Tomato plants resistant to resistant breakthrough TSWV strains and corresponding resistance genes

Abstract

The present invention relates to a Sw-5b protein of a variant of a wild type protein which confers recognition of an NSm protein against tomato spotted wilf virus (TSWV), in particular resistant to a breakthrough type TSWV, and wherein the variant comprises a variation relative to the Sw-5b protein at one or more of the following positions: 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and 759. The invention also relates to a gene sequence encoding the variant protein, preferably to a mutated sw-5b gene, and to plants, in particular tomato plants, comprising in its genome said mutated gene conferring resistance to RB-TSWV. The invention also relates to parts and progeny of these plants and to the use of these sequences for providing TSWV resistance.

Description

TECHNICAL FIELD

[0001] The present invention relates to the resistance of tomato (Solanum lycopersicum) plants, also known as tomatoes (Lycopersicum esculentum), to tospoviruses, in particular to Tomato Spotted Wilt virus (TSWV), including resistance-breaking (RB)-TSWV. More specifically, the present invention relates to tomato plants, cells, seeds and fruits comprising resistance genes and / or proteins conferring resistance to at least TSWV and RB-TSWV. According to the present invention, the resistance gene conferring resistance to these tospoviruses is a variant or allele of the wild-type sw-5b gene. The resistance gene can be present homozygously or heterozygously in the genome of the tomato plant. The present invention also relates to the resistance gene and its parts, the polypeptides and proteins encoded thereby, and the use of these sequences and proteins to obtain resistant plants. The present invention also relates to the seeds and progeny of such plants, to the propagation material for obtaining such plants, and to the different uses of these plants. BACKGROUND OF THE INVENTION

[0002] All cultivated and commercial forms of tomatoes belong to the species most commonly known as tomato (Lycopersicon esculentum Miller). The genus Lycopersicon is a relatively small genus within the extremely large and diverse Solanaceae family, which is thought to consist of approximately 90 genera, including pepper, tobacco and eggplant. The genus Lycopersicon has been divided into at least two subgenera, the esculentum complex containing those species that can be easily hybridized with commercial tomatoes, and the peruvianum complex containing those species that are rather difficult to hybridize. Due to its value as a crop, tomatoes have been widely dispersed throughout the world.

[0003] Tomatoes are grown for their fruits and are widely used in the fresh market or processed products. As a crop, tomatoes are commercially grown anywhere environmental conditions allow for an economically viable yield. Most fresh market tomatoes are hand-harvested at the vine-ripe and mature green-ripe stages. Fresh market tomatoes are available year-round. Processed tomatoes are mainly mechanically harvested and used in a variety of forms, such as canned tomatoes, tomato juice, tomato paste, puree, tomato sauce or even tomato ketchup.

[0004] Tomatoes are generally simple diploid species with 12 pairs of differentiated chromosomes. However, polyploid tomatoes are also part of the present invention. Cultivated tomatoes are self-pollinating and are almost entirely self-pollinated. Tomato flowers are hermaphroditic. Commercial cultivars were initially open-pollinated. Since heterosis has been identified in tomatoes, with higher yields and more uniform plant characteristics, hybrids are becoming increasingly popular among farmers, and hybrids are replacing open-pollinated varieties. Due to its wide spread and high value, tomatoes have been intensively cultivated. This explains why there are now such a wide variety of tomato types. The range of shapes can be from small to large, and there are cherry, plum, pear, blocky, round, and beefsteak types. Tomatoes can be grouped by the amount of time it takes for the plant to reach a mature fruit ready for harvest, and cultivars are generally considered early-maturing, mid-maturing, or late-maturing. Tomatoes can also be grouped by the growth habit of the plant; grouped as determinate, semi-determinate, or indeterminate. Determinate plants tend to grow their foliage first and then flower, and if pollinated successfully the flowers will mature into fruit. All the fruit tends to mature on the plant at about the same time. Indeterminate tomatoes start by growing some foliage and then continue to produce foliage and flowers throughout the growing season. At any given time, these plants will tend to have tomato fruits at different stages of maturity. Semi-determinate tomatoes have a phenotype between determinate and indeterminate; they are typically of the determinate type, except that they grow larger than determinate cultivars. The latest developments in tomato breeding have led to a wider range of fruit colors. In addition to the standard red ripe color, tomatoes can be milky white, greenish, pink, yellow, gold, orange, or purple.

[0005] Hybrid commercial tomato seeds can be produced by artificial pollination. Pollen from the male parent is harvested and applied manually to the stigma surface of the female inbred line. Before and after artificial pollination, the flowers are covered so that insects do not bring in foreign pollen and create mixtures or impurities. The flowers are marked to identify the pollinated fruits, and the seeds will be harvested from the pollinated fruits.

[0006] A variety of pathogens affect the productivity of tomato plants, including viruses, fungi, bacteria, nematodes, and insects. Tomatoes are particularly vulnerable to many viruses, so virus resistance is of great significance in agriculture.

[0007] Viruses of the genus Tospovirus have a negative-sense, single-stranded RNA genome. The genome is divided into three segments called S (2.9 kb), M (5.4 kb), and L (8.9 kb). The M and S RNA segments encode proteins in an ambisense orientation. The name of the genus Tospovirus comes from the tomato spotted wilt virus (TSWV) species discovered in Australia in the 1920s.

[0008] Viruses of the genus Tospovirus, especially TSWV, are mainly vectorized by thrips, especially the tobacco thrips (Frankliniella fusca) and the western flower thrips (Frankliniella occidentalis).

[0009] The host range of TSWV is very wide, including hundreds of species, monocotyledonous and dicotyledonous plants, including tomatoes, peppers, lettuce, tobacco, etc.

[0010] In tomatoes, the main symptoms of TSWV infection are ring spot disease and mosaic disease / spots, followed by plant curling, stunted growth, chlorosis and lesions.

[0011] For tomatoes, the resistance gene encoding the CC-NBL-LRR protein Sw-5b (also known as Sw5b (SEQ ID NO:1)), namely the sw-5b (or sw5b) gene (SEQ ID NO:10), has been identified by plant breeders for many years as being able to provide resistance to TSWV and other viruses of the genus Tospovirus. Currently, there are TSWV-resistant tomato varieties containing the sw-5b gene.

[0012] Sw-5b is a single dominant resistance gene, initially identified in Solanum peruvianum and found on chromosome 9.

[0013] Alternative genes have also been identified in other species infected with TSWV, such as peppers.

[0014] Recently, TSWV strains that can infect tomatoes carrying sw5b have been identified; the emergence of these new resistance-breaking TSWV (or RB-TSWV) is considered a major global threat to tomato crops.

[0015] Multiple RB strains of TSWV have been characterized. The resistance-breaking mutations are located in the NSm gene of TSWV. In some commercial tomato growing operations, more than 50% of the plants have been infected with RB-TSWV, causing serious crop losses to growers (Crescenzi et al. 2015).

[0016] One of the most effective ways to combat RB-TSWV strain infection is to introduce genetic resistance genes. Therefore, the identification of resistance genes against these new resistance-breaking TSWV strains has become important and urgent for tomato breeders.

[0017] Recently, the characterization of putative resistance proteins and genes corresponding to Sw-5b protein variants has been published (WO2015 / 090468; ISI Sementi). However, it seems that these resistance proteins and genes do not seem to provide resistance, or are not sufficient to combat the currently prevalent RB-TSWV strains (see Example 3).

[0018] Plant NLR proteins have been widely evaluated. NLRs are proteins that trigger resistance responses in plants upon recognition of 'effector proteins'. NLRs have been classified into two main subclasses based on their N-terminal domains. The two main subclasses are CNL and TNL, referring to proteins containing a coiled-coil (C) or toll / interleukin-1 receptor (TIR) domain at their N-terminus, respectively. In addition to the N-terminal domain, NLRs also have a nucleotide-binding (NB) domain and a leucine-rich repeat (LRR) domain. It has been proposed that each common domain (NB, LRR, CC, and TIR) plays a role in the activation of NLR proteins (Wang et al. 2020).

[0019] In the case of the NLR protein Sw-5b, the TSWV movement protein NSm is the avirulence protein (elicitor) recognized by Sw-5b (Hallwass et al., 2014; Peiro et al., 2014). Co-expression of Sw-5b and TSWV NSm has been shown to induce a strong hypersensitive response (HR) in Nicotiana benthamiana and tomato leaves.

[0020] When the NSm protein of the RB-TSWV isolate was used in this test, co-expression of Sw-5b was no longer sufficient to induce HR.

[0021] Huang et al. (2021) recently disclosed two Sw-5b mutants using a two-step artificial mutagenesis protocol, which are alleged to be effective against TSWV RB carrying the NSmC118Y or NSmT120N mutations. They found that a mutation in the LRR domain of Sw-5b was particularly important, namely R927A (wild-type sequence represented by reference SEQ ID NO:1), but was not sufficient to induce HR of the whole protein. Further mutations in the N-terminal solanaceae domain (SD) region were necessary, that is, in addition to the R927A mutation, simultaneous mutations of L33P and K319E were necessary for inducing HR.

[0022] However, to confirm that these mutations are viable in the solanum genetic background and effectively confer resistance to the RB-TSWV strain without losing resistance to wild-type TSWV, they did not disclose tomato plants containing these mutations. In addition, as long as these mutants are obtained artificially, the corresponding mutant genes cannot be introgressed into commercial varieties.

[0023] Therefore, there is an urgent need to identify improved resistance genes against multiple TSWV strains, including the RB-TSWV strain, to provide broader and more effective resistance to TSWV and can be easily introgressed into the tomato background.

[0024] The inventors of the present application unexpectedly found that variants of the sw-5b gene present in non-tomato wild germplasm can confer resistance to TSWV and RB-TSWV, and that this resistance gene is dominant. In addition, it has been demonstrated that this gene can be introgressed into the tomato background. It has also been unexpectedly found that, relative to the wild-type Sw-5b protein, this variation does not occur in the LRR domain, which is generally considered to be an interaction domain. Summary of the Invention

[0025] The present invention relates to a Sw-5b protein which is a variant of the wild-type Sw-5b protein having SEQ ID NO:1 and which is capable of recognizing the viral movement protein NSm of wild-type strains of TSWV and also the viral movement protein NSm of resistance-breaking strains of TSWV. This variant Sw-5b protein has at least 90% sequence identity with the wild-type Sw-5b protein (represented by SEQ ID NO:1) and comprises at least 1 variation or mutation relative to SEQ ID NO:1, including insertions and deletions, which are preferably present in one or more of the following 28 positions: positions 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and 759.

[0026] The numbering of the amino acids is relative to the wild-type sequence represented by SEQ ID NO:1.

[0027] The variation or mutation at one or more of these positions is preferably one or more of the following variations: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0028] Preferably, the variant Sw-5b protein of the present invention comprises at least one variation or mutation relative to SEQ ID NO:1:

[0029] - In the 386 - 772 domain of the Sw-5b protein, wherein the variation or mutation is preferably at positions 448, 522, 622, or 759, and more preferably is at least one of the following 4 variations relative to SEQ ID NO:1: N448S, P522Q, V622D, and L759I; and / or

[0030] - In the 136 - 147 domain of the Sw-5b protein, wherein the variation or mutation is preferably at positions 136, 138, 139, 140, 143, 147, and more preferably is at least one of the following variations relative to SEQ ID NO:1: K136E, R138S, F139L, F140C, R143I, and A147L.

[0031] Preferably, the variant Sw-5b protein of the present invention comprises the following variation or mutation relative to SEQ ID NO:1:

[0032] - Variations in the 386 - 772 domain of the Sw-5b protein, wherein the variations include at least the following 4 variations relative to SEQ ID NO:1: N448S, P522Q, V622D, and L759I; and / or

[0033] - Variations in the 136 - 147 domain of the Sw-5b protein, wherein the variations include at least 3 or at least 4 of the following variations: K136E, R138S, F139L, F140C, R143I, and A147L.

[0034] The present invention also relates to different variants of the Sw-5b protein comprising additional variations or mutations, and nucleotide sequences encoding said proteins and polypeptides.

[0035] The present invention also relates to a resistance gene encoding a mutant or allelic variant of the wild type represented by SEQ ID NO:10, said resistance gene conferring resistance to multiple Tospovirus viruses in tomatoes, and more specifically resistance to TSWV (including strains considered to be the RB-TSWV strain).

[0036] The newly discovered resistant Sw-5b protein of the present invention confers resistance to TSWV (including RB-TSWV), due to at least one mutation or variation in the wild type protein, preferably at least one mutation or variation at one of the above 28 positions, more preferably due to at least one of the 28 substitutions or deletions, even more preferably due to:

[0037] - At least 1, 2, 3, or all four substitutions at positions 448, 522, 622, and 759 in the 386-772 domain of the wild-type Sw-5b protein, namely, the asparagine (N) at position 448 is replaced by serine (S); the proline (P) at position 522 is replaced by glutamine (Q); the valine (V) at position 622 is replaced by aspartic acid (D), and the leucine (L) at position 759 is replaced by isoleucine (I); and / or

[0038] - At least 1, 2, 3, or 4 substitutions in the 136-147 domain, selected from the following substitutions: the lysine (K) at position 136 is replaced by glutamic acid (E), the arginine (R) at position 138 is replaced by serine (S), the phenylalanine (F) at position 139 is replaced by leucine (L), the phenylalanine (F) at position 140 is replaced by cysteine (C), the arginine (R) at position 143 is replaced by isoleucine (I), and the alanine (A) at position 147 is replaced by leucine (L); and / or

[0039] - Combinations of these substitutions.

[0040] As detailed below, additional substitutions are preferably in the 386-772 domain or the 136-147 domain and may improve the resistance conferred by the substitutions disclosed above.

[0041] The present invention also provides plants, especially tomato plants that are resistant to TSWV, including tomato plants that are resistant to RB-TSWV, especially commercial plants, lines, and hybrids, as well as methods for producing or identifying plants, especially tomato plants or populations (germplasm) that are resistant to TSWV and RB-TSWV. The present invention also discloses molecular genetic markers linked to the newly discovered resistance genes. The present invention also provides plants obtained by the methods and uses of such molecular markers.

[0042] The present invention also provides a variety of methods, including methods for identifying TSWV- and RB-TSWV-resistant plants, methods for increasing tomato production in environments infected with different viruses of the genus Tospovirus (including TSWV, especially RB-TSWV), methods for protecting tomato fields from infection by viruses of the genus Tospovirus (including TSWV, especially RB-TSWV), and methods for identifying, detecting, and / or selecting mutants or variants of the Sw-5b protein that confer resistance to at least TSWV and RB-TSWV.

[0043] Definition:

[0044] The term "resistance" is defined by the Vegetables and Ornamental Crops Section of the ISF (International Seed Federation) and is used to describe the response of plants to pests or pathogens, as well as abiotic stresses in the vegetable seed industry. Specifically, resistance refers to the ability of a plant variety to limit the growth and development of a specific pest or pathogen and / or the damage they cause, compared to a susceptible plant variety under similar environmental conditions and pest or pathogen pressure. Under severe pest or pathogen pressure, resistant varieties may exhibit some disease symptoms or damage. Two levels of resistance are defined:

[0045] High resistance: Plants that highly limit the growth and / or development of a specific pest and / or the damage it causes compared to susceptible plants under normal pest pressure. However, these plants may exhibit some symptoms or damage under heavy pest pressure.

[0046] Moderate resistance: Plants that highly limit the growth and / or development of a specific pest and / or the damage it causes, but may exhibit a wider range of symptoms or damage compared to highly resistant plants. When grown under similar environmental conditions and / or pest pressure, moderately resistant plants still exhibit less severe symptoms or damage than susceptible plants.

[0047] The term "tolerance" is generally used to describe the ability of plants to tolerate abiotic stresses without serious consequences for growth, appearance, and yield.

[0048] However, in the literature and patents, the term is also used to denote the phenotype of a plant in which at least some disease symptoms remain absent when the plant is exposed to an infectious dose of a virus, whereby there is systemic or local infection, virus multiplication, and at least under certain culture conditions, the presence and / or genomic integration of the viral genomic sequence can be determined in the plant cells. Thus, tolerant plants are resistant to symptom manifestation, but are asymptomatic virus carriers. Sometimes, viral sequences can be present in or even multiply in the plant without causing disease symptoms. It should be understood that although tolerant plants are infected with a virus, they are generally able to at least moderately limit the growth and development of the virus. For this reason, the best-characterized tolerant plants according to this definition are intermediate-resistant plants.

[0049] Symptoms of TSWV infection typically include ringspot and mosaic / dotting, followed by curling, stunting, chlorosis, and lesions.

[0050] Susceptibility: Plants are unable to limit the growth and development of a specific pest or pathogen; susceptible plants exhibit harmful symptoms associated with virus infection, i.e., foliage damage and fruit damage in the case of TSWV infection.

[0051] Tomato plants susceptible to Tomato spotted wilt virus (TSWV) are, for example, the commercially available variety Tresor.

[0052] The resistance-breaking TSWV, or RB-TSWV, or RB-TSWV strain is a mutant of wild-type TSWV and can infect plants containing the wild-type resistance gene sw-5b that expresses the resistance Sw-5b protein. Such viruses are disclosed in Huang et al. (2021) and WO2015 / 090468; ISI Sementi. For example, the commercially available variety YOKO is a tomato plant that is resistant to Tomato spotted wilt virus (TSWV) but is susceptible to RB-TSWV infection.

[0053] As used herein, the terms "progeny" or "offspring" refer to any plant that is a progeny resulting from vegetative or sexual reproduction from one or more parent plants or their progeny. For example, progeny plants can be obtained by cloning or selfing of a parent plant, or by crossing two parent plants, and include selfing as well as F1 or F2 or further generations. F1 is the first generation of progeny produced by the parents, where at least one parent is used as a donor of a trait for the first time, while the progeny of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples produced from the selfing of F1', F2', etc. Thus, F1 can be (and usually is) a hybrid produced by crossing two true-breeding parents (true-breeding is homozygous for the trait), while F2 can be (and usually is) the progeny produced by self-pollination of the said F1 hybrid.

[0054] As used herein, the terms "cross", "crossing", "outcrossing" or "cross-breeding" refer to the process of applying (either artificially or naturally) the pollen of a flower on one plant to the ovule (stigma) of a flower on another plant.

[0055] As used herein, the term "genotype" refers to the genetic constitution of a single cell, cell culture, tissue, organism (e.g., a plant) or group of organisms.

[0056] As used herein, the term "grafting" is the operation of grafting a scion onto a rootstock. The main motivation for grafting is to avoid damage caused by soil-borne pests and pathogens when genetic or chemical methods of disease management are not available. Grafting a susceptible scion onto a resistant rootstock can provide a resistant cultivar without breeding resistance into the cultivar. In addition, grafting can enhance tolerance to abiotic stresses, increase yield and result in more efficient water and nutrient use.

[0057] As used herein, the term "heterozygote" refers to a diploid or polyploid individual cell or plant that has different alleles (forms of a given gene, genetic determinant or sequence) present at least at one locus.

[0058] As used herein, the term "heterozygous" refers to the presence of different alleles (forms of a given gene, genetic determinant, or sequence) at a particular locus.

[0059] As used herein, the term "homozygous" refers to an individual cell or plant having the same alleles at one or more loci on all homologous chromosomes.

[0060] As used herein, the term "homozygous" refers to the presence of the same alleles at one or more loci in a homologous chromosomal segment.

[0061] As used herein, the term "hybrid" refers to any individual cell, tissue, or plant resulting from a cross between parents that are different at one or more genes.

[0062] As used herein, the term "locus" (plural: "loci") refers to any genetically defined site, which can be a single position (nucleotide) or a chromosomal region. A locus can be a gene, genetic determinant, part of a gene, or DNA sequence, and can be occupied by different sequences. A locus can also be defined by an SNP (single nucleotide polymorphism), multiple SNPs, or two flanking SNPs.

[0063] As used herein, the term "rootstock" is the lower part of a plant that is capable of receiving a scion during the grafting process.

[0064] As used herein, the term "scion" is the upper part of a plant that is capable of being grafted onto a rootstock during the grafting process.

[0065] The present invention includes plants at different ploidy levels, mainly diploid plants, but also includes triploid plants, tetraploid plants, etc. Detailed Description

[0066] The inventors of the present application have identified variants of the Sw-5b protein such that the proteins comprising variations or mutations relative to the wild-type protein Sw-5b corresponding to SEQ ID NO: 1 recognize and / or bind to the movement proteins (MPs) of a variety of Tospovirus viruses, including at least the NSm protein of TSWV (Tomato spotted wilt virus), and also preferably at least a portion of the NSm protein of resistance-breaking TSWV (RB-TSWV), and most preferably the entire NSm protein of RB-TSWV.

[0067] Accordingly, the present invention relates to variants of the Sw-5b protein, i.e., variants of SEQ ID NO:1, which corresponds to the sequence of the wild-type Sw-5b protein. The variants according to the present invention have at least 90% sequence identity with SEQ ID NO:1 at the amino acid level, and if expressed in tomato plants, the variants confer the ability to recognize and / or bind at least the movement protein NSm of TSWV, and preferably also confer the ability to recognize and / or bind at least the movement protein NSm of at least a part or all of RB-TSWV.

[0068] In contrast, the wild-type Sw-5b protein, i.e., SEQ ID NO:1 or a similar sequence, confers the ability to bind only the NSm of wild-type TSWV, but does not confer significant binding or recognition of the NSm of RB-TSWV. The Sw-5b protein variants of the present invention may be interchangeably referred to as the Sw-5b protein of the present invention, Sw-5b protein variants, mutants or alleles.

[0069] If the Sw-5b protein directly or indirectly (but preferably directly) binds to the TSWV NSm, the Sw-5b protein is considered to recognize the NSm protein of TSWV; the direct or indirect binding may be at the level of the Sw-5b protein, or at the level of one or more domains of the Sw-5b protein, such as the SD domain or the LRR domain, or may involve the entire protein. Such recognition can be tested in Nicotiana benthamiana by the assays disclosed in the examples.

[0070] Such a Sw-5b protein is also referred to hereinafter as the resistance protein of the present invention.

[0071] The Sw-5b protein variants or alleles according to the present invention comprise variations in their amino acid sequences relative to the wild-type sequence represented by SEQ ID NO:1, i.e., one or more variations, which are preferably found at one or more of the following 28 positions within the protein: positions 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and 759. These positions are unexpectedly not in the LRR domain of the Sw-5b protein.

[0072] Preferably, the Sw-5b protein according to the present invention is a variant of the wild-type sequence, which contains at least 1 (but preferably at least 2, 3 or 4) variations relative to SEQ ID NO:1, and the variations will be found at the above 28 positions. In some embodiments, the protein of the present invention contains more than 4 variations at the 28 positions, preferably at least 5 variations at the positions, preferably at least 8 or 10 variations at the positions relative to SEQ ID NO:1. The protein of the present invention may also contain one or more variations relative to SEQ ID NO:1 at other positions.

[0073] The variation or mutation at one or more of the 28 positions is preferably one or more of the following variations: relative to SEQ ID NO:1, substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0074] According to other embodiments, the protein of the present invention contains at least 2, 3 or 4 of the above variations, whether substitution or deletion. According to some embodiments, the protein of the present invention contains more than 4 of these variations relative to SEQ ID NO:1, preferably at least 5, 8 or 10 of the variations, or more.

[0075] Preferably, the variant Sw-5b protein of the present invention contains at least 1 variation or mutation relative to SEQ ID NO:1:

[0076] - in the 386-772 domain of the Sw-5b protein, wherein the variation or mutation is preferably at position 448, 522, 622 or 759, and more preferably at least one of the following 4 variations relative to SEQ ID NO:1: N448S, P522Q, V622D and L759I; and / or

[0077] - in the 136-147 domain of the Sw-5b protein, wherein the variation or mutation is preferably at positions 136, 138, 139, 140, 143, 147, and more preferably at least one of the following variations relative to SEQ ID NO:1: K136E, R138S, F139L, F140C, R143I and A147L.

[0078] According to a preferred embodiment, therefore, a Sw-5b protein variant or allele according to the present invention comprises a variation in its amino acid sequence relative to the wild-type sequence represented by SEQ ID NO:1, said variation being found in the 386-772 domain, or in the 136-147 domain, or in both domains. Preferably, there is no variation in the LRR domain.

[0079] 386 - 772 domain of Sw-5b

[0080] It has actually been confirmed in WO2015 / 090468 that the 386-772 domain (numbered based on SEQ ID NO:1) is involved in the recognition of TSWV, and according to WO2015 / 090468, modifications in this region provide resistance to RB-TSWV. The corresponding sequence of the mutant Sw-5b2 protein described in WO2015 / 090468 is as Figure 3 shown and corresponds to SEQ ID NO:5.

[0081] However, the inventors of the present application have noted that, contrary to the teachings of WO2015 / 090468, plants containing the Sw-5b2 protein do not have significantly higher resistance to the tested RB-TSWV isolates compared to plants that are considered susceptible to RB-TSWV containing the wild-type Sw-5b protein. However, they have identified other mutations or variants relative to the wild-type sequence in this region, which, alone or in combination with other variations (such as some of the variations disclosed in WO2015 / 090468), provide resistance to RB-TSWV.

[0082] In particular, variants of the wild-type Sw-5b protein that confer resistance to TSWV (including RB-TSWV) contain at least one variation or mutation, preferably 2, 3, or 4, in the 386-772 domain. Preferred positions in this domain are positions 448, 522, 622, and 759. Preferred substitutions at these positions are as follows:

[0083] - Substitute asparagine (N) at position 448 with serine (S);

[0084] - Substitute proline (P) at position 522 with glutamine (Q);

[0085] - Substitute valine (V) at position 622 with acidic aspartic acid (D), and

[0086] - Substitute leucine (L) at position 759 with isoleucine (I).

[0087] In a preferred embodiment, a variant of the wild-type Sw-5b protein conferring resistance to TSWV (including RB-TSWV) contains at least the following four mutations at positions 448, 522, 622, and 759 in the 386-772 domain:

[0088] - replacing asparagine (N) at position 448 with serine (S);

[0089] - replacing proline (P) at position 522 with glutamine (Q);

[0090] - replacing valine (V) at position 622 with acidic aspartic acid (D), and

[0091] - replacing leucine (L) at position 759 with isoleucine (I).

[0092] The Sw-5b protein according to the present invention, which is a variant of the wild-type sequence SEQ ID NO:1, preferably contains at least 1, preferably 2, 3, or all of the four mutations, hereinafter referred to as primary mutations in the 386-772 domain, which may be combined with other mutations, although variants containing only 3 or fewer of the mutations are also covered by the present invention.

[0093] According to an alternative embodiment, the Sw-5b protein according to the present invention further contains additional modifications with respect to the wild-type sequence, in particular one of the following mutations, referred to as secondary mutations:

[0094] - replacing alanine (A) at position 477 with valine (V),

[0095] - replacing valine (V) at position 489 with leucine (L),

[0096] - replacing acidic aspartic acid (D) at position 659 with serine (S), and

[0097] - replacing glutamate (E) at position 707 with acidic aspartic acid (D).

[0098] Preferably, the Sw-5b protein according to the present invention contains at least 1 of these other mutations, preferably 2 or 3 of these other mutations, and most preferably all four of these other mutations.

[0099] Although some of these mutations can sometimes be found in plants susceptible to RB-TSWV, when combined with one or more of the first four mutations, i.e., with one or more of N448S, P522Q, V622D, and L759I, and preferably in the presence of the combination of the four mutations, they are considered to improve resistance to RB-TSWV.

[0100] Thus, a preferred Sw-5b protein according to the present invention comprises the N448S, P522Q, V622D and L759I mutations, and at least one, preferably 2, 3 or 4 of the A477V, V489L, D659S and E707D mutations.

[0101] In another embodiment, the present invention also relates to a Sw-5b protein which, in addition to the primary substitutions, comprises one or more of the following mutations relative to the wild-type sequence SEQ ID NO:1, which mutations are referred to as tertiary mutations in the 386-772 domain:

[0102] - replacing asparagine (N) at position 393 with aspartic acid (D);

[0103] - replacing serine (S) at position 461 with alanine (A);

[0104] - replacing aspartic acid (D) at position 614 with asparagine (N);

[0105] - replacing leucine (L) at position 623 with valine (V), and

[0106] - replacing isoleucine (I) at position 661 with asparagine (N).

[0107] Preferably, the Sw-5b protein according to the present invention comprises at least 1, 2, 3, 4 or 5 of the above-mentioned other mutations. These mutations are also found in the sw-2b2 protein disclosed in WO2015 / 090468. As mentioned above, contrary to the disclosure of WO2015 / 090468, these mutations alone do not confer resistance to RB-TSWV. However, in Sw-5b protein variants that already contain at least one or more of the four primary mutations N448S, P522Q, V622D and L759I, preferably contain all four primary mutations, they are presumed to increase resistance to RB-TSWV.

[0108] In a preferred embodiment, the Sw-5b protein according to the present invention comprises four primary mutations in combination with at least one, preferably at least 2 or 3, or all 4 secondary mutations, and at least one tertiary mutation, preferably more than one tertiary mutation.

[0109] Preferably, the Sw-5b protein according to the present invention is a variant of SEQ ID NO:1, comprising all primary, secondary and tertiary mutations relative to that sequence, but containing one secondary or tertiary mutation, or alternatively containing all mutations except for two mutations (which are two secondary mutations, or two tertiary mutations, or one of each).

[0110] In another embodiment, in addition to secondary and / or tertiary variations in the 386-772 domain, the Sw-5b protein according to the invention may also comprise a variation at position 613 of the protein relative to SEQ ID NO:1, where serine (S) is replaced with valine or cysteine, preferably valine.

[0111] According to a preferred embodiment, the Sw-5b protein according to the invention thus comprises at least the following 13 variations relative to the wild-type Sw-5b protein represented by SEQ ID NO:1: N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D and L759I; or at least 10, 11 or 12 of these variations, provided that it comprises N448S; P522Q; V622D and L759I. According to another embodiment, the Sw-5b protein according to the invention comprises the following 14 variations relative to SEQ ID NO:1: N393D, N448S, S461A, A477V, V489L, P522Q, D659S, S613V, D614N, V622D, L623V, I661N, E707D and L759. The corresponding 386-772 domain of this variant is represented by SEQ ID NO:4.

[0112] In a preferred embodiment, the Sw-5b protein of the invention comprises the 386-772 domain having SEQ ID NO:4. This protein may or may not comprise other variations in other domains of Sw-5b. Regardless of these variations, at the whole protein level, the percentage of sequence identity between the protein of the invention and SEQ ID NO:1 is preferably at least 90%.

[0113] 136 - 147 domain of Sw-5b

[0114] The 136-147 domain (numbered based on SEQ ID NO:1) is considered to be directly involved in the interaction between the NSm protein of wild-type TSWV and the Sw-5b protein. In fact, according to Li et al. (2019) and Seong et al. (2022), the interaction site between Sw-5 and the NSm of wild-type strains of TSWV is allegedly mapped between positions 136 and 147 (NterSD domain).

[0115] The inventors of the present application have identified mutations or variations in this region and in the extended regions 134 - 151 relative to the wild - type sequence, which mutations or variations, alone or in combination with other variations (such as some of the variations disclosed in WO2015 / 090468), provide resistance to RB - TSWV.

[0116] In one embodiment, a variant of the wild - type Sw - 5b protein that confers resistance to TSWV (including RB - TSWV) contains at least 1 variation or mutation, preferably 2, 3, or 4, in the 134 - 151 domain, more preferably in the 136 - 147 domain. Preferred positions in said domain are positions 134, 135, 136, 138, 139, 140, 143, 147, 148, and 152, more preferably positions 136, 138, 139, 140, 143, and 147. Preferred substitutions at these positions are substitution of L134F, substitution of Q135H, substitution of K136E, substitution of R138S, substitution of F139L, substitution of F140C, substitution of R143I, substitution of A147L, substitution of K148I, and substitution of S151T.

[0117] In particular, a variant of the wild - type Sw - 5b protein that confers resistance to TSWV (including RB - TSWV) contains at least 1, 2, 3, or preferably at least 4 of the following primary variations in the 136 - 147 domain:

[0118] - replacement of lysine (K) at position 136 with glutamic acid (E);

[0119] - replacement of arginine (R) at position 138 with serine (S);

[0120] - replacement of phenylalanine (F) at position 139 with leucine (L);

[0121] - replacement of phenylalanine (F) at position 140 with cysteine (C);

[0122] - replacement of arginine (R) at position 143 with isoleucine (I); and

[0123] - replacement of alanine (A) at position 147 with leucine (L).

[0124] Preferably, the Sw - 5b protein according to the invention contains at least 3, at least 4, preferably 5, or more preferably 6 of the above - mentioned primary variations. If so, the preferred Sw - 5b protein according to the invention contains the K136E, R138S, F139L, F140C, R143I, and A147L variations relative to the wild - type Sw - 5b sequence represented by SEQ ID NO:1.

[0125] In another embodiment, the Sw-5b protein of the present invention further comprises a mutation at position 137, i.e., aspartic acid (D) is replaced by glycine (G).

[0126] In addition to the 6 primary variations in the 136-147 domain, the corresponding 136-147 domain of the variant containing the said variation at position 137 is represented by SEQ ID NO:7.

[0127] According to other preferred embodiments, the Sw-5b protein according to the present invention further comprises additional secondary variations very close to the 136-147 domain. Therefore, the preferred protein comprises at least one of the following variations:

[0128] - replacing leucine (L) at position 134 with phenylalanine,

[0129] - replacing glutamine (Q) at position 135 with histidine (H),

[0130] - replacing lysine (K) at position 148 with isoleucine (I), and

[0131] - replacing serine (S) at position 151 with threonine (T).

[0132] In addition to at least 3 or at least 4 primary variations disclosed above, the Sw-5b protein according to the present invention preferably comprises at least two or three of these additional secondary variations; and in addition to at least 4 primary variations, preferably comprises these 4 additional secondary variations.

[0133] Therefore, a particularly preferred Sw-5b protein of the present invention comprises one of the following variation combinations in the 134-151 domain:

[0134] (1) K136E, R138S, F139L, F140C, R143I, A147L and L134F;

[0135] (2) K136E, R138S, F139L, F140C, R143I, A147L and Q135H;

[0136] (3) K136E, R138S, F139L, F140C, R143I, A147L and K148I; and

[0137] (4) K136E, R138S, F139L, F140C, R143I, A147L and S151T.

[0138] The Sw-5b protein according to the present invention may also comprise at least 3, at least 4, preferably 5 or more of the following variations relative to the wild-type Sw-5b sequence represented by SEQ ID NO: 1: L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I and S151T.

[0139] According to another embodiment, the sw-5b protein of the present invention comprises a 134-151 domain that incorporates all of the above variations (primary, secondary and position 137) in this region; such a region is shown in SEQ ID NO: 9.

[0140] In a preferred embodiment, the Sw-5b protein of the present invention comprises a 136-147 domain or a 134-151 domain having SEQ ID NOs: 7 and 9, respectively. The protein may or may not contain other variations in other domains of Sw-5b. Preferably, the protein further comprises a 386-772 domain having SEQ ID NO: 4. Regardless of these variations, at the whole protein level, the percentage of sequence identity between the protein of the present invention and SEQ ID NO: 1 is preferably at least 90%.

[0141] Further modification:

[0142] Thus, the Sw-5b protein according to the present invention comprises variations relative to the wild-type sequence, preferably variations at one or more of the 28 identified positions, and / or variations in the 136-147 domain, wherein the variations are as disclosed above, or variations in the 386-772 domain, wherein the variations are also as disclosed above, or variations in both domains.

[0143] In addition to these variations that are preferably present in one or both of the two domains, the protein of the present invention may also advantageously comprise variations in other segments of the protein, which can increase the interaction between the protein and the NSm protein of TSWV (especially RB-TSWV).

[0144] Such advantageous other variations are preferably the deletion of the amino acid at position 82 (threonine) and / or the amino acid at position 83 (asparagine) in the variant according to the present invention. In this case, the Sw-5b protein according to the present invention comprises one less, preferably two less amino acids than the wild-type sequence, namely the amino acid at position 82 or 83, or both at positions 82 and 83. According to another independent embodiment, even in the absence of the above other variations in the 386-772 or 136-147 domain or 134-151 domain, such deletions may result in resistance to RB-TSWV.

[0145] Another other variation that is advantageous in the context of the present invention is the replacement of lysine (K) at position 319 with glutamate (E). This replacement has been shown in Huang et al. to be involved in TSWV resistance.

[0146] At the amino acid level, the Sw-5b protein according to the present invention preferably has at least 90% sequence identity with the wild-type protein represented by SEQ ID NO:1 and contains at least the above-mentioned variation, preferably in the 136-147 domain or the 386-772 domain.

[0147] Sequence identity between two amino acid sequences is generally defined as defined in the domains of the present invention. For example, Clustal Omega is a program suitable for defining sequence identity.

[0148] According to a preferred embodiment, at the protein level, the Sw-5b protein of the present invention has at least 92% sequence identity with the entire sequence of the protein; preferably at least 94% sequence identity with the entire sequence.

[0149] According to a particularly preferred embodiment, the sequence of the Sw-5b protein according to the present invention has at least 95% sequence identity with the wild-type sequence represented by SEQ ID NO:1.

[0150] Except for the variations or mutations defined at the 28 identified positions and / or in the 136-147 domain and / or in the 386-772 domain, other variations or mutations present in the Sw-5b protein of the present invention are preferably conservative amino acid substitutions, preferably retaining the 3D structure of the protein.

[0151] That is, basic amino acids (such as lysine or arginine) are preferably replaced by another basic amino acid; acidic amino acids (such as aspartic acid or glutamic acid) are preferably replaced by another acidic amino acid. Small non-polar amino acids (such as glycine, alanine, proline, cysteine or valine) are preferably replaced by another small non-polar amino acid. Large non-polar amino acids (such as leucine, isoleucine, phenylalanine, methionine or tryptophan) are preferably replaced by another large non-polar amino acid. Small polar amino acids (such as serine or threonine) are preferably replaced by another small polar amino acid. Large polar amino acids (such as asparagine or glutamine) are preferably replaced by another large polar amino acid. Aromatic amino acids (such as tyrosine, phenylalanine or tryptophan) are preferably replaced by another aromatic amino acid.

[0152] However, advantageously, this protein does not contain a variation or mutation at the position corresponding to position 33 in SEQ ID NO:1, i.e., the protein contains leucine at the position corresponding to L33 in SEQ ID NO:1. Similarly, the protein according to the invention preferably does not contain a mutation at the position corresponding to position 927 in SEQ ID NO:1, i.e., it contains arginine (R) at the position corresponding to R927 in SEQ ID NO:1. More generally, the protein according to the invention preferably does not contain mutations or variations in the LRR domain.

[0153] A particularly preferred Sw-5b protein according to the invention, which is a variant of the wild-type sequence SEQ ID NO:1, is SEQ ID NO:3, or a sequence having at least 99% sequence identity with SEQ ID NO:3, especially at the above-mentioned positions.

[0154] As described above, the Sw-5b protein of the invention recognizes and actually interacts with the viral movement protein NSm of TSWV, and the viral movement protein NSm of TSWV includes the movement protein NSm of the RB-TSWV strain, especially the movement protein NSm of the RB-TSWV strain characterized by the mutation at position C118 of the virus, such as the C118Y or C118F variant of TSWV called RB-TSWV, and the strains characterized by the mutations at positions 137 and 153 (such as V137I and V153I mutations) or the mutation at position 120 (such as T120N mutation). These strains are called RB-TSWV, or have been proven by the inventors to be RB-TSWV.

[0155] The Sw-5b protein according to the invention differs from the wild-type Sw-5b in that it is capable of interacting with these RB-TSWV mutants, especially with the C118Y and C118F mutants, and the mutants containing the mutations V137I and V153I. The experimental section discloses simple assays to demonstrate the interaction between the wild-type or variant Sw-5b protein and the NSm protein of wild-type or RB-TSWV TSWV. The ability of the Sw-5b protein to confer recognition and / or binding to the movement protein NSm of TSWV can indeed be easily tested by the transient expression assay disclosed in the examples, i.e., by transiently expressing the Sw-5b protein containing the variant to be tested in Nicotiana benthamiana in the presence of the NSm protein of TSWV and testing by screening for the hypersensitive necrosis reaction (HR).

[0156] Notably, the detection of a hypersensitive necrotic response in transient expression assays in alternative plants such as Nicotiana benthamiana is equivalent to the detection of resistance in tomato. There is indeed a correlation between the ability of the Sw-5b protein to trigger a strong HR response in Nicotiana benthamiana leaves (in the presence of the Tospovirus NSm protein) and virus resistance in tomato. This correlation has been confirmed in another tomato virus, namely ToBRFV. Indeed, in WO 2022 / 117884, it has been shown that the hypersensitivity in an alternative assay of transient expression of the Tm-2-2 gene variant and the movement protein of ToBRFV in Nicotiana benthamiana indicates ToBRFV resistance in tomato plants containing the Tm-2-2 gene variant.

[0157] The variant proteins according to the invention provide resistance not only to TSWV (including RB-TSWV), but also to different Tospovirus viruses, like the wild-type Sw-5b protein. Thus, the variants according to the invention preferably represent the acquisition of a new resistance (i.e., resistance against RB-TSWV) without any loss, i.e., all the resistances provided by the wild-type Sw-5b protein are retained.

[0158] Such variant proteins, once expressed in the corresponding plants, can provide resistance to Tospovirus viruses in tomato plants, more specifically to TSWV and RB-TSWV, but also in other species and varieties susceptible to TSWV, especially commercial plants. In particular, the variant proteins according to the invention can provide resistance to TSWV infection in tomato, pepper, potato, and / or lettuce, including resistance to resistance-breaking TSWV strains containing the C118Y or C118F mutation and strains containing the V137I and V153I mutations.

[0159] The invention also relates to nucleotide sequences encoding the Sw-5b protein or protein variants as defined above. Given the degeneracy of the genetic code, very different nucleotide sequences encoding the proteins of the invention (i.e., variants encoding SEQ ID NO:1) can be envisaged, the variants being characterized mainly by variations at one or more of the 28 identified positions and / or in the 136-147 domain and / or in the 386-772 domain and / or in both domains.

[0160] Suitable nucleotide sequences are, for example, the sequences corresponding to SEQ ID NO:11, i.e., the sequences encoding specific variants of the Sw-5b protein identified by the inventors.

[0161] The present invention also relates to a sequence derived from SEQ ID NO: 10, which corresponds to the wild-type sequence of the sw-5b gene and has at least 70% sequence identity with the wild-type sequence, preferably at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity. Irrespective of the percentage of sequence identity with the sequence of SEQ ID NO: 10, the sequences of the present invention encode the Sw-5b protein of the present invention as defined above. Given the degeneracy of the genetic code, sequences encoding the Sw-5b variants of the present invention may still have less than 70% sequence identity with SEQ ID NO: 10 and are also within the scope of the present invention.

[0162] The nucleotide sequence according to the present invention includes at least DNA, single-stranded DNA, RNA, double-stranded RNA, and mixtures of DNA and RNA, as well as their complementary sequences. The sequence may or may not be isolated.

[0163] Another aspect of the present invention also relates to a resistance gene encoding a Sw-5b protein, which is a variant, mutant or allele of the Sw-5b protein, conferring at least resistance to TSWV in plants (especially tomatoes, as well as peppers, potatoes and / or lettuce), and preferably also conferring resistance to at least one or more RB-TSWV. Preferably, such a resistance gene confers resistance to RB-TSWV containing at least the C118Y or C118F mutation or containing the V137I and V153I mutations.

[0164] Mutants, variants or alleles of the Sw-5b protein are variants of the wild-type Sw-5b protein as disclosed above, which contain at least one or more of the variations disclosed at one or more of the 28 positions of SEQ ID NO: 1 identified by the inventors, and preferably in the 386-772 domain, or in the 136-147 domain, or in both. The resistance gene encoding the Sw-5b protein of the present invention defined above also refers to the resistance allele of the sw5b gene or sw5b allele of the present invention.

[0165] The newly discovered resistance protein and the resistance gene encoding the protein confer resistance to TSWV, especially to some or all TSWV variants called RB-TSWV, due to the aforementioned substitutions in one or both of the domains mentioned or at one or more of the identified positions.

[0166] According to yet another aspect, the present invention also relates to a nucleic acid construct comprising a sequence encoding the Sw-5b protein according to the present invention, i.e., a variant of the wild-type sequence represented by SEQ ID NO: 1, or comprising the resistance gene according to the present invention.

[0167] The nucleotide sequence encoding the Sw-5b protein of the present invention or such a sequence comprising a resistance gene may hereinafter be interchangeably referred to as the nucleotide sequence or resistance gene of the present invention, or the variant sw-5b gene or sw-5b allele.

[0168] Such a sequence encoding a protein, polypeptide or variant of interest is preferably under the control of a promoter, which is a constitutive or inducible promoter. Preferably, the promoter is a promoter active in plant cells. According to an embodiment, the promoter is not the wild-type promoter of the sw-5b gene. However, preferably, the promoter of the sw-5b variant gene is the natural promoter of the sw-5b gene.

[0169] Thus, the nucleic acid construct according to the present invention can be a vector, plasmid or T-DNA plasmid. The presence of the construct of the present invention in a cell can, under appropriate conditions, result in the expression of a protein, polypeptide Sw-5b variant or resistance protein according to the present invention and as defined above.

[0170] The present invention also includes an expression vector or construct adapted to express a polypeptide, protein, Sw-5b variant or resistance protein according to the present invention, preferably in plant cells.

[0171] According to another embodiment, the present invention also relates to the use of the defined sequence encoding the Sw-5b allele of the present invention, or a construct comprising such a sequence, for conferring resistance to TSWV on tomato plants or for obtaining transgenic tomato plants resistant to TSWV, which TSWV includes RB-TSWV and comprises at least the C118Y or C118F mutation or the V137I and V153I mutations. Indeed, as shown in the examples, the sw-5b variant recognizes TSWV NSm as well as RB-TSWV NSm, thus triggering the HR response associated with TSWV resistance.

[0172] According to another aspect, the present invention also relates to a cell comprising the nucleotide sequence or resistance gene according to the present invention, or the DNA construct as described above, or the resistance protein or Sw-5b protein variant of the present invention.

[0173] The cell is preferably a plant cell, preferably from the Solanaceae family, such as from the genus Solanum, and even more preferably a cell of a tomato plant, or a cell of the genus Capsicum or Nicotiana, preferably a cell of Nicotiana tabacum, Nicotiana benthamiana or Capsicum annuum.

[0174] The cell transiently or constitutively expresses the Sw-5b protein according to the present invention, which is thus a variant of the wild-type Sw-5b protein, as described above.

[0175] According to a preferred embodiment, the cell contains, in its genome and preferably in its nuclear genome, the nucleotide sequences, resistance genes, DNA or nucleic acid constructs according to the invention and described above. The presence of these sequences confers the desired phenotype, namely the expression of a protein that interacts with at least the TSWV (including RB-TSWV) NSm protein, which triggers a resistance or HR response under suitable conditions. Based on the sequence, the presence of these sequences can be revealed by any technique well known to those skilled in the art.

[0176] Particularly preferred cell types are cells of the Solanum, Nicotiana or Capsicum genera, more preferably tomato cells.

[0177] The cell according to the invention can be any type of cell, especially a tomato cell, especially an isolated cell and / or a cell capable of regenerating an entire plant, especially a tomato plant carrying the nucleotide sequence or resistance gene of the invention. Thus, the cell can be a renewable cell or a non-renewable cell.

[0178] The target nucleotide sequence or resistance gene can be present homozygously or heterozygously in the cell of the invention. Preferably, the cell according to the invention contains a resistance gene or a nucleotide sequence as defined above in a heterozygous state. The Sw-5b gene is indeed considered a dominant gene. However, the presence of the resistance gene of the invention may be preferred because it provides greater resistance under certain conditions of TSWV infection or RB-TSWV infection.

[0179] The invention also relates to a tissue culture of non-renewable or renewable cells as defined above according to the invention; preferably, the renewable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons and / or hypocotyls, and the cells contain in their genome a nucleotide sequence or a resistance gene conferring resistance to at least TSWV. Preferably, such sequences or resistance genes also provide resistance to one or more RB-TSWVs, and preferably resistance to RB-TSWV strains containing the C118Y or C118F mutation or the V137I and V153I mutations in at least the TSWV genome.

[0180] The invention also relates to any plant part, especially a tomato plant part, especially seeds, explants, reproductive materials, scions, cuttings, seeds, fruits, roots, rhizomes, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles or flowers, wherein the plant part contains at least one cell as described above.

[0181] The invention also provides a protoplast containing the nucleotide sequence or resistance gene of the invention.

[0182] According to another aspect, the invention relates to a plant, more preferably to a tomato or a pepper plant, which comprises in its genome a nucleotide sequence or a resistance gene as defined above encoding the Sw-5b protein of the invention, said Sw-5b protein being a variant of the wild-type Sw-5b protein as described above. Thus, such a nucleotide sequence or resistance gene encodes a variant of the Sw-5b protein which comprises at least one variation relative to SEQ ID NO:1, preferably found at one or more of the following 28 positions: positions 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and 759. The variation or mutation at one or more of these positions is preferably one or more of the following variations: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0183] In a preferred embodiment, the nucleotide sequence or resistance gene encodes a variant of the Sw-5b protein, comprising

[0184] (1) at least 1, 2, 3, or preferably at least 4 variations in the 136-147 domain of SEQ ID NO:1, selected from K136E, R138S, F139L, F140C, R143I and A147L, or

[0185] (2) 1, 2, 3 or preferably 4 variations N448S, P522Q, V622D and L759I relative to SEQ ID NO:1, or

[0186] (3) a combination of variations (1) and (2);

[0187] and confers resistance against TSWV infection, preferably including RB-TSWV infection, in particular by infection with an RB-TSWV strain or isolate comprising at least the C118Y or C118F mutation, or the V137I and V153I mutations.

[0188] Preferably, the nucleotide sequence or resistance gene encodes a variant of the Sw-5b protein, further comprising other mutations or variations as described above, especially the dinucleotide deletion at positions 82 and 83 of the wild-type sequence, and / or variations around the 136-147 domain, i.e., positions 134-151 relative to SEQ ID NO:1.

[0189] As already mentioned in the previous section and applicable to all aspects of the present invention, particularly preferred nucleotide sequences or resistance genes encode variants of the Sw-5b protein corresponding to or comprising SEQ ID NO:3, or proteins having at least 95% or preferably at least 99% sequence identity with SEQ ID NO:3. A suitable nucleotide sequence encoding such a protein is SEQ ID NO:11.

[0190] In the plants of the present invention, the defined resistance gene or nucleotide sequence can be present homozygously or heterozygously. It can provide resistance to TSWV (including RB-TSWV) in the heterozygous state. These sequences can also be present as multiple copies.

[0191] The said resistance gene or nucleotide sequence of the present invention is preferably present in the genomic DNA of the plants of the present invention, such as tomato plants.

[0192] Accordingly, the present invention also encompasses tomato plants resistant to TSWV, said tomato plants comprising an allele of the Sw-5b gene encoding a Sw-5b protein having at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1), and comprising at least 1, 2, preferably at least 3, more preferably at least 4 of the following variations relative to SEQ ID NO:1: K136E, R138S, F139L, F140C, R143I, and A147L; preferably at least 5 of these variations, even more preferably 6 variations. One or more other variations in close proximity may also be present, especially at positions 134, 135, 148, and 151.

[0193] According to another embodiment, the present invention also encompasses a tomato plant resistant to TSWV, comprising an allele of the Sw-5b gene encoding a Sw-5b protein having at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1), and comprising at least 1, 2, 3, more preferably all of the following 4 variations relative to SEQ ID NO:1: N448S, P522Q, V622D, and L759I; preferably comprising some other variations in the 386-772 domain already disclosed in the previous part.

[0194] Such plants exhibit resistance to TSWV, particularly to RB-TSWV, such as RB-TSWV containing mutations at positions 118 and / or 137 and 153.

[0195] The plants of the invention, particularly tomato plants, can advantageously combine variations in the 136 - 147 domain and the 386 - 772 domain.

[0196] The resistance gene or nucleotide sequence of the invention can also encode a Sw-5b protein comprising variations relative to SEQ ID NO:1 at one or more of 28 identified positions, but not in the 136 - 147, 134 - 151 or 386 - 772 domains, and these variations may or may not be combined with one or more variations in these domains.

[0197] The resistance gene or nucleotide sequence of the invention preferably exists stably in the nuclear genome of the plant cell. It can be stably integrated into the nuclear genome, for example, after transformation, or it can be generated by mutagenesis processes such as Tilling. The presence of these sequences conferring resistance to TSWV (including RB-TSWV) can also be caused by introgression of genes from resistant parents. These sequences conferring resistance to viruses of the genus Tospovirus (including at least TSWV and RB-TSWV) preferably but not necessarily exist at the locus of the sw-5b gene on chromosome 9. Other positions in the genome, such as those generated by random integration, are also suitable and are covered by the invention.

[0198] According to a specific embodiment, this resistance gene or nucleotide sequence of the invention is integrated into a plant, particularly a tomato plant, by introgression or homologous recombination, and in this case, the nucleotide sequence or resistance gene is usually present on chromosome 9 of the tomato plant.

[0199] The resistance phenotype can be tested and scored by natural infection with thrips or by artificial mechanical inoculation as described in the experimental section.

[0200] The invention also relates to tissues of the plants of the invention; said tissues can be undifferentiated tissues or differentiated tissues. Such tissues contain one or more cells containing the resistant sw-5b gene of the invention.

[0201] The invention also relates to propagation material capable of producing resistant plants according to the invention, particularly resistant tomato plants, which contain a resistance gene or a mutated sw-5b gene encoding the Sw-5b protein of the invention as defined above. The invention particularly relates to seeds of such resistant plants, which contain the resistance gene, especially tomato seeds and seeds that can grow into tomato plants according to the invention.

[0202] These plants are indeed particularly valuable as they are resistant to TSWV infection and especially to infection by breakthrough strains of TSWV.

[0203] Such tomato seeds are preferably coated or granulated with a single or combined active species such as phytohormones, enhanced microorganisms or products for disinfecting seeds and the plant environment. Such species and chemicals can be products that promote plant growth, such as hormones, or products that increase their resistance to environmental stress, such as elicitors, or products that stabilize the pH of the substrate and its surrounding environment, or optionally nutrients.

[0204] They can also be products for protecting seedlings from reagents that are adverse to seedling growth, including the viruses and pathogenic microorganisms described herein, such as fungicidal, bactericidal, nematicidal, insecticidal or herbicidal products that act by contact, ingestion or gas diffusion; for example, any suitable essential oil, such as an extract of thyme. All these products enhance the plant's resistance response and / or disinfect or condition the plant's environment. They can also be living biomaterials, such as non-pathogenic microorganisms, such as at least one fungus, or bacterium, or virus, with a culture medium to ensure their viability if required; and such microorganisms, such as types of Pseudomonas, Bacillus, Trichoderma, Clonostachys, Fusarium, Rhizoctonia, etc., stimulate plant growth or protect it from pathogens.

[0205] The plants, cells or seeds of the present invention can be heterozygous or homozygous for the resistance gene of the present invention or the sw-5b allele that confers resistance to TSWV. Accordingly, the present invention also encompasses plants, cells or seeds that are heterozygous in their genome for the resistance gene of the present invention as defined above.

[0206] Preferably, the tomato plants according to the present invention are commercial plants or lines. Such commercial plants or lines preferably also exhibit one or more of the following other characteristics: ToMV / TMV resistance (Tm-2 or Tm-22 resistance genes), nematode resistance traits (Mi-1 or mi-j), Fusarium resistance, Verticillium resistance and / or TYLCV resistance.

[0207] Other resistances or tolerances are also contemplated according to the present invention.

[0208] In addition, the commercial tomato plants of the present invention preferably bear fruits under suitable conditions, which are at least 10 grams, preferably 25 grams, preferably at least 100 grams at full maturity, and / or even more preferably at least 150 grams or at least 200 grams at full maturity. In addition, the number of fruits per plant is essentially not affected by the presence of the resistance gene of the present invention, that is, the productivity of the plants according to the present invention is not less than 20% better than that of plants with the same genotype but lacking the resistance gene.

[0209] Still according to another embodiment, the tomato plants of the present invention are determinate, indeterminate or semi-determinate plants or their seeds or cells, that is, corresponding to determinate, indeterminate or semi-determinate growth habits.

[0210] For determinate ones, it refers to tomato plants that tend to grow branches and leaves first, then flower, and if pollination is successful, the flowers will mature into fruits. All fruits tend to mature on the plant almost simultaneously. Indeterminate tomatoes start by growing some branches and leaves, and then continue to produce branches, leaves and flowers throughout the growing season. At any time, these plants will tend to have tomato fruits at different stages of maturity. Semi-determinate tomatoes have a phenotype between determinate and indeterminate. They are typically determinate, except that they grow larger than determinate varieties.

[0211] Still according to yet another embodiment, the plants of the present invention are used as scions or rootstocks in the grafting process. Grafting is a process that has been used for many years in crops such as cucurbitacea, etc., but has only recently been used for tomatoes. Grafting can be used to provide a certain level of resistance to telluric pathogens such as Phytophthora or certain nematodes. Therefore, grafting tends to prevent the plants or varieties to be cultivated from coming into contact with infected soil. Varieties interested in being used as grafts or scions can be selected as F1 hybrids and grafted onto resistant plants used as rootstocks. The resistant rootstocks remain healthy and provide a normal supply from the soil for the grafts isolated from the disease.

[0212] As described above, the present invention more particularly relates to tomato plants exhibiting TSWV resistance (including RB-TSWV resistance), and seeds that produce these plants, and cells of these plants or seeds, or other plant parts, which contain the resistance gene in their genomes, and progeny of the plants of the present invention containing the resistance gene.

[0213] Progeny include first-generation offspring, second-generation offspring and all other offspring obtained by crossing with the plants according to the present invention, where the crossing includes self-crossing or crossing with another plant. Preferred progeny correspond to the first and second generations.

[0214] It should be noted that the seeds or plants of the present invention can be obtained by different treatments, not exclusively by essentially biological treatments. The resistance gene encoding the Sw-5b protein of the present invention can actually be introduced, incorporated or "obtained intracellularly" by different techniques. Therefore, the plants, cells or seeds according to the present invention can be transgenic or non-transgenic, and they are preferably obtained by technical methods that are not essentially biological treatments, as detailed in other parts of this specification and the examples. According to a preferred embodiment, the plants, cells or seeds according to the present invention are not exclusively obtained by essentially biological treatments.

[0215] The resistance gene encoding the disclosed Sw-5b protein variant can advantageously be obtained by gene editing techniques, base editing or prime editing techniques (such as mutagenesis, especially targeted mutagenesis such as TILLING) or by other gene editing techniques (such as the CRISPR / Cas system) or by custom endonucleases or by base editing or prime editing with Cas9, Cas12a or other Cas proteins.

[0216] These techniques are well known to those skilled in the art.

[0217] The genetic engineering means that can be used include using all techniques called New Breeding Techniques, which are various new techniques developed and / or used to create new traits in plants through genetic variation, aiming at targeted mutagenesis, targeted introduction of new genes or gene silencing (RdDM). Examples of such new breeding techniques are to promote targeted sequence changes by using the following techniques: Zinc Finger Nuclease (ZFN) technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Patent No. 9,145,565), Oligonucleotide Directed Mutagenesis (ODM), Cisgenesis and Intragenesis, grafting (on GM rhizomes), reverse breeding, Agro-infiltration (Agro-infiltration "in the strict sense", Agrobacterium inoculation, floral dip method), Transcription Activator-Like Effector Nucleases (TALENs, see U.S. Patent Nos. 8,586,363 and 9,181,535), CRISPR / Cas systems (see U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641), engineered meganucleases, reengineered homing endonucleases, DNA-guided genome editing (Gao et al., Nature Biotechnology (2016)) and synthetic genomics. The main part of targeted genome editing, which is another name for new breeding techniques, is the application of inducing DNA double-strand breaks (DSBs) at selected positions in the genome where modification is expected. Such applications can be used to generate mutations (e.g., targeted mutations or precise native gene editing) as well as precise insertion of genes (e.g., homologous genes, within endogenous genes or heterologous genes). Applications that result in mutations are generally identified as Site-Directed Nuclease (SDN) techniques, such as SDN1, SDN2 and SDN3. For SDN1, the result is a targeted, non-specific gene deletion mutation: the position of the DNA DSB is precisely selected, but the DNA repair of the host cell is random and results in small nucleotide deletions, additions or substitutions. For SDN2, SDN is used to generate a targeted DSB and a DNA repair template (a short DNA sequence identical to the target DSB DNA sequence except for one or a few nucleotide changes) is used to repair the DSB: this results in a targeted and predetermined point mutation in the desired target gene. For SDN3, SDN is used together with a DNA repair template containing a new DNA sequence (e.g., a gene). The result of this technique is the integration of this DNA sequence into the plant genome.The most likely applications of using SDN3 are the insertion of cisgenic, intragenic or transgenic expression cassettes at selected genomic positions. A full description of each technique can be found in the report "New Plant Breeding Techniques - State of the Art and Perspectives for Commercial Development" published in 2011 by the Institute for Prospective Technological Studies of the Joint Research Centre of the European Commission.

[0218] By transformation, in particular Agrobacterium transformation, defined resistance genes can also be introduced into the plants, cells or seeds of the present invention, thereby producing transgenic plants, cells or seeds.

[0219] This application relates to plants, seeds or cells containing the resistance genes or sw-5b alleles of the present invention as already defined, regardless of the manner in which these sequences are provided; thus, it relates indifferently to transgenic and non-transgenic plants.

[0220] In an embodiment, the plants, cells or seeds of the present invention are transgenic plants, cells or seeds, or genetically modified plants, cells or seeds, to integrate the resistance genes or sw-5b alleles of the present invention.

[0221] On the other hand, the present invention also relates to different methods for obtaining, breeding or producing plants (especially tomato plants) resistant to TSWV (including RB-TSWV).

[0222] Therefore, the present invention encompasses methods for producing plants (especially tomato plants) resistant to TSWV (preferably also resistant to RB-TSWV), comprising the following steps:

[0223] a) Treating M0 seeds of the plant to be modified with a mutagen to obtain M1 seeds, the plant preferably being a tomato plant, and the mutagen preferably comprising the wild-type sw-5b gene encoding the wild-type Sw-5b protein;

[0224] b) Growing plants from the M1 seeds thus obtained to obtain M1 plants;

[0225] c) Producing M2 seeds by self-pollination of the M1 plants; and

[0226] d) Optionally repeating steps b) and c) n times to obtain M1+n seeds.

[0227] Growing the M1 or M2 seeds into plants and subjecting them to TSWV and / or RB-TSWV infection or screening to identify variants of the sw-5b gene.

[0228] In this method, the M1 seeds of step a) can be obtained by chemical mutagenesis (such as EMS mutagenesis) or by other chemical mutagens or physical means (such as radiation), and the physical means are, for example, selected ionizing radiation (X-rays, γ-rays, α-particles...), heavy ion beam radiation, ultraviolet radiation, radioactive decay or fast neutron radiation.

[0229] Another method for producing tomato plants resistant to TSWV (preferably including RB-TSWV) includes introducing a mutation of the sw-5b gene into a plant that already contains the sw-5b gene on chromosome No. 9 to produce a mutant sw-5b gene according to the present invention, that is, encoding the Sw-5b protein according to the present invention, showing variations relative to SEQ ID NO:1 that has been detailed above.

[0230] When the starting material is a tomato plant containing the sw-5b gene, the preferred method can advantageously include introducing a mutation into the sw-5b gene, preferably by mutagenesis, by TILLING or by genome editing, base editing or primer editing, especially by mutagenesis induced by physical or chemical reagents, especially by techniques selected from the following: ethyl methanesulfonate (EMS) mutagenesis, N-methyl-N-nitrosourea (MNU) mutagenesis or sodium azide (NaN3, SA) mutagenesis, oligonucleotide-directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, Cas9, Cas12a or other Cas proteins, engineered meganucleases, recombinant homing endonucleases and DNA-guided genome editing, wherein the mutation causes at least a substitution in the protein encoded by the sw-5b gene that has been detailed above, and the substitution occurs at one or more of the 28 identified positions, preferably in the 136-147 domain or in the 386-772 domain, or in both domains, especially

[0231] (1) causing at least 1, 2, 3, preferably all of the following 4 mutations in the protein: N448S, P522Q, V622D and L759I; or

[0232] (2) causing at least 1, 2, 3, preferably 4 or more of the following mutations in the protein: K136E, R138S, F139L, F140C, R143I and A147L, or

[0233] (3) causing a combination of (1) and (2).

[0234] The method can include introducing further mutations into the sw-5b gene, thereby causing other mutations in the Sw-5b protein, as described above.

[0235] Other mutations can be introduced as long as the recognition of the TSWV and RB-TSWV NSm proteins is not lost.

[0236] The present invention also encompasses different methods for obtaining transgenic plants according to the present invention, in particular transgenic tomato plants resistant to TSWV (including the RB-TSWV strain), by introducing the resistance genes according to the present invention. These methods may include the following steps:

[0237] - Obtaining a DNA construct as defined in the foregoing aspects of the present invention, i.e., comprising a resistance sw-5b gene or allele encoding the Sw-5b protein according to the present invention,

[0238] - Introducing the construct into a cell, in particular into a tomato cell

[0239] - Regenerating a transgenic plant, and

[0240] - Optionally, propagating the obtained plant.

[0241] According to another aspect, the present invention also relates to the use of tomato seeds or plants of the present invention homozygously containing the resistance gene of the present invention as breeding partners in a breeding program for obtaining tomato plants having a resistant phenotype to TSWV and preferably also to RB-TSWV. In fact, such a breeding partner homozygously contains in its genome the resistance gene conferring the target phenotype. By crossing this plant with a tomato plant (in particular a line), the resistance gene of the present invention conferring the desired phenotype can be transferred to the offspring. Thus, the plants according to the present invention can be used as breeding partners for introgressing the resistance gene into tomato plants or germplasm. Although, as detailed above, plants or seeds carrying the target resistance gene can also be used as breeding partners, the segregation of the phenotype may complicate the breeding program.

[0242] Therefore, the present invention also relates to a method for cultivating tomato plants resistant to TSWV and preferably also to RB-TSWV, including strains having mutations at position C118 and at positions V137 and V153, including:

[0243] (a) Crossing a tomato plant containing the resistance gene according to the present invention with an initial tomato plant lacking the resistance gene but possibly containing the wild-type sw-5b gene,

[0244] (b) Screening the offspring thus obtained for plants carrying the resistance gene of the present invention,

[0245] (c) Optionally, self-pollinating the plants obtained in step (b) one or more times and screening the offspring thus obtained for plants carrying the resistance gene.

[0246] Screening can be carried out by any suitable means well-known to those skilled in the art, in particular by using markers specific for the resistance gene or mutant gene.

[0247] The present invention also relates to a method for producing a tomato plant resistant to TSWV, said TSWV including mutant strains containing mutations such that they are considered RB-TSWV, said method comprising obtaining a part of a plant according to the present invention, thereby containing the resistance gene as defined, and asexually propagating said plant part to produce a plant from said plant part.

[0248] In all methods and processes according to the present invention, the tomato plant is determinate, indeterminate or semi-determinate.

[0249] As already disclosed, the tomato plants according to the present invention preferably are also resistant to nematodes, TMV, ToMV, TYLCV, Fusarium and / or Verticillium.

[0250] The present invention also relates to tomato plants and seeds obtainable or obtained by any of the methods and processes disclosed above. Such plants are indeed tomato plants expressing the Sw-5b variant of the present invention, which variant confers resistance to viruses of the genus Tomato spotted wilt virus, in particular resistance to TSWV and RB-TSWV.

[0251] According to a further aspect, the present invention also relates to a method for genotyping a plant, preferably a tomato plant or tomato germplasm, for detecting the presence of the resistance gene or mutant sw5b gene according to the present invention associated with resistance to TSWV infection (including RB-TSWV), wherein said method comprises determining or detecting in the genome of the test plant a nucleic acid comprising or corresponding to at least one of the following variations, substitutions or deletions: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0252] Preferably, the method comprises determining or detecting in the genome of a test plant a nucleic acid comprising or corresponding to a mutation or substitution of K136E, R138S, F139L, F140C, R143I, A147L, N448S, P522Q, V622D, and L759I in the sw-5b gene encoding the Sw-5b protein. Preferably, the method comprises the step of identifying in a sample of a plant to be tested a specific sequence associated with at least one of these variations, which is specific for the sw-5b allele identified by the inventors. According to a preferred embodiment, the method comprises determining or detecting in the genome of a test plant

[0253] (1) causing in the protein at least 1, 2, 3, preferably all, of the following 4 mutations: N448S, P522Q, V622D, and L759I; or

[0254] (2) causing in the protein at least 1, 2, 3, preferably 4 or more, of the following mutations: K136E, R138S, F139L, F140C, R143I, and A147L, or

[0255] (3) causing a combination of mutations of (1) and (2),

[0256] conferring resistance to TSWV (including RB-TSWV).

[0257] Similarly, the present invention also relates to a method for identifying, detecting, and / or screening tomato plants resistant to RB-TSWV in plants known to be resistant to TSWV, the method comprising detecting a mutant allele of the sw-5b gene in the genome of the plant, wherein the mutant allele comprises at least one mutation or variation selected from mutations causing the following substitutions or deletions: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D, and L759I relative to SEQ ID NO:1.

[0258] According to a preferred embodiment, the mutant allele comprises at least one mutation or variation selected from mutations causing mutations of K136E, R138S, F139L, F140C, R143I, A147L, N448S, P522Q, V622D, and L759I in the encoded protein relative to SEQ ID NO:1.

[0259] In this regard, molecular markers are also envisaged in the context of the present invention, which are specifically designed to detect or select tomato plants resistant to TSWV resistance-breaking strains in plants resistant to TSWV; said markers being capable of distinguishing at least one of the following variations in the sequence encoded by SEQ ID NO:10: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D and L759I; and preferably at least one of the following variations: K136E, R138S, F139L, F140C, R143I, A147L, N448S, P522Q, V622D and L759I and L759I. Suitable markers are typically less than 50 nucleotides in length and have at least 95% identity with the corresponding part of SEQ ID NO:10. The design of suitable markers or primers is routine for those skilled in the art. For designing a KASP assay, preferably three primers are used, two allele-specific forward primers and one common reverse primer. These two allele-specific primers differ only at the 3'-end, one being specific for the "resistant" allele and the other being specific for the "susceptible" or wild-type allele. The allele-specific primers are advantageously labeled with fluorescent dyes.

[0260] Other techniques are also known to those skilled in the art for designing markers that distinguish between the two alleles of a given polymorphism.

[0261] Given the ability of the resistant plants of the present invention to limit the damage caused by infections with different Tospovirus viruses (including TSWV and RB-TSWV infections), they are advantageously grown in an environment infected or potentially infected or infested with TSWV and RB-TSWV; under these conditions, the resistant plants of the present invention produce more marketable tomatoes than susceptible plants. Accordingly, the present invention also relates to a method for improving the yield of tomato plants in an environment infected with TSWV and RB-TSWV, comprising growing tomato plants comprising in their genome a resistance gene as defined in the foregoing aspects of the present invention and conferring resistance to TSWV and RB-TSWV on said plants.

[0262] Preferably, the method includes a first step of selecting or screening tomato plants containing the target resistance gene. The method can also be defined as a method for increasing the productivity of tomato fields, tunnels or greenhouses, or as a method for reducing the intensity or amount of chemical or fungicide application in tomato production.

[0263] The present invention also relates to a method for reducing tomato production losses under TSWV infection or infestation conditions, more specifically under RB-TSWV infection conditions, including growing tomato plants as defined above.

[0264] These methods are particularly valuable for tomato plant populations in fields, tunnels or greenhouses.

[0265] Alternatively, the method for improving yield or reducing tomato production losses may include a first step of identifying tomato plants resistant to TSWV (including RB-TSWV) and containing in their genome the resistance gene of the present invention that confers at least TSWV resistance (including resistance to RB-TSWV) to the plant, and then growing the resistant plants in an environment infected or likely to be infected with the virus.

[0266] The resistant plants of the present invention are also capable of restricting the growth of TSWV, especially RB-TSWV, thereby restricting the infection of other plants and the propagation of the virus, especially those mutant viruses that break through the resistance provided by the wild-type sw5b protein. Therefore, the present invention also relates to a method for protecting fields, tunnels or greenhouses or any other type of planting land from RB-TSWV infection, or at least restricting the level of RB-TSWV infection in the fields, tunnels or greenhouses, or restricting the spread of RB-TSWV in the fields, tunnels or greenhouses, especially in tomato fields. Such a method preferably includes the step of growing the resistant plants of the present invention, i.e., plants containing the resistance gene in their genome, conferring RB-TSWV resistance to the plant, especially resistance to mutant strains containing mutations at C118 and at positions V137 and V153.

[0267] The present invention also relates to the use of plants resistant to RB-TSWV for controlling RB-TSWV infection or infestation in fields, tunnels or greenhouses or other planting lands; such plants are the plants of the present invention and contain the resistance gene as defined above in their genome. This use or method is also a method for disinfecting fields, tunnels or greenhouses by reducing the virus population.

[0268] In another aspect, the present invention also relates to a method for producing tomatoes, including:

[0269] a) growing the tomato plants of the present invention, containing the resistance gene as defined above;

[0270] b) causing the plant to bear fruit; and

[0271] c) harvesting the fruit of the plant, preferably at maturity and / or before maturity.

[0272] All preferred embodiments regarding the resistance gene have been disclosed in the context of the foregoing aspects of the present invention. The method may advantageously include a further step of processing the tomato into tomato processed food.

[0273] The present invention also relates to a method for producing tomatoes in a transgenic tomato plant, comprising introducing a nucleic acid molecule encoding the Sw-5b protein of the present invention into the tomato plant, wherein the Sw-5b protein has substitutions in the 386-772 or / and 136-147 domains, or has the said variations at one or more of the 28 identified positions. The method may further include the steps of regenerating the transgenic plant and causing the plant to bear fruit. The method may further include the step of harvesting the fruit of the transgenic plant.

[0274] Sequence:

[0275] SEQ ID NO:1: Amino acid sequence of the Sw-5b protein of Mospomor (wild type);

[0276] SEQ ID NO:2: Amino acid sequence of the 386-772 domain of SEQ ID NO:1;

[0277] SEQ ID NO:3: Amino acid sequence of the Sw-5b protein of strain 111 (resistant);

[0278] SEQ ID NO:4: Partial sequence of SEQ ID NO:3 corresponding to the 386-772 domain of SEQ ID NO:1;

[0279] SEQ ID NO:5: Amino acid sequence of sw-5b2 (corresponding to the 386-772 domain of SEQ ID NO:1).

[0280] SEQ ID NO:6: Amino acid sequence of the 136-147 domain of SEQ ID NO:1;

[0281] SEQ ID NO:7: Partial sequence of SEQ ID NO:3 corresponding to the 136-147 domain of SEQ ID NO:1;

[0282] SEQ ID NO:8: Amino acid sequence of the 134-151 domain of SEQ ID NO:1;

[0283] SEQ ID NO:9: The partial sequence of SEQ ID NO:3 corresponding to the 134 - 151 domain of SEQ ID NO:1.

[0284] SEQ ID NO:10: The nucleotide sequence of the sw - 5b gene of Mospomor (wild - type).

[0285] SEQ ID NO:11: The nucleotide sequence of the sw - 5b gene of line 111 (resistant). Description of the Drawings

[0286] Figure 1 : This figure shows the percentage of plants containing virus detected 4 weeks after inoculation. Each plant has been subjected to an ELISA test. Two RB - TSWV strains (one strain (strain 1) has the same characteristics and mutations as strains prevalent especially in South America, etc.; and one strain (strain 2) has the same characteristics and mutations as strains prevalent especially in Italy, etc.) and one TSWV strain have been tested against 3 genotypes: the line homozygous for sw5b (sw5b line), the line without sw5b (no - sw5b line), and the wild (non - tomato) germplasm line 111.

[0287] Figure 2 : This figure shows the presence of virus in the inoculated plants after inoculation, as demonstrated by ELISA test. RB - TSWV strain 1 has been tested against 3 genotypes: the line homozygous for sw5b (sw5b line), the line without sw5b (no - sw5b line), and two different hybrids containing the sw5b2 variant allele of sw5b.

[0288] Figure 3: This figure is an alignment of different Sw-5b protein sequences. The Sw5b amino acid sequences are from wild accession line 111 (SEQ ID NO:3), wild-type sw5b Mospomor line (resistant only to TSWV strains) (SEQ ID NO:1), and the sequence from WO2015 / 090468 (Sw5b2, which corresponds to a partial sequence of full-length sw-5b, SEQ ID NO:5). Six amino acids in line 111 and Sw5b2 (which are the same in these two lines and different from the wild-type sw5b Mospomor line) are underlined; eight amino acids in line 111 (which are different from those of Sw5b2 corresponding to SEQ ID NO:1 and wild-type sw-5b) are shown in italics and lowercase letters. Twenty-eight substitutions in wild accession line 111 (wild accession Line 111), which are not found in the wild-type Sw-5b sequence, in the Sw5b2 variant, or in other susceptible variants, are double-underlined. The LLR domain is shown in italics and uppercase.

[0289] Figure 4 : This photograph shows the HR response when the Sw5b and NSm proteins of TSWV are co-expressed or expressed alone. The expression of Tm22 and the movement protein of TMV are used as positive controls for HR.

[0290] Figure 5 : This photograph shows the HR response. The picture was taken 6 days after infiltration. The spots on the left half (L) of the leaf were infiltrated with Sw5b from Mospormor (wild-type), and the spots on the right half (R) of the leaf were infiltrated with Sw5b from accession 111. Spot 1 (L1, R1) corresponds to the NSm wild-type protein (TSWV strain), spot 2 (L2, R2) corresponds to the NSm variant with the mutation C118Y, spot 3 (L3, R3) corresponds to the NSm variant not identified as RB-TSWV, and spot 4 (L4, R4) corresponds to the NSm variant with the mutations V137I and V153I.

[0291] Figure 6 : This figure shows the presence of the virus in the inoculated plants after inoculation, as confirmed by ELISA tests. The plants tested are F1, three different BC1F1, and susceptible controls EP7 and Mospomor.

[0292] Example

[0293] The present inventors screened more than 50 wild Solanum germplasms from their proprietary lines, which are different from tomatoes, for resistance to the RB-TSWV strain. After identifying the mutations in these strains (Example 2), mechanical inoculation tests were used to make them resistant to the sw-5b resistance gene. The wild germplasms tested were from species known to contain resistance, although this resistance is usually difficult to introgress, such as Solanum habrochaites.

[0294] One such wild germplasm (line 111) showed a good level of resistance to 2 different RB-TSWV strains or isolates (Example 3).

[0295] To better understand this resistance, the sw-5b gene of line 111 (pJL 491 clone) was cloned and sequenced and compared with the wild-type sw-5b allele (pJL488 clone) currently used in breeding programs that does not have resistance to the RB-TSWV strain (Example 4).

[0296] The results showed that there was 95.3% homology between the two protein alleles of Sw-5b at the amino acid level.

[0297] Therefore, the present inventors hypothesized that the improved resistance was due to the improved ability of the newly identified Sw-5b allele to respond to the NSm protein from the RB-TSWV isolate.

[0298] Hallwass et al. have demonstrated that transient expression of the Sw-5b (wild-type) protein and the NSm (wild-type) protein in Nicotiana benthamiana leaves results in a hypersensitive response (HR). This HR is triggered by the binding of the NSm protein to the Sw-5b protein. In Example 5, the inventors have demonstrated using this system that the Sw-5b protein of line 111 detects the NSm protein variant from the RB-TSWV strain and triggers a strong HR response. In contrast, the Sw-5b (wt) protein cannot recognize the NSm protein from these same RB-TSWV strains.

[0299] Then, the inventors transferred the sw-5b gene from the wild germplasm 111 to a commercial tomato line (Example 6).

[0300] Example 1: Materials and Methods

[0301] Virus Strains:

[0302] Two RB-TSWV strains have been tested (one strain has the same characteristics and mutations as the strains prevalent especially in regions such as South America, hereinafter referred to as "RB-TSWV strain 1", and one strain has the same characteristics and mutations as the strains prevalent especially in regions such as Italy, hereinafter referred to as "RB-TSWV strain 2"). Isolates of RB-TSWV strain 1 contain C118F mutation and V137I mutation. Isolates of RB-TSWV strain 2 contain at least C118Y mutation.

[0303] Virus inoculation:

[0304] The RB-TSWV strain or isolate multiplies in the leaves of susceptible plants. The symptomatic leaves are harvested and stored at -80 °C.

[0305] Optimal results are obtained by using Datura plants as maintainer lines. The frozen and ground tissue infected with the RB-TSWV strain is mechanically inoculated onto fresh Datura plants. The newly infected Datura plant leaves correspond to the 3rd generation inoculum. The tomato plants are inoculated with the freshly inoculated Datura plant leaves. This corresponds to the 3rd generation (the 1st generation: the original material frozen at -80 °C, the 2nd generation: Datura plant leaves, the 3rd generation: tomato plants). A second inoculation can be carried out.

[0306] Mechanical inoculation The protocol has been internally optimized by the inventors and used with two different RB-TSWV strains. The cotyledons or 1 to 2 true leaves of the seedlings are gently wiped with the inoculum. The inoculation is repeated weekly for 3 weeks.

[0307] Scoring is carried out 15 days and 21 days after the first inoculation. The main symptoms for scoring are ring spot and mosaic / dots, followed by curling, stunting, chlorosis and lesions.

[0308] For the wild germplasm line 111, consistent results have been obtained for these RB-TSWV strains.

[0309] It has also been tested on Inoculation with thrips (Frankliniella occidentalis) and compared with mechanical inoculation.

[0310] For the susceptible control, both inoculation methods (thrips-mediated inoculation and mechanical inoculation) showed 100% plant infection, thus confirming the effectiveness of thrips-mediated inoculation, that is, it was proved that thrips successfully acquired and transmitted the virus through the adopted protocol.

[0311] The present inventors have also concluded that mechanical inoculation seems to be stronger than thrips-mediated inoculation, such that the resistance identified by mechanical inoculation is expected to be converted into resistance to natural thrips infection.

[0312] Elisa

[0313] ELISA tests have been performed in all inoculated plants to evaluate the presence of the virus in the plants, usually 4 weeks after inoculation.

[0314] Example 2: Sequencing of virus strains:

[0315] The NSm gene of different RB-TSWV strains has been sequenced. Two strains representing three of the most common mutations have been retained (see Table 1), including the well-known C118 (Table 1).

[0316]

[0317] Table 1: The RB-TSWV strains used in this test.

[0318] Example 3: Identification of resistant lines and comparison with other resistant plants

[0319] The inventors have screened more than 50 different wild Solanum germplasms with the aim of identifying potential sources of resistance to TSWV (including RB-TSWV).

[0320] These plants were mechanically inoculated with the virus TSWV strains (including the two RB-TSWV strains disclosed in Example 1) as described in Example 1.

[0321] Plants showing milder symptoms were retained and retested, including a thrips inoculation protocol that is closer to natural conditions.

[0322] A germplasm, namely line 111, showed significant and reproducible resistance in these initial tests and was retained for further characterization.

[0323] To confirm the resistance of the identified line, Elisa tests have been performed in inoculated plants with wild-type TSWV strains and the two RB-TSWV isolates described in Example 2. Plants of the identified line 111, as well as plants containing the sw-5b gene (wild type) and plants not containing said gene at all, have been tested.

[0324] The results are shown in Figure 1 .

[0325] It can be inferred from this figure that for the RB-TSWV strains RB-TSWV strain 1 and strain 2, compared with the two control lines (sw5b line and line without sw5b) showing 100% infection, a 60% to 75% reduction in infected plants was observed in germplasm 111.

[0326] For wild-type TSWV strains, there was no resistance breakthrough, and both line 111 and plants containing the sw-5b gene showed complete resistance (no infected plants).

[0327] These results confirm that the wild-type sw-5b gene confers resistance only against wild-type TSWV strains and not against RB-TSWV strains. The identified line 111 is resistant to all types of TSWV (both wild-type and resistance breakthrough strains), confirming the broad applicability of the resistance present in this strain.

[0328] The resistance of two hybrids (Centus and Performer) from ISI Sementi, which contain alleles of the sw-5b gene allegedly conferring resistance to RB-TSWV, was tested with the RB-TSWV strain 1 disclosed in Example 2. At the seedling stage, the plants were inoculated three times with the RB-TSWV strain 1. Phenotypic scoring was performed at 3 weeks and 4 weeks after inoculation, as well as TSWV ELISA to determine the percentage of plants infected and not infected with the disease.

[0329] The results are as Figure 2 shown and show that these hybrids did not exhibit a high level of resistance.

[0330] Example 4: Resistance protein sequencing:

[0331] The Sw5b gene of the wild germplasm line 111 disclosed in Example 3 has been sequenced and compared with the sw5b gene from the Mospomor line and the sequence sw5b2 disclosed in ISI Sementi in Application WO2015 / 090468.

[0332] The Mospomor line is homozygous for sw5b and is resistant to TSWV strains. The Sementi patent discloses a partial sequence of the sw5b gene called Sw5b2. The patent claims a new sw5b sequence that allegedly confers moderate resistance to resistance breakthrough strains in Italy.

[0333] These hybrids have been tested with the RB-TSWV strain 1 disclosed in Example 2 and did not show a high level of resistance (see Figure 2 ).

[0334] Interestingly, compared with the WT sw5b gene and the patent sequence, the sw5b gene from line 111 presents multiple mutations (for the sequence alignment in this region, see Figure 3 ). Six mutations are conserved with the Sw5b2 sequence disclosed by Sementi (underlined), and eight are new mutations specific to the sw5b of line 111 in this region (Figure 3 in italics and lowercase).

[0335] Example 5: Confirmation of the recognition between the NSm protein of the RB strain and the SW5B protein variant

[0336] The full sequence of sw5b of line 111 has been used to test the recognition between this new version of sw5b and the common mutations observed in the RB-TSWV Nsm protein, especially the two RB-TSWV strains disclosed in Example 2.

[0337] Agrobacterium co-infiltration transient assays have been performed on Nicotiana benthamiana leaves.

[0338] The applicability of the test was first tested with wild-type Sw-5b protein and NSm protein, as disclosed by Hallwass et al. TMV (MP-TMV) and TM2-2 proteins, which are known to trigger HR, were used as positive controls. The results are as Figure 4 shown.

[0339] The detection of HR completely matched the expectation existing between the Sw-5b protein and the NSm protein.

[0340] Then the same protocol was used to test the interaction between the NSm proteins of 4 different TSWV strains (i.e., wild-type sequences and known variants not identified as resistance breakers) and the two RB-TSWV characterized in Example 2 and the wild-type Sw-5b protein or variant alleles identified in line 111. The results are as Figure 5 shown.

[0341] These transient assay results showed that HR was present in all NSm variants and NSm WT (point 1) with the Sw5b sequence from wild germplasm line 111 (R). In the presence of sw5b from line Mospomor, the binding recognition (or HR) was lost in the presence of the NSm variant with the C118Y mutation (point L2) and the NSm variant with the V137I and V153I mutations (point L4), and these variants are called resistance-breaking strains.

[0342] These results confirm that the resistance observed in germplasm 111 (Example 3) indeed comes from the allele of the sw5b gene of this germplasm.

[0343] Example 6: Transfer of genes in a commercial context

[0344] The inventors successfully crossed the resistant plants of line 111 identified in the foregoing examples with tomato plants.

[0345] The following steps were carried out:

[0346] 1. F1 plants were generated by crossing plants from resistant line 111 with elite lines of processing tomatoes. Since the seeds were non-viable, embryo rescue was performed.

[0347] 2. Molecular markers were used to confirm that the F1 plants were hybrids between the tomato genome and wild germplasm (non-tomato).

[0348] 3. The F1 from line 111 was crossed to obtain BC1F1 plants. Since the BC1F1 plants were still non-viable at this stage, embryo rescue was performed to obtain BC1F1 plants.

[0349] Although there are protocols for tomato embryo rescue using the tomato genome and wild germplasm for rescue breeding programs, different adjustments are needed to obtain hybrid plants.

[0350] The BC1F1 plants were tested for resistance.

[0351] This test was performed in a growth chamber using the RB-TSWV strain 1 (similar to the strain that spreads especially in South America, etc.). Sixteen plants (from one BC1 embryo rescue plant) were sown for each genotype.

[0352] The tomato plants were inoculated twice. Scoring was performed on the 25th and 32nd days after inoculation, and ELISA was performed on the 35th day after inoculation.

[0353] The tomato plants were inoculated twice (on the 27th and 38th days after sowing). Scoring was performed on the 24th and 31st days after the first inoculation. An ELISA test was performed on the 31st day after inoculation.

[0354] Mospomor and EP7 were used as susceptible controls.

[0355] All F1 (111.4), BC1F1, and controls were from cuttings, except Figure 6 the controls labeled "Mosp-S" and "EP7-S" were from seeds. The BC1F1 tested were 111.4-9, 111.4-27, and 111.4-31. They were obtained by embryo rescue and cuttings. The results are as Figure 6 shown and confirm that the resistant phenotype has been properly transferred to the progeny.

[0356] The BC1F1 line 111-4-31 was selected based on molecular verification of the cross and its high resistance level. This line has been used to cross with multiple tomato sources. A BC2F2 population was generated and used for phenotyping and pathology tests.

[0357] To obtain tomato plants with target agronomic traits and resistance to TSWV (including the RB-TSWV strain), further selfing was performed.

[0358] Example 7: RB-TSWV resistance assay was conducted in seedlings in the context of tomatoes.

[0359] To evaluate the level of RB-TSWV resistance in the source tomatoes, a growth chamber phenotyping test was conducted on the BC2F2 population.

[0360] Virus strain:

[0361] Strain 1 (South America) has been used in this example.

[0362] Virus inoculation:

[0363] The RB-TSWV strain multiplies in the leaves of susceptible plants. The symptomatic leaves are harvested and stored at -80 °C.

[0364] Optimal results were obtained by using Datura plants as maintainer lines. The frozen ground tissue infected with the RB-TSWV strain was mechanically inoculated onto fresh Datura plants. The newly infected Datura plant leaves corresponded to the 3rd generation inoculum. The tomato plants were inoculated with the freshly inoculated Datura plant leaves. This corresponded to the 3rd generation (1st generation: the original material frozen at -80 °C, 2nd generation: Datura plant leaves, 3rd generation: tomato plants). A second inoculation could be performed.

[0365] Mechanical inoculation The protocol has been internally optimized and used by the inventors. The cotyledons of the seedlings were gently wiped with the inoculum. The inoculation was repeated weekly for 2 weeks.

[0366] Scoring was performed 15 days and 21 days after the first inoculation. The main symptoms for scoring were ringspot and mosaic / dots, followed by curling, stunting, chlorosis, and lesions.

[0367] The controls in this experiment were as follows:

[0368] Susceptible check:

[0369] - Mospomor: Homozygous sw5b inbred line

[0370] - Hybrid carrying sw5b heterozygote

[0371] Resistance source: 111

[0372] ELISA

[0373] ELISA tests have been conducted in all inoculated plants to evaluate the presence of the virus in the plants, usually 4 weeks after inoculation.

[0374] Results

[0375] Table 2 reports the test results, including scores at 21 days post inoculation (foliar symptoms) and ELISA assays.

[0376]

[0377] Results of this test showed that the resistance phenotype from source 111 has been transferred in the tomato background. Among the BC2F2 generation, 11 out of 29 plants were free of RB-TSWV at about 4 weeks post inoculation.

[0378] The present invention more particularly relates to the following aspects:

[0379] 1. A Sw-5b protein that recognizes the viral movement protein NSm of TSWV, wherein the protein has at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1) and comprises at least 1, preferably 2, 3 or 4 variations relative to SEQ ID NO:1 found at the following positions: position 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and / or 759.

[0380] 2. The Sw-5b protein according to aspect 1, wherein the at least one variation is selected from the following mutations relative to SEQ ID NO:1: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K(136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D and L759I.

[0381] 3. The Sw-5b protein according to aspect 1 or 2, comprising at least 1, preferably 2, 3 or the following 4 variations relative to SEQ ID NO:1: N448S; P522Q; V622D and L759I.

[0382] 4. The Sw-5b protein according to any one of aspects 1 to 3, comprising at least the following 4 variations relative to SEQ ID NO:1: N448S; P522Q; V622D and L759I.

[0383] 5. The Sw-5b protein according to any one of aspects 1 to 4 further comprises one or more of the following variations relative to SEQ ID NO: 1: A477V; V489L; D659S; and E707D.

[0384] 6. The Sw-5b protein according to any one of aspects 1 - 5 further comprises one or more of the following mutations relative to SEQ ID NO: 1: N393D; S461A; D614N; L623V; and I661N; and possibly also S613V or S613C.

[0385] 7. The Sw-5b protein according to any one of aspects 1 - 6 comprises the following variations relative to SEQ ID NO: 1: N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D, and L759I.

[0386] 8. The Sw-5b protein according to aspect 1 that recognizes the viral movement protein NSm of TSWV, which comprises at least one of the following variations relative to SEQ ID NO: 1: K136E; R138S; F139L; F140C; R143I; and A147L.

[0387] 9. The Sw-5b protein according to aspect 8 comprises at least four of the following variations relative to SEQ ID NO: 1: K136E, R138S, F139L, F140C, R143I, and A147L; preferably at least 5, or 6 of these variations.

[0388] 10. The Sw-5b protein according to aspect 8 or 9 further comprises the variation D137G.

[0389] 11. The Sw-5b protein according to any one of aspects 8 to 10 further comprises one or more of L134F, Q135H, K148I, and S151T.

[0390] 12 The Sw-5b protein according to any one of aspects 1 to 11 further comprises a deletion of amino acids 82 (T) and / or 83 (N) of SEQ ID NO: 1.

[0391] 13 The Sw-5b protein according to any one of aspects 1 to 12 further comprises the variation K319E relative to SEQ ID NO: 1.

[0392] 14. The Sw-5b protein according to any one of aspects 1 to 13, wherein the amino acids corresponding to L33 and / or R927 in SEQ ID NO: 1 are not modified.

[0393] 15. The Sw-5b protein according to any one of aspects 1 to 14, having at least 92%, preferably at least 94% sequence identity with SEQ ID NO: 1 throughout the sequence.

[0394] 16. The Sw-5b protein according to any one of aspects 1 to 15, having at least 95% sequence identity with SEQ ID NO: 1.

[0395] 17. The Sw-5b protein according to any one of aspects 1 to 16, having the sequence shown in SEQ ID NO: 3, or a sequence having more than 99% sequence identity with SEQ ID NO: 3.

[0396] 18. The Sw-5b protein according to any one of aspects 1 to 17, recognizing and interacting with the viral movement protein NSm of the resistance-breaking TSWV strain, said resistance-breaking TSWV strain including strains containing the C118Y mutation and strains containing the V137I and V153I mutations.

[0397] 19. The Sw-5b protein according to any one of aspects 1 to 18, recognizing and interacting with the viral movement protein NSm of a TSWV strain (referred to as RB-TSWV), said viral movement protein NSm not interacting with SEQ ID NO: 1.

[0398] 20. The Sw-5b protein according to any one of aspects 1 to 19, conferring resistance to TSWV infection on tomato, pepper, potato, and / or lettuce, said TSWV including the resistance-breaking TSWV strain containing the C118Y mutation and the strain containing the V137I and V153I mutations.

[0399] 21. A resistance gene encoding the Sw-5b protein according to any one of aspects 1 to 20, said resistance gene conferring resistance to TSWV infection on tomato plants.

[0400] 22. A nucleic acid construct comprising a sequence encoding the Sw-5b protein according to any one of aspects 1 to 20 or comprising the resistance gene according to claim 21, preferably under the control of a promoter (such as a vector, plasmid, or T-DNA plasmid).

[0401] 23. The nucleic acid construct according to aspect 21 or 22, comprising a sequence having at least 75% sequence identity with SEQ ID NO: 11, preferably at least 90%, more preferably at least 99%.

[0402] 24. A cell of tomato, tobacco (Nicotinia tabacum), Nicotiana benthamiana or Capsicum annuum that transiently or constitutively expresses the Sw-5b protein according to any one of aspects 1 to 20.

[0403] 25. A cell of tomato, tobacco, Nicotiana benthamiana or Capsicum annuum that contains the resistance gene according to claim 21 or the nucleic acid construct according to aspect 22 or 23, preferably homozygously or heterozygously within its genomic DNA.

[0404] 26. The cell or tissue culture of the cell according to aspect 25, wherein the cell is derived from embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon and / or hypocotyl, and contains the nucleic acid construct or the resistance gene in its genome.

[0405] 27. A tomato, tobacco, Nicotiana benthamiana or Capsicum annuum plant that is resistant to TSWV infection (including resistance-breaking strains), wherein the plant contains, preferably on chromosome 9, homozygously or heterozygously, the resistance gene according to aspect 21 in its genome.

[0406] 28. A tomato plant resistant to TSWV, the tomato plant containing an allele of the Sw-5b gene encoding the Sw-5b protein, the Sw-5b protein having at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1) and containing at least one of the following variations relative to SEQ ID NO:1: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0407] 29. A tomato plant resistant to TSWV according to aspect 28, comprising an allele of the Sw-5b gene encoding the Sw-5b protein, said allele comprising at least one of the following variations relative to SEQ ID NO: 1: K136E; R138S; F139L; F140C; R143I and A147L, preferably at least 2, 3 or 4 of these variations.

[0408] 30. A tomato plant resistant to TSWV according to aspect 29, comprising at least 4 of the variations K136E, R138S, F139L, F140C, R143I and A147L, preferably at least 5 or 6 of these variations.

[0409] 31. A tomato plant resistant to TSWV according to aspect 28, comprising an allele of the Sw-5b gene encoding the Sw-5b protein, said Sw-5b protein comprising at least one of the following variations relative to SEQ ID NO: 1: N448S, P522Q, V622D and L759I, preferably 2 or 3.

[0410] 32. The tomato plant according to aspect 31, comprising the following 4 variations relative to SEQ ID NO: 1: N448S, P522Q, V622D and L759I.

[0411] 33. The tomato plant according to any one of aspects 28 to 32, wherein the allele of the Sw-5b gene is obtained by gene editing, base editing or prime editing techniques, preferably by mutagenesis, by TILLING and / or the CRISPR / Cas system.

[0412] 34. A plant part of a tomato plant according to any one of aspects 27 to 33, in particular a seed, explant, reproductive material, scion, cutting, seed, fruit, root, rhizome, pollen, ovule, embryo, protoplast, leaf, anther, stem, petiole or flower, wherein the plant part comprises at least one cell according to aspect 24, 25 or 26.

[0413] 35. A tomato seed that grows into a tomato plant according to any one of claims 27 to 33 or comprises at least one cell according to any one of aspects 24 to 26.

[0414] 36. A method for obtaining a transgenic tomato plant resistant to TSWV, said TSWV including the RB-TSWV strain, the method comprising:

[0415] - Obtain a construct comprising a sequence encoding the Sw-5b protein according to any one of aspects 1 to 20 or comprising the resistance gene according to claim 21,

[0416] - Introduce the construct into tomato cells,

[0417] - Regenerate transgenic plants;

[0418] - Optionally, propagate the obtained plants.

[0419] 37. A method for producing a tomato plant resistant to TSWV, said TSWV including the RB-TSWV strain, said method comprising introducing at least one mutation into the Sw-5b gene on chromosome 9 of a tomato plant containing Sw-5b, wherein said at least one mutation is introduced by mutagenesis, by TILLING, by genome editing, base editing or primer editing, in particular by a technique selected from: N-methyl-N-nitrosourea (MNU) mutagenesis, sodium azide (NaN3, SA) mutagenesis, oligonucleotide-directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALENs), CRISPR / Cas system, Cas9, Cas21a, engineered meganucleases, recombinant homing endonucleases and DNA-guided genome editing; and

[0420] wherein said mutation causes at least the following amino acid substitutions or deletions in the protein encoded by the Sw-5b gene: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

[0421] 38. The method according to aspect 37, wherein said mutation causes:

[0422] - At least one of the following amino acid substitutions in the protein encoded by the Sw-5b gene: N448S, P522Q, V622D and L759I; and preferably 2, 3 or 4 of said substitutions, or

[0423] -At least one of the following amino acid substitutions in the protein encoded by the -Sw-5b gene: K136E, R138S, F139L, F140C, R143I, A147L, and preferably at least 2, 3, or at least 4 of said substitutions.

[0424] 39. A method for breeding tomato plants resistant to TSWV, said TSWV including the RB-TSWV strain, said method comprising:

[0425] a. Crossing a tomato plant comprising the resistance gene according to aspect 21 with an initial tomato plant lacking said resistance gene,

[0426] b. Selecting plants carrying the resistance gene among the offspring thus obtained,

[0427] c. Optionally, self-pollinating the plants obtained in step (b) one or more times and selecting plants carrying said resistance gene among the offspring thus obtained.

[0428] 40. A method for producing tomato plants resistant to TSWV, comprising:

[0429] a. Obtaining a part of a plant according to any one of aspects 27 - 33,

[0430] b. Asexually propagating said plant part to generate plants from said plant part.

[0431] 41. Use of the sequence according to aspect 21 or the construct according to aspect 22 or 23 for conferring resistance to TSWV in tomato plants or for obtaining transgenic tomato plants resistant to TSWV, said TSWV including resistance-breaking TSWV strains.

[0432] 42. Use of a tomato plant or seed or a part thereof or its offspring carrying the resistance gene according to aspect 21 or the construct according to aspect 22 or 23 as a breeding partner in a breeding program for conferring resistance to TSWV in tomato plants, said resistance to TSWV including resistance to resistance-breaking TSWV strains.

[0433] 43 A method for improving the yield of tomato plants or reducing tomato yield losses in an environment infected or potentially infected with TSWV, in particular an environment infected with resistance-breaking TSWV strains, said method comprising growing tomato plants comprising the resistance gene according to aspect 21 in their genome.

[0434] 44. A method for reducing tomato yield loss under TSWV infection, said TSWV being in particular a resistance-breaking TSWV strain, said method comprising growing tomato plants comprising in their genome the resistance gene according to aspect 21.

[0435] 45. A method for producing tomatoes, comprising:

[0436] a. growing tomato plants according to any one of aspects 27 - 33;

[0437] b. allowing said plants to bear fruit; and

[0438] c. harvesting the fruits of said plants, preferably before or at the mature stage.

[0439] 46. A method for identifying, detecting and / or selecting tomato plants resistant to resistance-breaking TSWV strains in plants resistant to TSWV, said method comprising detecting in the genome of said plants an allele of the Sw-5b gene, wherein said allele comprises at least one variation that causes at least one amino acid substitution in the Sw-5b protein encoded by the Sw-5b gene relative to the wild-type sequence SEQ ID NO:1, said variation being selected from the group consisting of the D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D and L759I substitutions or deletions in the Sw-5b protein,

[0440] and preferably, selected from the group consisting of the K136E, R138S, F139L, F140C, R143I, A147L, N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D and L759I substitutions in the Sw-5b protein.

[0441] 47. A molecular marker for identifying, detecting, and / or selecting tomato plants resistant to a resistance-breaking TSWV strain in plants resistant to TSWV, wherein the marker has a length of less than 50 nucleotides, has at least 95% identity with the corresponding part of SEQ ID NO: 10, and is capable of distinguishing at least one of the following variations of the sequence encoded by SEQ ID NO: 11 relative to SEQ ID NO: 10: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D, and L759I; and preferably, K136E, R138S, F139L, F140C, R143I, A147L, N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D, and L759I.

[0442] 48. The molecular marker according to aspect 47, which can distinguish sequences encoding the Sw-5b protein having at least one of the following variations relative to SEQ ID NO: 1: K136E, R138S, F139L, F140C, R143I, and A147L.

[0443] 49. The molecular marker according to aspect 47, which can distinguish sequences encoding the Sw-5b protein having at least one of the following variations relative to SEQ ID NO: 1: N448S, P522Q, V622D, and L759I.

[0444] 50. The molecular marker according to aspect 47, 48, or 49, wherein the marker is an amplification probe or a detection probe.

[0445] References:

[0446] - Crescenzi, A., Fanigliulo, A., and Viggiano, A. (2015). RESISTANCE BREAKING TOMATO SPOTTED WILT VIRUS ISOLATES ON RESISTANT TOMATO CULTIVARS IN ITALY, Acta Hortic. 1069, 95 - 98

[0447] - Hallwass M., de Oliveira A.S., de Campos Dianese E., Lohuis D., Boiteux L.S., Inoue - Nagata A.K. et al. (2014). The Tomato spotted wilt virus cell - to - cell movement protein (NSm) triggers a hypersensitive response in Sw - 5 - containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw - 5b resistance gene copy. Mol. Plant Pathol. 15 871–880.

[0448] - Huang, H., Huang, S., Li, J., Wang, H., Zhao, Y., Feng, M., Dai, J., Wang, T., Zhu, M. and Tao, X. (2021) Stepwise artificial evolution of an Sw - 5b immune receptor extends its resistance spectrum against resistance - breaking isolates of Tomato spotted wilt virus. Plant Biotechnol. J., 1 - 13

[0449] - Li et al., 2019. A Plant Immune Receptor Adopts a Two-Step Recognition Mechanism to Enhance Viral Effector Perception. Mol. Plant. 12, 248–262

[0450] - Peiro A., Canizares M.C., Rubio L., Lopez C., Moriones E., Aramburu J. et al. (2014). The movement protein (NSm) of Tomato spotted wilt virus is the avirulence determinant in the tomato Sw-5 gene-based resistance. Mol. Plant Pathol. 15 802–813.

[0451] - Seong et al., 2022. Evolution of NLR resistance genes with noncanonical N-terminal domains in wild tomato species. New Phytologist (2020) 227:1530–1543

[0452] - Stevens J.M. 1964. Tomato Breeding. Project report W-Vv1, Department of Agricultural Technical Services, Republic of South Africa

[0453] - Wang, J., Chen, T., Han, M., Qian, L., Li, J., Wu, M., Han, T., Cao, J., Nagalakshmi, U., Rathjen, J. P., Hong, Y., and Liu, Y. 2020. Activation of the plant NLR immune receptor Tm-22 requires NB-ARC domain-mediated self-association of the CC domain. PLoS Pathog 16: e1008475.

Claims

1. A tomato (S. lycopersicum) plant resistant to Tomato spotted wilt virus (TSWV), said tomato plant comprising an allele of the Sw-5b gene encoding a Sw-5b protein, said Sw-5b protein having at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1) and comprising at least 5 of the following variations relative to SEQ ID NO:1: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D, and substitution L759I.

2. The tomato plant resistant to TSWV according to claim 1, comprising an allele of the Sw-5b gene encoding a Sw-5b protein, said Sw-5b protein comprising at least 3 of the following variations relative to SEQ ID NO:1: K136E, R138S, F139L, F140C, R143I, and A147L, preferably at least 4, at least 5, or 6 variations.

3. The tomato plant according to claim 1, comprising the following 4 variations relative to SEQ ID NO:1: N448S, P522Q, V622D, and L759I.

4. The tomato plant according to any one of claims 1 - 3, comprising an allele of the Sw-5b gene encoding the Sw-5b protein, wherein, The amino acids corresponding to L33 and / or R927 in SEQ ID NO:1 are not modified.

5. The tomato plant according to any one of claims 1 to 4, wherein The allele of the Sw-5b gene encodes a Sw-5b protein having at least 95% sequence identity with the wild-type Sw-5b protein (SEQ ID NO:1).

6. The tomato plant according to any one of claims 1 - 5, wherein, The allele of the Sw-5b gene is obtained by gene editing, base editing, or prime editing techniques, preferably by mutagenesis, by TILLING, and / or by the CRISPR / Cas system.

7. The tomato plant according to any one of claims 1-6, wherein, The plant is resistant to TSWV, said TSWV including resistance-breaking TSWV strains containing the C118Y mutation and strains containing the V137I and V153I mutations.

8. A plant part of a tomato plant according to any one of claims 1 to 7, in particular a seed, an explant, reproductive material, a scion, a cutting, a seed, a fruit, a root, a rhizome, a pollen, an ovule, an embryo, a protoplast, a leaf, an anther, a stem, a petiole or a flower, wherein The plant part comprises at least one cell containing the allele of the Sw-5b gene.

9. A tomato (S. lycopersicum) seed capable of growing into a tomato plant according to any one of claims 1-8.

10. A Sw-5b protein that recognizes the viral movement protein NSm of TSWV, wherein, The protein has at least 90% sequence identity with the wild-type Sw-5b protein (SEQ ID NO: 1) and contains at least 1, preferably 2, 3 or 4 variations relative to SEQ ID NO: 1 found at the following positions: positions 23, 26, 28, 29, 30, 75, 82, 83, 90, 134, 135, 136, 138, 139, 140, 143, 147, 148, 151, 203, 209, 214, 325, 351, 448, 522, 622 and / or 759.

11. The Sw-5b protein according to claim 10, wherein, The at least 1 variation is selected from the following mutations relative to SEQ ID NO: 1: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D and L759I.

12. The Sw-5b protein according to claim 10 or 11, comprising at least 1, preferably 2, 3 or the following 4 variations relative to SEQ ID NO: 1: N448S, P522Q, V622D and L759I.

13. The Sw-5b protein according to any one of claims 10-12, comprising the following variations relative to SEQ ID NO: 1: N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D and L759I.

14. The Sw-5b protein according to claim 10, comprising at least 3 of the following variations relative to SEQ ID NO: 1: K136E, R138S, F139L, F140C, R143I and A147L; preferably at least 4, or at least 5, or 6 variations of these.

15. The Sw-5b protein according to any one of claims 10 to 14, wherein The amino acids corresponding to L33 and / or R927 in SEQ ID NO: 1 are not modified.

16. The Sw-5b protein according to any one of claims 10-15, having an entire sequence with at least 92%, preferably at least 94% or at least 95% sequence identity with SEQ ID NO: 1; or having the sequence shown in SEQ ID NO: 3, or a sequence having more than 99% sequence identity with SEQ ID NO:

3.

17. The Sw-5b protein according to any one of claims 10-16, wherein the Sw-5b protein recognizes and interacts with the viral movement protein NSm of a resistance-breaking TSWV strain, or interacts with the viral movement protein NSm of an RB-TSWV strain that does not interact with SEQ ID NO:1, and the resistance-breaking TSWV strains include strains containing the C118Y mutation and strains containing the V137I and V153I mutations.

18. The Sw-5b protein according to any one of claims 10-17, which confers resistance to TSWV infection in tomato, pepper, potato, and / or lettuce, and the TSWV includes resistance-breaking TSWV strains containing the C118Y mutation and strains containing the V137I and V153I mutations.

19. A resistance gene encoding the Sw-5b protein according to any one of claims 10-18, and the resistance gene confers resistance to TSWV infection in tomato plants.

20. A nucleic acid construct comprising a sequence encoding the Sw-5b protein according to any one of claims 10-18 or comprising the resistance gene according to claim 19, preferably under the control of a promoter such as a vector, plasmid, or T-DNA plasmid.

21. The nucleic acid construct according to claim 20, comprising a sequence having at least 75% sequence identity with SEQ ID NO:11, preferably at least 90%, more preferably at least 99%.

22. A cell of tomato, tobacco (Nicotinia tabacum), Nicotiana benthamiana, or Capsicum annuum, comprising the resistance gene according to claim 19 or the nucleic acid construct according to claim 20 or 21, preferably homozygously or heterozygously in its genomic DNA.

23. A tomato, tobacco, Nicotiana benthamiana, or Capsicum annuum plant resistant to TSWV infection, and the TSWV includes resistance-breaking strains. Among them, The plant homozygously or heterozygously contains the resistance gene according to claim 19 in its genome, preferably on chromosome 9.

24. A method for obtaining a transgenic tomato plant resistant to TSWV, and the TSWV includes the RB-TSWV strain, and the method comprises: - obtaining a construct comprising a sequence encoding the Sw-5b protein according to any one of claims 10-18 or comprising the resistance gene according to claim 19; - introducing the construct into a cell of tomato; - regenerating a transgenic plant; - optionally propagating the obtained plant.

25. A method for producing a tomato plant resistant to TSWV, said TSWV including the RB-TSWV strain, said method comprising introducing at least one mutation into the Sw-5b gene on chromosome 9 of a tomato plant containing Sw-5b, wherein, The at least one mutation is introduced by mutagenesis, by TILLING, by genome editing, base editing or prime editing, in particular by a technique selected from: N-methyl-N-nitrosourea (MNU) mutagenesis, sodium azide (NaN3, SA) mutagenesis, oligonucleotide-directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALENs), CRISPR / Cas systems, Cas9, Cas21a, engineered meganucleases, recombinant homing endonucleases and DNA-guided genome editing; and wherein, the mutation causes at least the following amino acid substitutions or deletions in the protein encoded by the Sw-5b gene: substitution D23H, substitution D26N, substitution L28F, substitution R29Q, substitution I30L, substitution R75I, deletion T82DEL, deletion N83DEL, substitution E90L, substitution L134F, substitution Q135H, substitution K136E, substitution R138S, substitution F139L, substitution F140C, substitution R143I, substitution A147L, substitution K148I, substitution S151T, substitution V203L, substitution E209G, substitution E214K, substitution G325V, substitution I351V, substitution N448S, substitution P522Q, substitution V622D and substitution L759I.

26. The method according to claim 25, wherein The mutation causes: - at least one of the following amino acid substitutions in the protein encoded by the Sw-5b gene: N448S, P522Q, V622D and L759I; and preferably 2, 3 or 4 of said substitutions, or - at least one of the following amino acid substitutions in the protein encoded by the Sw-5b gene: K136E, R138S, F139L, F140C, R143I, A147L, and preferably at least 2, 3 or at least 4 of said substitutions.

27. A method for breeding tomato plants resistant to TSWV, said TSWV including the RB-TSWV strain, said method comprising: a. crossing a tomato plant comprising the resistance gene according to claim 19 with an initial tomato plant lacking said resistance gene, b. selecting, in the progeny thus obtained, plants carrying the resistance gene, c. optionally, self-pollinating the plants obtained in step (b) one or more times and selecting, in the progeny thus obtained, plants carrying said resistance gene.

28. A method for producing tomato plants resistant to TSWV, comprising: a. obtaining a part of a plant according to any one of claims 1-7 and 23, b. asexually propagating the part of the plant to generate a plant from the part of the plant.

29. Use of the sequence according to claim 19 or of the construct according to claim 20 or 21 for conferring resistance to TSWV on tomato plants or for obtaining transgenic tomato plants resistant to TSWV, said resistance to TSWV including resistance to resistance-breaking TSWV strains.

30. Use of a tomato plant, or a seed, or a part thereof, or a progeny thereof carrying the resistance gene according to claim 19 or the construct according to claim 20 or 21 as a breeding partner in a breeding program for conferring resistance to TSWV on tomato plants, said resistance to TSWV including resistance to resistance-breaking TSWV strains.

31. A method for improving the yield of tomato plants or reducing tomato yield losses in an environment infected or potentially infected with TSWV, in particular with resistance-breaking TSWV strains, said method comprising growing tomato plants comprising in their genome the resistance gene according to claim 19.

32. A method for producing tomatoes, comprising: a. growing a tomato plant according to any one of claims 1-7 and 23; b. allowing the plant to bear fruit; and c. harvesting the fruits of the plant, preferably before or at the mature stage.

33. A method for identifying, detecting, and / or selecting tomato plants resistant to a resistance-breaking TSWV strain in plants resistant to TSWV, the method comprising detecting an allele of the Sw-5b gene in the genome of the plant, wherein, The allele comprises at least one variation that causes at least one amino acid substitution in the Sw-5b protein encoded by the Sw-5b gene relative to the wild-type sequence SEQ ID NO:1, selected from the group consisting of the D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D and L759I substitutions or deletions in the Sw-5b protein, and preferably, selected from the group consisting of the K136E, R138S, F139L, F140C, R143I, A147L, N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D and L759I substitutions in the Sw-5b protein.

34. A molecular marker, which is used to identify, detect, and / or select tomato plants resistant to resistance-breaking TSWV strains in plants resistant to TSWV, wherein, The length of the marker is less than 50 nucleotides, has at least 95% identity with the corresponding part of SEQ ID NO: 10, and is capable of distinguishing at least one of the following variations of the sequence encoded by SEQ ID NO: 11 relative to SEQ ID NO: 10: D23H, D26N, L28F, R29Q, I30L, R75I, T82DEL, N83DEL, E90L, L134F, Q135H, K136E, R138S, F139L, F140C, R143I, A147L, K148I, S151T, V203L, E209G, E214K, G325V, I351V, N448S, P522Q, V622D, and L759I; and preferably, K136E, R138S, F139L, F140C, R143I, A147L, N393D, N448S, S461A, A477V, V489L, P522Q, D659S, D614N, V622D, L623V, I661N, E707D, and L759I.

35. The molecular marker according to claim 34, wherein the molecular marker is capable of distinguishing a sequence encoding a Sw-5b protein having at least one of the following variations relative to SEQ ID NO: 1: K136E, R138S, F139L, F140C, R143I, and A147L.

36. The molecular marker according to claim 34, wherein the molecular marker is capable of distinguishing a sequence encoding a Sw-5b protein having at least one of the following variations relative to SEQ ID NO: 1: N448S, P522Q, V622D, and L759I.

37. The molecular marker according to claim 34, 35 or 36, wherein, The molecular marker is an amplification probe or a detection probe.

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