Plants having improved pathogen resistance

Abstract

The present invention relates to novel tomato plants having improved resistance to lesion forming pathogens due to reduced Pub17 protein levels, activity or expression. The level, activity, or expression of the Pub17 protein can be reduced by modifying the Pub17 allele to contain an SNP identified to be associated with increased lesion forming pathogen resistance. The invention further relates to plant parts and seeds derived from said tomato plant, and to methods of making said tomato plant or increasing the resistance of a tomato plant to lesion-forming pathogens, as well as to the use of an SNP as a marker and related kits.

Description

Technical Field

[0001] The present invention relates to novel tomato plants with improved resistance to lesion forming pathogens. The invention further relates to plant parts and seeds derived from said tomato plants, and to methods of making said tomato plants or increasing the resistance of tomato plants to lesion forming pathogens. Additional aspects of the invention relate to modified Pub17 nucleic acid and Pub17 protein sequences that are associated with such improved resistance to lesion forming pathogens. Background Art

[0002] Valuable crop plants such as tomatoes are hosts to a wide variety of pests and pathogens from more than 200 species. In plant breeding practice, one of the most prominent issues since the 1950s has been breeding for resistance to the most destructive pests and pathogens by transferring disease resistance (R) genes from wild relatives to cultivated plants. Today, about 20 pathogens can be genetically controlled by R-genes, which are derived from a relatively small number of wild species. In most cases, monogenic resistance controlled by a single dominant gene is introgressed into cultivated varieties (Bai et al., 2018). Most of the dominant R-genes cloned to date can be divided into two groups: (1) plasma membrane receptors, including receptor-like kinases (RLKs, encoded by I-3 genes) and receptor-like proteins (RLPs, encoded by Cf genes and Ve-1 genes); and (2) in most cases, intracellular receptors, which represent proteins with a nucleotide binding site and a leucine-rich repeat domain (NBS-LRR). These plant receptors are able to recognize pathogen molecules known as pathogen-associated molecular patterns and effectors, leading to pathogen-induced resistance (Dangl et al., 2013).

[0003] For (semi) biotrophic microbial pathogens, dominant R-genes have been very successful infiltrating cultivars from wild species. The mode of action of such R-genes has been extensively studied over the past two decades, and in most cases has been shown to rely on a mechanism known as effector-triggered immunity (ETI). The effector molecules of biotrophic pathogens are considered to be important in suppressing the so-called PAMP-triggered immunity (PTI). Plant receptor proteins (products of R-genes) induce programmed cell death ("hypersensitive response", HR) to the recognition of microbial effector molecules, thereby preventing further invasion of pathogens.

[0004] However, dominant R-genes that confer resistance to lesion-forming pathogens, especially necrotrophic pathogens, including the common tomato pathogens Botrytis cinerea and Alternaria solani (Adhikari et al., 2017; Bai et al., 2018), have not been identified. The above immune responses are ineffective against such microbial pathogens. Instead, necrotrophs are thought to "hijack" host cell death pathways in response to effector molecules (Mengiste, 2012; Vleeshouwers and Oliver 2014; Shi et al., 2016). Therefore, it is crucial to develop alternative breeding strategies that circumvent the use of plant receptor genes that recognize effectors.

[0005] Most of the currently available resistance to lesion-forming pathogens is quantitative and is conferred by a number of quantitative trait loci (QTLs, Poland et al., 2009; Bai et al., 2018). In contrast to R-gene-mediated qualitative resistance, the molecular mechanisms of quantitative resistance conferred by QTLs are not yet understood. It has been proposed that resistance QTLs may be regulated by genes involved in defense signaling, genes regulating morphological traits, and genes encoding components of chemical warfare agents (Poland et al., 2009; Roux et al., 2014). In breeding, QTLs are difficult to use due to the impact of small individual QTLs on resistance. For example, in some wild tomato relative germplasm, there is a good level of disease resistance to Botrytis cinerea and Alternaria solani (ten Have et al., 2007; Smith et al., 2014), however, once introgressed into the tomato (S. lycopersicum) background, the resistance level drops, indicating that the genetic background of resistance in wild species is very complex (Finkers et al., 2007; Smith et al., 2014).

[0006] In 2010, a new strategy for crop breeding for resistance was proposed: the use of impaired plant susceptibility (S) genes (Pavan et al., 2010). S-genes are plant genes that encode proteins that pathogens exploit for their own benefit during infection (Pavan et al., 2010). These S-genes can be divided into three groups (Van Schie and Takken, 2014): (i) genes that allow basic plant-pathogen compatibility, facilitating host recognition and penetration; (ii) genes that encode negative regulators of immune signaling; and (iii) genes that allow sustained compatibility and pathogen proliferation, fulfilling metabolic or structural needs of the pathogen. When such genes become dysfunctional due to mutation or loss of expression, they hinder the pathogen from colonizing the plant. Thus, impaired S-genes contribute primarily to recessive resistance traits, in contrast to recognition-based resistance dominated by dominant R-genes. Strategies using S-genes offer fundamentally different opportunities for controlling diseases caused by lesion-forming microorganisms. However, many S-genes remain undiscovered, and the concept of using S-genes to generate pathogen-resistant plants has not yet been tested or realized in key crop plants such as tomatoes.

[0007] The present invention aims to solve one or more of the above problems in the art by providing an alternative means of increasing plant resistance to lesion-forming pathogens, particularly in tomato plants. Summary of the invention

[0008] According to a first aspect of the present invention there is provided a tomato plant or plant material having reduced Pub17 protein levels, activity or expression thereby conferring increased resistance to lesion forming pathogens relative to a reference tomato plant or plant material.

[0009] In one embodiment, the tomato plant or plant material has been modified to reduce the level, activity or expression of Pub17 protein. Thus, in one embodiment, there is a tomato plant or plant material that has been modified to reduce the level, activity or expression of Pub17 protein, thereby conferring increased resistance to lesion forming pathogens relative to a reference tomato plant or plant material.

[0010] In one embodiment, the tomato plant or plant material comprises a modified Pub17 allele. In one embodiment, the plant or plant material comprises a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof, wherein the Pub17 allele comprises a mutation. In one embodiment, the modified Pub17 allele confers increased resistance to lesion-forming pathogens relative to a reference tomato plant or plant material. Thus, in one embodiment, there is a tomato plant or plant material comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof, wherein the Pub17 allele comprises a mutation that reduces Pub17 protein levels, activity or expression, thereby conferring increased resistance to lesion-forming pathogens relative to a reference tomato plant or plant material.

[0011] According to a second aspect of the present invention there is provided a method of increasing resistance of a tomato plant or plant material to a lesion forming pathogen, the method comprising reducing the level, activity or expression of Pub17 protein in the tomato plant or plant material.

[0012] In one embodiment, the method comprises modifying a tomato plant to reduce the level, activity or expression of a Pub17 protein in the tomato plant or plant material. Thus, in one embodiment, there is a method of increasing resistance of a tomato plant or plant material to a lesion forming pathogen, the method comprising modifying a tomato plant to reduce the level, activity or expression of a Pub17 protein in the tomato plant or plant material.

[0013] In one embodiment, the method comprises obtaining a mutant population of tomato plants and selecting a tomato plant comprising a modified Pub17 allele. In one embodiment, the method comprises selecting a tomato plant comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation. Thus, in one embodiment, there is a method of increasing resistance of a tomato plant or plant material to a lesion-forming pathogen, the method comprising obtaining a mutant population of tomato plants and selecting a plant comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation that reduces Pub17 protein levels, activity or expression.

[0014] In one embodiment, the increased resistance may be relative to a reference tomato plant or plant material.

[0015] According to a third aspect of the present invention there is provided a method of producing a tomato plant with increased resistance to lesion forming pathogens, the method comprising reducing the level, activity or expression of Pub17 protein in a tomato plant or plant material.

[0016] In one embodiment, the method comprises modifying a tomato plant to reduce the level, activity or expression of a Pub17 protein in a tomato plant or plant material. Thus, in one embodiment, there is a method of producing a tomato plant having increased resistance to a lesion forming pathogen, the method comprising modifying the plant to reduce the level, activity or expression of a Pub17 protein in a tomato plant or plant material.

[0017] In one embodiment, the method comprises obtaining a mutant population of tomato plants and selecting plants comprising a modified Pub17 allele. In one embodiment, the method comprises selecting a tomato plant comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation. Thus, in one embodiment, there is a method for producing a tomato plant having increased resistance to a lesion-forming pathogen, the method comprising obtaining a mutant population of tomato plants and selecting a modified tomato plant comprising a modified Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation that reduces Pub17 protein levels, activity or expression.

[0018] In one embodiment, the increased resistance may be relative to a reference tomato plant or plant material.

[0019] According to a fourth aspect of the present invention, there is provided a method for enhancing the growth of tomato plants by increasing the resistance of tomato plants or plant materials to lesion-forming pathogens, the method comprising reducing the level, activity or expression of Pub17 protein in the tomato plants or plant materials.

[0020] In one embodiment, the method comprises modifying a tomato plant to reduce the level, activity or expression of a Pub17 protein in the tomato plant or plant material. Thus, in one embodiment, there is a method of enhancing growth of a tomato plant by increasing resistance of a tomato plant or plant material to a lesion forming pathogen, the method comprising modifying a tomato plant to reduce the level, activity or expression of a Pub17 protein in the tomato plant or plant material.

[0021] In one embodiment, the method comprises obtaining a mutant population of tomato plants and selecting plants comprising a modified Pub17 allele. In one embodiment, the method comprises selecting a tomato plant comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation. Thus, in one embodiment, there is a method for enhancing tomato plant growth by increasing resistance of tomato plants or plant material to lesion-forming pathogens, the method comprising obtaining a mutant population of tomato plants and selecting a modified plant comprising a modified Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation that reduces Pub17 protein levels, activity or expression.

[0022] In one embodiment, the increased resistance may be relative to a reference tomato plant or plant material.

[0023] According to a fifth aspect of the present invention, there is provided a method for identifying a tomato plant having increased resistance to a lesion-forming pathogen relative to a reference tomato plant or plant material, the method comprising: determining the level, activity or expression of Pub17 protein in one or more tomato plants, and comparing it with the level, activity or expression of Pub17 protein in a reference tomato plant, and selecting a tomato plant having a reduced level, activity or expression of Pub17 protein compared to the reference tomato plant, wherein a reduction in the level, activity or expression of Pub17 protein indicates increased resistance to lesion-forming pathogens relative to the reference tomato plant.

[0024] In one embodiment, the method comprises the step of obtaining a mutant population of tomato plants. In one embodiment, a method for identifying a tomato plant having increased resistance to a lesion forming pathogen relative to a reference tomato plant or plant material is provided, the method comprising: obtaining a mutant population of tomato plants, determining the level, activity or expression level of a Pub17 protein in one or more tomato plants in the tomato plant population, and comparing it to the level, activity or expression of the Pub17 protein in a reference tomato plant, selecting a plant having a reduced level, activity or expression of the Pub17 protein compared to the reference tomato plant, wherein a reduced level, activity or expression of the Pub17 protein indicates increased resistance to a lesion forming pathogen relative to the reference tomato plant.

[0025] In one embodiment, the method comprises obtaining a mutant population of tomato plants and screening for tomato plants comprising a modified Pub17 allele. In one embodiment, the method comprises screening for tomato plants comprising a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation. Thus, in one embodiment, there is a method for identifying a tomato plant having increased resistance to a lesion-forming pathogen relative to a reference tomato plant or plant material, the method comprising: obtaining a mutant population of tomato plants, screening the tomato plant population for the presence of a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof and comprising a mutation that reduces Pub17 protein levels, activity or expression, and selecting a tomato plant having the Pub17 allele.

[0026] According to a sixth aspect of the present invention, there is provided a plant part obtained from the tomato plant of the first aspect.

[0027] In one embodiment, the plant part is a fruit. In one embodiment, the plant part comprises a modified Pub17 allele. In one embodiment, the plant part comprises a Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof, and comprising a mutation.

[0028] According to a seventh aspect of the present invention, there is provided a seed capable of producing the tomato plant of the first aspect.

[0029] In one embodiment, the seed comprises a modified Pub17 allele. In one embodiment, the seed comprises a Pub17 allele that is at least 70% identical to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof, and comprises a mutation.

[0030] According to the eighth aspect of the present invention, there is provided an isolated polynucleotide sequence having at least 70% identity with SEQ ID NO:1 (wild type) or its direct homolog or homolog, wherein the sequence comprises a mutation at position 1477 of SEQ ID NO:1 or at a position corresponding thereto.

[0031] In one embodiment, the isolated polynucleotide comprises or consists of the sequence according to SEQ ID NO:2.

[0032] In one embodiment, the isolated polynucleotide sequence is capable of conferring increased resistance to lesion forming pathogens. Suitably, it is capable of conferring increased resistance to lesion forming pathogens when expressed in a plant or plant material.

[0033] According to the ninth aspect of the present invention, there is provided an isolated polypeptide sequence encoded by the polynucleotide sequence of the eighth aspect.

[0034] In one embodiment, the isolated polypeptide sequence consists of the amino acid sequence according to SEQ ID NO: 4 (truncated protein sequence) or a portion thereof, or an amino acid sequence having at least 70% identity thereto.

[0035] In one embodiment, the isolated polypeptide sequence is capable of conferring increased resistance to lesion forming pathogens. Suitably, when present in a plant or plant material, it is capable of conferring increased resistance to lesion forming pathogens.

[0036] According to the tenth aspect of the present invention, a vector or expression construct comprising the polynucleotide sequence of the eighth aspect is provided.

[0037] According to the eleventh aspect, a host cell is provided, comprising the polynucleotide sequence according to the eighth aspect, the vector according to the tenth aspect, or the polypeptide according to the ninth aspect.

[0038] According to a twelfth aspect, there is provided a method of producing hybrid seeds, the method comprising crossing a first tomato plant of the first aspect with a second tomato plant and obtaining seeds therefrom.

[0039] According to the thirteenth aspect, a kit for detecting Pub17 alleles resistant to lesion-forming pathogens in tomato plants is provided, the kit comprising a PCR oligonucleotide primer pair, wherein the primer pair comprises: a forward primer SEQ ID NO:25 and a reverse primer SEQ ID NO:24; or a first forward primer SEQ ID NO:31, a second forward primer SEQ ID NO:30, and a reverse primer SEQ ID NO:32.

[0040] In one embodiment, the kit is for use in gene-specific PCR and comprises a forward primer of SEQ ID NO:25 and a reverse primer of SEQ ID NO:24.

[0041] In one embodiment, the kit is for use in allele-specific PCR and comprises a first forward primer SEQ ID NO:31, a second forward primer SEQ ID NO:30, and a reverse primer SEQ ID NO:32.

[0042] The present invention exploits the principle of a mutated S-gene to achieve resistance against lesion-forming pathogens, rather than using classical R-genes or introgression of several (minor) effector QTLs.

[0043] The present inventors first identified a novel S-gene, Pub17, which was previously not known to be a susceptibility gene in Solanaceae species. The inventors further discovered that Solanaceae plants carrying a mutated dysfunctional allele of this S-gene have increased pathogen resistance, particularly resistance to lesion-forming pathogens. Examples demonstrate that Solanaceae plants homozygous for this mutated Pub17 allele have significantly reduced susceptibility to necrotrophic pathogens (including Botrytis and Alternaria) and hemibiotrophic pathogens (Phytophthora infestans). The inventors have shown that this is the case in several different genetic backgrounds. Advantageously, the Pub17 allele can be used in crop breeding to obtain populations of Solanaceae plants that are less susceptible to severe disease from necrotrophic pathogens than existing cultivars. Thus, the present invention provides an alternative solution to the problem of lesion-forming pathogen control in Solanaceae crops that is much simpler than using QTLs or R-genes. Given that Solanaceae is one of the largest crop plant families, containing not only tomatoes but also potatoes and peppers, such resistant plants of the present invention are of economic interest. The present invention can be used to limit the damage caused by these pathogens and increase the yield of Solanaceae crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 shows: Pedigrees of identified M2042 mutants generated by EMS mutagenesis. A. Selfing generation of M2042 in Micro-Tom (MT) background. B. Hybrid generation after crossing resistant M4 plant M2042-1-2-12 with Moneymaker (MM). IR, moderately resistant; S, susceptible.

[0045] Figure 2 Shows :The structure of the mutant M2042 candidate gene PUB17 with reduced susceptibility to Botrytis cinerea is provided. Tomato PUB17 (Solyc02g072080) has three domains: the U-box N-terminal domain UND (amino acids 20-171), the U-box domain (amino acids 297-364) and the ARM armadillo repeat sequence (amino acids 429-682) (derived by comparison with potato StPUB17, Ni et al. 2010). A SNP at position 1477 was identified, which produces a premature stop codon at amino acid R493.

[0046] Figure 3 Shows : Relative gene expression levels of mutant PUB17 candidate genes in wild-type Little Tom (MT) and Botrytis-resistant mutant plants M2042-1-1-17 and M2042-1-2-12 after infection with Botrytis cinerea. hpi, hours post infection.

[0047] Figure 4 Shows : Location of selected RNAi fragments used to silence PUB17 expression.

[0048] Figure 5 It shows: Relative expression levels of PUB17 in RNAi-transformed tomatoes compared to untransformed Moneymaker (MM) were determined by qPCR using EF1α as a reference gene. T1 plants 3-5 and 3-29 (indicated by arrows) were selected for further analysis.

[0049] Figure 6 Shows : Box plots of lesion diameter size on leaves from the PUB17 RNAi T3 family, with two negative controls (MM and TV24) on the left side of each sub-plot. Left sub-plot, results at 3 days post-inoculation (dpi); right sub-plot, results at 4dpi. Different letters above the box plots indicate significant differences, as calculated by the Tukey HSD method.

[0050] Figure 7 Shows: Box plots of lesion diameter size on leaves of TV181105 (TV05, plants containing NPTII) from PUB17 RNAi T2 family, with two negative controls (MM and TV181105 plants without NPTII) on the left side of each sub-plot. Left sub-plot, results at 3 days post-inoculation (dpi); right sub-plot, results at 4dpi. Different letters above the box plots indicate significant differences, as calculated by the Tukey HSD method.

[0051] Figure 8 Shows : Location of four sgRNAs targeting the tomato PUB17 gene using CRISPR / Cas9.

[0052] Fig. 9 Shows : Four CRISPR / Cas9 PUB17 transformants, which showed smaller bands than the expected wild-type Moneymaker (MM) control, indicating that cleavage had occurred. PUB17 was amplified using primers FWD_MR_GX_CRISPR+REV_MR_GX_CRISPR (A) or AWPUB17_F1+REV_MR_GX_CRISPR (B). The PCR products were run on a 1% agarose gel with TAE. The size of the marker bands is indicated in bp.

[0053] Fig.10 Shows : CRISPR-induced mutations in the tomato PUB17 gene. Schematic representation of the tomato PUB17 genomic sequence. Individual exons are shown as solid arrows with the locations of the four sgRNA target sites shown as asterisks. The expected PCR product for the wild-type allele is shown below the exon. The size of the deletion in the CRISPR transformant is indicated by the line above the exon.

[0054] Fig.11 Shows : CRISPR-induced single nucleotide mutations at the target sites of sgRNA3 and sgRNA4 in the tomato PUB17 gene in CRISPR / Cas9 transformant 7.

[0055] Fig.12 Shows : Box plots of lesion diameter size on leaves from the PUB17 CRISPR mutant T3 family compared to the negative control Moneymaker (MM) and the non-mutant T2 family TV181133. A. Results at 3 days post inoculation (dpi). B. Results at 4 dpi.

[0056] FIG. 13 shows: Shown at 10 days after inoculation (A) and 5 days after inoculation (B), compared with the control Moneymaker (MM) and Little Tom (MT), in F3, F4 and M5 EMS-induced mutant PUB17 plants infected with Alternaria solani reduced lesion diameter size. (C) Box plots of lesion diameters of leaves from PUB17 CRISPR mutant T3 families TV192007 (TV07) and TV192023 (TV23) at 7dpi (days after inoculation). MM was used as a susceptible control. Different letters above the box plots indicate significant differences (P < 0.05), as calculated by the Tukey HSD method.

[0057] Fig.14 It shows: Results of the Botrytis cinerea stem assay. A. Disease scores of the PUB17 RNAi T3 family at 10 days post inoculation (dpi), with Moneymaker (MM) and wild-type T3 family TV192024 as negative controls (NC). B. Disease scores of the PUB17 CRISPR T3 family TV192008, TV192012, TV192007, TV192009, TV192016, TV192019, TV192014, TV192023 6 days after stem inoculation with Botrytis cinerea, with Moneymaker (MM) and wild-type T3 family TV181133 as negative controls (NC). "abs, 0 to 3" is the Disease Severity Index (DSI) score: high numbers indicate large damage, or "abs" indicates shedding of the petiole stump. Six days after inoculation, the plants began to show disease symptoms.

[0058] Fig.15 Shown: Photographic representation of the disease index scores in the Botrytis stem assay. abs, abscission. The petiole stump has fallen off. 0, Petiole stump unchanged. Comparable reaction to the treated mock. 1, Petiole stump reaction. The petiole stump is partially or completely thinning and brown. 2, Outward main stem infection begins. Small brown rings are visible on the main stem around the axils of the inoculated petiole stump. 3, Outward main stem infection spreads. The brown rings become irregular and spread up and down the main stem. 4, Complete main stem infection and wilting of the plant. The main stem is invaded, followed by internal browning and collapse of the stem tissue. The stem is drying up and eventually the plant tip overturns.

[0059] Fig.16 Shown are: protein domains in mutant PUB17 alleles. WT, wild-type tomato PUB17 protein; M2042, EMS PUB17 mutant allele; alleles 1-4, CRISPR PUB17 mutant alleles. Output obtained from the ScanProsite tool.

[0060] Figure 17 shows:EMS-mutant pub17 pre-breeding results. A. Scheme for introgressing the pub17 mutation into breeding lines H1 and H2. B. Scheme for introgressing the pub17 mutation into pre-breeding lines A and B of F1 hybrids. C. Observation of self-necrosis of pre-breeding line B. No necrotic spots were shown on the leaves of F1 hybrids and pre-breeding line A with pub17 mutation (pub17pub17) (left panel). WT, control carrying the normal PUB17 allele. Necrotic spots were observed on the leaves of pre-breeding line B (right panel) 120 days after sowing. Pre-breeding line B-1 showed the least necrosis and was therefore selected for producing F1 hybrids by crossing with pre-breeding line A. D. There were no significant differences in fruit environment between wild-type (WT) hybrids and hybrids carrying pub17. Upper panel, average fruit weight of the first fruit cluster 40 days after pollination (±SD, n=6 plants). Lower panel, average number of fruits in the first, second and third fruit clusters 20 days after pollination (± SD, n = 6 plants).

[0061] Further features and embodiments of the invention will now be described under the heading sections.Any feature in any section may be combined with any of the above-described aspects or embodiments of the invention in any feasible combination. DETAILED DESCRIPTION

[0062] definition

[0063] If not otherwise indicated below, the technical terms and expressions used within the scope of the present application are generally given the meanings commonly applied thereto in the fields related to plant breeding and cultivation.

[0064] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes one or more plants, and reference to "a cell" includes mixtures of multiple cells, tissues, etc.

[0065] As used herein, the term "about," when referring to a value or to an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of ±20% from the stated amount in some embodiments, ±10% from the stated amount in some embodiments, ±5% from the stated amount in some embodiments, ±1% from the stated amount in some embodiments, ±0.5% from the stated amount in some embodiments, and ±0.1% from the stated amount in some embodiments, as such variations are appropriate in the context of the present invention.

[0066] "Cultivated" plants are understood within the scope of the present invention to mean plants that are no longer in their natural state, but have been developed and domesticated by human care and are used for agricultural purposes and / or for human consumption, and do not include wild germplasm. For example, in an embodiment, a "cultivated plant" is a hybrid plant.

[0067] In the scope of the present invention, "allele" is understood to refer to the alternative or variant forms of different genetic units that are identical or relevant to the different forms of a gene, which are alternative in heredity because they are at the same locus of a homologous chromosome. Such alternative or variant forms can be the result of a single nucleotide polymorphism, insertion, inversion, translocation or disappearance, or the result of gene regulation (caused by, for example, chemical or structural modification), transcriptional regulation or post-translational modification / regulation. In diploid cells or organisms, two alleles of a given gene or genetic element typically occupy a corresponding locus on a homologous chromosome.

[0068] The term "trait" refers to a characteristic or phenotype. In the context of the present invention, a nematode resistance trait is an improved nematode resistance trait. Traits can be inherited in a dominant or recessive manner or in a partially or incompletely dominant manner. Traits can be monogenic or polygenic, or can be produced by the interaction of one or more genes with the environment. Traits in plants can be homozygous or heterozygous.

[0069] The terms "hybrid," "hybrid plant," and "hybrid progeny" refer to individuals produced from genetically different parents (eg, individuals that are genetically heterozygous or predominantly heterozygous).

[0070] The term "inbred strain" refers to a population that is homozygous or nearly homozygous genetically. For example, an inbred strain can be obtained by several cycles of brother / sister breeding or selfing or doubled haploid production.

[0071] The term "doubled haploid line" refers to a stable inbred line derived from anther culture. Some pollen grains (haploid) cultured in a specific culture medium and environment can develop into plantlets containing n chromosomes. These plantlets are then "doubled" and contain 2n chromosomes. The offspring of these plantlets are called "doubled haploids" and are essentially no longer segregating (stable).

[0072] The term "cultivar" or "variety" refers to a horticulturally derived variety that is different from the naturally occurring variety. In some embodiments of the invention, the cultivar or variety is commercially valuable.

[0073] The term "stock" refers to a plant used as a scion plant receptacle. Typically, the stock plant and the scion plant have different genotypes. In an embodiment, a plant according to the present invention is used as a stock plant.

[0074] The term "genetically fixed" refers to a genetic element that has been stably incorporated into a plant genome that does not normally contain the genetic element. When genetically fixed, the genetic element can be spread to other plants in an easy and predictable manner through sexual hybridization.

[0075] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0076] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0077] A "plant organ" is a distinct and distinct structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0078] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Any plant tissue in a plant or in culture is included. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with any specific type of plant tissue as listed above or otherwise covered by this definition or alone is not intended to exclude any other type of plant tissue.

[0079] As used herein, the term "breeding" and its grammatical variants refer to any process of producing progeny individuals. Breeding can be sexual or asexual, or any combination thereof. Exemplary non-limiting breeding types include hybridization, selfing, generation of doubled haploid derivatives, and combinations thereof.

[0080] As used herein, phrase " breeding population established " refers to the set of potential breeding partners produced by the parent in a breeding program (for example, a commercial breeding program) and / or used as a parent. The members of the breeding population established are typically fully characterized in terms of gene and / or phenotype. For example, several phenotypic traits of interest may have been evaluated, for example under different environmental conditions, in multiple locations and / or at different times. Alternatively or in addition, one or more genetic loci relevant to the expression of phenotypic traits may have been identified, and one or more members of the breeding population may have been genotyped with respect to the one or more genetic loci and with respect to the one or more gene markers relevant to the one or more genetic loci.

[0081] As used herein, the phrase "diploid individual" refers to an individual having two sets of chromosomes, typically one set from each of its two parents. However, it should be understood that in some embodiments, a diploid individual may receive its "maternal" and "paternal" sets of chromosomes from the same single organism, such as when a plant self-pollinates to produce successive generations of plants.

[0082] "Homozygous" is understood within the scope of the present invention to mean identical alleles at one or more corresponding loci on homologous chromosomes.

[0083] "Heterozygous" is understood within the scope of the present invention to mean different alleles at one or more corresponding loci on homologous chromosomes.

[0084] A "dominant" allele is understood within the scope of the present invention to mean an allele which determines the phenotype when present in the heterozygous or homozygous state.

[0085] A "recessive" allele is one that determines the phenotype when present only in the homozygous state.

[0086] "Genetic locus" is understood within the scope of the present invention to mean a region on a chromosome that contains a gene or any other genetic element or factor that contributes to a trait.

[0087] As used herein, "marker locus" refers to a region on a chromosome that contains nucleotides or polynucleotide sequences that are present in an individual's genome and are associated with one or more loci of interest, which may contain genes or any other genetic determinants or factors that contribute to a trait."Marker locus" also refers to a region on a chromosome that contains a polynucleotide sequence that is complementary to a genomic sequence (such as a sequence of a nucleic acid used as a probe).

[0088] As used herein, the phrases "sexual crossing" and "sexual reproduction" in the context of the presently disclosed subject matter refer to the fusion of gametes to produce offspring (e.g., by fertilization, such as in plants by pollination to produce seeds). In some embodiments, "sexual crossing" or "cross-fertilization" is the fertilization of one individual by another individual (e.g., cross-pollination in plants). The term "selfing" in some embodiments refers to the production of seeds by self-fertilization or self-pollination; that is, the pollen and ovules are from the same plant.

[0089] As used herein, phrase "genetic marker" refers to the feature (for example, present in the nucleotide or polynucleotide sequence in the individual genome) associated with one or more loci of interest in an individual genome. In certain embodiments, gene marker is polymorphic in a colony of interest, or loci are occupied by polymorphism, depending on context.Genetic marker includes for example single nucleotide polymorphism (SNP), indel (that is, insertion / deletion), simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), cutting amplified polymorphic sequence (CAPS) mark, diversity array technology (DArT) mark and amplified fragment length polymorphism (AFLP) and many other examples.Genetic marker can be for example used for locating the genetic locus of the allele that comprises the variability that contributes to phenotypic traits on chromosome.Phrase "genetic marker" can also refer to the polynucleotide sequence complementary to genomic sequence (such as the sequence of the nucleic acid used as probe).

[0090] "Gene marker" can be physically located on a chromosome with a genetic locus associated therewith inside or outside (i.e., correspondingly inside or outside a gene). In other words, although the position of a gene or functional mutation on a chromosome corresponding to a locus of interest (e.g., inside a control element outside a gene) has not yet been identified and a non-zero recombination ratio is typically used between a gene marker and a locus of interest, the subject matter disclosed by the present invention can also use a gene marker physically located within the border of a genetic locus (e.g., inside a genomic sequence corresponding to a gene, such as, but not limited to, a polymorphism within an intron or exon of a gene). In some embodiments of the subject matter disclosed by the present invention, one or more gene markers are included in between one and ten markers, and in some embodiments, one or more gene markers include more than ten gene markers.

[0091] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. "A set of gene-marked genotypes" of an individual include the specific alleles of one or more gene marker loci present in the haplotype of the individual. As known in the art, genotypes can relate to a single locus or multiple loci, whether these loci are related or unrelated, and / or are linked or non-linked. In certain embodiments, the genotype of an individual relates to one or more related genes, because one or more of these genes participate in the expression of a phenotype of interest (e.g., quantitative traits as defined herein). Thus, in certain embodiments, genotypes include the summary of one or more alleles present at one or more genetic loci of an individual intrinsic quantitative trait. In certain embodiments, genotypes are represented by haplotypes (defined below).

[0092] As used herein, the term "germplasm" refers to the totality of the genotype of a population or other individual group (e.g., species). The term "germplasm" can also refer to plant material, e.g., a group of plants that serve as a repository of different alleles. The phrase "adapted germplasm" refers to plant material that has been confirmed to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "exotic germplasm" refer to plant material with unknown or unconfirmed genetic value; for example, for a given environment or geographic region; like this, the phrase "unadapted germplasm" refers in some embodiments to plant material that does not belong to an established breeding population and has no known relationship with a member of an established breeding population.

[0093] As used herein, the phrase "nucleic acid" refers to any physical monomer unit string that can correspond to a string of nucleotides, including polymers of nucleotides (e.g., typical DNA, cDNA, or RNA polymers), modified oligonucleotides (e.g., oligonucleotides containing bases that are not typical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc. In some embodiments, nucleic acids can be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise indicated, a specific nucleic acid sequence of the subject matter disclosed herein optionally includes or encodes a complementary sequence in addition to any sequence explicitly indicated.

[0094] As used herein, the term "plurality" refers to more than one. Thus, "plurality of individuals" refers to at least two individuals. In some embodiments, the term plurality refers to more than half of a whole. For example, in some embodiments, "plurality of a population" refers to more than half of the members of that population.

[0095] As used herein, the term "progeny" refers to one or more offspring of a particular cross. Typically, progeny are produced by the breeding of two individuals, but some species (particularly some plants and hermaphroditic animals) can be selfed (i.e., the same plant serves as the donor of both male and female gametes). The one or more offspring can be, for example, F 1 、F 2 or any succession.

[0096] The term "recipient plant" is used herein to indicate a plant that will receive DNA obtained from a donor plant, the DNA comprising a modified allele to improve lesion-forming pathogen resistance.

[0097] "Donor plant" is understood within the scope of the present invention to mean a plant that provides modified alleles associated with improved resistance to lesion-forming pathogens. As used herein, the phrase "quality trait" refers to a phenotypic trait that is controlled by one or a few genes that exhibit most of the phenotypic effects. Thus, quality traits are usually simply inherited.

[0098] "Marker-based selection" is understood within the scope of the present invention to mean the detection of one or more nucleic acids from a plant (wherein the nucleic acid is associated with a desired trait), for example using a genetic marker, to identify plants carrying the gene for the desired (or undesirable) trait, so that those plants can be used (or avoided) in a selective breeding program.

[0099] "Single nucleotide polymorphism (SNP)" is a DNA sequence variation that occurs when a single nucleotide in a genome (or other consensus sequence) - A, T, C or G - differs between paired chromosomes of members of a biological species or individuals. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case, there are two alleles: C and T. The basic principles of SNP arrays are the same as DNA microarrays. These are a fusion of DNA hybridization, fluorescence microscopy, and DNA capture. The three components of SNP arrays are an array containing nucleic acid sequences (i.e., amplified sequences or targets), one or more labeled allele-specific oligonucleotide probes, and a detection system that records and interprets hybridization signals.

[0100] "PCR (polymerase chain reaction)" is understood within the scope of the present invention to mean a method for generating relatively large amounts of specific regions of the genomic DNA or one or more subsets, thereby allowing for possible different analyses based on those regions.

[0101] "PCR primer" is understood within the scope of the present invention to mean a relatively short single-stranded DNA fragment used in PCR amplification of a specific region of DNA.

[0102] "Phenotype" is understood within the scope of the present invention to mean one or more distinguishable characteristics of a genetically controlled trait.

[0103] As used herein, the phrase "phenotypic trait" refers to an outward or other detectable characteristic in an individual resulting from the interaction of the individual's genome, proteome, and / or metabolome with the environment.

[0104] "Polymorphism" is understood within the scope of the present invention to mean the presence in a population of two or more different forms of a gene, a genetic marker or a genetic trait or a gene product obtainable, for example, by alternative splicing, DNA methylation, etc.

[0105] "Selective breeding" is understood within the scope of the present invention to mean a breeding program which uses plants having or showing desirable traits as parents.

[0106] "Tester" plants are understood within the scope of the present invention to mean plants used to genetically characterize traits in test plants. Typically, the test plant is crossed with a "tester" plant and the segregation ratio of the trait in the hybrid progeny is scored.

[0107] As used herein, "probe" refers to a group of atoms or molecules that can recognize and bind to a specific target molecule or cell structure and thus allow the detection of the target molecule or structure. In particular, "probe" refers to a labeled DNA or RNA sequence that can be used to detect the presence of a complementary sequence and quantify it by molecular hybridization.

[0108] As used herein, the term "hybridization" refers to conventional hybridization conditions, preferably the following hybridization conditions, wherein 5xSSPE, 1% SDS, 1 times Denhardt solution (Denhardts solution) are used as solutions, and / or the hybridization temperature is between 35°C and 70°C, preferably 65°C. After hybridization, preferably washing is first performed with 2xSSC, 1% SDS, and then washing with 0.2xSSC at a temperature between 35°C and 75°C (especially between 45°C and 65°C, especially 59°C) (for the definition of SSPE, SSC and Denhardt solution, see Sambrook et al. in the above citation). High stringency hybridization conditions such as described by Sambrook et al. above are particularly preferred. If hybridization and washing occur at 65°C as described above, for example, there are particularly preferred stringent hybridization conditions. Non-stringent hybridization conditions such as hybridization and washing performed at 45°C are less preferred and are even less preferred at 35°C.

[0109] According to the present invention, the term "position corresponding to position X" (X being any number that can be found in the corresponding context of the present application) includes not only the corresponding position in the subsequently mentioned SEQ ID NO, but also any sequence corresponding to a Pub17 allele or encoding a Pub17 protein, wherein, after alignment with the reference SEQ ID NO, the corresponding position may have a different number, but corresponds to the number indicated by the reference SEQ ID NO. Alignment of Pub17 alleles or Pub17 protein sequences can be achieved by applying various alignment tools in a reasonable manner, for example by applying the tools described below.

[0110] "Sequence identity". In the context of two or more nucleic acids or protein sequences, the term "identical" or "identity" refers to, as measured using one of the following sequence comparison algorithms or by visual estimation, when comparing and aligning maximum correspondence, two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides. If the two sequences compared to each other are of different lengths, sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence with the nucleotide residues of the longer sequence having identity. As used herein, the number of spaces and the length of each space (need to introduce them in the optimized comparison of the two sequences) are considered, and the percentage identity / homology between the two sequences is a function of the number of identical positions shared by these sequences (that is, the total number of the number / position of % identity=identical positions x 100). The comparison of sequences and the determination of the percentage identity between the two sequences can be completed using a mathematical algorithm, as described below. For example, sequence identity can be routinely determined by using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489 to find the segment with the maximum sequence identity between two sequences. When Bestfit or another sequence alignment program is used to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and homology gaps are allowed to account for up to 5% of the total number of nucleotides in the reference sequence. When using Bestfit, preferably the so-called optional parameters are kept at their preset ("default") values. Deviations occurring in a comparison between a given sequence and the above-mentioned sequences of the invention may be caused by, for example, additions, deletions, substitutions, insertions or recombination.Preferably, such sequence comparisons can also be performed using the program "fasta20u66" (version 2.0u66, September 1998, written by William R. Pearson and the University of Virginia; see also WRPearson (1990), Methods in Enzymology 183, 63-98, the examples attached, and http: / / workbench.sdsc.edu / ). For this purpose, the "default" parameter settings can be used.

[0111] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase: "specifically hybridizes" refers to a molecule that binds, duplexes or hybridizes only to a specific nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binds" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and encompasses minor mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0112] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is found in the following literature: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes [Biochemistry and Molecular Biology Laboratory Technology-Hybridization with Nucleic Acid Probes] Chapter 2 Part I "Overview of principles of hybridization and the strategy of nucleic acid probe assays [Hybridization principles and nucleic acid probe assay strategy overview]" Elsevier [Elsevier Group], New York. Usually, for a specific sequence at a defined ionic strength and pH, highly stringent hybridization and washing conditions are selected to be about 5°C lower than the thermal melting point. Typically, under "stringent conditions", the probe will hybridize to its target subsequence, but not to other sequences.

[0113] "Thermal melting point" is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes with a fully matched probe. Very stringent conditions are selected to be equal to the melting temperature (T.sub.m) of a specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids (which have more than 100 complementary residues on filter paper in a DNA or RNA blot) is 50% formamide with 1 mg of heparin, at 42 ° C, and the hybridization is carried out overnight. An example of high stringency washing conditions is 0.15 M NaCl, which lasts for about 15 minutes at 72 ° C. An example of stringent washing conditions is 0.2x SSC washing at 65 ° C for 15 minutes (for a description of SSC buffer, see Sambrook, below). Typically, low stringency washing is performed before high stringency washing to remove background probe signals. An example of medium stringency washing for a duplex of, for example, more than 100 nucleotides is 1x SSC at 45 ° C for 15 minutes. An example of low stringency washing for a duplex of, for example, more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. Typically, in a specific hybridization assay, a signal-to-noise ratio of 2 times (or higher) observed for non-related probes indicates that specific hybridization has been detected. If the proteins encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, the nucleic acids remain substantially identical. For example, when a nucleic acid copy is generated using the maximum codon degeneracy allowed by the genetic code, this occurs.

[0114] As used herein, "homologue" refers to a functionally equivalent protein, i.e., having the same activity as the Pub17 protein having the amino acid sequence defined herein, but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. Homologues may have a lower sequence identity with the Pub17 protein identified herein, such as at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or higher sequence identity, but are able to perform the same function.

[0115] As used herein, an "orthologue" is a protein that is a homologue and therefore functionally equivalent but is found in a different species, ie has the same activity as the Pub17 protein defined herein but is present in a different plant species.

[0116] Tomato Plants

[0117] As used herein, a "plant" is any plant at any stage of development. Suitably, in most embodiments of the invention, the plant is a tomato plant. Suitably, in most embodiments of the invention, the plant is any one of the following tomato species: Solanum lycopersicum, Solanum habrochaites, Solanum pimpinellifolium, Solanum pennellii, Solanum arcanum, Solanum cheesmaniae, Solanum chilense, Solanum chmielewskii, Solanum corneliomulleri, Solanum galapagense, Solanum neorickii, or Solanum peruvianum. Suitably, in most embodiments of the invention, the plant is a tomato plant.

[0118] Suitably, the plant may be any variety or cultivar of tomato such as, for example: alicante, adoration, azoychka, beefsteak, better boy, black krim, brandywine, campari, celebrity, cherokee, early girl, fourth of July, garden peach, gardeners delight, germajohnson, guilette F1, granadero, great white, green zebra, hanover, hillbilly, japanese black trifele, jersey boy, jubilee, Juliet, lillians yellow, matt's wildcherry, micro-tom, moneymaker, monterosa, montserrat, mortgage lifter, mr. stripey, pantano romanesco, plum, Raf, rebellion, currant, roma, Rutgers, san Marzano, Santorini, super sweet, tomacco, yellow pear, zebra. Suitably, the plant may be a cultivated tomato plant.

[0119] However, in some embodiments, the plant can be any Solanaceae plant. Suitably, the plant can be selected from any Solanaceae plant, such as tomato, tobacco, pepper, potato or eggplant. Suitably, the plant can be selected from any Solanaceae genus of plant, for example: Lycianthes, Cestrum, Nolana, Physalis, Lycium, Solanum, Brunfelsia and Nicotiana. Suitably, the plant may be selected from any Solanaceae species of plant, for example: potato (Solanum tuberosum), tomato (Solanum lycopersicum), bell pepper (Capsicum annuum), pepper species (Capsicum sp.), chili pepper (Capsicum frutescens), eggplant (Solanum melongena), cape gooseberry (Physalis peruviana), mint golden lantern (Physalis pruinosa), tomatillo (Physalis philadelphica), Nicotiana rustica (Nicotiana rustica) and common tobacco (Nicotiana tabacum).

[0120] Suitably, any reference to a tomato plant, part or material thereof as used herein may be replaced with another Solanaceae plant, part or material thereof.

[0121] In one embodiment, the plant is a crop, or an economically and / or agriculturally valuable plant. In one embodiment, the plant is a Solanum crop.

[0122] In some embodiments, the plant is an inbred plant, a doubled haploid plant, or a hybrid plant.

[0123] Plant parts or materials

[0124] The term "plant" or "plant part" or "plant material" refers hereinafter to a plant part, organ or tissue obtainable from a tomato plant according to the invention, including but not limited to leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristems, callus, seeds, cuttings, cells or tissue cultures. Suitably, any reference to a "plant" herein also encompasses a plant part or material.

[0125] Suitably the plant part or material may be any plant part, organ or tissue obtainable from a cultivated tomato plant, suitably from a cultivated tomato plant, suitably from a cultivated tomato plant of the present invention.

[0126] Suitably, the tomato plant part or material still exhibits improved resistance to lesion-forming pathogens compared to a reference tomato plant part or material. In some embodiments, this resistance may only exist when the part or material is grown into a tomato plant. Thus, suitably, the tomato plant part or material exhibits a reduction in the level, activity or expression of the Pub17 protein compared to a reference tomato plant part or material. Suitably, the tomato plant part comprises a modified Pub17 allele and is suitably capable of expressing the modified Pub17 allele.

[0127] Suitably, the term plant material may include propagation material obtainable from a tomato plant according to the present invention. Suitable propagation material may be cuttings, roots, fruits, tubers, bulbs, rhizomes, meristems, and the like. Suitably, the propagation material still exhibits improved resistance to lesion-forming pathogens compared to a reference propagation material. Thus, suitably, the propagation material exhibits a reduction in the level, activity, or expression of Pub17 protein compared to a reference propagation material. Suitably, the propagation material comprises a modified Pub17 allele and is suitably capable of expressing the modified Pub17 allele. Suitably, the propagation material may be propagated into a tomato plant, suitably propagated into a tomato plant having improved resistance to lesion-forming pathogens compared to a reference tomato plant; suitably propagated into a tomato plant having reduced levels, activity, or expression of Pub17 protein compared to a reference tomato plant; suitably propagated into a tomato plant comprising a modified Pub17 allele and capable of expressing the modified Pub17 allele. "Propagation" refers to the process of growing a plant from a plant part or material (e.g., a plant protoplast or explant). Such regeneration techniques rely on the manipulation of certain plant hormones in the tissue culture growth medium. The choice of propagation step method is not important. See, for example, Ammirato et al., Handbook of Plant Cell Culture—Crop Species [Plant Cell Culture Handbook—Crop Species] Macmillan Publ. Co. [Macmillan Publishing Company] (1984).

[0128] The invention also extends to fruit. In another aspect of the invention there is provided fruit produced by a tomato plant according to the invention.

[0129] Suitably, the fruit is a tomato fruit. Suitably, the fruit may be obtained from a cultivated tomato plant, more preferably a cultivated tomato plant of the invention. Suitably, the tomato fruit still exhibits improved resistance to lesion-forming pathogens compared to a reference tomato fruit. Suitably, the tomato fruit exhibits a reduction in Pub17 protein levels, activity or expression compared to a reference tomato fruit. Suitably, the tomato fruit comprises a modified Pub17 allele, and suitably is capable of expressing the modified Pub17 allele.

[0130] The invention also extends to one or more seeds. In another aspect of the invention there is provided a tomato seed produced by a plant according to the invention.

[0131] As used herein, "plant seeds" are seeds that grow into plants, suitably into tomato plants according to the present invention. The term "seed" encompasses all types of seeds and plant propagules, including but not limited to true seeds, seed pieces, suckers, grains, bulbs, fruits, tubers, grains, cuttings, cuttings, etc.

[0132] Suitably, the seed is capable of producing a tomato plant exhibiting improved resistance to lesion forming pathogens compared to a reference tomato plant. Suitably, the seed exhibits a reduction in Pub17 protein level, activity or expression compared to a reference tomato seed. Suitably, the seed comprises a modified Pub17 allele and is suitably capable of growing into a tomato plant expressing the modified Pub17 allele.

[0133] In one embodiment, the seed is a tomato seed that produces a tomato plant according to the present invention. Suitably, the tomato seed can be obtained from a cultivated tomato plant, more preferably a cultivated tomato plant of the present invention. Suitably, the tomato seed comprises a modified Pub17 allele and is suitably capable of growing into a tomato plant expressing the modified Pub17 allele.

[0134] Seed can be treated or untreated seed. For example, seed can be treated to improve germination, for example by initiating seed, or by sterilizing to prevent seed-borne pathogens. In another example, seed can be coated with any available coating to improve, for example, plantability, seed emergence and protection for seed-borne pathogens. Seed coating can be any form of seed coating, including but not limited to pelleting, film coating and encrustation.

[0135] Methods for reducing Pub17 protein levels, activity or expression

[0136] Suitably, the level, activity or expression of Pub17 protein may be reduced in the tomato plants of the invention by any means. However, suitably it is not reduced by an essentially biological process.

[0137] Suitably, the term "reducing the level, activity or expression of Pub17 protein" may refer to the underexpression, inhibition or temporal or spatial misexpression of Pub17 polypeptide in a plant or plant material and / or a reduction in the biological effect or activity of Pub17 protein in a plant or plant material. This can be achieved by various standard techniques well known in the art. Suitably, the reduction in the level of Pub17 protein in a plant may be a reduction in the amount of Pub17 protein. Suitably, it is a reduction in the amount of Pub17 protein located in plant cells (e.g., leaf tissue cells) compared to the amount of Pub17 protein in the same tissue of a native plant of the same species (i.e., an unmodified reference plant) at the same stage grown under the same conditions and where the expression level is not intentionally altered.

[0138] Suitably, the level, activity or expression of Pub17 protein is reduced by modifying the tomato plant. Suitably by genetic modification of the tomato plant. Suitably, the genetic modification of the tomato plant may be transient or stable modification. In one embodiment, the resulting tomato plant is stably modified.

[0139] Suitably, stable transformation refers to the incorporation of a polynucleotide into a plant host chromosome, such that the host genetic material can be permanently and heritably altered, and the transformed cell can continue to express the trait caused by this genetic material, even after several generations of cell division. Suitably, transient transformation of a plant cell refers to the cell containing heterologous DNA or RNA and being able to express the trait conferred by the heterologous genetic material without the genetic material being fully incorporated into the cell's DNA.

[0140] Suitably, genetic modification of tomato plants can be achieved by any means known in the art, such as, for example, by random mutagenesis, transformation, homologous recombination or gene editing. Suitable random mutagenesis techniques can be chemical mutagenesis, gamma-ray mutagenesis, UV mutagenesis or X-ray mutagenesis. For example, suitable gene editing techniques can be by CRISPR-Cas systems (particularly CRISPR-Cas9 or CRISPR-Cas13, any reference to Cas9 hereinafter may also refer to other Cas proteins, such as Cas13), zinc finger nucleases or TALENs.

[0141] Alternatively, the level, activity or expression of Pub17 protein is reduced by inhibition. Suitably, the expression of Pub17 protein is inhibited by inhibiting the expression of Pub17 gene. Suitably, inhibition of Pub17 gene expression can be achieved using any means known in the art, such as by using RNAi, miRNA, siRNA, nuclease-deficient CRISPR / Cas system, i.e., for example CRISPRi, modified TALE or zinc finger.

[0142] Suitably, the resulting tomato plant or plant material may be transgenic or non-transgenic. In one embodiment, the tomato plant or plant material is non-transgenic.

[0143] The plant, part or plant material of the present invention may be transgenic, i.e. it is produced by a method involving a gene transfer event of some degree; that is, genetic material from one species has been isolated and transferred and incorporated into the genetic material of a recipient plant using gene transfer methods well known to those skilled in the art. The method may also include synthetic nucleic acid sequences produced according to design.

[0144] Alternatively, the plants or plant materials of the invention may be non-transgenic, i.e. the genetic material of the plant, part or cell has been modified by methods involving, for example, Crispr-Cas based gene editing, whereby a modification of the identity of a single nucleotide base or multiple bases is achieved in the genome. Likewise, methods of gene editing of plant genetic material and regeneration of whole plants from the starting point of modified plant protoplasts, plant cells or plant tissues are well known to those skilled in the art.

[0145] In one embodiment, the modification is used to reduce the level, expression or activity of Pub17 protein in a tomato plant. Suitably, the modification is performed by chemical mutagenesis or CRISPR / Cas9-mediated gene editing. Suitably, in such an embodiment, the Pub17 gene sequence is modified. Suitably, the Pub17 gene sequence comprises one or more modifications as a result of the modification method.

[0146] Suitably, chemical mutagenesis can be carried out by exposing the tomato plant to a chemical mutagen, such as ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), NMU (nitrosyl methyl urea), methyl methanesulfonate (MMS), ethidium bromide, psoralen, acridine orange or sodium azide. Suitably, the tomato plant is exposed to EMS. Suitably, the seed of the tomato plant is exposed to EMS, and then the tomato plant is grown from the seed. Suitably, the seed can be pre-soaked in distilled water. Suitably, the seed can be pre-soaked for 2 to 15 hours, suitably for about 8 hours. Suitably, the seed is treated with an EMS dilution of 0.5% to 10%, suitably 1%-5%, suitably 1% EMS dilution. Suitably, the seed is treated for 6 to 48 hours, suitably 12 to 245 hours, suitably for about 12 hours.

[0147] Suitably, CRISPR / Cas9 gene editing is performed by introducing components of the CRISPR / Cas9 system into tomato plants. Suitably, the CRISPR-Cas system allows for target-specific cleavage of genomic DNA under the guidance of a Cas9 endonuclease complexed with a guide RNA (gRNA) that complementarily binds to a target DNA sequence. Suitably, the components of the CRISPR / Cas9 system are a Cas9 endonuclease protein and a suitable guide RNA that is complementary to a target sequence in the plant genome.

[0148] As used herein, the term "guide RNA" or "gRNA" generally refers to an RNA molecule (or group of total RNA molecules) that can bind to a CRISPR system effector (such as a Cas or Cpf1 protein) and help target the Cas or Cpf1 protein to a specific location within a target polynucleotide (such as DNA). The guide RNA can be an engineered single RNA molecule (sgRNA), wherein, for example, the sgRNA comprises a crRNA segment and optionally a tracrRNA segment. The guide RNA can also be a dual-guide system, in which the crRNA and tracrRNA molecules are physically distinct molecules that then interact to form a duplex for recruiting a CRISPR system effector (such as Cas9) and for targeting the protein to a target polynucleotide.

[0149] As used herein, the term "crRNA" or "crRNA segment" refers to an RNA molecule or a portion of an RNA molecule that includes a polynucleotide targeting guide sequence, a stem sequence involved in protein binding, and optionally a 3'-overhang sequence. The polynucleotide targeting guide sequence is a nucleic acid sequence that is complementary to a sequence in a target DNA (e.g., Pub17 allele). This polynucleotide targeting guide sequence is also referred to as a "pre-spacer sequence". In other words, the polynucleotide targeting guide sequence of the crRNA molecule interacts with the target DNA via hybridization (i.e., base pairing) in a sequence-specific manner. In this way, the nucleotide sequence of the polynucleotide targeting guide sequence of the crRNA molecule can vary, and determines the position in the target DNA where the guide RNA and the target DNA will interact.

[0150] The polynucleotide targeting guide sequence of crRNA molecules can be modified (e.g., by genetic engineering) so as to hybridize with any desired sequence in target DNA. The polynucleotide targeting guide sequence of crRNA molecules of the present invention can have a length from about 12 nucleotides to about 100 nucleotides. For example, the polynucleotide targeting guide sequence of crRNA can have the following length: from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt. For example, the polynucleotide targeting guide sequence of crRNA can have a length from about 17 nt to about 27 nt.

[0151] As used herein, the term "tracrRNA" or "tracrRNA segment" refers to an RNA molecule or a portion thereof that includes a protein binding segment (e.g., the protein binding segment is capable of interacting with a CRISPR-associated protein such as Cas9).

[0152] Suitably, the CRISPR / Cas9 complex can be introduced into a tomato plant as one or more polynucleotides and / or proteins. Suitably, the CRISPR / Cas9 complex can be introduced into a tomato plant as one or more polynucleotides encoding components of the complex. Suitably, the one or more polynucleotides can be contained on one or more vectors. Suitably, the CRISPR / Cas9 complex can be introduced into a tomato plant by any known means of transformation. Those skilled in the art will appreciate that the technology of CRISPR / Cas9 gene editing in plants is well known, for example, see Wada, N. et al., (2020) "Precision genome editing in plants: state-of-the-art in CRISPR / Cas9-based genome engineering" BMC Plant Biology [BMC Plant Biology] Vol. 20, Article No.: 234.

[0153] "Transformation" refers to the process of introducing an exogenous nucleic acid molecule (e.g., a recombinant polynucleotide) into a cell or protoplast, and the exogenous nucleic acid molecule is incorporated into the host cell genome or organelle genome (e.g., chloroplast or mitochondria) or is capable of autonomous replication. "Transformed" or "transgenic" refers to a cell, tissue, organ or organism into which an exogenous nucleic acid, such as an expression vector or a recombinant nucleic acid molecule, has been introduced. Suitably, the means of transformation is, or for example, gene transfer by a disarmed Ti plasmid vector carried by Agrobacterium tumefaciens, transformation mediated by Agrobacterium species, vacuum infiltration, floral dip, spraying, particle or microparticle bombardment, protoplast transformation, electroporation, microinjection, electrophoresis, pollen tube pathway, silicon carbide or liposome-mediated transformation, root uptake, direct injection into the xylem or phloem or other forms of direct DNA uptake.

[0154] Suitably, the change of the guide RNA sequence allows the Cas9 endonuclease to be programmed to cut DNA at a site complementary to the guide RNA. Suitable guide RNAs for use in the present invention can be selected from those complementary to or targeting sequences in the Pub17 gene. Suitably, the guide RNA can be complementary to or targeting sequences in the UND domain, U box domain, or ARM repeat domain of the Pub17 gene of suitably SEQ ID NO:1 or its ortholog or homolog. Suitable guide RNAs can be designed using widely available bioinformatics tools to target specific sequences in the Pub17 gene. Suitably, the guide RNA is a single guide RNA. In one embodiment, the guide RNA is selected from one or more of the following sequences: sgRNA1 of SEQ ID NO:17, sgRNA2 of SEQ ID NO:18, sgRNA3 of SEQ ID NO:19, and sgRNA4 of SEQ ID NO:20. Suitably, in some embodiments, more than one guide RNA can be used in combination to guide the CRISPR / Cas9 complex to cut the Pub17 gene at multiple locations. Suitably, in one embodiment of the invention, all four guide RNAs of SEQ ID NOs: 17-20 are used.

[0155] Suitably, the method involving modification of tomato plants preferably introduces one or more modifications into the Pub17 allele of the tomato plant. Thus suitably, the tomato plant comprises a modified Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type allele), the modified Pub17 allele having a mutation such that the level, activity or expression of the Pub17 protein is reduced compared to a reference tomato plant. Suitably, the modified Pub17 allele comprises a specific mutation, which is suitably defined below with respect to a modified Pub17 allele.

[0156] In another embodiment, inhibition is used to reduce the level, expression or activity of Pub17 protein in a tomato plant. Suitably, the inhibition is performed by RNA interference (also known as RNAi). Suitably, in such an embodiment, the Pub17 gene sequence is not modified. Suitably, in such an embodiment, the expression of the Pub17 gene sequence is inhibited or suppressed. Suitably, in such an embodiment, the expression of the Pub17 gene sequence is silenced.

[0157] Suitably, RNAi suppression is carried out by introducing one or more polynucleotide sequences of the coding RNAi agent complementary to the target DNA sequence into tomato plants. Two types of small RNA molecules are the core of RNA interference, i.e., microRNA (miRNA) and small interfering RNA (siRNA). These small RNAs can guide the enzyme complex to degrade messenger RNA (mRNA) molecules, and therefore reduce their activity by preventing translation via post-transcriptional gene silencing. In addition, transcription can be suppressed by the silencing mechanism before transcription of RNA interference, by which the enzyme complex catalyzes DNA methylation at the genomic position complementary to the compound siRNA or miRNA.

[0158] Therefore, in some embodiments of the invention, an inhibitory RNA is used, such as siRNA, miRNA or another RNAi for inhibiting the expression of Pub17 protein. The inhibitory RNA can be synthesized and delivered to the plant, or it can be expressed in the plant from a suitable expression construct.

[0159] RNAi and its implementation methods are well known in the art. RNAi agents can be chemically or enzymatically synthesized extracellularly and subsequently delivered to cells (see, e.g., Fire et al., Nature [Nature], 391:806-11 (1998); Tuschl et al., Genes and Dev. [Gene and Development], 13:3191-97 (1999); and Elbashir et al., Nature [Nature], 411:494-498 (2001)); or can be expressed in vivo by appropriate vectors in cells (see, e.g., U.S. Patent No. 6,573,099).

[0160] Suitably, the RNAi agent is a miRNA or siRNA. Suitably, the RNAi agent comprises a polynucleotide sequence complementary to a target sequence in the Pub17 gene. Suitable means for transforming a tomato plant with such a polynucleotide sequence or a vector comprising the polynucleotide sequence are described above.

[0161] Suitable RNAi agent sequences can be selected from those that are complementary to or target sequences in the UND domain or U box domain of the Pub17 gene of SEQ ID NO:1 or its ortholog or homolog. In one embodiment, the sequence of the RNAi agent is selected from any one of the following sequences: RNAi3 of SEQ ID NO:11 and RNAi7 of SEQ ID NO:12. Suitably, in some embodiments, more than one RNAi agent can be used in combination. Suitably, in one embodiment of the invention, RNAi agents of SEQ ID NO:11 and 12 are used.

[0162] Modified Pub17 alleles and Pub17 protein

[0163] In some embodiments, the tomato plant comprises a modified Pub17 allele that reduces the level, expression or activity of the corresponding Pub17 protein. Suitably, the modified Pub17 allele is not the result of a substantially biological process. Suitably, the modified Pub17 allele is artificially produced. In some embodiments, the tomato plant according to the present invention, or any plant part, seed or product thereof, is not obtained solely by a substantially biological process. Suitably, the modified Pub17 allele causes increased resistance to lesion forming pathogens.

[0164] In one embodiment, the tomato plant comprises two copies of the modified Pub17 allele and is therefore homozygous for the modified Pub17 allele.

[0165] Suitably, the modified Pub17 nucleic acid sequence is at least 70% identical to SEQ ID NO: 1 (wild-type allele) or an orthologue or homologue thereof, wherein the nucleic acid sequence comprises a mutation which reduces the level, activity or expression of the Pub17 protein. Suitably, the modified Pub17 nucleic acid sequence is a modified Pub17 allele.

[0166] Suitably, the modified Pub17 nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1 (wild-type allele) or its orthologue or homologue. Suitably, at any level of identity, the sequence still comprises a mutation that reduces the level, activity or expression of the Pub17 protein. Suitably, the modified Pub17 nucleic acid sequence is a modified Pub17 allele.

[0167] Suitably, the modified Pub17 nucleic acid sequence may comprise one or more than one mutation. Suitably, each mutation reduces the level, activity or expression of the Pub17 protein. Suitably, the modified Pub17 nucleic acid sequence is a modified Pub17 allele.

[0168] Suitably, the modified Pub17 nucleic acid sequence comprises a mutation in the second half of the nucleic acid sequence according to SEQ ID NO: 1 or in the second half of its orthologous or homologous nucleic acid sequence. Suitably, the modified Pub17 nucleic acid sequence comprises a mutation in the 3' region of the nucleic acid sequence according to SEQ ID NO: 1 or in the 3' region of its orthologous or homologous nucleic acid sequence. Suitably, the 3' region of the nucleic acid sequence is considered to be the half closest to the 3' end of the nucleic acid sequence. Suitably, the 3' region can be the last 50%, last 40%, last 30%, last 20%, last 10% or last 5% of the nucleic acid sequence (when reading in the 5' to 3' direction).

[0169] Suitably, the modified Pub17 nucleic acid sequence comprises a mutation in the ARM region of SEQ ID NO:1 (wild-type allele), or a mutation in a region corresponding thereto in a direct homologue or homologue thereof. Suitably, the ARM region is a region of the nucleic acid sequence encoding one or more ARM (Armadillo) repeat sequences. Suitably, with reference to a Pub17 nucleic acid sequence, such as a nucleic acid sequence of SEQ ID NO:1, the ARM region is a region encoding four ARM (Armadillo) repeat sequences. Suitably, the modified Pub17 nucleic acid sequence comprises a mutation in one or more of the regions encoding the first, second, third and / or fourth ARM repeat sequences of SEQ ID NO:1 (wild-type allele), or a mutation in a region corresponding thereto in a direct homologue or homologue thereof.

[0170] Suitably, the mutation is a SNP. Suitably, the SNP is an A to T SNP. Suitably, the mutation is at nucleotide position 1477 of SEQ ID NO:1 (wild-type allele) or at a position corresponding thereto, for example in its ortholog or homolog. Therefore suitably, the modified Pub17 nucleic acid sequence comprises an A to T SNP at position 1477 of SEQ ID NO:1 (wild-type allele) or at a position corresponding thereto. Suitably, the modified Pub17 nucleic acid sequence comprises a mutation in the region encoding the second ARM repeat sequence. Suitably, the modified Pub17 nucleic acid sequence comprises an SNP in the region encoding the second ARM repeat sequence. Suitably, the modified Pub17 nucleic acid sequence comprises an A to T SNP in the region encoding the second ARM repeat sequence. Suitably, the modified Pub17 nucleic acid sequence is a modified Pub17 allele.

[0171] Thus suitably, the modified Pub17 nucleic acid sequence is at least 70% identical to SEQ ID NO: 1 (wild-type allele) or an orthologue or homologue thereof, wherein the nucleic acid sequence comprises an A to T SNP at position 1477 of SEQ ID NO: 1 (wild-type allele) or a position corresponding thereto, such that the level, activity or expression of the Pub17 protein is reduced. Suitably, the modified Pub17 nucleic acid sequence is a modified Pub17 allele.

[0172] In one embodiment, the modified Pub17 allele comprises SEQ ID NO: 2 (modified allele). In one embodiment, the modified Pub17 allele consists of SEQ ID NO: 2 (modified allele).

[0173] Further aspects of the invention relate to an isolated nucleic acid sequence according to SEQ ID NO: 2 (modified allele), and to vectors, expression cassettes and host cells comprising said sequence.

[0174] Alternatively, in some embodiments, the mutation is a deletion. Suitably, a deletion of a portion of the ARM region. Thus suitably, the modified Pub17 nucleic acid sequence comprises a deletion in the ARM region, suitably encoding one or more of the regions of the first, second, third and / or fourth ARM repeat sequences of SEQ ID NO: 1 (wild-type allele) or a deletion in a region corresponding thereto in its ortholog or homolog. In one embodiment, the modified Pub17 nucleic acid sequence comprises a deletion in a region encoding the first, second and third ARM repeat sequences of SEQ ID NO: 1 (wild-type allele) or a deletion in a region corresponding thereto in its ortholog or homolog.

[0175] Suitably, the Pub17 protein is encoded by a Pub17 nucleic acid sequence. Thus, suitably, the Pub17 protein is also modified. Suitably, the Pub17 protein modification is caused by modification of the Pub17 nucleic acid sequence of the Pub17 allele as described above.

[0176] Suitably, the Pub17 protein is truncated. Suitably, the truncation is caused by a premature stop codon in the Pub17 nucleic acid sequence. Suitably, the premature stop codon is caused by a mutation in the Pub17 nucleic acid sequence, suitably caused by a SNP mutation in the Pub17 nucleic acid sequence. Suitably, the mutation causing the premature stop codon is present in the 3' region of the Pub17 nucleic acid sequence as described above. Suitably, the mutation causing the premature stop codon is present in the last 50%, last 40%, last 30%, last 20%, last 10% or last 5% of the 3' end of the Pub17 nucleic acid sequence. Suitably, the mutation causing the premature stop codon is present in the ARM region, particularly in the region encoding the second ARM repeat sequence.

[0177] Therefore, suitably, the modified Pub17 protein is truncated at the C-terminus. Suitably, the Pub17 protein is not modified in the UND domain or the U box domain. Therefore, suitably, the modified Pub17 protein is truncated at the C-terminus until within the ARM region, suitably until the fourth, third, second or first ARM repeat sequence. Suitably, the ARM region of the PUB17 protein is a region comprising one or more ARM (Armadillo) repeat sequences. Suitably, the ARM region of the PUB17 protein is a region comprising four ARM (Armadillo) repeat sequences. Suitably, with reference to the Pub17 protein sequence of SEQ ID NO:3, the ARM region is between amino acids 429 and 682, or within the corresponding amino acids in its orthologs or homologs. Suitably, the modified Pub17 protein is truncated at the C-terminus until the second ARM repeat sequence, or until the first ARM repeat sequence. Therefore, suitably, the modified Pub17 protein does not contain a complete fourth, third or second ARM repeat sequence. Thus, suitably, the modified Pub17 protein comprises only the intact first ARM repeat sequence.

[0178] In one embodiment, the Pub17 protein is truncated up to position R493 of SEQ ID NO: 3 (wild-type protein) or a position corresponding thereto, such as in an orthologue or homologue thereof.

[0179] Alternatively, as described above, the Pub17 protein may comprise a deletion. Suitably, the Pub17 protein may comprise a deletion of one or more of the first, second, third or fourth ARM repeat sequences. In one embodiment, the Pub17 protein may comprise a deletion of the first, second and third ARM repeat sequences. Thus, in one embodiment, the Pub17 protein may comprise only the fourth ARM repeat sequence.

[0180] Suitably, the Pub17 protein comprises an amino acid sequence according to SEQ ID NO: 3 (wild-type protein) or a portion of its orthologue or homologue. Suitably, the Pub17 protein comprises an amino acid sequence according to SEQ ID NO: 3 (wild-type protein) or a portion of its orthologue or homologue. Suitably, the Pub17 protein comprises an amino acid sequence according to at least 50%, 60%, 70%, 80% or 90% of the total length of its orthologue or homologue. Suitably, the Pub17 protein does not consist of SEQ ID NO: 3 or a orthologue or homologue thereof. Suitably, the Pub17 protein comprises an amino acid sequence according to about 70%, more specifically 72% of the total length of SEQ ID NO: 3 (wild-type) or a orthologue or homologue thereof.

[0181] Suitably, the modified Pub17 protein consists of an UND domain, a U-box domain and a first ARM repeat sequence. Suitably, the modified Pub17 protein consists of amino acids 1-493 of SEQ ID NO: 3 (wild-type protein).

[0182] Suitably, the modified Pub17 protein consists of an amino acid sequence according to SEQ ID NO: 4 (modified protein) or a portion thereof having at least 70% identity thereto. Suitably, the modified Pub17 protein consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto with SEQ ID NO: 4 (modified protein) or a portion thereof.

[0183] Suitably, the modified Pub17 protein consists of the amino acid sequence according to SEQ ID NO: 4 (modified protein) or a part thereof.

[0184] Suitably, the portion of SEQ ID NO: 4 may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total length of SEQ ID NO: 4 (the modified protein).

[0185] Suitably, the modified Pub17 protein may comprise one or more than one further mutation. Suitably, each mutation results in a reduction in the level, activity or expression of the Pub17 protein.

[0186] Further aspects of the invention relate to an isolated polypeptide sequence according to SEQ ID NO: 4 (modified protein) or a portion thereof, and a host cell comprising the polypeptide.

[0187] In another aspect of the present invention there is provided a plant or plant part or seed comprising a modified Pub17 protein as defined herein.

[0188] Lesion-forming pathogens

[0189] Suitably, the lesion forming pathogen may be any pathogen that forms one or more lesions on the tissue of the tomato plant. Suitably on the stems, leaves and / or fruits of the tomato plant. Suitably, the lesions may be localized necrosis or chlorotic areas of diseased tissue.

[0190] Suitably, the lesion forming pathogen may be a biotrophic, hemibiotrophic or necrotrophic pathogen.In one embodiment, the lesion forming pathogen is a necrotrophic pathogen.

[0191] Suitably, the pathogenesis pathogen may be a bacterium, fungus, virus, protozoa or archaea.

[0192] Suitably, the pathogenesis pathogen is a fungus or a virus or an oomycete, or any combination thereof.

[0193] Suitably, the lesion forming pathogen is an oomycete. Suitably, the lesion forming oomycete may be selected from: Phytophthora infestans, Hyaloperonospora arabidopsidis, Phytophthora aramorum, Phytophthora sojae, Phytophthora capsici, Plasmopara viticola, Phytophthora cinnamomi, Pythium ultimum, Albugo candida and Phytophthora parasitica.

[0194] Suitably, the lesion-forming pathogen is a virus. Suitable lesion-forming viruses may be selected from one or more of the following: for example, tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), pomegranate mosaic virus (PepMV), cucumber mosaic virus (CMV), potato virus Y (PVY), combined leaf streak TMV+CMV, tobacco etch virus (TEV), tomato ringspot virus (TRSV), tomato sterility virus (TAV), tomato yellow leaf curl virus (TYLCV) and tomato brown rugose fruit virus (ToBRFV).

[0195] Suitably, the lesion forming pathogen is a fungus. Suitable lesion-forming fungi may be selected from any of the following species: Heterospora, Anthospora anthracis, Victoria victoria, Alternaria alternata, Alternaria solani, Alternaria brassicae, Periconia circinata, Pyrenophora tritici-repentis, Bipolaris sacchari, Phyllostictamaydis, Stagnospora nodorum, Stemphylium vesicarium, Botrytis fabae, Botrytis elliptica, Botrytis cinerea, Sclerotinia sclerotiorum, Mollinia fructicola, Fusarium graminearum, Septoria tritici, Cercospora zeae-maydis, Exserohilum turcicum, Leptosphaeria maculans, Ascochyta rabiei, Diaporthe toxica, Phoma medicaginis, Leptosphaerulina trifoli, Pseudopeziza medicaginis, Stemphyllium botryosum, Stagonospora metiloti, Pleiochaetasetosa, Fusarium oxysporum, Rhizoctonia solani, and Pythium species.

[0196] Suitably, the lesion forming pathogen is a pathogen affecting tomato plants. Suitably, for this, the tomato plant is the host. Thus, suitably, the lesion forming pathogen is a tomato pathogen, suitably a necrotrophic tomato pathogen, suitably a necrotrophic fungal tomato pathogen or a necrotrophic viral tomato pathogen. Suitable necrotrophic fungi affecting tomato plants may be selected from Alternaria alternata, Alternaria solani, Botrytis cinerea, Sclerotinia sclerotiorum, Pseudomonas sclerotiorum, Fusarium oxysporum and Pythium species. Suitable necrotrophic viruses affecting tomato plants may be selected from Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Brown Rugose Fruit Virus (ToBRFV).

[0197] Suitably, the lesion forming pathogen belongs to the genus Botrytis or Alternaria. Thus, suitably, the lesion forming pathogen may be selected from the following species: Alternaria alternata, Alternaria solani, Alternaria brassicae, Botrytis viciae, Botrytis ellipsoidea and Botrytis cinerea.

[0198] Alternatively, the lesion forming pathogen may be a hemibiotrophic tomato pathogen, suitably a hemibiotrophic oomycete tomato pathogen. Suitable hemibiotrophic oomycetes affecting tomato plants may be Phytophthora infestans, Phytophthora capsici or Phytophthora parasiticus.

[0199] In one embodiment, the lesion forming pathogen is Botrytis cinerea or Alternaria solani. In one embodiment, the lesion forming pathogen is Tomato Brown Rough Fruit Virus (ToBRFV). In one embodiment, the lesion forming pathogen is Phytophthora infestans.

[0200] Suitably, the lesion-forming pathogen causes a disease in the plant. Suitable diseases may be selected from the group consisting of: wilt, botrytis wilt, gray mold, white mold, early blight, late blight, leaf blight, powdery mildew, rot, leaf spot, fruit rot, brown spot, black spot, brown spot, gray spot, head blight, ear rot, spot, stem canker, stem blight, black stem, crown rot, wilt, root rot and damping-off of seedlings.

[0201] Suitably, the lesion forming pathogen causes a disease that is a necrotic disease, suitably wherein cell death occurs. Suitably, the lesion forming pathogen causes a disease selected from: a wilt, such as Botrytis blight, early blight or late blight; a mildew, such as gray mold; and a rot. Thus, suitably, the plants of the invention have increased resistance or decreased susceptibility to necrotic diseases. Suitably, increased resistance or decreased susceptibility to: a wilt, such as Botrytis blight, early blight or late blight; a mildew, such as gray mold; or a rot. In one embodiment, the plants of the invention have increased resistance or decreased susceptibility to a wilt, suitably to Botrytis blight.

[0202] In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by lesion forming pathogens. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by lesion forming fungi or oomycetes or viral pathogens. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by necrotrophic or hemibiotrophic pathogens. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by necrotrophic or hemibiotrophic fungi or oomycete pathogens. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by Botrytis or Alternaria pathogens. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by Botrytis cinerea or Alternaria solani, suitably Botrytis cinerea. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by Phytophthora infestans. In one embodiment, the tomato plants of the present invention have increased resistance to wilt caused by Tomato Brown Rugose Fruit Virus (ToBRFV).

[0203] Suitably, the tomato plants of the invention may have increased resistance to more than one lesion forming pathogen, and thus may have increased resistance to more than one disease. Suitably, the tomato plants of the invention may have increased resistance to a combination of lesion forming pathogens as described herein, or any combination of diseases as described herein, which diseases suitably may be caused by lesion forming pathogens.

[0204] Suitably, the tomato plants of the invention may have increased resistance to any combination of fungal lesion forming pathogens, viral lesion forming pathogens and / or oomycete lesion forming pathogens. Suitably, the tomato plants of the invention may have increased resistance to any combination of the pathogens listed above. Suitably, the tomato plants of the invention may have increased resistance to any combination of Botrytis cinerea, Alternaria solani, Tomato Brown Rough Fruit Virus (ToBRFV) and / or Phytophthora infestans.

[0205] Increased resistance

[0206] Suitably, the tomato plants of the invention have increased resistance relative to a reference tomato plant, suitably increased resistance to lesion forming pathogens relative to a reference tomato plant.

[0207] A suitable reference tomato plant is a control plant. Suitably, such a reference tomato plant comprises the same genetic background as the tomato plant of the present invention, but it does not comprise a reduction in Pub17 protein levels, expression or activity. Suitably, the reference tomato plant may be a wild-type plant. Suitably, the reference tomato plant may be a tomato plant of the same plant variety as the plant of the present invention, and does not comprise a reduction in Pub17 protein levels, expression or activity. The term "plant variety" is understood herein according to the definition of UPOV. Suitably, the reference tomato plant has not been modified to reduce Pub17 protein levels, expression or activity. Suitably, the reference tomato plant does not contain a modified Pub17 allele. Suitably, the reference tomato plant contains a wild-type Pub17 allele. Suitably, the reference tomato plant is grown for the same length of time under the same conditions as the tomato plant of the present invention. Suitably, the reference tomato plant may be a near-isogenic line, an inbred line or a hybrid, provided that it has the same genetic background as the tomato plant of the invention, except that the reference tomato plant has not been modified to reduce Pub17 protein levels, expression or activity, and suitably does not contain the modified Pub17 allele of the invention.

[0208] For example, a reference tomato plant in the context of the present invention may comprise the tomato reference genome HEINZ or the tomato reference genome of Moneymaker (https: / / www.ebi.ac.uk / ena / browser / view / SAMEA2340764).

[0209] Suitably, the tomato plants of the invention have a statistically significant increase in resistance to one or more lesion forming pathogens compared to a reference tomato plant.

[0210] Suitably, the resistance to lesion-forming pathogens can be measured by a significant reduction in the number of lesions per tomato plant or plant material. Suitably, this can be measured using, for example, a Mann-Whitney test (α=1%, 2.5%, or 5%) or a Student's test (P<0.05). Suitably, plant of the present invention has at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% less lesions than reference tomato plants. Suitably, tomato plants of the present invention have 25%-50% less lesions than reference tomato plants.

[0211] Suitably, resistance to lesion forming pathogens can be measured by a significant reduction in the diameter of lesions on tomato plants or plant material, suitably a reduction in the average diameter of lesions on tomato plants. Suitably, this can be measured using, for example, a Mann-Whitney test (α=1%, 2.5% or 5%) or a Student's test (P<0.05). Suitably, the tomato plants of the present invention have lesions that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% smaller than the lesions on the reference tomato plants, suitably measured by the average diameter of the lesions on the tomato plants. Suitably, the plants of the present invention have lesions that are 20%-30% smaller than the lesions on the reference tomato plants, suitably measured by the average diameter of the lesions on the tomato plants.

[0212] Suitably, the measure of such resistance is calculated after the step of exposing the tomato plant or part thereof to the lesion forming pathogen for a suitable period of time. A suitable period of time is the amount of time sufficient for the lesion forming pathogen to infect the tomato plant or part thereof and cause lesions to appear. A suitable period of time may be 1-28 days, suitably 1-14 days, suitably 1-7 days after the tomato plant or part thereof is exposed to the lesion forming pathogen.

[0213] Screening methods

[0214] Aspects of the invention further relate to methods of identifying or selecting tomato plants having increased resistance to one or more lesion forming pathogens.

[0215] Suitably, these methods relate to tomato plants in identification groups, which have the desired character of increased resistance to one or more pathological pathogens. Suitably, the plant population can be a mutant population or a wild population of tomato plants. Suitably, the mutant population of tomato plants can be produced by mutagenesis, as described elsewhere herein. Suitably by chemical mutagenesis, suitably by using chemical mutagens such as EMS. Therefore, suitably, these methods can include the initial step of obtaining or producing a mutant population of tomato plants suitably by EMS mutagenesis.

[0216] Suitably, the tomato plants may be directly identified as being resistant to the lesion forming pathogen, alternatively or additionally, the tomato plants may be indirectly identified by having reduced Pub17 protein levels, expression or activity.

[0217] Suitably, the resistance of a tomato plant to lesion-forming pathogens can be determined by inoculation or exposure assays. Suitably, the tomato plant is exposed to lesion-forming pathogens, and its reaction is assessed compared to a reference tomato plant. The increased resistance to lesion-forming pathogens can be determined by such assays, such as detached leaf assays, such as those performed in the examples herein. Therefore, suitably, these methods may include the step of inoculating or exposing assays on the or each strain tomato plant, optionally on a tomato plant population. Suitably, such assays may include identifying plants that exhibit a reduction in lesion number and / or average size when exposed to lesion-forming pathogens compared to a reference plant. Suitable reduction levels are defined elsewhere herein.

[0218] Alternatively or additionally, resistance of tomato plants to lesion-forming pathogens can be determined by identifying tomato plants with modified Pub17 alleles. Suitably, tomato plants with modified Pub17 alleles are identified, and the modified Pub17 alleles have mutations as described above. Suitably, this can be determined by molecular methods such as PCR or genome sequencing of tomato plants. Suitably, this can be determined by genotyping of tomato plants. Genotypic evaluation of plants includes the use of techniques such as isoenzyme electrophoresis, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), arbitrary primer polymerase chain reaction (AP-PCR), allele-specific PCR (AS-PCR), DNA amplification fingerprint (DAF), sequence characteristic amplification region (SCAR), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) (also referred to as "microsatellite").

[0219] "DNA sequencing" refers to determining the nucleic acid sequence of a section of DNA (e.g., a gene). Standard methods and commercial services are known in the art. Basic methods for DNA sequencing include the Maxam-Gilbert method and the chain termination method. High-throughput technologies have also been developed and are preferably used in the methods of the present invention. These high-throughput technologies include, but are not limited to, massively parallel signature sequencing (MPSS), Polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, combined probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, and nanopore DNA sequencing.

[0220] Suitably, the presence or absence of the modified Pub17 allele can be determined by PCR, for example real-time PCR using a double-stranded DNA dye, or a fluorescent reporter probe. Suitably, a specific primer pair complementary to the modified Pub17 allele associated with the kit as described herein is used. Suitably, the primer pair detects the presence of a SNP at position 1477 of the Pub17 allele according to SEQ ID NO: 1 or at a corresponding position thereof. Thus, suitably, these methods may comprise the steps of performing PCR using suitable primers (such as those defined for the kit) and subsequently sequencing the resulting amplicon, or optionally performing genome sequencing of the or each tomato plant in a tomato plant population.

[0221] As used herein, the term "primer" refers to an oligonucleotide that can anneal to a nucleic acid target and serve as a starting point for DNA synthesis when placed under conditions that induce synthesis of primer extension products (e.g., in the presence of nucleotides and reagents for polymerization (e.g., DNA polymerase) and at a suitable temperature and pH). Primers (in some instances, extension primers, in some instances, amplification primers) can be single-stranded to achieve maximum efficiency for extension and / or amplification. Primers can be oligodeoxyribonucleotides. Primers are typically long enough to initiate the synthesis of extension and / or amplification products in the presence of reagents for polymerization. The minimum length of a primer can depend on many factors, including but not limited to the temperature and composition (A / T vs. G / C content) of the primer. In the case of amplification primers, these amplification primers are typically provided as a pair of bidirectional primers consisting of a forward and a reverse primer, or as a pair of forward primers commonly used in the field of DNA amplification (e.g., in PCR amplification).

[0222] Thus, suitably, a method of identifying or screening a tomato plant may comprise using a SNP at position 1477 of the Pub17 allele according to SEQ ID NO: 1 or a position corresponding thereto as a marker to identify the presence of resistance to a lesion forming pathogen in a tomato plant. Suitably identifying the presence of a modified Pub17 allele of the invention in a tomato plant.

[0223] Therefore, another aspect of the invention is the use of a SNP at position 1477 of the Pub17 allele of SEQ ID NO: 1 or a position corresponding thereto for identifying and / or diagnosing selection and / or genotyping lesion forming pathogen resistance alleles in tomato plants or parts thereof, suitably in cultivated tomato plants.

[0224] Suitably, the identification or screening method further comprises the step of selecting the or each tomato plant identified as having reduced Pub17 protein levels, expression or activity, and / or thereby selecting the or each tomato plant identified as having increased resistance to one or more lesion forming pathogens.

[0225] Suitably, the identification or screening method may further comprise the steps of breeding the selected tomato plant or the selected tomato plant of each strain, suitably to form a progeny. Suitably, the screening method may further comprise the steps of screening progeny and breeding a selected progeny with a desired trait in several additional rounds. Suitably, this may comprise the additional step of screening the progeny for the presence of the desired trait as explained above, and one or more additional steps of breeding a selected progeny.

[0226] Hybrids and breeding methods

[0227] A hybrid or cultivar may be produced by crossing a first plant of the invention with a second reference plant and obtaining progeny. Suitably, the hybrid or cultivar is a tomato plant.

[0228] Suitably, producing a hybrid tomato plant comprises crossing a first tomato plant according to the invention with a second reference tomato plant as defined above. Suitably, the reference tomato plant does not have a reduction in the level, expression or activity of the Pub17 protein, and suitably, the reference tomato plant does not have a modified Pub17 allele as described herein. However, suitably, the reference plant is of the same species as the tomato plant of the invention, and suitably is of the same variety as the tomato plant of the invention. Suitably, the reference plant and plant of the invention are tomato plants.

[0229] Suitably, the first plant comprises at least one copy, suitably two copies, of a modified Pub17 allele of the invention.

[0230] Suitably, there is provided a method of providing a cultivated tomato plant, preferably a cultivated tomato plant, plant part or seed, wherein the method comprises the following steps:

[0231] a) crossing a first plant according to any one of the preceding embodiments with a second plant that does not have the Pub17 allele of the present invention,

[0232] b) obtaining progeny plants, and

[0233] c) optionally selecting said progeny plants, characterised in that said plants exhibit improved resistance to lesion forming pathogens.

[0234] Suitably the tomato plants are hybridized to produce progeny, suitably hybrid progeny. Suitably, the method of producing a hybrid tomato plant may optionally further comprise selecting from the progeny a hybrid tomato plant that exhibits increased resistance to lesion forming pathogens and / or comprises reduced Pub17 protein levels, expression or activity. Suitably, the selection of hybrid tomato plants having the desired trait may be achieved using the screening techniques described above. Thus, suitably, the selection step may be performed by performing PCR with primer pairs of SEQ ID NO:24 and 25, suitably followed by sequencing of the resulting amplicon to detect the presence of the modified Pub17 allele of the present invention. Alternatively, the selection step may comprise selecting a plant in the progeny that exhibits a reduction in the number and / or average size of lesions when exposed to lesion forming pathogens compared to a reference plant. Suitable levels of reduction are defined elsewhere herein.

[0235] Another aspect of the present invention relates to a method for producing a cultivated tomato plant, preferably a cultivated tomato plant, which exhibits improved resistance to lesion-forming pathogens compared to a reference tomato plant, the method comprising the following steps:

[0236] a) providing seeds of tomato plants according to the invention;

[0237] b) germinating the seeds and growing mature fertile tomato plants therefrom;

[0238] c) inducing the tomato plant described in a) to self-pollinate, grow tomato fruits and harvest fertile seeds therefrom; and

[0239] d) Growing tomato plants from seeds harvested in c) and selecting tomato plants having increased resistance to lesion forming pathogens.

[0240] Suitably, selection of tomato plants having desirable traits may be achieved using the screening techniques described above.

[0241] Therefore, the plants exemplified herein can be used for breeding programs to develop other plants with at least partial resistance to lesion-forming pathogens, such as commercial varieties of such plants. According to such methods, the first parent plant can be used for hybridization with the second parent plant, wherein at least one of the first parent plant or the second parent plant contains at least one nucleic acid molecule encoding the modified Pub17 allele described herein. An application of this process is to produce F1 hybrid plants. Another aspect of this process is that this process can be used to develop new parents, double haploids or inbred strains. For example, plant strains as described herein can be hybridized with any second plant, and the resulting hybrid progeny are self-pollinated and / or sibling mated for about 5 to 7 generations or more, thereby providing a large number of different parent strains. These parent strains can then be hybridized with other strains, and the beneficial properties of the resulting hybrid progeny are analyzed. In this way, novel strains with desired characteristics can be identified. Various breeding methods can be used in this method, including haploid, pedigree breeding, single seed generation, improved single seed generation, recurrent selection and backcrossing.

[0242] use

[0243] Another aspect of the present invention relates to the use of tomato plants of the present invention or parts thereof, or seeds for growing tomato plants and producing and harvesting crop yields, seeds, and / or fruits therefrom. Suitably, methods for growing plants are known in the art. In one embodiment, crop yields are suitably tomato fruits.

[0244] Another aspect of the present invention relates to the use of the tomato plant of the present invention or its part, or seed for sowing fields, greenhouses, or greenhouses. In another embodiment, the present invention relates to the use of the cultivated plant (preferably cultivated tomato plant, more preferably cultivated tomato plant), plant part or seed according to any one of the preceding embodiments as a rootstock plant.

[0245] Another aspect of the invention relates to the use of lesion forming pathogen resistant propagation material obtainable from a tomato plant according to the invention for growing tomato plants. Suitably, the lesion forming pathogen resistance may be determined in an assay, suitably by assaying the propagation material. A suitable assay may be a detached leaf assay according to the examples herein. Alternatively, the lesion forming pathogen resistance of the propagation material may be determined by molecular methods to identify the presence of the modified Pub17 alleles described herein. Suitably growing tomato plants from the propagation material may be carried out by culturing the propagation material according to techniques known in the art.

[0246] Another aspect of the invention relates to the use of a modified Pub17 allele of the invention to confer increased resistance to lesion-forming pathogens on a tomato plant that does not have the allele. Suitably, further details of the modified Pub17 allele are provided above. Suitably, the modified Pub17 allele may be introduced into a plant, or the plant may be modified to comprise the modified Pub17 allele of the invention. Suitable techniques for providing a plant having a modified Pub17 allele of the invention are described above.

[0247] Another aspect of the invention relates to the use of a tomato plant according to the invention for introgressing a lesion forming pathogen resistance trait into a tomato plant not having said trait. Suitably, the trait is conferred by a modified Pub17 allele. Suitably, further details of the modified Pub17 allele are defined above. Suitably, methods of introgressing traits into plants are known in the art.

[0248] Reagent test kit

[0249] The present invention further provides a kit for detecting a lesion forming pathogen resistance trait allele in a tomato plant. Suitably, it is a kit for detecting a modified Pub17 allele of the present invention in a plant. In one embodiment, the plant is a tomato plant.

[0250] Suitably, such a kit may be used in the screening methods described above.

[0251] Suitably, the kit comprises at least one PCR primer pair having a forward primer and a reverse primer that specifically bind to the Pub17 coding sequence. Suitably, the primers used can specifically bind to the Pub17 gene, or specifically bind to a modified Pub17 allele. Suitably, the primers that bind to the Pub17 gene can bind to the region of the gene that is flanked by modifications, suitably flanking the SNP at position 1477 of SEQ ID NO:1. Therefore, suitably, in a PCR using such primers, subsequent sequencing is used to identify the modified allele (if present). Suitably, primers that specifically bind to the modified Pub17 allele can directly detect the presence of the modification. Suitably, a subsequent sequencing step may not be required.

[0252] Suitably, the kit may comprise a PCR primer pair comprising a forward and reverse primer complementary to the Pub17 coding sequence. Suitably, the forward primer consists of SEQ ID NO:25. Suitably, the reverse primer consists of SEQ ID NO:24. Thus, suitably, the kit may be used to detect modified Pub17 alleles, suitably by subsequently sequencing the resulting amplicon. Suitably, in such embodiments, the kit is for use in a gene-specific PCR.

[0253] Suitably, the resulting amplicon is produced from PCR. Suitably, the amplicon is sequenced, the amplicon being included in position 1477 of SEQ ID NO:1 (wild-type Pub17 allele) or in its corresponding position (e.g., in a straight homologue or homologue sequence) comprising a SNP from A to T. Therefore, suitably, the SNP is used as a marker, suitably the A1477T mutation is used as a marker, suitably as a marker for resistance to lesion-forming pathogens.

[0254] Suitably, the kit may alternatively comprise a PCR primer pair comprising a forward primer and a reverse primer that specifically binds to a modified Pub17 allele of the present invention. Suitably, the forward primer specifically binds to a modified Pub17 allele of the present invention. Suitably, the forward primer is complementary to the modified Pub17 sequence. Suitably, the forward primer consists of SEQ ID NO:31. Suitably, the reverse primer consists of SEQ ID NO:32. Suitably, the kit may further comprise a second forward primer that specifically binds to a wild-type Pub17 allele. Suitably, the second forward primer is complementary to an unmodified Pub17 sequence. Suitably, the second forward primer consists of SEQ ID NO:30. Suitably, the kit may comprise all three primers: a first forward primer according to SEQ ID NO:31, a reverse primer according to SEQ ID NO:32, and a second forward primer according to SEQ ID NO:30. Suitably, the kit may comprise all three primers: a first forward primer according to SEQ ID NO:31, a reverse primer according to SEQ ID NO:32, and a second forward primer according to SEQ ID NO:30. Thus, suitably, the kit can detect both the modified Pub17 allele and the wild-type Pub17 allele (if present).

[0255] Suitably, in such an embodiment, the kit is for use in allele-specific PCR, suitably competitive allele-specific PCR (also known as KASP PCR), which is for example described in (Semagn et al. 2014). Thus, suitably, each forward primer comprises an indicator molecule, such as a fluorescent molecule. Suitably, the indicator molecule is tethered to the forward primer, suitably tethered to the first and second forward primers. Suitable fluorescent molecules may be FAM or HEX. Suitably, in an embodiment, the first forward primer comprises FAM and the second forward primer comprises HEX.

[0256] Suitably, the resulting amplicon is produced from PCR. Suitably, the amplicon is sequenced, the amplicon being included in position 1477 of SEQ ID NO:1 (wild-type Pub17 allele) or in its corresponding position (e.g., in a straight homologue or homologue sequence) comprising a SNP from A to T. Therefore, suitably, the SNP is used as a marker, suitably the A1477T mutation is used as a marker, suitably as a marker for resistance to lesion-forming pathogens.

[0257] Therefore, suitably, the present invention further discloses the use of the SNP markers according to the present invention for diagnostic selection and / or genotyping of lesion forming pathogen resistance trait alleles in cultivated plants, particularly cultivated tomato plants, more particularly cultivated tomato plants.

[0258] The present invention further discloses the use of the SNP markers according to the present invention for identifying the presence of lesion forming pathogen resistance trait alleles in plants (especially cultivated tomato plants, more especially tomato plants according to the present invention) and / or for monitoring the introgression of lesion forming pathogen resistance trait alleles in cultivated plants (especially cultivated tomato plants, more especially tomato plants according to the present invention and as described herein).

[0259] Suitably, the SNP markers are identified by one of the above PCR methods, suitably using the above primers.

[0260] Suitably, the kit may further comprise other components suitable for performing PCR, such as polymerase, salts, buffers, instructions, etc.

[0261] Another aspect of the invention relates to an amplification product obtained from PCR involving said primer pair which is associated with a lesion forming pathogen resistance trait and thus co-segregates with the lesion forming pathogen resistance trait or with a disclosed marker. Suitably, the amplification product is a nucleic acid.

[0262] Another aspect of the invention is a polynucleotide having at least 70% identity to or hybridizing to the amplified product, suitably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the amplified product. Suitably, the amplified product can be used to generate new primers and / or probes for identifying modified Pub17 alleles. Suitably, these are derived markers or probes genetically associated with lesion forming pathogen resistance traits. Suitably, such derived markers or probes can also be used to identify plants with increased resistance to lesion forming pathogens.

[0263] Examples

[0264] Materials and Methods

[0265] Plant materials

[0266] Two different tomato cultivars were used in this experiment: cv. Little Tom (MT) and cv. Moneymaker (MM). MT seeds were obtained from Beekenkamp Plants BV company (Maasdijk, The Netherlands).

[0267] Development of the Little Tom EMS Group

[0268] Considering the advantages offered by the tomato cultivar Little Tom (MT), such as small size, the possibility of growing at high density, and having a short life cycle (Meissner et al. 1997), MT was selected as the tomato cultivar for the EMS population in our laboratory (Yan et al. 2021). A total of five batches of approximately 1000 MT seeds (M 0 ) were pre-soaked in distilled water for 8 hours and treated with 1% EMS dilution overnight. 1 Seeds were washed thoroughly with distilled water and sown in a greenhouse. Plants were grown at 21°C / 19°C day / night temperatures at 60% relative humidity during a 16h day / 8h night regime. Three-week-old seedlings were transplanted individually into 14 cm pots and grown until ripe fruits could be harvested. The collected M 2 Seeds were surface sterilized in 2% HCl (hydrogen chloride) and subsequently treated with 10% trisodium phosphate (TSP) solution for at least one hour and then air dried. From the first two batches, 5 to 10 fruits / plant were harvested. However, for the last three batches, all developed fruits were harvested to collect more seeds.

[0269] Botrytis cinerea disease test

[0270] The potato detached leaf assay (DLA) was performed according to a modified version of the one described by Sun et al. (2017). The DLA of MT plants consisted of taking the middle leaflet of the third leaf from each plant, 20 per M2 family, and placing them on a water agar medium ( The DLA of MM consisted of taking the third leaf (3 terminal leaflets, left, middle and right) from 6-week-old plants and placing all three leaflets in a square Petri dish prepared as above. The adaxial side of the leaf was inoculated with 5-6 drops of 2 μl of Botrytis cinerea strain B05.10 (Amselem et al. 2011). The spores were suspended in a mixture of PDA (potato dextrose agar) and PDB (potato dextrose broth) (final concentration was half-strength PDB (12 g / l)) and 0.3% agar at a density of 1 x 10 6 Spores / ml. After inoculation, the culture dishes were grouped (16-18 culture dishes per group) and each group was assembled into a tray containing a damp filter paper. The tray was placed in a plastic bag to obtain 100% humidity. They were maintained at 18°C ​​(16h light / 8h dark). The lesion diameter on the leaf was measured using a caliper with a digital display (Mitutoyo nr 500-161-30, Mitutoyo Nederland BV, Veenendaal, The Netherlands) at 3 and 4 days (dpi) after inoculation.

[0271] The stem assay was performed by cutting the 3rd, 4th and 5th leaves from 6-week-old plants, leaving a petiole stump of approximately 2.5 cm. The petiole surface was inoculated with 10 μl of Botrytis cinerea strain B05.10 at a density of 1 x 10 6 spores / ml. Plants were kept in a plastic tent with high humidity for 24 hours. Symptoms were scored 3, 6, 10, 14, 17 and 21 days after inoculation. Scoring was based on a 0-4 scale, where 0: unchanged petiole stumps comparable to mock treatment, 1: petiole stumps partially or completely thinned and brown; 2: outward stem infection begins, with small brown rings visible on the main stem around the axils of the inoculated petiole stumps, 3: infection spreads throughout the entire stem, with brown rings becoming irregular and spreading up and down the stem, and 4: complete main stem infection and wilting of the plant, internal browning, collapse of stem tissue, and eventual collapse and overturning of the plant apex ( Fig.15 ). In addition, petiole abscission was recorded.

[0272] Alternaria solani disease test

[0273] The potato detached leaf assay (DLA) was performed following a modified version of the one described by Sun et al. (2017). The DLA for MT followed the same procedure as for Botrytis cinerea. The DLA for MM consisted of taking the terminal leaflets of the 3rd, 4th and 5th true leaves from 6-week-old plants and placing all three leaflets in a square Petri dish prepared as above. Five to six 10 μl drops of the Alternaria solani isolate “altNL03003” (accession number CBS143772) were inoculated on the adaxial side of the leaves. Spore collection was performed as previously reported (Wolters et al. 2019). The spores were suspended in a mixture of PDA (potato dextrose agar) and PDB (potato dextrose broth) (final concentration was half-strength PDB (12 g / l)) and 0.3% agar at a density of 1x10 5 Spores / ml. After inoculation, the culture dishes were grouped (16-18 culture dishes per group) and each group was assembled into a tray containing a damp filter paper. The tray was placed in a plastic bag to obtain 100% humidity. They were maintained at 18°C ​​(16h light / 8h dark). The lesion diameter on the leaf was measured using a caliper with a digital display (Mitutoyo nr500-161-30, Mitutoyo Nederland BV, Veenendaal, The Netherlands) at 5 and 7 days (dpi) after inoculation.

[0274] Additional disease determinations

[0275] Screening for altered susceptibility to tomato powdery mildew (Pseudoidium neolycopersici) Wageningen isolate On-Ne was performed as described by Bai et al. (2003). Disease assays with Phytophthora infestans isolates PIC99177 or C65 were performed using DLA as described by Sun et al. (2016). Testing for resistance to Tomato Yellow Leaf Curl Virus (TYLCV) was performed using Agrobacterium inoculation as described by Verlaan et al. (2011). Tomato Brown Rough Fruit Virus (ToBRFV) resistance was determined by manually infecting 10-day-old seedlings with the ToBRFV-IL isolate (Luria et al. 2017; Genbank accession number KX619418). Leaves were rubbed with emery and subsequently inoculated with juice. Disease symptoms were monitored weekly and scored four weeks after inoculation.

[0276] The development of a separate group

[0277] The mutant M2042 with reduced susceptibility to Botrytis cinerea was selfed until the M4 line was obtained. The M4 line was fixed for the mutation that resulted in lower susceptibility. The confirmed mutant M4 line M2042-1-2-12 was crossed with MM and the F1 seeds were collected. Subsequently, five F1 plants were selfed and the F2 seeds were collected.

[0278] Screening for d and sp mutations in Little Tom

[0279] The tomato cultivar Little Tom contains at least two mutations in the genes Self-Pruning (Sp; Solyc06g074350) and Dwarf (D; Solyc02g089160) (Martí et al. 2006) that are responsible for the small size of the plants. A high-resolution melting (HRM) assay was developed to identify the causal Little Tom SNP for the determinant (sp, self-pruning) phenotype. Forward and reverse primers SP_F (TGAGACGGACAAGATGACATGA) SEQ ID NO:33 and SP_R (TGTCATTTCCCCTTCCAAAGT) SEQ ID NO:34 flanking exon 2 were designed, generating a 218 bp PCR product. These primers were used to generate a PCR product with Phire TM Hot Start DNA Polymerase (ThermoFischer) and LCGreen TM In the PCR of Plus+ (BioChem), System (Idaho Technology), amplification conditions were as follows: 98° C. for 30 s; 98° C. for 5 s, 40 cycles; 57° C. for 5 s; and 72° C. for 15 s, followed by 72° C. for 30 s, 94° C. for 30 s, 25° C. for 30 s, and then cooling to 10° C. Three different genotypes (homozygous SpSp, heterozygous Spsp, and homozygous spsp) could be distinguished by melting curve analysis.

[0280] To identify the causal Tom SNP for the dwarf (d) phenotype in the F2 population, the CAPS marker was used with primer C in exon 8 (GGAACTTGGTGTAGCAGAAATTT CCACATTTC) SEQ ID NO: 5 and primer D in exon 9 (TTAGTGAGCTGAAACTCTAATCCGTAGAC) SEQ ID NO: 6 (Martí et al. 2006). PCR was performed using DreamTaq polymerase with a melting temperature of 60°C. The 243 bp PCR product was then incubated with restriction enzyme HpyCH4V at 47°C for 4 hours. Subsequently, the product was run on a 1.5% TBE gel at 110V for 1 hour. Digestion of the PCR product into 152 bp and 91 bp fragments indicated the presence of the wild-type (MM-like) allele, while the undigested product indicated the presence of the MT allele.

[0281] Identification of mutant genes by cluster segregation analysis combined with whole genome sequencing (BSA-WGS)

[0282] In total, 200 F2 plants from the cross of EMS mutant M2042 and wild-type susceptible Moneymaker were tested for resistance to Botrytis cinerea. Plants showing very small or very large lesion diameter sizes were selected and re-inoculated to confirm resistance or susceptibility. Finally, 18 plants with the smallest lesions and 18 plants with the largest lesions were selected. DNA was isolated from leaf samples of these plants and 10 individual wild-type MT plants using the DNeasy Plant Mini Kit (Qiagen). and (Thermo Fisher Scientific). DNA concentration was determined by equimolar pooling of DNA from resistant and susceptible F2 plants and wild-type MT plants to generate three DNA pools, M2042R, M2042S and MTWT. These DNA pools were sequenced (whole genome resequencing, WGS) by Novogene Ltd (Hong Kong). For this purpose, a 350 bp insert DNA library was prepared. Paired-end sequencing was performed on the HiSeq platform, with a read length of 150 bp (PE150) at each end, and a genome coverage of approximately 35x for each sample. The reads were mapped to the tomato Heinz reference genome (SL2.50 version), and SNP detection was performed using SAMtools.

[0283] A total of 2,659,728 SNPs were identified. For each library, the number of reads containing the reference (Heinz) allele and the number of reads containing alternative alleles at each SNP position were recorded. Many calculations were performed: 1) the sum of the number of reference alleles (REF) and alternative alleles (ALT) reads at each SNP position in each library (total number of reads = coverage = read depth); 2) the percentage of alternative alleles at each SNP position in each library; % ALT = (ALT / [REF + ALT]) * 100; 3) the difference in the percentage of alternative alleles at each SNP position between the resistance library and the susceptible library (% ALT [R] -% ALT [S]). Next, the filtering of each chromosome SNP was performed as follows: 1) only SNPs with a total number of reads ≥ 35 in the resistance library (M2042R) were retained (coverage of at least 35); 2) only SNPs with % ALT [R] ≥ 80 were retained. In theory, alternative alleles should be 100% present in the resistance pool. However, for safety reasons, a lower percentage was selected. Thereafter, the occurrence of more or less continuous regions of each chromosome was examined, where the percentage difference of alternative alleles at each SNP position between M2042R and M2042S was very large (% ALT[R]-% ALT[S]>50). On chromosome 2, there was a 6.6Mbp region between positions 40900926 and 47531242 (SL2.50 reference genome) that met this criterion. The next filtering consisted of the following: SNP positions were selected in the exons of annotated genes, for which alternative alleles were present in the resistance pool and the susceptible pool (M2042R and M2042S), but not in the wild-type Little Tom pool (MTWT). In the chromosome 2 region, under the conditions of filtering SNP position ≥35 coverage, %ALT[R]≥80 and %ALT[R]-%ALT[S]>50, the SNP was observed: [T / A] SNP at position SL2.50ch02:41352738 in gene Solyc02g072080 (SlPUB17), producing a premature stop codon R493*.

[0284] Association of PUB17 mutations with resistance to Botrytis cinerea

[0285] For individual F2 plants of the M2042R and M2042S pools, the sequence of the candidate gene PUB17 was examined to determine the presence and homozygosity of the alternative SNP. The progeny were tested for Botrytis resistance to examine whether segregation for disease resistance occurred in order to link resistance to the mutation.

[0286] Determination of gene expression levels by RT-qPCR

[0287] Gene expression levels were determined by RT-qPCR of plant cDNA synthesized by iScript cDNA synthesis kit (BioRad) using RNA extracted by RNeasy plant mini kit (Qiagen). Specific primers PUB17_qPCR_Fw1 (5'-GGAAGTGAAGGTGTTGCGA-3') SEQ ID NO:7 and PUB17_qPCR_Rv1 (5'-CTACTGCCATTTCCTCATTGC-3') SEQ ID NO:8 were developed for PUB17, generating a 100 bp PCR product. Elongation factor 1 alpha (Ef1α) was used as a reference gene, with primers Ef1a-Fw (5'-ATTGGAAACGGATA TGCCCCT-3') SEQ ID NO:9 and Ef1a-Rv (5'-TCCTTACCTGAA CGCCTGTCA-3') SEQ ID NO:10, generating a 101 bp PCR product. RT-qPCR was performed using a CFX96 real-time PCR instrument (Bio-Rad), with two technical replicates for each sample. The relative expression of PUB17 was expressed as ΔΔC T Calculated according to the method (Livak & Schmittgen 2001).

[0288] RNAi and CRISPR transformation for candidate gene validation

[0289] Two PUB17 RNAi constructs were generated using the binary vector pHellsgate12 (Helliwell and Waterhouse 2003). The vector contains the CaMV 35S promoter driving the expression of the inverted repeat and a kanamycin resistance gene as a selection marker. Primers were designed for PUB17 to amplify a fragment of the tomato gDNA sequence from cv. Moneymaker. The primer sequences are shown in Table 6. RNAi fragment 7 was amplified using the forward primer caccGGTGTGGGAAATTGATGGCA (SEQ ID NO: 15) and the reverse primer AAACGGCAGCCTTTTACCTG (SEQ ID NO: 16), generating a 176 bp product targeting the UND domain of the PUB17 protein. RNAi fragment 3 was amplified using the forward primer caccAGCCCACATCCTCAGTTCTC (SEQ ID NO: 13) and the reverse primer CATATGTCTGCCCTGTTGCC (SEQ ID NO: 14), generating a 240 bp product targeting the U box domain. The forward primer contained CACC at the 5' end for directional cloning into the pENTR / D-TOPO (ThermoFisher) vector. The primers were used to clone the DNA using Phusion TM High-fidelity DNA polymerase (Thermo Fisher Scientific) was used for blunt-end PCR, and the PCR product was purified using QIAquick PCR purification kit (Qiagen). The resulting DNA was cloned into pENTR / D-TOPO and transformed into Escherichia coli DH5α. The culture was plated on LB medium containing spectinomycin (100 μg / ul) and grown overnight at 37°C. The plasmid DNA of these clones was sequenced to verify the presence of the correct insert.

[0290] CRISPR / Cas9 constructs were designed to generate deletions within the PUB17 coding sequence using four sgRNAs with the Cas9 endonuclease gene and the NPTII plant selection marker. sgRNAs were designed using the CCTop-CRISPR / Cas9 target online predictor tool (https: / / crispr.cos.uni-heidelberg.de / ; Stemmer et al. 2015), with the tomato genome (Tomato cv. Heinz SL2.50) as a reference for target site evaluation. From the sgRNA table provided by the online predictor, only sgRNAs without exonic off-target sites were selected. The selected sgRNAs were further controlled by verifying that their GC content was between 30% and 80% (http: / / www.endmemo.com / bio / gc.php), and their secondary structures were evaluated according to Liang et al. (2016) (http: / / unafold.rna.albany.edu / ?q=mfold / RNA-Folding-Form; Zuker, 2003). Additional scoring tools were used to compare, validate and select the best four sgRNAs (https: / / sgrnascorer.cancer.gov / ; Chari et al. 2017), (https: / / portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-de sign; Sanson et al., 2018), (http: / / crispr.wustl.edu / ; Wong et al. 2015). The distance between sgRNA target sites was approximately 600 bp. sgRNAs were selected to target different protein domains, with guide 1 (GGAAATGACCTGAAATCGAA) SEQ ID NO: 17 located in the UND domain, guide 2 (TTCTATATCGAGGTGGATGG) SEQ ID NO: 18 located in the U box domain, and guide 3 (GAGATTTGGGCACACCACAG) SEQ ID NO: 19 and guide 4 (CAGGAACAAAGCGCGCAAGG) SEQ ID NO: 20 located in the ARM repeat domain. Constructs were assembled using the Golden Gate cloning system (Engler et al. 2008).The selected sgRNA was developed by using primers containing the sequence (5'-TGTGGTCTCA [sgRNA sequence] GTTTAGAGCTAGAAATAGCAAG-3') SEQ ID NO: 21 (forward primer) and (5'-TGTGGTCTCAAGCGTAATGCCAACTTTGTAC-3') SEQ ID NO: 22 (reverse primer). Each forward and reverse primer pair was used in a 0-stage reaction with plasmid plCH86966 containing a kanamycin resistance gene as a template. The 0-stage product was then PCR cleaned up (QIAquick PCR purification kit, Qiagen), and the cleaned-up product was used to assemble the 1-stage reaction. The 1-stage reaction consisted of combining plasmid pICHSL01009 (AtU6 promoter), plasmids designated for each guide position (pICH47751, pICH47761, pICH47772, and pICH47781), and the cleaned-up product from the 0-stage reaction. Reactions were performed by digesting the indicated plasmids with BsaI / Eco31I, ligating with T4 DNA (Thermo Scientific, Bleiswijk, The Netherlands) and cloning into E. coli DH5α as follows: pICH47751 (sgRNA guide 1 position 1), pICH47761 (sgRNA guide 2 position 2), pICH47772 (sgRNA guide 3 position 3), and pICH47781 (sgRNA guide 4 position 4). Plasmids were purified using a plasmid preparation kit (Qiagen Benelux BV, Venlo, The Netherlands). The Level 1 construct was assembled into the Level 2 binary vector pAGM4723 along with NPTII (pICH47732), Cas9 (pICH47742) and a linker (pICH41822) by digestion with BpiI / BpsI and ligation with T4 DNA and cloned into E. coli DH5α. The Level 2 construct was purified and sequenced for verification.

[0291] Two RNAi constructs and one CRISPR / Cas9 construct of PUB17 were transformed into electrocompetent Agrobacterium tumefaciens AGL1+virG cells. Transformation of tomato cv.MM was performed as previously described by Huibers et al. (2013).

[0292] Analysis of CRISPR and RNAi transformants

[0293] To determine the presence of mutations in CRISPR transformants, DNA was isolated from young leaves using CTAB buffer (1M Tris-HCl pH 7.5, 0.5M EDTA pH 8.0, 5M NaCl, 2% CTAB). Gene-specific PCR was then performed on genomic DNA using DreamTaq DNA polymerase (Thermo Scientific, Bleiswijk, The Netherlands). Two different forward primers, FWD_MR_GX_CRISPR (5'-ACGGCGTTATCTTCTGAGCT-3') SEQ ID NO: 23 and AWPUB17_F1 (5'-AGAGAGTGGGACGCAGATT-3') SEQ ID NO: 25, were used, paired with reverse primer REV_MR_GX_CRISPR (5'-CATGCTCACACCGTTGGAAT-3') SEQ ID NO: 24, respectively, to generate PCR products of 1942 bp and 827 bp for the wild-type (WT) allele, respectively. The PCR products were sent to Macrogen Europe (Amsterdam, The Netherlands) for sequencing.

[0294] To confirm the integration of the T-DNA of the silencing construct in the genome of the RNAi transformant, PCR was performed to detect the presence of the NPTII gene and the 35S promoter. The forward and reverse primers NPTII_421_Fw (5'-GAAGGGACTGGCTGCTATTG-3') SEQ ID NO: 26 and NPTII_421_Rv (5'-AATATCACGGGTAGCCAACG-3') SEQ ID NO: 27 for detecting the NPTII gene produced a PCR product of 421 bp. The forward and reverse primers 35S_597_Fw (5'-TACAAAGGCGGCAACAAAC-3') SEQ ID NO: 28 and 35S_597_Rv (5'-AGCAAGCCTTGAATCGTCC-3') SEQ ID NO: 29 for detecting the 35S promoter amplified a region of 597 bp.

[0295] Statistical analysis

[0296] ANOVA F test was performed on the data points of each DLA experiment using R studio v 1.1.463 (2016). The ANOVA test was followed by post hoc test using Tukey HSD method for multiple pairwise comparisons.

[0297] Development of KASP markers for pub17 mutations

[0298] Developing KASP TMMarking assay (Semagn et al. 2014) was used to track EMS-induced mutations in the PUB17 gene in the F2 population. Two forward primers were designed: K_RTWT_For1 5'-GAAGGTGACCAAGTTCATGCTGTC TGGCTTTGATAGTTGGAGTTTTGT-3' SEQ ID NO:30 for the wild-type allele and K_RTmut_For1:GAAGGTCGGAGTCAA CGGATTGTCTGGCTTTGATAGTTGGAGTTTTGA SEQ ID NO:31 for the pub17 mutant allele. The reverse primer K_RT_Rev70 5'-GTTGCTGCAGCATTTTC CCGTG-3' SEQ ID NO:32 was used in combination with the forward primer. The forward primer of the WT sequence was labeled with HEX dye, while the forward primer of the mutant sequence was labeled with FAM dye. For PCR KASP V4.0 2X Master mix 96 / 384, Low Rox (LGC Group) was used. PCR was performed according to the KASP thermal protocol provided by the manufacturer (LCG Group). The plate was read with a plate reader (Bio-Rad C1000 thermal cycler) and the data were analyzed using Bio-Rad CFX Maestro 1.1.

[0299] result

[0300] Reduced susceptibility to Botrytis cinerea in tomato mutant M2042

[0301] To identify S genes targeting necrotrophic fungi, the Little Tom EMS population developed at Wageningen University - Plant Breeding (Yan et al. 2021) was screened. The EMS population consisted of 4500 M2 families, of which 692 were screened for phenotypic changes including: dwarfing, light green leaves, changes in leaf shape, changes in flower morphology and color, and changes in fruit color. The M2 families were tested for diseases including late blight (Phytophthora infestans isolate C65 or PIC99177), gray mold (Botrytis cinerea strain B05.10), and powdery mildew (Pseudoidium neolycopersici strain On-Ne), where each plant was tested with three pathogens. The Botrytis cinerea disease assay allowed the identification of M2 family M2042 ( Figure 1A), with plant 1 showing reduced susceptibility compared to the WT control and less mycelial growth 9 days after inoculation. In addition, when the same plants from M2042 were tested for P. infestans, plant 1 showed smaller lesions as well as necrotic spots and less / no mycelial growth 14 days after inoculation compared to the wild type. The mutation in M2042 was fixed in the M4 line, and subsequent Botrytis disease assays confirmed moderate resistance, showing a 20%-30% reduction in lesion diameter compared to Little Tom.

[0302] Early stop codon mutation in PUB17 (Solyc02g072080) in mutant M2042

[0303] The mutations found in M2042 responsible for the reduced lesion size after infection with Botrytis cinerea (termed intermediate resistance, IR) were mapped by cluster segregation analysis and whole genome sequencing (BSA-WGS) approach. Figure 1B ) were subjected to initial phenotypic analysis in order to establish two different pools of resistant and susceptible plants to Botrytis cinerea. First, DLA was performed on all 200 plants and visual inspection was performed on plants showing the smallest and largest lesion diameters. This was followed by a second confirmation and further selection of plants found in extreme cases. Plants with extreme phenotypes were selected for both the resistant and susceptible pools, with 18 plants selected for each pool. As a control, a third pool consisting of wild-type Little Tom plants was developed. As described in the Materials and Methods, an interesting non-synonymous mutation was initially identified by whole genome sequencing and further filtering of SNPs. The mutation is an A→T SNP at position 1477 of the coding region of the gene Solyc02g072080, which produces an early stop codon R493*( Figure 2 ). This gene is the tomato ortholog of PUB17.

[0304] To determine the relative expression levels of candidate genes, RT-qPCR was performed using wild-type MT plants and M4 progenies showing moderate resistance (M2042-1-1-17 and M2042-1-2-12). Leaves were mock-inoculated or inoculated with Botrytis cinerea and sampled at three time points (0, 24, and 48 hours post infection (hpi)). The expression of PUB17 was significantly induced in wild-type MT after infection with Botrytis cinerea ( Figure 3 ). However, PUB17 expression was not induced in mutants M2042-1-1-17 and M2042-1-2-12.

[0305] To examine whether the PUB17 mutation was associated with Botrytis resistance, disease assays were performed using progeny of selected M4 and F2 plants derived from a cross between MM and the M4 plant M2042-1-2-12 ( Figure 1B , Table 1). F3 and M5 progeny plants were tested for resistance to Botrytis cinerea to evaluate whether they segregated phenotypically.

[0306] Table 1. PUB17 genotypes of M2042 M4 control and F2 plants, and the results of Botrytis disease assays of progeny. WT, homozygous for the wild-type allele of PUB17; H, heterozygous for PUB17 SNP; M, homozygous mutant for PUB17 SNP; R, resistant; S, susceptible.

[0307] plant PUB17 Botrytis resistance in progeny Control M4 plants M4 Plant M2042-1-20-19 WT All S M4 Plant M2042-1-2-7 M Both R M4 Plant M2042-1-2-12 M Both R F2 plants F2 Plant 1-66 M Both R F2 Plant 2-59 M Both R F2 Plant 3-10 M Both R F2 Plant 3-26 M Both R F2 Plant 3-39 H Separation

[0308] The progeny of M4 plants M2042-1-2-7 and M2042-1-2-12 and F2 plants 1-66, 2-59, 3-10 and 3-26 (homozygous mutants for PUB17) all showed smaller lesion sizes than the wild-type control M2042-1-20-19. On the other hand, the progeny of F2 plant 3-39, which was heterozygous for the PUB17 gene (one mutant allele, one wild-type allele), showed segregation in response to Botrytis infection (Table 1).

[0309] The F3 progeny of 3-39 was genotyped for the PUB17 gene. In total, 4 homozygous mutant plants were observed in 24 progeny plants (Table 2). All four homozygous mutant plants produced smaller lesions than heterozygous and homozygous wild-type progeny. In short, these results support the hypothesis that the mutation that causes the early stop codon in PUB17 is the causal change of the moderate resistance of M2042 to Botrytis cinerea.

[0310] Table 2. Genotyping and phenotyping results of F3 progeny plants of recombinant 3-39. WT, wild type.

[0311] genotype PUB17 SNP 1477 Plant No. Response to Botrytis Homozygous mutant TT 4 Moderate resistance Heterozygous AT 17 Susceptible Homozygous WT AA 3 Susceptible

[0312] Silencing of PUB17 by RNAi increases resistance to Botrytis cinerea

[0313] To analyze whether silencing of the identified PUB17 gene is sufficient to obtain reduced susceptibility to Botrytis cinerea and does not involve other (mutated) genes, two RNAi constructs targeting PUB17 were prepared, with RNAi fragment 7 (176 bp) targeting the UND domain and RNAi fragment 3 (239 bp) targeting the U-box domain ( Figure 4A total of 50 RNAi transformants were obtained. After the transformants were transferred to the greenhouse, the relative expression levels of PUB17 in 24 RNAi transformants containing fragment 3 and 19 RNAi transformants containing fragment 7 were determined ( Figure 5 ).

[0314] RNAi fragment 3 appeared to be slightly more effective in silencing compared to RNAi fragment 7 ( Figure 5 ). The transformant T1 RNAi3-5 (T2 progeny TV181088, Table 3) with the lowest PUB17 gene expression was selected as the main candidate for further testing. At the same time, it was noted that some of these RNAi transformants showed slight autonecrosis on the leaves, namely one RNAi3 transformant (3-29, TV181105) and one RNAi7 transformant (7-33, TV181136; not included in Figure 5 We thought that this might be the result of silencing PUB17. Therefore, these two RNAi families, TV181105 and TV181136, were also selected for further testing.

[0315] From the isolated T2 families TV181088 and TV181136, individual plants were selected based on the presence of a clear and strong fragment after PCR with NPTII primers (indicating the presence of T-DNA). T3 progeny were obtained from these selected plants (Table 3). Subsequently, the T3 RNAi-silenced transformants of PUB17 were inoculated with strain B05.10 of Botrytis cinerea in stem assays and detached leaf assays (DLA). MM plants and RNAi families showing the absence of NPTII (TV192024) were used as susceptible controls.

[0316] For the stem assay, petiole stumps were inoculated and monitored for 21 days. The Disease Severity Index (DSI) was scored from 0-4, indicating an increase in observed damage or "abs" when the petiole stump fell off. Plants began to show disease symptoms 6 days after inoculation. The two negative controls, MM and TV192024, showed susceptibility to Botrytis cinerea, indicating a relatively high percentage of petioles with DSI 3 or 4. At the same time, PUB17 RNAi-silenced transformants showed a lower level of susceptibility to Botrytis cinerea based on a low percentage of stems showing a DSI of 3 or 4, with families TV192027 and TV192029 having the least sensitive phenotype ( Fig.14 ).

[0317] Table 3. RNAi PUB17 transformants and coding of T2 and T3 progeny. Transformants with RNAi silencing fragments 3 or 7 were obtained ( Figure 4 ).

[0318]

[0319] At the same time, DLA was performed on T3 lines silenced by PUB17 RNAi of T1 transformants 3-5 and 7-33 (Table 3). The lesion diameter on infected leaves was measured at 3 and 4 days post inoculation (dpi). Figure 6 ).

[0320] The results of the DLA test showed that the two controls, MM and TV192024, showed similar Botrytis cinerea lesion diameter sizes ( Figure 6 ). In contrast, significantly smaller Botrytis cinerea lesions were observed on leaves of PUB17 RNAi-silenced families TV192025, TV192026, TV192027, TV192028, and TV192029 at 3 and 4 dpi. Tukey HSD multiple pairwise comparisons were performed on lesion diameters of all groups. When compared to negative control MM and T3 family TV192024, leaves of all PUB17 RNAi-silenced families TV192025, TV192026, TV192027, TV192028, and TV192029 showed significant differences in mean lesion diameter (p<0.05), while negative controls did not show significant differences from each other at both 3 dpi and 4 dpi time points.

[0321] From another RNAi-silenced family, T2 TV181105, T3 progeny were obtained only later. Therefore, the isolated T2 families were tested for Botrytis cinerea, including stem and leaf assays. The T2 plants were genotyped for the presence of NPTII to distinguish transgenic from non-transgenic plants. Based on the low percentage of stems showing DSI 3 and the absence of stems showing DSI 4, the stem test results indicated that the transgenic TV181105 plants had a reduced level of susceptibility to Botrytis cinerea compared to non-transgenic T2 plants and the negative control MM. In the detached leaf assay, the control, MM and plants without NPTII were compared to T2 plants containing NPTII (TV05, Figure 7 ). As with the previously tested PUB17 RNAi family, significant differences were observed between the lesion diameters of transgenic TV181105 plants and controls. Tukey HSD multiple pairwise comparisons confirmed that transgenic T2 TV181105 plants had smaller lesion diameters than the negative controls (p<0.05). Meanwhile, the negative controls also showed statistical differences between each other. CRISPR / Cas9 mutation of wild-type PUB17 results in increased resistance to Botrytis cinerea

[0322] To further test whether mutation of PUB17 in EMS mutant M2042 is sufficient to reduce susceptibility to Botrytis cinerea, CRISPR / Cas9-targeted mutagenesis of PUB17 was performed using a construct with four sgRNAs ( Figure 8 ).

[0323] This construct was used to transform the tomato cv. Moneymaker and 56 transformants were obtained. Fig. 9 A) or only the last 2 sgRNAs ( Fig. 9 Primary transformants were genotyped using specific primer pairs for CRISPR PUB17 (primers are provided in Table 7). Among 56 CRISPR PUB17 transformants, 4 transformants with obvious mutant alleles were identified by PCR and electrophoresis ( Fig. 9 ).

[0324] For CRISPR transformants 9, 21, and 36, in addition to the PCR product with the size of the wild-type allele, smaller PCR products appeared in two PCRs. For plant 46, the small PCR product of about 700 bp indicated that a large deletion occurred between the first and last sgRNA target sites. Therefore, it was not possible to amplify the mutant allele with the primer combination used in group B. At the same time, plant 36 showed 3 clear PCR fragments, which may indicate a mosaic mutant. The bands were cut from the gel and sequenced using the primers used to obtain the PCR products. The sequence of the mutant allele was aligned with the WT sequence to find the exact size of each deletion. The results are shown in Fig.10 shown.

[0325] Small deletion or insertion mutations cannot be identified by gel electrophoresis. Therefore, the PCR products of all transformants showing a band of approximately the WT size were also sequenced. This ultimately identified an additional bi-allelic mutant plant 7, which had a 1 bp deletion and a 1 bp insertion in one mutant allele, while the second mutant allele had two separate 1 bp insertions ( Fig.11 ).

[0326] A summary of the mutations identified is provided in Table 4. The size of the deletion and the position relative to the PUB17 sgRNA selected for the construct are recorded. The majority of mutations occurred in the region targeted by sgRNA3.

[0327] Table 4. CRISPR-induced mutations in the PUB17 gene analyzed in primary transformants

[0328]

[0329] The effect of the mutation on the predicted protein sequence is determined for each mutant allele.

[0330] The deletions in plants 21, 46 and the 5 bp deletion in one of the alleles of plant 36 resulted in a premature stop codon, while the others resulted in out-of-frame mutations.

[0331] T3 progeny can be obtained from T2 plants carrying deletions as small as 345 bp (Table 5). T3 PUB17 CRISPR transformants were simultaneously subjected to the Botrytis cinerea stem assay and DLA using strain B05.10. All transformants tested were homozygous mutants for the indel mutation in PUB17. MM plants and T2 PUB17 CRISPR transformant family TV181133 (which showed no presence of mutation observed in its T1 parent plant 21) were used as susceptible controls.

[0332] Table 5. Homozygous mutant T3 progeny of PUB17 CRISPR mutants.

[0333]

[0334]

[0335] For the stem assay, inoculated petioles were monitored over 21 days and disease symptoms were detected at 6 dpi. Negative control MM and T2 PUB17 transformant family TV181133 showed the highest susceptibility levels to Botrytis cinerea, followed by T3 families TV192008 and TV192012, as indicated by the relatively high percentage of stems with a DSI of 4. On the other hand, T3 PUB17 CRISPR families TV192007, TV192009, TV192016, TV192019, TV192014, and TV192023 all exhibited reduced susceptibility to Botrytis cinerea (based on the low percentage of stems displaying a DSI of 3 or 4) ( Fig.14 ).

[0336] In the detached leaf assay, lesion diameters on infected leaves were measured on days 3 and 4 after inoculation ( Fig.12 ). The two control groups, MM and TV181133, showed similar Botrytis cinerea lesion diameters. Significantly smaller Botrytis cinerea lesion diameters were observed on all eight PUB17 CRISPR mutant T3 families compared to the two negative controls (p<0.001).

[0337] The consequences of mutations in the structure / construction of the PUB17 protein

[0338] To compare the sequence features of PUB17 proteins in stable CRISPR mutants and original PUB17 EMS mutants, a multiple sequence alignment of predicted PUB17 proteins was performed. Protein alignment was performed using Clustal Omega, a multiple sequence alignment program provided by the European Bioinformatics Institute (EMBL-EBI) (Madeira et al., 2022). Protein domains were predicted using the ScanProsite tool (De Castro et al., 2006). The alignment revealed that all mutant alleles contained complete UND (amino acids 20-171) and U-box domains (amino acids 297-364). Mutations in CRISPR mutant alleles 1, 2, and 3 caused frameshift mutations and early stop codons, and CRISPR mutant allele 4 contained a large deletion (345bp), which resulted in the removal of amino acids 454 to 568, but retained the C-terminal region of the coding sequence.

[0339] Given that the Armadillo repeat (ARM, amino acids 429-682) domain is compromised in all mutant alleles, we analyzed the predicted effects of the mutation on the protein domain ( Fig.16 ). The WT allele contains four ARM repeats, while CRISPR mutant alleles 1-3 and the EMS mutant M2042 allele contain only the first ARM repeat, with the second ARM repeat truncated and ARM repeats 3-4 completely missing. CRISPR mutant allele 4 lost the first three ARM repeats but retained the last predicted ARM repeat (ARM4).

[0340] Increased resistance to other pathogens

[0341] As mentioned previously, the PUB17 mutant M2042 was found to show reduced susceptibility to the hemibiotrophic oomycete Phytophthora infestans as well as the necrotrophic fungus Botrytis cinerea. To analyze whether mutations in the PUB17 gene affect susceptibility to other tomato pathogens, the PUB17 mutant was subjected to disease assays with the necrotrophic fungus Alternaria solani. We observed that the original mutant as well as the F3 mutant plants obtained after crossing with MM showed a significant reduction in susceptibility to this fungus, as indicated by a reduction in lesion diameter size (Figure 13). In addition, DLA of Alternaria solani was performed using two PUB17 CRISPR T3 families, TV192007 and TV192023. These CRISPR mutants also showed a significant reduction in lesion diameter compared to the MM control ( Fig. 13C ).

[0342] In addition, the mutant was tested against two important tomato-infecting viruses, the DNA virus TYLCV and the RNA virus ToBRFV. The PUB17 CRISPR mutant plants showed reduced ToBRFV symptoms compared to wild-type plants.

[0343] Breeding value of EMS pub17 mutant

[0344] The original EMS mutant plant M2042 has a leaf slightly smaller than the wild-type MT, and the leaf is slightly wrinkled. After the EMS mutant is hybridized with MM, backcrossed and the progeny is selfed, a plant with an MM sample phenotype can be obtained. However, self-necrosis on the leaf often occurs in the progeny derived from the EMS mutant. In order to evaluate the breeding value of the EMS pub17 mutation, the F3 plant obtained after the EMS mutant is hybridized with the MM from the F2 family 1-66 is backcrossed twice with different tomato breeding lines H1 and H2 and selfed subsequently (BC2S1)( Fig.17A ). Plants of BC2S1_H1 had a normal phenotype comparable to that of the parental line H1. Meanwhile, plants of BC2S1_H2 had small autonecrotic spots on their leaves and could not be used for further breeding. This suggests that the occurrence of autonecrotic death in the pub17 mutant depends on the genetic background. Plants of BC2S1_H1 carrying the pub17 mutation were crossed with the two parental lines (A and B) of the hybrid and backcrossed ( Fig. 17B ). Different levels of autonecrosis were observed in some BC2S1 plants derived from line B, which were homozygous for the pub17 mutation ( Fig. 17C ). However, the F1 hybrids prepared between Line A-pub17pub17 and Line-B-1-pub17pub17 did not show autonecrosis. In addition, the F1 plants of Line A x Line B carrying the homozygous pub17 allele showed normal plant phenotype and fruit condition compared to the F1 without pub17 mutation ( Fig.17D ). Taken together, these results demonstrate that the EMS mutant allele of pub17 can be used for breeding without pleiotropic effects.

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[0394] Madeira F,Pearce M,Tivey RNA,Basutkar P,Lee J,Edbali O,MadhusoodananN,Kolesnikov A,Lopez R.2022.Sequence analysis tools services fromEMBL-EBI in 2022.Nucleic Acids Research50:W276-W279.

[0395] Brief Description of the Sequence

[0396] Pub17 Small Tom Wild-Type Allele Sequence (SEQ ID NO: 1)

[0397] ATGGCATCTGCTGCAATTTTCTCATCGTTGAGAAGACAAAGGTCG

[0398] CCGACACTGGAAGCGTTCTTGGCGCCGGTGGATCTGACAGATGT

[0399] TGGGTTGTTGCAAACTTTAACGGCGTTATCTTCTGAGCTGATTTC

[0400] TGCATATTCAGGTAAAAGGCTGCCGTTTTATCAGCGGAAGAATTG

[0401] TAAGTCTTTGTTAAGGAAAATTCAAGTATTTTCTGTTCTCTTGGAG

[0402] TGTCTTCTTGAGAATAAAAAGAACAGAAGTAGTGGTTCTTCAGAT

[0403] TTGCCGTTTACAGCTTTTTTGTGCTTCAAGGAGTTGTATTTATTGC

[0404] TTTACCGGTCGAAAATCTTGCTTGATTATTGCTCTTATTCAAGTAA

[0405] GTTGTGGCTGTTGCTACAAAACCATTCGATTTCAGGTCATTTCCA

[0406] TGATTTGAACCAAGAAATCTCGACCCTTTTGGATGTTTTCCCCTT

[0407] AAAGGATTTAAAAAATTTATCTGAAGATGTTAGGGAACAGGTTGA

[0408] GTTGTTGAAGAAACAAGCAAGAAAATCTCAGTTGTTTGTTGATAA

[0409] ATATGATGAGATGCTAAGGTTGAAATTGTTTTCTTTCTTGAATGA

[0410] GTTTGAGAATGGTGGTGTTCCTGACTATGCTCAGTTGTACTCTTT

[0411] TTTTGTGGAAAAATTGGGGATTTGTAATCCTAGGAGTTGCAGAGT

[0412] TGAGATTGAGTTTTTGGAGGAGCAGATTGTGAACCATGAAGGAG

[0413] ATATTGAGCCCACATCCTCAGTTCTCAACGGGTTTGTGGCGTTGA

[0414] TGCGATACTGCAGGTTTTTGCTATTTGGCTTTGAAGAGGATGATA

[0415] TGGGGTTGAGATTGGGTAAGCATAAGAAGCCGAAGAGAGGGCTG

[0416] ATTAGTCAAGAGATTGCAGAGACATTCATTTCTGTACCAAAGGAC

[0417] TTCTGTTGTCCGATATCGTTGGATTTGATGAGGGATCCAGTTATT

[0418] GTGGCAACAGGGCAGACATATGATCGAGCTTCTATATCGAGGTG

[0419] GATGGAGGAGGGTCACTGTACTTGCCCAAAGACAGGGCAGTTGC

[0420] TTGATCATACCCGGCTTGTGCCAAACAGGGCTCTTAGGAATTTGA

[0421] TTATGCATTGGTGTGCTGCTCGCAAAATTCCCTATGACCCTCTGG

[0422] AGAGTGGGGATCCATGTGTTGAATGTTTTCCATCAGCTTCACCTA

[0423] GCAGGGCTGCACTAGAAGCTAATAAAGCCACAGCAGCTCTTCTG

[0424] ATTAAGCAGCTAGAGAGTGGGACGCAGATTGCAAAAACTATTGCT

[0425] GCTCAGGAGATAAGACTTTTAGCTAAAACTGGTAAGGAGAATCGT

[0426] GCATACATAGCTGAGGCTGGTGCAATCCCACATTTGAAGAATTTG

[0427] CTTTCATCTCCAGATGCTGTGGCACAAGAAAATTCCGTCACTGCA

[0428] ATGCTTAACTTATCGATTTTTGATAAGAATAAAGGCCGAATTATTGATGAAGTAGGGTGTCTGGCTTTGATAGTTGGAGTTTTGAGATTTGGGCACACCACAGAGGCACGGGAAAATGCTGCAGCAACATTATTCAGTCTGTCAGCTGTTCATGACTATAAGAGGCAAATAGCAAAAGAAGATGGGGCAGTCGAGGCCTTAGCGGGTCTGTTGCGAGAAGGTTCTCCCAGAGGGAAGAAAGATGCAGTAACTGCTCTATTTAATTTATCCACCCACACAGATAATTGTGCGAGGATGATAGAGTCTGGAGCTGTTACTGCTCTAGTTGGAGCTTTGGGAAGTGAAGGTGTTGCTGAAGAAGCTGCTGGTGCATTGGCGCTGATTGTTAGGCAGCAAGTTGGTGCTACAGCTGTTGGCAATGAGGAAATGGCAGTAGCAGGGCTCATTGCAATGATGCGATGTGGGACACCAAGAGGGAAGGAGAATGCTGTTGCTGCATTACTTGAATTATGCCGCGGTGGTGGAGCAGCTGCTACTGAGAGGGTCTTGAAGGCGCCGTCATTAGCAAGTTTACTTCAGACGTTGCTCTTTACAGGAACAAAGCGCGCAAGGAGGAAAGCAGCATCGCTTGCTAGAGTATTCCAACGGTGTGAGCATGCAGCAGTTCATTATAGTGGGTTTGGTGTAGGATATGCATTTGCTGGAAACTCAGCTGCTGCTAGGGATTCAACTTTTCCTGGTGATGTCTCAGTGTCCATGTCCATTTCAGTTCCAGTATTATAG

[0429] Modified allele sequence of Pub17 Little Tom: SNP A to T at position 1477, shown in bold (SEQ ID NO: 2)

[0430]

[0431]

[0432] Wild-type amino acid sequence of Pub17 Little Tom (SEQ ID NO: 3)

[0433] MASAAIFSSLRRQRSPTLEAFLAPVDLTDVGLLQTLTALSSELISAYS

[0434] GKRLPFYQRKNCKSLLRKIQVFSVLLECLLENKKKNRSSGSSDLPFTA

[0435] FLCFKELYLLLYRSKILLDYCSYSSKLWLLLQNHSISGHFHDLNQEIS

[0436] TLLDVFPLKDLKNLSEDVREQVELLKKQARKSQLFVDKYDEMLRLK

[0437] LFSFLNEFENGGVPDYAQLYSFFVEKLGICNPRSCRVEIEFLEEQIVN

[0438] HEGDIEPTSSVLNGFVALMRYCRFLLFGFEEDDMGLRLGKHKKPKR

[0439] GLISQEIAETFISVPKDFCCPISLDLMRDPVIVATGQTYDRASISRWME

[0440] EGHCTCPKTGQLLDHTRLVPNRALRNLIMHWCAARKIPYDPLESGD

[0441] PCVECFPSAPSRAALEANKATAALLIKQLESGTQIAKTIAAQEIRLL

[0442] AKTGKENRAYIAEAGAIPHLKNLLSSPDAVAQENSVTAMLNLSIFDK

[0443] NKGRIIDEVGCLALIVGVLRFGHTTEARENAAATLFSLASAVHDYKRQ

[0444] IAKEDGAVEALAGLLREGSPRGKKDAVTALFNLSTHTDNCARMIES

[0445] GAVTALVGALGSEGVAEEAAGALALIVRQQVGATAVGNEMAVAG

[0446] LIAMMRCGTPRGKENAVAALLELCRGGGAATERVLKAPSLASLLQ

[0447] TLLFTGTKRARRKAASLARVFQRCEHAAVHYSGFGVGYAFAGNSAA

[0448] ARDSTFPGDVSVSMSISVPVL

[0449] Pub17 LittleTom modified amino acid sequence: premature stop codon at position 493, shown in bold (SEQ ID NO: 4)

[0450]

[0451] Primer C (SEQ ID NO: 5)

[0452] GGAACTTGGTGTAGCAGAAATTTCCACATTTC

[0453] Primer D (SEQ ID NO: 6)

[0454] TTAGTGAGCTGAAACTCTAATCCGTAGAC

[0455] PUB17_qPCR_Fw1 Primer (SEQ ID NO:7)

[0456] GGAAGTGAAGGTGTTGCGA

[0457] PUB17_qPCR_Rv1 Primer (SEQ ID NO: 8)

[0458] CTACTGCCATTTCCTCATTGC

[0459] Ef1a-Fw (SEQ ID NO:9)

[0460] ATTGGAAACGGATATGCCCCT

[0461] Ef1a-Rv (SEQ ID NO: 10)

[0462] TCCTTACCTGAACGCCTGTCA

[0463] Sequence of RNAi3 PUB17 silencing fragment (SEQ ID NO: 11)

[0464] AGCCCACATCCTCAGTTCTCAACGGGTTTGTGGCGTTGATGCGAT

[0465] ACTGCAGGTTTTTGCTATTTGGCTTTGAAGAGGATGATATGGGGT

[0466] TGAGATTGGGTAAGCATAAGAAGCCGAAGAGAGGGCTGATTAGT

[0467] CAAGAGATTGCAGAGACATTCATTTCTGTACCAAAGGACTTCTGT

[0468] TGTCCGATATCGTTGGATTTGATGAGGGATCCAGTTATTGTGGCA

[0469] ACAGGGCAGACATATG

[0470] Sequence of the RNAi7 PUB17 silencing fragment (SEQ ID NO:12)

[0471] GGTGTGGGAAATTGATGGCATCTGCTGCAATTTTCTCATCGTTGA

[0472] GAAGACAAAGGTCGCCGACACTGGAAGCGTTCTTGGCGCCGGTG

[0473] GATCTGACAGATGTTGGGTTGTTGCAAACTTTAACGGCGTTATCT

[0474] TCTGAGCTGATTTCTGCATATTCAGGTAAAAGGCTGCCGTTT

[0475] Table 6. RNAi primers

[0476]

[0477]

[0478] sgRNA1 (SEQ ID NO:17)

[0479] GGAAATGACCTGAAATCGAA

[0480] sgRNA2 (SEQ ID NO:18)

[0481] TTCTATATCGAGGTGGATGG

[0482] sgRNA3 (SEQ ID NO: 19)

[0483] GAGATTTGGGCACACCACAG

[0484] sgRNA4 (SEQ ID NO: 20)

[0485] CAGGAACAAAGCGCGCAAGG

[0486] sgRNA forward primer (SEQ ID NO: 21)

[0487] TGTGGTCTCA[sgRNA sequence]GTTTTAGAGCTAGAAATAGCAAG

[0488] sgRNA reverse primer (SEQ ID NO: 22)

[0489] TGTGGTCTCAAGCGTAATGCCAACTTTGTAC

[0490] Table 7. Primers used for genotyping

[0491]

[0492] NPTII_421_Fw primer (SEQ ID NO: 26)

[0493] GAAGGGACTGGCTGCTATTG

[0494] NPTII_421_Rv primer (SEQ ID NO: 27)

[0495] AATATCACGGGTAGCCAACG

[0496] 35S_597_Fw Primer (SEQ ID NO: 28)

[0497] TACAAAGGCGGCAACAAAC

[0498] 35S_597_Rv Primer (SEQ ID NO: 29)

[0499] AGCAAGCCTTGAATCGTCC

[0500] KASP assay K_RTWT_For1 primer (SEQ ID NO: 30)

[0501] GAAGGTGACCAAGTTCATGCTGTCTGGCTTTGATAGTTGGAGTTTT

[0502] GT

[0503] KASP assay K_RTmut_For1 primer (SEQ ID NO: 31)

[0504] GAAGGTCGGAGTCAACGGATTGTCTGGCTTTGATAGTTGGAGTTTT

[0505] GA

[0506] KASP assay K_RT_Rev70 primer (SEQ ID NO: 32)

[0507] GTTGCTGCAGCATTTTCCCGTG

[0508] Primer SP_F (SEQ ID NO: 33)

[0509] TGAGACGGACAAGATGACATGA

[0510] Primer SP_R (SEQ ID NO: 34)

[0511] TGTCATTTCCCCTTCCAAAGT

Claims

1. A tomato plant or plant material having reduced Pub17 protein levels, activity or expression, thereby conferring increased resistance to lesion forming pathogens relative to a reference tomato plant or plant material.

2. The tomato plant or plant material according to claim 1, wherein the tomato plant or plant material has been modified to reduce the level, activity or expression of Pub17 protein.

3. The tomato plant or plant material according to claim 1 or 2, wherein the tomato plant or plant material comprises a modified Pub17 allele.

4. The tomato plant or plant material according to claim 3, wherein the modified Pub17 allele has at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an orthologue or homologue thereof, and wherein the Pub17 allele comprises a mutation.

5. The tomato plant or plant material according to claim 4, wherein the mutation is a SNP, preferably A to TSNP.

6. A tomato plant or plant material according to claim 4 or 5, wherein the mutation is located in the 3' region of SEQ ID NO: 1 (wild-type allele), preferably in the ARM region of SEQ ID NO: 1 (wild-type allele), more preferably in nucleotide position 1477 of SEQ ID NO: 1 (wild-type allele) or a position corresponding thereto.

7. A plant part obtained from the tomato plant according to any one of claims 1 to 6.

8. A seed capable of producing a tomato plant according to any one of claims 1 to 6.

9. A method of increasing the resistance of a tomato plant or plant material to a lesion-forming pathogen, the method comprising: Reducing the level, activity or expression of Pub17 protein in the tomato plant or plant material.

10. A method of producing a tomato plant having increased resistance to lesion forming pathogens, the method comprising: Reducing the level, activity or expression of Pub17 protein in the tomato plant or plant material.

11. The method of claim 9 or 10, wherein the method comprises modifying the tomato plant or plant material to reduce the level, activity or expression of Pub17 protein in the tomato plant or plant material.

12. The method according to any one of claims 9 to 11, wherein the method comprises obtaining a mutant population of tomato plants and selecting a modified tomato plant comprising a modified Pub17 allele having at least 70% identity to SEQ ID NO: 1 (wild-type Pub17 allele) or an ortholog or homolog thereof, and wherein the modified Pub17 allele comprises a mutation that reduces the level, activity or expression of Pub17 protein in the tomato plant or plant material.

13. A method of identifying a tomato plant having increased resistance to a lesion forming pathogen relative to a reference tomato plant or plant material, the method comprising: (a) determining the level, activity or expression of Pub17 protein in one or more tomato plants and comparing it to the level, activity or expression of Pub17 protein in a reference tomato plant; and (b) selecting tomato plants having reduced level, activity or expression of Pub17 protein relative to the reference tomato plants; wherein reduced level, activity or expression of Pub17 protein indicates increased resistance to lesion forming pathogens relative to the reference tomato plants.

14. The method according to claim 13, wherein the method comprises the following steps: (a) obtaining a mutant population of tomato plants; (b) screening the tomato plant population for the presence of a Pub17 allele, wherein the Pub17 allele is at least 70% identical to SEQ ID NO: 1 (wild-type Pub17 allele) or a direct homologue or homologue thereof, and comprises a mutation that reduces the level, activity or expression of Pub17 protein in the tomato plants or plant material; and (c) selecting tomato plants having the Pub17 allele.

15. The tomato plant or plant material according to any one of claims 1 to 6, the plant part according to claim 7, the seed according to claim 8 or the method according to any one of claims 9 to 14, wherein the lesion forming pathogen is a necrotrophic fungal pathogen, preferably selected from the group consisting of Alternaria alternata, Alternaria solani, Botrytis cinerea, Sclerotinia sclerotiorum, Stipes stolonifer, Fusarium oxysporum and Pythium species, preferably wherein the lesion forming pathogen is Botrytis cinerea.

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