WO2025233367A1 - Corynespora resistant plant - Google Patents

Abstract

The present invention relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1, wherein said modified WAK gene leads to Corynespora cassiicola resistance in a plant. The function of the encoded WAK protein is reduced, changed or absent. The invention further relates to plants comprising the modified gene and showing resistance to a Corynespora cassiicola.

Description

CORYNESPORA RESISTANT PLANT

The present invention relates to a modified gene which leads to Corynespora cassiicola resistance in a plant. The invention further relates to a plant comprising said modified gene. The invention further relates to a method for producing such a plant and methods for identification and selection of such a plant. The invention also relates to progeny, seed and fruit of the Corynespora cassiicola resistant plant, to propagation material suitable for producing the plant, and to a food product comprising such fruit or part thereof. The invention also relates to a method for the identification of the presence of the modified gene in such a plant.

Corynespora cassiicola leads to a fungal disease called “target leaf spot” which affects a broad range of plant species such as cucumber. Symptoms of the infection usually start with small and irregular spots on the leaves having a yellow margin. As the disease progresses, spots enlarge, and the affected leaves fall out. Severe infection can result in plant defoliation, which directly or indirectly leads to a reduction in yield since this will cause the premature death of the plant. The spots can also affect the stem and the fruit of the infected plant.

The main Corynespora cassiicola control in many regions remains the repeated application of fungicides to minimize the impact of the disease but the fungus develops resistances against some of these fungicides. There is thus a need for the provision of genes that lead to Corynespora cassiicola resistance. The search for new genetic resources for resistance is permanently ongoing in plant breeding research. Developing new varieties that help the growers cope with the ever-present disease challenges requires constant adaptation to new or stronger variants of fungi.

It is an object of the present invention to provide new genetics for resistance to Corynespora cassiicola in a plant. In the research leading to the invention, it was surprisingly found that a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence confers resistance to Corynespora cassiicola in a plant, in particular in a cucumber plant.

Wall-associated kinases (WAK) are known to be localized in the cell wall and to act as a link for signals between the extracellular matrix and the cytoplasm.

The WAK gene of the invention was identified through fine-mapping of various internally developed cucumber populations that segregated for Corynespora cassiicola resistance. As described in Example 2, various internally developed cucumber populations that segregated for Corynespora cassiicola resistance were fine-mapped to a small region on chromosome 6 comprising potential genes which were likely to contribute to the Corynespora cassiicola resistance. Whole genome sequences were available in-house for the backgrounds of the resistant and susceptible lines that were used in the development of these populations. Therefore, by comparing the sequences of the resistant lines to the sequences of the susceptible lines and by determining the differences, unique polymorphisms in the region were identified. Among the genes in the region of interest was a WAK gene, which had various polymorphisms between susceptible and resistant material.

Through analysis of the correlation in segregation of phenotypes and genotypes it was determined that a modification in an WAK gene as compared to the wild type was the cause of the Corynespora cassiicola resistance of the resistant cucumber plants.

The localization of the corresponding wild -type WAK gene of the invention in cucumber (Cucumis sativus) is cs9930_v3_BRKR.chr6.0203730 (Cucumber_9930_V3), which is published in the NCBI database and can for example be found at https://www.ncbi.nlm.nih.gOv/datasets/genome/GCF_000004075.3/. The wild-type coding sequence (CDS) of the WAK gene comprises SEQ ID No. 1. The wild-type genomic sequence of the WAK gene comprises SEQ ID No. 6. The encoded WAK wild-type protein comprises SEQ ID No. 2. As used herein the “wild-type WAK gene of the invention” comprises SEQ No. 1 and encodes the protein having SEQ ID No. 2. The wild-type sequence does not confer resistance to Corynespora cassiicola to a cucumber plant.

A homologous sequence of a gene as used herein defines genes which encode proteins having the same or similar function. As used herein, sequence identity is the percentage of nucleotides or amino acids that are identical between two sequences after proper alignment of those sequences. As used herein, sequence similarity is the percentage of amino acids that are identical between two sequences after proper alignment of those sequences. The person skilled in the art is aware of how to align sequences, for example by using a sequence alignment tool such as BLAST®, which can be used for both nucleotide sequences and protein sequences. To obtain the most significant result, the best possible alignment that gives the highest sequence identity or similarity score should be obtained. The percentage sequence identity or similarity is calculated through comparison over the length of the shortest sequence in the assessment, wherein in the present case a sequence that is included in such an assessment represents a gene that at least comprises a start codon and a stop codon, or a complete protein encoded by such a gene. Sequence similarity is used to compare amino acid sequences, wherein conservative amino acid substitutions are deemed to be similar and are calculated herein based on the BLOSUM62 scoring matrix.

The deletion, and/or the substitution, and/or the insertion are thus not included in the calculation of the 80% or more sequence identity.

The invention relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1.

The invention relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1, wherein said modified WAK gene leads to Corynespora cassiicola resistance in a plant. Said modified gene is the “modified WAK gene of the invention”. In one embodiment, the WAK gene of this invention is a nucleic acid, in particular a nucleic acid molecule, more in particular an isolated nucleic acid molecule.

The modified WAK gene of the invention confers resistance to a fungus to a cucumber plant. The fungus can be selected from the group consisting of Alternaria cucumerina, Colletotrichum orbiculare, Rhizoctonia solani, Cercospora citrulline, Pseudoperonospora cubensis, Fusarium oxysporum, Erysiphe cichoracearum, Septaria cucurbitacearum, Corynespora cassiicola and Verticillium dahlia. Preferably, the modified WAK gene of the invention confers resistance to Corynespora cassiicola [(Berk. & M.A. Curtis) C.T. Wei, (1950)] to a cucumber plant.

The invention relates in particular to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1, wherein said modified WAK gene leads to Corynespora cassiicola resistance in a cucumber plant.

The invention relates in particular to a modified WAK gene comprising a deletion and/or a substitution, and an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1.

The invention relates in particular to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1, wherein said modified WAK gene leads to Corynespora cassiicola resistance in a cucumber plant and wherein the deletion, substitution and insertion are at separate locations of the gene. As used herein, a modified WAK gene of the invention is a WAK gene comprising a deletion and/or a substitution and/or an insertion when compared to the wild-type sequence. As used herein, a gene comprises exonic sequences and regulatory sequences such as a promoter sequence, UTR and polyadenylation signals and if present it also comprises intronic sequences.

Modifications of the WAK gene leading to a reduction, change or absence of the function of the encoded WAK protein can be the absence of the gene, an amino acid change caused by a nucleotide substitution, a modification resulting in a premature stop codon, or a modification resulting in a frameshift. The modifications lead to a protein with an altered expression level and/or function of the encoded protein: the function of the encoded WAK protein is reduced, changed or absent. The modifications described herein are loss-of-function mutations.

The invention relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , wherein the modification is that the WAK gene is absent, i.e. the modification comprises the absence of SEQ ID No. 1 leading to a knocked out WAK gene and wherein said modified WAK gene leads to Corynespora cassiicola resistance in a plant.

The invention also relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , wherein the modified WAK gene comprises a mutation resulting in a frame shift or the modified WAK gene comprises a premature stop codon leading to a knocked out WAK gene and wherein said modified WAK gene leads to Corynespora cassiicola resistance in a plant.

An insertion changes the number of DNA bases in a gene by adding a piece of DNA. A deletion changes the number of DNA bases by removing one or more nucleotides, or even an entire gene. The gene is absent when the entire gene is removed from the genome of a plant. As used herein when the WAK gene is absent, SEQ ID No. 1 is absent. When the WAK gene is absent the encoded WAK protein comprising SEQ ID No. 2 is not expressed in the plant.

A nucleotide substitution of one or more nucleotides in the coding sequence can result in a different codon, often encoding a different amino acid and leading to an amino acid substitution in the encoded protein sequence. Mutations resulting in an amino acid substitution are called non-synonymous or missense mutations. Due to the redundancy of the genetic code not all point mutations lead to amino acid changes. Such mutations are termed silent mutations. Some amino acid changes are conservative, i.e. they lead to the replacement of one amino acid with another amino acid with comparable properties, such that the mutation is unlikely to change the folding of the mature protein or influence its function. Other amino acid changes are more likely to affect protein function: non-conservative amino acid changes in domains that play a role in substrate recognition, the active site of enzymes, interaction domains or in major structural domains (such as transmembrane helices) may partly or completely destroy the functionality of an encoded protein, without thereby necessarily affecting the expression level of the encoding gene. Whether an amino acid substitution is conservative or non-conservative may be predicted on the basis of chemical properties, for example similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity or amphipathic nature of the amino acids.

Frame shift mutations are caused by insertion or deletion of one or more nucleotides in a DNA sequence encoding a protein. When the number of inserted or deleted nucleotides at a certain position within the coding sequence is not a multiple of 3, the codon encoding the individual amino acids of the protein sequence becomes shifted relative to the original open reading frame, and then the encoded protein sequence changes. Protein translation will result in an entirely different amino acid sequence than that of the originally encoded protein, and very often a frameshift leads to a premature stop codon in the open reading frame.

A deletion, insertion, nucleotide substitution and/or frame shift mutation may result in a knocked out gene. A knocked out gene is a nucleic acid molecule encoding a protein wherein a codon is changed into a premature stop codon resulting in expression of a truncated protein. How much of the protein is lost determines whether or not the protein is still functional. Especially when all or part of the conserved functional domains are lacking from the truncated protein it is likely protein function is affected. If a premature stop codon is introduced early in the gene sequence, it is more likely that the gene is not even expressed. When the gene is absent it also results in a knocked out gene.

The modification to the gene affects the expression and/or function of the encoded protein. The expression of the modified gene can be inhibited leading to the modified gene not being expressed into a protein. Due to the modification, the encoded protein has a changed function, a reduced function, or it is non-functional. The function of the protein is also affected when the expression of the protein is changed, reduced or absent.

The invention relates to a modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , wherein said modified WAK gene is not expressed, which leads to Corynespora cassiicola resistance in a plant.

In particular, the invention relates to a modified WAK gene comprising a deletion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1 , wherein said modified WAK gene is absent, resulting in the WAK gene not being expressed, which leads to Corynespora cassiicola resistance in a plant. During the research for the present invention, it was identified that the susceptible wild-type cucumber plants comprise a functional WAK gene with a coding sequence comprising SEQ ID No. 1. Two cucumber plants that are resistant to Corynespora cassiicola were identified and it was found that in these plants said WAK gene is absent. The involvement of the WAK gene was confirmed in the experiment performed in Example 3; plants with a modified WAK gene of the invention had a lower phenotypic score as compared to plants with the wild-type WAK gene.

In one embodiment, the invention relates to the complete deletion of the WAK gene. A cucumber plant of the invention, comprising a complete deletion of the wild-type WAK gene homozygously can be grown from seed deposited as NCIMB 44368 or NCIMB 44369. Such plants show Corynespora cassiicola resistance.

In a particular embodiment, the invention relates to a deletion of 16765 bp from the genome of the cucumber plant, which comprises the deletion of SEQ ID No. 1. The said deletion leads to the presence of SEQ ID No. 4 on Chromosome 6 of the cucumber plant. SEQ ID No. 4 comprises the sequences upstream and downstream from the deletion that are now adjacent to each other. The location of the deletion in SEQ ID No. 4 is indicated with [...]. This deletion is as comprised in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44368. A cucumber plant that has said deletion homozygously can be grown from seed deposited as NCIMB 44368. Such plants show Corynespora cassiicola resistance.

The invention relates to a cucumber plant comprising a deletion of 16765 bp from its genome as found in a in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44368, wherein said cucumber plant is resistant to Corynespora cassiicola.

Optionally, the modified WAK gene of the invention is combined with the presence of a gene which CDS sequence comprises SEQ ID No. 3 in a cucumber plant. The modified WAK gene of the invention is combined with the presence of a gene which CDS sequence comprises SEQ ID No. 3 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 3, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 3. The combination of the gene comprising SEQ ID No. 3 and the absence of the wild-type WAK gene of the invention is as comprised in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44369. A cucumber plant that comprises the absence of the wild-type WAK gene of the invention homozygously can be grown from seed deposited as NCIMB 44369.

The invention relates to a cucumber plant comprising the deletion of the WAK gene as found in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44369, wherein said cucumber plant is resistant to Corynespora cassiicola.

The WAK gene can be modified by using different methods. Two examples are described in Example 4. In the examples modifications are introduced in seed of a plant of interest in which resistance to a Corynespora cassiicola is needed. The modification is introduced through mutagenesis, such as an EMS treatment, through radiation means, or through a specific targeted approach, such as a CRISPR/Cas system. The radiation means comprising fast neutron (FN) exposure is a known method that leads to deletions varying from a deletion of one single nucleotide to a deletion of several hundred nucleotides. This method can be used to introduce the deletion as described herein into a cucumber plant. When a non-targeted approach such as EMS is used, this is combined with an identification technique such as TILLING. In this way, both for mutagenesis as well as a targeted modification means, a modification in a WAK gene can be generated and identified. The skilled person is familiar with these means for introducing modifications into the genome of a plant of interest. Modified seed is then germinated and plants are grown, which are crossed or selfed to generate M2 or further generation seed. Subsequently a plant screen is performed to identify the modifications in a WAK gene, based on comparison to the wild-type sequence of the WAK gene of that species. For cucumber for example, comparison to SEQ ID No. 1 should be done. The skilled person is familiar with TILLING to identify mutations in specific genes (McCallum et. Al. (2000) Nature Biotechnology, 18: 455-457), and with techniques for identifying nucleotide changes such as DNA sequencing, amongst others. Plants with a modified WAK gene are homozygous or made homozygous by selfing, crossing, or the use of doubled haploid techniques which are familiar to the skilled person. Plants identified and selected on the basis of a modification in a WAK gene can then be tested for resistance to a Corynespora cassiicola. A plant that is produced, identified and selected in this way is confirmed to have the resistance as a result from one or more modifications in the WAK gene.

In one embodiment, the modification of the present invention is in particular an induced modification. An induced modification is not naturally present, and leads to a non-natural modified WAK gene, and thereby to a non-natural modified WAK protein.

In particular for cucumber plants, the resistance is tested by the Corynespora cassiicola resistance test as further described in Example 1.

In short, ten plants are sown in trays filled with potting soil in a climate cell under normal cucumber growing conditions. Multiplication of the Corynespora cassiicola pathogen is started on standard agar plates and when the first leaf has a size of 2-3 cm (12-13 days after sowing), plants are inoculated with 40.000 spores/ml using a high pressure inoculum pump. After inoculation plants are covered with plastic and grown at regular greenhouse temperatures. The plants are assessed 8-10 days after inoculation using the table 1. Ventura RZ or a plant grown from the deposits NCIMB 44369 and NCIMB 44368, can be used as resistant variety and Kaspian RZ can be used as a susceptible variety.

As used herein, a resistant plant is a plant having a phenotypic score of 1 or 2 when using Table 1 for scoring. In particular, a resistant cucumber plant is a plant having a phenotypic score of 1 or 2 when using Table 1 for scoring. A plant comprising the modified WAK gene of the invention homozygously has a phenotypic score of at least 2, preferably 1 , when using Table 1 for scoring.

A plant of the invention is identified by verifying the sequence of the WAK gene and performing a resistance test. If the identified plant comprises a modification such as a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence and said modification leading to an encoded protein having reduced, changed or absence of function or expression, and the tested plant is resistant to Corynespora cassiicola the plant is a plant of the invention. The identified plant is in particular a cucumber plant.

Alternatively, determining the presence of a modification can be done through sequence comparison, which is known to the skilled person.

The present invention relates to a plant comprising a modified WAK gene of the invention. The present invention relates in particular to a Corynespora cassiicola resistant cucumber plant (Cucumis sativus) comprising a modified WAK gene of the invention, more in particular an absent WAK gene. A plant of the invention is preferably a cultivated plant which is non-wild and has agronomical value, and is in particular agronomically elite.

The invention also relates a method for the production of a plant, in particular a cucumber plant, that is resistant to Corynespora cassiicola by growing a seed, in particular a cucumber seed, lacking the WAK gene, comprising SEQ ID No. 1 or a sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1, into a plant.

The invention further relates to a seed that comprises a modified WAK gene of the invention, in particular an absent WAK gene, which seed can grow into a plant of the invention. The invention also relates to use of said seed for the production of a plant of the invention, by growing said seed into a plant. The invention also relates to a part of a plant, which comprises a modified WAK gene in its genome which plant part comprises a fruit or a seed. The seed is in particular a cucumber seed. The fruit is in particular a cucumber fruit.

The invention further relates to a method for seed production comprising growing a plant from a seed of the invention, allowing the plant to produce a fruit with seed, harvesting the fruit, and extracting those seed. Production of the seed is suitably done by selfing or by crossing with another plant that is optionally also a plant of the invention. The seed that is so produced has the capability to grow into a plant that is resistant to Corynespora cassiicola. The produced seed is in particular a cucumber seed.

The invention further relates to hybrid seed and to a method for producing said hybrid seed, comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and/or the second parent plant is a plant of the invention comprising a modified WAK gene of the invention. The resulting hybrid plant that can be grown from the hybrid seed, comprising a modified WAK gene of the invention, which hybrid plant has the modified WAK gene of the invention, is also a plant of the invention. The hybrid seed is in particular a cucumber seed. The parent that provides the resistance to Corynespora cassiicola, can be a plant grown directly from the deposited seed. The parent can also be a progeny plant from the deposited seed which is a direct or further descendant obtained by crossing with itself or with another plant one or more times, that has retained the modified WAK gene, or a progeny plant from seed that is identified to have obtained a modified WAK gene of the invention and thereby the resistance to a Corynespora cassiicola, by other means.

The present invention relates to a method for producing a Corynespora cassiicola resistant plant, comprising introducing a modification in a WAK gene, comprising SEQ ID No. 1 or a sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1 , which modification leads to resistance to Corynespora cassiicola. Said method comprises the introduction of a deletion and/or a substitution, and/or an insertion in the coding sequence and/or the promoter sequence of a WAK gene. The produced plant is in particular a cucumber plant.

In one embodiment the introduced modification in the WAK gene is a nucleotide change leading to an amino acid change in the encoded protein.

In a particular embodiment, the introduced modification in the WAK gene is a nucleotide substitution.

In another particular embodiment, the introduced modification in the WAK gene is a nucleotide deletion.

In a particular embodiment, the introduced modification in the WAK gene is a nucleotide insertion.

In a further embodiment, the introduced mutation comprises any combination of one or more substitutions, one or more deletions and one or more insertions.

In another embodiment, the introduced modification is a deletion of said WAK gene from the genome of the plant. In a particular embodiment, the introduced modification is a deletion of the sequence comprising SEQ ID No. 1. In a particular embodiment, the introduced modification is a deletion comprising nucleotide 1 to nucleotide 2253 of SEQ ID No. 1. In another embodiment the introduced modification in the WAK gene is a premature stop codon.

In another embodiment the introduced modification in the WAK gene results in a frame shift.

In another embodiment the introduced modification in the introduction of a deletion of 16765 bp, which deletion is as found in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44368.

The introduction of such a modification can be done by a mutagenesis approach using a chemical compound, such as ethyl methane sulphonate (EMS); or by using physical means, such as UV-irradiation, fast neutron exposure, or other irradiation techniques.

Introduction of a modification can also be done using a more specific, targeted approach including targeted genome editing by means of homologous recombination, oligonucleotide -based mutation introduction, zinc-finger nucleases (ZFN), transcription activatorlike effector nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems.

Introduction of a modified WAK gene of the invention can also be done through introgression from a Corynespora cassiicola resistant plant and comprising said modified WAK gene. Breeding methods such as crossing and selection, backcrossing, recombinant selection, or other breeding methods that result in the transfer of a genetic sequence from a resistant plant to a susceptible plant can be used. A resistant plant can be of the same species or of a different and/or wild species. Difficulties in crossing between species can be overcome through techniques known in the art such as embryo rescue, or cis-genesis can be applied. Progeny of a deposit can be sexual or vegetative descendants of that deposit, which can be selfed and/or crossed, and can be of an Fl, F2, or further generation, as long as the descendants of the deposit still comprise a modified WAK gene of the invention. A plant produced by such method is also a part of the invention. The produced plant is in particular a cucumber plant.

Transgenic techniques used for transferring sequences between plants that are sexually incompatible can also be used to produce a plant of the invention, by transferring a modified WAK gene from one species to another. Techniques that can suitably be used comprise general plant transformation techniques known to the skilled person, such as the use of an Agrobacterium-mediated transformation method.

The invention relates to a method for producing a Corynespora cassiicola resistant plant, comprising introducing a modified WAK gene of the invention into a plant.

The invention also relates to a method for the production of a Corynespora cassiicola resistant plant, said method comprising: a) crossing a plant comprising a modified WAK gene with another plant; b) optionally performing one or more rounds of selfing and/or crossing a plant resulting from step a) to obtain a further generation population; c) selecting from the population resulting from the cross of step a), or from the further generation population of step b), a plant that comprises a modified WAK gene as defined herein, which plant is a Corynespora cassiicola resistant plant.

The produced plant is in particular a cucumber plant. In one embodiment, the plant of the invention used in the method for the production of a Corynespora cassiicola resistant cucumber plant, is a plant grown from seed deposited under NCIMB accession number NCIMB 44368 or a plant grown from seed deposited under NCIMB accession number NCIMB 44369, or a progeny plant thereof which is a direct or further descendant through crossing a plant grown from the deposited seed with itself or with another plant for one or more subsequent generations.

The invention also relates to a method for the production of a Corynespora cassiicola resistant plant, said method comprising: a) crossing a first parent plant comprising a modified WAK gene with a second parent plant, which is another plant not comprising a modified WAK gene, or is another plant that comprises a different modification in a WAK gene; b) backcrossing the plant resulting from step a) with the second parent plant for at least three generations; c) selecting from the third or higher backcross population a plant that comprises at least the modified WAK gene of the first parent plant of step a).

The produced plant is in particular a cucumber plant. In one embodiment, the plant used in the method for the production of a Corynespora cassiicola resistant cucumber plant, is a plant grown from seed deposited under NCIMB accession number NCIMB 44368 or a plant grown from seed deposited under NCIMB accession number NCIMB 44369, or a progeny plant thereof which is a direct or further descendant through crossing a plant grown from the deposited seed with itself or with another plant for one or more subsequent generations.

The invention additionally provides for a method of introducing another desired trait into a Corynespora cassiicola resistant plant, comprising: a) crossing a plant comprising a modified WAK gene with a second plant that comprises the other desired trait to produce Fl progeny; b) optionally selecting in the Fl for a plant that comprises the resistance and the other desired trait; c) crossing the optionally selected Fl progeny with one of the parents for at least three generations, to produce backcross progeny; d) selecting backcross progeny comprising the resistance and the other desired trait; and e) optionally repeating steps c) and d) one or more times in succession to produce selected fourth or higher backcross progeny that comprises the resistance to Corynespora cassiicola and the other desired trait.

In one embodiment, the plant used in the method for the production of a Corynespora cassiicola resistant cucumber plant, is a plant grown from seed deposited under NCIMB accession number NCIMB 44368 or a plant grown from seed deposited under NCIMB accession number NCIMB 44369, or a progeny plant thereof which is a direct or further descendant through crossing a plant grown from the deposited seed with itself or with another plant for one or more subsequent generations.

Optionally, selfing steps are performed after any of the crossing or backcrossing steps. Selection of a plant comprising resistance and the other desired trait can alternatively be done following any crossing or selfing step of the method. The other desired trait can be selected from, but is not limited to, the following group: resistance to bacterial, fungal or viral diseases, insect or pest resistance, improved germination, plant size, plant type, improved shelf-life, water stress and heat stress tolerance, and male sterility. The invention includes a plant produced by this method and a fruit obtained therefrom. The produced plant is in particular a cucumber plant.

The invention further relates to a method for the production of a plant comprising a modified WAK gene, which plant is resistant to Corynespora cassiicola, by using tissue culture or by using vegetative propagation. The produced plant is in particular a cucumber plant.

The present invention relates to a method for identification of a plant comprising a modified WAK gene, which plant is resistant to Corynespora cassiicola, wherein the identification comprises determining the presence of a modification in the WAK gene of SEQ ID No. 1 , or a sequence having at least 80% sequence identity to SEQ ID No. 1, preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to SEQ ID No. 1 , and optionally analyzing if the plant comprising the modification is resistant to Corynespora cassiicola. Determining the presence of a modification in a WAK gene comprises identification of any of the modifications as described herein. Modifications can be identified by identified by sequence comparison or by using a marker that is designed to identify such modification as its sequence comprises that particular modification. The method can be used to identify in particular a cucumber plant.

The present invention further relates to a method of selection of a plant which is resistant to Corynespora cassiicola, the method comprising identification of a modified WAK gene of the invention in a plant and subsequently selecting said plant as a plant which is resistant to Corynespora cassiicola. Optionally the resistance can be confirmed by performing a test as described in Example 1. The selected plant obtained by such a method is also a part of this invention. The method can be used to select in particular a cucumber plant. The present invention further relates to a method for identifying a Corynespora cassiicola resistant plant, wherein the method comprises the following steps: a) detecting in the genome of a plant the modified WAK gene as defined herein, and b) optionally testing of the plant comprising the modified WAK gene for exhibiting resistance to Corynespora cassiicola.

The invention also relates to propagation material suitable for producing a plant of the invention, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, pollen, an ovary, an ovule, an embryo sac, or an egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem cell, or a protoplast, or is suitable for tissue culture of regenerable cells, and is in particular selected from a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed and a stem, and wherein the plant produced from the propagation material comprises a modified WAK gene of the invention that provides resistance to Corynespora cassiicola. A plant of the invention may be used as a source of the propagation material. A tissue culture comprising regenerable cells also forms a part of this invention. The propagation material is in particular of a cucumber plant.

The invention further relates to a cell of a plant of the invention. Such a cell may either be in isolated form or a part of the complete plant or parts thereof and still forms a cell of the invention because such a cell comprises a modified WAK gene of the invention. Each cell of a plant of the invention carries a modified WAK gene . A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention. The cell is in particular from a cucumber plant.

The invention further relates to plant tissue of a plant, which comprises a modified WAK gene. The tissue can be undifferentiated tissue or already differentiated tissue. Undifferentiated tissue is for example a stem tip, an anther, a petal, or pollen, and can be used in micropropagation to obtain new plantlets that are grown into new plants of the invention. The tissue can also be grown from a cell of the invention. The plant tissue is in particular of a cucumber plant.

The invention moreover relates to progeny of a plant, a cell, a tissue, or a seed of the invention, which progeny comprises a modified WAK gene. As used herein, progeny comprises the first and all further descendants from a cross with a plant of the invention, wherein a cross comprises a cross with itself or a cross with another plant, and wherein a descendant that is determined to be progeny comprises a modified WAK gene. Descendants can be obtained through selfing and/or further crossing of a plant comprising the modified WAK gene which can be for instance a plant grown from the deposited seed. Progeny also encompasses material that is obtained by vegetative propagation or another form of multiplication such as tissue culture of regenerable cells. The progeny plant is in particular a cucumber plant.

DEPOSITS

Seed of cucumber (Cucumis sativus) comprising a modified WAK gene of the invention homozygously, resulting in a Corynespora cassiicola resistant plant, was deposited with NCIMB Ltd, Wellheads Place, Dyce, Aberdeen, AB21 7GB Scotland under deposit accession numbers NCIMB 44368 and NCIMB 44369.

SEQUENCES

The wild- type and modified sequences of the cucumber (Cucumis sativus WAK genes and proteins

SEQ ID No. 1: Wild-type CDS atggagcgtttgaggaagactcttgtgggtctcacggtgatcatcttattatcaacactggcctcagcagcctcacaagccaaacctgattgtgat gaatggtgtggcgacttacgaattccatatccatttggagtaaaacaaggatgttatttcaaccaagcattcttaattacatgtgacaaagcctttaa ccctccaaaggcgtttctaaaggacacaaacattagcgttaccaatatatcactcaatggtgagctccacatgttgcagcctatagtccgatattg ctacgaggatgtgcaattagtgagtggtactccttttatccccaacacaaccaacctttctgcaccggcgacattaccgattgcggatggcaaaa acaagttcatcgccatcggttgcaatacgttcggtttattcacagggatgctaaagggaggtgaatttctaactggttgtgttgcgatatgtacaaat aatagtattatagttgatgggtcgtgttctgggactggatgttgtgagttggatattccaaatgggttgagtgatttgagtttggctgtgggtccagtg ttacctgatactaatcgtagtttagtgaagaataattcatgtgggtatgcttttgtggttggagaagaagggtttaagtttaaatcaagttttattgataa ttttgaagataaggaagttgaggttgtggttgattggagtattggaaatgaaacaataattgatgtttgtggaataaatagtaaaaggaatagtagtt tctctgatgatagatctcaataccgttgccaatgtccggatggttacgaaggaaatccatatctccctcaaggatgtgatcaagatataaatgaatg cgagcataaagagctgaatgactgcacgcacgaatgcattaacacaaatggaagctatacttgcaaatgtcctaaaaactataaaggagatgg aagacgaggggaagatggacacggctgcactcgagattccaaggctattcccatcataatcggaattggagtagggttcactgttttattaattg ctagcacatggatattcttgggttacaaaaagtggaagttcatcaaaaggaaagagaaattttttaaagaaaatggaggtttcatacttcaacaaca actttctcaatggcaatcctccccaaatgaaatggtcagaattttcacccaagaagaattggagaaggccacaaacaactacgaccatagcact attgttggcaaaggtgggtacggtactgtttacaaaggagtcttagaagatggcttggcagtggcaatcaagaaatcgaaacttatagaccaatc ccaaactgatcaattcattaatgaagttattgttttgtctcaaatcaaccatcgcaacgtggtcagactcttgggatgttgtttagagacacaagtccc gttgttggtgtatgagtttgtaaccaatggcaccctctttgaacacatccatgacaaaaccaagcatgcttcactttcgtgggaagcccgcttaaaa atagcattggagactgcaggtgtgctttcgtatttacattcgtcagcttccactccaattattcatagagatgtcaagacgaccaacatacttttagat aataattacactgcaaaggtatccgattttggagcgtcaaagttggttccaatggatcaaacacaagtatccacgttggtacaagggacattagg gtatttagacccagagtacttattgacaagcgagttgacagagaagagcgacgtttatagttttggaatagtgttgttagagcttataactgggaag aaagcggtgagttttgatgggccagaagaggagaggaacctagcaatgtatgtgctttgtgcaatgaaagaagatcggttggaagaagttgtg gagaaagcgatgatggtgaaagaggcaagttttgaggaagctgttaaacaagtggctaaggtagcaatgaaatgcttgagaattaaagggga agagcggcccagcatgaaagaagtggctatggagttggagggagtgcgatcaatgcaagttcaacattcatgggctaataataatgattcgtcc aactatgaggaaacgatatgtttattggatgtggaagcttcagactcgaacaattttgcttcgcgtggcactacgagtatcgttggggatagcata aaagcttcaattttgccgcacattcaccatggaagatga

SEQ ID No. 2: Wild-type protein

MERLRKTLVGLTVIILLSTLASAASQAKPDCDEWCGDLRIPYPFGVKQGCYFNQAFLITCD KAFNPPKAFLKDTNISVTNISLNGELHMLQPIVRYCYEDVQLVSGTPFIPNTTNLSAPATLPI ADGKNKFIAIGCNTFGLFTGMLKGGEFLTGCVAICTNNSIIVDGSCSGTGCCELDIPNGLSD

LSLAVGPVLPDTNRSLVKNNSCGYAFVVGEEGFKFKSSFIDNFEDKEVEVVVDWSIGNETII DVCGINSKRNSSFSDDRSQYRCQCPDGYEGNPYLPQGCDQDINECEHKELNDCTHECINTN GSYTCKCPKNYKGDGRRGEDGHGCTRDSKAIPIIIGIGVGFTVLLIASTWIFLGYKKWKFIK RKEKFFKENGGFILQQQLSQWQSSPNEMVRIFTQEELEKATNNYDHSTIVGKGGYGTVYK GVLEDGLAVAIKKSKLIDQSQTDQFINEVIVLSQINHRNVVRLLGCCLETQVPLLVYEFVTN GTLFEHIHDKTKHASLSWEARLKIALETAGVLSYLHSSASTPIIHRDVKTTNILLDNNYTAK VSDFGASKLVPMDQTQVSTLVQGTLGYLDPEYLLTSELTEKSDVYSFGIVLLELITGKKAV SFDGPEEERNLAMYVLCAMKEDRLEEVVEKAMMVKEASFEEAVKQVAKVAMKCLRIKG EERPSMKEVAMELEGVRSMQVQHSWANNNDSSNYEETICLLDVEASDSNNFASRGTTSIV GDSIKASILPHIHHGR

SEQ ID No. 3 atggagcgtttgaggaagactcttgtgggtctcacggtgatcatcttattatcaacactggcctcagcagcctcacaagccaaacct gattgtgatgaatggtgtggcgacttacgaattccatatccatttggagtaaaacaaggatgttatttcaaccaagcattcttaattacat gtgacaaagcctttaaccctccaaaggcgtttctaaaggacacaaacattagcgttaccaatatatcactcaatggtgagctccatat cttgcagcccatagtccgattttgcaacgaggatgtgtcattagtgaatcgttcttttatccccaacacaaccaaccttcctgcaacgg cgacattcccgattgcggatggcaaaaacaagttcatcgccatcggttgcaatacgttcggttttttcacagggaagctgaagggag gtgatcaatttctaactggttgtattgcggtatgtccaaataataataagaataatacttggtcgtgttctgggaatggatgttgtaagttg gatattccagatgggtcgagtgatttgaatttgactgtggctccagcgttacttgatactgatcgtaatttagtgcagaataaaccgtgt gggtatgcttttgtggttggagaagaagggtttgagtttaaacaaagttatattgataattttgaagatacggaagttgaggttgtggtt gattggagtactgaaagtgaaataattgatgtttgtagaaaagatactaaaaggaatagtaatttctctgatgatagatctcaatatcgtt gccaatgtccggatggttacgaaggaaatccatatctccctcaaggatgtgatcaagatataaatgaatgcgagcataaagagctg aatgactgcacgcacgaatgcattaacacaaatggaagctatacttgcaaatgtcctaaaaactataaaggagatggaagacgag gggaagatggacacggctgcactcgagattccaaggctattcccatcataatcggaattggagtagggttcactgttttattaattgct agcacatggatattcttgggttacaaaaagtggaagttcatcaaaaggaaagagaaattttttaaagaaaatggaggtttcatacttca acaacaactttctcaatggcaatcctccccaaatgaaatggtcagaattttcacccaagaagaattggagaaggccacaaacaact acgaccatagcactattgttggcaaaggtgggtacggtactgtttacaaaggagtcttagaagatggcttggcagtggcaatcaag aaatcgaaacttatagaccaatcccaaactgatcaattcattaatgaagttattgttttgtctcaaatcaaccatcgcaacgtggtcaga ctcttgggatgttgtttagagacacaagtcccgttgttggtgtatgagtttgtaaccaatggcaccctctttgaacacatccatgacaaa accaagcacgcttcactttcgtgggaagcccgcttaaaaatagcattggagactgcaggtgtgctttcgtatttacattcgtcagcttc cactccaattattcatagagatgtcaagacgaccaacatacttttagataataattacactgcaaaggtatccgattttggagcgtcaa agttggttccaatggatcaaacacaagtatccacgttggtacaagggacattagggtatttagacccagagtacttattgacaagcg agttgacagagaagagcgacgtttatagttttggaatagtgttgttagagcttataactgggaagaaagcggtgagttttgatgggcc agaagaggagaggaacctagcaatgtatgtgctttgtgcaatgaaagaagatcggttggaagaagttgtggagaaagcgatgatg gtgaaagaggcaagttttgaggaagctgttaaacaagtggctaaggtagcaatgaaatgcttgagaattaaaggggaagagcgg cccagcatgaaagaagtggctatggagttggagggagtgcgatcaatgcaagttcaacattcatgggctaataataatgattcgtcc aactatgaggaaacgatatgtttattggatgtcgaagcttcagactcgaacaatgttgcttcgcgtggcactacgagtatcgttgggg atagcataaaagcttcaattttgccgcacattcaccatggaagatga

SEQ ID No. 4: Modified sequence ggaacgaaaaacaagaaacaagaaacaaaatacataattattaaacatgcccttaataatcaagtgcatgtaatgaatttttatgacc atgaatatttatcaaagttttaaaggttaattagagcaatgctagggaagaaaatgatttcgatgcatcgacagatttagaataggtgat ctggagggctcgagcctctgtcaattttatattttatatatatgtatatataaattataatatcaatactaatcactaactcagtggtgaaac aagctttcagctcattgtatactcaaagttaagtttcacttctaaattttttttcaatatcattttcggtgaattataaagttttgtcaataaata gtaagccctaagtttgtcggtgtctatatagatgaaaaggaaaaagaaattatatataggaagattaatttggtaatggaagtgttaat gtttgtaatgactttattgttaaatttaattttgtggatttgtttagatataaatgaatgcgagcataaagagctgaatgactgcacgcacg aatgcattaacacaaatggaagctatacttgcaaatgtcctaaaaactataaaggagatggaagacgaggggaagatggacacgg ctgcactcgagattccaaggctattcccatcataatcggtatgtttttatgttctctaatacttatcaaatctttttgtatgctttaattctccat atatatgctttgaatataaataatgatatacgtacccaagcaaaatgtaggaattggagtagggttcactgttttagtaattgctagcac atggatattcttgggttacaaaaagtggaagttcatcaaaaggaaagagaaattttttaaagaaaatggaggtttcatacttcaacaac aactttctcaatggcaatcctccccaaatgaaatggtcagaattttcacccaagaagaattggagaaggccacaaacaactacgac catagcactattgttggcaaaggtgggtacggtactgtttacaaaggagtcttagaagatggcttggcagtggcaatcaagaaatcg aaacttatagaccaatcccaaactgatcaattcattaatgaagttattgttttgtctcaaatcaaccatcgcaacgtggtcagactcttgg gatgttgtttagagacacaagtcccgttgttggtgtatgagtttgtaaccaatggcaccctctttgaacacatccatgacaaaaccaag catgcttcactttcgtgggaagcccgcttaaaaatagcattggagactgcaggtgtgctttcgtatttacattcgtcagcttccactcca attattcatagagatgtcaagacgaccaacatacttttagataataattacactgcaaaggtatccgattttggagcgtcaaagttggtt ccaatggatcaaacacaagtatccacgttggtacaagggacattagggtatttagacccagagtacttattgacaagcgagttgaca gagaagagcgacgtttatagttttggaatagtgttgttagagcttataactgggaagaaagcggtgagttttgatgggccagaagag gagaggaacctagcaatgtatgtgctttgtgcaatgaaagaagatcggttggaagaagttgtggagaaagcgatgatggtgaaag aggcaagttttgaggaagctgttaaacaagtggctaaggtagcaatgaaatgcttgagaattaaaggggaagagcggcccagcat gaaagaagtggctatggagttggagggagtgcgatcaatgcaagttcaacattcatgggctaataataatgattcgtccaactatga ggaaacgatatgtttattggatgtcgaagcttcagactcgaacaatgttgcttcgcgtggcactacgagtatcgttggggatagcata aaagcttcaattttgccgcacattcaccatggaagatgattttatatcttatttatatacatacttccttaaatataaagagcagaattttcg tatagaatttagatttataagttagattatatgtatatgctacattattctgaaacttgatatatattattgttgtataatgcttaacgggttgat ttatatttcgttta[ ... ]cgaatgcttttgggagaccaaaatgcaactttagaaaattttaaccatttttagatgttcggtaaaatagatggt tccttaattttttaaaataacttttttttttctaatttatcatatcaattaacaagtgttttaaactaaaatctcatccttttatatcccttcaaaaga agagtatcttcacaatagctaaaacttgttaaattaaaatttctctagtagtgttacaccccacctcacaaaatgtccttatcctcgcgttg tggcactcgaacacgacttgaggagccttggtaatatatcttcacaatagctaaaagttgttaaattaaaattgctaaaagttgttaaatt aaaatttctcgagtagtgttacaccccacctcacaaaatgtccttatcctcgcgttgtggcactcgaacacaacttgaggagccttga cgtctgttggcaaaacaccataacactcgattttaaacctttgaaccttgaactttacacttaaagcttagctcgaactaggactcttaa aactactaagcgataagggaaaaacaacaacgcaagataaataaacgaactctttattaacttagtaacattcgttgaagatgcataa tacattctttcttgaatagaaagatttataacaaacttcaaacttcgaacgtgtagcatagcttcttcaaattcaaactttagaacacacct ggctatcgccttaacacgtagcagctcaccttactcttctcaacttgacctcagcgcctttgtaggtagcttagtgggtaactgtgcttg tcagcccaaccccccttccaaccttagttcagatcttacttgtgtagccttttttttagccgtcaataaattttatgaatctactactacttgt aacctgtaaaaggaaaacttaaaacatagatcaattacacaatgagtgaggttttaaaaatcatttttgggacataaaaatgaatcaca taagttcattttcataaacaagctcacaacacctctttcaaaacatatacacacacgttaacttctacttatttcttttacccaagaacatg aactattcacatgcataaactattgcgatttctcatattcttccagaattttctaattcaggtggttaactcaatacactacctatcaagcaa tccagtgccaagaataaagattaagtttatgagattagttgacattttttatgtgtaaaaaattgttttcttaacatgttttacacatcaaga atgtgtttatacttaacatgttttacgtgtcaataacttgtttaaaacaggtcaaattttgtttattatttccaagacttttttcttcaaatgaaa agttgtaccgttatattgacccaagacttgtttatgtgtattcttaacatgttttacacagttatattgcaccacctgttttgaagcacaaatt tgctagggcacttgcaaatatagaaaaaaaaattatgataatatgacccatatcactacattttctaaattgtaaaagtataaaatttaaa attaataatcatatgatagccctatgatatcactaattacttatcatatttgtcatagtcacaatatgcaaaaaataggtgtcataggctat atttttctaaatgtttggcatctgatgcaatttctcatttgttaaactacgcatattaattgaacaattgaatcaattctaactttccatcaaag aactaatattgtaacaacttgcatatatcacttaaccatccatatagttggtcattacatatgcttaattacaatagtccaaacttctaccat atgaaacaat

SEQ ID No. 6: wild-type genomic

ATTGAATCTAATTGCTAAATTTGAAACTATCTCTTTTTATATTTATCATTGTGGGTCTAT GGATTTTTTTCGTTTTCGAAATTGTTCTATACAAAGTAAATATTTTACCGGTTTGTTACA _{TTTTTTAAAAAAAGCCTATTAATAATGTTATTATCAACATAAATGTCTTTTAAAGAATA} AAAATAAAAGATACATAATTTTATAACTGTGTATATTATACTGAAGTTATTGCAAATG AAGTTAGATTGTAAAGTCATAAAAACTTATCTTGATTTTCCTAATAAAAAACCAAAAT ATTTAAACCAATTTTTTGTATCAGTTTTATTAAAAAAAAAAAAAAACCAAAGCAATAG ACATACATGTTTTTTATTGGGGTTTTTTAAAAAATATAGAAATGCGACAAAATATTTGC ACTATATAGAACTAATTCAAAAAAACAAAAAAATATTCATAGATCTACAATGTAAAA CCAAAAATTGCATCGTGTATCAATATTCTTAACGATTTTTTATCTTTTATAGTTGATCTA

AGCCAATATCCAATGATTTTTGTTCACATCGTGTACTAATTTATATTTAATTTTTGTTCA

TATCGTGTACTAGTTTATATTTCACATAATCTTGAACCAGATAACATTTTGAAGAAAAA

AAATACAAGATGACCATGATTAAAAAAATATAAAAGAAAAAAAAAACTGCAAAAGA

AGAGAAGGACGTTGGAAAGGAAATAAAAAAAAATGGCAGAAGAGAAAATGATGGGA

AGAAAGTCAAACGTGAAATACTTTTTAAAAATGGCCGATTTCATGGATCTTTTGATTTT

GTTGCCTAAACTATAAATATTTTGATGTTTTATTATATTTATGAAAATTAACATTTTTTG

TTTTTGCTTTTGATTTTTTTAAACAACTATAACACATAAATTATATGGAAGGACAAAAC

TACTAAAAATATTTACAATTATAAGAAAATGTCACAGTCTATTTGTGTGATAGAATAA

GATAGATCAAGATAGACTATTACATAAATCTATCATGATCTACACATATTAAAGTCTA

TCTTGATCTATCGCAGTCTATTGTAAATAAATTTTGAATACTTTGATTTATTTTGCTATA

TTTGAAAATAACACAAATTAATTTCTTTTTTAATTACAAACTGAATTTCTGAAATGGGG

CTAAGTTAATGTAGGGTTATATTTTTGGAAAAATAAAAGGGGAATGTTTTAGTATGAT

AACCAATTTCTTACTATTATTTTCCAATGGGCATGCTTCAGTTGACCATGTTTATGGAC

CTTTTCGTTTTCTTTTTCTAATCTTTGAAATGTTAAACCTTGAGTCTAGGGTCTTAATAT

CTGTCTATTTTTTAAATGTGAACATGAGAAAAATCAAACTCGAGAAAAACTGTATTAT

TCCATGAAGCAGACATAAATAGTGACGGTTGTGGTAACCATTTCAGATTTTGTATGAA

TAGAATAGTTGTTATTGTTTATATATCTATTATTCTGGTTCTTATTTATATATCTATTAT

TCTTGTTTTTTTTAAAGGTATTTTATTGCTAAAAGTAGAGAGGGAATTGAACCTCATGG

CCTTGGCACAACCAACAAAACCATCAAACAAACTAACCTAAAAAATAATTACCTAAC

ATAACACACAAATATATAATACTTATTTGACTTATCTTTATGATGTTTTTATAACTCAT

ATGTGGTTTCCAATTTTGATTGTGTGCCACCTTAAAAGACTATGGCAGATTGACTTTTG

TTTGTATTAAATAAAATAATGGAACCAAATACTAACAAGAGATCCTAGGTTGAGTAAT

TAAAAAATTAATAAATAATGAAAACAATGTTTCTCCAAACTTTTGTCATTTAATTAATG

TGAAAGTAGAAAATGGCGACCATTGAAAGCTTGAAATGTTCAATATTGAAGTCTTCCC

ATCATCCCAACATCTTAAATATACTTTCAGTTTCTCTCGATCTAATTTATTAATTCAGCT

TTAAGATGGAGCGTTTGAGGAAGACTCTTGTGGGTCTCACGGTGATCATCTTATTATC

AACACTGGCCTCAGCAGCCTCACAAGCCAAACCTGATTGTGATGAATGGTGTGGCGAC

TTACGAATTCCATATCCATTTGGAGTAAAACAAGGATGTTATTTCAACCAAGCATTCTT

AATTACATGTGACAAAGCCTTTAACCCTCCAAAGGCGTTTCTAAAGGACACAAACATT

AGCGTTACCAATATATCACTCAATGGTGAGCTCCACATGTTGCAGCCTATAGTCCGAT

ATTGCTACGAGGATGTGCAATTAGTGAGTGGTACTCCTTTTATCCCCAACACAACCAA

CCTTTCTGCACCGGCGACATTACCGATTGCGGATGGCAAAAACAAGTTCATCGCCATC

GGTTGCAATACGTTCGGTTTATTCACAGGGATGCTAAAGGGAGGTGAATTTCTAACTG

GTTGTGTTGCGATATGTACAAATAATAGTATTATAGTTGATGGGTCGTGTTCTGGGACT

GGATGTTGTGAGTTGGATATTCCAAATGGGTTGAGTGATTTGAGTTTGGCTGTGGGTC CAGTGTTACCTGATACTAATCGTAGTTTAGTGAAGAATAATTCATGTGGGTATGCTTTT

GTGGTTGGAGAAGAAGGGTTTAAGTTTAAATCAAGTTTTATTGATAATTTTGAAGATA

AGGAAGTTGAGGTTGTGGTTGATTGGAGTATTGGAAATGAAACAATAATTGATGTTTG

TGGAATAAATAGTAAAAGGAATAGTAGTTTCTCTGATGATAGATCTCAATACCGTTGC

CAATGTCCGGATGGTTACGAAGGAAATCCATATCTCCCTCAAGGATGTGATCAAGGTA

TGAATACCTTACTTTTCTCTACTAATTTTCCTTCATATCTTACAATTTCATCATTCAAGC

TACTCAATTTTCATCTTACAATTTCATTATTTTTTAAAAGAATTAATAATGATGTTCGTT

ATTATAAAGGTTAGTGAGAGACAACAGGAAAATAGATGGAACGAAACTAAAGACTAC

AAAAATTAAGACAAAATAGATCGATCAAGAATCGACCAATAAATAGAAAATGGTAAA

GATTAAGGACTTGTTTGATAACCTTTTTATTTCTTCTTCTCTTTTTGTTTATCATCTTTTA

ACAAACACAATAGTCGTTTTCAAATTTTAAGAAATATTTTTTAAAATTGAACAATAATT

GAGAAAATATGAAAAGTAGTTTCCTCCTAGTTATCTTTTCGTCTTATTTTATTTCAATTT

TTTTGTAATCTATTATTGTTTGTTTGCTCATCGGCAGAGACACGCTTAAAATCAATTTTT

CCTCCATTTCTTGAGTTCAACTCCCATTCTCTTTTTCGGAAATTTTCAAAAATAGTAAA

TTTTATAAAATACTTTCAACTTATAACAAATTTTATCACTAATATGCTAGTGATAATAG

ATTAAAAATTTTTGCTATAATTTGTATATATTTTAATTTATTTTACTAGGTTTAGAAATC

TCTCTTCTCTTTTTTCTTCTTTTATTAATTTTTCATATATGTTTAATATTTTTTCCTTCTTT

GACTAATTCTTCAGATACGTTTAATATTTTTTTATTTCTTTTATTTGTTTTTCATGTATGT

TTAATATTTTTTCCTTTCTTTATTATTTTTTATATTTATATATTTAATTTAATTTTAAATT

TAAATTTTTAATATTTAATTAATGATTATTTGATTTCAATTTTCATTTTTAATTTTAAAG

TTTCTTTTCTATTTAGCTACATTTATTTATTTTTCGTATTTACATCTTTAATTCAATTTTA

AATTTAAAACGGTTGAATATTTAAATTTATATATTATTTTGACCTTTCAAATTTAGATA

TTTAATATGTTTGTTTAAATATTTCCTGAATTGTCAATTTTAAAATTTTGCAATAGACAT

GTTTATATGTTATTTTAAATTTAAATTTATTACTTTTAGTTCCTTCTAAATTAGTTAGAA

TTTTATCTTGTATAATTTTTTTTATTAACATATTCATTTTGGTTATTTTTAGGAATTTACA

ATCGGTTGAATTAGAATCAAAATTTGTATTGTAACTTGTTATTAATTTTAATTTTGCAA

CAAGGATTTATGTATATTTGAAAATGTTGAGGTTGTATAACTACTCTTTTATATTTTAA

TGAATTCTATATACTAGAAAAATAATGGAAAATGTTATGAAAAATAACACAAACAAC

GAGAAACAAAAAATAATTATCAAACAAAATTATGCTTTTAGTTCAATTTACAAGAACA

ATAAACATAAATAGTTATCGAACATAGTTTTGTTTCTTGTTTTTTTTTTTAAAAAAAAA

GAAAGAAACAAGGAACGAAAAACAAGAAACAAGAAACAAAATACATAATTATTAAA

CATGCCCTTAATAATCAAGTGCATGTAATGAATTTTTATGACCATGAATATTTATCAAA

GTTTTAAAGGTTAATTAGAGCAATGCTAGGCAAGAAAATGATTTCGATGCATCGACAG

ATTTAGAATAGGTGATCTGGAGGGCTCGAGCCTCTGTCAATTTTATATTTTATATATAT

GTATATATAAATTATAATATCAATACTAATCACTAACTCAGTGGTGAAACAAGCTTTC

AGCTCATTGTATACTCAAAGTTAAGTTTCACTTCTAAATTTTTTTTCAATATCATTTTCG GTGAATTATAAAGTTTTGTCAATAAATAGTAAGCCCTAAGTTTGTCGGTGTCTATATAG

ATGAAAAGGAAAAAGAAATTATATATAGGAAGATTAATTTGGTAATGGAAGTGTTAA

TGTTTGTAATGACTTTATTGTTCAATTTAATTTTGTGGATTTGTTTAGATATAAATGAAT

GCGAGCATAAAGAGCTGAATGACTGCACGCACGAATGCATTAACACAAATGGAAGCT

ATACTTGCAAATGTCCTAAAAACTATAAAGGAGATGGAAGACGAGGGGAAGATGGAC

ACGGCTGCACTCGAGATTCCAAGGCTATTCCCATCATAATCGGTATGTTTTTATGTTCT

CTAATACTTATCAAATCTTTTTGTATGCTTTAATTCTCCATATATATGCTTTGAATATAA

ATAATGATATACGTACCCAAGCAAAATGTAGGAATTGGAGTAGGGTTCACTGTTTTAT

TAATTGCTAGCACATGGATATTCTTGGGTTACAAAAAGTGGAAGTTCATCAAAAGGAA

AGAGAAATTTTTTAAAGAAAATGGAGGTTTCATACTTCAACAACAACTTTCTCAATGG

CAATCCTCCCCAAATGAAATGGTCAGAATTTTCACCCAAGAAGAATTGGAGAAGGCC

ACAAACAACTACGACCATAGCACTATTGTTGGCAAAGGTGGGTACGGTACTGTTTACA

AAGGAGTCTTAGAAGATGGCTTGGCAGTGGCAATCAAGAAATCGAAACTTATAGACC

AATCCCAAACTGATCAATTCATTAATGAAGTTATTGTTTTGTCTCAAATCAACCATCGC

AACGTGGTCAGACTCTTGGGATGTTGTTTAGAGACACAAGTCCCGTTGTTGGTGTATG

AGTTTGTAACCAATGGCACCCTCTTTGAACACATCCATGACAAAACCAAGCATGCTTC

ACTTTCGTGGGAAGCCCGCTTAAAAATAGCATTGGAGACTGCAGGTGTGCTTTCGTAT

TTACATTCGTCAGCTTCCACTCCAATTATTCATAGAGATGTCAAGACGACCAACATACT

TTTAGATAATAATTACACTGCAAAGGTATCCGATTTTGGAGCGTCAAAGTTGGTTCCA

ATGGATCAAACACAAGTATCCACGTTGGTACAAGGGACATTAGGGTATTTAGACCCAG

AGTACTTATTGACAAGCGAGTTGACAGAGAAGAGCGACGTTTATAGTTTTGGAATAGT

GTTGTTAGAGCTTATAACTGGGAAGAAAGCGGTGAGTTTTGATGGGCCAGAAGAGGA

GAGGAACCTAGCAATGTATGTGCTTTGTGCAATGAAAGAAGATCGGTTGGAAGAAGT

TGTGGAGAAAGCGATGATGGTGAAAGAGGCAAGTTTTGAGGAAGCTGTTAAACAAGT

GGCTAAGGTAGCAATGAAATGCTTGAGAATTAAAGGGGAAGAGCGGCCCAGCATGAA

AGAAGTGGCTATGGAGTTGGAGGGAGTGCGATCAATGCAAGTTCAACATTCATGGGC

TAATAATAATGATTCGTCCAACTATGAGGAAACGATATGTTTATTGGATGTGGAAGCT

TCAGACTCGAACAATTTTGCTTCGCGTGGCACTACGAGTATCGTTGGGGATAGCATAA

AAGCTTCAATTTTGCCGCACATTCACCATGGAAGATGATTTTATATCTTATTTATATAC

ATACTTCCTTAAATATAAAGAGCAGAATTTTCGTATAGAAATTTAGATTTATAAGTTA

GATTATATGTAATATGCTACATTATTCTGAAACTTGATATATATTATTGTTGTATAATG

CTTAACGGGTTGATTTATATTTTCGTTTACGAATGCTTTTGGGAGACCAAAATGCAACT

TTTAGAAAATTTTAACCATTTTTAGATGTTCGGTAAAATAGATGGTTCCTTAATTTTTT

AAAATAACTTTTTTTTTTCTAATTTATCATATCAATTAACAAGTGTTTTAAACTAAAAT

CTCATCCTTTTATATCCCTTCAAAAGAAGAGTATCTTCACAATAGCTAAAACTTGTTAA

ATTAAAATTTCTCTAGTAGTGTTACACCCCACCTCACAAAATGTCCTTATCCTCGCGTT GTGGCACTCGAACACGACTTGAGGAGCCTTGGTAATATATCTTCACAATAGCTAAAAG TTGTTAAATTAAAATTGCTAAAAGTTGTTAAATTAAAATTTCTCGAGTAGTGTTACACC CCACCTCACAAAATGTCCTTATCCTCGCGTTGTGGCACTCGAACACAACTTGAGGAGC CTTGACGTCTGTTGGCAAAACACCATAACACTCGATTTTAAACCTTTGAACCTTGAACT TTACACTTAAAGCTTAGCTCGAACTAGGACTCTTAAAACTACTAAGCGATAAGGGAAA AACAACAACGCAAGATAAATAAACGAACTCTTTATTAACTTAGTAACATTCGTTGAAG ATGCATAATACATTCTTTCTTGAATAGAAAGATTTATAACAAACTTCAAACTTCGAAC GTGTAGCATAGCTTCTTCAAATTCAAACTTTAGAACACACCTGGCTATCGCCTTAACAC GTAGCAGCTCACCTTACTCTTCTCAACTTGACCTCAGCGCCTTTGTAGGTAGCTTAGTG GGTAACTGTGCTTGTCAGCCCAACCCCCCTTCCAACCTTAGTTCAGATCTTACTTGTGT AGCCTTTTTTTTAGCCGTCAATAAATTTTATGAATCTACTACTACTTGTAACCTGTAAA AGGAAAACTTAAAACATAGATCAATTACACAATGAGTGAGGTTTTAAAAATCATTTTT GGGACATAAAAATGAATCACATAAGTTCATTTTCATAAACAAGCTCACAACACCTCTT TCAAAACATATACACACACGTTAACTTCTACTTATTTCTTTTACCCAAGAACATGAACT ATTCACATGCATAAACTACTGCGATTTCTCATATTCTTCCAGAATTTTCTAATTCAGGT GGTTAACTCAATACACTACCTATCAAGCAATCCAGTGCCAAGAATAAAGATTAAGTTT

ATGAGATTAGTTGACATTTTTTATGTGTAAAAAATTGTTTTCTTAACATGTTTTACACA TCAAGAATGTGTTTATACTTAACATGTTTTACGTGTCAATAACTTGTTTAAAACAGGTC

AAATTTTGTTTATTATTTCCAAGACTTTTTTCTTCAAATGAAAAGTTGTACCGTTATATT

GACCCAAGACTTGTTTATGTGTATTCTTAACATGTTTTACACAGTTATATTGCACCACC

TGTTTTGAAGCACAAATTTGCTAGGGCACTTGCAAATATAGAAAAAAAAAATTATGAT

AATATGACCCATATCACTACATTTTCTAAATTGTAAAAGTATAAAATTTAAAATTAAT

AATCATATGATAGCCCTATGATATCACTAATTACTTATCATATTTGTCATAGTCACAAT ATGCAAAAAATAGGTGTCATAGGCTATATTTTTCTAAATGTTTGGCATCTGATGCAATT TCTCATTTGTTAAACTACGCATATTAATTGAACAATTGAATCAATTCTAACTTTCCATC AAAGAACTAATATTGTAACAACTTGCATATATCACTTAACCATCCATATAGTTGGTCA

TTACATATGCTTAATTACAATAGTCCAAACTTCTACCATATGA

The present invention will be further illustrated in the Examples that follow and that are for illustration purposes only. The Examples are not intended to limit the invention in any way. EXAMPLES

EXAMPLE 1

Corynespora cassiicola resistance test

Ten plants were sown in trays filled with potting soil in a climate cell under normal cucumber growing conditions.

Multiplication of the Corynespora cassiicola pathogen was started on standard agar plates and when the first leaf has a size of 2-3 cm (12-13 days after sowing), plants were inoculated with 40.000 spores/ml using a high pressure inoculum pump. After inoculation plants are covered with plastic and grown at regular greenhouse temperatures. The plants were assessed 8-10 days after inoculation using Table 1. Ventura RZ was used as resistant variety and Kaspian RZ was used as a susceptible variety.

Table 1: Scoring Corynespora cassiicola symptoms

4 are categorized to be susceptible.

EXAMPLE 2

Identification of the WAK gene

Various internally developed cucumber populations that segregated for Corynespora cassiicola resistance were fine-mapped to a small region on chromosome 6, comprising potential genes which were likely to contribute to the Corynespora cassiicola resistance. Whole genome sequences were available in-house for the backgrounds of the resistant and susceptible lines that were used in the development of these populations. Therefore, by comparing the sequences of the resistance lines to the sequences of the susceptible lines and by determining the differences, unique polymorphisms in the region were identified.

Among the genes in the region of interest was a WAK gene, which had various polymorphisms between susceptible and resistant material. The susceptible cucumber plants comprise the wild-type WAK gene and in the two resistant cucumber plants the WAK gene was absent. One of the resistant cucumber plant comprised a complete deletion of the WAK gene: A deletion of 16 735 bp which comprises the deletion of the SEQ ID No. 1. The presence of the deletion leads to the presence of SEQ ID No. 4 on chromosome 6 of the cucumber plant. This deletion is as comprised in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44368. In the other resistant cucumber plant the wildtype WAK gene was also absent, which is as comprised in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44369.

Through analysis of the correlation in segregation of phenotypes and genotypes it was determined that a modification in an WAK gene resulting in a protein having a changed, reduced or absent function was the cause of the Corynespora cassiicola resistance of the resistant cucumber plants.

EXAMPLE 3

Determination of the contribution of a modified WAK gene

To determine the contribution of the WAK gene, in the resistance test described in Example 1 cucumber plants were genotyped: 11 plants with the deletion of the WAK gene (Plant 1), 9 plants had a similar background but without the deletion and with a wild- type WAK gene (Plant 3) and 3 plants with the absence of WAK as found in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44369. (Plant 2). The results are shown in Table 2.

Table 2

Scoring Corynespora cassiicola symptoms in plants where the WAK gene is absent or present

To confirm the contribution of the WAK gene, in the resistance test described in Example 1 a large number of plants were genotyped: four populations with a deletion comprising the deletion of the WAK gene (Plant 1-4), four populations had a similar background but without the deletion and with a wild-type WAK gene (Plants 5-8) and one population with the absence of WAK as found in the genome of a cucumber plant representative seed of which was deposited under accession number NCIMB 44369 (Plant 9). The test comprised four repetitions (A, B, C and D) and 10 plants of each population were tested per repetition. The results are shown in Table 3. Table 3

Scoring Corynespora cassiicola symptoms in plants where the WAK gene is absent or present

In both experiments, plants where the wild- type WAK gene was absent had a lower phenotypic score than plants with the wild- type WAK gene.

EXAMPLE 4

Modification of the WAK gene through EMS treatment

Seeds of the cucumber breeding line are treated with EMS by submergence of approximately 5000 seeds into an aerated solution of 0.07% (w/v) EMS during 24 hours at room temperature. The treated Ml seeds are germinated and the resulting Ml plants are grown and selfpollinated in a greenhouse to produce M2 seeds . After maturation, M2 seeds are harvested and bulked in one pool. The resulting pool of M2 seeds is used as starting material to identify the individual M2 plants that show Corynespora cassiicola resistance.

Modification the WAK gene through radiation

A population of cucumber mutant plants is obtained by performing a fast neutron (FN) treatment on 10.000 seeds of a cucumber line at a dose of 17 Gy. The seeds are subsequently grown to plants and the M2 seeds obtained through self-fertilization were harvested in bulk. The resulting pool of M2 seeds was used as starting material to identify M2 plants that has Corynespora cassiicola resistance. (Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

(Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)

FOR RECEIVING OFFICE USE ONLY

FOR INTERNATIONAL BUREAU USE ONLY

Claims

1. A modified WAK gene comprising a deletion and/or a substitution, and/or an insertion when compared to the wild-type sequence comprising SEQ ID No. 1 or compared to a homologous sequence having at least 80% sequence identity to SEQ ID No. 1.

2. A modified WAK gene as claimed in claim 1 , wherein said modified WAK gene leads to Corynespora cassiicola resistance in a plant.

3. A modified WAK gene as claimed in claim 1 or 2, wherein the modified WAK gene is a knocked out gene.

4. A modified WAK gene as claimed in any one of the claims 1 to 3, wherein the modified WAK gene comprises a premature stop codon.

5. A modified WAK gene as claimed in any one of the claims 1 or 3, wherein the modification is that the WAK gene is absent.

6. A modified WAK gene as claimed in claim 5, wherein the modification is the absence of SEQ ID No. 1.

7. A modified WAK gene as claimed in claim 1 or 2, wherein the modified WAK gene comprises a mutation resulting in a frame shift.

8. A modified WAK gene as claimed in claim 1, wherein the modified WAK gene comprises a deletion, a substitution and an insertion in separate locations of the gene.

9. A modified WAK gene as claimed in any one of the claims 1 to 8, wherein the WAK gene is not expressed.

10. A modified WAK gene as claimed in any one of the claims 1 to 9, wherein the function of the encoded WAK protein is reduced, changed or absent.

11. A plant comprising the modified WAK gene as claimed in any one of the claims 1 to 10.

12. The plant as claimed in claim 11, wherein the plant is a Corynespora cassiicola resistant cucumber plant.

13. A seed comprising a modified WAK gene as defined in any of the claims 1 to 10, wherein the plant grown from the seed is a plant as claimed in claim 11 or 12.

14. Method for identifying a Corynespora cassiicola resistant plant, wherein the method comprises the following steps: a) detecting in the genome of a plant the modified WAK gene as claimed in any one of the claims 1 to 10, and b) optionally testing of the plant comprising the gene of the invention for exhibiting resistance to Corynespora cassiicola.

15. Method for producing a Corynespora cassiicola resistant plant, comprising introducing a modification in a WAK gene, wherein the modification leads to a modified WAK gene as defined in any of the claims 1 to 10.

16. Method for producing a Corynespora cassiicola resistant plant, comprising introducing a modified WAK gene as claimed in any one of the claims 1 to 10 into a plant.

17. Method for the production of a Corynespora cassiicola resistant plant, comprising: a) crossing a plant as claimed in claim 11 or 12, with another plant; b) optionally performing one or more rounds of selfing and/or crossing of the plant resulting from the cross of step a) to obtain a further generation population; c) selecting from the population resulting from the cross of step a), or from the further generation population of step b), a plant that comprises the modified WAK gene as defined in any of the claims 1 to 10, which plant is resistant against Corynespora cassiicola.

18. Method for the production of a hybrid seed, comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and/or the second parent plant is a plant as claimed in claim 11 or 12, and wherein the hybrid plant that is grown from the seed has the modified WAK gene as claimed in claim 1 to 10.

19. Method as claimed in claim 18 for the production of a hybrid cucumber seed, and wherein said seed has the modified WAK gene as claimed in any of the claims 1 to 10.

20. The hybrid seed produced by the method of claim 18 or 19.

21. Method for identification of a plant comprising a modified WAK gene as claimed in any of the claims 1 to 10, wherein the identification comprises determining the presence of a modification in a WAK gene comprising SEQ ID No. 1 or a sequence having at least 80% sequence identity to SEQ ID No. 1 , and optionally analyzing if the plant comprising the modification has resistance to Corynespora cassiicola.

22. Propagation material suitable for producing a plant as claimed in claim 11 or 12, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, pollen, an ovary, an ovule, an embryo sac, and an egg cell; or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem cell, and a protoplast; or is suitable for tissue culture of regenerable cells, and is in particular selected from a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed, and a stem; wherein the plant produced from the propagation material comprises the modified WAK gene as claimed in any of the claims 1 to 10 that provides resistance to a Corynespora cassiicola.

23. Method for seed production, comprising growing a plant from a seed as claimed in claim 11 or 12 that comprises a modified WAK gene as claimed in any of the claims 1 to 10, allowing the plant to produce a fruit with seed, harvesting the fruit, and extracting those seed.

24. Method for producing a plant as claimed in claim 11 or 12, comprising growing a seed as claimed in claim 13 into a plant.