Lettuce plant resistant to downy mildew and resistance gene

Abstract

The present invention relates to a lettuce plant that is resistant to downy mildew, more specifically to a lettuce plant that comprises one or more MRC4 resistance gene(s) that confers broad spectrum resistance to oomycetes in lettuce, more specifically Bremia lactucae. Furthermore, the present invention relates to a MRC4 resistance gene and a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the step of introducing said MRC4 resistance gene into a lettuce plant.

Description

[0001] LETTUCE PLANT RESISTANT TO DOWNY MILDEW AND RESISTANCE GENE

[0002] Description

[0003] The present invention relates to a lettuce plant that is resistant to downy mildew, more specifically to a lettuce plant that comprises one or more MRC4 resistance gene(s) that confers broad spectrum resistance to oomycetes in lettuce, more specifically Bremia lactucae (B. lactucae). Furthermore, the present invention relates to a MRC4 resistance gene and a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the step of introducing said MRC4 resistance gene into a lettuce plant.

[0004] Downy mildew disease is a significant problem in lettuce production as it can cause significant yield losses, reduce the quality of lettuce, and increase production costs. Downy mildew refers to several types of oomycete microbes that are pathogens of plants. Disease is spread from plant to plant by airborne spores. It infects lettuce plants through spores that are released from infected plants and then carried by wind or water to healthy plants. Once the spores land on a plant, they germinate and penetrate the leaves, where they develop into structures called sporangia, which release more spores. Downy mildew can originate from various species, but mainly of Peronospora, Plasmopcirci and Bremia. Downy mildew is a problem in many food crops, for example in lettuce caused by B. lactucae, affecting the production of this crop worldwide. Plants that are being affected include food crops such as brassicas (e.g. cabbage), grape, spinach, lettuce, onion and cucumber.

[0005] Downy mildew infection shows symptoms of discoloured areas on upper leaf surfaces in combination with white, grey or purple mould located on the lower side of the leaf facing the floor. Further symptoms of downy mildew in lettuce include yellowish-green to brownish-gray lesions on the upper surface of the leaves, and a grayish-white, downy growth on the lower surface of the leaves. The infected leaves may also curl or twist, and the plants may become stunted or wilted. In severe cases, the disease can lead to complete defoliation of the plants.

[0006] Downy mildew is a major challenge for lettuce growers because the pathogen can develop resistance to fungicides, making control measures difficult. Additionally, the disease can spread rapidly in wet and humid conditions, which are common in many lettuce-growing regions. Therefore, it is crucial for growers to implement a variety of integrated pest management strategies, such as crop rotation, use of resistant cultivars, and timely application of fungicides, to minimize the impact of downy mildew disease in lettuce production. Fungicides can be used to control B. lactucae, but eventually B. lactucae becomes immune to these chemicals, because over time the pathogen also acquires resistance to fungicides. Letuce, mostly Lactuca sativa, but also including Lactuca species such as L. serriola, L. saligna or L. virosa, is a very important crop worldwide. Some of the most popular varieties available belong to the Iceberg, Romaine, Buterhead, Batavia and Oakleaf letuce types. There are many plant pathogens that affect L. sativa of which the most important disease is downy mildew, which is caused by the B. lactucae, an oomycete pathogen that belong to Peronosporaceae . For lettuce, some cultivars with resistance to downy mildew are available. However, the pathogen under pressure will mutate over time and / or new species are generated due to e.g. (a-)sexual recombination to break the disease resistance and new disease resistance in lettuce is needed to control the infection. Especially in lettuce the occurrence of downy mildew resistance is particularly complex as there are many different races, and new downy mildew resistant species emerging all the time, as found in European and the USA markets. For example, there are currently 37 races of B. lactucae that have been identified and characterized and that cause downy mildew disease in letuce. These races are distinguished based on their ability to infect different lettuce cultivars that carry different resistance genes. Multiple letuce varieties are available that are resistant to B. lactucae but resistance is quickly overcome because new races develop rapidly. The identification of new races of B. lactucae is an ongoing process, as the pathogen continues to evolve and adapt to changing environmental conditions and crop management practices. Therefore, it is of the utmost importance to find other methods to control B. lactucae infection. Most preferably is to identify a resistance gene that gives broad resistance against B. lactucae and to provide for lettuce plants that are resistant to downy mildew. Therefore, identification of resistance genes is a promising alternative.

[0007] During the development of new disease or pathogen resistant plants often traits are being combined, for example by introgression of a genetic locus comprising one or more resistance gene, thereby combining multiple resistance genes to combat the pathogen being able to overcome the resistance. For example, it is known that major resistance clusters are present in lettuce on e.g. chromosome 1, 2, and 9 comprising several disease resistance genes. It is highly desired to have disease resistance genes present on different chromosomes, e.g. to facilitate gene stacking and providing improved disease resistance in a crop. Alternatively, or additionally, the introgression fragments often comprise, apart from the resistance gene of interest, other genetic elements that may negatively affect the plant in terms of yield, growth, vitality, and seed production. For example, introgression of (additional) new resistance genes in letuce often result in a severe reduction in seed production as a result of linkage drag. Therefore, apart from the generation of improved disease resistance in plant, there is also great benefit to reduce this so-called linkage drag which becomes an increasing hurdle in plant breeding. Single event introgression as well as marker-aided selection techniques in the flanking regions of the resistance gene to reduce the introgression segment can play an important role here, with the objective of minimizing residual genetics (apart from the resistance gene) being transferred to the parent plant and to eliminate linkage drag effects. Gene editing and the use of sequence information in genome-wide selection will further add to the precision of reduction of linkage drag.

[0008] Considering the above, there is a need in the art to provide plants that are resistant to downy mildew and wherein plants have a broad-spectrum resistance against this pathogen. Furthermore, it is an object of present invention to provide plants having a broad-spectrum downy mildew resistance, and to provide a method to obtain such downy mildew resistant plants.

[0009] It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0010] Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a downy mildew resistant lettuce plant, wherein said lettuce plant comprises one or more MRC4 resistance gene(s) encoding a MRC4 resistance protein having at least 70% sequence identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, preferably at least 92%, more preferably at least 95% even more preferably at least 96%, most preferably at least 99% with amino acid sequence of SEQ ID No. 6 providing downy mildew resistance, wherein said MRC4 resistance protein comprises a protein motif A represented by SEQ ID No. 9 and a protein motif B represented by SEQ ID No. 10, wherein said lettuce plant is resistant to B. lactucae races Bl: 16-37EU.

[0011] The downy mildew resistance conferring gene MRC4 is a dominant resistance trait and may be homozygous or heterozygous present in a downy mildew resistant lettuce plant, preferably homozygous. The resistance gene against B. lactucae has been found on chromosome 4 in lettuce, present in the Major Resistance Cluster 4. This MRC4 resistance gene of the present invention gives resistance to B. lactucae races Bl: 16-37EU and preferably also to Bl: 1-15EU wherein said B. lactucae races have been characterized and classified according to the SEXTET code by IBEB (International Bremia Evaluation Board). Therefore, the one or more MRC4 resistance gene(s) provides full spectrum resistance to Bl: 1-37EU.

[0012] As disclosed herein, the percentage (%) sequence identity is known to the person skilled in the art. Preferably it is to be understood in relation to a query sequence having at least 90% of the sequence length of the gene or protein sequence as claimed herein, preferably at least 95%, more preferably 98%, even more preferably at least 99%, most preferably 100% sequence length. Alternatively, or additionally, the gene sequence alignment is performed from start(codon) to stop(codon) of the coding sequence or protein sequence. For example, Geneious Prime (Clustal Omega algorithm) can be used to align sequences and calculate the % sequence identity.

[0013] The one or more MRC4 resistance gene(s) encode for a MRC4 resistance protein that comprises a protein motif A represented by SEQ ID No. 9 (CLEELHMANECQKL), and a protein motif B represented by SEQ ID No. 10 (SKLRTFDLGMTPN). Protein motif A represents a Zinc finger (Znf) motif or domain. Preferably the MRC4 resistance protein providing the downy mildew resistant lettuce plant according to present invention, comprises at least two protein motifs

[0014] A, preferably at least 6, more preferably at least 8, most preferably at least 10 protein motifs A. Protein motif B represents a Leucine Rich Repeat (LLR) motif, and preferably the MRC4 resistance protein providing the downy mildew resistant lettuce plant according to present invention, comprises at least six motifs B, preferably at least 8, more preferably at least 9, most preferably at least 10 protein motifs B. A known resistance gene DM11 (L. Parra et al. , Theoretical and Applied genetics (2021) 134:519-528) which is also present on chromosome 4 and provides downy mildew resistance (see figure 2) in lettuce, does not comprise any of these motif A or motif

[0015] B.

[0016] The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat proteins, also known as NBS-LRR proteins (encoded by R genes). These proteins are characterized by nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains as well as variable amino- and carboxy-terminal domains and are involved in the detection of diverse pathogens, including bacteria, viruses, fungi, nematodes, insects and oomycetes. There are three major subfamilies of plant NBS-LRR proteins defined by the Toll / interleukin-1 receptor (TIR) also called TNLs, the coiled-coil (CC) motifs in the amino-terminal domain containing NBS- LRRs also called CNLs and RPW8-NLTRs also called RNLs. A typical R gene contains an NB- ARC domain which is proposed to regulate activity of the R protein. The MRC4 gene is initially picked up by fine mapping and VIGS experiments based on the L. sativa genome. The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat proteins, also known as NBS-LRR proteins (encoded by R genes). These proteins are characterized by nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains as well as variable amino- and carboxy-terminal domains and are involved in the detection of diverse pathogens, including bacteria, viruses, fungi, nematodes, insects, and oomycetes. The MRC4 gene comprises a TIR domain and several, preferably at least six LRR motifs (i.e. protein motif B represented by SEQ ID No. 10 (SKLRTFDLGMTPN) within the gene that may be beneficial and contributes to generate a new R gene that confers broad spectrum B. lactucae resistance. The identified MRC-4 proteins herein all comprise highly homologue three-dimensional protein structures, including the sheets in a horseshoe structure LLR domains and the TIR domain.

[0017] The MRC4 resistance genes included herein are unique since it comprises features that are typical for a plant resistance gene, more specifically the MRC4 resistance gene comprises a TIR domain and has LRR domains. Surprisingly, it was found that the MRC4 genes contains multiple repeats, preferably at least two of Zinc finger (Znf) motifs features (i.e. motif A represented by SEQ ID No. 9 (CLEELHMANECQKL) present within the LRR domain which could serve as a decoy domain and providing disease resistance. Znf domains or motifs are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Their binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. The MRC4 resistance gene comprises CysCysHisCys (CCHC) type zinc finger domains and has the sequence C-X4-H-X4-C in repeat where X can be any amino acid, and the number indicates the number of residues. And although it is not the signature motif of a typical ZnF motif (which is C-X2-C-X4-H-X4-C) it is picked up by in silico prediction analysis as ZnF C2HC motifs.

[0018] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein said MRC4 resistance protein comprises protein motif A at least two times, preferably at least six times, more preferably at least eight times, most preferably at least ten times.

[0019] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein said MRC4 resistance protein comprises protein motif B at least six times, preferably at least eight times, more preferably at least nine times, most preferably at least ten times.

[0020] The presence of the MRC4 resistance gene will provide broad spectrum B. lactucae resistance to lettuce plants. To decrease the chances of the pathogen overcoming the resistance, as often seen with R genes, multiple R genes can be combined to enhance the durability of disease resistance. For example, the downy mildew resistant lettuce plant of the present invention may further comprise one or more resistance genes located on chromosome 4 at a significant distance from the MRC4 resistance gene or with R genes located at different linkage groups. Additionally, or alternatively the MRC4 resistance gene may be stacked with other resistance genes on other chromosomes. MRC4 is present on the MRC4 locus or cluster (major resistance cluster 4) and could be used to combine with another active B. lactucae resistance gene. As such, stacking of multiple resistance genes will enable broad and durable B. lactucae resistance in lettuce.

[0021] To demonstrate that the MRC4 resistance genes disclosed herein provide B. lactucae resistance, the MRC4 resistance genes were silenced by tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to induce susceptibility to B. lactucae infection in resistant L. sativa lines containing one of the MRC4 resistance genes. With VIGS it was demonstrated that the MRC4 resistance genes are associated with downy mildew resistance, since VIGS induced gene silencing was used to create B. lactucae susceptibility in resistant Lactuca accessions containing only MRC4 resistance. Resistant lettuce plants were transiently transformed with a silencing construct specific against the MRC4 resistance gene which resulted in the silencing of the resistance gene and consequently made the plant or plant organs susceptible to B. lactucae infection, thus by “removing” or silencing the MRC4 resistance gene via virus induced gene silencing.

[0022] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein said one or more MRC4 resistance gene(s) comprises a coding sequence having at least 74%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 92%, more preferably at least 95% even more preferably at least 96%, most preferably at least 99% sequence identity with SEQ ID No. 5. Said MRC4 resistance gene is preferably located on chromosome 4 of the resistant lettuce plant.

[0023] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein said one or more MRC4 resistance gene(s) comprise a coding sequence having at least 95% sequence identity with SEQ ID No. 1.

[0024] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein the MRC4 resistance protein comprises an amino acid sequence having at least 92%, preferably having at least 99% sequence identity with SEQ ID No. 2.

[0025] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the protein encoded by said one or more MRC4 resistance gene(s) comprises the amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, preferably SEQ ID No. 6.

[0026] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein said one or more MRC4 resistance gene(s) comprises the coding sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7, preferably SEQ ID No. 5.

[0027] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the lettuce plant is further resistant to one or more of B. lactucae races selected from the group consisting of races Bl: 1-15EU. Recent experiments show that a lettuce plant of the present invention comprising one of the MRC4 resistant genes is resistant to B. lactucae races from Bl: 16 to B1:37EU. Previous disease resistance tests on less recent B. lactucae races Bl: 1-15EU (data not shown) confirmed that the MRC4 resistance gene further provides resistance to these B. lactucae races. Resistance to B. lactucae in the lettuce of present invention comprises full spectrum resistance to B. lactucae races Bl: 1 to B1:37EU.

[0028] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa. According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the one or more MRC4 resistance gene(s) is at least heterozygous present in the lettuce plant, preferably homozygous present.

[0029] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the MRC4 resistance gene is obtainable, derived, or originates from a lettuce plant of L. serriola, L. sciligna or L. virosa, preferable L. serriola. Most preferably the present invention relates to the lettuce plant, wherein the MRC4 resistance gene is obtainable, derived, or originates from a lettuce plant, more specifically an L. sativa plant, deposited under number NCIMB 44113, NCIMB 44114, NCIMB 44115 or NCIMB 44116. Seeds comprising the MRC4-1 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44113. Seeds comprising the MRC4-2 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44114. Seeds comprising the MRC4-3 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44115. Seeds comprising the MRC4-4 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44116.

[0030] The present invention, according to a second aspect, relates to a seed, plant tissue, plant cell or plant part of of a lettuce plant of the present invention, comprising one or more MRC4 resistance gene(s) encoding a protein as described above. The seed preferably comprises a seed coating, wherein the seed coating preferably comprises one or more selected from the group consisting of clay, wood fibres, peat, coconut fibre, calcium carbonate, cellulose, activated carbon, or a mixture thereof, preferably wherein said clay comprises one or more selected from the group consisting of hydrous aluminium phyllosilicates, halloysite, lizardite, chrysotile, pyrophyllite-talc, illite, celadonite, vermiculite, montmorillonite, nontronite, saponite, sudoite, clinochlore, chamosite, sepiolite-palygorskite, rectorite, corrensite, tosudite, allophane-imogolite, and mixtures thereof.

[0031] The present invention, according to a further aspect, relates to an MRC4 resistance gene that confers resistance to downy mildew in lettuce plants, wherein the resistance gene encodes for a protein that has at least 70% sequence identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, preferably at least 92%, more preferably at least 95% even more preferably at least 96%, most preferably at least 99% sequence identity with SEQ ID No. 6, and wherein said MRC4 resistance protein comprises a protein motif A represented by SEQ ID No. 9 and a protein motif B represented by SEQ ID No. 10. The MRC4 resistant gene is a dominant trait.

[0032] According to yet another preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein said MRC4 resistance protein comprises protein motif A at least two times, preferably at least six times, more preferably at least eight times, most preferably at least ten times and / or wherein said MRC4 resistance protein comprises protein motif B at least six times, preferably at least eight times, more preferably at least nine times, most preferably at least ten times.

[0033] According to another preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein said one or more MRC4 resistance gene(s) comprises a coding sequence having at least 74% sequence identity, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 92%, more preferably at least 95% even more preferably at least 96%, most preferably at least 99% with SEQ ID No. 5.

[0034] According to a preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein said one or more MRC4 resistance gene(s) comprise a coding sequence having at least 95% sequence identity with SEQ ID No. 1.

[0035] According to another preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein the MRC4 resistance protein comprises an amino acid sequence having at least 92%, preferably having at least 99% sequence identity with SEQ ID No. 2.

[0036] According to yet another preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein the protein encoded by said MRC4 resistance gene comprises an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, preferably SEQ ID No. 6.

[0037] According to another preferred embodiment, the present invention relates to the MRC4 resistance gene, wherein said MRC4 resistance gene comprises the coding sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7, preferably SEQ ID No. 5.

[0038] According to yet another preferred embodiment, the present invention relates to a resistance gene that confers resistance to B. lactucae in lettuce plants, wherein the resistance gene provides resistance to at least B. lactucae races Bl: 16-37EU, preferably B. lactucae races Bl: 1- 37EU in lettuce.

[0039] According to yet another preferred embodiment, the present invention relates to the resistance gene that confers resistance to B. lactucae in lettuce plants, wherein the plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

[0040] The present invention, according to a further aspect, relates to a method for identifying (i) a downy mildew resistant lettuce plant or (ii) a seed of said plant as disclosed herein, wherein the method comprises the step of establishing, in the genome of said plant or seed, the presence of one or more MRC4 resistance gene encoding a protein as defined above. The step of establishing, in the genome of the seed or resistant plant, the presence of any genetic information, including the presence of the MRC4 resistance gene encoding the protein as defined above, may suitably involve allowing the seed to grow into a plant and establishing the presence of the genetic information in the genome of the plant grown from the seed.

[0041] According to yet another preferred embodiment, the present invention relates to the method for identifying a downy mildew resistant lettuce plant of present invention, wherein the step of establishing, comprises establishing the presence of the resistance gene is achieved by using one or more markers selected from the group consisting of SEQ ID No.l 1, SEQ ID No.12, SEQ ID No. 13 and SEQ ID No.14, preferably by using all of said markers and / or by establishing the presence of one or more sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7. A downy mildew resistant plant of present invention comprising the MRC4 resistance gene can be identified by the presence in the genome of said plant of one or more MRC4 sequences or by using specific SNP markers for the identification of resistant plants comprising at least one MRC4 resistance gene. Most preferably the plant can be identified by the presence of SEQ ID No.5 in the plant.

[0042] The present invention, according to a further aspect, relates to a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the steps of, a) crossing a lettuce plant comprised of the resistance gene of the present invention with a lettuce plant susceptible to downy mildew and which does not comprise said resistance gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to downy mildew.

[0043] The selecting of step c may for example be done by the method for identifying a downy mildew resistant lettuce plant as disclosed above.

[0044] A plant having this resistant phenotype can be obtained via use of gene editing and / or mutation techniques, such as EMS mutagenesis or CRISPR / Cas in concert with cloning techniques on the MRC4 resistance gene to generate disease resistant crops. A resistance gene can be brought into the plant by known means including e.g. transgenic techniques or by introgression, wherein the resistance providing sequence(s) are introduced into the plant.

[0045] The present invention, according to a further aspect, relates to the use of a gene construct or plasmid for introducing an MRC4 resistance gene into the genome of a plant or plant cell and providing broad spectrum resistance to downy mildew caused by one or more of B. lactucae races selected from the group of race Bl: 16 - 37EU wherein the gene construct is comprised of the MRC4 resistance gene operably linked to expression providing sequences in said plant. The resistance gene of present invention may be transferred (e.g. by transformation or transfection) into plants, such as lettuce plants, using a plasmid or vector or linear gene construct that comprises the resistance gene of present invention. The MRC4 resistance gene, after being transferred into the lettuce plant will provide resistance to B. lactucae, i.e. resistance to at least B. lactucae of race Bl: 16-37EU, preferably Bl: 1-37EU.

[0046] The present invention, according to a further aspect, relates to the use of an MRC4 resistance gene as disclosed herein for conferring or improving resistance against Bremia lactucae in a plant of L. sativa, wherein the MRC4 resistance gene is preferably obtainable from L. serriola, L. virosa or L. saligna.

[0047] The present invention will be further detailed in the following examples and figures wherein:

[0048] Figure 1: shows the % of susceptible leaves of lettuce that have been infected with B. lactucae B1:22EU, after VIGS silencing of a lettuce plant comprising one of the MRC4 resistance genes of present invention; more specifically either MRC4-1, MRC4-2, MRC4-3, or MRC4-4. The specific MRC4 resistance gene was silenced using the VIGS gene silencing constructs of Table 2 and subsequently infected with B. lactucae. As expected with transient gene silencing, VIGS gene silencing does not result in fully 100% silencing of the gene in all plants, as reflected by their level of susceptibility. However, the leaves from plants wherein the MRC4 gene (one of MRC4-1 to MRC4-4) has been silenced by VIGS silencing, all showed a significant percentage of susceptible leaves (between 10 to 25%) when infected with B. lactucae as compared to plants where the MRC4 gene was not silenced, i.e. by the PDS silencing construct on the plant comprising the MRC4 gene.

[0049] Figure 2: shows an overview of the disease test performed with the most recent isolates of B. lactucae B1: 16-37EU on A. sativa line “Cobham Green”, line “Captain” comprising Dml 1 resistance gene (both public available), and plants of present invention comprising one of the MRC4 resistance genes; more specifically MRC4- 1, MRC4-2, MRC4-3, or MRC4-4. The plants of present invention shows to be resistant (-) to all tested B. lactucae isolates, Bl: 16-37EU, providing broad spectrum resistance. Cobham Green was susceptible (+) to all tested isolates, as well as DM11 which was susceptible for B122, -29, -33, -34, and -37. “ND” = not determined indicating that no tests were performed with DM11 and the indicated B. lactuccie isolates.

[0050] Examples

[0051] Gene Mapping of MRC4 resistance genes in lettuce

[0052] Gene mapping experiments were done to identify resistance gene(s) involved in full spectrum Bremia (B. lactucae) resistance in lettuce (L. sativa). A resistance locus indicated as Major Resistance Cluster 4 (MRC4 locus) was identified by fine mapping an F2 population of about 12,000 lettuce plants by gene mapping, which comprised one or more MRC4 resistance candidate genes mapped on chromosome 4, providing B. lactucae resistance in lettuce. Candidate genes were subsequently screened using gene silencing (VIGS silencing, see below) to confirm their role in B. lactucae resistance, wherein silencing of the MRC4 resistance gene in a resistant plant resulted in a susceptible phenotype.

[0053] The MRC4 resistance gene, of which four alleles have confirmed as providing B. lactucae resistance. SNP markers (See Table 1) were used to identify plants comprising the specific resistance providing alleles. Marker 2 (SEQ ID No. 12) is used for the identification of plants comprising the MRC4-2 gene. The SNPs (susceptible / resistant) are indicated in N bold and underlined on which the resistant plants could be selected. For MRC4-2, in B. lactucae susceptible lettuce plants the indicated SNP nucleotide was an “A” as indicated in SEQ ID No. 12 and a “T” for lettuce plant showing full spectrum B. lactucae resistance, i.e. comprising the MRC4-2 gene. Similar, marker 3 (SEQ ID No. 13) was used for the identification of MRC4-3, wherein a “G” = susceptible and “A” resistant genotype. For Marker 1 (SEQ ID No. 11) a “G” = susceptible and a “A” is resistant and Marker 1 detects MRC4-1, but also MRC4-2 and MRC4-4. For Marker 4 (SEQ ID No. 14) a “G” = susceptible and a “A” is resistant and Marker 4 detects MRC4-2 and MRC4-4. Using one or more of these markers the plants were screened for the presence of the specific MRC4 gene, and subsequently confirmed by sequencing.

[0054] Table 1. Marker sequences for identification of MRC4 genes

[0055] MRC4 resistance gene silencing experiment using Virus Induced Gene Silencing (VIGS)

[0056] To demonstrate that the identified MRC4 resistance genes provides B. lactucae resistance, the specific MRC4 resistance gene was silenced by tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to induce susceptibility to B. lactucae infection in L. sativa lines containing the MRC4 resistance gene. Tobacco rattle virus (TRV)-derived VIGS vectors have been abundantly described to study gene function in Arabidopsis thaliana, Nicotiana benthamiana, Solarium esculentum and other plants (see for example Huang C, Qian Y, Li Z, Zhou X.: Virus- induced gene silencing and its application in plant functional genomics. Sci China Life Sci. 2012;55(2):99-108). With VIGS it was demonstrated that the MRC4 resistance gene was associated with downy mildew resistance, since VIGS induced gene silencing was used to create B. lactucae susceptibility in resistant Lactuca accessions comprising the MRC4 gene. Resistant lettuce plants were transiently transformed with a silencing construct specific against the resistance MRC4 gene which will result in the silencing of the resistance gene.

[0057] Briefly, lettuce plants containing one of the identified MRC4 resistance genes were silenced for MRC4 resistance gene by VIGS using different silencing constructs to identify if the MRC4 resistance gene was indeed responsible for the observed resistance. VIGS -constructs were used that results in silencing of the MRC4 resistance gene (MRC4). Furthermore, independent of resistance gene silencing the PDS gene was silenced that served as positive control to indicate if VIGS is working and to determine the efficiency. The PDS gene is involved in carotenoid biosynthesis and is the first step in lycopene biosynthesis. This step is catalyzed by the enzyme phytoene desaturase (PDS). When silencing of the PDS gene is achieved, this results in bleached leaves. Experiments showed bleached leaves indicating that the VIGS silencing was achieved and performed correctly (data not shown). All plants that were VIGS inoculated were harvested and put in a tray and sprayed with B. lactucae to test the effect of the gene silencing on disease resistance. The VIGS constructs were cloned in the K20 vector. The constructs were transformed and transiently expressed into a lettuce plant of present invention that is resistant to B. lactucae, using co-cultivation with agrobacterium (GV3101) to study the resistance gene function in relation to B. lactucae resistance. The % of susceptible B. lactucae leaves was observed in both groups and both silencing constructs. With the leaves of VIGS -experiments independent disease tests (see below) were performed to observe that when MRC4 resistance gene was silenced, plants became susceptible to B. lactucae.

[0058] Results (Figure 1) indicate that when MRC4 was silenced by VIGS with the MRC4 construct the plants became susceptible after B. lactucae infection (B1:22EU) confirming that the resistance gene is linked to a resistance gene that provides the plant resistance against B. lactucae. The PDS control plants remained resistant to the B. lactucae infection, all leaves were unaffected.

[0059] Table 2. VIGS construct

[0060] Disease test and biotest for downy mildew in Lettuce

[0061] Leaves of resistant plants transiently transformed with the above described VIGS constructs, were put in trays with moistened paperboard and infected with B. lactucae race 22. Infected seedlings are suspended in 20 ml water, filtered by cheesecloth and the flow-through is collected in a spray flask. The trays are spray-inoculated with the B. lactucae suspension. The trays are covered with a glass plate and stored in a climate chamber at 15°C (12 hours of light). A black, opaque foil is placed over the trays for one day to improve growth of B. lactucae. After one day, the foil is removed. Experiments were performed in triple, and eight to ten days after infection leaves are phenotypically scored by eye on the presence of B. lactucae, i.e. being susceptible or resistant. Disease resistance tests show that the MRC4 resistance genes encoding the MRC4 resistance proteins, more specifically MRC4-1 to MRC4-4 (SEQ ID No. 1 to SEQ ID No. 8), provide resistance to B. lactuccie races from B1: 16EU to B1:37EU (See Figure 2). In comparison, a related and known R gene DM11, also present on chromosome 4, provides incomplete spectrum disease resistance, at least not providing resistance to Bl:22, 33,34 and 37EU. Previous disease resistance test have shown that the MRC4 resistance genes also provide resistance to Bl: 1EU to Bl: 15EU (results not shown), therefore the MRC4 resistance genes provides full spectrum resistance to Bl: 1-37EU.

[0062] A single gene line comprising the MRC4 resistance gene was used internally to test B. lactuccie diagnostically. Seeds of a lettuce (L. sativa) comprising the MRC4-1 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44113. Seeds of a lettuce (L. sativa) comprising the MRC4-2 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44114. Seeds of a lettuce (L. sativa) comprising the MRC4-3 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44115. Seeds of a lettuce (L. sativa) comprising the MRC4-4 gene are deposited at NCIMB Ltd, Aberdeen, Scotland on 2 February 2023 under the number NCIMB 44116.

[0063] MRC4 resistance genes and protein sequence alignments

[0064] The similarity of the novel MRC4 resistance genes (Table 3) and resistance proteins (Table 4) was determined using multiple alignment software (Geneious Prime, Clustal Omega algorithm) using the coding sequences of MRC4-1 (SEQ ID No. l (cDNA) and SEQ ID No. 2 (protein)), MRC4-2 (SEQ ID No. 3 (cDNA) and SEQ ID No. 4 (protein)), MRC4-3 (SEQ ID No. 5 (cDNA) and SEQ ID No. 6 (protein)) and MRC4-4 (SEQ ID No. 7 (cDNA) and SEQ ID No. 8 (protein)).

[0065] Table 3. Percent Identity Matrix on coding sequence of the MRC4 resistance genes.

[0066] Table 4. Percent Identity Matrix on amino acid sequence of the MRC4 resistance proteins

[0067] (Original in Electronic Form)

[0068] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0069] (This sheet is not part of and does not count as a sheet of the international application)

[0070] FOR RECEIVING OFFICE USE ONLY PCT

[0071] (Original in Electronic Form)

[0072] (This sheet is not part of and does not count as a sheet of the international application)

[0073] FOR INTERNATIONAL BUREAU USE ONLY

Claims

Claims1. A downy mildew resistant lettuce plant, wherein said lettuce plant comprises one or more MRC4 resistance gene(s) encoding a MRC4 resistance protein having at least 70% sequence identity with amino acid sequence of SEQ ID No. 6 providing downy mildew resistance, wherein said MRC4 resistance protein comprises a protein motif A represented by SEQ ID No. 9 and a protein motif B represented by SEQ ID No. 10, wherein said lettuce plant is resistant to Bremia lactucae races Bl: 16-37EU.

2. Lettuce plant according to claim 1, wherein said MRC4 resistance protein comprises protein motif A at least two times, preferably at least six times.

3. Lettuce plant according to claim 1 or 2, wherein said MRC4 resistance protein comprises protein motif B at least six times, preferably at least eight times.

4. Lettuce plant according to any one of the claims 1 to 3, wherein said one or more MRC4 resistance gene(s) comprises a coding sequence having at least 74% sequence identity with SEQ ID No. 5.

5. Lettuce plant according to any one of the claims 1 to 4, wherein said one or more MRC4 resistance gene(s) comprise a coding sequence having at least 95% sequence identity with SEQ ID No. 1.

6. Lettuce plant according to any one of the claims 1 to 5, wherein the MRC4 resistance protein comprises an amino acid sequence having at least 92%, preferably having at least 99% sequence identity with SEQ ID No. 2.

7. Lettuce plant according to any one of the claims 1 to 6, wherein the MRC4 resistance protein comprises the amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, preferably SEQ ID No. 6.

8. Lettuce plant according to any one of the claims 1 to 7, wherein said one or more MRC4 resistance gene(s) comprise the coding sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7, preferably SEQ ID No. 5.

9. Letuce plant according to any one of the claims 1 to 8, wherein the lettuce plant is further resistant to one or more of Bremia lactucae races selected from the group consisting of races Bl: 1-15EU.

10. Letuce plant according to any one of the claims 1 to 9, wherein the lettuce plant is selected from the group consisting of Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactucci serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, and Lactuca viminea, preferably Lactuca sativa.

11. Letuce plant according to any one of the claims 1 to 10, wherein the one or more MRC4 resistance gene(s) is obtainable, derived, or originates from a letuce plant deposited under number NCIMB 44113, NCIMB 44114, NCIMB 44115 or NCIMB 44116.

12. Seed, plant tissue, plant cell or plant part of a letuce plant according to any one of the claims 1 to 11, comprising one or more MRC4 resistance gene(s) encoding the MRC4 resistance protein as defined in said claims.

13. Seed according to claim 12, wherein the seed comprises a seed coating, wherein the seed coating preferably comprises one or more selected from the group consisting of clay, wood fibres, peat, coconut fibre, calcium carbonate, cellulose, activated carbon, or a mixture thereof, preferably wherein said clay comprises one or more selected from the group consisting of hydrous aluminium phyllosilicates, halloysite, lizardite, chrysotile, pyrophyllite-talc, illite, celadonite, vermiculite, montmorillonite, nontronite, saponite, sudoite, clinochlore, chamosite, sepiolite-palygorskite, rectorite, corrensite, tosudite, allophane-imogolite, and mixtures thereof.

14. An MRC4 resistance gene that confers resistance to downy mildew in letuce plants, wherein the resistance gene encodes for a MRC4 resistance protein that has at least 70% sequence identity with SEQ ID No. 6 and wherein said MRC4 resistance protein comprises a protein motif A represented by SEQ ID No. 9 and a protein motif B represented by SEQ ID No. 10.

15. MRC4 resistance gene according to claim 14, wherein said MRC4 resistance protein comprises protein motif A at least two times, preferably at least six times.

16. MRC4 resistance gene according to claim 14 or 15, wherein said MRC4 resistance protein comprises protein motif B at least six times, preferably at least eight times.

17. MRC4 resistance gene according to any one of the claims 14 to 16, wherein said one or more MRC4 resistance gene(s) comprises a coding sequence having at least 74% sequence identity with SEQ ID No. 5.

18. MRC4 resistance gene according to any one of the claims 14 to 17, wherein said one or more MRC4 resistance gene(s) comprise a coding sequence having at least 95% sequence identity with SEQ ID No. 1.

19. MRC4 resistance gene according to any one of the claims 14 to 18, wherein the MRC4 resistance protein comprises an amino acid sequence having at least 92%, preferably having at least 99% sequence identity with SEQ ID No. 2.

20. MRC4 resistance gene according to any one of the claims 14 to 19, wherein the MRC4 resistance protein comprises an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, preferably SEQ ID No. 6.

21. MRC4 resistance gene according to any one of the claims 14 to 20, wherein said MRC4 resistance gene comprises the coding sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7, preferably SEQ ID No. 5.

22. MRC4 resistance gene according to any one of the claims 14 to 21, wherein the MRC4 resistance gene provides resistance to at least Bremia lactucae races Bl: 16 to Bl:37, preferably to Bremia lactucae races Bl: 1-37EU in lettuce.

23. Method for identifying or selecting (i) a downy mildew resistant lettuce plant or (ii) a seed of said plant according to any one of the claims 1 to 13, wherein the method comprises the step of establishing, in the genome of said plant or seed, the presence of a resistance gene encoding a protein as defined in any one of the claims 14 to 22.

24. Method according to claim 23, wherein the step of establishing, comprises establishing the presence of the resistance gene is achieved by using one or more markers selected from the group consisting of SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No.14, preferably by using all of said markers and / or by establishing the presence of one or moresequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, or SEQ ID No. 7.

25. Method for providing a lettuce plant that is resistant to downy mildew, wherein the method comprises the steps of, a) crossing a lettuce plant comprising a resistance gene according to any one of the claims 14 to 22 with a lettuce plant that is susceptible to downy mildew and does not comprise said resistance gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to downy mildew.

26. Method according to claim 25, wherein the selecting of step c is done by the method of claim 23 or 24.

27. Use of a gene construct or plasmid for introducing an MRC4 resistance gene into the genome of a plant or plant cell and providing broad spectrum resistance to downy mildew caused by one or more of B. lactuccie selected from the group of race Bl: 1-37EU, preferably Bl: 16-37EU, wherein the gene construct is comprised of the MRC4 resistance gene according to any one of the claims 14 to 22 operably linked to expression providing sequences in said plant.

28. Use of an MRC4 resistance gene of any one of claims 14 to 22 for conferring or improving resistance against Bremia lactuccie in a plant of L. sativa, wherein the MRC4 resistance gene is preferably obtainable from A. serriola, L. virosa or A. saligna.

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