is determined compared to a wild-type plant. As used herein, the term "wild-type" (also written as WT) refers to the typical form of a plant or gene that serves as a control plant not containing the inactivated gene of the present invention, a preferred version of which is a near-isogenic line. "Wild-type plant" refers to a plant having a phenotype corresponding to plants in natural populations that do not contain the inactivated gene of the present invention.

[0022] Although identified as a source of reduced susceptibility to PepMV infection or improved phenotypes of resistance to PepMV infection in tomato plants, the genes of the present invention and the proteins they encode have orthogonal homologs with similar structural protein characteristics, as shown by the inventors (see Examples). To identify protein homologs, those skilled in the art typically use the identity of amino acid sequences between two proteins. The term "identity" as used herein refers to the proportion of identical amino acids or nucleotides along their full-length sequence between two compared peptide / protein or nucleotide sequences. Methods for comparing sequences are known in the art and include, but are not limited to, procedures BLASTP or BLASTN, EMBOSS Needle, ClustalW, and FASTA. It can be assumed that peptide, protein, or nucleotide sequences having an identity percentage of at least 60%, 70%, 80%, or 90% will retain the same characteristics as the sequences they are compared to.

[0023] Identification of homologs of the genes of the present invention indicates that inactivation of said homologs can be used to create plants resistant to PepMV infection or with an improved phenotype of resistance to PepMV infection. Therefore, in another preferred embodiment, the plant or part thereof, or the reproductive or propagating plant material of the present invention, or the plant cells of the present invention belong to the Solanaceae family. In another preferred embodiment, the plant or part thereof, or the reproductive or propagating plant material of the present invention, or the plant cells of the present invention belong to a species of the genera *Solanum*, *Capsicum*, *Nicotiana*, or *Physalis*. In a more preferred embodiment, the species are selected from a list consisting of the following: tomato (S. lycopersicum), potato (S. tuberosum), eggplant (S. melongena), Pennell's tomato (S. pennellii), pimpinellifolium, Peruvian eggplant (S. peruvianum), Cheesmani tomato (S. cheesmanii), Galapagos tomato (S. galapagense), Chilean tomato (S. chilense), red eggplant (S. aethiopicum), prickly eggplant (S. quitoense), water eggplant (S. torvum), and cantaloupe eggplant (S. The species include *S. muricatum*, *S. betaceum*, *S. chmielewskii*, *S. arcanum*, *S. cornelliomulleri*, *S. habrochaiti*, *S. huaylasense*, *S. neorickii*, *S. dulcamara*, *S. lycopersicoides*, *S. sitiens*, *S. juglandifolium*, *S. ochranthum*, and *S. cheesmaniae*. This invention also relates to synonyms for these species, such as tomato (*Lycopersicon esculentum*, *Lycopersicon esculentum* Mill., *Lycopersicon esculentum var. esculentum*), and eggplant (*Solanum esculentum*, *Solanumesculentum* Dunal).

[0024] It should be understood that a species also includes all its subspecies and varieties or cultivars. The term "variety" as used herein refers to a group of plants within a species that share characteristics that distinguish them from other possible varieties within that species. Such distinguishing traits or sets of traits must be stable post-reproduction and sufficiently homogeneous among their individuals and offspring. For self-fertile or self-pollinating species (such as in the Solanaceae family), most commercial varieties are pure lines or inbred lines, and F1 hybrids between two inbred lines. The term "cultivar" as used herein refers to a plant having a biological state other than "wild," which indicates the original, uncultivated, or natural state of the plant or accession. The term “cultivation” includes, but is not limited to, semi-natural, semi-wild, weed, traditional and ancestral cultivation, local varieties, breeding materials, research materials, breeding lines, synthetic populations, hybrids, original cultivars / base populations, inbred lines (parents of hybrid cultivation), segregating populations, mutant / genetic cultivars, and advanced / improved cultivation.

[0025] Therefore, in another preferred embodiment of the plant or part thereof of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, the species of tomato includes the following varieties / cultivars: AnnaRussian, Applause, Aussie, Baladere, Bella Rosa, Black cherry, Black pear, Black russian, Blondkopfchen, Brandywine, Cabri, Caracas, Carbón, Ceylan, Cherokee purple, Cherry, Comanche, Costoluto genovese, Ditmarcher, Dombito, Estrella, Eros, Gallician, Glacier, Gartenperle, Green sausage, Grushovka, Harzfeuer, Hugh, Jersey devil, Juboline, Kosovo, Crimean black. Black), Kumato, Liguria, Limachino, Lime green salad, Manitoba, Marvel stripe, Moneymaker, Marglobe, Meltine, Monserrat, Muchamiel, Nemato, Opalka, Pera de Girona, Hawaiian pineapple ( Hawaiana), Rio grande, RAF, Roma, Siberian, Sprite, Sugary, Sun sugar, Sobeto, Sonatine, Tigerella, Terrades, Vergel, White Queen, Raf Claudia, Roma, Valenciano, Adoration, Alicante, Azoychka, Better Boy, Big Beef, Big Rainbow, Blaby Special, Black Krim, Branywine, Campari, Celebrity, Canario, Tomkin, Early Girl, Enchantment, Ferris Wheel, Flamenco, Fourth of July, Garden Peach, Gardener's Delight, Granadero, Great White White), Green Zebra, Hanover Tomato, Japanese Black Trifele, Jubilee, Juliet, Lillian's Yellow, Matt's Wild Cherry, Micro-Tom, Moneymaker, Monterosa, Mortgage Lifter, Mr.Stripey, Pantano Romanesco, Plum tomato, Raf tomato, Rebellion, Red Currant, Rosa de Barbastro, San Marzano, San Pedro, Sasha Altai, Tiny Tim, Cherry Bambelo, Cherry Nebula, Santorini, Tomaccio, Yellow Pear, White Queen, Corazón de Buey, Angela, Colgar en Rama, Ciruela Negro, Optima, PataNegra, Copia, Velasco, Montenegro, Vertyco, Ventero, Ramyle, Pitenza, Paladium, Mayoral, Razymo, Motto, Caniles, Byelsa, Royalty, Trujillo, Delizia, DumasDuratom, Larguero, Torry, Tovistar, Pintón, Grueso, Larga Vida, Marenza, Window boxRoma, Ninette, Retinto, Boludo, Anairis, Tobi Star, Myla, Guarapo, Atago, Jawara, Velasco, Manitu, Colbi, Duraton, Patriarca, Danubio, Intense, Pera Fitto, Vernal, Cecilio, Cherry Kumato, Amarillo Cherry (Cherry Amarillo), Cherry Redondo, Cherry Ministar, Cherry Guindos, Cherry Marinica and Angel Cherry.

[0026] In another preferred embodiment of the plant or part thereof of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, the species of potato includes the following varieties / cultivars: Kennebec, Monalisa, Desirée, Bintje, and Cape Arawa. Palogan, Pedro Roja Duchess (Duquesa), Goya, Olalla, Turia, Víctor, Lora, Gauna, Alda, Belda, Buesa, Iturrieta, Diba, Phoenix (Fénix), Wave (Onda), Arene, Asun, Ayala, Edurne, Gorbea, Idoia, Iker, Inca, Isla, Mayka, Mikel, Montico, Nagore, Nerea, Zadorra, Zarina, and Zela.

[0027] To obtain the plants of the present invention, the reproductive or propagating plant material of the present invention, or the plant cells of the present invention as mentioned herein, purely biological methods and non-purely biological methods or procedures are used. Therefore, the statement "not obtained solely through substantially biological processes" means that the host organism, such as plant cells, seeds, plants, or parts of plants, has had its genome or proteome modified by methods other than substantially biological methods (e.g., hybridization, intervarietal hybridization, selective breeding, introgression, self-pollination, or other biological processes that do not involve technical steps involving modification of the genome or proteome). Examples of methods other than substantially biological processes for obtaining plants in which the genes of the present invention are inactivated include, but are not limited to, somatic cell hybridization, mutagenesis with mutagens (e.g., but not limited to: ionizing radiation, such as X-rays, fast neutrons, UV radiation, etc.; or chemical reagents, such as, but not limited to: ethyl methanesulfonate (EMS), diethyl sulfate (DES), acridine (EI), propanesulfonate lactone, ethyl N-methyl-N-nitrosocarbamate (MNU), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (ENU), sodium azide), and genetic engineering. Examples of the latter methods include, but are not limited to, the use of microbial vectors to insert exogenous nucleic acids into the genome of target plants, microparticle bombardment, electroporation, microinjection, transformation of transposons or endogenous nucleic acids, or genome editing technologies, including CRISPR / Cas technology or other technologies, such as, but not limited to, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), etc.

[0028] For the purposes of this invention, the phrase "not obtained solely through substantially biological processes" refers to a plant whose genetic material has been intentionally modified to alter a gene encoding a protein, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, or wherein the amino acid sequence of the protein is composed of SEQ ID NO:1. Preferably, the protein is a high molar osmotic concentration-gated calcium permeable channel 4.1 (OSCA4.1). In particular, the plants of this invention have been modified by substantially and non-substantially biological processes to inactivate the expression of the genes of this invention described herein and / or modify their expression products, thereby rendering the products functionally reduced or nonfunctional.

[0029] Like all other plants, the plant of the present invention comprises cells, tissues, and organs that fall within the scope of the invention. Therefore, in another preferred embodiment, the reproductive or propagation material is selected from cells, fruits, seeds, tubers, or progeny. In another preferred embodiment of the plant of the present invention, the plant portion is selected from a list consisting of: leaves, stems, flowers, ovaries, or callus tissue. These components of the plant of the present invention (reproductive or propagation material and portions of the plant of the present invention) are further characterized in that they contain genes encoding proteins, wherein the proteins comprise amino acid sequences having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, and the genes are inactivated.

[0030] To obtain the plant of the present invention, the reproductive or propagating plant material of the present invention, the plant cell of the present invention, or any component of the plant of the present invention, several methods can be used by those skilled in the art, as they will know the best practices for applying the chosen method to obtain the gene-inactivated plant of the present invention. Therefore, another aspect of the present invention relates to a method for producing the plant of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, or a component of the plant of the present invention, wherein said plant or part thereof, reproductive or propagating plant material, or plant cell exhibits resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection, said method (hereinafter referred to as the method of the present invention) comprising:

[0031] a) subjecting the plant or parts thereof, reproductive or propagating plant material, or plant cells to random or directed mutagenesis, and

[0032] b) Detecting a mutation in a gene encoding a protein in the plant or its parts, reproductive or propagating plant material, or plant cells, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, and wherein the mutation results in the inactivation of the gene.

[0033] In a preferred embodiment of the method of the present invention, the plant of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, or the plant component of the present invention belongs to the Solanaceae family. In another preferred embodiment of the method of the present invention, the plant of the present invention, or the reproductive or propagating plant material of the present invention, or the plant cell of the present invention, or the plant component of the present invention belongs to a species of the genera *Solanum*, *Capsicum*, *Nicotiana*, or *Physalis*. In a more preferred embodiment of the method of the present invention, the plant of the present invention, or the reproductive or propagating material of the present invention, or the plant cell of the present invention, or the plant component of the present invention belongs to a species selected from a list consisting of the following: tomato, potato, *Tomato pennisetum*, *Tomato spp. ... and *Tomato spp.*, *Tomato spp.*, *Tomato spp.*, and *Tomato spp.*, *Tomato spp.*, and *Tomato spp.*, *Tomato spp.*, and *Tomato spp.*, *Tomato spp.*, and *Tomato spp.*, *Tomato spp.*, and *Tomato spp.*, *Tomato It should be understood that a species includes all subspecies and varieties or cultivars therein. Examples of varieties / cultivars of several species are listed earlier in the specification (but are not limited to), and such varieties / cultivars are valid for the present aspect and its implementation.

[0034] Methods for obtaining plants, reproductive or propagation material, plant cells, progeny, or parts of plants (particularly where genes are inactivated) are widely known in the art, and experts will be able to discern the best methods for applying them to obtain the desired plant. Such methods include directed mutagenesis and random mutagenesis strategies. Random mutagenesis strategies are primarily based on (but not limited to) techniques for inducing mutations in the DNA of cells, such as contact with a mutagen, which is, for example, a chemical substance (e.g., ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons, e.g., in fast neutron mutagenesis, etc.), alpha rays, gamma rays (e.g., those provided by a cobalt-60 source), X-rays, UV radiation, etc., or any combination thereof. Directed mutagenesis strategies include, but are not limited to, gene targeting based on homologous recombination, antisense RNA, directed transposon insertion, virus-induced gene silencing, and genome editing techniques, including but not limited to CRISPR / Cas technology.

[0035] Therefore, in a preferred embodiment of the method of the present invention, the gene of the present invention is inactivated by mutagenesis with a mutagen, mutagenesis with a chemical reagent, genetic engineering, or genome editing technology (including CRISPR / Cas technology).

[0036] The plant of the present invention has agricultural uses. Among these uses, the most widespread is the growth of the plant to obtain or produce products for feeding or consumption. Therefore, another aspect of the invention relates to the use of the plant of the present invention, the reproductive or propagating plant material of the present invention, the plant cells of the present invention, or components of the plant of the present invention for the production of agro-industrial products, preferably said agro-industrial products being food or feed.

[0037] The intended use is closely related to the method for producing the agricultural industrial product. Therefore, another aspect of the invention relates to a method for producing an agricultural industrial product, preferably wherein the agricultural industrial product is food or feed, the method comprising:

[0038] a) Cultivating the plant of the present invention, the reproductive or propagating plant material of the present invention, the plant cells of the present invention, or components of the plant of the present invention.

[0039] b) Harvesting the fruits, seeds, tubers, or edible parts of plants for the production of agricultural and industrial products, and

[0040] c) Optionally, prepare agricultural and industrial products for consumption, whether fresh or modified.

[0041] The invention set forth in this specification and the following claims relates to plants, wherein inactivation of the gene of the invention confers resistance to PepMV infection or an improved phenotype of resistance to PepMV infection. This specification further states that plants possessing resistance to PepMV infection or having said improved phenotype can be identified by screening for changes in the nucleotide sequence of the gene of the invention (which may affect the expression level of said gene) or by screening for changes in the expression level of the product of said gene. Therefore, another aspect of the invention relates to the use of a gene as a biomarker for selecting plants having resistance to PepMV infection or having an improved phenotype in terms of resistance to PepMV infection (from this point onward, the use of a biomarker in the invention), wherein said gene encodes a protein comprising an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1. In another preferred embodiment of the biomarker of the invention, said gene comprises a nucleotide sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:4. If the detected gene is altered in a manner that ensures its inactivation, then the plant possesses resistance to PepMV infection. Therefore, in another embodiment of the use of the biomarker of the present invention, the biomarker is inactivated. In another preferred embodiment of the use of the biomarker of the present invention, the biomarker is inactivated and it encodes a protein comprising the amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3. In another preferred embodiment of the use of the biomarker of the present invention, the biomarker is inactivated and comprises the nucleotide sequence according to SEQ ID NO:5 or SEQ ID NO:6. The use of the biomarker of the present invention in marker-assisted breeding projects to select inbred lines, progeny, and / or plants with PepMV resistance traits is also part of this specification.

[0042] As used herein, the term "biomarker" includes any measurable substance in a plant whose presence indicates a biological state or intended condition. In this invention, the biomarker relates to the nucleotide sequence of a gene of the invention, the expression product of the gene, or a nucleotide sequence linked thereto. Therefore, in a preferred embodiment of the use of the biomarker of the invention, the selection is performed by determining the nucleotide sequence of the gene or a fragment of the nucleotide sequence and identifying alterations therein. In another embodiment, the selection is performed by detecting or quantifying the expression product of a gene of the invention, wherein the product is selected from a list consisting of complementary DNA or fragments thereof, messenger RNA or fragments thereof, and proteins or fragments thereof. In another preferred embodiment of the biomarker of the invention, the selection is performed by further identifying a biomarker locus co-isolated with SEQ ID NO:4, preferably wherein the biomarker locus is located within 100,000 nucleotides upstream or downstream of the biomarker of the invention.

[0043] Another aspect of the invention relates to a marker locus for selecting plants that have resistance to PepMV infection or have an improved phenotype in terms of resistance to PepMV infection, wherein the marker locus is co-separated with SEQ ID NO:4 and is located within 100,000 nucleotides upstream or downstream of SEQ ID NO:4.

[0044] As used herein, the term "marker locus" refers to a specific, fixed location on a chromosome where a particular gene or genetic marker is located, cosegregating with SEQ ID NO:4. The term "cosegregation" as used herein refers to two or more genetic markers on a chromosome that are inherited together due to their close physical proximity, i.e., they are linked. The marker locus may comprise, or consist of, any genetic nucleotide sequence or common trait, such as, but not limited to, genes, introns, exons, enhancers, promoters, single nucleotide polymorphisms, small-scale insertions / deletions, transposable elements, microsatellites, or simply nucleotide fragments of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more base pairs of the nucleotide sequence.

[0045] As used herein, the terms “upstream” and “downstream” refer to the location in the 5' or 3' direction of the biomarker of the present invention, respectively.

[0046] Methods for detecting biomarkers are common knowledge in the art. For example, but not limited to, the identification of alterations in the nucleotide sequence of the gene or a fragment thereof of the present invention can be performed by electrophoretic analysis or sequencing analysis of polymerase chain reaction (PCR) products of the whole gene or a fragment thereof. As used herein, “alteration in nucleotide sequence” refers to a mutation in the nucleotide sequence, whether a substitution, insertion, or deletion, that, when present in the protein-coding region of the gene of the present invention, can result in a missense mutation, in which one amino acid is replaced by another, or a nonsense mutation, in which an early stop codon is formed. Such alterations are more likely to result in a functionally reduced or nonfunctional expression of the gene of the present invention, and thus lead to inactivation of the gene or more severe silencing of the gene of the present invention. The expression “alteration in nucleotide sequence” as used herein also covers alterations occurring in the gene of the present invention outside the protein-coding region, such as, but not limited to, promoter and / or enhancer regions; or in sequences linked to the gene of the present invention, such as, but not limited to, enhancers, which can affect the expression level of the product of the gene of the present invention.

[0047] As is known to those skilled in the art, alterations to the nucleotide sequence of the biomarker can be detected in one or more small fragments of the entire sequence, if the fragments can be clearly identified and mapped to native or wild-type sequences, in order to identify the alterations and determine their potential to inactivate the biomarker. The fragments can range from 10 base pairs to variations of the full length of the biomarker. Therefore, in a preferred embodiment, the biomarker comprises fragments of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 base pairs of the gene sequence.

[0048] The detection of the expression product of the gene of the present invention can be performed by detecting or quantifying the level of messenger RNA (mRNA) transcribed from the gene or a fragment thereof, wherein the analysis of the mRNA level can be performed, for example (but not limited to), by means of: polymerase chain reaction (PCR) amplification, reverse transcription PCR (RT-PCR), reverse transcription combined with ligase chain reaction (RT-LCR) or any other nucleic acid amplification method; DNA microarrays generated using oligonucleotides deposited by any mechanism; DNA microarrays made of oligonucleotides synthesized in situ by photolithography or any other mechanism; in situ hybridization using specific probes labeled by any labeling method; electrophoresis gel; membrane transfer and hybridization with specific probes; nuclear magnetic resonance or any other imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with DNA / RNA probes, antibodies or any other means.

[0049] As used herein, the term "mRNA fragment" refers to a nucleotide sequence obtained by transcription of the gene of the present invention, wherein the sequence is missing one or more nucleotides from the 5' initial and / or 3' initial regions or any of those regions compared to the complete nucleotide sequence obtained by transcription of the gene of the present invention.

[0050] In addition to detecting mRNA, the detection of the biomarkers of the present invention can also be performed by detecting and / or quantifying the protein products of the biomarkers of the present invention or fragments thereof. As mentioned above, the methods are well known in the art and include, but are not limited to, Western blotting, protein arrays, ELISA, immunohistochemistry, or immunoprecipitation.

[0051] As used herein, the term "protein fragment" refers to a protein that, compared to a normal full-length protein, lacks one or more amino acids from its N-terminus and / or C-terminus or any portion thereof, wherein the fragment does not retain the original function of the full-length protein. In this invention, the protein is the OSCA4.1 protein obtained through the translation of the gene of this invention.

[0052] One of the more common methods for detecting proteins or protein fragments is the use of antibodies and techniques employing said antibodies. As used herein, the term "antibody" refers to immunoglobulin molecules and immunoactive fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to proteins (respond to an immune response). There are five main classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE).

[0053] Regarding the uses described herein, another aspect of the invention relates to a method for selecting plants that are resistant to PepMV infection or have an improved phenotype in terms of resistance to PepMV infection compared to WT (hereinafter referred to as the selection method of the invention), the method comprising the following steps:

[0054] a) Detecting a gene encoding a protein, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, and

[0055] b) Determine whether the gene described in step a) is inactivated.

[0056] The inactivated genes indicate resistance to PepMV infection compared to WT, or an improved phenotype in terms of resistance to PepMV infection.

[0057] In a preferred embodiment of the selection method of the present invention, the detection in step (a) is performed by determining the nucleotide sequence of the gene or a fragment thereof. In another preferred embodiment of the selection method of the present invention, the detection in step (a) is performed by detecting or quantifying the expression product of the gene of the present invention, wherein the product is selected from a list consisting of: complementary DNA or a fragment thereof, messenger RNA or a fragment thereof, and protein or a fragment thereof.

[0058] The terms and expressions “plant”, “resistance to PepMV infection”, “gene”, “protein”, “amino acid sequence”, “identity”, “inactivated”, “silenced”, “gene fragment”, “protein fragment”, “messenger RNA”, “mRNA fragment”, “complementary DNA”, and “cDNA fragment” have been previously defined in relation to the foregoing aspects of the present invention, and such definitions are equally valid for this aspect and its embodiments.

[0059] Methods for detecting genes by determining their nucleotide sequence or fragments thereof, or their expression products (e.g., mRNA, cDNA, or protein or fragments thereof), have previously been described in relation to the use of biomarkers of the present invention, relating to methods and techniques for detecting changes in the nucleotide sequence of the biomarker or in the expression product of the biomarker. These methods and techniques are equally effective for this aspect and its embodiments.

[0060] Other uses and methods for generating the plants or parts thereof, the reproductive or propagating plant material of the present invention, or the plant cells of the present invention rely on significant human intervention in biological processes using biomarkers of the present invention to achieve desired outcomes. Therefore, another aspect of this specification is the use of biomarkers of the present invention in assisted breeding projects to select plants that are resistant to PepMV infection or have an improved phenotype of PepMV infection resistance compared to the wild type. Another aspect of the invention relates to the use of biomarkers of the present invention for screening plant populations for the presence of inactivating alleles of genes of the present invention, wherein the presence indicates increased resistance to PepMV infection or an improved phenotype of PepMV infection resistance compared to the wild type.

[0061] As used herein, the term "marker-assisted breeding program" (also known as "marker-assisted selection") refers to a selection process in which a target trait is selected based on a marker associated with the trait rather than the trait itself. In this invention, the marker is the biomarker of this invention, and the trait is resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection.

[0062] Another aspect of the invention relates to a method for producing hybrids of the plants or parts thereof of the invention, hybrids of the reproductive or propagating plant material of the invention, or hybrids of the plant cells of the invention, wherein said hybrid plants or parts thereof, hybrid reproductive or propagating plant material, or hybrid plant cells exhibit resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection (hereinafter referred to as the hybridization method of the invention), the method comprising:

[0063] a) Hybridizing the plant or a portion thereof, the reproductive or propagating plant material of the present invention, or the plant cell of the present invention with a second plant; and

[0064] b) Harvest the hybrid offspring of the hybridization.

[0065] As used herein, the terms "hybrid," "hybrid plant," or "hybrid offspring" refer to individuals derived from genetically distinct parents (e.g., individuals that are genetically heterozygous or predominantly heterozygous). In a preferred embodiment of the hybridization method of the present invention, the second plant in step a) belongs to a species of the genera *Solanum*, *Capsicum*, *Nicotiana*, or *Physalis*. In another preferred embodiment, the second plant is selected from a list consisting of: tomato, potato, *Tomato pennisetum*, *Tomato spp.*, *Solanum arcanum*, *Solanum cheesmaniae*, *Solanum guaiacum ...

[0066] In another embodiment of the hybridization method of the present invention, the second plant in step a) is an inbred line, and the hybrid offspring in step b) is a single-cross F1 hybrid. As used herein, the term "inbred line" refers to a population that is genetically homozygous or nearly homozygous. For example, an inbred line can be obtained by several cycles of sibling breeding, self-pollination, or by producing double haploids. As used herein, the expression "single-cross F1 hybrid" refers to the first generation (or "F1") hybrid produced by crossing two inbred lines. In some preferred embodiments of the hybridization method of the present invention, the inbred line achieves purebred propagation for one or more desired phenotypic traits. In another further preferred embodiment of the hybridization method of the present invention, the inbred line is an elite line. As used herein, the term "elite line" refers to a plant line that provides a constant quality of product. An elite line is the result of many years of inbreeding and combines a variety of desirable traits, such as high yield, fruit quality, and resistance to pests, diseases, or abiotic stresses. These improved lines typically yield significantly higher average yields than the original wild (local) registered varieties. These improved lines can be used directly as crop plants or for producing single-cross F1 hybrids.

[0067] In a more preferred embodiment of the hybridization method of the present invention, the method further includes an additional step (c), wherein those hybrids harvested in step (b) that exhibit inactivation of a gene encoding a protein are selected by artificial intervention, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1.

[0068] Another aspect of the invention relates to plants or parts thereof, reproductive or propagating plant material, or plant cells obtained by the hybrid method of the invention.

[0069] Another aspect of the invention relates to a method for producing the plant or a portion thereof, the reproductive or propagating plant material of the invention, or the plant cells of the invention, wherein the plant or a portion thereof, the reproductive or propagating material, or the plant cells have, compared to the wild type, resistance to PepMV infection or an improved phenotype in terms of resistance to PepMV infection (from this point onward, the infiltration method of the invention), the method comprising:

[0070] a) Hybridize the breeding plant or part thereof of the present invention, the reproductive or propagating plant material of the present invention, or the breeding plant cell of the present invention with a second plant;

[0071] b) Select offspring plants resulting from the hybridization in step a) having introgression from the breeding plant or part thereof of the present invention, the breeding reproductive or propagating plant material of the present invention, or the breeding plant cells of the present invention, which are associated with resistance to PepMV or an improved phenotype in terms of resistance to PepMV infection.

[0072] c) Self-crossing and / or backcrossing the offspring plants selected in step (b), wherein the breeding plant or part thereof of the present invention, the breeding reproductive or propagating plant material of the present invention, the breeding plant cell line of the present invention or a second plant is used as a parent, as in (a).

[0073] d) Select progeny plants resulting from the hybridization in step c) that have introgression from the breeding plant or a portion thereof, the breeding reproductive or propagating plant material of the present invention, or the breeding plant cells of the present invention, which are associated with resistance to PepMV or with an improved phenotype in terms of resistance to PepMV infection; and

[0074] e) Repeat steps (c) and (d) for self-crossing and / or backcrossing, and the selected steps, to provide a plant breeding line that is substantially homozygous for the introgression, wherein at least one selection performed in step (b) or (d) is performed by marker-assisted selection.

[0075] The infiltration described therein comprises a mutation in a gene encoding a protein, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, and wherein the mutation results in the inactivation of the gene.

[0076] As used herein, the term "introgression" is intended to mean the introduction of a genetic determinant into a plant that does not carry the genetic determinant through hybridization and selection, starting from the first generation in which the trait becomes visible or detectable. For dominant traits, selection can begin once segregation for the trait begins in the F1 progeny of a cross between a plant exhibiting the trait and a plant that does not exhibit the trait (e.g., F2 or the first backcross [BC1] generation). For recessive traits, this can also begin from F2. Alternatively, and particularly for polygenic traits, selection can be performed using molecular markers associated with the trait. Marker-assisted selection can be carried out in any generation or population that may include plants carrying the marker.

[0077] As used herein, the term "hybridization" refers to the fertilization of a female plant (or gamete) by a male plant (or gamete). The term "gamete" refers to a haploid reproductive cell (egg or sperm) produced in a plant through mitosis from the gametophyte and involves sexual reproduction, during which two heterosexual gametes fuse to form a diploid zygote. The term generally includes references to pollen (including sperm cells) and ovules (including eggs). Thus, "hybridization" generally refers to the fertilization of an individual's ovules with pollen from another individual, while "self-fertilization" refers to the fertilization of an individual's ovules with pollen from the same individual. When hybridization is mentioned in the context of achieving introgression of genomic regions or segments, those skilled in the art will understand that, in order to achieve the introgression of only a portion of the chromosome of one plant into the chromosome of another plant, what is required is that random portions of the genomes of the two parental lines are recombined during hybridization because an exchange event occurs during the production of gametes in the parental lines. Therefore, the genomes of both parents must be combined in a single cell through hybridization, which will result in an introgression event after gametes are produced from the cell and they fuse during fertilization.

[0078] The term "backcross" refers to the process of crossing a plant resulting from hybridization between two parent lines with one of its parent lines, where the parent line used in the backcross is called the recurrent parent. Repeated backcrosses result in increasingly homozygous or inbred genomes.

[0079] Another aspect of the invention relates to a plant or part thereof, reproductive or propagating plant material or plant cell obtained by the infiltration method of the invention, characterized in that it comprises a gene encoding a protein, wherein the protein comprises an amino acid sequence having at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1, and wherein the gene has been inactivated.

[0080] All terminology definitions previously described in connection with other aspects of the invention are equally valid for all aspects and embodiments of the invention.

[0081] Attached Figure Description

[0082] Figure 1- Symptoms induced by PepMV-H30 in the leaves of wild-type and mutant tomato plants. Wild-type (WT) and 2F531 mutant plants of tomato cultivar M82 were inoculated with the invasive isolate PepMV-H30 (or uninoculated (-) for healthy controls), which induced bright yellow mottling in the leaflets of the infected plants. The photographs were taken 16 days after inoculation.

[0083] Figure 2 - Resistance of mutant 2F531 tomato plants to PepMV isolates from different strains. Viral load was measured by RT-qPCR using specific primers, and a reference calibration curve was used for absolute PepMV RNA quantification. A sharp decrease in PepMV accumulation was observed in 2F531 compared to WT plants. The PepMV isolates were: PepMV-Sp13, PepMV-H30 (EU strain), PepMV-PS5, and PepMV-KLP2 (CH2 strain). Mean and SD of four replicates from each of the three plants are shown. Total RNA was extracted at 16 days post-inoculation. *Indicates significant differences (p<0.05, Student's T test).

[0084] Figure 3 - Durability of resistance to mutant 2F531. Up to five passages of two PepMV isolates were performed in WT and mutant plants to assess the probability of resistance interruption: (A) a planning scheme for establishing six lineages of PepMV-Sp13 and six lineages of PepMV-PS5 in WT and 2F531 plants. (B) evolution of viral load (log10 ng viral RNA / 100 ng total RNA, at 16 days post-inoculation (dpi)) for isolates PepMV-Sp13 (top) and PepMV-PS5 (bottom).

[0085] Figure 4- A plot of mutations in 2F531 that confer resistance to PepMV. (A) Susceptibility of BC1F1 to PepMV-H30 compared to WT and 2F531: viral load accumulation (ng viral RNA / 100 ng total RNA), at 16 days post-inoculation (dpi); mean and SD of 8 replicates from 3 plants each. a and b indicate significantly different levels of PepMV accumulation (p < 0.05, LSD test). (B) Phenotypic analysis of BC1F3: histogram of the BC1F3 family, % of its susceptible individuals. (C) Manhattan plot depicting the differences in allele frequencies between the WT and R aggregates (y-axis) across all variants detected along the tomato chromosome according to the reference genome (Heinz 1706, SL2.50). Dashed lines show the threshold of differences in allele frequencies above 0.7, which is an indicator of association with resistance. (D) Genetic mapping and association analysis of SNPs in candidate regions of chromosome 2: linkage maps of 12 markers in candidate regions after segregation analysis of 200 BC1F2 individuals, with genetic distances expressed as cM (centers); and probability maps of loss of susceptibility associated with the markers, expressed as LOD scores (scales at the top of the graph, on the right).

[0086] Figure 5 - The number of paired reads mapped to candidate genes. The number of normalized paired reads (Y-axis) mapped to each candidate gene after RNA-seq, in 3 aggregates, each aggregate having 6 F2 plants whose offspring exhibit either a resistance (R) or susceptibility (WT) phenotype.

[0087] Figure 6 - The SlOSCA4.1 gene was edited using CRISPR / Cas9. (A) Schematic diagram of the coding sequence of the wild-type (WT) SlOSCA4.1 gene, indicating the location of the gRNA designed for gene editing via CRISPR / Cas9, and the edited gene slosca4.1_2 sequenced from the edited plant, which has a 1192 bp deletion relative to the WT gene. (B) PepMV symptom presentation in WT and slosca4.1_2 knockout tomato plants infected with PepMV-H30 (or uninfected, for healthy controls). (C) Accumulation of PepMV-Sp13 in WT tomato plants and slosca4.1_2 knockout mutants at 16 days post-inoculation. Data are the mean and standard deviation from 6 infected plants. Asterisks indicate significant differences, where one-way ANOVA was used (***p<0.001).

[0088] Figure 7 - Relative expression of PepMV in leaf protoplasts of WT and 2F531 mutants. Protoplasts from leaves of WT and 2F531 mutants were infected with purified PepMV-SP13 virus particles. Relative expression of PepMV at 0, 17, and 24 hours post-infection was analyzed by RT-qPCR. Expression at 17 and 24 hours was relativized to expression at 0 hours. Data are the mean and standard deviation of 8 replicates for WT and 7 replicates for mutants. Asterisks indicate significant differences, where t-statistics were used (p < 0.05). Example

[0089] Example 1

[0090] Example 1.1: Method

[0091] Tomato mutant screening

[0092] The mutant population consisted of 1000 M2 families, provided after treating seeds of tomato cultivar M82 with ethyl methanesulfonate (EMS). Twenty-five plants / families were sown in a nursery (Murcia, Spain) and transplanted to a greenhouse (Murcia, Spain) 33 days post-sowing, where windows and entrances were protected with thrips netting. WT tomato cultivar M82 plants were included as susceptible controls and for the boundary line. The plant density was 4 plants / m². On the day of transplanting, the plants were inoculated with the invasive isolate PepMV-KLP2 (Agüero et al., 2018, Front Plant Sci 9, 1-12). Nicotina benthamiana plants were used as inoculum for propagation: 14 days post-inoculation (dpi), symptomatic leaves above the inoculated leaves were harvested, mixed with 30 mM phosphate buffer (pH 8) to a concentration of 100 g / L, and stored at -80°C; this stock solution was diluted 5-fold for inoculation. Tomato plants were sprayed under autoclave with a suspension of carborundum powder (0.037 mm particle size; 10 g / L) in the inoculum solution. A total of 28 L of diluted inoculum solution was used for the entire tomato mutant population. A second round of inoculation was performed 28 days later to ensure infection. Forty-two days post-inoculation, each M2 plant was scored for symptom severity. A symptom severity scale of 0-2 was defined as follows: 0, no symptoms; 1, scattered bright yellow spots in new leaves; 2, bright yellow mottling affecting all new leaves. Plants scoring 0 and 1 were selected. To examine the association between symptoms and actual infection, PepMV was tested in 10 plants / family; we used molecular hybridization in tissue imprints of petiole cross sections, as described in Marco et al. (2003, Phytopathology 93, 844-852). The crop was managed according to standard practices, except for extreme sanitary measures for personnel working in the greenhouse. Once evaluated, fruits were harvested and seeds extracted from plants with mild or no symptoms, thus preserving over 600 M3 family members.

[0093] In the second round of selection, 10–12 plants / M3 family were evaluated; seeds were sterilized with 4% H2O2 for 30 minutes to eliminate contamination with PepMV, then sown in 40 seedling trays for inoculation and growth in an experimental greenhouse (CEBAS-CSIC, Murcia, Spain). The virus isolate used for inoculation was PepMV-H30, which, like PepMV-KLP2, induces bright yellow spots but has a more stable infection phenotype (Agüero et al., 2018, Front Plant Sci 9, 1–12). Inoculum was generated in tomato plants (Moneymaker) in this case, and mechanical inoculation was performed manually 21 days after sowing, as in Agüero et al. (2018, Front Plant Sci 9, 1–12). Plants were re-inoculated 14 days after the first inoculation. Symptom display was annotated at 25 dpi, recording the percentage of symptomatic plants for each family. Following evaluation, 3–6 plants from the selected family were transplanted into coconut fiber bags and grown under standard cultivation conditions in a greenhouse (Finca “La Matanza”, CEBAS-CSIC, Murcia, Spain) until fruit maturity. Controlled pollination was performed on the selected plants to obtain two rounds of self-pollination (M4 and M5 seeds).

[0094] Measure the viral load in 2F531 plants

[0095] Four PepMV isolates were identified on WT and 2F531 (M5 seed) plants: PepMV-Sp13 and PepMV-H30, belonging to the EU strain, and PepMV-PS5 and PepMV-KLP2, belonging to the CH2 strain. PepMV-Sp13 and PepMV-PS5 were attenuated isolates that induced mild symptoms, while PepMV-H30 and PepMV-KLP2 were invasive isolates (Agüero et al., 2018, Front Plant Sci 9, 1-12). Inoculum was revived in Tobacco Benedict plants following standard practice. Three to four replicates of each virus or virus isolate were inoculated on three to four tomato plants of each genotype. In all cases, plants with two true leaves were mechanically inoculated as previously described (Gómez et al., 2009a, J Virol 83, 12378-12387), and sampling was performed at 16 dpi. Plants were grown in 1.1L pots filled with a mixture of peat and coconut fiber (2:1) in a crystal greenhouse (CEBAS-CSIC) with climate control (daytime temperature set at 24-25°C, nighttime temperature set at 16-18°C, and 16 hours of light). Viral RNA quantification was performed after total RNA extraction. All leaves from each replicate of plants were harvested and homogenized in a homogenizer with 4 mL TNA buffer / g plant tissue (TNA: 2% SDS, 100 mM Tris HCl pH 8, 10 mM EDTA pH 8); 500 μL of the homogenate was sampled and compared with the same volume (RNA Isolation Reagent, Sigma Chemical Co, USA) was mixed; RNA extraction was performed according to the manufacturer's instructions. The final precipitate was dissolved in 50 μl of sterile RNase-free water and extracted using TURBO DNA-free reagent according to the manufacturer's protocol. TM The kit (Invitrogen, USA) is used to treat and remove any residual DNA. In Nano RNA levels were estimated using One (Thermo Scientific, USA). RT-qPCR was used for viral RNA quantification. Standard curves were generated for each of the different viruses detected using serial 1:10 dilutions of known concentrations of viral RNA. KAPA was used. The FAST Universal One-Step RT-qPCR kit (KAPA Biosystems, USA) was used, with 2 μl of purified viral RNA dilution or extracted plant RNA in a 20 μl reaction volume, and specific primers (Gómez et al., 2009a, J Virol 83, 12378-12387). Triple technical replicates / biological replicates were analyzed using a StepOnePlus thermal cycler (Applied Biosystems, USA).

[0096] Continuous passaging experiment

[0097] The stability of resistance to PepMV in 2F531 plants was characterized in a series of passage experiments. This experiment included two plant types (2F531 and WT) and two viral genotypes (PepMV-Sp13 and PepMV-PS5). M5 seeds were used for 2F531. Three plants of each genotype and each PepMV isolate were used to establish 12 lineages. Figure 3 A). First, the initial inoculum of the two isolates was regenerated in WT plants, quantified as described above, and prepared for use in subculture 0 to approximately 10. 7 The concentration of viral copies / ng total RNA was determined. 50 μL of inoculum was mechanically inoculated into each of the founder plants of each lineage at the two-true-leaf stage, followed by five successive passages; for this purpose, four 10.7 mm discs were removed from the second leaf above the inoculated leaf at 16 dpi and used as a new source of inoculum for the next passage in that lineage. Figure 3 A). Four additional discs from the same leaf were kept frozen at -80°C for quantifying viral load, as described above. All plants were grown in 1.1L pots filled with a mixture of peat and coconut fiber (2:1) in a crystal greenhouse (CEBAS-CSIC) with climate control (daytime temperature set at 24-25°C, nighttime temperature at 16-18°C, and 16 hours of light).

[0098] Plotting populations and phenotypic analysis

[0099] Controlled pollination was performed to obtain backcrosses to M82 (BC1F1). BC1F2 was obtained through controlled self-pollination of BC1F1. 204 BC1F2 individuals were grown and used as a mapping population, and self-pollinated to generate 204 BC1F3 progeny. In all cases, plants were grown in coconut fiber bags in a PVC greenhouse (Finca “La Matanza”, CEBAS-CSIC). Phenotypic values ​​for any given BC1F2 were determined by analyzing the susceptibility of 10–12 of its BC1F3 progeny to PepMV-H30. The method used for this progeny testing was similar to that previously described regarding the second round of selection in large-scale screening, except for re-inoculation at 7 dpi and final scoring at 14 dpi.

[0100] Segregating population grouping analysis and high-throughput genotyping

[0101] Two bulk clusters were generated: the WT cluster, with 18 BC1F2 individuals whose BC1F3 individuals exhibited 100% symptomatic progeny; and the R cluster, with 18 individuals exhibiting 0% symptomatic progeny. Leaf tissue from each BC1F2 individual was used for nucleic acid extraction. This followed the guidelines for "PureFood GMO and Authentification Kit for Food, Feed and Seed samples". Automated DNA extraction was performed using the CSC (Promega Corp., USA) operating protocol. Minor modifications were made to improve yield; specifically, 60 mg of ground tissue was used as the starting material, along with 600 μL of CTAB and 30 μL of proteinase K, incubated at 65°C for 2 hours, and the final volume was 80 μL. The 2.0 fluorometer (Lifetechnologies, USA) uses Qubit TM The dsDNA BR Assay Kit is used to quantify DNA, and... In One, quality was checked by electrophoresis on 1% agarose gel. DNA from selected individuals was pooled to ensure each was equally represented, and both pools were deep sequenced by Macrogen Inc. (South Corea). TruSeq DNA PCR-Free libraries with a fragment size of 350 bp were generated, and... The study was conducted on a 2500-High Throughput HORM (Illumina Inc., USA) with paired end reads to achieve approximately 50X coverage depth. Raw data were analyzed as follows: read quality was tested using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ); read mapping against the tomato reference genome (see Heinz 1706, version SL2.50; http: / / solgenomics.net / organism / Solanum_lycopersicum / genome; TomatoGenome Consortium, 2012) was performed using the BWA aligner (Li and Durbin, 2009, Bioinformatics 25, 1754-1760); and the M82 sequence was retrieved from public databases (Bolger et al., 2014a, Nat Genet 46, 1034-1038). The Freebayes procedure (Garrison and Marth, 2012, ArXiv:1207.3907[q-bio.GN]) was used for variant calling of the two pools and M82; after filtering, allele frequencies were calculated in the pools and M82 (criteria: available data in the two pools, coverage equal to or greater than 20 / sample, variant quality of at least 25, and total allele frequency less than 0.9). Finally, the differences in allele frequencies between the pools were calculated and plotted as a Manhattan plot using R software. It was estimated that such differences would not be less than 0.7 for Mendelian recessive mutations associated with the observed loss of function.

[0102] RNA-Seq

[0103] The same 18 BC1F2 individuals / clusters were further subdivided into 3 replicates / 6 plants. As described above, equal amounts of leaf tissue from each of the 6 plants / aggregates were used for RNA extraction, except by using a kit. The final RNA preparation was obtained using an RNA plant (Macherey-Nagel GmbH, Germany). This was achieved through the use of... The resulting RNA preparations were evaluated using a One and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). All samples showed RNA integrity numbers higher than 7.4. After library construction using the TruSeq Stranded mRNA LT kit (Illumina Inc.), the RNA was analyzed using NovaSeq. TM In the 6000 platform (Illumina Inc.), six pools (Macrogen Inc.) were sequenced, yielding 151 bp paired reads. The raw data (adaptor and 10 nucleotides at the 5' end) were trimmed using the Trimmomatic program (Bolger et al., 2014b), and filtered for quality using FastQC (minimum QC of 30 and a length of at least 70 bp). Reads were paired using BBMap (www.sourceforege.net / projects / bbmap), and then mapped against a reference genome using BWA's MEM algorithm (Li and Durbin, 2009, Bioinformatics 25, 1754-1760), with Qualimap (bampc) used to check mapping quality. Variant identification and allele frequency calculations were performed as described for DNA resequencing. The number of reads mapped to annotated genes was calculated using the featureCounts function of SubRead, its quality was determined using DESeq2, and it was normalized using rlog. DESeq2 also allows for the study of differential expression between R and WT samples, taking into account two factors: genotype and duplication. Goseq was used for enrichment analysis of GO. Finally, SnpEff (Cingolani et al., 2012, SnpEff.Fly 5, 29-30) was used to predict the biological effects of variants.

[0104] CRISPR / Cas9 editor

[0105] Three gRNAs complementary to the coding sequence of SlOSCA4.1 were designed using the bioinformatics tool BreakingCas (Oliveros et al., 2016). The targeted sequences in SlOSCA4.1 were 5'-ACTTCAATTACGACGTCGCT-3' (SEQ ID NO:7), 5'-CAGAGCTGCCGCCCTCAATA-3' (SEQ ID NO:8), and 5'-ATAAGGCTGTCCAGGACCTC-3' (SEQ ID NO:9). Following the... The protocol described in The Plant Cell, 2017, 29(6), 1196-1217, involved annealing sense and antisense oligonucleotides (Integrated DNA Technologies, Inc.) and cloning them into the pDIRECT_22C (Addgene ref. #91135) binary plasmid. The resulting plasmid was used to transform Agrobacterium tumefaciens strain GV3101, which was used to transform explants of the tomato cultivar Micro-Tom following the protocol described in Van Eck et al., 2006, 343, 459-473. Plants rooted in selective media were transferred to the substrate and acclimated in a growth chamber. To examine whether SlOSCA4.1 editing occurred in T0 plants, PCR amplification of the targeted region within the gene was performed using the Phire Tissue direct PCR kit (Thermo Scientific) following the manufacturer's instructions, followed by Sanger sequencing. The edited plants were self-pollinated to obtain T1 seeds. Genotyping was performed on the T1 plants grown in the substrate, selecting those with the mutation in a homozygous state.

[0106] Example 1.2: Loss of susceptibility to PepMV in a batch of tomato mutants

[0107] A population of 25,000 tomato mutants from 1,000 M2 families was screened. The mutant plants were inoculated with invasive PepMV isolates (which induce distinct bright yellow mottling). Symptom severity was scored for each plant using a 0-2 scale, where 0 represents no symptoms, 1 represents scattered bright yellow spots in new leaves, and 2 represents bright yellow mottling affecting all new leaves. Figure 1Invasive symptoms (score 2) were observed in 97.5% of the plants, which were recorded as susceptible. Plants with scores of 0 or 1 were present in 379 families. Subpopulations of symptomatic and asymptomatic plants were tested for PepMV infection, revealing a perfect correlation between infection and symptom presentation. One to four plants / families with scores of 0 or 1 were selected and self-crossed to generate more than 600 M3 families. From these, 10 to 12 individuals from each of the 453 M3 families were inoculated with PepMV and the symptom score was annotated again. The 2F531 family showed 100% asymptomatic plants, pointing to a homozygous mutation causing loss of susceptibility to PepMV; plants from this family were self-crossed to generate M4 seeds and backcrossed into the wild-type registered plant M82 to obtain BC1F1. From now on, we will refer to the loss of susceptibility phenotype in these plants as resistance.

[0108] Compared to the wild type, tomato plants carrying the inactivated gene of this invention did not exhibit phenotypic differences, except for resistance to PepMV infection or an improved phenotype in PepMV infection resistance. In the absence of PepMV infection, mutant 2F531 or slosca4.1_2-knockout small plants ( Figure 6 B) Both fully grown plants and WT plants are indistinguishable from WT plants: they have similar well-defined growth habits, leaf size, color and shape, fruit appearance, number or fruit set, and seed production (yield and germination capacity), indicating that the mutation does not alter the health of the tomato plant.

[0109] The breadth and persistence of resistance in mutant 2F531

[0110] PepMV accumulation in wild-type (WT) and 2F531 plants was compared after inoculation with PepMV-Sp13, PepMV-H30 (both EU strains), PepMV-PS5, or PepMV-KLP2 (CH2 strain). For all four isolates, a sharp and significant reduction in viral load was observed in the mutants compared to the WT plants. Figure 2 This was more pronounced for EU isolates (9 to 10 times) than for CH2 isolates (3 to 5 times), and also more drastic for invasive isolates (PepMV-H30 and PepMV-KLP2) than for mild isolates (PepMV-Sp13 and PepMV-PS5).

[0111] Durability of resistance is crucial for deploying sustainable pathogen control strategies in the field. To test whether PepMV could readily overcome 2F531 resistance, passage experiments were conducted. After initial inoculation with PepMV-Sp13 or PepMV-PS5, three virus lines were established on plants of each M82 or 2F531 genotype, and five successive passages were performed. Figure 3 A). Viral load was measured for each passage. For PepMV-PS5 passages, viral load fluctuated, but it was always lower in 2F531 than in WT plants (in most cases, almost an order of magnitude). Figure 3 B). For PepMV-Sp13, viral load fluctuations with passages are more pronounced, and in fact, by the 3rd, 4th, and 5th passages, the viral population in each of the 2F531 lineages becomes almost extinct. Figure 3 B).

[0112] Sequencing mapping of mutations associated with loss of susceptibility to PepMV.

[0113] BC1F1 plants showed similar PepMV accumulation and symptoms to WT plants. Figure 4 A), indicating that the 2F531 resistance is recessive. BC1F1 plants were self-pollinated to obtain over 200 BC1F2 plants, which were then allowed to grow and self-pollinated to obtain their BC1F3 progeny, and samples were taken individually for DNA and RNA extraction. Phenotypic analysis was performed on 204 BC1F3 families by inoculating 12 plants / families with PepMV. In 50 and 54 BC1F3 families, respectively, all individuals were either resistant or susceptible, while intrafamilial segregation was observed in 100 cases. Figure 4 B). These frequencies almost perfectly match the expected gene segregation in models where resistance is monogenic and recessive (χ² statistics, 2df = 0.23; α > 0.001).

[0114] Bulked Segregant Analysis (BSA), coupled with high-throughput sequencing (HTS), was used to map mutations associated with PepMV resistance. Two clusters were established: one containing 18 BC1F2 individuals (i.e., 0% of susceptible plants in BC1F3) in the R pool and another 18 BC1F2 individuals (i.e., 100% of susceptible plants in BC1F3) in the WT pool. For each cluster, the pooled DNA was sequenced at a depth of 50X. After quality filtering and alignment with the reference genome (Heinz1706, SL2.50), 99% of the reads were mappable, with 78% having a MAPQ quality score >57. After filtering for low coverage, 1,285,278 variants were identified against the reference genome. Most variants can be attributed to natural polymorphism between M82 and Heinz 1706, and only 6302 variants (0.49%) are attributed to EMS-induced mutagenesis; this indicates an approximate ratio of 1 mutation per 150 kbp in mutant 2F531. Allele frequencies were calculated in each aggregate for all variants. For the 10 SNPs and 1 insertion / deletion located distal to chromosome 2, the difference in allele frequencies was greater than 0.7, indicating an association between variant alleles and aggregate type. Figure 4 (C) These 11 variants span a 2.02 Mb region, from position 45,135,056 to 47,155,034; five of the mutations are located in annotated genes. This region includes 448 other low-coverage variants and covers 145 annotated genes.

[0115] To refine the gene mapping, recombination analysis was performed. Twenty-four SNPs identified within or near the target genomic region were selected and analyzed from 200 BC1F2 individuals; only 12 of the markers segregated in the population. Linkage maps were constructed for those markers, and association analysis was performed to correlate marker genotype with susceptibility. Figure 4D shows the genetic map and probability density along it, and the diagram illustrates the strong association between resistance and the markers C18 and C19. Analysis of recombinants further indicates that loss of susceptibility to PepMV is only demonstrated by individuals with alternative alleles in a homozygous state at a region framed by C16 and C20 (spanning 734,632 bp). Within this region, BSA-HTS analysis identified four variants; the predicted functional effects of these variants are low to moderate, except for mutations A through T at nucleotide position 1803 within the unique Solyc02g083430 exon, which corresponds to position 1660 within the major open reading frame of Solyc02g083430. The protein encoded by Solyc02g083430 has 831 amino acids, and this mutation affects the lysine at position 554, instead introducing an premature stop codon.

[0116] To validate and supplement the above data, RNA-Seq analysis was performed using pooled RNA from the R and WT clusters. Transcripts were sequenced, mapped to a reference genome, and filtered to identify variants, and allele frequencies were compared across pools. Again, the only locus with an allele frequency difference greater than 0.7 was located in the same genomic region previously identified, and variants were specifically detected at three loci: Solyc02g081200, Solyc02g082660, and Solyc02g083430. Some novel SNPs could be identified in this region, but with low coverage. When comparing the number of reads mapped to each of the three RNA-Seq candidates for the R vs WT pools (… Figure 5 Differences were observed for Solyc02g083430; although these were not statistically significant, reads of Solyc02g083430 appeared to be fewer for the resistant cluster than for the susceptible cluster, consistent with the nature of the mutations observed for this gene. On another front, gene ontology (GO) enrichment analysis identified 27 overrepresented GO entries. Nearly 9% of the dysregulated genes were associated with ATPase complexes within the cellular component category. Within the molecular function category, ATP binding (GO:0005524), aspartate endopeptidase activity (GO:0004190), and oxidoreductase activity (GO:0016491) were the most enriched. Finally, within the biological function category, only two enriched biological processes were identified: the response to injury (GO:0009611) and alcohol metabolism (GO:0006066).

[0117] Solyc02g083430 encodes SlOSCA4.1, a protein involved in vacuolar transport and a member of the high molar osmotic pressure gated calcium permeable channel 1 (OSCA) family.

[0118] The protein encoded by Solyc02g083430 (genomic sequence SEQ ID NO:4), SEQ ID NO:1, has three conserved domains: a transmembrane domain, which is part of a calcium-permeable cation exchange channel 1 activated by physical signals (e.g., osmotic stress) (Csc1_N); a phosphotransporter domain, which is predicted to be cytoplasmic (PHM7_cyt); and a region with seven transmembrane domains, which are part of a putative phosphotransporter (RSN1_7TM) (Zhu et al., 2008, Nat Genet 40, 854-861). The premature introduction of the stop codon at amino acid 554 leads to the loss of many of the transmembrane domains of RSN1_7TM, which can cause complete or partial loss of protein function, resulting in a mutant phenotype. Its closest Arabidopsis ortholog encodes AtOSCA4.1, which belongs to the high molar osmotic concentration-gated / mechanically activated calcium permeable channel (OSCA) family (Yuan et al., 2014, Nature 514, 367-371) and shares 69% amino acid identity with it. As in Arabidopsis, tomato OSCA4.1 (SlOSCA4.1) belongs to a small family of 12 members, phylogenetically organized into the same four clades as in Arabidopsis. AtOSCA4.1 has been definitively identified as a vacuolar sorting factor in two independent reports (Fuji et al., 2007, Plant Cell 19, 597-609; Delgadillo et al., 2020, PNAS 117, 9884-9895). Further searching for orthologs of the protein encoded by the gene Solyc02g083430 in important agricultural species using similar structural organization yielded two groups of proteins: one group is highly conserved within the Solanaceae family, with a minimum identity of 91.29% across the entire protein (Table 1); and the second group is highly conserved outside the Solanaceae family (Table 2), with a minimum identity of 62.77% across the entire protein. The structural similarity and domain distribution of these two groups of proteins suggest that these proteins should possess functions conserved for the protein encoded by Solyc02g083430.

[0119] Editing SlOSCA4.1 in the tomato cultivar Micro-Tom confirmed its proviral function against PepMV.

[0120] To confirm the involvement of SlOSCA4.1 in PepMV susceptibility, we used the CRISPR / Cas9 genome editing technology to generate the tomato cultivar Micro-Tom mutant at the Solyc02g083430 locus. The guide RNA was programmed to target the sequence at the beginning of the unique Solyc02g083430 exon. Homozygous mutations were observed in T1 generation individuals inoculated with PepMV. Figure 6 A). A similar infection phenotype to that of mutant 2F531 plants was observed: no disease symptoms were observed in the edited plants. Figure 6 B), and compared to WT plants, PepMV accumulation was significantly reduced in the mutant ( Figure 6 C), thus verifying the initial hypothesis.

[0121] Table 1: Orthologs of SEQ ID NO:1 in Solanaceae species

[0122]

[0123] Table 2: Orthologs of SEQ ID NO:1 in non-Solanaceae species

[0124]

[0125]

[0126] Example 2

[0127] Example 2.1: Method

[0128] Protoplast isolation and inoculation

[0129] Protoplasts were isolated from leaves of WT and 2F531 mutant tomato plants using a procedure described by Tan et al. (1987, Plant Cell Reports, 6(3), 172-175). Approximately 2 g of tomato leaves were harvested from WT and 2F531 mutants and subjected to protoplast isolation. Protoplasts were inoculated with 50 μg of purified PepMV virus particles containing approximately 2 × 10⁻⁶ particles using the PEG 4000 method. 6 Each protoplast sample of each cell was incubated in a growth chamber at 26°C under humid and constant light conditions for 24 hours. Protoplasts were sampled at 0, 17, and 24 hours post-infection. Total RNA was isolated from the protoplasts using Trizol reagent. Genomic DNA was removed from the RNA samples, the RNA was normalized, and used for expression analysis by RT-qPCR. PepMV expression was normalized using elongation factor 1-α as an endogenous gene.

[0130] Example 2.2: PepMV infection of tomato WT and 2F531 protoplasts confirmed its role in intracellular PepMV replication.

[0131] To supplement data on the involvement of SLOSCA4.1 in PepMV susceptibility and replication, we used leaf protoplasts from tomato cultivar M82 (WT and mutant 2F531). Protoplasts were isolated from leaves of WT and mutant plants, infected with purified PepMV virus particles, and sampled at 0, 17, and 24 hours post-infection. Viral expression was measured from total RNA by relative RT-qPCR using α-elongation factor 1 (EF-1α) as the endogenous gene for normalization. Results showed a significantly reduced relative accumulation of PepMV in protoplasts from mutant 2F531 plants compared to WT plants. Figure 7 This indicates that SlOSCA4.1 is involved in intracellular PepMV replication. sequence list <110> Abiopep, SL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (CSIC) <120> For plants resistant to the purslane mosaic virus infection <130> PCT4185.1 <150> ES P202130569 <151> 2021-06-18 <160> 9 <170> BiSSAP 1.3.6 <210> 1 <211> 831 <212> PRT <213> Tomato (Solanum lycopersicum) <400> 1 Met Ile Gln Ser Ser Phe Ser Ala Asp Ser Pro Ser Met Ala Ala Asn 1 5 10 15 Ser Thr Phe Ser Pro Pro Pro Ala Ala Gly Asp Gly Asp Phe Asn Tyr 20 25 30 Asp Val Ala Trp Tyr Gly Asn Ile Gln Tyr Leu Leu Asn Ile Ser Ala 35 40 45 Ile Gly Ala Leu Thr Cys Leu Leu Ile Phe Ile Phe Gly Lys Leu Arg 50 55 60 Ser Asp His Arg Arg Met Pro Gly Pro Thr Ala Ile Val Ser Lys Leu 65 70 75 80 Leu Ala Ala Trp His Ala Thr Gly Val Glu Ile Ala Arg His Cys Gly 85 90 95 Ala Asp Ala Ala Gln Tyr Leu Leu Ile Glu Gly Gly Ser Ser Ala Leu 100 105 110 Leu Leu Phe Leu Ala Leu Leu Ser Leu Ala Val Met Leu Pro Leu Asn 115 120 125 Ile Tyr Ala Gly Lys Ala Pro Met Ala Asp Gln Phe Ser Lys Thr Thr 130 135 140 Ile Asn His Ile Glu Lys Gly Ser Pro Leu Leu Trp Ile His Phe Ile 145 150 155 160 Phe Val Val Ile Val Val Val Leu Val His Tyr Gly Ile Ser Glu Ile 165 170 175 Gln Glu Arg Leu Lys Ile Thr Arg Leu Arg Asp Gly Tyr Gly Asn Pro 180 185 190 Ser Asn Ser Gly Thr Asn Val Ser Ala Ile Phe Ser Ile Met Val Gln 195 200 205 Gly Val Pro Lys Thr Leu Gly Phe Asp Lys Thr Pro Leu Val Glu Tyr 210 215 220 Phe Gln His Lys Tyr Pro Gly Lys Val Tyr Arg Val Val Val Pro Met 225 230 235 240 Asp Leu Cys Ala Leu Asp Asp Leu Ala Thr Glu Leu Val Lys Val Arg 245 250 255 Glu Asp Ile Ser Lys Leu Val Ser Arg Ile Glu Leu Arg Gly Tyr Leu 260 265 270 Asn Glu Gly Glu Glu Asp Glu Tyr Asn Asn Asp Ser Val Asn Gly Arg 275 280 285 Gly Leu Leu Glu Arg Leu Cys Phe Leu Trp Arg Lys Ala Lys Asp Thr 290 295 300 Trp Tyr His Val Val Asp Gln Leu Gly Phe Ser Asp Glu Glu Arg Leu 305 310 315 320 Arg Lys Leu Gln Glu Leu Arg Ala Asp Leu Glu Met Glu Met Ala Ser 325 330 335 Tyr Lys Glu Gly Arg Ala Arg Gly Ala Gly Val Ala Phe Val Val Phe 340 345 350 Lys Asp Val Phe Thr Ala Asn Lys Ala Val Gln Asp Leu Arg Asn Glu 355 360 365 Lys Arg Arg Arg Tyr Gly Arg Phe Phe Ser Val Ile Glu Leu Gln Leu 370 375 380 Gln Arg Asn Gln Trp Lys Val Glu Arg Ala Pro Leu Ala Thr Asp Ile 385 390 395 400 Tyr Trp Asn His Leu Gly Ser Thr Lys Phe Ser Leu Lys Leu Arg Arg 405 410 415 Val Leu Val Asn Thr Cys Leu Leu Leu Met Leu Leu Phe Cys Ser Ser 420 425 430 Pro Leu Ala Val Ile Ser Ala Ile Gln Ser Ala Gly Arg Ile Ile Asn 435 440 445 Ala Glu Ala Met Asp His Ala Gln Met Trp Leu Asn Trp Val Gln Gly 450 455 460 Ser Ser Trp Leu Ala Thr Ile Ile Phe Gln Phe Leu Pro Asn Val Leu 465 470 475 480 Ile Phe Val Ser Met Tyr Ile Val Val Pro Ser Val Leu Ser Tyr Leu 485 490 495 Ser Lys Phe Glu Gln His Leu Thr Val Ser Gly Glu Gln Arg Ala Glu 500 505 510 Leu Leu Lys Met Val Cys Phe Phe Leu Val Asn Leu Ile Leu Leu Arg 515 520 525 Ala Leu Val Glu Ser Thr Leu Glu Gly Ala Leu Leu Ser Met Gly Arg 530 535 540 Cys Tyr Leu Asp Gly Glu Asp Cys Lys Lys Ile Glu Gln Tyr Met Thr 545 550 555 560 Ala Ser Phe Leu Thr Arg Thr Cys Leu Ser Ser Leu Ala Phe Leu Ile 565 570 575 Thr Ser Ser Phe Leu Gly Ile Ser Phe Asp Leu Leu Ala Pro Ile Pro 580 585 590 Trp Ile Lys Lys Lys Leu Gln Lys Phe Arg Lys Asn Asp Met Leu Gln 595 600 605 Leu Val Pro Glu Arg Ser Glu Glu Tyr Pro Leu Glu Asn Gln Asp Ile 610 615 620 Asp Ser Leu Glu Arg Pro Leu Ile His Glu Arg Ser Ser Thr Val Ile 625 630 635 640 Ala Asp Asn Asn Gly Phe Leu His Asp Ala Ser Pro Asn Glu Ile Asp 645 650 655 Phe Pro Gly Gln Asp Leu Ser Glu Tyr Pro Pro Val Ser Arg Thr Ser 660 665 670 Pro Val Pro Lys Pro Lys Phe Asp Phe Ala Gln Tyr Tyr Ala Phe Asn 675 680 685 Leu Thr Ile Phe Ala Leu Thr Leu Ile Tyr Cys Ser Phe Ala Pro Leu 690 695 700 Val Val Pro Val Gly Ala Val Tyr Phe Gly Tyr Arg Tyr Leu Val Asp 705 710 715 720 Lys Tyr Asn Phe Leu Phe Val Tyr Arg Val Arg Gly Phe Pro Ala Gly 725 730 735 Asn Asp Gly Arg Leu Met Asp Thr Val Leu Ser Ile Met Arg Phe Cys 740 745 750 Val Asp Leu Phe Leu Leu Ser Met Leu Leu Phe Phe Ser Val Arg Gly 755 760 765 Asp Ser Thr Lys Leu Gln Ala Ile Phe Thr Leu Gly Leu Leu Val Val 770 775 780 Tyr Lys Leu Leu Pro Ser Asp Lys Asp Ser Phe Gln Pro Ala Leu Leu 785 790 795 800 Gln Gly Ile Gln Thr Ile Asp Asn Ile Val Glu Gly Pro Thr Asp Tyr 805 810 815 Glu Val Phe Ser Gln Pro Thr Phe Asp Trp Asp Thr Tyr Asn Ser 820 825 830 <210> 2 <211> 553 <212> PRT <213> artificial sequence <220> <223> Mutant EMS <400> 2 Met Ile Gln Ser Ser Phe Ser Ala Asp Ser Pro Ser Met Ala Ala Asn 1 5 10 15 Ser Thr Phe Ser Pro Pro Pro Ala Ala Gly Asp Gly Asp Phe Asn Tyr 20 25 30 Asp Val Ala Trp Tyr Gly Asn Ile Gln Tyr Leu Leu Asn Ile Ser Ala 35 40 45 Ile Gly Ala Leu Thr Cys Leu Leu Ile Phe Ile Phe Gly Lys Leu Arg 50 55 60 Ser Asp His Arg Arg Met Pro Gly Pro Thr Ala Ile Val Ser Lys Leu 65 70 75 80 Leu Ala Ala Trp His Ala Thr Gly Val Glu Ile Ala Arg His Cys Gly 85 90 95 Ala Asp Ala Ala Gln Tyr Leu Leu Ile Glu Gly Gly Ser Ser Ala Leu 100 105 110 Leu Leu Phe Leu Ala Leu Leu Ser Leu Ala Val Met Leu Pro Leu Asn 115 120 125 Ile Tyr Ala Gly Lys Ala Pro Met Ala Asp Gln Phe Ser Lys Thr Thr 130 135 140 Ile Asn His Ile Glu Lys Gly Ser Pro Leu Leu Trp Ile His Phe Ile 145 150 155 160 Phe Val Val Ile Val Val Val Leu Val His Tyr Gly Ile Ser Glu Ile 165 170 175 Gln Glu Arg Leu Lys Ile Thr Arg Leu Arg Asp Gly Tyr Gly Asn Pro 180 185 190 Ser Asn Ser Gly Thr Asn Val Ser Ala Ile Phe Ser Ile Met Val Gln 195 200 205 Gly Val Pro Lys Thr Leu Gly Phe Asp Lys Thr Pro Leu Val Glu Tyr 210 215 220 Phe Gln His Lys Tyr Pro Gly Lys Val Tyr Arg Val Val Val Pro Met 225 230 235 240 Asp Leu Cys Ala Leu Asp Asp Leu Ala Thr Glu Leu Val Lys Val Arg 245 250 255 Glu Asp Ile Ser Lys Leu Val Ser Arg Ile Glu Leu Arg Gly Tyr Leu 260 265 270 Asn Glu Gly Glu Glu Asp Glu Tyr Asn Asn Asp Ser Val Asn Gly Arg 275 280 285 Gly Leu Leu Glu Arg Leu Cys Phe Leu Trp Arg Lys Ala Lys Asp Thr 290 295 300 Trp Tyr His Val Val Asp Gln Leu Gly Phe Ser Asp Glu Glu Arg Leu 305 310 315 320 Arg Lys Leu Gln Glu Leu Arg Ala Asp Leu Glu Met Glu Met Ala Ser 325 330 335 Tyr Lys Glu Gly Arg Ala Arg Gly Ala Gly Val Ala Phe Val Val Phe 340 345 350 Lys Asp Val Phe Thr Ala Asn Lys Ala Val Gln Asp Leu Arg Asn Glu 355 360 365 Lys Arg Arg Arg Tyr Gly Arg Phe Phe Ser Val Ile Glu Leu Gln Leu 370 375 380 Gln Arg Asn Gln Trp Lys Val Glu Arg Ala Pro Leu Ala Thr Asp Ile 385 390 395 400 Tyr Trp Asn His Leu Gly Ser Thr Lys Phe Ser Leu Lys Leu Arg Arg 405 410 415 Val Leu Val Asn Thr Cys Leu Leu Leu Met Leu Leu Phe Cys Ser Ser 420 425 430 Pro Leu Ala Val Ile Ser Ala Ile Gln Ser Ala Gly Arg Ile Ile Asn 435 440 445 Ala Glu Ala Met Asp His Ala Gln Met Trp Leu Asn Trp Val Gln Gly 450 455 460 Ser Ser Trp Leu Ala Thr Ile Ile Phe Gln Phe Leu Pro Asn Val Leu 465 470 475 480 Ile Phe Val Ser Met Tyr Ile Val Val Pro Ser Val Leu Ser Tyr Leu 485 490 495 Ser Lys Phe Glu Gln His Leu Thr Val Ser Gly Glu Gln Arg Ala Glu 500 505 510 Leu Leu Lys Met Val Cys Phe Phe Leu Val Asn Leu Ile Leu Leu Arg 515 520 525 Ala Leu Val Glu Ser Thr Leu Glu Gly Ala Leu Leu Ser Met Gly Arg 530 535 540 Cys Tyr Leu Asp Gly Glu Asp Cys Lys 545 550 <210> 3 <211> 434 <212> PRT <213> artificial sequence <220> <223> CRISPR / Cas9 mutant <400> 3 Met Ile Gln Ser Ser Phe Ser Ala Asp Ser Gln Lys Phe Ser Leu Lys 1 5 10 15 Leu Arg Arg Val Leu Val Asn Thr Cys Leu Leu Leu Met Leu Leu Phe 20 25 30 Cys Ser Ser Pro Leu Ala Val Ile Ser Ala Ile Gln Ser Ala Gly Arg 35 40 45 Ile Ile Asn Ala Glu Ala Met Asp His Ala Gln Met Trp Leu Asn Trp 50 55 60 Val Gln Gly Ser Ser Trp Leu Ala Thr Ile Ile Phe Gln Phe Leu Pro 65 70 75 80 Asn Val Leu Ile Phe Val Ser Met Tyr Ile Val Val Pro Ser Val Leu 85 90 95 Ser Tyr Leu Ser Lys Phe Glu Gln His Leu Thr Val Ser Gly Glu Gln 100 105 110 Arg Ala Glu Leu Leu Lys Met Val Cys Phe Phe Leu Val Asn Leu Ile 115 120 125 Leu Leu Arg Ala Leu Val Glu Ser Thr Leu Glu Gly Ala Leu Leu Ser 130 135 140 Met Gly Arg Cys Tyr Leu Asp Gly Glu Asp Cys Lys Lys Ile Glu Gln 145 150 155 160 Tyr Met Thr Ala Ser Phe Leu Thr Arg Thr Cys Leu Ser Ser Leu Ala 165 170 175 Phe Leu Ile Thr Ser Ser Phe Leu Gly Ile Ser Phe Asp Leu Leu Ala 180 185 190 Pro Ile Pro Trp Ile Lys Lys Lys Leu Gln Lys Phe Arg Lys Asn Asp 195 200 205 Met Leu Gln Leu Val Pro Glu Arg Ser Glu Glu Tyr Pro Leu Glu Asn 210 215 220 Gln Asp Ile Asp Ser Leu Glu Arg Pro Leu Ile His Glu Arg Ser Ser 225 230 235 240 Thr Val Ile Ala Asp Asn Asn Gly Phe Leu His Asp Ala Ser Pro Asn 245 250 255 Glu Ile Asp Phe Pro Gly Gln Asp Leu Ser Glu Tyr Pro Pro Val Ser 260 265 270 Arg Thr Ser Pro Val Pro Lys Pro Lys Phe Asp Phe Ala Gln Tyr Tyr 275 280 285 Ala Phe Asn Leu Thr Ile Phe Ala Leu Thr Leu Ile Tyr Cys Ser Phe 290 295 300 Ala Pro Leu Val Val Pro Val Gly Ala Val Tyr Phe Gly Tyr Arg Tyr 305 310 315 320 Leu Val Asp Lys Tyr Asn Phe Leu Phe Val Tyr Arg Val Arg Gly Phe 325 330 335 Pro Ala Gly Asn Asp Gly Arg Leu Met Asp Thr Val Leu Ser Ile Met 340 345 350 Arg Phe Cys Val Asp Leu Phe Leu Leu Ser Met Leu Leu Phe Phe Ser 355 360 365 Val Arg Gly Asp Ser Thr Lys Leu Gln Ala Ile Phe Thr Leu Gly Leu 370 375 380 Leu Val Val Tyr Lys Leu Leu Pro Ser Asp Lys Asp Ser Phe Gln Pro 385 390 395 400 Ala Leu Leu Gln Gly Ile Gln Thr Ile Asp Asn Ile Val Glu Gly Pro 405 410 415 Thr Asp Tyr Glu Val Phe Ser Gln Pro Thr Phe Asp Trp Asp Thr Tyr 420 425 430 Asn Ser <210> 4 <211> 2496 <212> DNA <213> Tomato <400> 4 atgatccaat ccagcttctc tgcagactca ccttccatgg cagccaattc cactttctct 60 cctccgccgg ccgccggtga cggagacttc aattacgacg tcgcttggta tggtaacatc 120 cagtacctcc tcaatatc cgccattgga gctttgactt gccttcttat ttcatctttc 180 gggaagcttc gaagcgacca ccgtcgcatg cccggtccca ctgccattgt ctccaagctc 240 ttagctgcct ggcacgccac tggtgttgaa atcgcccgcc actgcggggc tgacgctgct 300 caatatctcc ttattgaggg cggcagctct gccctgctat tattcctcgc cttctttct 360 cttgctgtaa tgctgccgtt gaatatatat gctggtaagg ctcctatggc tgatcagttt 420 tcaaagacta caataaacca tatagaaaaa ggttctccat tactctggat tcactttata 480 tttgttgtta ttgttgttgt tttggtacat tatggtataa gtgaaataca agaaaggttg 540 aaaattacta gacttagaga cggctatgga aatccgagta attctggtac aaatgtcagt 600 gcaattttt ccattatggt gcagggtgta ctaagacct taggttttga taagacacct 660 ttagtggagt atttcagca taaatatccg gggaaggtgt atagagtagt tgtccctatg 720 gatttgtgtg ctctggatga tttagctaca gagttggtga aggttcgggga agatatctct 780 aaactagtct caagaattga gttacggggt tatttgaatg agggtgagga agacgagtat 840 aataatgata gtgtgaacgg gcggggcttg ttagaacgac tgtgcttttt gtggagaaag 900 gctaaggata catggtatca tgttgtggat caattaggtt tctcagatga agagagatta 960 agaaaattgc aagagttgag agctgatttg gagatggaaa tggcatctta taaagaaggg 1020 agggcaagag gtgctggtgt agcttttgtg gtatttaagg acgtattcac agctaataag 1080 gctgtccagg acctccggaa tgagaagagg aggcgatatg gtcgattctt ctcagtcatt 1140 gagttgcaac tacagaggaa ccagtggaaa gtggagagag ctcctttagc tactgacata 1200 tactggaacc acctgggatc aacaaagttc tccttaaagc tgcgcagagt gttggtgaac 1260 acatgcctat tgttgatgtt gctattctgc agttctccac tagctgtgat tagtgctatt 1320 caaagtgcag ggagaataat caatgctgaa gctatggatc atgctcagat gtggctgaac 1380 tgggtgcagg gctcgagctg gctagcaaca ataatatttc aatttttgcc caatgttctg 1440 atttttgtga gcatgtacat tgttgtccct tcagttcttt cttatctttc taaatttgaa 1500 caacatctta ctgtatctgg tgagcaaagg gctgagctac tgaaaatggt ttgcttcttt 1560 ctggtaaatc tcattctgct tagggctctg gtcgaatcta ctcttgaggg tgctctctta 1620 agtatgggtc ggtgttattt ggatggagaa gattgcaaaa agatcgagca gtacatgact 1680 gcttcctttt tgacaaggac atgcctctcg tctcttgcat ttttaattac aagcagtttt 1740 ttgggtatat cttttgattt attagctcca attccttgga ttaagaagaa gcttcaaaag 1800 ttccgtaaaa atgacatgct tcagttggta ccagaacgga gtgaggagta cccattggaa 1860 aatcaagaca ttgatagttt ggagaggcct ctgattcatg aaaggagttc aactgtgatt 1920 gctgacaaca atggattttt acacgatgcc tctccaaatg aaattgattt ccctggacaa 1980 gatttgtctg aataccctcc agtcagccga acctcaccag ttccaaagcc gaagtttgat 2040 tttgcacagt attatgcttt caatctgaca atatttgccc taaccctgat ctattgttcg 2100 tttgctcctc tggtggttcc tgttggtgca gtttactttg ggtaccggta tttagttgac 2160 aagtacaact tcctgtttgt atacagagtg cgaggtttcc ctgctggtaa tgatgggagg 2220 ttgatggata ctgtattatc tatcatgagg ttttgtgttg acttgttcct cctttcaatg 2280 ctacttttct tttctgtacg aggagactca actaagcttc aagccatatt cacacttgga 2340 ttgttagtgg tgtataaact cttgccctct gataaggatt cttttcagcc agcgttatta 2400 caaggcatac agactattga caacattgtc gaagggccaa ctgattatga ggttttctca 2460 caacctacat ttgattggga tacgtataat tcatga 2496 <210> 5 <211> 2496 <212> DNA <213> Artificial sequence <220> <223> EMS mutant <400> 5 atgatccaat ccagcttctc tgcagactca ccttccatgg cagccaattc cactttctct 60 cctccgccgg ccgccggtga cggagacttc aattacgacg tcgcttggta tggtaacatc 120 cagtacctcc tcaatatctc cgccattgga gctttgactt gccttcttat tttcatcttc 180 gggaagcttc gaagcgacca ccgtcgcatg cccggtccca ctgccattgt ctccaagctc 240 ttagctgcct ggcacgccac tggtgttgaa atcgcccgcc actgcggggc tgacgctgct 300 caatatctcc ttattgaggg cggcagctct gccctgctat tattcctcgc ccttctttct 360 cttgctgtaa tgctgccgtt gaatatatat gctggtaagg ctcctatggc tgatcagttt 420 tcaaagacta caataaacca tatagaaaaa ggttctccat tactctggat tcactttata 480 tttgttgtta ttgttgttgt tttggtacat tatggtataa gtgaaataca agaaaggttg 540 aaaattacta gacttagaga cggctatgga aatccgagta attctggtac aaatgtcagt 600 gcaattttt ccattatggt gcagggtgta ctaagacct taggttttga taagacacct 660 ttagtggagt atttcagca taaatatccg gggaaggtgt atagagtagt tgtccctatg 720 gatttgtgtg ctctggatga tttagctaca gagttggtga aggttcgggga agatatctct 780 aaactagtct caagaattga gttacggggt tatttgaatg agggtgagga agacgagtat 840 aataatgata gtgtgaacgg gcggggcttg ttagaacgac tgtgcttttt gtggagaaag 900 gctaaggata catggtatca tgttgtggat caattaggtt tctcagatga agagagatta 960 agaaaattgc aagagttgag agctgatttg gagatggaaa tggcatctta taaagaaggg 1020 agggcaagag gtgctggtgt agctttttgtg gtatttaagg acgtattcac agctaataag 1080 gctgtccagg acctccggaa tgagaagagg aggcgatatg gtcgattctt ctcagtcatt 1140 gagttgcaac tacagaggaa ccagtggaaa gtggagagag ctcctttagc tactgacata 1200 tactggaacc acctgggatc aacaaagttc tccttaaagc tgcgcagagt gttggtgaac 1260 acatgcctat tgttgatgtt gctattctgc agttctccac tagctgtgat tagtgctatt 1320 caaagtgcag ggagaataat caatgctgaa gctatggatc atgctcagat gtggctgaac 1380 tgggtgcagg gctcgagctg gctagcaaca ataatatttc aatttttgcc caatgttctg 1440 atttttgtga gcatgtacat tgttgtccct tcagttcttt cttatctttc taaatttgaa 1500 caacatctta ctgtatctgg tgagcaaagg gctgagctac tgaaaatggt ttgcttcttt 1560 ctggtaaatc tcattctgct tagggctctg gtcgaatcta ctcttgaggg tgctctctta 1620 agtatgggtc ggtgttattt ggatggagaa gattgcaaat agatcgagca gtacatgact 1680 gcttcctttt tgacaaggac atgcctctcg tctcttgcat ttttaattac aagcagtttt 1740 ttgggtatat cttttgattt attagctcca attccttgga ttaagaagaa gcttcaaaag 1800 ttccgtaaaa atgacatgct tcagttggta ccagaacgga gtgaggagta cccattggaa 1860 aatcaagaca ttgatagttt ggagaggcct ctgattcatg aaaggagttc aactgtgatt 1920 gctgacaaca atggatttt acacgatgcc tctccaaatg aaattgattt ccctggacaa 1980 gatttgtctg aataccctcc agtcagccga acctcaccag ttccaaagcc gaagtttgat 2040 tttgcacagt attatgcttt caatctgaca atattgccc taaccctgat ctattgttcg 2100 tttgctcctc tggtggttcc tgttggtgca gttactttg ggtaccggta tttagttgac 2160 aagtacaact tcctgtttgt atacagagtg cgaggttttcc ctgctggtaa tgatgggagg 2220 ttgatggata ctgtattac tatcatgagg ttttgtgttg acttgttcct cctttcaatg 2280 ctactttct tttctgtacg aggagactca actaagcttc aagccatatt cacacttgga 2340 ttgttagtgg tgtataaact cttgccctct gataaggatt cttttcagcc agcgttatta 2400 caaggcatac agactattga caacattgtc gaagggccaa ctgattatga ggttttctca 2460 caacctacat ttgattggga tacgtataat tcatga 2496 <210> 6 <211> 1305 <212> DNA <213> Artificial sequence <220> <223> CRISPR / Cas9 mutant <400> 6 atgatccaat ccagcttctc tgcagactca caaaagttct ccttaaagct gcgcagagtg 60 ttggtgaaca catgcctatt gttgatgttg ctattctgca gttctccact agctgtgatt 120 agtgctattc aaagtgcagg gagaataatc aatgctgaag ctatggatca tgctcagatg 180 tggctgaact gggtgcaggg ctcgagctgg ctagcaacaa taatatttca atttttgccc 240 aatgttctga tttttgtgag catgtacatt gttgtccctt cagttctttc ttatctttct 300 aaatttgaac aacatcttac tgtatctggt gagcaaaggg ctgagctact gaaaatggtt 360 tgcttctttc tggtaaatct cattctgctt agggctctgg tcgaatctac tcttgagggt 420 gctctcttaa gtatgggtcg gtgttatttg gatggagaag attgcaaaaa gatcgagcag 480 tacatgactg cttccttttt gacaaggaca tgcctctcgt ctcttgcatt tttaattaca 540 agcagttttt tgggtatatc ttttgattta ttagctccaa ttccttggat taagaagaag 600 cttcaaaagt tccgtaaaaa tgacatgctt cagttggtac cagaacggag tgaggagtac 660 ccattggaaa atcaagacat tgatagtttg gagaggcctc tgattcatga aaggagttca 720 actgtgattg ctgacaacaa tggattttta cacgatgcct ctccaaatga aattgatttc 780 cctggacaag atttgtctga ataccctcca gtcagccgaa cctcaccagt tccaaagccg 840 aagtttgatt ttgcacagta ttatgctttc aatctgacaa tatttgccct aaccctgatc 900 tattgttcgt ttgctcctct ggtggttcct gttggtgcag tttactttgg gtaccggtat 960 ttagttgaca agtacaactt cctgtttgta tacagagtgc gaggtttccc tgctggtaat 1020 gatgggaggt tgatggatac tgtattatct atcatgaggt tttgtgttga cttgttcctc 1080 ctttcaatgc tacttttctt ttctgtacga ggagactcaa ctaagcttca agccatattc 1140 acacttggat tgttagtggt gtataaactc ttgccctctg ataaggattc ttttcagcca 1200 gcgttattac aaggcataca gactattgac aacattgtcg aagggccaac tgattatgag 1260 gttttctcac aacctacatt tgattgggat acgtataatt catga 1305 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Guide RNA <400> 7 acttcaatta cgacgtcgct 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Guide RNA <400> 8 cagagctgcc gccctcaata 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Guide RNA <400> 9 ataaggctgt ccaggacctc 20

Claims

1. A plant, reproductive or propagating plant material, or plant cells, characterized in that, It contains a gene encoding a protein, wherein the protein consists of the amino acid sequence SEQ ID NO: 1, and the gene is inactivated; and wherein the plant, reproductive or propagating plant material, or plant cell belongs to the tomato family (Tomato). Solanum lycopersicum This species.

2. The plant, reproductive or propagating plant material, or plant cell according to claim 1, wherein the gene is inactivated and encodes a protein consisting of the amino acid sequence SEQ ID NO:

2.

3. The plant, reproductive or propagating plant material, or plant cell according to claim 1, wherein the gene is inactivated and encodes a protein consisting of the amino acid sequence SEQ ID NO:

3.

4. The plant, reproductive or propagating plant material, or plant cell according to any one of claims 1-3, wherein the reproductive or propagating plant material is selected from fruits, seeds, tubers, or offspring.

5. A method for producing a plant, reproductive or propagating plant material, or plant cell according to any one of claims 1-4, wherein the plant, reproductive or propagating plant material, or plant cell exhibits resistance to PepMV infection or an improved phenotype in terms of PepMV infection resistance, the method comprising: a) subjecting the plant, reproductive or propagating plant material, or plant cells to directed mutagenesis, and b) Detecting a mutation in a gene encoding a protein in the plant, reproductive or propagating plant material, or plant cell, wherein the protein consists of the amino acid sequence SEQ ID NO: 1, and wherein the mutation results in the inactivation of the gene.

6. The method of claim 5, wherein the directed mutagenesis is achieved by gene targeting dependent on homologous recombination, antisense RNA, directed transposon insertion, virus-induced gene silencing, or genome editing techniques.

7. The method of claim 6, wherein the genome editing technology is CRISPR / Cas technology.

8. Use of a gene as a biomarker for selecting plants that have resistance to PepMV infection or have an improved phenotype in resistance to PepMV infection, wherein the gene encodes a protein consisting of the amino acid sequence SEQ ID NO: 1, wherein the gene has been inactivated, and wherein the plant belongs to the species tomato.

9. The use according to claim 8, wherein the selection is performed by determining the nucleotide sequence of the gene.

10. A method for selecting plants that are resistant to PepMV infection or have an improved phenotype of resistance to PepMV infection compared to the wild type, said plants belonging to the species tomato, said method comprising the following steps: a) Detect the gene encoding a protein, wherein the protein consists of the amino acid sequence SEQ ID NO: 1, and b) Determine whether the gene described in step a) is inactivated. The inactivated genes indicate resistance to PepMV infection compared to the wild type, or an improved phenotype in terms of resistance to PepMV infection.

11. The method of claim 10, wherein the detection in step a) is performed by determining the nucleotide sequence of the gene or a fragment thereof.

12. A method for producing a hybrid of plant, reproductive or propagating plant material, or plant cells according to any one of claims 1-4, wherein the hybrid exhibits resistance to PepMV infection or an improved phenotype in terms of PepMV infection resistance, the method comprising: a) Hybridizing the plant, reproductive or propagating plant material, or plant cell according to any one of claims 1 to 4 with a second plant; and b) Harvest the hybrid offspring of the aforementioned hybridization.

13. A method for producing a plant, reproductive or propagating plant material, or plant cell according to any one of claims 1-4, wherein the plant, reproductive or propagating plant material, or plant cell exhibits resistance to PepMV infection or an improved phenotype of PepMV infection resistance compared to a wild type, the method comprising: a) Hybridize the breeding plant, breeding reproductive or propagating plant material, or breeding plant cell according to any one of claims 1-4 with a second plant; b) Select offspring plants resulting from the hybridization in step a) having introgression from breeding plants, breeding reproductive or propagating plant material or breeding plant cells according to any one of claims 1-4, which is associated with resistance to PepMV or an improved phenotype in terms of resistance to PepMV infection. c) Self-crossing and / or backcrossing the offspring plants selected in step (b), wherein the breeding plant, breeding reproductive or propagating plant material, breeding plant cell line or second plant is used as a parent as in (a); d) Select progeny plants resulting from the hybridization in step c) that have introgression from the breeding plant, breeding reproductive or propagating plant material, or breeding plant cells, which is associated with resistance to PepMV or with an improved phenotype in terms of resistance to PepMV infection; and e) Repeat steps (c) and (d) of self-crossing and / or backcrossing, as well as the selected steps, to provide a plant breeding line that is substantially homozygous for the introgression, wherein at least one selection performed in step (b) or (d) is performed by marker-assisted selection. The introgression involves a mutation in a gene encoding a protein, the protein being composed of the amino acid sequence SEQ ID NO: 1, and the mutation results in the inactivation of the gene, and the second plant in step a) belongs to the species tomato.

14. The method according to claim 12 or 13, wherein the second plant in step a) is an inbred line, and the hybrid offspring in step b) is a single-cross F1 hybrid.

15. The method according to claim 12 or 13, further comprising an additional step (c), wherein those hybrids harvested in step (b) showing inactivation of a gene encoding a protein are selected by artificial intervention, wherein the protein consists of the amino acid sequence SEQ ID NO:

1.

16. A plant, reproductive or propagating plant material, or plant cell obtained by the method according to any one of claims 12 to 15, wherein the plant, reproductive or propagating plant material, or plant cell contains a gene encoding a protein, wherein the protein consists of the amino acid sequence SEQ ID NO: 1, and wherein the gene has been inactivated.

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