PLANTS RESISTANT TO INFECTION BY PEPINO MOSAIC VIRUS

Abstract

The present invention relates to plants comprising in their genome a gene that has been inactivated rendering the plant resistant to Pepino mosaic virus (PepMV) infection. The present invention also refers to the inactivation of the gene required for PepMV infection. The invention encompasses parts of these plants and their progeny that show said gene inactivation and as a consequence an improved phenotype in terms of PepMV infection resistance. Methods for obtaining plants, or plant parts or seeds with resistance to PepMV infection are also part of this invention. The present invention further relates to the gene and sequences linked to it as markers for selecting plants resistant to PepMV infection. Therefore, the present invention belongs to the field of agriculture.

Claims

1. A plant or part thereof, reproductive or propagating plant material or a plant cell, characterized in that it comprises a gene which encodes for a protein, wherein said protein comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 1 and said gene has been inactivated.

2. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to claim 1, wherein the plant or part thereof, the reproductive or propagating plant material, or the plant cell is not exclusively obtained by means of an essentially biological process.

3. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to claim 1 or 2, wherein said gene is inactivated and encodes for a protein comprising the amino acid sequence SEQ ID NO: 2.

4. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to claim 1 or 2, wherein said gene is inactivated and encodes for a protein comprising the amino acid sequence SEQ ID NO: 3.

5. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to any one of claims 1 to 4 belonging to the Solanaceae family.

6. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to any one of claims 1 to 5 belonging to Solanum sp., Capsicum sp., Nicotiana sp. or Physalis sp.

7. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to any one of claims 1 to 6 belonging to a species selected from the list consisting of: Solanum lycopersicum, S. tuberosum, S. pennellii, S. pimpinellifolium, S. peruvianum, S. cheesmanii, S. galapagense, S. chilense, S. melongena, S. aethiopicum, S. quitoense, S. torvum, S. muricatum, S. betaceum, S. chmielewskii, S. arcanum, S. cornelliomulleri, S. habrochaiti, S. huaylasense, S. neorickii, S. dulcamara, S. lycopersicoides, S. sitiens, S. juglandifolium, S. ochranthum, and S. cheesmaniae.

8. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to any one of claims 1 to 7, wherein the part of the plant is selected from the list consisting of: a leaf, a stem, a flower, an ovary, or a callus.

9. The plant or part thereof, the reproductive or propagating plant material or the plant cell according to any one of claims 1 to 8, wherein the reproductive or propagating plant material is selected from a fruit, a seed, a tuber or a progeny.

10. A method for producing a plant or part thereof, a reproductive or propagating plant material, or a plant cell according to any one of claims 1 to 9, wherein said plant or part thereof, reproductive or propagating plant material, or plant cell shows resistance to infection by pepino mosaic virus (PepMV) or improved phenotype in terms of PepMV infection resistance, comprising: a) Subjecting the plant or part thereof, the reproductive or propagating plant material, or the plant cell to mutagenesis either random or directed, and b) detecting a mutation in a gene which encodes a protein, wherein said protein comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 1, in the plant or part thereof, the reproductive or propagating plant material, or the plant cell, and wherein said mutation leads to an inactivation of the gene.

11. The method according to claim 10, wherein the random mutagenesis is achieved by contacting the plant or part thereof, the reproductive or propagating plant material, or the plant cell with a mutagenic agent, preferably selected from the list consisting of: a chemical substance, ionizing radiation, alpha rays, gamma rays, X-rays, UV-radiation, or any combination thereof.

12. The method according to claim 10, wherein the directed mutagenesis is achieved by homologous recombination-dependent gene targeting, antisense RNA, directed transposon insertion, virus induced gene silencing or genome editing techniques, preferably wherein the genome editing technique is CRISPR/Cas technique.

13. A use of the plant or part thereof, the reproductive or propagating plant material, or the plant cell according to any one of claims 1 to 9 for producing an agro-industrial product, preferably wherein the agro-industrial product is a food or a feed.

14. An agro-industrial product production method, preferably wherein the agro-industrial product is a food or a feed, comprising: a) culturing the plant or part thereof, the reproductive or propagating plant material, or the plant cell, according to any of claims 1 to 9, b) harvesting the fruit, the seeds, the tubers or the edible part of the plant to produce the agro-industrial product, and c) optionally, preparing the agro-industrial product for consumption either fresh or transformed.

15. A use of a gene as a biomarker to select plants with resistance to infection by PepMV or with improved phenotype in terms of PepMV infection resistance, wherein said gene encodes for a protein which comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 1.

16. The use according to claim 15 wherein the selection is made by determining the nucleotide sequence of the gene.

17. The use according to claim 15 wherein the selection is made by detecting or quantifying the product of expression of the gene, wherein said products are selected from the list consisting of: complementary DNA or a fragment thereof, messenger RNA or a fragment thereof, and protein or a fragment thereof.

18. A use of a marker locus to select plants with resistance to infection by PepMV, wherein said marker locus co-segregates with SEQ ID NO: 4 and is localized in a range of 100000 nucleotides upstream or downstream of SEQ ID NO: 4.

19. The use according to claim 15 wherein the selection is made by further determining a marker locus according to claim 18.

20. A method for selecting plants with resistance to infection by PepMV or with improved phenotype in terms of PepMV infection resistance compared to the wild type comprising the steps of: a) Detecting a gene which encodes for a protein wherein said protein comprises an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with SEQ ID NO: 1, and b) Determining if said gene of step a) is inactivated, wherein an inactivated gene is indicative of resistance to infection by PepMV or improved phenotype in terms of PepMV infection resistance compared to the wild type.

21. The method according to claim 20 wherein the detection in step a) is made by determining the nucleotide sequence of the gene or a fragment thereof.

22. The method according to claim 20 wherein the detection in step a) is made by detecting or quantifying the product of expression of the gene, wherein said products are selected from the list consisting of: complementary DNA or a fragment thereof, messenger RNA or a fragment thereof, and protein or a fragment thereof.

23. A method for producing a hybrid of the plant or part thereof, of the reproductive or propagating plant material, or of the plant cell according to any one of claims 1 to 9, wherein said hybrid shows resistance to infection by pepino mosaic virus (PepMV) or improved phenotype in terms of PepMV infection resistance, comprising: a) Crossing the plant or part thereof, the reproductive or propagating plant material, or the plant cell according to any one of claims 1 to 9 with a second plant; and b) Harvesting the hybrid progeny of said crossing.

24. A method for producing the plant or part thereof, the reproductive or propagating plant material, or the plant cell according to any one of claims 1 or 3 to 9, wherein said plant or part thereof, reproductive or propagating plant material, or plant cell shows resistance to infection by pepino mosaic virus (PepMV) or improved phenotype in terms of PepMV infection resistance compared to the wild type, comprising: a) Crossing a breeding plant or part thereof, a breeding reproductive or propagating plant material, or a breeding plant cell according to any one of claims 1 to 9 with a second plant; b) selecting a progeny plant resulting from the crossing in step a) having an introgression from the breeding plant or part thereof, the breeding reproductive or propagating plant material, or the breeding plant cell according to any one of claims 1 to 9 associated with resistance to PepMV or improved phenotype in terms of PepMV infection resistance; c) selfing and/or backcrossing said progeny plant selected in step (b) using said breeding plant or part thereof, a breeding reproductive or propagating plant material, a breeding plant cell line or a second plant as in (a) as a parent; d) selecting a progeny plant resulting from the crossing in step c) having an introgression from the breeding plant or part thereof, the breeding reproductive or propagating plant material, or the breeding plant cell associated with resistance to PepMV or with improved phenotype in terms of PepMV infection resistance; and e) repeating said steps of selfing and/or backcrossing and selection of steps (c) and (d) to provide a plant breeding line essentially homozygous for said introgression, wherein at least one selection as performed in steps (b) or (d) is performed by marker-assisted selection, wherein the introgression comprises a mutation in a gene which encodes a protein, wherein said protein comprises an amino acid sequence with at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, and wherein said mutation leads to an inactivation of the gene.

25. The method according to claim 23 or 24, wherein the second plant in step a) belongs to Solanum sp., Capsicum sp., Nicotiana sp. or Physalis sp, preferably to Solanum lycopersicum, S. tuberosum, S. pennellii, S. pimpinellifolium, S. peruvianum, S. cheesmanii, S. galapagense, S. chilense, S. melongena, S. aethiopicum, S. quitoense, S. torvum, S. muricatum, S. betaceum, S. chmielewskii, S. arcanum, S, cornelliomulleri, S. habrochaiti, S. huaylasense, S. neorickii, S. dulcamara, S. lycopersicoides, S. sitiens, S. juglandifolium, S. ochranthum, or S. cheesmania.

26. The method according to any one of claims 23 or 25, wherein the second plant of step a) is an inbred line and the hybrid progeny of step b) is a single-cross F1 hybrid.

27. The method according to any one of claims 23, 25 or 26, which further comprises an additional step (c) in which those hybrids harvested in step (b) showing an inactivation of a gene which encodes for a protein, wherein said protein comprises an amino acid sequence with at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, are selected by human intervention.

28. A plant or part thereof, a reproductive or propagating plant material, or a plant cell obtained by the method according to any one of claims 23 to 27, wherein said plant or part thereof, reproductive or propagating plant material, or plant cell comprises a gene which encodes for a protein, wherein said protein comprises an amino acid sequence with at least 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1 and said gene has been inactivated.

Description

DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1Symptoms induced by PepMV-H30 in leaves of wild type and mutant tomato plants. Wild type (WT) and 2F531 mutant plants of the tomato cultivar M82 were inoculated (or non-inoculated (?) for the healthy controls) with the aggressive isolate PepMV-H30, which induces bright yellow mosaics in the leaflets of the infected plants. The picture was taken after 16 days post inoculation.

[0074] FIG. 2Resistance of mutant 2F531 tomato plants to PepMV isolates from different strains. Viral load was measured by RT-qPCR using specific primers and referred to a calibration curve for absolute PepMV RNA quantification. A sharp decrease in PepMV accumulation was observed for 2F531 with respect to WT plants. PepMV isolates were: PepMV-Sp13, -H30 (EU strain), -PS5 and -KLP2 (CH2 strain). Average and SD of 4 replicates of 3 plants each. Total RNA was extracted at 16 days post inoculation. * indicates significant differences (p<0.05, T Student test).

[0075] FIG. 3Durability of the resistance of mutant 2F531. Up to five passages of two PepMV isolates were carried out in WT and mutant plants to estimate the probability of resistance breaking: (A) Scheme of the set up for stablishing 6 lineages of PepMV-Sp13 and 6 of PepMV-PS5 in WT and 2F531 plants. (B) Viral load evolution along the experiment (log.sub.10 ng of viral RNA/100 ng total RNA, at 16 days post inoculation (dpi)), for the isolates PepMV-Sp13 (top) and PepMV-PS5 (bottom).

[0076] FIG. 4Mapping of the mutation conferring resistance to PepMV in 2F531. (A) Susceptibility of BC.sub.1F.sub.1 to PepMV-H30, compared to WT and 2F531: viral load accumulation (ng of viral RNA/100 ng total RNA), at 16 days post inoculation (dpi); averages and SD of 8 replicates of 3 plants each. a and b indicate levels of PepMV accumulation significantly different (p<0.05, LSD test). (B) Phenotyping of BC.sub.1F.sub.3: Histogram of BC.sub.1F.sub.3 families regarding their % of susceptible individuals. (C) Manhattan plot representing the difference in allelic frequencies between the WT and R pools (y axis), in all detected variants along the tomato chromosomes according to the reference genome (Heinz 1706, SL2.50). The dotted line shows the threshold for difference in allelic frequencies higher than 0.7, indicative of association with resistance. (D) Genetic map and association analysis of SNPs in the candidate region of Chromosome 2: Linkage map of 12 markers in the candidate region after segregation analysis of 200 BC.sub.1F.sub.2 individuals, genetic distances in cM (center); and probability plot of loss-of-susceptibility being associated with the markers, represented as LOD score (scale on top of the graph, right).

[0077] FIG. 5Number of paired reads mapping to candidate genes. Number of normalized paired reads after RNA-seq (Y axis) that mapped to each candidate gene, in 3 pools of six F2 plants each, whose offspring displays either the resistant (R) or the susceptible (WT) phenotype.

[0078] FIG. 6Editing the SIOSCA4.1 gene using CRISPR/Cas9. (A) Schematic representation of the coding sequence of the wild type (WT) SIOSCA4.1 gene indicating the positions of the designed gRNAs used for gene editing by CRISPR/Cas9 and the edited gene slosca4.1_2 sequenced from edited plants, with a 1192 bp deletion respect to the WT gene. (B) PepMV-symptom expression in WT and slosca4.1_2-knockout tomato plants infected (or not for the healthy control) with PepMV-H30. (C) Accumulation of PepMV-Sp13 in tomato WT plants and slosca4.1_2 knockout mutants at 16 days post inoculation. Data are means and standard deviation from 6 infected plants. The asterisks indicate significant differences using the One-way ANOVA statistical test (*** p<0.001).

[0079] FIG. 7Relative expression of PepMV in WT and 2F531 mutants leaves protoplasts. Protoplast from leaves of both WT and 2F531 mutants were infected with PepMV-SP13 purified virion. Relative expression of PepMV at 0, 17 and 24 hours post infection was analyzed by RT-qPCR. The expression at times 17 and 24 hours were relativized to time 0 hours. Data are the means and standard deviation of 8 experimental repetitions for the WT and 7 for the mutant. The asterisks indicate significant differences using the t statistical test (p<0.05).

EXAMPLES

Example 1

Example 1.1: Methods

Tomato Mutants Screening

[0080] The mutant population consisted of 1,000 M.sub.2 families supplied after treating seeds of the tomato cv. M82 with ethyl methanesulfonate (EMS). Twenty-five plants per family were seeded in a nursery (Murcia, Spain) and 33 days after seeding transplanted to greenhouses (Murcia, Spain) with windows and entrances protected with anti-thrips mesh. WT tomato cv. M82 plants were included as susceptible controls and for the border lines. Plant density was 4 plants per square meter. Plants were inoculated with the aggressive isolate PepMV-KLP2 (Ag?ero et al., 2018, Front Plant Sci 9, 1-12) the same day of transplantation. Nicotina benthamiana plants were used to propagate the inoculum: 14 days after inoculation (dpi), symptomatic leaves above the inoculated ones were harvested, blended with 30 mM phosphate buffer pH 8 to a concentration of 100 g/L, and preserved at ?80? C.; for inoculations, this stock was diluted 5 times. Tomato plants were sprayed at high pressure with a suspension of carborundum powder (0.037 mm particle size; 10 g/L) in the inoculum solution. We used a total of 28 L of diluted inoculum solution for the whole tomato mutant population. A second round of inoculation was performed 28 days later, to ensure infection. Each M.sub.2 ?lant was scored for symptoms 42 days after the initial inoculation. A 0-2 symptom severity scale was defined as follows: 0, no symptoms; 1, sporadic bright yellow spots in newly emerged leaves; 2, bright yellow mosaic affecting all newly emerged leaves. Plants scoring 0 and 1 were selected. To check the association of symptoms with actual infection, PepMV detection was performed in 10 plants per family; we used molecular hybridization in tissue prints of petioles cross sections as in Marco et al. (2003, Phytopathology 93, 844-852). Crops were managed following conventional practices, except that extreme hygiene measures were adopted for personnel working in the greenhouses. Once evaluated, fruits from plants with mild or no symptoms were harvested and seeds extracted, thus preserving over 600 M.sub.3 families.

[0081] In a second round of selection, 10-12 plants per M.sub.3 family were evaluated; seeds were disinfected with 4% H.sub.2O.sub.2 for 30 minutes to eliminate contaminating PepMV, then sown in 40 seedling trays, inoculated and grown in an experimental greenhouse (CEBAS-CSIC, Murcia, Spain). The viral isolate used for inoculations was PepMV-H30 which, like PepMV-KLP2, induces bright yellow mosaics but has a more stable infection phenotype (Ag?ero et al., 2018, Front Plant Sci 9, 1-12). In this case, inoculum was produced in tomato plants (cv. Moneymaker) and mechanical inoculations were performed manually as in Ag?ero et al. (2018, Front Plant Sci 9, 1-12) at 21 days after seeding. Plants were reinoculated 14 days after the first inoculation. Symptom display was annotated at 25 dpi, recording the percentage of symptomatic plants per family. After evaluation, 3-6 plants of the selected families were transplanted to coconut fiber sacs and grown in greenhouses (Finca La Matanza, CEBAS-CSIC, Murcia, Spain) under standard cultivation conditions until fruit maturation. For selected plants, controlled pollinations were conducted to obtain two rounds of selfings (M.sub.4 and M.sub.5 seed).

Measuring Viral Load in 2F531 Plants

[0082] Four PepMV isolates were assayed on WT and 2F531 (M.sub.5 seed) plants: PepMV-Sp13 and PepMV-H30 belonging to the EU strain, and PepMV-PS5 and PepMV-KLP2 to the CH2 strain. PepMV-Sp13 and -PS5 are attenuated isolates inducing mild symptoms, while PepMV-H30 and -KLP2 are aggressive isolates (Ag?ero et al., 2018, Front Plant Sci 9, 1-12). Inocula were revived in N. benthamiana plants following standard practices. Three to four replicates of 3 tomato plants per genotype were inoculated with each virus or viral isolate. In all cases, plants with 2 true leaves were inoculated mechanically as previously described (G?mez et al., 2009a, J Virol 83, 12378-12387) and sampling was carried out at 16 dpi. Plants were grown in 1.1 L pots filled with a mix of peat and coconut fiber (2:1) in a crystal greenhouse (CEBAS-CSIC) with climatic control (day temperature set up at 24-25? C., night temperature at 16-18? C., 16 h of light). Viral RNA quantification was performed after extracting total RNA. All leaves from plants from each replicate were harvested and homogenized in a blender with 4 mL of TNA buffer per g of plant tissue (TNA: 2% SDS, 100 mM Tris HCl pH 8, 10 mM EDTA pH 8); 500 ?l of the homogenates were sampled and mixed with the same amount of TRI-Reagent? (RNA Isolation Reagent, Sigma Chemical Co, USA); RNA extraction was carried out according to the manufacturer's instructions. The final precipitate was dissolved in 50 ?l of sterile RNase free water and any residual DNA was eliminated by treatment with the TURBO DNA-free TM kit (Invitrogen, USA), according to the manufacturer's protocol. Quantity of RNA was estimated in a Nano-Drop? One (Thermo Scientific, USA). RT-qPCR was used for quantification of viral RNA. Standard curves were generated for each of the different viruses assayed, with 1:10 serial dilutions of a viral RNA of known concentration. The KAPA SYBR? FAST Universal One-Step RT-qPCR Kit (KAPA Biosystems, USA) was used, with 2 ?l of the purified viral RNA dilution or extracted plant RNA, in a reaction volume of 20 ?l, and with specific primers (G?mez et al., 2009a, J Virol 83, 12378-12387). Three technical replicates per biological replicate were analyzed using a thermocycler StepOnePlus (Applied Biosystems, USA).

Serial Passaging Experiment

[0083] The stability of resistance to PepMV in 2F531 plants was characterized in a serial-passaging experiment. Two plant (2F531 and WT) and 2 virus (PepMV-Sp13 and PepMV-PS5) genotypes were included in this experiment. M.sub.5 seeds were used for 2F531. Three plants per genotype and per PepMV isolate were used to establish 12 lineages (FIG. 3A). Initial inocula for both isolates were first refreshed in WT plants, quantified as described above, and prepared for passage 0 to a concentration of approximately 107 virus copies/ng of total RNA. Fifty ?L of inoculum was used to mechanically inoculate each of the founder plants of each lineage at the 2 true leaves stage, and then 5 successive passages were carried out; for that, 4 discs of 10.7 mm were taken from the second leaf above the inoculated one at 16 dpi, and used as the new inoculum source for the next passage on that lineage (FIG. 3A). Another 4 discs from the same leaf were kept frozen at ?80? C. for quantifying viral load as described above. All the plants were grown in 1.1 L pots filled with a mix of peat and coconut fiber (2:1), in a crystal greenhouse (CEBAS-CSIC) with climatic control (day temperature set up at 24-25? C., night temperature at 16-18? C., 16 h of light).

Mappinq Populations and Phenotypinq

[0084] Controlled pollinations were conducted to obtain the backcross to M82 (BC.sub.1F1). BC.sub.1F2 was obtained by controlled selfing of BC.sub.1F1. Two-hundred and four BC.sub.1F.sub.2 individuals were grown and used as the mapping population, and selfed to generate 204 BC.sub.1F.sub.3 progenies. In all cases, plants were grown in coconut fiber sacs in a PVC greenhouse (Finca La Matanza, CEBAS-CSIC). The phenotyping value of any given BC.sub.1F.sub.2 was determined by analyzing the susceptibility to PepMV-H30 of 10-12 of its BC.sub.1F.sub.3 descendants. The methodology for this progeny test was similar to the one described previously for the second round of selection in the massive screening, except for re-inoculation, that took place at 7 dpi, and final scoring at 14 dpi.

Bulked Segregant Analysis and High Throughput Genotyping

[0085] Two bulks were generated, the WT bulk with 18 BC.sub.1F.sub.2 individuals whose BC.sub.1F.sub.3 displayed 100% of symptomatic descendants, and the R bulk with 18 individuals with 0% symptomatic descendants. Leaf tissue of each BC.sub.1F.sub.2 individual was used for nucleic acids extraction. Automated DNA extraction was performed following the Maxwell? CSC (Promega Corp., USA) protocol for PureFood GMO and Authentification Kit for Food, Feed and Seed samples. Minor modifications were used to improve yield; namely, 60 mg of ground tissue as starting material, 600 ?L of CTAB, 30 ?L of Proteinase K, a 2 h incubation at 65? C., and a final volume of 80 ?L. DNA was quantified with Qubit? dsDNA BR Assay Kit in a Qubit? 2.0 fluorimeter (Life technologies, USA), and its quality checked by electrophoresis in 1% agarose and in a Nano-Drop? One. DNA of the selected individuals was pooled so that each of them was equally represented, and both pools were deep sequenced by Macrogen Inc. (South Korea). TruSeq DNA PCR-Free libraries were generated, with a fragment size of 350 pb, and run in a HiSeq?2500-High Throughput HORM (Illumina Inc., USA) with paired-end reads, to achieve a depth of coverage of around 50?. The raw data was analyzed as follows: Reads quality was tested with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/); reads mapping against the tomato reference genome (cv Heinz 1706, version SL2.50; http://solgenomics.net/organism/Solanum_lycopersicum/genome; Tomato Genome Consortium, 2012) was done with BWA aligner (Li and Durbin, 2009, Bioinformatics 25, 1754-1760); M82 sequence was retrieved from public databases (Bolger et al., 2014a, Nat Genet 46, 1034-1038). The program Freebayes (Garrison and Marth, 2012, ArXiv:1207.3907 [q-bio.GN]) was used for variant calling of both pools and M82; allelic frequencies were calculated in pools and M82 after filtering (criteria: available data in both pools, coverage equal or higher than 20 per sample, variant quality of at least 25, and total frequency of an allele lower than 0.9). Finally, the difference of allelic frequencies between pools was calculated, and represented as a Manhattan plot using R software. It was estimated that for a mendelian recessive mutation associated with the observed loss of function, such difference would be no less than 0.7.

RNA-Seq

[0086] The same 18 BC.sub.1F.sub.2 individuals per bulk were further subdivided in 3 replicates of 6 plants. Equivalent amounts of leaf tissue from each of the 6 plants per pool was used for RNA extractions as described above, except that the final RNA preparations were obtained using the kit Nucleo-Spin? RNA plant (Macherey-Nagel GmbH, Germany). The resulting RNA preps were evaluated using a Nano-Drop? One and an Agilent 2100 bioanalyzer (Agilent Technologies, USA). All the samples showed RNA integrity numbers above 7.4. The six pools were sequenced (Macrogen Inc.) following library construction with the kit TruSeq Stranded mRNA LT (Illumina Inc.), in the NovaSeq? 6000 platform (Illumina Inc.) with 151 bp paired read reads. Raw data were trimmed (adapters and 10 nucleotides of the 5 end) with the program Trimmomatic (Bolger et al., 2014b), and filtered for quality (a minimum QC of 30 and a length of at least 70 bp) with FastQC. Reads were paired with BBMap (www.sourceforege.net/projects/bbmap), and then mapped against the reference genome using the MEM algorithm of BWA (Li and Durbin, 2009, Bioinformatics 25, 1754-1760), checking the quality of the mapping with Qualimap (bampc). Variant calling and calculation of allelic frequencies was performed as described for DNA re-sequencing. The number of reads that mapped to annotated genes was calculated with the function featureCounts of SubRead, its quality with DESeq2 and their normalization with rlog. DESeq2 allowed also to study the differential expression between the R and WT samples, considering two factors, genotype and replicate. Goseq was used for the enrichment analysis GO. Finally, the biological impact of the variants was predicted with SnpEff (Cingolani et al., 2012, SnpEff. Fly 5, 29-30).

CRISPR/Cas9 Editing

[0087] Three gRNAs complementary to the SIOSCA4.1 coding sequence were designed using the BreakingCas bioinformatics tool (Oliveros et al., 2016). The targeted sequences in SIOSCA4.1 were 5-ACTTCAATTACGACGTCGCT-3 (SEQ ID NO: 7), 5-CAGAGCTGCCGCCCTCAATA-3 (SEQ ID NO: 8), and 5-ATAAGGCTGTCCAGGACCTC-3 (SEQ ID NO: 9). Sense and antisense oligonucleotides (Integrated DNA Technologies, Inc.) were annealed and cloned into the pDIRECT_22C (Addgene ref. #91135) binary plasmid following the protocol described in ?erm?k et al. (The Plant cell, 2017 29(6), 1196-1217). The resulting plasmid was used to transform Agrobacterium tumefaciens strain GV3101, which on its turn was used to transform explants of tomato cv. Micro-Tom following the protocol described in Van Eck et al. (Methods in molecular biology, 2006, 343, 459-473). Plants rooted in selective medium were transferred to substrate and acclimatized in growth chambers. To check if edition of SIOSCA4.1 took place in TO plants, the targeted region within the gene was PCR-amplified by direct tissue PCR using the Phire Tissue direct PCR kit (Thermo Scientific) following the manufacturer's specifications, and Sanger sequenced. Edited plants were selfed to obtain the T1 seed. T1 plants grown in substrate were genotyped, selecting those that had the mutation in homozygosis.

Example 1.2: Loss-of-Susceptibility to PepMV in a Collection of Tomato Mutants

[0088] A screening was conducted on a population of 25,000 tomato mutants from 1,000 M2 families. Mutant plants were inoculated with an aggressive PepMV isolate which induces obvious bright yellow mosaics. Symptom severity was scored for each plant according to a 0-2 scale, were 0 is absence of symptoms, 1 is sporadic bright yellow spots in newly emerging leaves, and 2 is bright yellow mosaic affecting all newly emerged leaves (FIG. 1). Aggressive symptoms (score 2) appeared in 97.5% of the plants, which were recorded as susceptible. There were plants scoring 0 or 1 in 379 families. A subpopulation of symptomatic and asymptomatic plants was tested for PepMV infection, revealing a perfect correlation between infection and symptoms display. One to 4 plants per family scoring 0 or 1 were selected and selfed, giving rise to more than 600 M.sub.3 families. From these, 10 to 12 individuals from each of 453 M.sub.3 families were inoculated with PepMV, and symptom scoring was again annotated. Family 2F531 showed 100% of symptomless plants, pointing to a homozygous mutation causing loss-of-susceptibility to PepMV; plants of this family were selfed to generate M.sub.4 seeds and backcrossed to the wild type accession M82 to obtain BC.sub.1F1. From now on, we will refer to the loss-of-susceptibility phenotype of these plants as resistance.

[0089] Tomato plants bearing the inactivated gene of the invention display no phenotypic differences relative to the wild type, other than the resistance to infection by PepMV or improved phenotype in terms of PepMV infection resistance. Neither plantlets (FIG. 6B) nor fully grown plants of mutants 2F531 or the slosca4.1_2-knockout could be distinguished from the WT plants in absence of infection by PepMV: they have similar determinate growth habit, size, color and shape of leaves, as well as fruits appearance, number or setting, and seed production (yield and germinability) indicating, that the mutation does not alter the fitness of the tomato plants.

Breadth and Durability of Resistance in Mutant 2F531

[0090] PepMV accumulation in wild type (WT) and 2F531 plants was compared after inoculation with PepMV-Sp13, PepMV-H30 (both belonging to the EU strain), PepMV-PS5 or PepMV-KLP2 (belonging to the CH2 strain). There was a sharp and significant decrease in viral load in the mutant with respect to the WT plants for all four isolates (FIG. 2), which was more pronounced for the EU (9 to 10-fold) than for the CH2 isolates (3 to 5-fold), and also sharper for the aggressive (PepMV-H30 and -KLP2) than for the mild (PepMV-Sp13 and -PS5) isolates.

[0091] Resistance durability is key for deploying sustainable pathogen control strategies in the field. To test if PepMV could easily overcome the 2F531 resistance, a passaging experiment was carried out. After initial inoculation with PepMV-Sp13 or PepMV-PS5, three viral lineages were set up on plants of each M82 or 2F531 genotypes, and 5 successive passages were carried out (FIG. 3A). Viral load was measured for each passage. For PepMV-PS5 passages, viral load fluctuated but it was always smaller (almost one order of magnitude in most cases) in the 2F531 than in the WT plants (FIG. 3B). For PepMV-Sp13, viral load fluctuations with passaging were more pronounced and, indeed, by the 3.sup.rd, 4.sup.th and 5.sup.th passages, viral populations in each of the 2F531 lineages, respectively, became almost extinct (FIG. 3B).

Mapping-by-Sequencing the Mutation Associated to Loss-of-Susceptibility to PepMV

[0092] BC.sub.1F.sub.1 plants showed similar PepMV accumulation and symptoms than WT plants (FIG. 4A), indicating that the 2F531 resistance has a recessive nature. BC.sub.1F.sub.1 plants were selfed to obtain over 200 BC.sub.1F.sub.2 plants, which were grown, selfed to obtain their BC.sub.1F.sub.3 progenies, and individually sampled for DNA and RNA extractions. Two hundred and four BC.sub.1F.sub.3 families where phenotyped by inoculating 12 plants per family with PepMV. In 50 and 54 BC.sub.1F.sub.3 families, respectively, all individuals were either resistant or susceptible, while within-family segregation was observed in 100 cases (FIG. 4B). These frequencies fit almost to perfection to the expected gene segregation in a model in which resistance is monogenic and recessive (?2 statistic, 2 df=0.23; ?>0.001).

[0093] Bulked Segregant Analysis (BSA) coupled to High Throughput Sequencing (HTS) was adopted to map the mutation associated with PepMV resistance. Two bulks were built, with 18 BC.sub.1F.sub.2 individuals in the R pool (i.e., 0% susceptible plants in BC.sub.1F.sub.3) and another 18 BC.sub.1F.sub.2 individuals in the WT pool (i.e., 100% susceptible plants in BC.sub.1F.sub.3). Pooled DNAs were sequenced for each bulk to a 50?depth. After quality filtering and alignment onto the reference genome (Heinz 1706, SL2.50), 99% of the reads could be mapped, 78% of them having a quality score MAPQ>57. Variant calling against the reference genome detected 1,285,278 variants after filtering for low coverage. Most of the variants could be attributed to natural polymorphisms between M82 and Heinz 1706, and only 6,302 (0.49%) to the EMS-induced mutagenesis; this indicates an approximate rate of 1 mutation each 150 Kbp in mutant 2F531. Allelic frequencies in each pool were calculated for all variants. The difference in allelic frequencies was greater than 0.7 for 10 SNPs and 1 Insertion/Deletion located at the distal end of chromosome 2, indicating an association between variant alleles and pool type (FIG. 4C). These 11 variants span a region of 2.02 Mb, from position 45,135,056 to 47,155,034; five of the mutations are located in annotated genes. This region includes 448 other variants of low coverage and encompasses 145 annotated genes.

[0094] To refine gene mapping, an analysis of recombination was carried out. Twenty-four SNPs identified inside or adjacent to the genomic region of interest were selected and analyzed in 200 BC.sub.1F.sub.2 individuals; only 12 of the markers segregated in the population. A linkage map was constructed for those markers and an association analysis was carried out to correlate marker genotypes and susceptibility. FIG. 4D displays the genetic map and the probability density along it and illustrates the strong association of resistance with markers C18 and C19. An analysis of recombinants further indicates that the loss-of susceptibility to PepMV is only displayed by individuals with alternative alleles in homozygosis at the region framed by C16 and C20, spanning 734,632 bp. Within this region, the BSA-HTS analysis identified 4 variants; the predicted functional effect of these variations was low to moderate except for mutation A to T at nucleotide position 1803 within the only Solyc02g083430 exon, which corresponds to position 1660 within the Solyc02g083430 main open reading frame. The protein encoded by Solyc02g083430 has 831 amino acids, and the mutation affects lysine at position 554, introducing a premature stop codon instead.

[0095] To validate and complement the above data, an RNA-Seq analysis was carried out using pooled RNAs from the R and WT bulks. Transcripts were sequenced, mapped to the reference genome and filtered, variants were identified, and allelic frequencies between pools were compared. Again, the only loci where allelic frequencies differed by more than 0.7 were located in the same genomic region as previously found, and variants were specifically detected in three loci, Solyc02g081200, Solyc02g082660 and Solyc02g083430. Some new SNPs could be identified in the area, but with low coverages. When comparing the number of readings mapping to each of the three RNA-Seq candidates for the R vs the WT pool (FIG. 5), differences were observed for Solyc02g083430; although these were not statistically significant, readings for Solyc02g083430 seemed to be less for the resistant than for the susceptible bulk, in agreement with the nature of the mutation observed for this gene. On the other hand, a gene ontology (GO) enrichment analysis identified 27 over-represented GO terms. Almost 9% of the deregulated genes are related to ATPase complexes within the category of cellular component. Within the molecular function category, ATP binding (GO: 0005524), Aspartic-type endopeptidase activity (GO: 0004190) and Oxidoreductase activity (GO: 0016491) were the most enriched. Finally, within the biological function category, only two enriched biological processes were identified: Response to wounding (GO: 0009611) and Alcohol metabolic process (GO: 0006066).

Solyc02g083430 Encodes SIOSCA4.1, a Protein Involved in Vacuolar Trafficking and Member of the Hyperosmolality-Gated Calcium-Permeable Channel 1 (OSCA) Family

[0096] The protein encoded by Solyc02g083430 (genomic sequence SEQ ID NO: 4), SEQ ID NO: 1, has 3 conserved domains: A transmembrane domain that is part of a calcium permeable cation exchange channel 1 (Csc1_N) activated by physical signals such as osmotic stress; a phosphate transporter domain, predicted as cytosolic (PHM7_cyt); and a region of 7 transmembrane domains that are part of a putative phosphate transporter (RSN1_7TM) (Zhu et al., 2008, Nat Genet 40, 854-861). The premature introduction of the stop codon at amino acid 554 results in the loss of much of the RSN1_7TM transmembrane domain, which could cause the total or partial loss of the protein function, resulting in the mutant phenotype. Its closest Arabidopsis ortholog encodes AtOSCA4.1, which belongs to the hyperosmolality-gated/mechanically activated calcium-permeable channels (OSCA) family (Yuan et al., 2014, Nature 514, 367-371) and with which it shares 69% amino acid identity. As in Arabidopsis, the tomato OSCA4.1 (SIOSCA4.1) belongs to a small family composed of 12 members phylogenetically organized in the same four clades as in Arabidopsis. The AtOSCA4.1 has been clearly identified as a vacuolar sorting factor in two independent reports (Fuji et al., 2007, Plant Cell 19, 597-609; Delgadillo et al., 2020, PNAS 117, 9884-9895). A further search for orthologs of the protein encoded by the gene Solyc02g083430, in agricultural important species, with similar structural organization resulted in two sets of proteins: one extremely conserved inside the Solanaceae with a minimum identity of 91.29% across the whole protein (Table 1); and a second one highly conserved outside Solanaceae (Table 2) with a minimum identity of 62.77% across the whole protein. The structural similarity and domain distribution of both sets of proteins, indicated that the proteins must have a conserved function to the protein encoded by Solyc02g083430.

Editing SIOSCA4.1 in Tomato Cv. Micro-Tom Confirms its Proviral Function for PepMV

[0097] To confirm the implication of SIOSCA4.1 in PepMV susceptibility, we used the genome editing technology CRISPR/Cas9 to produce tomato cv. Micro-Tom mutants in the Solyc02g083430 locus. Guide RNAs were designed targeting sequences at the beginning of the only Solyc02g083430 exon. Homozygous mutations were observed in individual plants of the T1 generation (FIG. 6A), which were inoculated with PepMV. An infection phenotype similar to that of mutant 2F531 plants was observed: no disease symptoms were seen in edited plants (FIG. 6B) and PepMV accumulation was significantly reduced in the mutant compared to the WT plants (FIG. 6C), thus validating the starting hypothesis.

TABLE-US-00003 TABLE 1 Orthologs of SEQ ID NO: 1 in Solanaceae species Species (Solanaceae) % id Nicotiana_benthamiana_1 91.29 Nicotiana_benthamiana_2 92.27 Capsicum_annuum_cv 93.98 Capsicum_annuum_glabriusculum 94.26 Capsicum_annuum_zunla 94.38 Solanum_melongena 93.98 Solanum_lycopersicum 100.00 Solanum_pimpinellifolium 100.00 Solanum_pennellii 98.32 Solanum_tuberosum 97.56

TABLE-US-00004 TABLE 2 Orthologs of SEQ ID NO: 1 in non-Solanaceae species Species (Non Solanaceae) % id Musa_acuminata 65.04 Zea_mays 62.96 Oryza_sativa_Indica 62.77 Triticum_aestivum 63.12 Brassica_rapa 69.33 Brassica_rapa_2 62.90 Daucus_carota 75.55 Actinida_chinensis 76.78 Sesamum_indicum 79.34 Olea_europaea 78.16 Solanum_lycopersicum 100.00 Coffea_canephora 80.37 Phaseolus_vulgaris 71.73 Glycine_max 70.85 Malus_domestica 72.29 Manihot_esculenta 73.79 Cucumis_sativus 71.11

Example 2

Example 2.1: Method

Protoplast Isolation and Inoculation

[0098] Protoplasts were isolated from leaves of WT and 2F531 mutant tomato plants by procedures described by Tan et al. (1987, Plant cell reports, 6(3), 172-175). Approximately 2 g of tomato leaves were harvested from both WT and 2F531 mutant and subjected to protoplast isolation. Each protoplast sample containing approximately 2?10.sup.6 cells was inoculated with 50 ?g of PepMV purified virion by PEG 4000 method and incubated 24 h at 26? C. with humidity and constant light in growth chamber. Protoplasts were sampled at 0, 17 and 24 h after infection. Total RNA was isolated from protoplast by Trizol reagent. Genomic DNA was removed from RNA samples, RNA was normalized and use for expression analysis by RT-qPCR. PepMV expression was standardized using elongation factor 1-alpha as endogenous gene.

Example 2.2: Infection of Tomato WT and 2F531 Protoplasts with PepMV Confirms its Role in PepMV Replication within the Cell

[0099] To complement data on the implication of SLOSCA4.1 in PepMV susceptibility and replication, we used leaves protoplasts of tomato cv. M82 both WT and mutant 2F531. Protoplast were isolated from leaves of WT and mutant plants, infected with PepMV purified virion and sampled at 0, 17 and 24 hours post infection. Viral expression was measured from total RNA by relative RT-qPCR using alpha elongation factor 1 (EF-1alpha) as endogenous gene for normalization. The results showed that relative accumulation of PepMV in protoplast from the mutant 2F531 plants was significantly reduced compared to the WT plants (FIG. 7), thus indicating that SIOSCA4.1 is involved in PepMV replication within the cell.