A cucurbitaceae plant resistant to cucurbit aphid-borne yellows disease

Abstract

The present invention relates to a Cucurbitaceae plant that comprises a CABYV resistance locus providing resistance to Cucurbit aphid-borne yellows disease and / or the Cucurbitaceae plant wherein said plant comprises a stay green (SGR) gene providing improved fruit shelf life and reduced leaf yellowing. The present invention further relates to a CABYV resistance gene and / or an SGR gene, the use of said gene(s) for providing a Cucurbitaceae plant resistant to Cucurbit aphid-borne yellows disease and methods for identifying or selecting a Cucurbitaceae plant resistant to Cucurbit aphid-borne yellows disease and / or leaf yellowing.

Description

[0001] A CUCURBITACEAE PLANT RESISTANT TO CUCURBIT APHID-BORNE YELLOWS

[0002] DISEASE

[0003] Description

[0004] The present invention relates to a Cucurbitaceae plant that comprises a CABYV resistance locus providing resistance to Cucurbit aphid-borne yellows disease and / or the Cucurbitaceae plant wherein said plant comprises a stay green (SGR) gene providing improved fruit shelf life and reduced leaf yellowing. The present invention further relates to a CABYV resistance gene and / or an SGR gene, the use of said gene(s) for providing a Cucurbitaceae plant resistant to Cucurbit aphid-borne yellows disease and methods for identifying or selecting a Cucurbitaceae plant resistant to Cucurbit aphid-borne yellows disease and / or leaf yellowing.

[0005] Cucurbit aphid-borne yellows virus (CABYV) is a worldwide spread virus and a limiting factor for cucumber production in key markets. CABYV belongs to the group of Polero virus and is transmitted by aphids, including Aphis gossypii, Myzus persicae and Macrosiphum euphorbiae. Being systemic and persistent in infected plants, CABYV can be accidentally introduced by the importation of infected plant material, or of plants infested by viruliferous aphids. It can lead to considerable damage in cucurbit crops including cucumber, melon, and squash and can lead to reduced growth and fruit loss. Besides all major cucurbit crops, CABYV has been reported to infect other annual crops including lettuce, faba bean and chickpea. Ultimately, a virus infection in a greenhouse can result in a significant loss of production.

[0006] Symptoms of CABYV vary depending on the plant species and age, but generally include yellowing and curling of leaves, stunted growth, and reduced fruit production. Infected plants may also have a lower tolerance for stressors such as drought or extreme temperatures. In melon, cucumber, squash or watermelon the first CABYV symptoms are yellow mottling and interveinal chlorotic patches which eventually coalesce, leaves becoming yellow, thickened and brittle. Also, a reduced number of fruits are often observed in CABYV infected plants. Reports describe a significant reduction in fruit number per plant (-50%) was observed in cucumber and a 40% reduction in melon. Furthermore, the CABYV virus causes basal yellowing symptoms easily mistaken to other important virus Cucurbit yellow stunting disorder virus (CYSDV), making necessary to combine resistance to both diseases in order to obtain a durable control of the symptoms.

[0007] CABYV is readily transmitted by ubiquitous aphids that provide an efficient means of invasiveness worldwide. CABYV is becoming more frequent in areas where it was reported earlier (France, Spain and parts of Asia) and has recently been introduced into new areas (Northern Europe, South America). Therefore, CABYV is becoming an increasing threat in cucurbit plants. CABYV prevalence is increasing due the expansion of its geographical distributions from the Mediterranean to Northern Europe in the past years and the limitations on the treatments against its aphid vector.

[0008] CABYV is a serious disease that can cause significant losses in cucurbit crops. There is no known cure for the disease, and management strategies typically involve using virus-free seed, controlling aphid populations through cultural practices and insecticides, and practicing crop rotation to prevent the build-up of virus reservoirs in the soil. Control or prevention of CABYV infection is at present focussed on limiting the spread of CABYV including limiting virus sources and vector populations or interfering with vector activities to regulate the population of aphid vectors and / or their virus load. Breeding for resistance is the most attractive method of controlling CABYV and resistance to CABYV has been identified in melon, cucumber, squash and bitter gourd, however in view of the recent outbreaks and increasing CABYV reports, resistance seems to be overcome by the virus or at least resistance to the virus in cucurbit crops is decreasing, wherein as from 2021 onward a surge of the CABYV disease has been observed.

[0009] Furthermore, several crops in the Cucurbitaceae family, such as cucumber, zucchini, are mainly eaten in the “unripe” green form since the ripe yellow form normally becomes less tasty, such as for cucumber it becomes bitter and sour. Disease such as CABYV also result in the unappealing yellowing of the crop which is commercially undesired and further reducing the shelf life and agronomical properties of the fruits.

[0010] Considering the above, there is a need in the art to develop a more diverse and durable resistance in cucurbit crops against CABYV. Furthermore, it is an object of present invention to provide a method to obtain such CABYV resistant plants and / or providing plants showing reduced or retarded leaf yellowing following CABYV infection. There is a need for new or a higher diversity of genes and / or alleles so that more genetic variation can be achieved in commercial crops, making it harder for CABYV to adapt. Furthermore, there is a need to provide for cucurbit plants providing fruits having increased shelf life in relation to increased disease tolerance and increased agronomical performance.

[0011] It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0012] Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein said plant comprises a CABYV resistance gene that encodes for a CABYV resistance protein, wherein said CABYV resistance gene comprises one or more mutations providing said resistance to Cucurbit aphid-borne yellows disease, wherein said CABYV resistance protein comprises an amino acid sequence having at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.6. The Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease comprises a CABYV resistance locus that comprises a CABYV resistance gene, wherein the CABYV resistance locus comprises one or more genomic sequences selected from the group consisting of SEQ ID No.l, SEQ ID No.4 SEQ ID No.7, SEQ ID No.10, SEQ ID No.13, SEQ ID No.16, SEQ ID No.19, and SEQ ID No.22, or having at least 90%, more preferably at least 95%, even more preferably at least 98%, preferably at least 99%, most preferably 100% sequence identity with any one of the SEQ ID No’s, preferably SEQ ID No.4. The Cucurbitaceae plant comprising the CABYV resistance locus and resistance gene shows to be resistant to Cucurbit aphid-borne yellows disease, more specifically resistant to CABYV infection. The CABYV resistance locus is flanked by marker of SEQ ID No. 25 and marker of SEQ ID No. 26 and comprised the genomic sequences indicated. The sequence homology among CABYV resistance proteins of various Cucurbitaceae plants were analysed using multiple alignment software and showed that that the proteins share a sequence homology of at least 88%, and preferably at least 90%, more preferably at least 92%, even more preferably at least 93, most preferably at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity between Cucurbitaceae plants (See also Table 4 and 5). Preferably the Cucurbitaceae plant is Cucumis sativus.

[0013] According to yet another preferred embodiment the present invention relates to the Cucurbitaceae plant, wherein expression of the mutated CABYV resistance gene or protein activity of the mutated CABYV resistance protein is reduced or even absent as compared to the expression or the protein activity of a CABYV resistance gene or CABYV resistance protein, respectively, in a Cucurbitaceae plant not resistant to Cucurbit aphid-borne yellows disease. The CABYV resistance gene seems to being used by CABYV during infection and promote disease development and / or spreading of the virus. The current hypothesis of the mode of action is that the CABYV resistance protein plays a role in the RISC-complex formation / AGO mediated gene silencing involved in the viral infection and / or plant’s viral defense mechanism, for example as formation promotion factor or RISC loading / affinity protein. A reduced protein activity of the CABYV resistance protein, as observed in the VIGS silencing experiments included herein, benefits the plant becoming resistant to CABYV infection.

[0014] According to yet another preferred embodiment the present invention relates to the Cucurbitaceae plant, wherein said CABYV resistance gene encodes for a coding sequence comprised of SEQ ID No.5, or having at least 95% sequence, preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with said SEQ ID No. 5.

[0015] According to yet another preferred embodiment the present invention relates to the Cucurbitaceae plant, wherein said reduced or even absent protein activity or said reduced or even absent gene expression is provided by said one or more mutations in the coding sequence and / or in the promotor sequences and / or regulatory sequences of said CABYV resistance gene. Rresistance to Cucurbit aphid-borne yellows disease may be provided by one or more mutations in the regulatory regions or non-coding sequences of the CABYV resistance gene. Examples of regulatory regions of the present genes are promotor and terminator regions and examples of noncoding regions are introns and especially splicing influencing motifs therein.

[0016] According to yet another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said one or more mutations in said CABYV resistance gene result in an amino acid substitution, frame-shift or pre-mature stop codon, preferably amino acid substitutions in the CABYV resistance protein. Preferably, the one or more mutations in the CABYV resistance gene results in a non-functional or truncated CABYV resistance protein.

[0017] According to yet another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said one or more mutations result in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No.42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D). 3D protein modeling was performed using the susceptible ‘non mutated” CABYV protein represented by SEQ ID No.42 and the resistance CABYV resistant gene (SEQ ID No. 6) of present invention that comprises the amino acid substitution of G166D. It was found that the mutation resulting in the resistant phenotype is due to a change in the polarity from non-polar to polar in amino-acid. Furthermore, 3D modelling shows that this amino-acid is located on the outside of the protein structure which may lead to different protein-protein interactions, as observed in the homolog protein of Arabidopsis, indicated as Arabidopsis RBV which is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGON AUTE1 loading. Here it was also observed that 1 amino-acid change resulted in a suppression of leaf bleaching in Arabidopsis. This amino-acid change was also from a non-polar amino-acid to a polar amino-acid and was also on the outside structure of the protein resulting in a phenotypic effect in the plant.

[0018] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein the CABYV resistance gene is present on Chr6 and encodes for a coding sequence of SEQ ID No. 5, wherein said coding sequence comprises an “A” on genomic position 22536842, based on genome 9930 v2 (http: / / cucurbitgenomics.Org / organism / 2 or also available at https: / / www.ncbi.nlm.nih.gOv / nuccore / ACHR00000000.2, Chinese Long inbred line “9930” version 2 (Huang et al. 2009)1, also known as Chinese Long v2. Fine mapping of CABYV resistant plants indicated that the resistance gene is present on chromosome 6, mapped to a genomic position of between 22533586 - 22545054 (11468 bp) based on genome 9930 v2. In this area only a single gene was present indicated herein as Csa6G486680. The identified CABYV resistance gene comprised a genomic sequence, coding sequence, and protein sequence of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively. The identified resistance gene comprises one amino acid change in comparison to the susceptible counterpart on position: 22536842. The resistant plant comprises an “A”, whereas the susceptible plant has a “G” on this position. In the coding sequence (cDNA) this mutation is a G497A mutation, corresponding to the coding sequence of SEQ ID No. 5. Thus, a susceptible plant comprises a “G” on position 497 in view of SEQ ID. No.5, whereas the resistant plant comprises an “A” on this position (in correspondence with SEQ ID No.5). On protein level, this mutation causes an amino acid change from a Glycine (Gly) to Aspartic Acid (Asp) in the amino acid sequence, referred to herein as G166D, and corresponding to the protein sequence of SEQ ID No.6 encoding the CABYV resistance protein of present invention. Therefore, said CABYV resistance gene encodes for a CABYV resistance protein of SEQ ID No. 6, wherein the protein providing CABYV disease resistance in said plant differs in an amino acid change from a Glycine (Gly) to Aspartic Acid (Asp) in the amino acid sequence in respect to the gene encoding the protein not providing CABYV resistance in the Cucurbitaceae plant.

[0019] According to a preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said one or more mutations result in an amino acid substitution of G166D in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

[0020] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said one or more mutations is a G497A mutation in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

[0021] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said CABYV resistance locus and / or CABYV resistance gene is present in heterozygous or homozygous form, preferably homozygous.

[0022] According to yet another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said CABYV resistance locus and / or gene is obtainable from deposit number NCIMB 43857. Seeds comprising the CABYV resistance locus and / or gene are deposited at NCIMB Ltd, Aberdeen, Ferguson Building, Craibstone Estate, Bucksburn, AB21 9 YA Scotland on 15 September 2021 under the number NCIMB 43857.

[0023] In a further or alternative aspect, the present invention relates to a Cucurbitaceae plant wherein said plant further comprises or comprises, respectively, a stay green (SGR) gene comprising one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein comprising an amino acid sequence having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32. The Cucurbitaceae plant comprises the stay green (SGR) gene comprising one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing in addition to the CABYV resistance locus and / or gene providing resistance to Cucurbit aphid-borne yellows disease. Similarly, as done for the CABYV resistance gene, the sequence homology among SGR coding sequences of various Cucurbitaceae plants were analysed using multiple alignment software and showed that that SGR amino acid sequence shares a sequence homology of at least 81%, and preferably at least 91%, most preferably at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity between Cucurbitaceae plants (See also Table 6). Preferably the Cucurbitaceae plant is Cucumis sativus.

[0024] The mutated SGR gene and resulting protein of present invention provides Cucurbitaceae plants that provides fruits with an improved shelf life, or preferably fruits which stay green during storage for a period of at least 4 or 5 weeks. Furthermore, the mutated SGR protein provides a plant the ability to maintain a green phenotype under stress conditions for a longer period in time, in comparison to plants that do not comprise the mutated SGR gene and protein. The SGR gene has been described earlier in WO2016 / 012346 and WO2022 / 207114. Experiments showed that the SGR gene in a plant of present invention providing increased disease tolerance, for example a Cucumber, differs from the SGR gene not providing this effect in that it comprises a mutation in its sequence resulting in a non-functional (knock-out) SGR protein product. The non-functional SGR protein results in turn in a lower chlorophyll degradation rate resulting in fruit and foliage with increased period of time in which the green colour is retained. For fruits, chlorophyll degradation is one of the traits related to shelf life, others being for example related to fruit flesh softening, cellular degradation, water loss.

[0025] Under stress conditions, such as disease pressure, Cucurbitaceae plant senescence is delayed resulting in that leaves maintain their green phenotype for longer periods which positively affects plants health and crop yield. Plant comprising both the CABYV resistance gene and the SGR gene show a synergistic beneficial effect of providing disease resistance to the Cucurbit aphid-borne yellows disease and at the same time masking disease effects (for example due to other diseases than CABYV, such as Downy Mildew) resulting in a greatly improved shelf life and a stay green phenotype of the plant and its fruit. Approximately an improved shelf life of at least 4 to 5 weeks is observed for fruits from a Cucurbitaceae plant comprising the SGR gene encoding the present protein.

[0026] A plant according to the present invention is deposited wherein the mutated SGR gene and CABYV resistance locus and / or gene are obtainable from E23L.16642 plant deposit and / or ES2018.62116 plant deposit. Seeds comprising the mutated SGR gene together with the CABYV resistance locus and / or CABYV resistance gene are deposited at NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland on 9 August 2023 under the number NCIMB 44206 (E23L.16642). Seeds comprising the mutated SGR gene are deposited at NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland on 9 August 2023 under the number NCIMB 44212 (ES2018.62116).

[0027] According to a preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein the expression or the protein activity of said mutated SGR gene is reduced or even absent as compared to the expression or the protein activity of a SGR gene in a Cucurbitaceae plant not providing fruits having an improved shelf life, wherein said reduced or even absent protein activity or gene expression is provided by one or more mutations in the genomic or coding sequence of said stay green gene. The SGR protein has a chlorophyll decarboxylase function wherein, amongst other reactions, a carboxyl group is removed, and carbon dioxide is released. Accordingly, the present reduced or even absent activity can be determined using an assay measuring compounds being either the starting compounds or the resulting compounds of the protein reaction. As a suitable alternative, protein levels, being inherently indicative of a reduced or even absent activity, of the present proteins can be determined by, for example, ELISA or protein hybridization both being techniques commonly known to the skilled person.

[0028] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant wherein said one or more mutations is selected from the group consisting of;

[0029] - a G1410T mutation in the SGR sequence represented by SEQ ID No.27; Seeds comprising the G1410T mutated SGR gene are deposited as NCIMB 44212 or NCIMB 44206.

[0030] - a C457T mutation in the SGR sequence represented by SEQ ID No.28,

[0031] - a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,

[0032] - a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

[0033] The above specific recited mutations in the SGR sequences as disclosed herein may be equivalent present in all Cucurbitaceae plants, more specifically one or more selected from the group consisting of Cucumis sativus, Cucumis melo, Citrullus lanatus, Cucurbita maxima, Cucurbita pepo, Cucurbita moschata, Lagenaria siceraria, Cucurbita argyrosperma and Momordica charantia, preferably Cucumis sativus.

[0034] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said plant is one or more selected from the group consisting of;

[0035] Cucumis Sativus wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of G to T at position 1410 (G1410T) in the SGR sequence represented by SEQ ID No.27 and / or a nucleotide mutation of C to T at position 457 (C457T) in the SGR sequence represented by SEQ ID No.28, and / or

[0036] Cucurbita pepo wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.33 and / or

[0037] Cucumis melo wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 161 (C161T) and / or a nucleotide mutation of G to A at position 221 (G221A) in the SGR sequence represented by SEQ ID No.34, and / or

[0038] Cucurbita moschata wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.37, and / or

[0039] Cucurbita maxima wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.36, and / or

[0040] Cucurbita argyrosperma wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.35, and / or

[0041] Lagenaria siceraria wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.38, and / or

[0042] Citrullus lanatus wherein said mutated stay green gene encodes for a sequence corresponding with a nucleotide mutation of C to T at position 155 (C155T) and / or a nucleotide mutation of G to A at position 215 (G215A) in the SGR sequence represented by SEQ ID No.39.

[0043] Preferably the Cucurbitaceae plant is Cucumis sativus.

[0044] The present Cucurbitaceae plants, for example cucumber plants, can be obtained by mutagenesis of cucumber plants. For example, mutations, either at the expression level or the protein level, can be introduced in these plants by using mutagenic chemicals such as ethyl methane sulfonate (EMS) or by irradiation of plant material with UV or gamma rays or fast neutrons or by CRISPR-Cas gene editing. Mutagenized plants carrying mutations in the present gene can be readily identified by using the TILLING (Targeting Induced Local Lesions IN Genomes) method.

[0045] According to a preferred embodiment of the present invention the present plants detailed above are not plants exclusively obtained by means of an essentially biological process. According to yet another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said one or more mutations results in a non-functional or truncated SGR protein, preferably the SGR protein of SEQ ID No.32.

[0046] According to a preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said SGR gene is present in heterozygous or homozygous form, preferably homozygous.

[0047] According to another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein the Cucurbit aphid-borne yellows disease is caused by one of more selected from the group consisting of Cucurbit aphid-borne yellows disease virus (CABYV), Suakwa aphid-borne yellows disease virus (SABYV), Melon aphid-borne yellows disease virus (MABYV), Cucumber yellow stunting disorder virus (CYSDV), Beet pseudo-yellows virus (BPYV), Cucumber vein yellowing virus (CVYV), and Cucurbit chlorotic yellows virus (CCYV), preferably CABYV, SABYV and / or MABYV.

[0048] According to yet another preferred embodiment, the present invention relates to the Cucurbitaceae plant, wherein said plant is one or more selected from the group consisting of Cucumis sativus, Cucumis melo, Citrullus lanatus, Cucurbita maxima, Cucurbita pepo, Cucurbita moschata, Lagenaria siceraria, Cucurbita argyrosperma and Momordica charantia, preferably Cucumis sativus.

[0049] The present invention, according to a further aspect, relates to a seed, plant cell, plant tissue, fruit, or plants parts of a Cucurbitaceae plant of the present invention comprising a CABYV resistance gene providing resistance to Cucurbit aphid-borne yellows disease, and / or an SGR gene providing improved fruit shelf life and / or reduced leaf yellowing.

[0050] The present invention, according to a further aspect, relates to a resistance gene for providing CABYV resistance in a Cucurbitaceae plant, wherein the resistance gene encodes for a CABYV resistance protein, wherein said CABYV resistance gene comprises one or more mutations providing said resistance to Cucurbit aphid-borne yellows disease, wherein said CABYV resistance protein comprises an amino acid sequence having at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.6.

[0051] According to a preferred embodiment, the present invention relates to the resistance gene, wherein the CABYV resistance gene encodes for a coding sequence of SEQ ID No. 5 or having at least 95% sequence, preferably at least 98%, even more preferably at least 99%, most preferably 100% identity with SEQ ID No. 5.

[0052] According to a preferred embodiment, the present invention relates to the resistance gene, wherein said one or more mutations result in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

[0053] According to another preferred embodiment, the present invention relates to the resistance gene, wherein said one or more mutations results in an amino acid substitution G166D in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6 According to yet another preferred embodiment, the present invention relates to the resistance gene, wherein said one or more mutations is a G497A mutation in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

[0054] The present invention, according to a further aspect, relates to a protein for providing CABYV resistance in a Cucurbitaceae plant, wherein the protein comprises SEQ ID No.6, or having at least 95% sequence, preferably at least 98% identity with SEQ ID No.6. Preferably the Cucurbitaceae plant is Cucumis sativus.

[0055] The present invention, according to a further aspect, relates to a stay green (SGR) gene for providing fruits having an improved shelf life and / or reduced leaf yellowing in a Cucurbitaceae plant, wherein the stay green (SGR) gene comprising one or more mutations or the encoded SGR protein. For example said SGR gene encodes a SGR protein comprising an amino acid sequence having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32, or wherein said one or more mutations is selected from the group consisting of;

[0056] - a G1410T mutation in the SGR sequence represented by SEQ ID No.27,

[0057] - a C457T mutation in the SGR sequence represented by SEQ ID No.28,

[0058] - a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,

[0059] - a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

[0060] The present invention, according to a further aspect, relates to a combination of a CABYV resistance gene and an SGR gene as disclosed herein in a Cucurbitaceae plant providing CABYV resistance in said plant and providing fruits having an improved shelf life and / or reduced leaf yellowing. The plants of present invention comprising the combination of the SGR gene of for example SEQ ID No.27 and the CABYV resistance locus and / or CABYV resistance gene, for example SEQ ID No.4 show to have an increased shelf life, reduced leaf yellowing, increased disease tolerance and increased agronomical performance by means of a reduction in chlorophyll degradation during plant development, disease challenge or fruit storage. The present invention, according to a further aspect, relates to a combination of a CABYV resistance protein and an SGR protein as defined herein in a Cucurbitaceae plant providing CABYV resistance in said plant and providing fruits having an improved shelf life and / or reduced leaf yellowing. Preferably the Cucurbitaceae plant is Cucumis sativus.

[0061] The present invention, according to a further aspect, relates to a method for identifying or selecting a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease and / or having an improved shelf life and / or reduced leaf yellowing, wherein the method comprises the step of establishing the presence of a CABYV resistance gene in said Cucurbitaceae plant that encodes for a CABYV resistance protein comprising SEQ ID No.6, or having at least 95% sequence, preferably at least 98% sequence identity with said SEQ ID No.6. in said plant, and / or, wherein the method comprises the step of establishing the presence of a CABYV resistance gene comprising SEQ ID No. 5 in said Cucurbitaceae plant or at least 95% sequence identity, preferably at least 98% sequence identity with identity SEQ ID No. 5. Optionally the method may also comprise the step of establishing the presence of a CABYV resistance locus that comprises a CABYV resistance gene in said Cucurbitaceae plant, wherein said method comprises the step of establishing the presence of one or more genomic sequences selected from the group consisting of SEQ ID No.l, SEQ ID No.4 SEQ ID No.7, SEQ ID No.10, SEQ ID No.13, SEQ ID No.16, SEQ ID No.19, and SEQ ID No.22, or having at least 90% sequence identity with any one of the SEQ ID No’s, preferably SEQ ID No.l or SEQ ID No.4 in said plant.

[0062] According to another preferred embodiment, the present invention relates to the method, wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence of an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

[0063] According to a preferred embodiment, the present invention relates to the method, wherein the step of the presence of a CABYV resistance locus that comprises a CABYV resistance gene comprises the determination of the presence of one or more SNP on Chr6 as described in Table 3, preferably the determination of a G to A on position 22536842, preferably the determination of an “A” on genomic position 22536842, based on genome 9930 v2 accessible on http: / / cucurbitgenomics.Org / organism / 2. According to a preferred embodiment, the present invention relates to the method, wherein the method wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence of an Aspartic Acid on position 166 in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

[0064] According to another preferred embodiment, the present invention relates to the method, wherein the method wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence a “A” on position 497 in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

[0065] According to yet another preferred embodiment, the present invention relates to the method, wherein the method further comprises the step of establishing the presence of a stay green (SGR) gene as defined herein in the Cucurbitaceae plant.

[0066] According to yet another preferred embodiment, the present invention relates to the method, wherein said SGR gene comprises one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein comprising an amino acid sequence having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32.

[0067] According to a preferred embodiment, the present invention relates to the method, wherein said one or more mutations is selected from the group consisting of;

[0068] - a G1410T mutation in the SGR sequence represented by SEQ ID No.27,

[0069] - a C457T mutation in the SGR sequence represented by SEQ ID No.28,

[0070] - a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,

[0071] - a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

[0072] According to another preferred embodiment, the present invention relates to the method, wherein said method comprises the step of establishing the presence of the CABYV resistance gene and / or a CABYV resistance protein as defined herein in said Cucurbitaceae plant and / or comprises the step of establishing the presence of the SGR gene and / or SGR protein as defined herein in said Cucurbitaceae plant.

[0073] The present invention, according to a further aspect, relates to a method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, said method comprises the step of introducing one or more mutations in a CABYV resistance gene encoding a CABYV resistance protein comprising SEQ ID No.6, or having at least 95% sequence, preferably at least 98% sequence identity with said SEQ ID No.6. in said plant, and / or, wherein the method comprises the step of introducing one or more mutations in a CABYV resistance gene comprising SEQ ID No. 5 in said Cucurbitaceae plant or at least 95% sequence identity, preferably at least 98% sequence identity with identity SEQ ID No. 5, and wherein the expression of the mutated CABYV resistance gene is reduced as compared to the expression of said CABYV resistance gene in a Cucurbitaceae plant not being resistant to Cucurbit aphid-borne yellows disease.

[0074] According to another preferred embodiment, the present invention relates to the method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

[0075] According to another preferred embodiment, the present invention relates to the method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an amino acid change in said CABYV resistance protein resulting in an Aspartic Acid on position 166 in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

[0076] According to yet another preferred embodiment, the present invention relates to the method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an “A” on position 497 in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

[0077] According to a preferred embodiment, the present invention relates to the method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease wherein the method further comprises the step the step of introducing one or more mutations in an SGR gene, wherein the SGR gene comprises one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32.

[0078] According to another preferred embodiment, the present invention relates to the method for providing a Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein said one or more mutations in the SGR gene is selected from the group consisting of;

[0079] - a G1410T mutation in the SGR sequence represented by SEQ ID No.27,

[0080] - a C457T mutation in the SGR sequence represented by SEQ ID No.28, - a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,

[0081] - a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

[0082] According to yet another preferred embodiment, the present invention relates to the method, wherein said one or more mutations in the SGR gene are provided by gene editing techniques, preferably by chemical mutagenesis, UV- or y- radiation, or by CRISPR-Cas.

[0083] The present invention, according to a further aspect, relates to a method for providing a Cucurbitaceae plant of any one of the claims 1 to 12, wherein the method comprises the steps of, a) crossing a Cucurbitaceae plant comprising a CABYV resistance gene and / or an SGR gene as defined herein with a Cucurbitaceae plant that is susceptible to Cucurbit aphid-borne yellows disease and does not comprise said CABYV resistance gene and / or SGR gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to Cucurbit aphid-borne yellows disease and / or show reduced leaf yellowing, wherein said plant preferably comprises said CABYV resistance gene and SGR gene. Preferably the Cucurbitaceae plant is Cucumis sativus.

[0084] According to yet another preferred embodiment, the present invention relates to the method, wherein the selecting of step c is done as described by the step of establishing the presence of a CABYV and / or stay green (SGR) gene in the Cucurbitaceae plant resistant to Cucurbit aphid- borne yellows disease.

[0085] The present invention, according to a further aspect, relates to the use of a CABYV resistance gene as defined herein for providing CABYV resistance in a Cucurbitaceae plant.

[0086] The present invention, according to a further aspect, relates to the use of a resistance gene and / or a SGR gene in Cucurbitaceae plant for providing CABYV resistance in a Cucurbitaceae plant and providing fruits having an improved shelf life.

[0087] The present invention, according to a further aspect, relates to the use of a CABYV resistance gene and an SGR gene as defined herein in Cucurbitaceae plant for providing CABYV resistance in a Cucurbitaceae plant and providing fruits having an improved shelf life or reduced leaf yellowing.

[0088] The present invention, according to a further aspect, relates to the use of a gene construct or plasmid for introducing the CABYV resistance locus and / or CABYV resistance gene providing resistance to Cucurbit aphid-borne yellows disease, and / or an SGR gene providing improved fruit shelf life and / or reduced leaf yellowing, into the genome of a plant or plant cell. The resistance locus and / or genes of present invention may be transferred (e.g. by transformation or transfection) into plants, such as lettuce plants, using a plasmid or vector or linear gene construct that comprises the resistance gene(s) or locus of present invention.

[0089] The present invention will be further detailed in the following examples and figures wherein: Figure 1: Shows a susceptible cucumber plant (Tyria variety, not comprising the CABYV resistance gene) and a cucumber plant comprising the CABYV resistance gene, approximately three weeks after inoculation with CABYV. The susceptible cucumber plant shows severe yellowing of the leaves, resulting in a disease score of 1 (upper panel). The cucumber plant comprising the CABYV resistance gene remains green and free of disease symptoms, resulting in a disease score of 9 (lower panel).

[0090] Figure 2: Shows the results of phenotype (resistant or susceptible) in respect to and linked with marker selection; plants homozygous (resistant) for the CABYV resistance gene are showing full resistance to CABYV (disease score of on average = 8.8 on 1-9 scale). The CABYV resistance gene is not fully recessive but seems incomplete dominant, since the heterozygous plants perform somewhat variably, with an average disease score of 4.8.

[0091] Figure 3: Shows cucumber plants according to present invention (indicated as E23L.16642) comprising the mutated SGR gene homozygous (and the CABYV resistance gene) providing the “stay green” effect and reduced leaf yellowing, masking Downy mildew, see Figure 3 upper panels. The right upper panel shows aspects of fruit from E23L.16642, which is unaffected showing total absence of yellowing. Figure 3 lower panels show a control plant (Marumba variety, which does not comprise the SGR gene) that is affected by Downy Mildew as observed by the yellowing and curled leaves. The right lower panel shows the fruit from the control plant heavily affected showing yellowing.

[0092] Figure 4: Shows the synergistic “masking effect” in CABYV infected cucumber plants comprising the mutated SGR gene and CABYV resistance gene. When infected with CABYV, the plant of present invention comprising the mutated SGR gene and CABYV resistance gene, showed absence leaf yellowing (right panel) in comparison to plant that do not comprise the mutant SGR gene and CABYV resistance gene (left panel). Therefore, the plants of present invention remain healthy and green as compared to the plants which do not comprise the SGR and CABYV genes.

[0093] Example 1 - Disease tests and identification of novel CABYV resistance locus

[0094] RECTIFIED SHEET (RULE 91 ) ISA / EP Cucumber plants were screened to identify for CABYV resistance in tests performed in controlled inoculation (controlled aphid test) with an infective clone of the virus and scored for disease symptoms. This test was confirmed by a controlled infection using the aphid vector, wherein the disease symptoms were scored by rating on a scale of 1 being susceptible to 9 being resistant. Plants having a score of 9 were identified as fully resistant and selected for further analysis.

[0095] Briefly, young cucumber plants at the 2-4 true leaf stage, were infected with CABYV using Aphis Gossypii in a Greenhouse (~24 °C) inside a net cage for inoculation. Disease observations was initiated at about 21 days post inoculation and continued for additional 3 to 4 weeks or until no new disease progression was observed, detecting yellowing of the leaves. Disease symptoms are scored according to a scale 1 to 9 (see also Figure 1), wherein;

[0096] Level 9: Total absence of yellowing in basal leaves;

[0097] Level 8: Faint yellowing spots that could be attributed to other factors but are present;

[0098] Level 7: Faint yellowing or light green spots clearly associated with the disease;

[0099] Level 6: Clear yellowing spots that form small patches in the leave leaving green areas between them;

[0100] Level 5: Clear yellowing spots that form patches starting to connect between them forming a continuous area;

[0101] Level 4: Yellowing or clear green spots that covers almost full basal leaves but do not form an uniform clear are on affected leaves;

[0102] Level 1 to 3: Yellowing that covers all affected leaves and form a uniform area, symptoms level are modulated by yellowing intensity between levels 1 and 3 according to the pictures below.

[0103] Thirty-eight individuals that showed CABYV resistance out of a population of about 200 plants in total were genotyped using marker analysis. Based on these results we identified a possible QTL on Chromosome 6 that is involved in CABYV resistance, and it was concluded that the CABYV resistance gene is located on chromosome 6 between position 22368282 and 23274171 ((based on genome 9930 v2 accessible on http: / / cucurbitgenomics.Org / organism / 2)), making the CABYV resistance locus total size of 905889 bp.

[0104] Example 2 -Determination of dominance of the CABYV resistance and identification of CABYV resistance gene

[0105] To determine the dominance of the underlying genetics in total 87 individuals were phenotyped for CABYV resistance in the controlled aphid test and were genotyped in the CABYV resistance locus located on position Chr6:22583735. The phenotypic distribution alone indicates a single gene underlying the incomplete dominant resistance (Figure 2). In combination with marker results, the conclusion is that the gene acts as incompletely dominant.

[0106] The CABYV resistance locus was subsequently screened for the identification of candidate resistance gene(s) involved, i.e. genes located within the 22505855 - 22568996 region and results allowed to further reduce the size of the CABYV resistance locus on Chr6, between positions 22505855 - 22568996 (63141bp), as identified by flanking markers M4 and M5 (Table 2). The region comprises at least 8 candidate resistance genes; see Table 1 for their genomic position (based on genome 9930 v2 accessible on http: / / cucurbitgenomics.Org / organism / 2) and the genomic-, coding- and protein sequences of the identified candidate resistance genes, as included in the sequence listing herein. Of these candidate resistance genes the most promising candidates are Csa6G486670 and Csa6G486680, represented by their genomic sequences of SEQ ID No.l and 4, respectively.

[0107] Table 1. The CABYV resistance locus on Chr6 comprises eight candidate resistance genes.

[0108] Table 2. Marker sequences used to further reduce the size of the CABYV resistance locus on Chr6. Lowercase and underlined letters represent the SNPs for selection of the CABYV resistance locus.

[0109] Seeds of a cucumber plant according to present invention comprising the CABYV resistance locus on Chr6 including the sequences of SEQ ID No.1 to SEQ ID No.24. have been deposited at NCIMB Ltd, Aberdeen, Scotland on 15 September 2021 under reference NCIMB 43857.

[0110] Several of the identified gene candidates comprise SNPs that are linked to the CABYV resistant phenotype in cucumber. Table 3 provides an overview of the identified SNP describing the SNP position per gene candidate in view of the CABYV resistant and susceptible phenotype. Table 3. Overview of SNPs positions on Chr6 that are linked to the CABYV resistant phenotype in cucumber

[0111] Further fine mapping was done of recombinant plants using further markers on chromosome 6, reducing the region comprising the resistance to CABYV to genomic positions of between 22533586 - 22545054 (11468 bp) based on genome 9930 v2. In this area only a single gene was annotated, indicated herein as Csa6G486680. The identified CABYV resistance gene comprised a genomic sequence, coding sequence, and protein sequence of SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively. The identified resistance gene comprises nonsynonymous SNP, encoding amino acid change, in comparison to the susceptible counterpart on position: 22536842. The resistant plant comprises an “A”, whereas the susceptible plant has a “G” on this position, as also indicated in Table 3 (see underlined). This SNP causes an amino acid change from a Glycine (Gly) to Aspartic Acid (Asp) in the amino acid sequence, referred to herein as G166D, and corresponding to the protein sequence of SEQ ID No.6 encoding the CABYV resistance protein of present invention.

[0112] Example 4 - Identification of SGR gene in cucumber, stay green effect in cucumber

[0113] Fl hybrid referenced herein as E23L.16642, was observed as having superior shelf life and disease tolerance characteristics. The shelf life of the fruits of E23L.16642 was measured during approximately 5 weeks using the following scale: (1) Good colour (green); (2) Acceptable colour (somewhat lighter green); (3) Unacceptable colour (first occurrence of yellowing); (4) Unacceptable colour (yellowing); (5) Unacceptable colour (extreme yellowing). A conventional cucumber fruit (Marumba variety) was also measured for approximately 5 weeks.

[0114] Molecular marker analysis revealed that a genetic factor influencing the value of the observed traits is located on Chr5 of the cucumber genome. E23L.16642 is sequenced using Illumina short read sequencing technology (2x125 bp) on whole genome DNA processed for sequencing with Illumina TruSeq sample preparation. Sequencing results were processed according to standard procedure and aligned to public reference genome sequence 9930 v2 (http: / / cucurbitgenomics.Org / organism / 2), along with larger set of 284 other diverse cucumber germplasm samples. A single position was identified that is unique to E23L.16642 linked to the stay green and masking phenotype observed. The position unique to E23L.16642 results in the loss of a splice site. The possible splice site abolishing effect of the E23L.16642 variant Chr5:5464327 G>T (referred to herein as G1410T mutation) was further investigated by producing cDNA sequence results from E23L.16642. The splicing acceptor sequence “AG”, typically present at the 3’ end of an intron, is found as “AT” in E23L.16642. This AT will not function as acceptor and the next “AG” site downstream of the original will gain the function of splicing acceptor. This next possible splice acceptor site is located 25 bp downstream of the original, resulting in an additional 25 bp deletion from the cDNA sequence, compared to the wildtype sequence. The 25 bp deletion in the coding sequence for SGR effectively results in a frameshift mutation, causing the translational frame to jump from frame 1 to frame 3. In effect, this frameshift results in a premature stop just 9 amino acids downstream and this premature stop results in a non-functional truncated SGR protein. Seeds of the cucumber plants with a mutated SGR gene of SEQ ID No. 30 (i.e. comprising the G1410T mutation) in homozygous form and wild type plants (i.e. not comprising the mutated SGR gene) were grown till the cotyledon stage. The cotyledons were removed from the plantlets and placed in a plastic container covered with a glass plate. It can be considered that there was no air exchange possible between the content of the container and the environment. After 2 weeks of storage the leaves of the plant comprising the mutated SGR gene remained green, whereas the wild type plant yellowing of the leaves is observed.

[0115] Example 5 - Masking effect in Downy Mildew affected cucumber plants comprising the mutated SGR gene and CABYV resistance gene

[0116] Leaves of the cucumber plant which are in the lower part of the plant, become old and yellow, start to disintegrate when they are covered by upper leaves. The disintegration of the leaf is, amongst others, caused by the inactivity and degradation of the chlorophyll. Chlorophyll is giving the green color to the leaf. Downy mildew (caused by Pseudoperonospora citbensis} in cucumber causes light green to yellow angular spots on the upper surfaces of leaves. With time these lesions turn brown and dry up.

[0117] Plants of the wild type (lacking the present mutant stay green gene), plants with the present stay green gene in heterozygous form, and plants with the present stay green gene comprising the mutation in homozygous form have been planted in the greenhouse and subjected to Downy Mildew infection. At mature stage of the plants (i.e. after 10 weeks) were placed next to each other and infected by Downy Mildew. The result is shown in Figure 3. It is clear that the plant (Upper panels are plant of E23L.16642, a plant according to present invention) with the present mutated SGR gene in homozygous form (and CABYV resistance gene), can maintain its chlorophyll for a longer period as compared to the control plant (Marumba variety) not comprising the mutated SGR gene. Likewise, it is expected that the plant containing this mutated SGR gene in homozygous form can be photosynthetic active for a longer period. The E23L.16642 provides a “stay green” effect by masking Downy mildew infection showing total absence of yellowing, see Figure 3 upper panels. Figure 3 lower panels show the control that is affected by Downy Mildew as observed by the yellowing and curled leaves.

[0118] When infected with CABYV, the E23L.16642 plant comprising the mutated SGR gene and CABYV resistance gene, showed absence (or strongly reduced) leaf yellowing. Therefore, the plants remain healthy and green as compared to the wild type plants which do not comprise the SGR and CABYV genes (Figure 4). The plants on the left panel are the control plant not comprising the mutated SGR gene and CABYV resistance gene. The plants on the right panel are the plants comprising the mutated SGR gene and CABYV resistance gene. Example 6 - Sequence homology among the newly identified CABYV resistance protein and SGR gene in Cucurbitaceae plants.

[0119] The sequence homology among the in cucumber (Cucumis sativus) newly identified CABYV resistance protein (SEQ ID No. 3) of various Cucurbitaceae plants were analyzed using multiple alignment software CABYV resistance protein of, Cucumis melo (2), Cucurbita pepo (3), Cucurbita moschata (4), Cucurbita argyrosperma (5), Lagenaria siceraria (6), Cucurbita maxima (7), Citrullus lanatus (8), and Momordica charantia (9) were analyzed and it was shown that the Cucurbitaceae plants all comprise proteins that share a high sequence homology of at least 89%, and in most cases more than 93% sequence identity between Cucurbitaceae plants (Table 4) with the newly identified CABYV resistance protein. Similarly, Table 5 shows the homology of CABYV resistance protein of SEQ ID No.6, having at least 95% homology.

[0120] Table 4. % sequence homology among CABYV resistance protein SEQ ID No.3 in Cucurbitaceae plants.

[0121] Table 5. % sequence homology among CABYV resistance protein SEQ ID No.6 in Cucurbitaceae plants.

[0122] Similar as for the newly identified CABYV resistance protein, protein alignments were made on the SGR protein, that show that the SGR is highly conserved within the Cucurbitaceae family; Lagenaria siceraria (2), Cucurbita argyrosperma (3), Cucurbita maxima (4), Cucurbita moschata (5), Cucurbita pepo (6), and Momordica charantia (7), Cucumis melo (8), Citrullus lanatus (9). Since the G1410T mutation leads to a non-functional protein in cucumber, the protein sequences of up to the potential splice for each of the included Cucurbitaceae plants was included, as it is to be expected on these sequences that the effect of the nonfunctional truncated protein (knock out) is the same for the included plants. The Cucurbitaceae plants comprise the SGR protein that share a high sequence homology of at least 81%, and in most cases more than 91% sequence identity (Table 6) with the mutated SGR resistance protein in cucumber.

[0123] Table 6. Shows the % sequence homology among SGR protein sequences in Cucurbitaceae plants

[0124] Example 7 - CABYV resistance locus VIGS gene silencing and virus titer ELISA

[0125] Virus-induced gene silencing (VIGS) is a method used to study gene function in plants. By modifying a virus to carry a short piece of the plant’s genetic material, any gene homologous to this sequence will be “silenced” (partially turned off) once the virus infects and spreads through the plant. By silencing specific genes, we can study their involvement in the development of specific phenotypes, in this case disease resistance (reverse genetics).

[0126] After gene mapping the CABYV resistance gene present on the CABYV resistance locus, VIGS gene silencing was done to silence the identified candidate gene in both the resistant and susceptible plants. Therefore, a VIGS-construct (SEQ ID No. 40) was made targeting the candidate CABYV resistance gene (i.e. targeting SEQ ID No.5, see Table 7 for the VIGS sequences). Furthermore, a VIGS construct targeting the coding sequence encoding for the red fluorescent protein (RFP), was included as negative control (SEQ ID No. 41). The constructs were transformed by viral vectors into cucumber to study the CABYV resistance gene function.

[0127] Two plants were included in this test; Plant 1 is a susceptible cucumber ( Cucumis sativus) plant comprising the “wild type” non mutated CABYV resistance gene (i.e. not comprising the G166D mutation as previously identified in SEQ ID No.5), Plant 2 is a cucumber ( Cucumis sativus) plant according to present invention comprising a CABYV resistance gene comprising a mutation, more specifically a G497A mutation in view of SEQ ID No.5, resulting in a G166D amino acid change, as represented by SEQ ID No.6. Plant 1 is susceptible to CABYV, plant to is resistant to CABYV.

[0128] The candidate CABYV resistance gene is silenced in VIGS independent experiments. With the plants of the VIGS -experiments independent disease tests were performed on both resistant and susceptible plants (as control) to observe that when the candidate CABYV resistance gene was silenced in susceptible plant, these plants became resistant to CABYV.

[0129] Furthermore, viral titers are measured in the VIGS treated plants to monitor and quantify viral infection. ELISA measures the CABYV virus titers, indicated as relative absorbance. Furthermore, all plants that were VIGS inoculated and infected with CABYV were sampled. Each sample that did not show any gene silencing was discarded from the test. Disease resistance tests and ELISA were performed on the VIGS treated plants, wherein plants were disease scored as described previously in example 1.

[0130] Table 7. VIGS constructs

[0131] Results (Table 8) show that when the CABYV resistance gene was silenced by VIGS, plant 1 became resistant to viral infection, as indicated by increased disease score and reduced virus titers. Surprisingly, this seems to suggest that the gene is being used by the CABYV during infection and promote disease development and / or spreading of the virus. The exhibited resistant phenotype of the CABYV resistance gene VIGS silenced plant 1 was comparable to the untreated (no VIGS) plant 2, which is known to be CABYV resistant. Results show that the CABYV resistance gene provides resistance to CABYV when silenced (expression is reduced or absent), as indicated by the VIGS results. When the CABYV resistance gene in Plant 2 was silenced, the disease score remained high and virus titers remained low. The disease scoring corresponded with the ELISA results. ELISA values above 0.3 were considered as susceptible and <0.3 as resistant to CABYV.

[0132] Table 8. Disease score and ELISA values of CABYV infected plants after VIGS silencing.

[0133] Example 8 - 3D modeling of the CABYV resistance protein

[0134] An in-silico protein folding experiment with artificial intelligence in YAS ARA was performed to determine the effect of the mutations and monitor differences between a susceptible variant (SEQ ID No.42) and the resistance CABYV resistant gene (SEQ ID No. 6) that comprises the amino acid substitution of G166D. We modelled both the susceptible and resistant form of the protein. We found that this mutation does not lead to changes in the structure of the protein per se, but there is a change is the polarity from non-polar to polar. Furthermore, 3D modelling shows that this amino-acid is located on the outside of the protein structure which may lead to different protein-protein interactions. In support of this hypothesis, a similar observation was found in the homolog protein of Arabidopsis, indicated as Arabidopsis RBV, as disclosed in C. Liang et al., 2022, Nature communications, Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGON AUTE1 loading. Here it was also observed that 1 amino-acid change resulted in a suppression of leaf bleaching in Arabidopsis. This amino-acid change was also from a non-polar amino-acid to a polar amino-acid and was also on the outside structure of the protein resulting in a phenotypic effect in the plant. Therefore and in view of the above, the known G166D mutation resulting in the resistant phenotype in combination with the data of 3D modelling, in the plant of present invention, it was observed that, wherein said one or more mutations preferably result in an amino acid substitution of the non-polar amino acid Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No.42, in a polar amino acid substitution preferably selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

Claims

Claims1. A Cucurbitaceae plant that is resistant to Cucurbit aphid-borne yellows disease, wherein said plant comprises a CABYV resistance gene that encodes for a CABYV resistance protein, wherein said CABYV resistance gene comprises one or more mutations providing said resistance to Cucurbit aphid-borne yellows disease, wherein said CABYV resistance protein comprises an amino acid sequence having at least 95%, more preferably at least 98% sequence identity with SEQ ID No.6.

2. Cucurbitaceae plant according to claim 1, wherein expression of the mutated CABYV resistance gene or protein activity of the mutated CABYV resistance protein is reduced as compared to the expression or the protein activity of a CABYV resistance gene or CABYV resistance protein, respectively, in a Cucurbitaceae plant not resistant to Cucurbit aphid-borne yellows disease.

3. Cucurbitaceae plant according to claim 1 or 2, wherein said CABYV resistance gene encodes for a coding sequence comprised of SEQ ID No.5, or having at least 95% sequence, preferably at least 98% sequence identity with said SEQ ID No. 5.

4. Cucurbitaceae plant according to any one of the claims 1 to 3, wherein said reduced protein activity or said reduced gene expression is provided by said one or more mutations in the coding sequence and / or in the promotor sequences and / or regulatory sequences of said CABYV resistance gene.

5. Cucurbitaceae plant according to any one of the claims 1 to 4, wherein said one or more mutations in said CABYV resistance gene result in an amino acid substitution, frame-shift or premature stop codon, preferably amino acid substitutions in the CABYV resistance protein.

6. Cucurbitaceae plant according to any one of the claims 1 to 5, wherein said one or more mutations results in a non-functional or truncated CABYV resistance protein.

7. Cucurbitaceae plant according to any one of the claims 1 to 6, wherein said one or more mutations result in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No.42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid(E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

8. Cucurbitaceae plant according to any one of the claims 1 or 7, wherein said one or more mutations result in an amino acid substitution of G166D in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

9. Cucurbitaceae plant according to any one of the claims 1 or 8, wherein said one or more mutations is a G497A mutation in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

10. Cucurbitaceae plant according to any one of the claims 1 to 9, wherein said CABYV resistance CABYV resistance gene is present in heterozygous or homozygous form, preferably homozygous.

11. Cucurbitaceae plant according to any one of the claims 1 to 10, wherein said CABYV resistance gene is obtainable from deposit number NCIMB 43857.

12. Cucurbitaceae plant according to any one of the claims 1 to 11, wherein said plant further comprises a stay green (SGR) gene comprising one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein comprising an amino acid sequence having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32.

13. Cucurbitaceae plant according to claim 1 to 12, wherein the expression or the protein activity of said mutated SGR gene is reduced as compared to the expression or the protein activity of a SGR gene in a Cucurbitaceae plant not providing fruits having an improved shelf life, wherein said reduced protein activity or gene expression is provided by one or more mutations in the genomic or coding sequence of said stay green gene.

14. Cucurbitaceae plant according to claim 12 or 13, wherein said one or more mutations in the SGR gene is selected from the group consisting of;- a G1410T mutation in the SGR sequence represented by SEQ ID No.27,- a C457T mutation in the SGR sequence represented by SEQ ID No.28,- a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,- a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

15. Cucurbitaceae plant according to any one of the claims 12 to 14, wherein said one or more mutations in the SGR gene results in a non-functional or truncated SGR protein, preferably the SGR protein of SEQ ID No.32.

16. Cucurbitaceae plant according to any one of the claims 12 to 15, wherein said SGR gene is present in heterozygous or homozygous form, preferably homozygous.

17. Cucurbitaceae plant according to any one of the claims 1 to 16, wherein the Cucurbit aphid- borne yellows disease is caused by Cucurbit aphid-borne yellows disease virus (CABYV).

18. Cucurbitaceae plant according to any one of the claims 1 to 17 wherein said plant is one or more selected from the group consisting of Cucumis sativus, Cucumis melo, Citrullus lanatus, Cucurbita maxima, Cucurbita pepo, Cucurbita moschata, Lagenaria siceraria, Cucurbita argyrosperma and Momordica charantia, preferably Cucumis sativus.

19. A Seed, plant cell, plant tissue, fruit, or plants parts of a Cucurbitaceae plant according to any of claim 1 to 18 comprising a CABYV resistance gene providing resistance to Cucurbit aphid- borne yellows disease, and / or an SGR gene providing improved fruit shelf life and / or reduced leaf yellowing.

20. A resistance gene for providing CABYV resistance in a Cucurbitaceae plant, wherein the resistance gene encodes for a CABYV resistance protein, wherein said CABYV resistance gene comprises one or more mutations providing said resistance to Cucurbit aphid-borne yellows disease, wherein said CABYV resistance protein comprises an amino acid sequence having at least 95%, more preferably at least 98% sequence identity with SEQ ID No.6.

21. Resistance gene according to claim 20, wherein the CABYV resistance gene encodes for a coding sequence of SEQ ID No. 5 or having at least 95% sequence, preferably at least 99% identity with SEQ ID No.5.

22. Resistance gene according to claim 20 or 21, wherein said one or more mutations result in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consistingof Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

23. Resistance gene according to any one of the claims 20 to 22, wherein said one or more mutations results in an amino acid substitution G166D in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.

624. Resistance gene according to any one of the claims 20 to 23, wherein said one or more mutations is a G497A mutation in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

25. A protein for providing CABYV resistance in a Cucurbitaceae plant, wherein the protein comprises SEQ ID No.6, or having at least 95% sequence, preferably at least 98% identity with SEQ ID No.6.

26. A combination of a CABYV resistance gene as defined in any one of the claims 1 to 11, 20 to 24 and an SGR gene as defined in any one of the claims 12 to 16 for providing CABYV resistance in in a Cucurbitaceae plant and for providing fruits of said plant having an improved shelf life and / or reduced leaf yellowing.

27. Method for identifying or selecting a Cucurbitaceae plant of any one of the claims 1 to 18 that is resistant to Cucurbit aphid-borne yellows disease and / or having an improved shelf life and / or reduced leaf yellowing, wherein the method comprises the step of establishing the presence of a CABYV resistance gene in said Cucurbitaceae plant that encodes for a CABYV resistance protein comprising SEQ ID No.6, or having at least 95% sequence, preferably at least 98% sequence identity with said SEQ ID No.

6. in said plant, and / or, wherein the method comprises the step of establishing the presence of a CABYV resistance gene comprising SEQ ID No. 5 in said Cucurbitaceae plant or at least 95% sequence identity, preferably at least 98% sequence identity with identity SEQ ID No. 5.

28. Method according to claim 27, wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence of an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented byamino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

29. Method according to claim 27 or 28, wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence of an Aspartic Acid on position 166 in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

30. Method according to any one of claim 27 to 29, wherein the step of establishing the presence of said CABYV resistance gene in said Cucurbitaceae plant comprises the determination of the presence a “A” on position 497 in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

31. Method according to any one of the claims 27 to 30, wherein the method further comprises the step of establishing the presence of a stay green (SGR) gene as defined in any one of the claims 12 to 16 in the Cucurbitaceae plant.

32. Method according to claim 31 , wherein said SGR gene comprises one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32.

33. Method according to claim 32, wherein said one or more mutations is selected from the group consisting of;- a G1410T mutation in the SGR sequence represented by SEQ ID No.27,- a C457T mutation in the SGR sequence represented by SEQ ID No.28,- a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,- a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

34. Method for providing a Cucurbitaceae plant of any one of the claims 1 to 18 that is resistant to Cucurbit aphid-borne yellows disease, said method comprises the step of introducing one or more mutations in a CABYV resistance gene encoding a CABYV resistance protein comprising SEQ IDNo.6, or having at least 95% sequence, preferably at least 98% sequence identity with said SEQ ID No.

6. in said plant, and / or, wherein the method comprises the step of introducing one or more mutations in a CABYV resistance gene comprising SEQ ID No. 5 in said Cucurbitaceae plant or at least 95% sequence identity, preferably at least 98% sequence identity with identity SEQ ID No. 5, and wherein the expression of the mutated CABYV resistance gene is reduced as compared to the expression of said CABYV resistance gene in a Cucurbitaceae plant not being resistant to Cucurbit aphid-borne yellows disease.

35. Method according to claim 34, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an amino acid substitution of a Glycine (G) on any one of the positions 19, 30, 50, 60, 80, 92, 100, 103, 166, 176, 184, 216, 224, 250 and 292 in the CABYV resistance protein as represented by amino acid sequence of SEQ ID No. 42, preferably wherein said amino acid substitution of the Glycine (G) is an amino acid substitution to a polar amino acid selected from the group consisting of Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glutamic acid (E), Histidine (H), Lysine (K), Serine (S), Threonine (T), and Tyrosine (Y), more preferably a G to Q on position 30 (G30Q) and / or a G to D on position 166 (G166D).

36. Method according to claim 34 or 35, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an amino acid change in said CABYV resistance protein resulting in an Aspartic Acid on position 166 in the CABYV resistance protein, as represented by amino acid sequence of SEQ ID No.6.

37. Method according to any one of the claims 34 to 36, wherein the step of introducing one or more mutations in the CABYV resistance gene in said Cucurbitaceae plant results in an “A” on position 497 in the coding sequence of the CABYV resistance gene, as represented by coding sequence of SEQ ID No.5.

38. Method according to any one of the claims 34 to 37, wherein the method further comprises the step the step of introducing one or more mutations in an SGR gene, wherein the SGR gene comprises one or more mutations providing improved fruit shelf life and / or reduced leaf yellowing, wherein said SGR gene encodes an SGR protein having at least 81%, more preferably at least 91% sequence identity with SEQ ID No.32.

39. Method according to claim 38, wherein said one or more mutations is selected from the group consisting of;- a G1410T mutation in the SGR sequence represented by SEQ ID No.27,- a C457T mutation in the SGR sequence represented by SEQ ID No.28,- a C155T and / or a G215A mutation in the SGR sequence represented by SEQ ID No.33, 35, 36, 37, 38, or 39,- a C161T and / or G221A mutation in the SGR sequence represented by SEQ ID No.34.

40. Method according to any one of the claims 34 to 39, wherein said one or more mutations in the CABYV resistance gene and / or SGR gene are provided by gene editing techniques, preferably by chemical mutagenesis, UV or gamma radiation, or by CRISPR-Cas.

41. Method for providing a Cucurbitaceae plant of any one of the claims 1 to 18, wherein the method comprises the steps of, a) crossing a Cucurbitaceae plant comprising a CABYV resistance gene as defined in any one of the claims any one of the claims 1 to 11 , 20 to 24 and an SGR gene as defined in any one of the claims 12 to 16 with a Cucurbitaceae plant that is susceptible to Cucurbit aphid-borne yellows disease and does not comprise said CABYV resistance gene and / or SGR gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to Cucurbit aphid-borne yellows disease and / or show reduced leaf yellowing, wherein said plant preferably comprises said CABYV resistance gene and SGR gene.

42. Method according to claim 41, wherein the selecting of step c is done by the method of any one of claim 27 to 33.

43. Use of a CABYV resistance gene as defined in any one of the claims 1 to 11, 20 to 24 for providing CABYV resistance in a Cucurbitaceae plant.

44. Use of a CABYV resistance gene as defined in any one of the claims 1 to 11, 20 to 24 and an SGR gene as defined in any one of the claims 12 to 16 in Cucurbitaceae plant for providing CABYV resistance in a Cucurbitaceae plant and providing fruits having an improved shelf life or reduced leaf yellowing.

Images