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 a resistance gene that confers resistance to oomycetes in lettuce, more specifically B. lactucae. Furthermore, the present invention relates to a resistance gene and a method for selecting a lettuce plant that is resistant to downy mildew.

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 a resistance gene that confers resistance to oomycetes in lettuce, more specifically Bremia lactucae. Furthermore, the present invention relates to a resistance gene and a method for selecting a lettuce plant that is resistant to downy mildew comprising said resistance gene.

[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, Plasmopara and Bremia. Downy mildew is a problem in many food crops, for example in lettuce caused by Bremia lactucae (B. lactucae), affecting the production of this crop worldwide. Plants that are being affected include food crops such as cabbage, grape, spinach, lettuce, onion, and cucumber.

[0005] Lettuce, mostly known as 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, Butterhead, Batavia and Oakleaf lettuce types. There are many plant pathogens that affect L. sativa, and some of the diseases caused by these pathogens are downy mildew, sclerotinia rot, powdery mildew, fusarium wilt of which the most important disease is lettuce downy mildew, which is caused by the B. lactucae.

[0006] 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 41 races of B. lactucae that have been identified and characterized and that cause downy mildew disease in lettuce. These races are distinguished based on their ability to infect different lettuce cultivars that carry different resistance genes. Multiple lettuce 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. 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.

[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. However, 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 / or seed production. For example, introgression of (additional) new resistance genes in lettuce often result in a severe reduction in growth providing lettuce that are reduced in size, and / or have a reduced 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 or genetic 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 lettuce plants that are resistant to downy mildew and wherein plants have a broad-spectrum resistance against this pathogen, while maintaining agronomical value of the lettuce, especially in view of its growth, size and / or seed production levels, e.g. by reducing the linkage drag often associated with the generation of new Bremia resistant lettuce plants. 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 a V04 resistance gene encoding a protein having at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with amino acid sequence of SEQ ID No. 2 providing downy mildew resistance, wherein said lettuce plant is resistant to Bremia lactucae race Bl:40 EU, preferably also Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, and B1:34EU. The downy mildew resistance conferring gene V04 is a dominant resistance trait and may be homozygous or heterozygous present in a downy mildew resistant lettuce plant. The resistance gene against B. lactucae has been found on chromosome 2 in lettuce, present in the Major Resistance Cluster 2. This V04 resistance gene of the present invention gives resistance to B. lactucae races Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, Bl:34 and Bl:40 EU wherein said Bremia races have been characterized and classified according to the SEXTET code by IBEB (International Bremia Evaluation Board). Furthermore, previous disease resistance tests show that the V04 resistance gene further provides resistance to the “older”, less recent Bremia races Bl:l to Bl:15.

[0011] 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.

[0012] 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 V04 resistance gene is characterized as an NB-ARC-LLR gene.

[0013] To demonstrate that the V04 resistance gene provides Bremia resistance, this V04 resistance gene was 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 the V04 resistance gene. With VIGS it was demonstrated that the V04 resistance gene was associated with downy mildew resistance, since VIGS induced gene silencing was used to create Bremia susceptibility in resistant Lactuca accessions containing only V04 resistance. Resistant lettuce plants were transiently transformed with a silencing construct specific against the V04 resistance gene which resulted in the silencing of the resistance gene and as a consequence made the plant or plant organs susceptible to B. lactucae infection, thus by “removing” or silencing the V04 resistance gene via virus induced gene silencing.

[0014] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein said V04 resistance gene comprises a coding sequence having at least 95%, more preferably at least 98%, most preferably 100% sequence identity with SEQ ID No. 1.

[0015] 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: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, and B1:34EU. Recent experiments show that a lettuce plant of the present invention comprising the V04 resistant gene is resistant to Bremia races from Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, B1:34EU and Bl:40 EU. Previous disease resistance tests on less recent Bremia races Bl:l to 15EU (data not shown) show that the V04 resistance gene further provides resistance to these Bremia races.

[0016] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the V04 resistance gene comprises SEQ ID No.5 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance.

[0017] 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.

[0018] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the V04 resistance gene is at least heterozygous present in the lettuce plant, preferably homozygous present.

[0019] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein said plant comprises SEQ ID No.9 having a “A” on the indicated SNP position, as indicated in Table 2. Said plant has an improved phenotype as compared to a plant comprising the V04 resistance gene and being resistant to said Bremia race(s) but which does not have the “A” on the indicated SNP position in SEQ ID No. 9. Preferably the plant which comprises SEQ ID No.9 having a “A” on the indicated SNP position, as indicated in Table 2 has an agronomical elite phenotype not affected by growth reduction, and / or is of comparable size to said plant not comprising the V04 resistance gene and being susceptible to said Bremia race(s). In Bremia resistance lettuce comprising the V04 resistance gene according to the present invention, genetic drag was observed that was previously linked to the introgression fragment that comprises the V04 resistance gene. The genetic drag was especially observed under hydric stress conditions and resulted in dwarfism of the lettuce plant, i.e. significantly smaller lettuce crops. Surprisingly, it was found that by reducing the introgression fragment comprising the V04 an uncoupling was observed wherein a Bremia resistant plant was obtained and under hydric stress an agronomical elite plant was obtained, i.e. no dwarfism was observed. The uncoupling of the genetic drag associated with the V04 is correlated to the presence of marker #5 V04 (SEQ ID No.9, see also Table 2, wherein at the SNP position a “A” corresponds to a plant which is absent of the above described linkage drag. In case an “T” at the SNP position of SEQ ID No.9 is observed than the plant comprises the linkage drag resulting in the described dwarfism phenotype.

[0020] 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. The presence or carry-over of these additional unwanted genetics together with the desired gene during breeding is known as linkage drag It was observed that lettuce plants carrying V04 resistance would also have linkage drag which in the presence of hydric stress would block the Bremia resistant plants in their growth and remain too small for commercialisation. Hydric stress is a temporary lack of water at any stage during the first three weeks after planting, which may especially occur if the lettuce plants are planted in heavy soils, such as soils which are rich in clay, and wherein watering takes place in a discontinuous manner e.g. by irrigating once per 2 days. The earlier in the three weeks the hydric stress occurs the smaller the plants will be. However, surprisingly it was also observed that resistant plants with a reduced introgression fragment comprising the V04 resistance gene of present invention did not react to hydric stress in the same way and obtain a commercial size of a lettuce head. We were able to reduce the size of the introgression fragment comprising the V04 resistance gene providing Bremia resistance in lettuce, while at the same time maintaining agronomical properties of the lettuce, i.e. reduced or even no dwarfism was observed, even under conditions of hydric stress

[0021] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the V04 resistance gene is obtainable, derived, or originates from a lettuce plant of L. virosa. Most preferably the present invention relates to the lettuce plant, wherein the V04 resistance gene is obtainable, derived, or originates from a lettuce plant deposited under number NCIMB 42897.

[0022] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the lettuce plant further comprises one or more genes selected from the group consisting of

[0023] - SE17 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said plant comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:31, Bl:33, Bl:35, Bl:37 and Bl:38 EU, the SE17 resistance gene is obtainable from a lettuce plant deposited under number NCIMB 44201; or

[0024] - DM3 resistance gene wherein said plant comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:33, Bl:35, Bl:37 and Bl:38 EU, the DM3 resistance gene is obtainable from a lettuce plant deposited under number NCIMB 42897 ; or

[0025] - SA12 resistance gene wherein said plant comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30 and B1:31EU, the SA12 resistance gene is obtainable from a lettuce plant deposited under number NCIMB 44202, preferably wherein the lettuce plant further comprises said SEI 7 resistance gene. Disease resistance tests further show that the SE17, DM3 and SA12 resistance genes may further provide resistance to the “older”, less recent Bremia races Bl:l to Bl: 15. Preferably the V04 is combined with SEI 7 providing full spectrum Bremia resistance. The combinations of V04 with any of SE17, DM3, SA12 in coupling phase (e.g. the gene combinations are located on the same chromosome arm) and according to the invention have reduced or no linkage drag associated with hydric stress.

[0026] The presence of the V04 resistance gene will provide 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 and improve the spectrum 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 2. Additionally, or alternatively the V04 resistance gene may be stacked with other resistance genes on other chromosomes. As such, stacking of multiple resistance genes will enable broad and durable B. lactucae resistance in lettuce. Combining the resistances provided further resistance to Bremia resulting for example in a broader, preferably full spectrum resistance to Bremia races up to Bl:40 and Bl:41 for the lettuce plant comprising the V04 and SE17, or the V04 and DM3 resistance gene combinations. Combining the V04 and SA12 resistance genes in lettuce provides resistance to Bremia races up to Bl:40 and preferably also Bl:41.

[0027] The V04 and DM3 resistance genes are obtainable, derived, or originates from a lettuce plant deposited under number NCIMB 42897, deposited on 15thNovember 2017, NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland. The V04 and SE17 resistance genes are obtainable, derived, or originates from a lettuce plant deposited under number NCIMB 44201, deposited on 4thAugust 2023, NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland.

[0028] The V04 and SA12 resistance genes are obtainable, derived, or originates from a lettuce plant deposited under number NCIMB 44202, deposited on 4thAugust 2023, NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland.

[0029] The present invention, according to a second aspect, relates to seed, plant parts, fruits, and / or a plant cell of a lettuce plant of the present invention, comprising a V04 resistance gene encoding a protein as described above. The seed, plant parts, fruits, and / or a plant cell comprises the V04 resistance gene as described above.

[0030] According to a preferred embodiment, the present invention relates to a resistance gene V04 that confers resistance to B. lactucae in lettuce plants, wherein the resistance gene encodes for a protein that has at least 95%, more preferably at least 98%, most preferably 100% sequence identity with SEQ ID No. 2. The V04 resistance gene is a dominant trait.

[0031] According to another preferred embodiment, the present invention relates to a resistance gene that confers resistance to B. lactucae in lettuce plants, wherein the coding sequence of said resistance gene has at least 95%, more preferably at least 98%, most preferably 100% sequence identity with SEQ ID No. 1.

[0032] 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 race Bl:40 EU in lettuce, preferably at least Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, Bl:34 and Bl:40 EU. The V04 resistance gene comprises SEQ ID No.5 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance.

[0033] 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.

[0034] The present invention, according to a further aspect, relates to a combination of the V04 resistance gene in coupling phase with one or more genes SE17, DM3 or SA12 for providing a downy mildew resistant plant having an agronomical elite phenotype not affected by growth reduction under hydric stress, and / or wherein said plant is of comparable size to a lettuce plant not comprising the V04 resistance gene. The present invention, according to a further aspect, relates to a method for selecting (i) a downy mildew resistant lettuce plant or (ii) a seed of said plant, the method comprises the step of establishing, in the genome of a plant or seed, plant parts, fruits, and / or a plant cell, the presence of a V04 resistance gene encoding a protein as defined above. The step of establishing, in the genome of the seed, the presence of any genetic information, including the presence of the V04 resistance gene encoding the protein as defined above, may suitably involve allowing the seed to grow into a plant, plant parts, fruits, and / or a plant cell and establishing the presence of the genetic information in the genome of the plant or plant parts grown from the seed.

[0035] According to yet another preferred embodiment, the present invention relates to the method for selecting a downy mildew resistant lettuce plant of present invention, wherein the step of establishing, comprises establishing the presence of SEQ ID No. 1 in the genome of a plant or seed, or the presence in the genome of a plant or seed of SEQ ID No. 5 having a “T” on the indicated SNP position, as indicated in Table 1. The selection method will provide a Bremia resistant lettuce plant of present invention comprising the V04 resistance gene.

[0036] According to yet another preferred embodiment, the present invention relates to the method for selecting a downy mildew resistant lettuce plant of present invention, wherein the method further comprises a further step of selecting for a plant that comprise SEQ ID No.9 having a “A” on the indicated SNP position, as indicated in Table 2, and wherein said plant has an agronomical elite phenotype not affected by growth reduction, and preferably wherein said plant is of comparable size to said plant not comprising the V04 resistance gene. This further selection step will provide a Bremia resistant lettuce plant of present invention comprising the V04 resistance gene that is free of genetic drag due to a reduced introgression fragment that uncouples the genetic drag from the resistance gene. This is to be selected using the marker of SEQ ID No.9 based on the specified SNP position, as indicated in Table 2.

[0037] According to yet another preferred embodiment, the present invention relates to the method for selecting a downy mildew resistant lettuce plant of present invention, wherein the method further comprises the step of establishing, in the genome of a plant or seed the presence of one or more genes selected from the group consisting of

[0038] - SEI 7 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said plant comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, or

[0039] - DM3 resistance gene wherein said plant comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, or

[0040] - SA12 resistance gene wherein said plant comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, preferably establishing the presence of said SEI 7 resistance gene in the genome of a plant or seed.

[0041] The V04 resistance gene can be stacked with other resistance genes on chromosome two in lettuce. Using the above identified markers and the SNP positions identified also in Table 1, plants can be selected having stacked resistance genes. According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the V04 resistance gene comprises SEQ ID No.5 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance.

[0042] 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 V04 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.

[0043] 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.

[0044] The present invention, according to a further aspect, relates to the use of a gene construct or plasmid for introducing a V04 resistance gene into the genome of a plant or plant cell and providing resistance to downy mildew caused by one or more of B. lactucae selected from the group of race Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, Bl:34 and Bl:40 EU, wherein the gene construct is comprised of at least the 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 V04 resistance gene, after being transferred into the lettuce plant will provide resistance to B. lactucae.

[0045] According to yet another preferred embodiment, the present invention relates to the use, wherein the gene construct or plasmid further comprises one or more genes selected from the group consisting of - SE17 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said gene construct or plasmid comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:31, Bl:33, Bl:35, Bl:37 and Bl:38 EU ; or

[0046] - DM3 resistance gene wherein said gene construct or plasmid comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:33, Bl:35, Bl:37 and Bl:38 EU; or

[0047] - SA12 resistance gene wherein said gene construct or plasmid comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30 and B1:31EU, preferably wherein the gene construct or plasmid further comprises said SEI 7 resistance gene. Combining the resistances provided further resistance to Bremia resulting for example in a broader, preferably full spectrum resistance to Bremia races up to Bl:40 and Bl:41 for the lettuce plant comprising the V04 and SE17, or the V04 and DM3 resistance gene combinations. Combining the V04 and SA12 resistance genes in lettuce provides resistance to Bremia races up to Bl:40 and preferably also Bl:41.

[0048] The present invention will be further detailed in the following examples and figures wherein:

[0049] Figure 1: shows the % of susceptible leaves of lettuce that have been infected with Bremia lactucae B1:22EU, after VIGS silencing of the V04 resistance gene of present invention of a lettuce plant of present invention comprising the V04 resistance gene using 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. However, the leaves from plants wherein the V04 resistance gene has been silenced by VIGS silencing (Fl silencing construct), showed a significant percentage of susceptible leaves (about 30%) when infected with Bremia as compared to plants where the V04 gene was not silenced (i.e. by F2 or PDS silencing construct on the plant comprising the V04 gene). Figure 2: shows an overview of the disease test performed with the most recent isolates of B. lactucae Bl: 16 to Bl:40EU on L. sativa lines Cobham Green (=control plant that is known to be susceptible), and the plant of present invention comprising the V04 resistance gene and combinations of V04 with other resistance genes V04 + SE17, DM3 or SA12. The Bl:20, 27, 36 and 39 races were not tested (indicated as “ND”). The plant of present invention shows to be resistant to most of tested downy mildew isolates, and also showed a reduced effect of the genetic drag associated with V04 (see also figure 3 below) .

[0050] Figure 3: shows the genetic drag associated with the V04 resistance gene in lettuce. The left lettuce is an example of when the introgression fragment comprising the V04 resistance gene is reduced, the genetic drag is not present, as shown by normal crop size (i.e. an increased crop size in view of the V04 lettuce suffering from genetic drag). The left side lettuce comprises an recombinant introgression fragment comprising the V04 and DM3 resistance genes that is reduced in size in comparison to the right side lettuce. The right lettuce crop comprises the larger introgression fragment comprising the V04 resistance gene, suffering from genetic drag resulting in a dwarfism phenotype. Both lettuce are resistant to Bremia, as tested in figure 2. The crop size of the lettuce comprising the V04 reduced introgression (lettuce on the left side) is comparable to the size of lettuce which does not comprise the V04 resistance gene (and is susceptible to Bremia).

[0051] Examples

[0052] Gene Mapping of V04 resistance gene in lettuce

[0053] Gene mapping experiments were done to identify a resistance gene that is involved in Bremia (B. lactucae) resistance in lettuce (L. sativa). The resistance gene was originally isolated from L. virosa and was mapped on chromosome 2, more specifically major resistance cluster 2 (MRC2), providing Bremia resistance in lettuce. This MRC2 area comprises other important resistance genes, and may include DM3, SE17 and SA12 and the herein annotated V04 gene.

[0054] The identified resistance locus comprises a single SNP marker for selecting plants comprising the V04 resistance gene; the marker #1 V04 (SEQ ID No.5), providing a resistance locus which comprises a novel resistance gene identified as V04. After fine mapping in a population of about 1 ,500 plants there was one putative resistance gene present in the identified resistance locus. The SNP is indicated in bold and underlined as N on which the resistant plants could be selected. In Bremia susceptible lettuce plants the indicated SNP nucleotide was an “C” in respect to a susceptible plant and a “T” for a Bremia resistant plant in respect to SEQ ID No. 5, see also table 1 below.

[0055] Furthermore, via additional marker assisted breeding using markers #2 SE17, marker #3 DM3 or marker #4 SA12 of Table 1, further plants were obtained combining or stacking the V04 resistance in coupling phase with other resistance genes known to be located on chromosome 2, major resistance cluster 2, including SE17, DM3, and SA12, and thereby reducing the genetic drag linked to the V04 resistance gene. This resulted in a Bremia resistant lettuce with improved hydric stress resistance, i.e. no reduction in crop size was observed anymore due to hydric stress, in contrast to previous Bremia resistant lettuce crops.

[0056] Table 1. Marker sequences for selecting Bremia resistance in lettuce.

[0057] Furthermore marker #5 V04 (SEQ ID No.9) was developed for the selection of Bremia resistant lettuce plant comprising the V04 resistance gene and not suffering from genetic drag. The SNP is indicated in bold and underlined as N on which the resistant plants free of genetic drag could be selected; in respect to a plant free of genetic drag the indicated SNP nucleotide was an “A” and an “T” for a Bremia resistant plant suffering genetic drag, in respect to SEQ ID No. 9, see also table 2 below. Furthermore, marker analysis using the markers in Tables 1 and 2, confirmed that lettuce plants comprised of the V04 resistance gene in combination with any one of SE17, DM3 or SA12, provided Bremia resistance and did not show the genetic drag i.e. the reduction in crop size (dwarfism phenotype) anymore.

[0058] Table 2. Marker sequences for selecting agronomical elite Bremia resistant lettuce free of genetic drag.

[0059] Assessment of linkage drag associated with V04 resistance gene

[0060] Hydric stress in lettuce plants, also known as water stress, refers to the condition where the plant experiences an imbalance between the water it absorbs and the water it loses. This stress can occur due to both a lack of water (drought stress) and an excess of water (waterlogging stress). Hydric stress referred to herein is a temporary lack of water during a period of strong growth of the plant in the first 2 to 3 weeks after planting. Hydric stress mainly occurs in heavy soils where watering is done once every 2 days compared to daily watering in light soils or drop irrigation, where water supply is regular. The genetic drag observed in view of the Bremia resistance linked to V04, seem to result and express as a blockage of the growth of the lettuce crop caused by hydric stress in the first 3 weeks after planting. The earlier the hydric stress takes places after planting the smaller the plants will be.

[0061] We have been able to clone the V04 resistance gene, and to bring the V04 resistance gene in coupling phase with each of DM3, SE17 and SA12 resistance genes. As a result, the linkage drag observed during hydric stress conditions has been removed. In general, lettuce crops could be observed by eye and when suffering from dwarfism these crops are easily differentiated from lettuce crops not suffering from the linkage drag, as shown in Figure 3. The left lettuce comprises the recombinant; V04 resistance gene in combination with the Dm3 resistance gene, resulting in a introgression fragment having a reduced size, in comparison to the right lettuce crop, comprising the V04 resistance gene in the original larger introgression fragment resulting in the linkage drag associated dwarfism phenotype, as confirmed by marker analysis. V04 resistance gene silencing experiment using Virus Induced Gene Silencing (VIGS)

[0062] To demonstrate that the V04 resistance gene provides Bremia resistance, the V04 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 V04 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 V04 resistance gene was associated with downy mildew resistance, since VIGS induced gene silencing was used to create Bremia susceptibility in resistant Lactuca accessions comprising the V04 gene. Resistant lettuce plants were transiently transformed with a silencing construct specific against the resistance V04 gene which will result in the silencing of the resistance gene.

[0063] Briefly, lettuce plants containing the V04 resistance gene were silenced for V04 resistance gene by VIGS using different silencing construct to identify if this V04 resistance gene was indeed responsible for the observed resistance. Two VIGS -constructs were used, one (Fl) that results in specific silencing of the V04 resistance gene and a control construct (F2) that targets a region on chromosome 2 in close proximity of the V04 resistance gene. Furthermore, independent of resistance gene silencing the PDS gene was silenced as well 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 Bremia to test the effect of the gene silencing on disease resistance.

[0064] The VIGS constructs were cloned in the K20 vector (See Table 2 for sequences Fl and F2, respectively SEQ ID No. 10, SEQ ID No. 11). The constructs were transformed and transiently expressed into a lettuce plant of present invention that is resistant to Bremia, using cocultivation with agrobacterium (GV3101) to study the resistance gene function in relation to Bremia resistance. The % of susceptible Bremia 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 V04 resistance gene was silenced, plants became susceptible to Bremia. Results (Figure 1) indicate that when V04 was silenced by VIGS with the Fl construct the plants became susceptible (30% of the leaves showed infection) after Bremia infection (B1:22EU) confirming that the resistance gene is linked to a resistance gene that provides the plant resistance against Bremia. The PDS and F2 controls plants remained resistant to the Bremia infection, all leaves were unaffected. Also, B1:27EU was tested and similar results were obtained.

[0065] Table 2. VIGS constructs

[0066] Disease test and biotest for downy mildew in Lettuce

[0067] Leaves of resistant plants transiently transformed with the above described VIGS constructs, were put in trays with moistened paperboard and infected with Bremia (race Bl:22 EU). 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 Bremia, i.e. being susceptible or resistant.

[0068] Disease resistance tests show that the V04 resistance gene provides resistance to Bremia races from Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, Bl:34, and Bl:40 EU (See Figure 2). Furthermore, disease resistance test show that the V04 resistance gene further provides resistance to US Bremia races Bl:l to Bl: 15 EU (results not shown). Furthermore, lettuce plants comprising the V04 resistance gene in combination with further resistance genes SE17, DM3 and SA12 were also screened in the disease test. Combining the resistances provided further resistance to Bremia resulting for example in a broader, preferably full spectrum resistance to Bremia races up to Bl:40 and Bl:41 for the lettuce plant comprising the V04 and SE17, or the V04 and DM3 resistance gene combinations. Combining the V04 and SA12 resistance genes in lettuce provides resistance to Bremia races up to Bl:40 and preferably also Bl:41.

Claims

Claims1. A downy mildew resistant lettuce plant, wherein said lettuce plant comprises a V04 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 2 providing downy mildew resistance, wherein said lettuce plant is resistant to at least Bremia lactucae race Bl:40 EU.

2. Lettuce plant according to claim 1, wherein said V04 resistance gene comprises a coding sequence having at least 95% sequence identity with SEQ ID No. 1.

3. Lettuce plant according to any one of the claims 1 or 2, wherein the lettuce plant is further resistant to one or more of Bremia lactucae races selected from the group consisting of races Bl: 16-19, BI:21-26, BI:28, BI:29, BI:32, and B1:34EU.

4. Lettuce plant according to any one of the claims 1 to 3, wherein the lettuce plant is a Lactuca sativa.

5. Lettuce plant according to any one of the claims 1 to 4, wherein said plant comprises SEQ ID No.9 having a “A” on the indicated SNP position, as indicated in Table 2.

6. Lettuce plant according to any one of the claims 1 to 5, wherein the V04 resistance gene is obtainable, derived, or originates from a lettuce plant deposited under number NCIMB 42897.

7. Lettuce plant according to any one of the claims 1 to 6, wherein the lettuce plant further comprises one or more genes selected from the group consisting of- SEI 7 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said plant comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:31, Bl:33, Bl:35, Bl:37 and Bl:38 EU; or- DM3 resistance gene wherein said plant comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, BI:33, BI:35, BI:37 and BI:38 EU; or- SA12 resistance gene wherein said plant comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30 and B1:31EU, preferably wherein the lettuce plant further comprises said SEI 7 resistance gene.

8. Plant according to any one of the preceding claims, wherein the V04 resistance gene is in coupling phase with the one or more genes of claim 7, and / or preferably wherein said plant has an agronomical elite phenotype not affected by growth reduction under hydric stress, and / or wherein said plant is of comparable size to a lettuce plant not comprising the V04 resistance gene.

9. Seed of a Bremia resistant lettuce plant comprising a V04 resistance gene encoding a protein as defined in any one of the claims 1 to 8.

10. A resistance gene V04 that confers resistance to downy mildew in lettuce plants, wherein the V04 resistance gene encodes for a protein that has at least 95% sequence identity with SEQ ID No. 2.

11. Resistance gene according to claim 10, wherein the coding sequence of said V04 resistance gene has at least 95% sequence identity with SEQ ID No. 1.

12. Resistance gene according to claim 9 or 10, wherein the resistance gene provides resistance to at least Bremia lactucae race Bl:40 EU in lettuce, preferably at least Bl: 16- 19, BE21-26, BI:28, BI:29, BI:32, BI:34 and BI:40 EU.

13. Resistance gene according to any one of the claims 10 to 12, wherein the V04 resistance gene comprises SEQ ID No.5 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance.

14. A combination of a V04 resistance gene of any one of claim 10 to 13 in coupling phase with one or more genes of claim 7 for providing a downy mildew resistant plant having an agronomical elite phenotype not affected by growth reduction under hydric stress, and / or wherein said plant is of comparable size to a lettuce plant not comprising the V04 resistance gene.

15. Method for selecting (i) a downy mildew resistant lettuce plant according to any one of the claims 1 to 8 or (ii) a seed of said plant, the method comprises the step of establishing, in the genome of a plant or seed the presence of a V04 resistance gene encoding a protein as defined in any one of the claims 1 to 8.

16. Method according to claim 15, wherein the step of establishing, comprises establishing the presence of SEQ ID No. 1 in the genome of a plant or seed, and / or the presence in the genome of a plant or seed of SEQ ID No. 5 having a “T” on the indicated SNP position, as indicated in Table 1.

17. Method according to claim 15 or 16, wherein the method further comprises a further step of selecting for a plant that comprise SEQ ID No.9 having a “A” on the indicated SNP position, as indicated in Table 2, and preferably wherein said plant has an agronomical elite phenotype not affected by growth reduction, wherein said plant is of comparable size to said plant not comprising the V04 resistance gene.

18. Method according to any one of the claims 15 to 17, wherein the method further comprises the step of establishing, in the genome of a plant or seed the presence of one or more genes selected from the group consisting of- SEI 7 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said plant comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, or- DM3 resistance gene wherein said plant comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, or- SA12 resistance gene wherein said plant comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, preferably establishing the presence of said SEI 7 resistance gene in the genome of a plant or seed.

19. Use of a gene construct or plasmid for introducing a V04 resistance gene into the genome of a plant or plant cell and providing resistance to downy mildew caused by one or more of B. lactucae selected from the group of race Bl: 16-19, Bl:21-26, Bl:28, Bl:29, Bl:32, Bl:34 and Bl:40 EU, wherein the gene construct is comprised of at least the resistance gene according to any one of the claims 10 to 14 operably linked to expression providing sequences in said plant.

20. Use according to claim 19, wherein the gene construct or plasmid further comprises one or more genes selected from the group consisting of- SE17 resistance gene encoding a protein having at least 95% sequence identity with amino acid sequence of SEQ ID No. 4, and / or wherein said gene construct or plasmid comprises SEQ ID No.6 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of B. lactucae races Bl:30, Bl:31, Bl:33, Bl:35, Bl:37 and Bl:38 EU ; or- DM3 resistance gene wherein said gene construct or plasmid comprises SEQ ID No.7 having a “T” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30, Bl:33, Bl:35, Bl:37 and Bl:38 EU; or- SA12 resistance gene wherein said gene construct or plasmid comprises SEQ ID No.8 having a “A” on the indicated SNP position, as indicated in Table 1, providing downy mildew resistance, wherein said lettuce plant is furthermore resistant to at least one or more of Bremia lactucae races Bl:30 and B1:31EU, preferably wherein the gene construct or plasmid further comprises said SEI 7 resistance gene.

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