Resistance genes and lettuce plants resistant to Fusarium wilt

Abstract

The present invention relates to a lettuce plant resistant to Fusarium wilt, and more particularly to a lettuce plant containing a mutant gene that confers resistance to F. oxysporum in lettuce. Further, the present invention relates to a resistance gene and a method for obtaining a lettuce plant resistant to Fusarium wilt, the method including a step of introducing a mutation into the gene.

Description

Technical Field

[0001] The present invention relates to lettuce plants resistant to Fusarium wilt, and more particularly to lettuce plants containing a mutant gene conferring resistance to fungi infecting lettuce. Further, the present invention relates to a method for obtaining a resistant gene and a lettuce plant resistant to Fusarium wilt, the method including the step of introducing a mutation into the gene.

Background Art

[0002] Fusarium wilt is a fungal disease caused by Fusarium oxysporum (F. oxysporum). Fusarium wilt symptoms include wilting, albinism, necrosis, early leaf drop, brown discoloration of the vascular system, growth inhibition, and damping-off. Among them, vascular wilting is the most important symptom, showing vein transparency in younger leaves and drooping of older lower leaves, followed by growth inhibition, yellowing of lower leaves, leaf drop, marginal necrosis, and plant death. Fusarium wilt is a problem in many food crops that affects the production of crops worldwide, including tomatoes, tobacco, cucurbitaceae plants, sweet potatoes, bananas, and lettuce. In lettuce, Fusarium wilt is caused by F. oxysporum f.sp. lactucae, which affects only lettuce. Fusarium wilt caused by F. oxysporum f.sp. lactucae affects all lettuce types, but is more prevalent in crisphead, iceberg, and leaf types.

[0003] F. oxysporum is a soil pathogen that can survive in soil for long periods, and thus crop rotation is not a useful control method. F. oxysporum can also spread through infected dead plant material, and thus end-of-season sanitation is important. The fungus is thought to be sensitive in the laboratory to many broad-spectrum fungicides, but these products have limited effectiveness in the field. It has been found difficult to find effective biological control methods because extrapolation from greenhouse studies to the field is not possible. The best control method against F. oxysporum is the development and use of resistant variants, but with little success, as most result in tolerance to Fusarium wilt but not resistance. Multiple tests have shown that the romaine type may be more resistant to F. oxysporum infection. In contrast, crisphead, iceberg, or leaf-type lettuce show no resistance at all. Furthermore, some lettuce varieties show tolerance to Fusarium, but the pathogen mutates to overcome disease resistance under pressure, and new resistance in the crop is needed to control Fusarium wilt infection.

[0004] Fusarium wilt caused by Fusarium oxysporum f. sp. lactucae (Fol) is the second most threatening pathogen in lettuce after Bremia lactucae. Fields infested with Fol are often abandoned and lost for lettuce production. Fol is present worldwide. Currently, four different Fol races have been reported. Fol:1 is widespread worldwide, while Fol:2 and Fol:3 are limited to Japan, Taiwan, and South Korea. For Fol:1, Fol:2, and Fol:3, official isolates are used by the industry to claim resistance. Fol:4 is present throughout Western Europe (Portugal, Spain, Italy, France, Belgium, the Netherlands, the United Kingdom, Germany, Ireland, and Poland). Internally (Enza Zaden, Enkhuizen, NL), two additional Fol:4 isolates are used internally: Fol:4_enza-AD035 (collected in NL) and Fol:4_enza-AA032 (collected in IT). In recent years, the industry has agreed to use the same type of isolates to claim Fol:4 resistance. Fol:4_official was collected in NL and published by Gilardi et al. in 2017, Plant Pathol. 66, 677 - 688. Fol isolates characterized as Fol:4 are thought to have the same pathogenic spectrum but different levels of pathogenicity. Fol:4_enza-AD035 is more pathogenic than Fol:4_official and Fol:4_enza-AA032.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In view of the above, there is a need in the art to provide a plant resistant to Fusarium wilt, wherein the plant has resistance to this pathogen. Furthermore, it is an object of the present invention to provide a method for obtaining such a Fusarium wilt-resistant plant.

Brief Description of the Drawings

[0006]

Figure 1

Mode for Carrying Out the Invention

[0007] Among other objects, it is an object of the present invention to address the above - mentioned needs in the art. Among other objects, the objects of the present invention are fulfilled by the present invention as outlined in the appended claims.

[0008] In particular, among other objects, the above object is fulfilled by the present invention, in a first aspect, by a lettuce plant resistant to Fusarium wilt, said plant comprising one or more mutations in a KINASE gene that confers said resistance to Fusarium wilt, said KINASE gene encoding a KINASE protein sequence having at least 99% sequence identity, preferably 100% sequence identity, to SEQ ID NO: 2, and the plant further comprising a resistance gene LG7 encoding a resistance protein LG7 having at least 99% sequence identity, preferably 100% sequence identity, to SEQ ID NO: 8, preferably wherein said resistance protein LG7 has valine (V) at amino acid position 111. The KINASE gene containing one or more mutations and conferring resistance by the present invention will hereinafter be referred to as the KINASE R gene.

[0009] Genes conferring resistance to Fusarium in tomatoes are known to include gene I encoding a leucine-rich repeat receptor-like protein (LRR-RLP), I-2 encoding an NBS-LRR protein, I-3 encoding an S-receptor-like kinase, and I-7 encoding a leucine-rich repeat receptor-like protein (LRR-RLP). The KINASE R gene is expected to contain the NBS-LRR mechanism known in plants. The presence of the KINASE R resistance gene reduces the opportunity for pathogens to overcome resistance, as is often seen with resistance genes. Even so, when combined with other resistance genes in lettuce, disease resistance (e.g., to Fusarium wilt) may be further improved.

[0010] The identification of the novel candidate recessive resistance gene, herein designated as the KINASE R gene, was obtained by genetic mapping of multiple independent Fusarium wilt resistance genes in lettuce. Resistance to Fusarium was identified in the lettuce cultivar L. sativa. In a mapping population for Fusarium race 4, more specifically Fol:4_enza-AD035, resistance was mapped to a region narrowed down to 22,922 bp where a single recessive candidate gene, Lsa030992.1, a serine threonine protein kinase, herein referred to as KINASE, is located. For the first time in lettuce, a KINASE gene potentially related to plant disease resistance has been found.

[0011] According to another preferred embodiment, the present invention relates to a lettuce plant in which a mutation in the KINASE gene results in an amino acid change consisting of an amino acid substitution at amino acid positions 205 and / or 231 in the KINASE protein represented by SEQ ID NO: 2.

[0012] According to yet another preferred embodiment, the present invention relates to a lettuce plant in which a mutation in the KINASE gene results in an amino acid substitution of cysteine to tyrosine at amino acid position 205, C205Y, and / or an amino acid substitution of tyrosine to asparagine at amino acid position 231, Y231N.

[0013] According to another preferred embodiment, the present invention relates to a lettuce plant in which a KINASE gene containing one or more mutations encodes a protein sequence represented by SEQ ID NO: 4. The mutant KINASE gene that confers Fusarium resistance in lettuce is herein referred to as the KINASE R gene. Sequencing experiments have shown that the protein encoded by the KINASE R gene from resistant plants differs by two amino acid substitutions, more specifically an amino acid substitution of cysteine to tyrosine at amino acid position 205, C205Y, and / or an amino acid substitution of tyrosine to asparagine at amino acid position 231, Y231N, compared to the protein encoded by the KINASE gene of susceptible plants. The resistance gene (KINASE R) encoding the protein containing the above mutations is represented by SEQ ID NO: 3. The mutant protein is represented by SEQ ID NO: 4.

[0014] SEQ ID NO: 1 represents the coding sequence encoded by the KINASE gene of susceptible Lactuca sativa. SEQ ID NO: 2 represents the protein sequence encoded by the KINASE gene of susceptible Lactuca sativa. SEQ ID NO: 3 represents the coding sequence encoded by the KINASE R gene of the Fusarium wilt-resistant Lactuca sativa plant of the present invention. SEQ ID NO: 4 represents the protein sequence encoded by the KINASE R gene of the Fusarium wilt-resistant Lactuca sativa plant of the present invention. SEQ ID NO: 7 represents the coding sequence encoded by the resistance gene LG7 of the Fusarium wilt-resistant Lactuca sativa plant of the present invention. SEQ ID NO: 8 represents the protein sequence encoded by the resistance gene LG7 of the Fusarium wilt-resistant Lactuca sativa plant of the present invention.

[0015] According to a preferred embodiment, the present invention relates to a lettuce plant in which the KINASE gene and the resistance gene LG7 that confer the resistance to Fusarium wilt exist independently in the plant either in a heterozygous or homozygous form, preferably, both the KINASE gene and the resistance gene LG7 exist in a homozygous form. The results indicate that when both genes exist in a homozygous form, the resistance to Fusarium wilt in lettuce is the most potent.

[0016] According to yet another preferred embodiment, the present invention relates to a lettuce plant selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

[0017] According to another preferred embodiment, the present invention relates to a lettuce plant that is one or more selected from the group consisting of crisphead, butterhead, romaine, oakleaf, and batavia. More preferably, the present invention relates to a lettuce plant that is one or more selected from the group consisting of crisphead, butterhead, oakleaf, and batavia. Most preferably, the present invention relates to a cabbage lettuce plant, and the cabbage lettuce plant is preferably selected from the group consisting of crisphead and butterhead lettuce. As is known to those skilled in the art, crisphead lettuce is equivalent to iceberg lettuce in this specification.

[0018] According to a preferred embodiment, the present invention relates to a lettuce plant in which a mutation in the KINASE gene is obtained by a gene editing technique, preferably mutagenesis or CRISPR / Cas.

[0019] According to another preferred embodiment, the present invention relates to a lettuce plant in which Fusarium wilt is caused by Fusarium oxysporum (F. oxysporum), more preferably F. oxysporum forma specialis lactucae.

[0020] According to another preferred embodiment, the present invention relates to a lettuce plant that is resistant to Fusarium wilt caused by one or more of F. oxysporum selected from the group consisting of Fol:4_forma specialis, Fol:4_enza-AD035, and Fol:4_enza-AA032, preferably at least Fol:4_forma specialis, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032. The F. oxysporum isolates have been deposited and are obtained from deposit numbers NCIMB; Fol:4_forma specialis (NCIMB43967), Fol:4_Enza-AD035 (NCIMB43968), and Fol:4_Enza-AA032 (NCIMB43969). All of the F. oxysporum deposits were made on March 30, 2022, at NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland.

[0021] According to a further aspect, the present invention relates to F. oxysporum selected from the group of Fol:4_enza-AD035 and / or Fol:4_enza-AA032, wherein Fol:4_Enza-AD035 is obtained from the deposit NCIMB43968 and Fol:4_Enza-AA032 is obtained from the deposit NCIMB43969. Fol:4 enza-AD035 and / or Fol:4 enza-AA032 can be used to screen and / or select lettuce plants resistant to Fusarium wilt, more particularly lettuce plants resistant to Fusarium wilt according to the present invention as disclosed herein.

[0022] According to a preferred embodiment, the present invention relates to a lettuce plant in which the resistance gene LG7 is contained in the resistance locus LG7, and the resistance locus LG7 is adjacent to the sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6. Experiments have shown that when KINASE R is combined with the resistance locus LG7 in lettuce, which contains the resistance gene LG7, the resistance to Fusarium wilt is synergistically enhanced, resulting in improved resistance to more virulent Fusarium wilt types. Similar to KINASE R, LG7 may also be present in the plant as heterozygous or homozygous, preferably homozygous.

[0023] According to yet another preferred embodiment, the present invention relates to a lettuce plant in which the mutant KINASE gene, the resistance gene LG7 and the resistance locus LG7 are obtained from the deposit number NCIMB43971. Seeds containing the mutant KINASE gene, the resistance gene LG7 and the resistance locus LG7 were deposited under the number NCIMB43971 on April 6, 2022 with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland.

[0024] According to a further aspect of the present invention, it relates to seeds, plant tissues, plant cells or plant parts produced by or obtained from the lettuce plants of the present invention. The present invention relates to seeds, plant tissues, plant cells or plant parts of the lettuce plants of the present invention, which contain the KINASE gene, the resistance gene and / or the locus LG7 that confer the above-mentioned resistance to Fusarium wilt disease described above.

[0025] According to a further aspect of the present invention, it relates to the resistance gene LG7 that confers resistance to Fusarium wilt disease in lettuce plants, which contains a coding sequence having at least 99% sequence identity to SEQ ID NO: 7, or encodes a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO: 8.

[0026] According to a preferred embodiment, the present invention relates to the resistance gene LG7, in which the resistance protein LG7 has valine (V) at amino acid position 111.

[0027] According to a further aspect of the present invention, it is a combination of the resistance gene KINASE R and the resistance locus LG7 or the resistance gene LG7 described above that confers resistance to Fusarium wilt disease to the lettuce plants in the lettuce plants, wherein the KINASE R gene contains a coding sequence having at least 99%, preferably 100% sequence identity to SEQ ID NO: 3, the LG7 resistance locus contains the resistance gene LG7, and the resistance gene LG7 contains a coding sequence having at least 99%, preferably 100% sequence identity to SEQ ID NO: 7, or The KINASE R gene encodes a KINASE protein sequence having at least 99%, preferably 100% sequence identity to SEQ ID NO: 4, the LG7 resistance locus contains the resistance gene LG7, and the resistance gene LG7 encodes a resistance protein LG7 having at least 99%, preferably 100% sequence identity to SEQ ID NO: 8. Relates to the combination.

[0028] Combining KINASE R with the resistance locus LG7 in lettuce results in improved resistance against more virulent Fusarium wilt types, particularly Fol:4_enza-AD035 and / or Fol:4_enza-AA032.

[0029] According to another preferred embodiment, the present invention relates to a combination in which the KINASE R gene encodes a KINASE R protein represented by SEQ ID NO: 4 having tyrosine (Y) at amino acid position 205 and asparagine (N) at amino acid position 231.

[0030] According to yet another preferred embodiment, the present invention relates to a combination in which the resistance gene LG7 encodes a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO: 8, and the resistance protein LG7 has valine (V) at amino acid position 111.

[0031] According to a preferred embodiment, the present invention relates to a combination in which the resistance locus LG7 is adjacent to the sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6.

[0032] According to another preferred embodiment, the present invention relates to a combination in which the resistance to Fusarium wilt in the lettuce plant includes resistance to F. oxysporum selected from the group consisting of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032.

[0033] According to a preferred embodiment, the present invention relates to a resistance gene KINASE R that encodes a KINASE R protein having at least 85%, preferably at least 90%, more preferably at least 95%, most preferably at least 98%, most preferably 100% sequence identity to SEQ ID NO: 4.

[0034] According to another preferred embodiment, the present invention relates to the resistance gene KINASE R, wherein the resistance to wilting resistance in lettuce includes resistance to Fusarium oxysporum selected from the group of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032.

[0035] According to yet another preferred embodiment, the present invention relates to the resistance gene KINASE R, wherein the lettuce plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeata, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

[0036] According to a further aspect of the present invention, it relates to seeds produced by the lettuce plants of the present invention.

[0037] According to a further aspect of the present invention, it is a method for obtaining a lettuce plant resistant to Fusarium wilt, comprising: a) crossing a lettuce plant composed of the resistance gene KINASE R and the resistance locus LG7 or the combination of the resistance gene LG7 disclosed herein with a lettuce plant not resistant to Fusarium wilt; b) optionally, selfing the plant obtained in step a) at least once; c) selecting a plant resistant to Fusarium wilt, more preferably resistant to Fusarium oxysporum selected from the group of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032. Relates to a method, including. In the method of the present invention, lettuce plants capable of obtaining resistance to Fusarium wilt are Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeata, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably selected from Lactuca sativa. The selection may involve genotyping for the presence of the KINASE R gene in the plant, in (a) or (b), by sequence identification of SEQ ID NO: 4 or SEQ ID NO: 5 in the Fusarium wilt-resistant plant. The selection may also involve a phenotypic test including a disease resistance test against one or more of the shown Fol4 isolates. Following determination of the KINASE R gene, the lettuce plants may be screened for the presence of the resistance locus LG7, and the resistance locus LG7 is adjacent to the sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6. Experiments have shown that when KINASE R is combined with the resistance locus LG7 in lettuce, the resistance to Fusarium wilt is synergistically enhanced, resulting in improved resistance to more virulent Fusarium wilt types. Similar to KINASE R, LG7 may also be present in the plant as heterozygous or homozygous, preferably as homozygous.

[0038] According to a further aspect, the present invention provides a method for obtaining a lettuce plant resistant to Fusarium wilt, comprising causing one or more mutations in the KINASE gene of the lettuce plant to produce an amino acid change consisting of amino acid substitutions at positions 205 and 231 in a KINASE protein having at least 99%, preferably 100% sequence identity to SEQ ID NO: 2, wherein the mutation in the KINASE gene results in an amino acid substitution from cysteine to tyrosine at position 205 (C205Y) and an amino acid substitution from tyrosine to asparagine at position 231 (Y231N), and the lettuce plant further comprises a resistance gene LG7 encoding a resistance protein LG7 having at least 99%, preferably 100% sequence identity to SEQ ID NO: 8, and the resistance protein LG7 has valine (V) at position 111. SEQ ID NO: 2 represents the KINASE protein sequence of susceptible Lactuca sativa. This protein sequence does not contain mutations compared to the KINASE R protein of the present invention. Therefore, L. sativa expressing the protein of SEQ ID NO: 2 is susceptible to Fusarium wilt. The mutant KINASE protein (KINASE R) is represented by SEQ ID NO: 4 and consists of amino acid substitutions at positions 205 and / or 231 from the perspective of the KINASE protein represented by SEQ ID NO: 2.

[0039] According to another preferred embodiment, the present invention relates to a method wherein the mutation in the KINASE gene results in a protein represented by SEQ ID NO: 4.

[0040] The lettuce plants consisting of amino acid substitutions resulted in a phenotype of high resistance to Fusarium wilt. Plants having this resistant phenotype can be obtained by gene editing and / or mutagenesis techniques, such as EMS mutagenesis or the use of CRISPR / Cas, in cooperation with cloning techniques for the KINASE gene, for creating disease-resistant crops.

[0041] According to a preferred embodiment of the present invention, the plant detailed above is not a plant obtained exclusively by substantially biological methods.

[0042] According to yet another preferred embodiment, the present invention relates to a method in which a mutation in the KINASE gene is obtained by gene editing techniques, preferably mutagenesis and / or CRISPR / Cas. Alternatively, the KINASE R gene may be introduced into the plant by transgenic techniques or introgression.

[0043] According to another preferred embodiment, the present invention relates to a method in which a mutation in the KINASE gene is a non-natural mutation. Mutations induced by gene editing techniques, such as mutagenesis, CRISPR / Cas, transgenic techniques, or others, are considered non-natural mutations.

[0044] According to a further aspect of the present invention, there is provided a method for identifying or selecting (i) a Fusarium wilt-resistant lettuce plant or (ii) seeds of said plant as disclosed herein, the method comprising establishing the presence in the genome of said plant or seeds of a resistance gene LG7, or a combination of a resistance gene KINASE R and a resistance locus LG7 or a resistance gene LG7, as defined herein, in a lettuce plant.

[0045] According to a further aspect of the present invention, there is provided the use of a plasmid for introducing a combination of a KINASE R resistance gene and a resistance gene LG7 or a resistance locus LG7 into the genome of a lettuce plant or a lettuce plant cell, wherein the plasmid comprises a resistance KINASE R gene, an LG7 resistance gene and / or a resistance locus LG7 as disclosed herein. The KINASE R resistance gene and / or resistance locus LG7 or resistance gene LG7 of the present invention may be transferred (e.g., by transformation or transfection) into a plant, such as a lettuce plant, using a plasmid comprising the KINASE R resistance gene of the present invention, and the gene comprises a coding sequence having at least 99% identity to SEQ ID NO: 3. The resistance gene KINASE R encodes a KINASE R protein having at least 99% sequence identity to SEQ ID NO: 4. After being transferred into a plant, the resistance gene KINASE R confers resistance to Fusarium wilt, i.e., resistance to Fusarium Fol:4_official, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_official, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032. When KINASE R is combined with the resistance gene LG7 in lettuce, the resistance to Fusarium wilt is synergistically enhanced, resulting in improved resistance to more virulent Fusarium wilt types.

[0046] The present invention is further described in detail in the following examples and figures.

Examples

[0047] Gene mapping of resistance genes A gene mapping experiment was conducted to identify the resistance genes involved in Fusarium (F. oxysporum f. sp. lactucae) resistance in lettuce (L. sativa). The resistance genes were originally isolated from L. sativa lettuce and mapped on chromosome 8. The bulk segregant analysis (BSA) method was used to map the resistance genes. RNA from multiple F3 families showing only Fusarium-resistant plants was pooled and compared to a pool of RNA from multiple F3 families showing only susceptible plants. When SNP markers were constructed and it was possible to identify the target region and flank it with markers, the fine mapping method was initiated. In approximately 20,000 samples of the F2 population, markers were used to identify plants containing recombination between markers based on specific SNPs (Table 1). For fine mapping on chromosome 8, the resistance range could potentially be reduced to 22,922 bp (L.sat_Salinas_v9) where a single recessive candidate gene, serine threonine protein kinase (KINASE R), is located. Following the identification of the KINASE R resistance gene on chromosome 8, a further resistance locus, more specifically LG7, was identified on chromosome 7.

[0048] Following the identified KINASE R gene (SEQ ID NO: 3), a resistance locus LG7 adjacent to two markers; marker 1 (SEQ ID NO: 5) having an SNP position C / T at 65092939 bp and marker 2 (SEQ ID NO: 6) having an SNP position G / A at 67158692 bp was identified, resulting in a resistance locus of approximately 2 Mbp containing several predicted resistance-conferring genes. Further fine mapping led to the identification of the target region of only 50 kpb, and a single resistance gene LG7 encoding the coding sequence of SEQ ID NO: 7 and a resistance protein LG7 represented by SEQ ID NO: 8. The resistance gene LG7 is a TIR-NBS-LRR class gene.

[0049]

Table 1

[0050] When comparing Fusarium wilt-resistant lettuce with susceptible lettuce, two amino acid changes that result in a resistant phenotype in L. sativa lettuce were identified in the KINASE gene, more specifically, base pair mutations that caused amino acid substitutions at positions 205 and / or 231 in the KINASE protein represented by SEQ ID NO: 2. The mutations in the KINASE gene (SEQ ID NO: 1) result in an amino acid substitution from cysteine to tyrosine at position 205, C205Y, and / or an amino acid substitution from tyrosine to asparagine at position 231, Y231N, conferring Fusarium resistance including the KINASE R gene (SEQ ID NO: 3).

[0051] When comparing Fusarium wilt-resistant lettuce with susceptible lettuce, one amino acid change related to the resistant phenotype in L. sativa lettuce was identified in the resistance gene and the resistance protein LG7 (its coding sequence represented by SEQ ID NO: 7, protein sequence represented by SEQ ID NO: 8), more specifically, a base pair mutation that caused an amino acid substitution at position A111V in the LG7 protein.

[0052] Disease tests and bioassays for Fusarium wilt in lettuce Spores were produced using 5-day-old cultures of F. oxysporum f. sp. lactucae in Czapek Dox liquid medium, more specifically, the Fol:4 isolate. The F. oxysporum isolates are deposited and obtained from deposit numbers NCIMB; Fol:4_official (NCIMB43967), Fol:4_Enza-AD035 (NCIMB43968), and Fol:4_enza-AA032 (NCIMB43969). After filtering the cultures through cheesecloth, the spore concentration was adjusted to 5x10 6 spores per ml. 10- to 14-day-old root-washed seedlings were immersed in the spore solution and transplanted into soil heated to 25°C and placed in a greenhouse where the air temperature was 22°C to 28°C. Disease phenotyping was performed between 10 and 14 days after inoculation using a qualitative scoring scale (disease score): 1 = plant death 3 = Severe growth reduction, plant withering, and cotyledon yellowing 5 = Moderate growth reduction, no chlorosis or withering 7 = Some growth reduction, plants smaller than mock-inoculated controls 8 - 9 = No symptoms, plant size and appearance comparable to mock-inoculated controls

[0053] The disease resistance test shows that the KINASE R gene confers resistance to Fusarium race 4, more specifically Fol:4_enza - AD035 and Fol:4_enza - AA032 (Figure 1). Furthermore, the resistance to Fol:4 official was also tested, and the results for Fol:4 official were found to be comparable to those found for FOL:4_enza - AA0032. Additionally, the disease resistance test shows that LG7 also confers resistance to these Fusarium race 4. Combining LG7 and KINASE R in lettuce plants further improves the resistance to Fusarium wilt against the more virulent races Fol:4_enza - AD035 and Fol:4_enza - AA032. The maximum level of Fusarium resistance was observed when both genes were present as homozygous (HO) in lettuce plants. Lettuce plants without either LG7 or KINASE R were used as positive controls.

[0054] Production of Fusarium-resistant lettuce plants using the prime editing (PE) mechanism on lettuce protoplast / cotyledon explants The inventors selected the KINASE gene encoding the protein sequence of SEQ ID NO: 2 in lettuce and created the resistant plants of the present invention containing C205Y and / or Y231N mutations by prime editing (PE).

[0055] PE is a new CRISPR-Cas9-based gene editing technology used to create specific mutations in the target genome. Prime editing can introduce any specific base change, even small ones, or defined deletions or insertions into a wider frame. In PE, to obtain a mutant resistance gene in lettuce, a reverse transcriptase (RT) and SpCas9H840A nickase fused to a 3'-extended guide RNA (pegRNA) with the desired mutation are used. This versatile pegRNA is a modified sgRNA with a reverse transcription template and primer binding site. This pegRNA anneals to the target locus and is used as a template by RT to introduce the desired mutation into the lettuce genome as previously described for plants (Lin et al., 2020, Nature Biotechnology, Xu et al. Molecular Plant 2020 and Xu et al. Nature Plants 2022).

[0056] The inventors used the PE-P2 (Prime Editing - Plant Version 2) plasmid, Cas9n (H840A), an M-MLV RT with multiple NLSs, and a plant-codon-optimized and resynthesized sequence via Twist Bioscience as described by Xu et al. To drive the Cas9H840A nickase, the ZmUbi1 promoter was replaced with the lettuce ubiquitin promoter (as described by Kawazu et al., 2019, The Horticulture Journal). Compared to a single guide RNA (sgRNA), the pegRNA has an additional 3' extension composed of a primer binding site and a reverse transcription template. To determine the best pegRNA sequences, the inventors used the previously described web tool pegFinder (Chow et al., 2020, Nature Biomedical Engineering) (http: / / pegfinder.sidichenlab.org). Subsequently, the sequences of KINASE with the desired C205Y and / or Y231N mutations and the sequences of KINASE without these were selected. The top-hit pegRNAs were fused to the AtU6 promoter and commercially synthesized via Twist Bioscience. To construct the binary vector, the PE-P2 and pegRNA cassettes were cloned into the same backbone as described by Xu et al. (2020). The binary plasmid was transformed into Agrobacterium tumefaciens strain GV2260, and the colonies were analyzed using PCR.

[0057] Next, lettuce cotyledon explants were transformed with Agrobacterium containing the plasmid as previously described (Sun et al., 2006, FEBS letters), and then selected and redifferentiated. To detect the targeted mutations, fragments spanning the target were amplified from genomic DNA and sequenced using the Illumina platform. Plants containing the desired mutant allele in either the homozygous or heterozygous state were self-pollinated. In the next generation, plants were selected for the presence of the homozygous mutant allele and the absence of the transgene.

[0058] Mutant plants were subjected to a Fusarium test using a detached leaf assay and scored for disease symptoms. In plants containing a mutant domain with the desired mutant allele in either the homozygous or heterozygous state, the expected resistant phenotype was observed and no Fusarium disease symptoms were observed. In the next generation, plants were selected for the presence of the homozygous mutant allele and the absence of the transgene.

[0059] Mutant plants were tested for Fusarium disease and scored for disease symptoms. In plants containing a mutant domain, the expected resistant phenotype was observed and no Fusarium disease symptoms were observed.

[0060] Verification of Fusarium-resistant lettuce plants Similar to the previous experiment above, gene editing (GE) was performed on lettuce plants containing the Kinase R gene as well as the resistance locus and gene LG7. In lettuce, GE mutants in the Kinase R gene (SEQ ID NO: 3) were generated that resulted in premature stop codons at positions around 655 bp (LT16-C4), around 690 bp (LT16-A6), or position 658 (LT16-A7).

[0061] Mutant plants were used in a Fusarium disease test and scored for disease symptoms. For Fusarium wilt resistance to Fol:4_enza-AD035, 24 plants per GE mutation were tested, and the parental strain was used as a positive (non-mutant lettuce, LAC) and negative control (Avidius, a lettuce plant without the resistance gene LG7 or KINASE R, used as a positive control known to be susceptible) in the fine mapping assay. See Table 2 for monitoring of the average disease score.

[0062]

Table 2

[0063] Plants having an early stop codon in the Kinase R gene show higher susceptibility to race 4 compared to the non-mutant lettuce plants (LAC) of the present invention containing both the Kinase R gene and the resistance locus and gene LG7, indicating that Kinase R significantly contributes to improved Fusarium wilt resistance. Furthermore, even when the Kinase R protein is not functionally present, the activity of the resistance protein LG7 against Fusarium wilt resistance in lettuce is observed.

Claims

1. A lettuce plant resistant to Fusarium wilt, wherein said plant comprises one or more mutations in a KINASE gene that confer said resistance to Fusarium wilt, said KINASE gene encoding a KINASE protein sequence having at least 99% sequence identity to SEQ ID NO: 2, and said plant further comprises a resistance gene LG7 encoding a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO:

8.

2. The lettuce plant according to claim 1, wherein said resistance protein LG7 has valine (V) at amino acid position 111.

3. The lettuce plant according to claim 1 or 2, wherein said mutation in the KINASE gene results in an amino acid change consisting of an amino acid substitution at amino acid position 205 and / or 231 in the KINASE protein represented by SEQ ID NO:

2.

4. The lettuce plant according to any one of claims 1 to 3, wherein said mutation in the KINASE gene results in an amino acid substitution C205Y from cysteine to tyrosine at amino acid position 205 and / or an amino acid substitution Y231N from tyrosine to asparagine at amino acid position 231.

5. The lettuce plant according to any one of claims 1 to 4, wherein said KINASE gene containing one or more mutations encodes a protein sequence represented by SEQ ID NO:

4.

6. The KINASE gene and the resistance gene LG7 that confer said resistance to Fusarium wilt are each present in said plant either heterozygously or homozygously, preferably both the KINASE gene and the resistance gene LG7 are present homozygously. The lettuce plant according to any one of claims 1 to 5.

7. The lettuce plant according to any one of claims 1 to 6, selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeata, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

8. The lettuce plant according to any one of claims 1 to 7, which is one or more selected from the group consisting of Crisphead, Butterhead, Romaine, Oakleaf, Batavia, preferably Crisphead or Butterhead lettuce.

9. The lettuce plant according to any one of claims 1 to 8, wherein the mutation in the KINASE gene is obtained by a gene editing technique, preferably mutagenesis or CRISPR / Cas.

10. The lettuce plant according to any one of claims 1 to 9, wherein the Fusarium wilt is caused by Fusarium oxysporum (F. oxysporum), more preferably F. oxysporum forma specialis lactucae.

11. The lettuce plant according to any one of claims 1 to 10, which is resistant to Fusarium wilt caused by F. oxysporum selected from the group consisting of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032.

12. The lettuce plant according to any one of claims 1 to 11, wherein the resistance gene LG7 is contained in the resistance locus LG7, and the resistance locus LG7 is adjacent to the sequences represented by SEQ ID NO:5 and SEQ ID NO:

6.

13. The lettuce plant according to any one of claims 1 to 12, wherein the mutant KINASE gene, the resistance gene LG7 and the resistance locus LG7 are obtained from the deposit number NCIMB43971.

14. Seeds, plants, plant cells, or plant parts produced or obtained from the lettuce plant according to any one of claims 1 to 13.

15. A resistance gene LG7 that confers resistance to Fusarium wilt in a lettuce plant, comprising a coding sequence having at least 99% sequence identity to SEQ ID NO:7, or encoding a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO:

8.

16. The resistance gene LG7 according to claim 15, wherein the resistance protein LG7 has valine (V) at amino acid position 111.

17. A combination of a resistance gene KINASE R and a resistance locus LG7 or a resistance gene LG7 that confers resistance to Fusarium wilt in a lettuce plant to the lettuce plant. The KINASE R gene comprises a coding sequence having at least 99% sequence identity to SEQ ID NO: 3, the LG7 resistance locus comprises the resistance gene LG7, and the resistance gene LG7 comprises a coding sequence having at least 99% sequence identity to SEQ ID NO: 7, or the KINASE R gene encodes a KINASE R protein sequence having at least 99% sequence identity to SEQ ID NO: 4, the LG7 resistance locus comprises the resistance gene LG7, and the resistance gene LG7 encodes a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO: 8, combination.

18. The combination according to claim 17, wherein the KINASE R gene encodes a KINASE R protein represented by SEQ ID NO: 4 having tyrosine (Y) at amino acid position 205 and asparagine (N) at amino acid position 231.

19. The combination according to claim 17 or 18, wherein the resistance gene LG7 encodes a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO: 8, and the resistance protein LG7 has valine (V) at amino acid position 111.

20. The combination according to any one of claims 17 to 19, wherein the resistance locus LG7 is adjacent to the sequences represented by SEQ ID NO: 5 and SEQ ID NO:

6.

21. The combination according to any one of claims 17 to 20, wherein the resistance to Fusarium wilt in the lettuce plant comprises resistance to Fusarium oxysporum selected from the group consisting of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032.

22. A method for obtaining a lettuce plant resistant to Fusarium wilt, comprising: a) crossing a lettuce plant comprising the combination of the resistance gene KINASE R and the resistance locus LG7 or the resistance gene LG7 according to any one of claims 17 to 21 with a lettuce plant not resistant to Fusarium wilt; b) optionally, self-breeding the plant obtained in step a) at least once. c) Selecting a plant resistant to Fusarium wilt, more preferably Fusarium oxysporum selected from the group consisting of Fol:4_formula, Fol:4_enza-AD035 and Fol:4_enza-AA032, preferably at least Fol:4_formula, more preferably at least Fol:4_enza-AD035 and / or Fol:4_enza-AA032 A method comprising the above steps **Claim 23** A method for obtaining a lettuce plant according to any one of claims 1 to 13, which is resistant to Fusarium wilt, comprising causing one or more mutations in the KINASE gene of the lettuce plant to result in an amino acid change consisting of amino acid substitutions at positions 205 and 231 in a KINASE protein having at least 99% sequence identity to SEQ ID NO:2, wherein the mutation in the KINASE gene results in an amino acid substitution C205Y from cysteine to tyrosine at position 205 and an amino acid substitution Y231N from tyrosine to asparagine at position 231, and the lettuce plant further comprises a resistance gene LG7 encoding a resistance protein LG7 having at least 99% sequence identity to SEQ ID NO:8, and the resistance protein LG7 has valine (V) at position 111. **Claim 24** The method according to claim 23, wherein the mutation in the KINASE gene results in a protein represented by SEQ ID NO:4 **Claim 25** The method according to claim 23 or 24, wherein the lettuce plant is selected from the group consisting of Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeata, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa **Claim 26** The method according to any one of claims 23 to 25, wherein the mutation in the KINASE gene is obtained by a gene editing technique, preferably mutagenesis or CRISPR / Cas **Claim 27** A method for identifying or selecting (i) a Fusarium wilt-resistant lettuce plant or (ii) seeds of said plant according to any one of claims 1 to 14, comprising establishing the presence in the genome of said plant or seeds of the resistance gene KINASE R or the resistance gene LG7 or the resistance locus LG7 in lettuce plants, or the combination of the resistance gene KINASE R and the resistance locus LG7 or the resistance gene LG7 according to any one of claims 15 to 21.

28. Use of a combination of genes and / or loci according to any one of claims 17 to 21 for conferring Fusarium resistance in lettuce plants.

29. Use of a plasmid for introducing into the genome of a lettuce plant or a lettuce plant cell a combination of the KINASE R resistance gene and the resistance gene LG7 or the resistance locus LG7, said plasmid comprising the resistance KINASE R gene and the LG7 resistance gene and / or the resistance locus LG7 according to any one of claims 17 to 21.

Images