New species of tobacco mosaic virus

Abstract

The invention provides a new species of tobacco mosaic virus, and its use for identifying plants comprising resistance against the virus.

Description

Technical Field

[0001] This invention relates to a novel species of the genus *tobamovirus* (family Virgaviridae) that infects plants of the family Solanaceae. The invention also relates to the use of said virus species for identifying and / or producing resistant Solanaceae plants (e.g., tomato, tobacco, pepper, and eggplant). The novel tobacco mosaic virus species is capable of carrying the Tomato Mosaic Virus (ToMV) resistance genes Tm1, Tm2, ​​and Tm2... 2 The virus reproduces and spreads on tomato plants, meaning that these resistance genes are ineffective against the novel virus, and infected plants exhibit a variety of symptoms, such as (mild) mosaic virus of leaves, leaf reddening, blistering, and leaf deformity. A novel virus species—referred herein to as Tomato Mosaic Heavy Virus (ToMSV or TMSV)—is provided, along with a method for diagnosing the presence of the novel tobacco mosaic virus species in plants and / or plant parts of Solanaceae species (particularly tomato, tobacco, pepper, and eggplant), and a method for screening plants and / or plant parts resistant to the virus using isolates of the novel virus (e.g., isolate VE484 (deposited on January 19, 2015, with accession number DSM 29970)). Background Technology

[0002] Historically, tobacco mosaic viruses in tomatoes were collectively classified as tobacco mosaic virus (TMV) strains. However, these tobacco mosaic viruses are now classified into different virus species based on sequence differences. Tobacco mosaic virus species include pepper mild spot virus (PMMV), tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and many other viruses (see, for example, the ICTVdB Index of Viruses).

[0003] Plant viruses can be devastating to fruit and vegetable production. Although many commercial varieties carry virus resistance genes, these genes can become ineffective because resistance-disrupting strains can evolve or new virus species can evolve. In tomatoes, three dominant ToMV resistance genes have been used for decades to control ToMV: Tm1 (introduced from the hairy tomato (S. habrochaites) gene; conferring resistance to ToMV strains 0 and 2), Tm2, ​​and Tm23. 2(All were infiltrated from the Peruvian tomato (S. peruvianum) gene, conferring resistance to ToMV strains 0 and 1, and 0, 1, and 2, respectively). The protein encoded by the Tm1 resistance gene binds to the ToMV replication protein and inhibits RNA-dependent RNA replication of ToMV. The replication protein in the ToMV resistance-disrupting mutant does not bind to Tm1, indicating that this binding is important for inhibition (see Ishibashi and Ishikawa, J Virol. 2013 July; 87(14):7933-7939). Tm2 2 The resistance gene encodes a protein that recognizes the ToMV mobile protein (particularly the carboxyl terminus of the mobile protein), and has a Tm2 amino acid variation at the carboxyl terminus of the mobile protein. 2 Resistance-destroying strains are no longer recognized, thus enabling the overcoming of resistance (see Weber and Pfitzner, 1998, MPMI Vol. 11, pp. 498-503). An ongoing evolutionary race exists between resistance genes and viral evolution. Therefore, rapid identification of new viruses is crucial to obtaining tools for identifying resistance genes effective against these new viruses.

[0004] One object of this invention is to identify a novel species of tobacco mosaic virus, referred herein as Tomato Mosaic Heavy Virus (ToMSV or TMSV), capable of infecting tomato plants carrying any one or a combination of these three resistance genes. Another object of this invention is to provide a method for diagnosing this novel viral species, and a method for screening new sources of resistance in germplasm using virulence isolates of said species. Summary of the Invention

[0005] As used herein, the term "comprising" and its variations are used in their non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned. However, those skilled in the art will understand that the term "comprising" also encompasses the term "consisting of." Furthermore, referring to an element by saying "a" or "an" does not preclude the possibility that more than one of that element may exist, unless the context clearly requires that there be only one of that element. Therefore, "a" or "an" generally means "at least one" or "one or more."

[0006] The term "cultivated species" (or "cultivated" plant) is used herein to refer to a plant having a biological state different from a "wild" state, where "wild" state refers to the original, uncultivated, undomesticated, or natural state of a plant or accession, and the term "cultivated" does not include such wild plants or weeds. The term "cultivated species" includes materials with favorable agronomic characteristics, such as breeding materials, research materials, breeding lines, superior breeding lines, synthetic populations, hybrids, creation of original / basic populations, inbred lines, cultivars (free-pollinated hybrid cultivars), segregating populations, mutant / genetic originals, and advanced / improved cultivars. In one embodiment, the term "cultivated species" also includes landraces, i.e., pepper plants (or populations) that have been artificially selected and locally cultivated over many years and modified to suit specific geographic environments and enjoy a shared gene pool. Compared to wild germplasm, cultivated species have favorable agronomic characteristics, such as higher yields, larger fruit size, higher fertility, and greater uniformity of plants and / or fruits.

[0007] As used herein, the term "plant" includes the whole plant or any part or derivative thereof, such as plant organs (e.g., harvested or unharvested fruits, leaves, seeds, flowers, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which a whole plant can be regenerated, plant callus, plant cell masses, and whole plant cells in a plant, or plant parts such as embryos, pollen, ovules, ovaries, fruits (e.g., harvested tissues or organs, such as harvested pepper fruits or parts thereof), flowers, leaves, seeds, asexually reproduced plants, roots, rootstocks, stems, root tips, etc. It also includes any developmental stage, such as immature and mature seedlings.

[0008] A "plant variety" is a group of plants belonging to the same, known lowest taxonomic group. A plant variety can be defined based on the expression of characteristics derived from a particular genotype or combination of genotypes (regardless of whether the identification criteria in the Plant Breeder's Rights apply). A plant variety can be distinguished from any other group of plants by the expression of at least one of those characteristics. A plant variety can also be considered a single entity because it can be propagated without alteration. Therefore, if a group of plants is characterized by the presence of a single locus or gene (or a series of phenotypic characteristics derived from that single locus or gene), but they can differ significantly from each other at other loci or genes, then even if they belong to the same class, the term "plant variety" cannot be used to describe that group of plants.

[0009] The Solanaceae family is a plant family that includes genera (especially Solanum and Capsicum), which include cultivated and bred fruit and plant species such as tomato (Solanum lycopersicum), pepper (Capsicum annuum), eggplant (Solanum melongena), and melon eggplant (Solanummuricatum).

[0010] "Tomato plant" or "cultivated tomato plant" refers to a tomato plant that is artificially cultivated and possesses favorable agronomic characteristics, including varieties, breeding lines, or cultivars of the tomato species; preferably, such plants are not "wild plants," which are plants that typically have much lower yields and agronomic characteristics than cultivated plants and grow naturally in wild populations, for example. "Wild plants" include, for example, wild germplasm or wild relatives of a species. In one aspect of the invention, so-called heirloom tomato varieties or cultivars—that is, freely pollinating varieties or cultivars that were typically cultivated in earlier periods of human history and typically adapted to suit specific geographic regions—are herein covered as cultivated tomato plants in one aspect of the invention. In one embodiment, the term "cultivar" also includes local varieties, i.e., plants (or populations) that have been artificially selected and locally cultivated over many years and adapted to suit specific geographic environments and enjoy a common gene pool.

[0011] The term "cultivated species" (or "cultivated" plant) is used herein to refer to a plant having a biological state different from that of a "wild" state, where "wild" state refers to the original, uncultivated, or natural state of the plant or germplasm, and the term "cultivated" does not include such wild or weed plants. The term "cultivated species" includes materials with good agronomic characteristics, such as breeding materials, research materials, breeding lines, superior breeding lines, synthetic populations, hybrids, creation of original / basic populations, inbred lines, cultivars (free-pollinated hybrid cultivars), segregating populations, mutant / genetic originals, and advanced / improved cultivars.

[0012] Wild relatives of tomatoes include wild tomato (S. arcanum), Kmerlyuski tomato (S. chmielewskii), small-flowered tomato (S. neorickii (=L. parviflorum)), Cheesmaniae tomato (S. cheesmaniae), glandular tomato (S. galapagense), narrow-leaved tomato (S. pimpinellifolium), Chilean tomato (S. chilense), S. corneliomulleri, hairy tomato (S. habrochaites (=L. hirsutum)), S. huaylasense, garlic mustard tomato (S. sisymbriifolium), Peruvian tomato (S. peruvianum), hairy tomato (S. hirsutum), Pennelli tomato (S. pennellii), cherry tomato (S. lycopersicoides), Riqui tomato (S. sitiens), or ochranthum tomato (S. ochranthum).

[0013] As used in this article, “chili plant” or “chili” refers to plants of the genus Capsicum or parts thereof (e.g., fruits). Chili peppers include all species of chili peppers, such as chili peppers and non-chili peppers (sweet peppers). The term includes both wild and domesticated chili peppers.

[0014] "Domesticated peppers" refers to the species *Capsicum annuum* L., *Capsicum chinense* Jacq., *Capsicum frutescens* L., *Capsicum baccatum* L., and *Capsicum pubescens* Ruiz & Pav. The term "cultivated peppers" refers to breeding lines and varieties of domesticated peppers that are cultivated artificially in the field or in protected environments (e.g., greenhouses or tunnels) to produce fruit. Compared to wild germplasm, cultivated varieties exhibit superior agronomical characteristics, such as higher yields, larger fruit size, higher fertility, and greater uniformity of plants and / or fruits. Examples of cultivated varieties include cultivars belonging to the species *Capsicum annuum*, *Capsicum chinense*, *Capsicum frutescens*, *Capsicum baccatum*, and *Capsicum pubescens*.

[0015] "Plant genotype" refers to plants that are genotype very similar, such as germplasm in seed banks (e.g., germplasm in the GRIN Collection in the United States; http: / / www.ars-grin.gov / npgs / acc / acc_queries.html or germplasm in the CGN (Center for Genetic Resources) Collection at Wageningen University and Research Centre in the Netherlands) and their offspring obtained through self-pollination, or plant lines, or plant varieties.

[0016] "Plant line" or "breeding line" refers to a plant and its offspring. The term "inbred line" used in this article refers to a plant line that has been repeatedly self-pollinated.

[0017] "F1, F2, etc." refers to the successive generations following a hybridization of two parent plants or parent lines. Plants that grow from seeds produced by hybridization of two plants or lines are called the F1 generation. Self-pollination of F1 plants produces the F2 generation, and so on. "F1 hybrid" plants (or F1 hybrid seeds) are the generations obtained by hybridizing two closely crossed parent lines.

[0018] "Hybrid" or "hybrid plant" is a plant produced by cross-pollination (cross-pollination) between at least two different plants or different parental lines. It should be understood that this article covers the seeds of such hybrids (hybrid seeds), as well as the hybrid plants grown from those seeds and the plant parts obtained from those grown plants.

[0019] The term "trait" refers to a heritable characteristic (such as ToMSV resistance), which can be transferred from one plant to another, for example, through hybridization and selection.

[0020] "ToMSV", "ToMSV strain", "ToMSV isolate" or "ToMSV pathogenic type" refers to a strain of Tomato Mosaic Severe Virus that can be serologically (using antibodies) by microscopy, sequence comparison and / or disease assay, all as described herein.

[0021] “VE484” refers to the virulence strain of ToMSV, and its representative sample is deposited in DSZM with accession number DSM29970.

[0022] The “Tm1 gene” or “Tm1 resistance gene” refers to a dominant resistance gene known in the art that confers resistance to ToMV strains 0 and 2, for example, from the commercial tomato variety Mobaci.

[0023] The “Tm2 gene” or “Tm2 resistance gene” refers to a dominant resistance gene known in the art that confers resistance to ToMV strains 0 and 2, for example, which can be obtained from the commercial tomato variety Moperon.

[0024] “Tm22 "Gene" or "Tm2" 2 "Resistance gene" refers to a dominant resistance gene known in the field that confers resistance to ToMV strains 0, 1, and 2, for example, which can be obtained from the commercial tomato variety Momor.

[0025] Plants that possess or contain "ToMSV resistance" or are "ToMSV resistant" (e.g., Solanaceae, such as tomato or wild relatives of cultivated tomato, wild or cultivated Capsicum species, eggplant, or Cantaloupe) are plants that do not develop systemic symptoms and in which, after inoculation or infection with one or more infectious strains of ToMSV (e.g., strain V484), the virus either develops or does not spread systemically (i.e., spread to uninoculated and / or uninfected parts of the plant, such as the upper leaves). If the virus does not develop systemic symptoms and cannot spread systemically, this resistance may be called "complete resistance," while if the virus does not develop systemic symptoms but can spread systemically, this resistance may also be referred to herein as "tolerance." Various methods can be used to test resistance (including complete resistance and tolerance). One example is the use of artificial mechanical inoculation assay, whereby, for example, one or two young leaves or cotyledons of a plant are mechanically inoculated with an infectious strain (e.g., Ve484), and the presence of the virus in uninoculated plant parts (e.g., upper leaves) is assessed one or more days after inoculation for systemic symptoms (e.g., mosaic, leaf deformity, blistering, and / or erythroplasia) and / or the presence of the virus in uninoculated plant parts (using, for example, ELISA, electron microscopy, etc.).

[0026] “Systemic symptoms” are symptoms visible on tissues or plant parts other than the inoculated / infected tissue or plant part (e.g., other leaves) (e.g., on upper leaves on which the virus has spread from the inoculated / infected leaf or cotyledon or stem or hypocotyl).

[0027] "Systemic spread" refers to the spread of the virus from inoculated / infected tissues or plant parts (e.g., leaves, cotyledons, stems, or hypocotyls) to uninoculated / uninfected tissues or plant parts (e.g., spread to upper leaves).

[0028] The term "allele" refers to any one or more alternative forms of a gene at a specific locus, all of which are associated with a trait or characteristic at that locus. In the diploid cells of an organism, the alleles of a given gene are located at a specific location on a chromosome or at a single locus (or multiple loci). One allele exists on each of a pair of homologous chromosomes. Diploid plant species (e.g., peppers and tomatoes) can contain a large number of different alleles at a given locus. These alleles can be the same allele of the gene (homozygous) or two different alleles (heterozygous).

[0029] The term "protein" refers to a polypeptide that has a mode of action, size, three-dimensional structure, or origin. Therefore, a "fragment" or "part" of a protein can still be called a "protein." "Isolated protein" is used to refer to a protein that no longer exists in its native environment.

[0030] Sequence identity and sequence similarity can be determined by aligning two peptide or nucleotide sequences using global or local alignment algorithms. Sequences are then said to be "substantially identical" or "substantially similar" when they share at least a minimum percentage of sequence identity (as further defined below) when optimally aligned using programs such as GAP, BESTFIT, or the Emboss program "Needle" (using default parameters, see below). These programs use the Needleman and Wunsch global alignment algorithms to align the full length of two sequences, maximizing the number of matches and minimizing the number of gaps. Typically, default parameters are used, where the gap creation penalty is 10 and the gap extension penalty is 0.5 (this applies to both nucleotide and protein alignments). For nucleotides, the default scoring matrix used is nwsgapdna, while for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). For example, computer programs (such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA, or EMBOSS (http: / / www.ebi.ac.uk / Tools / webservices / services / emboss)) can be used to determine sequence alignment and scores for percentage sequence identity. Alternatively, percentage similarity or identity can be determined by searching databases (such as FASTA, BLAST, etc.), but hits should be retrieved and paired for comparison of sequence identity.

[0031] In this article, "average" refers to the arithmetic mean.

[0032] “Strong hybridization conditions” can be used to identify nucleotide sequences that are substantially identical to a given nucleotide sequence (in methods known as nucleic acid hybridization). Strict conditions are sequence-dependent and vary depending on the specific circumstances. Typically, strict conditions are chosen to be approximately 5°C lower than the thermal melting temperature (Tm) of the particular sequence at a given ionic strength and pH. The Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Strict conditions are typically chosen where the salt concentration is approximately 0.02 mol at pH 7 and the temperature is at least 60°C. Decreasing the salt concentration and / or increasing the temperature both increase strictness. Strict conditions for RNA-DNA hybridization (using, for example, 100 nt probes) include, for example, those involving at least one 20-minute wash at 63°C in 0.2X SSC, or equivalent conditions. The stringent conditions for DNA-DNA hybridization (using, for example, DNA blotting with a 100nt probe) include, for example, those involving at least one (usually two) washes for 20 minutes in 0.2X SSC at a temperature of at least 50°C (typically about 55°C), or equivalent conditions. See also Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press; and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., ColdSpring Harbor Laboratory Press, NY; and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, vols. 1 and 2. Detailed Implementation

[0033] This invention provides a novel tobacco mosaic virus, referred herein as Tomato Mosaic Heavy Virus (ToMSV or TMSV), which was identified in leaf samples from tomato production fields and confirmed as a tobacco mosaic virus by ELISA, electron microscopy, and bioassays. Viral genome sequencing revealed it to be a novel species of tomato tobacco mosaic virus. The isolated strain of this virus, designated VE484, is deposited in DSMZ. Strain VE484 is capable of infecting plants containing the widely used resistance genes Tm1, Tm2, ​​and Tm3. 2 This strain can also infect other members of the Solanaceae family, particularly members of the Capsicum genus (such as cultivated peppers), and possibly other members of the Solanaceae genus, besides tomatoes, such as wild relatives of tomatoes, eggplants, and cantaloupe.

[0034] As with other tobacco mosaic viruses, the genome sequence of this positive-sense single-stranded RNA virus (SEQ ID NO:1 and SEQ ID NO:2) encodes four ORFs (open reading frames). The complete genome is 6402 bases long and shares only 82% sequence identity with its most similar virus—Genbank accession number FR878069.1 (Tobacco mosaic virus strain Ohio V, complete genome, genomic RNA). Based on this low percentage of genome sequence identity (below 90%), a new species name within the genus *Tobacco Mosaic Virus* is proposed, following the naming convention in the book "Virus Taxonomy: 9". th The species classification criteria proposed in the report of the International Committee on Virus Taxonomy, ISBN 978-0-12-384684-6, p1155, are presented here. This paper proposes classifying viruses with less than 90% sequence identity in their nucleotide genome sequences into a new genus.

[0035] Furthermore, the proteins encoded by the four ORFs are unique in public sequence databases. Using Emboss-needle (paired alignment, default parameters), the percentage of sequence identity with the most similar Genbank accession number is shown below:

[0036] Table 1 – ORF1 (SEQ ID NO:3), protein p126

[0037]

[0038]

[0039] The protein p126 (SEQ ID NO:3) is 1116 amino acids long and has a molecular weight of 126 kDa. It contains methyltransferase and helicase domains.

[0040] Table 2 – ORF2 (SEQ ID NO:4), protein p183

[0041] ORF2 (SEQ ID NO:4) CCC33060.1 ABN79257.1 ORF2 (SEQ ID NO:4) 100％ CCC33060.1 93.0％ 100％ ABN79257.1 92.9％ 98.8％ 100％

[0042] Protein p183 (SEQ ID NO:4) is 1609 amino acids long and has a molecular weight of 183 kDa. It contains methyltransferase and helicase domains (similar to p126) and an additional polymerase domain (RdRP). Protein p183 is produced by the repression of the p126 stop codon.

[0043] Table 3 – ORF3 (SEQ ID NO:5), a mobile protein

[0044] ORF3 (SEQ ID NO:5) AAY44881.1 CCC33061.1 ORF3 (SEQ ID NO:5) 100％ AAY44881.1 79.4％ 100％ CCC33061.1 79.4％ 96.3％ 100％

[0045] The protein SEQ ID NO:5 (ORF3) has a length of 266 amino acids and a molecular weight of 29.7 kDa, and is a viral mobile protein (MP).

[0046] Table 4 – ORF4 (SEQ ID NO:6), outer shell protein

[0047] ORF4 (SEQ ID NO:6) AIW42686.1 ABN13962.1 ORF4 (SEQ ID NO:6) 100％ ABN13962.1 81.8 100％ AIW42686.1 80.7 96.9 100％

[0048] The protein SEQ ID NO:6 (ORF4) is 176 amino acids long and has a molecular weight of 19.6 kDa. It is the viral capsid protein (CP).

[0049] Viruses are not transmitted by insects, but mechanically. They can also be transmitted through seeds (i.e., seeds produced on infected plants), as viruses have been found to originate from seeds.

[0050] Electron microscopy of infected leaves revealed that the virus particles were rod-shaped, approximately 300 nanometers (nm) in length and 18 nm in diameter. Mechanical inoculation of leaves allowed the virus to multiply on Solanaceae plants (e.g., tomato and Nicotiana benthamiana).

[0051] In one aspect, the present invention provides a novel species of tobacco mosaic virus whose genome sequence contains basic sequence identity with SEQ ID NO:1, i.e., at least 83% sequence identity with SEQ ID NO:1, preferably at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% or 100%. In another aspect, the present invention provides a novel species of tobacco mosaic virus whose nucleic acid genome sequence contains basic sequence identity with SEQ ID NO:1 (also shown in Figure 1), wherein basic sequence identity means at least 90%, preferably at least 91%, 92%, 93%, or higher (e.g., at least 94%, 95%, 96%, 97%, 98%, or even 99% or 100%) sequence identity with SEQ ID NO:1. Sequence identity is determined by comparing whole genome sequences, for example using the Emboss program 'Needle' (with default parameters). Other strains of this new species exist or will develop besides strain VE484 (a representative sample of which has been deposited). Skilled workers can readily isolate and identify these other strains according to the present invention (e.g., one or more criteria selected from sequence identity, bioassays, symptoms, antibody-based assays, etc.).

[0052] On the one hand, tobacco mosaic virus is provided that is not present in living plants (i.e., the virus is isolated from whole living plants such as those present in the field), but is provided in the form of, for example, cut plant parts (e.g., fresh leaf tissue, freeze-dried plant tissue), or extracted plant sap or solutions (e.g., buffers). The virus can also be provided in a container containing the virus.

[0053] The virus is infectious in Solanaceae species, particularly in the genera *Solanum* and *Capsicum*. In one aspect, the virus causes infection in *Solanum* species (e.g., at least in tomatoes, especially cultivated tomatoes lacking the Tm resistance gene or containing one or more Tm resistance genes selected from Tm1, Tm2, ​​and Tm3). 2 The virus causes systemic symptoms in tomato cultivars, for example, those with the following genotypes: Tm1 / Tm1 (Tm1 homozygous); Tm1Tm2 / Tm1Tm2 (Tm1 and Tm2 homozygous); Tm1 / Tm2 2 / Tm1 / Tm2 2 (Tm1 and Tm2) 2 (Homozygous); Tm2 / Tm2 (Tm2 homozygous); Tm2 2 / Tm2 2 (Tm2 2 (Homozygous type).

[0054] In other respects, the virus causes systemic symptoms in Capsicum plants (particularly cultivated Capsicum species).

[0055] Systemic symptoms refer to symptoms on parts of the plant other than the site of viral entry or inoculation. This indicates that the virus can spread from the site of infection (e.g., leaves) to other parts of the plant, such as other leaves like upper leaves (i.e., systemic spread of the virus). Systemic symptoms are varied and include one or more of the following: mosaic virus, leaf deformity, leaf blistering, and / or leaf reddish-brown discoloration. In some instances, plants may remain asymptomatic despite systemic viral spread. Therefore, in one respect, the ability of a virus to cause systemic symptoms indicates the presence of one or more systemic symptoms in at least 40%, 50%, 60%, preferably at least 70%, 80%, 85%, 90%, or more of plants of the same genotype that have been infected (e.g., in the field or through seed transmission) or inoculated (e.g., mechanically inoculated). Such plant genotypes are therefore susceptible to viral infection, although not all plants exhibit systemic symptoms.

[0056] Systemic spread of the virus in symptomatic and asymptomatic plants can be determined by a variety of methods that detect (and optionally quantify) the presence of the virus in uninfected or uninoculated plant parts (e.g., upper leaves). The virus can be detected by a variety of methods or combinations thereof, including microscopy (e.g., electron microscopy), antibody-based tests (e.g., ELISA or transverse flow apparatus tests), RT-PCT (reverse transcriptase PCR), sequencing, nucleic acid hybridization (using, for example, stringent hybridization conditions), and bioassays (e.g., inoculation of indicator plants such as Nicotiana tabacum var. Xanthi (containing a homozygous form of the N gene) and Nicotiana glutinosa).

[0057] In other respects, a container containing a novel species of tobacco mosaic virus is provided. Preferably, the container contains a virus strain, preferably an infectious strain. The container can be any type of container, such as a tube, vial, aperture, bottle, bag, etc. The virus can be present in the container in various forms, such as as cut plant tissue (e.g., fresh, dried, or freeze-dried tissue, such as leaves or leaf parts, stems or stem parts, seeds or seed parts, etc.). The virus can also be present outside of plant tissue, for example, in liquid form (e.g., extracted plant sap), or in a solution that does not contain plant cells and plant sap (e.g., water or buffer solution).

[0058] In one aspect, a sterile solution comprising the virus of the present invention is provided. In another aspect, the virus is a single infectious strain, such as VE484 or any other ToMSV strain. In yet another aspect, the solution is a buffer solution.

[0059] A method is also provided for identifying Solanaceae and Capsicum plants containing resistance (complete resistance or tolerance) to the virus using the virus of the present invention. The novel virus can be used to screen for resistant (complete resistance or tolerance) plant genotypes in different plant genotypes of the Solanaceae and / or Capsicum genera. In one aspect, this indicates that the virus does not cause systemic symptoms and does not spread systemically across the plant genotype. The virus may optionally cause localized damage on the inoculated plant parts (e.g., leaves) of the genotype. In another aspect, the virus does not cause systemic symptoms, but does spread systemically across the plant genotype (tolerance).

[0060] The method can be used to identify cultivated plants (e.g., cultivated tomato *S. lycopersicum* or cultivated pepper *Capsicum annuum*), or preferably wild plants of the *Solanum* or *Capsicum* genera containing ToMSV resistance. Thus, in one aspect, the method is used to identify ToMSV-resistant plants (e.g., VE484-resistant plants), wherein said plants are selected from the following species: tomato, wild tomato, *S. kmeleviskia*, *S. florida*, *S. chismanni*, *S. corneliomulleri*, *S. huaylasense ...

[0061] On the other hand, the method is used to identify ToMSV resistant plants (e.g., VE484 resistant plants), wherein the plants are selected from the following species: chili pepper, yellow lantern pepper, bird's eye chili, bell pepper and Capsicum pubescens.

[0062] On the other hand, the method is used to identify ToMSV resistant plants (e.g., VE484 resistant plants), wherein the plants are selected from the species eggplant and cantaloupe.

[0063] The method includes the following steps:

[0064] a) Provide one or more plants;

[0065] b) Provide an inoculum containing the virus of the present invention;

[0066] c) Use the inoculum of b) to inoculate one or more plant parts of the plant of a);

[0067] d) Cultivating inoculated plants.

[0068] Therefore, as mentioned, the one or more plants in step a) are preferably one or more plants from the genera *Solanum* or *Capsicum*, such as one or more plants from the species mentioned above. When referring to "one or more plants," it should be understood that it preferably refers to several plants of one or more plant genotypes. Thus, for example, if testing one or more genotypes (or germplasm), such as one or more wild germplasm of closely related to tomato, it is preferable to provide several plants of each genotype, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more plants of each genotype. Similarly, if testing one or more breeding lines or cultivars, several plants of each breeding line or cultivar are provided. Preferably, it also includes ToMSV-susceptible plant genotypes with known symptomology, such as tomato varieties Mobaci, Moperou, Momor, Mocimor, Philippos or others.

[0069] Viral inoculum can be prepared using known methods. Symptom-positive leaves or other infected plant tissues can be collected and ground in the presence of a buffer solution, or infectious plant sap can be used. Preserved viruses can also be used to prepare infectious inoculum. It should be noted that technicians do not need to use the preserved virus strains (VE484, DSM29970), but new tobacco mosaic virus strains can be identified in the field, their identity optionally verified by sequencing, and infectious inoculum can be prepared using these strains.

[0070] Step c) is preferably performed by mechanical inoculation, even if the plant surface is slightly damaged to allow the virus to enter. Thus, for example, silicon carbide powder can be sprinkled onto one or more leaves or cotyledons of each plant, and then the inoculum can be added to the leaf surface or cotyledon. Alternatively, other plant parts, such as stems, hypocotyls, petioles, or roots, can be inoculated. Clearly, there are different ways to slightly damage the plant surface. Therefore, in one aspect, the plant parts in c) are leaves, cotyledons, stems, hypocotyls, roots, or petioles.

[0071] In step d), the plants are then cultured at temperatures, light, and relative humidity (depending on the species) that allow for further plant growth. The plants are then periodically examined for systemic symptoms and / or localized damage, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and / or more days after inoculation.

[0072] Therefore, in one aspect, the method further includes step e): assessing symptoms on the plant or plant parts, particularly systemic symptoms and / or localized damage (on inoculated plant parts). "Localized damage" or "localized necrotic damage" is damage that forms as a defensive response of the plant and prevents the spread of the virus to uninoculated parts. Thus, systemic viral symptoms, such as mosaic virus, leaf deformity, blistering, and / or ochre, can be assessed, particularly on uninoculated parts of the plant (e.g., upper leaves). Symptom assessment can be performed visually. Optionally, the method further includes step f): determining (or assessing) the presence of virus particles in uninoculated plant parts (e.g., upper true leaves). This can be performed on one or more inoculated plants or all inoculated plants. In one aspect, it can be performed on one or more plants that do not exhibit systemic symptoms.

[0073] On the one hand, step e) above is missing, i.e., symptoms are not assessed, but the presence of virus particles in one or more uninoculated plant parts is assessed on one or more inoculated plants.

[0074] Following step e) or f), resistant or tolerant plants can be identified (selected), or all susceptible plants can be discarded. This identification can be performed in several ways:

[0075] In one aspect, the method also includes identifying plants (or plants in total) that do not exhibit systemic symptoms and in which virus particles are not present in uninoculated plant parts. Such plants can be considered to possess resistance to the virus.

[0076] In another aspect, the method further includes identifying plants (or plants) that have localized damage at the inoculated plant sites and / or where the virus particles are not present at the uninoculated plant sites. Such plants may be considered to possess resistance to the virus.

[0077] On the other hand, the method also includes identifying plants that do not exhibit systemic symptoms and in which viral particles are present in uninoculated plant parts. Such plants can be considered resistant to the virus, meaning that although the virus can spread systemically, it does not cause systemic symptoms.

[0078] On the other hand, the method also includes identifying plants that exhibit significantly reduced systemic symptoms compared to susceptible control plants and in which virus particles are present in uninoculated plant parts. Such plants can be considered to possess partial resistance to the virus. Significantly reduced systemic symptoms can be, for example, a statistically significantly lower percentage of genotype plants exhibiting one or more systemic symptoms than the percentage of susceptible control plants exhibiting one or more systemic symptoms. Thus, for example, if 100% of susceptible control genotype plants exhibit one or more systemic symptoms, significantly fewer plants in partially resistant genotypes exhibit one or more systemic symptoms, for example, less than 90%, 80%, 70%, 60%, 50%, or even less than 40% of inoculated plants exhibiting one or more systemic symptoms.

[0079] The presence of virus particles in uninoculated plant parts can be determined in various ways and optionally quantified. Thus, for example, the ELISA assay described in the examples can be performed on uninoculated parts (e.g., upper leaves of the plant) to determine the presence of the virus and whether it has thus spread systemically. Different methods can be used to detect the virus, such as microscopy (e.g., electron microscopy), antibody-based assays (e.g., ELISA or lateral flow device assays), or assays to detect viral RNA (or cDNA) such as polymerase chain reaction (PCR) methods, nucleic acid sequencing, nucleic acid hybridization, or bioassays (e.g., inoculation of indicator plants such as the Xanthi variety of tobacco (containing a homozygous form of the N gene) and Nicotiana spicata).

[0080] Optionally, plants that do not contain the virus in uninoculated plant parts may be selected and / or plants that contain the virus in uninoculated plant parts may be discarded. Therefore, in one aspect, the method includes determining the presence of virus particles in uninoculated plant parts.

[0081] The identified plants (or plant genotypes) can also be retested using the methods described above. This involves re-inoculating one or more identified plants with the virus and reculturing them to confirm their resistance or partial resistance to the ToMSV strain in a second or third assay. Different ToMSV strains can also be tested. Since only a few plants of one genotype (e.g., 1, 2, 3, or 4 plants) are tested in the initial screening, and only one or a few plants of one genotype can be identified and selected in the initial screening, it is preferable to retest a larger number of plants of the identified genotype in the second or third screening, such as at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 25, 26, 27, 28, 29, 30, or more plants.

[0082] When a larger number of plants of a given genotype are tested in any of the above methods, preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or most preferably 100% of the same genotype are identified as having complete resistance. Similarly, in one aspect, preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or most preferably 100% of the same genotype are identified as having tolerance.

[0083] Therefore, the methods described above can be used to identify plants, particularly wild relatives of tomatoes or wild germplasm of the genus *Capsicum*, or eggplant or melon plants, that possess resistance to one or more strains of ToMSV (e.g., to VE484). This article includes plants obtained using the methods described above. The resistance genes present in these plants can then be used to produce cultivated tomato plants, cultivated pepper plants, or cultivated eggplant or melon plants that possess resistance to one or more strains of ToMSV (e.g., at least to strain VE484). These plants can be produced using conventional breeding techniques.

[0084] In other respects, the present invention provides a method for assessing the presence of the virus of the present invention in a plant or plant part, the method comprising the following steps:

[0085] a) Determining the presence of nucleic acid molecules in a plant or plant part, said nucleic acid molecules containing at least 83% sequence identity with SEQ ID NO:1; and / or

[0086] b) Determining the presence of a protein or a nucleic acid molecule encoding the protein, said protein containing at least 93% sequence identity with SEQ ID NO:3; and / or

[0087] c) Determining the presence of a protein or a nucleic acid molecule encoding the protein, said protein containing at least 94% sequence identity with SEQ ID NO:4; and / or

[0088] d) Determining the presence of a protein or a nucleic acid molecule encoding the protein, said protein containing at least 80% sequence identity with SEQ ID NO:5; and / or

[0089] e) Determine the presence of a protein or a nucleic acid molecule encoding the protein, said protein containing at least 82% sequence identity with SEQ ID NO:6.

[0090] Since viruses are RNA viruses, and nucleic acid molecules are RNA molecules, RNA molecules are typically detected by first reverse transcribing the RNA into DNA (complementary DNA or cDNA), and then detecting the cDNA. RNA and cDNA have the same nucleotide base sequence, except that uracil (U) in RNA is replaced by thymine (T). Therefore, detecting cDNA is equivalent to detecting RNA molecules.

[0091] Technicians can easily determine the presence of nucleic acid molecules in any of steps a), b), c), d), or e) using known methods. For example, technicians can design primer pairs for RT-PCR and perform RT-PCR assays to amplify nucleic acid molecules or portions thereof. Nucleic acid hybridization methods (using, for example, stringent conditions) can also be used.

[0092] Regarding step a), primers can be designed to amplify any portion of SEQ ID NO:1 (the nucleic acid sequence of the VE484 genome), or any portion of a nucleic acid molecule containing at least 83% sequence identity with SEQ ID NO:1, such as any portion of a nucleic acid molecule containing at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97%, 98%, or 99% sequence identity with SEQ ID NO:1. Optionally, primer pairs of SEQ ID NO:7 and 8 or SEQ ID NO:9 and 10 can be used.

[0093] To detect sequence identity with at least 93% (e.g., at least 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:3; and / or sequence identity with at least 94% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:4; and / or sequence identity with at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:5; and / or sequence identity with at least 93% (e.g., at least 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:3 ... NO:6 is a nucleic acid coding sequence with at least 82% (e.g., at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity. Various methods can be used, such as designing primer pairs (or degenerate primer pairs) that amplify this type of nucleotide sequence and detect the presence of RNA molecules with that sequence in a sample. Similarly, RT-PCR can be used, for example. Nucleic acid hybridization methods (e.g., using stringent conditions) can also be used.

[0094] The nucleotide sequences encoding the proteins of SEQ ID NO:3, 4, 5, and 6 are shown in SEQ ID NO:1 and 2. The protein of SEQ ID NO:3 is encoded by nucleotides 77 to 3424 of SEQ ID NO:1. The protein of SEQ ID NO:4 is encoded by nucleotides 77 to 3424 and 3446 to 4921 of SEQ ID NO:1. The protein of SEQ ID NO:5 is encoded by nucleotides 4911 to 5708 of SEQ ID NO:1, and the protein of SEQ ID NO:6 is encoded by nucleotides 5671 to 6198 of SEQ ID NO:1.

[0095] For determining sequence identity with at least 93% (e.g., at least 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:3; and / or sequence identity with at least 94% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:4; and / or sequence identity with at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO:5; and / or sequence identity with at least SEQ ID NO:3; The presence of a protein with at least 82% (e.g., at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to NO:6 can be achieved using a variety of methods. In one aspect, antibody-based methods, such as ELISA or LFD, are preferred. Any capture antibody capable of binding to one of the aforementioned proteins (to form an antibody-antigen complex) can be used. The protein or protein moiety can be used as an antigen, i.e., to increase and generate antibodies that bind to the protein. Examples also show that TMV antibodies (catalog numbers CAB57400, ECA 57400, PSA 57400, and SRA57400) sold by Agdia can be used to detect strain VE484.

[0096] On the other hand, a separate nucleic acid molecule or a portion thereof is provided, comprising at least 83% (e.g., at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:1. The nucleic acid molecule may be an RNA or DNA molecule. The portion thereof may be a fragment, such as any molecule comprising at least 15, 20, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 6000 consecutive nucleotides of the nucleic acid molecule.

[0097] In another aspect, a separate nucleic acid molecule is provided that contains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with nucleotides 77 to 3424 of SEQ ID NO:1.

[0098] In another aspect, a separate nucleic acid molecule is provided that contains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with nucleotides 3446 to 4921 of SEQ ID NO:1.

[0099] In another aspect, a separate nucleic acid molecule is provided that contains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with nucleotides 4911 to 5708 of SEQ ID NO:1.

[0100] In another aspect, a separate nucleic acid molecule is provided that contains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with nucleotides 5671 to 6198 of SEQ ID NO:1.

[0101] Also provided are proteins selected from the following: proteins containing at least 93% (e.g., at least 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:3; proteins containing at least 94% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:4; proteins containing at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:5; and proteins containing the sequence identity with SEQ ID NO:3. The IDNO:6 protein has at least 82% (e.g., at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity.

[0102] Another aspect of the present invention is an antibody generated against a protein or protein motif selected from: proteins comprising at least 93% (e.g., at least 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identity with SEQ ID NO:3; proteins comprising at least 94% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identity with SEQ ID NO:4; proteins comprising at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identity with SEQ ID NO:5; and proteins comprising a sequence identity with SEQ ID NO:3. Proteins with at least 82% (e.g., at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to NO:6. These antibodies can be used to detect ToMSV strains in plant tissues or plant tissue extracts. Kits containing these antibodies are another aspect of the invention. Such kits can be, for example, ELISA kits or LFD kits.

[0103] This article also provides the use of the virus of the present invention for identifying resistant or partially resistant plants of the genera Solanaceae or Capsicum.

[0104] Similarly, the use of the nucleic acid molecule (or sequence), protein molecule (or sequence), or a portion thereof for detecting the virus of the present invention in a plant, plant part, or sample is provided.

[0105] Preservation Information

[0106] A representative sample of ToMSV strain Ve484 was deposited by Nunhems BV on January 19, 2015, in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany), accession number DSM 29970.

[0107] The applicant requests that, in accordance with the relevant regulations of Rule 32(1)EPC or a country or treaty with similar provisions and regulations, samples of the biological material and any material derived therefrom shall only be provided to designated professionals until 20 years from the date of patent grant announcement or filing (if the application is rejected, withdrawn or deemed withdrawn).

[0108] During the pending period of this application, the deposit may be requested and obtained by a qualified person as determined by the Director of the U.S. Patent and Trademark Office. Subject to 37 C.FR § 1.808(b), all restrictions imposed by the depositor regarding the public availability of the deposited material will be permanently removed upon patent grant. The deposit will be maintained for 30 years, or for 5 years after the most recent request, or for the effective life of the patent, whichever is longer, and will be replaced if the deposit becomes unviable during this period. The applicant does not waive any rights granted by this patent application or the Plant Variety Protection Act (7 USC 2321 et seq.).

[0109] The following non-limiting embodiments illustrate the production of the chili pepper plant, seeds, and fruits of the present invention. All references mentioned herein are incorporated by way of citation.

[0110] Example

[0111] Example 1 – Identification of ToMSV

[0112] Tomato plants exhibiting mosaic virus symptoms were identified from leaf samples taken from tomato production fields. These tomato plants carried the homozygous form of Tm2. 2 Resistance genes.

[0113] 1.1 Lateral Flow Device Testing

[0114] Collect leaves showing symptoms and use the testing kit provided by Agdia for TMV (Tobacco Mosaic Virus). The test (ISK 57400 / 0025) is performed using a transverse flow apparatus according to the manufacturer's instructions. In short, symptomatic leaf tissue is placed in an extraction bag (between the mesh liner) containing SEB1 buffer. Virus is extracted by rubbing the bag between the mesh liner. An immune strip is then placed into the so-called "channel" section of the bag for 30 minutes. If the test is performed adequately, the control line will become visible. If the virus is present, a pink / purple test line will also appear on the strip. The antibodies used in the test detect multiple viruses (but not all) of the tobacco mosaic virus group.

[0115] The test was positive, indicating that the mosaic symptoms were caused by members of the tobacco mosaic virus group.

[0116] 1.2 Virus detection in tomato seeds

[0117] Seeds were collected from infected plants found in the production fields.

[0118] Two assays were performed using seeds: an ELISA assay (Agdia) and a subsequent assay of localized damage on the tobacco plant, as described in the ISF protocol “Method for the detection of infectious tobamoviruses on tomato seed” (see worldseed.org / cms / medias / file / TradeIssues / PhytosanitaryMatters / SeedHealthTesting / ISHI-Veg / Tomato_Tobamo_Sept_2013.pdf).

[0119] 1.2.1 Sample Preparation

[0120] Twelve inoculum samples were prepared from 250 tomato seeds by grinding the seed samples in extraction buffer. Samples were also prepared from seeds of positive and negative controls. The inoculum samples were first used for ELISA and then for local lesion assay.

[0121] 1.2.1 ELISA

[0122] The ELISA assay was performed using the Agdia Tobacco Mosaic Virus ELISA Complete Kit (PSA57400 / 0288) according to the manufacturer's instructions. In short, the assay uses an antibody-coated 96-well microtiter plate that detects multiple (but not all) viruses in the Tobacco Mosaic Virus group. Tissue samples (e.g., leaf tissue, seeds, etc.) were ground and diluted in extraction buffer. The samples were then added to the microtiter wells along with the provided controls (positive control, negative control, and buffer only) and incubated. After incubation, the plate was washed and freshly prepared alkaline phosphatase conjugate was dispensed into the wells, followed by another incubation. The plate was washed again and PNP substrate was added to the wells, followed by another incubation. Results were examined visually and / or using a microplate reader at 405 nm. Visually, colored wells indicated a virus-positive result; and / or a 405 nm reading 2.5 times higher than the background (negative control) indicated a virus-positive result. The test results are valid only if the positive control is colored, the negative control is almost clear, and only the well containing the buffer solution is colorless.

[0123] 1.2.2 Local Damage Measurement

[0124] The local damage assay is a bioassay that uses mechanical inoculation of leaves from both the Xanthi variety of tobacco and Nicotiana spicata, both carrying the N gene for resistance to TMV, to detect infectious virus particles. 3000 tomato seeds were used in this assay.

[0125] Tobacco plants were allowed to grow to the 4-5 true leaf stage. Two leaves from each plant were sprinkled with silicon carbide powder and inoculated using one of 12 inoculum samples or a control inoculum sample. Inoculation was performed by rubbing the inoculum onto a sponge and then spreading it over the entire leaf surface. After inoculation, the plants were cultured at 20-25°C for 5-7 days (with at least 12 hours of light), and the number of necrotic lesions on the inoculated leaves was recorded.

[0126] This type of localized necrotic damage (hypersensitivity reaction) indicates that one or more seeds in the 250 seed samples contained infectious viral particles.

[0127] result

[0128] The tomato mosaic virus strain was detected in the seeds of infected tomato plants in both ELISA and local lesion assays. Therefore, the strain was seed-borne.

[0129] 1.3 Sequencing and Sequence Analysis

[0130] Tobacco leaves inoculated with seed extracts were used to prepare inoculum, which was then used for mechanical inoculation of *Nicotiana benthamiana* leaves. The leaves were mechanically inoculated (using silicon carbide as described above), and the presence of virus particles in the inoculated leaves was determined by electron microscopy. RNA was isolated and sequenced using the following primer combination for RT-PCR (reverse transcriptase PCR):

[0131] Tob-Uni 15'-ATTTAAgTggASggAAAAVCACT-3'(SEQ ID NO:7)

[0132] Tob-Uni 25'-GTYGTTGATGAGTTCRTGGA-3'(SEQ ID NO:8)

[0133] or

[0134] Tomato F 5'-GWCGCSGAKTCKGATTCGTWTTAAATATG-3'(SEQ ID NO:9)

[0135] Tomato R 5'-TGGGCCSCTACCSGSGG-3'(SEQ ID NO:10)

[0136] Sequencing was performed on several different samples and the sequences were compared.

[0137] The viral genome sequence is provided in SEQ ID NO:1 and SEQ ID NO:2 and in Figure 1.

[0138] ORF1 and ORF3 are shown in SEQ ID NO:1, while ORF2 (only the last 492 amino acids are shown) and ORF4 are shown in SEQ ID NO:2.

[0139] Using default parameters, BLAST (Basic Local Alignment Search Tool) was performed on the NCBI website, and pairwise alignment was performed using the Needle program, which had the best BLAST hit rate (using EMBOSS-needles, default parameters).

[0140] BLAST results and pairwise comparisons of the genome sequence of SEQ ID NO:1 showed that the most similar virus in the database shared only 82% sequence identity with the virus of this invention, indicating that the virus of this invention is a novel species of tobacco mosaic virus. The most similar virus is Genbank accession number FR878069.1 (Tobacco mosaic virus strain Ohio V, complete genome, genomic RNA). See ncbi.nlm.nih.gov / nuccore / FR878069.1. This virus also differs from the recently sequenced ToMMV (Tomato Mottle Mosaic Virus) (Genbank accession number KF477193) found in Mexico, the United States, and China, sharing only 80.8% sequence identity.

[0141] Four ORFs are present in the genome. ORF1 encodes protein p126, which is provided in SEQ ID NO:3. ORF2 encodes protein p183, which is provided in SEQ ID NO:4. The protein of SEQ ID NO:4 is produced by repression of the stop codon at the end of ORF1. ORF3 encodes a mobile protein, which is provided in SEQ ID NO:5. ORF4 encodes a shell protein, which is provided in SEQ ID NO:6.

[0142] The novel species of tobacco mosaic virus mentioned herein is referred to as Tomato Mosaic Heavy Virus (ToMSV or TMSV), and the sequenced and preserved infectious strain is referred to as VE484.

[0143] Example 2 – Bioassay of a novel ToMSV virus on tomatoes and peppers (results not shown)

[0144] Different genotypes of tomato varieties containing different TMV / ToMV resistance genes were mechanically inoculated using the preserved virus strain VE484. The genotypes inoculated for bioassay were:

[0145] Tomato varieties genotype Mobaci Tm1 / Tm1 (Tm1 homozygous type) Moperou Tm2 / Tm2 (Tm2 homozygous type) Momor <![CDATA[Tm2 2 / Tm2 2 (Tm2 2 Homozygous type) Mocimor <![CDATA[Tm2 2 Tm1 / Tm2 2 Tm1 (Tm1 and Tm2) 2 Homozygous type) Philippos <![CDATA[Tm2 2 / Tm2 2 (Tm2 2 Homozygous type)

[0146] All plants were inoculated during a 15-day period, and a visual assessment was performed 15 days post-inoculation when systemic symptoms were recorded. Twelve plants were inoculated for each genotype.

[0147] These different genotypes exhibit a range of systemic symptoms: leaf mosaic, leaf deformity, blistering, and reddish-brown discoloration. The LFD test is used to confirm the presence of the virus in symptomatic leaves.

[0148] All plants exhibited symptoms and were therefore susceptible to the virus. Therefore, in Tm1 and / or Tm2 or Tm2 2 In plants with homozygous resistance genes, viruses can cause systemic symptoms.

[0149]

[0150] The results were further confirmed in ELISA tests, including healthy, uninfected tomato varieties as negative controls, and the susceptible variety Monalbo (lacking the Tm resistance gene) as a positive control (data not shown). The ELISA results also confirmed that the new ToMSV virus strain VE484 is capable of overcoming all known tobacco mosaic virus resistance genes Tm1 (also known as Tm-1), Tm2 (also known as Tm-2), and Tm2... 2 (Also known as Tm-22).

Claims

1. A tobacco mosaic virus having the genome sequence SEQ ID NO: 1, and wherein said virus causes tomato ( Solanum lycopersicum Systemic symptoms of the plant, wherein the tomato plant is selected from Tm1, Tm2 and Tm2. 2 One or more Tm resistance genes are homozygous.

2. The virus of claim 1, a representative sample of which has been deposited under accession number DSM 29970.

3. A container or sterile solution containing the virus of claim 1 or 2, or a non-renewable cut plant part.

4. A method for identifying Solanaceae or Capsicum genus plants containing resistance to the virus of claim 1 or 2.

5. The method of claim 4, wherein the method comprises the following steps: a) Provide one or more plants; b) Provide an inoculation containing the virus of claim 1 or 2; c) Use the inoculum of b) to inoculate one or more plant parts of the plant of a); d) Cultivating inoculated plants.

6. The method of claim 5, further comprising: e) Determine the presence of virus particles in uninoculated plant parts.

7. The method of claim 5, further comprising: e) Assess symptoms on the plant or plant parts, especially systemic symptoms and / or local lesions on the inoculated plant parts.

8. The method of claim 7, further comprising: f) Determine the presence of virus particles in uninoculated plant parts.

9. The method of claim 7 or 8, further comprising identifying plants that do not have systemic symptoms and wherein viral particles are not present in uninoculated plant parts.

10. The method of claim 7 or 8, further comprising identifying plants having localized damage in the inoculated plant parts and / or wherein virus particles are not present in the uninoculated plant parts.

11. The method of claim 7 or 8, further comprising identifying plants that do not have systemic symptoms and wherein viral particles are present in uninoculated plant parts.

12. The method of claim 5, wherein the plant part of c) is a leaf, cotyledon, hypocotyl, stem, petiole, or root.

13. The method of claim 5, wherein the plant in step a) is selected from the following species: tomato ( Solanum lycopersicum ),chili( Capsicum annuum ),eggplant( Solanum melongena ), cantaloupe eggplant ( Solanum muricatum ) 、 Wild tomatoes ( Solanum arcanum Kmerliuschi tomatoes Solanum chmielewskii ), small flower tomatoes ( Solanum neorickii Chesmanni tomatoes ( Solanum cheesmaniae ), multi-glandular tomato ( S. galapagense ), Narrow-leaf tomato ( Solanum pimpinellifolium Chilean tomatoes Solanum chilense ), S. corneliomulleri Hairy tomatoes ( Solanum habrochaites ), S. huaylasense Garlic mustard eggplant ( Solanum sisymbriifolium Peruvian tomatoes Solanum peruvianum Hairy tomatoes S. hirsutum Panali tomatoes Solanum pennellii ), cherry tomatoes Solanum lycopersicoides ), Riquito ( Solanum sitiens ), Ochre-yellow eggplant ( Solanum ochranthum Yellow lantern pepper () Capsicum chinense ), millet pepper ( Capsicum frutescens ), bell pepper ( Capsicum baccatum )and Capsicum pubescens .

14. A method for assessing the presence of the virus of claim 1 or 2 in a plant or plant part, the method comprising the following steps: a) Determining the presence of a nucleic acid molecule with the sequence SEQ ID NO: 1 in a plant or plant part; and / or b) Determine the presence of a protein or nucleic acid molecule encoding a protein with the sequence SEQ ID NO: 3-6 in a plant or plant part.

15. An isolated nucleic acid with the sequence SEQ ID NO:

1.

16. A protein assembly comprising proteins having sequences of SEQ ID NO: 3-6.

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