Tomato plants resistant to Tomato Brown Lugos Fruit Virus

Abstract

The present invention relates to tobamovirus-resistant S. lycopersicum plants comprising one or more genomic sequences. More particularly, the present invention relates to tomato plants (S. lycopersicum) that are resistant to Tomato Brown Rugos Fruit Virus (TBRFV). The present invention further relates to genomic sequences or loci that provide resistance to tobamoviruses. Additionally, the present invention relates to methods for providing S. lycopersicum plants that are resistant to tobamoviruses.

Description

Technical Field

[0001] The present invention relates to a plant of the species *Solanum lycopersicum* that is resistant to tobamoviruses and comprises one or more genomic sequences. More specifically, the present invention relates to tomato plants (*S. lycopersicum*) that are resistant to Tomato Brown Rugose Fruit Virus (TBRFV). The present invention further relates to genomic sequences or loci that provide resistance to tobamoviruses. Furthermore, the present invention relates to methods for producing *S. lycopersicum* plants that are resistant to tobamoviruses.

Background Art

[0002] Tobamovirus is a genus of the Virgaviridae family, which consists of viral species that infect plants including Solanaceous plants such as tobacco, potato, tomato and eggplant, and is one of the most serious threats to vegetable crops worldwide. Tobamoviruses are particularly problematic in tomato crops grown in protected environments; they are transmitted mechanically over long distances by external seed contamination, and from plant to plant through common cultivation practices via workers' hands, clothing, and tools, and have the ability to maintain infectivity in seeds and contaminated soil. Furthermore, common weeds that are often asymptomatic when infected with the virus constitute hidden reservoirs during the growing cycle.

[0003] Tobamovirus infection can have devastating effects on crops when contamination occurs. For example, prevention of infection by raising seedlings in virus-free environments is generally costly and / or environmentally unfavorable. In addition, these methods do not always provide satisfactory results.

[0004] Tobamoviruses are non-enveloped, have a helical rod-shaped geometry, and exhibit helical symmetry. Virus particles are rod-shaped, approximately 18 nm in diameter and 300–310 nm in length. Their positive sense single-stranded RNA genome is linear, unsegmented, and approximately 6.3–6.5 kb in length. This genus includes over 35 virus species, including tomato mosaic virus (ToMV) or tobacco mosaic virus (TMV), tomato mild mottle virus (ToMMV), and the recently discovered tomato brown lucos fruit virus (TBRFV).

[0005] In tomatoes, the four currently recognized strains of tobamovirus (more specifically ToMV) are designated as Tm-0, Tm-1, Tm-2, and Tm-2 2 ) are the resistance (R) genes Tm1, Tm2, ​​and Tm2, ​​which were introduced from the relevant wild species. 2 Based on the following: The Tm1 gene is introduced from Solanum habrochaites and is incompletely dominant. Tm2 and Tm2 2 The gene was introduced from Solanum peruvianum to confer dominant complete resistance to ToMV. However, as resistance has been overcome, new strains of tobamovirus have emerged, and resistance-breaking tobamovirus species have recently been reported in commercial fields in Mexico, Jordan, and Israel.

[0006] From late 2014 to early 2015, a new disease outbreak affecting tomatoes occurred in Israel and Jordan. Affected plants exhibited a mosaic pattern on their leaves, sometimes accompanied by narrowed leaves and yellow-spotted fruits. Studies revealed this new disease to be a novel tobamovirus, named TBRFV. TBRFV infection results in necrotic lesions on the leaves; tomato plants show mild leaf symptoms at the end of the season, but exhibit severe brown wilting on the fruit, rendering it unsuitable for consumption. Furthermore, TBRFV appears to have the ability to infect other members of the Solanaceae family, such as pepper plants, if planted in contaminated soil from the previous growth cycle of infected tomato plants at temperatures above 30°C.

[0007] In the fight against tobamovirus, R gene Tm2 and Tm2 2 Resistance was introduced into tomatoes through gene transfer, resulting in resistance to ToMV. However, different domains of the viral protein are composed of different protein structures, and different resistance mechanisms require new resistance mechanisms and / or resistance genes; therefore, these R genes do not exhibit resistance to new TBRFVs. Furthermore, viruses adapt and evolve, resulting in viral breakthroughs, so it is highly likely that resistance will be broken over time. Therefore, it is necessary to identify and / or combine new resistance genes to provide crops that are resistant to new TBRFVs in particular. [Overview of the project] [Problems that the invention aims to solve]

[0008] Considering the above, those skilled in the art have a need for TBR Fever-resistant tomato plants, and more specifically, TBR Fever-resistant S. lycopersicum. In addition, those skilled in the art have a need to provide methods and means for obtaining TBR Fever-resistant S. lycopersicum plants. [Means for solving the problem]

[0009] To solve the above-mentioned needs of those skilled in the art is, among other objectives, an objective of the present invention. Among other objectives, the objectives of the present invention are satisfied by the present invention as outlined in the appended claims.

[0010] In particular, among other objectives, the above objective is satisfied by the present invention with a plant of the S. lycopersicum species resistant to tobamovirus, comprising a TBR RIV resistance gene encoding a TBR RIV resistance protein, the protein having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, and most preferably 100% amino acid sequence identity with SEQ ID NO: 116. The TBR RIV resistance gene is predicted to encode an NBS-LRR resistance protein.

[0011] According to a preferred embodiment, the present invention relates to a plant in which the TBRFV resistance gene comprises a coding sequence having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, and most preferably 100% nucleotide sequence identity with SEQ ID NO: 115.

[0012] According to another preferred embodiment, the present invention relates to a plant comprising one or more genomic sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, or having at least 95% sequence identity with any of the aforementioned SEQ ID NOs. The genomic sequences encode one or more genes or genetic elements that provide resistance to tobamovirus. The sequences were examined for genetic homology using the publicly available database of the National Center for Biotechnology Information (NCBI). Six genomic sequences are homologous to sequences encoding NBS-LRR resistance proteins (SEQ ID NOs: 7, 8, 9, 10, 11, and 14). Four genomic sequences are homologous to LRR receptor-like serine / threonine protein kinases (SEQ ID NOs: 5, 6, 12, and 13). [Brief explanation of the drawing]

[0013] [Figure 1]This figure outlines the mapping of loci that provide resistance to tobamovirus, more specifically to S. lycopersicum, against TRBFV. To create two populations for mapping, F1 plants were generated by crossing S. habrokaites strain 90479-3, selected for its resistant phenotype, with S. lycopersicum strains OT9 and OT1317. Over 700 plants were tested for TRBFV resistance, and several recombinant plants (21 plants) were selected, with disease test results combined with marker data. Several molecular markers (M1-M42) were used to determine the location and size of genomic sequences that provide resistance to TRBFV. Clear segregation was observed between resistant (R) and infective (S) plants. The results indicate that a genomic region located between markers M16 and M17 provides TRBFV resistance; the corresponding locus (named the LYC4943 R locus) is 133,515 bp long and contains several putative genes. Based on further detailed mapping, the size and location of the genomic sequence possessing TRBFV resistance were determined to be between markers M33 and M38, approximately 68,000 bp larger than the S. lycopersicum SL2.40 reference genome (85,240 bp vs. 17,328 bp, respectively). Therefore, it is most likely that one or more genes are located within this region, referred to as the "TBRFV region," and provide TRBFV resistance. [Figure 2]This figure shows the results of qPCR for detecting TBRFV in infected and uninfected tomato plants (S. lycopersicum) and plants that do not contain the TBRFV resistance locus, according to the present invention. Low Ct values ​​(i.e., 30 or less) indicate the presence of a large amount of viral RNA in the sample. OT9 is a tomato plant that does not contain the TBRFV resistance locus. The control sample (uninfected OT9) showed a Ct value of 35-40 cycles, while the infected control sample (infected OT9) showed a Ct value of 20-25. Plants showing a Ct value of 30 cycles or more, preferably around 35 cycles, were considered resistant, and plants showing a Ct value of less than 30 were considered infectious. Tomato plants containing the TBRFV resistance locus, homozygous (B) or heterozygous (H), have a Ct value of 30 cycles or more and are considered resistant. Furthermore, the results indicate that resistance is dominant. Plants that did not contain the TBRFV resistance locus (A and infected OT9) showed Ct values ​​of 20-25, indicating that the plants were susceptible to TBRFV infection. [Figure 3] This figure shows a schematic overview of the genome sequences SEQ ID NOs: 1 to SEQ ID NOs: 18 of the present invention, which encode one or more genes or genetic elements that provide resistance to tobamovirus. [Figure 4]Figure 1, in addition to Figure 1, shows an overview of further mapping of the loci that provide resistance to tobamovirus. Further recombination selection was performed by further genotyping of plants at M33 and M38 to identify recombinant plants in the identified TBRFV region and further restrict this TBRFV region. Recombinant plants were further genotyped using markers (M-SEQ 10, M-SEQ 11-1, M-SEQ 11-2, and M-SEQ 14) specifically designed to cover the TBRFV locus and remove candidate regions within the TBRFV locus, i.e., one or more genes or genetic elements that provide resistance to tobamovirus. Recombinant plants 15321-02, 15321-03, and 15321-07 were screened for resistance upon inoculation with TBRFV isolate AE50. Based on these recombinant plants, combined with phenotyping, ELISA, and qPCR data to determine TBRFV infection, it was concluded that the resistance-constituting gene is the portion of genome sequence number 14. Plants 15321-02 and 15321-03, which did not contain SEQ ID NO: 14, showed infectivity to TBRFV with high ELISA scores and low qPCR Ct values, corresponding to those obtained in the infectious control strain OT9, indicating viral infection. Plant 15321-07, which contained SEQ ID NO: 14, showed resistance to TBRFV with low ELISA scores and higher qPCR Ct values. [Figure 5]This figure shows ELISA-based TBRFV infection in a homozygous TBRFV-resistant strain (15322-04) and an infectious control strain (OT9). Plants were infected with TBRFV (+TRBFV) and infiltrated with either VIGS-01a, a construct specifically targeting the TRBFV resistance gene, or VIGS-01b, a construct targeting different regions within the identified TRBFV region. ELISA readings were performed by measuring absorption at 405 nm. The TBRFV-infected control plant OT9 yielded absorption levels of 2000 abs or higher, while the TRBFV-infected resistant plant strain yielded absorption levels of approximately 500 abs. When the TRBFV resistance gene was silenced by VIGS-01a in the resistant plant strain, absorption levels of 1500–2250 abs were measured, indicating viral infection. Silencing with VIGS-01b in resistant plant strains resulted in absorption levels similar to those observed in infected resistant plant strains that were not silenced by VIGS. [Figure 6] This figure shows qPCR-mediated TBRFV infection in homozygous TBRFV-resistant strains (15322-04) and infectious control strains (OT9). Plants were infected with TBRFV (+TRBFV) and infiltrated with either VIGS-01a, a construct specifically targeting the TRBFV resistance gene, or VIGS-01b, a construct targeting different regions within the identified TRBFV region. Infected control samples showed Ct values ​​of approximately 12 or 13. Resistant plant strains infected with TRBFV showed a Ct value of approximately 30, indicating TBRFV resistance. When the TRBFV resistance gene was silenced by VIGS-01a in resistant plant strains, the Ct value was observed to decrease to approximately 12-20, higher than that of infected control cells, clearly indicating viral infection. Silencing of resistant plant strains with VIGS-01b resulted in Ct levels (Ct value approximately 30) similar to those observed in infected resistant plant strains that were not silenced by VIGS. [Modes for carrying out the invention]

[0014] Plant pathogen recognition occurs via two related groups of host receptors involving two major types of proteins: receptor-like kinases or proteins (RLKs or RLPs) and nucleotide-binding site leucine-rich repeat proteins (NBS-LRR-resistant proteins). The first group consists of pattern recognition receptors (PRRs) specializing in the recognition of pathogen-associated molecular patterns (PAMPs). RLPs or RLKs are cell membrane-bound and are extracellular immune receptors. Plant RLKs are involved in plant-pathogen interactions and defense responses, and plant receptor kinases (PRKs) can be defined as proteins containing an extracellular domain, a single-pass transmembrane domain, and a cytoplasmic serine / threonine (ser / thr) protein kinase domain. Plant LRR-RLKs (leucine-rich repeat-receptor-like kinases) possess a functional cytoplasmic kinase domain, and all plant LRR-RLKs analyzed to date possess ser / thr kinase activity. The resistance to pathogens obtained by these receptors is called PAMP-induced immunity (PTI). Other groups primarily consist of intracellular receptors called resistance proteins (R proteins). The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat proteins, also known as NBS-LRR proteins. These proteins are characterized by nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains, as well as variable amino and carboxyl-terminal domains, and are involved in the detection of a diverse range of pathogens, including bacteria, viruses, fungi, nematodes, insects, and oomycetes. The majority of identified genomic sequences providing tobamovirus resistance contain multiple LRR domains. These domains are thought to determine effector recognition and, therefore, disease infectivity / resistance.

[0015] Pathogens develop counter-strategies to overcome PTI by modifying or altering PAMP or MAMP. Plants then develop ways to recognize these effectors, inducing a faster, stronger secondary defense response known as effector-induced immunity (ETI). ETI is mediated by R proteins and involves localized cell death around the site of infection. The presence of these newly identified resistance genes and / or genomic regions encoding NBS-LRR proteins and / or plant receptor kinases reduces the opportunity for pathogens to overcome resistance, or, when combined with other resistance genes, disease resistance may be further improved.

[0016] According to a preferred embodiment, the present invention relates to a plant comprising a genomic sequence represented by Sequence ID No. 3. Genomic sequence Sequence ID No. 3 comprises a plurality of sequences homologous to sequences encoding an NBS-LRR resistance protein and an LRR receptor-like serine / threonine protein kinase.

[0017] According to yet another preferred embodiment, the present invention relates to the plant, including SEQ ID NOs: 8, 9, 10, and 11.

[0018] According to the present invention, plant tobamovirus resistance may be influenced by one or more genomic sequences encoding NBS-LRR proteins selected from the group of SEQ ID NOs: 8, 9, 10, 11, and 14, for example, SEQ ID NOs: 8 and SEQ ID NOs: 9, or combinations of SEQ ID NOs: 8 and SEQ ID NOs: 10, SEQ ID NOs: 8 and SEQ ID NOs: 11, SEQ ID NOs: 9 and SEQ ID NOs: 10, SEQ ID NOs: 10 and SEQ ID NOs: 11. Furthermore or alternatively, resistance may be influenced by one or more genomic sequences encoding LRR receptor-like serine / threonine protein kinases selected from the group of SEQ ID NOs: 12, SEQ ID NOs: 13, or SEQ ID NOs: 12 and SEQ ID NOs: 13.

[0019] According to yet another preferred embodiment, the present invention relates to said plant comprising the genomic sequences of SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 14.

[0020] According to a preferred embodiment, the present invention relates to said plant comprising SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0021] According to another preferred embodiment, the present invention relates to said plant which is resistant to tobamovirus strains Tm-0, Tm-1 and Tm-2. In tomato, four strains of tobamovirus have been identified: Tm-0, Tm-1, Tm-2 and Tm-2 2 .

[0022] According to yet another preferred embodiment, the present invention relates to said plant which is resistant to Tomato Brown Rugose Fruit Virus (TBRFV).

[0023] According to yet another preferred embodiment, the present invention relates to said plant, wherein said TBRFV is virus isolate AE050.

[0024] According to yet another preferred embodiment, the present invention relates to said plant, which is a tomato plant [Solanum lycopersicum].

[0025] According to yet another preferred embodiment, the present invention relates to said plant, wherein one or more genomic sequences and / or TBRFV resistance genes are present heterozygously or homozygously in the genome of said plant. From experimental data, it can be concluded that resistance is dominant, and the TBRFV resistance gene and / or genomic sequence must be present at least heterozygously in the genome of the plant to obtain resistance against tobamovirus.

[0026] In yet another preferred embodiment, the present invention relates to a plant in which the TBRFV resistance gene and / or one or more genomic sequences are found in deposit accession number NCIMB 43279. NCIMB 43279, Solanum lycopersicum seed, was deposited on November 16, 2018, at NCIMB, Ferguson Building, Craibstone Estate Bucksburn, AB21 9YA Aberdeen, United Kingdom.

[0027] According to a second aspect, the present invention relates to plants, plant parts, tissues, cells and / or seeds derived from the plant of the present invention.

[0028] The present invention, according to another aspect, relates to a resistance gene (TBRFV resistance gene) that provides resistance to tobamovirus in the plant S. lycopersicum, wherein the resistance gene is represented by a coding sequence having at least 90% nucleotide sequence identity with SEQ ID NO: 115.

[0029] The present invention, in another aspect, relates to a genome sequence that provides resistance to tobamovirus in the plant S. lycopersicum, and is selected from the group consisting of SEQ ID NOs. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, or has at least 95% sequence identity with any of the aforementioned SEQ ID NOs. Preferably, the genome sequence is SEQ ID NOs. 8, 11, or 14.

[0030] According to another aspect, the present invention relates to a resistance locus that provides resistance to tobamovirus in the plant S. lycopersicum, the locus being represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, preferably SEQ ID NO: 3.

[0031] According to a preferred embodiment of the present invention, a resistance gene, genome sequence, or resistance locus provides resistance to TBRFV.

[0032] According to another aspect, the present invention provides a method for providing a plant of the S. lycopersicum species that is resistant to tobamovirus, a) A step of selecting a S. habrokaites plant that is resistant to tobamovirus, which includes a step of confirming the presence of the resistance gene, genome sequence, or resistance locus of the present invention, The present invention relates to a method comprising the step of (b) transferring the identified genome sequence or locus from step (a) into a S. lycopersicum plant, thereby conferring tobamovirus resistance to the S. lycopersicum plant. The transfer may be carried out by crossing a selected S. habrokaites plant with S. lycopersicum. Subsequently, tobamovirus-resistant S. lycopersicum plants may be selected.

[0033] According to another preferred embodiment, the present invention relates to the method comprising, after step b), selecting a first S. lycopersicum plant that is resistant to tobamovirus, crossing it with a second S. lycopersicum plant that is not resistant to tobamovirus, and then selecting a S. lycopersicum plant that is resistant to tobamovirus.

[0034] According to a preferred embodiment, the present invention relates to the method wherein, in step a), the step of confirming the presence of a resistance gene (TBRFV resistance gene), a genomic sequence conferring resistance, or a resistance locus in a S. habrokaites plant is performed by one or more markers selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 94, 105, 106, 107, 108, 109, 110, 111, and 112, preferably 105, 106, 107, 108, 109, 110, 111, and 112.

[0035] The present invention, in a further aspect, relates to the use of a marker for confirming the presence of a TBR-R-V resistance gene, a genomic sequence conferring TBR-R-V resistance, or a resistance locus in the plant S. lycopersicum, wherein the marker is one or more selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 94, 105, 106, 107, 108, 109, 110, 111, and 112, preferably one or more selected from the group consisting of SEQ ID NOs: 105, 106, 107, 108, 109, 110, 111, and 112.

[0036] The present invention will be further described in the following examples and figures. [Examples]

[0037] Inoculation of tomato plants with TBRFV Disease assays were performed using TBRFV isolate AE050 (originating from Saudi Arabia). The virus was maintained using strain OT9, a plant strain infective to TBRFV, as plant material. Symptomatic leaves obtained from the original sample were used to mechanically inoculate strain OT9 with foliar sap. The virus was maintained on tomato plants OT9 that were systemically infected by monthly mechanical inoculation of foliar sap into new 3-week-old seedlings.

[0038] Tomato plants of the following species were screened (approximately 800 plants from a total of 912 wild Solanum strains): Solanum pennellii, S. peruvianum, S. chilense, S. habrokaites, S. pimpinellifolium, S. neorickii, S. corneriomulleri, S. chmielewskii, S. cheesemaniae, and S. galapagense. Twelve plants from each strain were infected with the TBRFV isolate AE050.

[0039] Seeds were sown in vermiculite, seedlings were transplanted to rockwool blocks, and inoculated four weeks after sowing. As starting material, infected OT9 symptomatic leaves were collected and ground in a mortar and pestle in cooled, desalinated water containing carborundum (1 gram / 100 mL). The oldest leaves of three-week-old seedlings of each test plant were mechanically inoculated with AE050 by gently rubbing the leaf once with one finger.

[0040] The phenotype of the plants was determined by visual inspection of the plants and leaves. Plants were scored for disease symptoms based on visual inspection at regular time intervals. Disease symptoms were visually assessed 2, 4, and 6 weeks after inoculation, and ELISA testing was performed on the remaining plants at one-month intervals starting from week 6. More than 50% of the plants already showed disease symptoms 2 weeks after inoculation.

[0041] Visual scoring was performed weekly. Plants were scored based on visually observed disease symptoms. The presence of yellow mosaic patterns on leaves and leaf deformation (narrowing, spotting) was recorded weekly at the plant level. The first symptoms were generally observed 12–14 days after inoculation. If no such symptoms were observed on the leaves, the plant was classified as resistant. Plants showing any of the symptoms on the leaves were classified as "infectious". Leaf samples were collected from symptomless plants (i.e., resistant) and tested for the presence of any of the viruses by ELISA.

[0042] Screening allowed for the selection of several candidates for resistance breeding, with the best candidate being LYC4943, a S. habrokaites strain originating from Peru. LYC4943 showed no symptoms for more than 15 weeks after inoculation and was tested for virus-free status by ELISA.

[0043] Determination of TBRFV infection by ELISA Infection was determined by ELISA. One apical leaf (fully extended) was collected from each plant. The leaves were crushed using a R302 D63N-472 machine (VECTOR aandrijftechniek BV, Rotterdam, Netherlands), and the leaf sap was collected by adding 2 mL of PBS-Tween buffer. 100 μL of the extract was used in ELISA with antibody against ToMV (supplier Prime Diagnostics, Wageningen, Netherlands). ELISA readings were performed by measuring the absorption at 405 nm using a FLUOstar Galaxy instrument. Plants showing absorption values ​​1.5 times or more than those of purebred control plants were considered infected (infectious).

[0044] Bioassay and mapping of TBRFV-resistant genome sequences The original LYC4943 (S. habrokaites) seed lot was isolated for resistance. Nine different F1 families were sown for bioassays to identify the F1 families that were fully resistant (resistance fixed) and would be used for further backcrosses. Four F1 families germinated and were tested in bioassays. F1 plants created from LYC4943 plant 3 (90479-3) were selected for their resistance phenotype, and S. lycopersicum strains OT9 and OT1317 were selected as backcrosses to create two populations for mapping. The markers M1-M42 (sequence codes 19-102, respectively) used for mapping are listed in Table 1.

[0045] 298 plants [(OT9×90479-3)×OT9] and 484 plants (OT1317×90479-3) = a total of 782 plants were inoculated with TBRVF isolate AE050. Two to three weeks after inoculation, TBRFV symptoms were present, and phenotypic determination was performed by eye. Clear segregation was observed between resistant (R) and infectious (S) plants, and the resistant phenotype may be linked to marker M1 (see Table 1), located on chromosome 8 at 2,673,609 bp in the reference genome SL2.40 (S. lycopersicum). 92 plants were genotyped using 26 markers and placed adjacent to QTLs (M2-M27, see Table 1). Based on these results, resistance can be mapped between 53,118,984 bp and 57,038,544 bp in the reference genome SL2.40 (between M8 and M20; see Table 1 and Figure 1). [Table 1] TIFF0007917980000002.tif221149 TIFF0007917980000003.tif183149

[0046] The entire population of 782 plants was genotyped using adjacent markers M8 and M20 to identify recombinant plants for further detailed mapping. This yielded 21 recombinant plants (see Figure 1). These 21 recombinant plants were selected and genotyped using 11 markers M9–M19 to further map the regions (Table 1). Resistance can be finely mapped on the reference genome SL2.40 between 56,920,720 and 56,990,004 (between markers M16 and M17).

[0047] By sequencing the resistant LYC4943 region using Oxford Nanopore sequencing technology, a 133,515 bp locus was obtained. Twenty-one recombinant plants were genotyped using additional markers (M28–M42) within this specific locus of LYC4943. Based on recombinant plants 594 and 608, the resistant region was determined to be located between positions 56,941,043–56,958,371 based on the reference genome SL2.40, corresponding to a position between positions 15,893–101,133 on the LYC4943 locus (between M33 and M38; see Figure 1).

[0048] Based on detailed mapping, the size and location of the genomic sequence possessing TBRFV resistance were determined to be between markers M33 and M38, approximately 68,000 bp larger than the S. lycopersicum SL2.40 reference genome (85,240 bp vs. 17,328 bp, respectively). Therefore, it is highly probable that one or more genes, indicated as Sequence ID No. 3 in this application, are located within this region, labeled “TBRFV region” in Figure 1, and provide TBRFV resistance. Based on the reference genome SL2.40 and in silico predictive analysis (ITAG 2.3), at least one gene encoding a CC-NBS-LRR resistance protein is located within the detailed-mapped region. By performing a blast search on the TBRFV region, which was precisely mapped to the database of the National Center for Biotechnology Information (NCBI), seven genome fragments were obtained. Five of these (SEQ ID NOs. 8, 9, 10, 11, and 14) showed homology to NBS-LRR resistance proteins, and two (SEQ ID NOs. 12 and 13) showed homology to LRR receptor-like serine / threonine protein kinases.

[0049] Next, further detailed mapping was performed, and recombinant selection was carried out by genotyping 668 BC2 plants [(OT9×90479-3)×OT9×OT9] using M33 and M38 to identify recombinant plants in the TBRFV region, thereby obtaining three plants 15321-02, 15321-03, and 15321-07 (see Figure 4). These three plants were tested for resistance by inoculation with TBRFV isolate AE50. Approximately three weeks after TBRFV inoculation, the plants were phenotyped by observation, and viral infection was monitored by ELISA and qPCR. The recombinant plants were genotyped using markers (M-SEQ 10, M-SEQ 11-1, M-SEQ 11-2, and M-SEQ 14, SEQ ID NOs. 105 to 112, respectively) specifically designed to cover the TBRFV locus and remove candidate genes within the TBRFV locus. This method provides insight into which of the candidate genome sequences of the present invention, SEQ ID NOs: 1 to 18, specifically provides resistance to TBRFV. Based on recombinant plants, disease testing, and phenotypic analysis by ELISA and qPCR, it was concluded that the resistance-constituting gene is encoded by the genome sequence of SEQ ID NO: 14, and more specifically, by the coding DNA sequence of SEQ ID NO: 115, which codes for the protein of SEQ ID NO: 16.

[0050] Verification of resistance of Tm0, Tm1, and Tm2 lines in plants containing the TBRFV resistance gene locus. The tomato plant (S. lycopersicum) of the present invention, containing the TBRFV resistance gene locus (SEQ ID NO: 1), was tested for resistance against Tm0, 1, and 2 lines. The presence of the TBRFV resistance gene locus was determined by markers M16, M17, and M33. 2 Further confirmation was made that the plants did not contain the genes (known genes that confer resistance to Tm0, 1, and 2 lines). In some cases, the plants contained the Tm1 resistance gene. As a control, plants that did not contain the TBRFV resistance locus were selected.

[0051] Eight plants (see Table 2, 1-8) containing the TRBV resistance locus (heterozygous) and two plants (7 and 8) not containing the TRBV resistance locus were inoculated with the Tm0 isolate. Eight plants (see Table 2, 9-16) containing the TRBV resistance locus (heterozygous) and two plants (15 and 16) not containing the TRBV resistance locus were inoculated with the Tm-1 isolate. Eight plants (see Table 2, 17-28) containing the TRBV resistance locus (two homozygous loci 17, 18, and two heterozygous loci 19, 20) and four plants not containing the TRBV resistance locus were inoculated with the Tm2 isolate. As a control, all three lines were also inoculated into the infectious cultivated tomato strain OT95.

[0052] The first symptom was generally observed 12–14 days after inoculation. Plants were classified as resistant (R) if no mosaic pattern of symptoms was observed on the leaves; plants showing any symptom on the leaves were classified as infective (S). The phenotype of each plant was compared to the TBRFV genotype. The results are summarized in Table 2 below. [Table 2]

[0053] Result Tm0 All plants containing the TBRFV resistance locus were resistant. Plants 7 and 8, which did not contain the TBRFV resistance locus, were resistant. A possible explanation for this result is that the Tm1 gene is responsible for resistance to the ToMV isolate Tm-0. In addition, plants 1, 2, and 3, which did not contain the Tm1 gene, contained the TBRFV resistance locus and showed resistance.

[0054] Result Tm1 The resistance phenotype is linked to the TBRFV genotype and results in resistance to the ToMV isolate Tm-1.

[0055] Result Tm2 The resistance phenotype (heterozygous, homozygous) is linked to the TBRFV genotype and results in resistance to the ToMV isolate Tm-2.

[0056] Determination of TBRFV infection in tomatoes (S. lycopersicum) by qPCR Tomato plants containing the TBRFV resistance locus (heterozygous or homozygous) and plants not containing this region were selected for TBRFV bioassays using markers (M16 and M17). The plants were infected with TBRFV, and infectious tomato strain OT9 (uninfected and infected OT9) were included as controls.

[0057] Three weeks after inoculation, one uppermost leaf from each plant was collected in a 2 mL tube containing a 6.35 mm metal sphere. The tube was frozen in liquid nitrogen. The tube was shaken vigorously to pulverize the plant material. After centrifugation, standard RNA extraction was performed using Machrey-Nagel® NucleoSpin® RNA Plant. RNA concentration was measured using DropSense 96 (Trinean) and diluted to a concentration of 100 ng / μL. 900 ng was used to synthesize cDNA using M-MLV reverse transcriptase (Invitrogen). 10 ng of cDNA was used for real-time PCR with LC Green as the intercalating dye. Two primer combinations were used to amplify the TBRFV line; see Table 3 (SEQ ID NOs. 103 and 104, respectively). [Table 3]

[0058] The more viral RNA present in the sample, the fewer PCR cycles are needed to amplify the cDNA (of the viral RNA) and pick up the signal, resulting in a lower Ct value in qPCR. Control samples (uninfected OT9) showed Ct values ​​of 35-40 cycles, while infected control samples (infected OT9) showed Ct values ​​of 20-25. Therefore, plants showing Ct values ​​of 30 or more cycles, preferably around 35 cycles, were considered resistant, while plants showing Ct values ​​below 30 were considered infectious (see Figure 2).

[0059] Tomato plants containing the TBRFV resistance locus, either homozygous (B) or heterozygous (H), all had a Ct value of 30 cycles or higher and could be considered resistant. The results indicate that resistance is dominant. Plants that did not contain the TBRFV resistance locus (A) showed a Ct value of 20-25, indicating that the plants were susceptible to TBRFV infection.

[0060] Genome sequencing of resistant tomato plants Genomic DNA was isolated from the resistant plant of the present invention (S. lycopersicum), i.e., a plant containing the TBRFV resistance locus, according to the protocol published on April 27, 2018, in Nature, Protocol Exchange (2018), by Rachael Workman et al., "High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing." A sequencing library was prepared using the PCR-free, non-multiplexed DNA Ligation Sequencing Kit-Promethion (SQK-LSK109). The isolation procedure yielded a high-quality sequencing library for use with the Oxford Nanopore System (ONT sequencing). Promethion Flowcell Packs (3000 pores / flowcell) version R9.4.1 were used for sequencing.

[0061] Furthermore, to further analyze the TBRFV locus and identify the gene providing TBRFV resistance, ONT sequencing was performed in the resistant strain (LYC4943). Complete transcript isoform sequencing of the resistant LYC4943 strain was performed using the Iso-Seq analysis application (Pacific Biosciences of California, PacBio). This yielded a single candidate resistant transcript / gene located between markers M33 and M38, more specifically in the region of the TBRFV resistance gene of SEQ ID NO: 115. This transcript was predicted to encode the CC-NBS-LRR resistant protein of SEQ ID NO: 116.

[0062] Genetic validation using VIGS To confirm that the TBRFV resistance gene (SEQ ID NO: 115) is indeed a gene that confers resistance to TBRFV, virus-induced gene silencing (VIGS) analysis was performed. VIGS vectors derived from tobacco stem necrosis virus (TRV) have been extensively documented for studying gene function in plants such as Arabidopsis thaliana, Nicotiana benthamiana, and Lycopersicon esculentum, as well as other plants [see, for example, Huang C, Qian Y, Li Z, and Zhou X.: Virus-induced gene silencing and its application in plant functional genomics. Sci China Life Sci. 2012; 55(2): pp. 99-108].

[0063] Therefore, we developed two VIGS constructs: one construct, VIGS-01a, to specifically target sequence number 115, and a control construct, VIGS-01b, to target sequence number 7, i.e., a sequence similarly located within the previously identified TBRFV locus (Table 4). [Table 4]

[0064] VIGS fragments were synthesized (IDT, gBlocks) and then cloned into TRV vectors. The DNA sequences were confirmed by Sanger sequencing. The vectors contain all the sequences encoding the proteins required for functional TRV particles, including the target sequences. VIGS vectors containing VIGS-01a and VIGS-01b constructs were used to transform Agrobacterium tumefaciens line GV3101, and these were used in VIGS experiments to reduce endogenous mRNA levels in the tomato plants used in the experiment. Homozygous TBRFV-resistant strains (15322-04) and infectious control strains (OT9) were used in VIGS experiments. The plants were inoculated with Agrobacterium at the seedling stage (cotyledon stage), and then inoculated with TBRFV isolate E50 three weeks after Agrobacterium inoculation. Two weeks after TBRFV inoculation, individual plants were phenotyped using ELISA and qPCR. Infectivity was observed in resistant plants treated with construct VIGS-01a, but not in resistant plants treated with construct VIGS-01b. The ELISA and qPCR results are shown in Figures 5 and 6, and the results are summarized in Table 5. [Table 5]

[0065] As expected, all OT9 plant strains were infectious. When the suspected TBRFV resistance gene was silenced using the VIGS-01a construct, which is designed to specifically target this gene, the R strain, which had previously been shown to be completely resistant, became infectious to TBRFV. On the other hand, silencing using the VIGS-01b construct (control construct) did not result in any infectivity in the tested plants. Based on these results, it can be concluded that gene sequence number 115 confers resistance to TBRFV.

Claims

1. A plant of the S. lycopersicum species resistant to tobamovirus, comprising a TBRFV resistance gene encoding a TBRFV resistance protein, wherein the protein has at least 90% amino acid sequence identity with SEQ ID NO:

116.

2. The plant according to claim 1, wherein the TBRFV resistance gene comprises a coding sequence having at least 90% nucleotide sequence identity with SEQ ID NO:

115.

3. The plant according to claim 1 or 2, wherein the tobamovirus is tomato brown lucos fruit virus (TBRFV).

4. The plant according to any one of claims 1 to 3, wherein the TBRFV resistance gene is present in the genome of the plant in a heterozygous or homozygous state.

5. The plant according to any one of claims 1 to 4, wherein the TBRFV resistance gene is found in deposit accession number NCIMB 43279.

6. A plant, plant part, tissue, cell and / or seed derived from a plant according to any one of claims 1 to 5 and having the TBRFV resistance gene.

7. A resistance gene that provides resistance to tobamovirus in S. lycopersicum plants, the resistance gene being represented by a coding sequence having at least 90% nucleotide sequence identity with sequence number 115.

8. The resistance gene according to claim 7, wherein the tobamovirus is TBRFV.

9. A method for providing a plant of the S. lycopersicum species that is resistant to tobamovirus, a) A step of selecting a S. habrocaites plant that is resistant to tobamovirus, comprising a step of confirming the presence of the resistance gene described in claim 7 or 8, b) A method comprising the step of introducing the resistance gene identified in step a) into a S. lycopersicum plant, for example by hybridization, thereby conferring tobamovirus resistance to the S. lycopersicum plant.

10. The method according to claim 9, wherein, after step b), a first S. lycopersicum plant resistant to tobamovirus is selected and crossed with a second S. lycopersicum plant not resistant to tobamovirus, and then a S. lycopersicum plant resistant to tobamovirus is selected.

11. The method according to claim 9 or 10, wherein in step a), the step of confirming the presence of the resistance gene in the S. habrocaites plant is performed by one or more markers selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 94, 105, 106, 107, 108, 109, 110, 111, and 112, preferably 105, 106, 107, 108, 109, 110, 111, and 112.

12. S. Use of a marker for confirming the presence of the TBRFV resistance gene described in claim 7 or 8 in Lycopersicum plants, wherein the marker is one or more selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 and 94, 105, 106, 107, 108, 109, 110, 111 and 112, preferably 105, 106, 107, 108, 109, 110, 111 and 112.

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