Lettuce plant resistant to downy mildew and resistance genes

Abstract

The present invention relates to a lettuce plant that is resistant to downy mildew, more specifically to a lettuce plant that comprises two or more downy mildew resistance genes that confer resistance to oomycetes in lettuce, more specifically Bremia lactucae (B. lactucae). Furthermore, the present invention relates two or more downy mildew resistance genes and a method for selecting a lettuce plant that is resistant to downy mildew comprising said two or more downy mildew resistance genes.

Description

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

[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 two or more downy mildew resistance genes that confer resistance to oomycetes in lettuce, more specifically Bremia lactucae (B. lactucae). Furthermore, the present invention relates two or more downy mildew resistance genes and a method for selecting a lettuce plant that is resistant to downy mildew comprising said two or more downy mildew resistance genes.

[0004] Lettuce, mostly known as Lactuca sativa (L. sativa), but also including Lactuca species such as Lactuca serriola (L. serriola), Lactuca saligna (L. saligna) or Lactuca virosa (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.

[0005] 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 downy mildew, but eventually downy mildew becomes immune to these chemicals, because over time the pathogen also acquires resistance to fungicides.

[0006] 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 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. Downy mildew can originate from various species, but mainly of Peronospora, Plasmopara and Bremia.

[0007] Some lettuce 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 in Europe. These races are distinguished based on their ability to infect different lettuce cultivars that carry different resistance genes. Furthermore, many of the introgression fragments used to obtain many of these resistant cultivars carry additional 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 lettuce may result in a severe reduction in seed production levels.

[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, and preferably while maintaining agronomical value of the plant, especially in view of its growth, size and / or seed production levels. 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 the 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 of Lactuca sativa, wherein the lettuce plant comprises two or more downy mildew resistance genes that have at least two different mechanisms of action for providing said downy mildew resistance, and / or the two or more downy mildew resistance genes originate from at least two different lettuce plants (or plant species) selected from the group consisting of Lactuca serriola, Lactuca virosa and Lactuca saligna, wherein the two or more downy mildew resistance genes are selected from the group consisting of FER, V10, V04, SA07, Dm3, SE17, SA12, SE7, V06, MACPF, SA23, RCB1, RCB2, SA17, SE20, SE28, SE29 and SE30, wherein each of said two or more downy mildew resistance genes encodes for a downy mildew resistance protein having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with an amino acid sequence, respectively; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or in case of DM3, SA12 or SA07 comprising a marker sequence respectively; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35. Combining multiple resistance genes from different sources also allows for a lettuce plant with broader spectrum, more durable B. lactucae resistance compared to a lettuce plant with only a single resistance gene, or multiple resistance genes from the same source. Furthermore, preferably two or more resistance gene in lettuce are combined wherein the two or more resistance genes have different mechanisms or modes of action and are derived from at least two different wild-type sources, such as Lactuca serriola, Lactuca virosa or Lactuca saligna. In the present invention the resistance genes are originally isolated or originate from Lactuca species consisting of Lactuca serriola, Lactuca virosa or Lactuca saligna. For example, a lettuce plant (Lactuca sativa) is provided with the downy mildew resistance gene V04, originally derived from Lactuca virosa lettuce source, and which is combined with resistance gene SE17, originally derived from Lactuca serriola lettuce source, thereby providing a broad spectrum and durable B. lactucae resistance in the lettuce plant (Lactuca sativa). By doing so the chance of the disease breaking resistance is decreased and the resistance is improved, so that the lettuce plant has broader spectrum and more durable B. lactucae resistance compared to a lettuce plant with only a single resistance gene using a single mode of action. Different mechanisms or modes of action may be combined with the genes included herein providing durable B. lactucae resistance and may comprise R genes (LRR, TIR, CC, Zinc Finger, NB(-Arc), or combinations thereof), FER genes ((pseudo)kinase) and MACPF (pore former) genes. R genes form the majority of disease resistance genes in plants and encode nucleotide- binding site leucine-rich repeat proteins, also known as NBS-LRR proteins. 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 two major subfamilies of plant NBS-LRR proteins defined by the Toll / interleukin-1 receptor (TIR) or the coiled-coil (CC) motifs in the amino-terminal domain and are both involved in pathogen recognition. Bremia resistance genes in lettuce comprise at least one of the LRR, TIR, CC, NB, or combinations thereof.

[0011] The FER, V10, V04, SE17, SL7, V06, MACPF, SA23, RCB1, RCB2, SA17, SE20, SE28, SE29 and SE30 downy mildew resistance genes comprise a coding sequence having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with a coding sequence, respectively; FER with SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5, V10 with SEQ ID No. 7, V04 with SEQ ID No. 9, SE17 with SEQ ID No. 11, SL7 with SEQ ID No. 13, V06 with SEQ ID No. 15, MACPF with SEQ ID No. 17, SA23 with SEQ ID No. 33, RCB1 with SEQ ID No. 19, RCB2 with SEQ ID No. 21, SE20 with SEQ ID No. 23, SE28 with SEQ ID No. 25, SE29 with SEQ ID No. 27 and SE30 with SEQ ID No. 29. FER originates from L. serriola (SEQ ID No.2) or L. saligna (SEQ ID No.4 or 6) and is located on Chromosome 1. V10 originates from L. virosa and is located on Chromosome 1. V04 originates from L. virosa and is located on Chromosome 2. SA07 originates from L. serriola and is located on Chromosome 2. SE17 originates from L. serriola and is located on Chromosome 2. SA12 originates from L. serriola and is located on Chromosome 2. SL7 originates from L. saligna and is located on Chromosome 3. V06 originates from L. virosa and is located on Chromosome 8. MACPF originates from L. serriola and is located on Chromosome 9. SA23 originates from L. serriola and is located on Chromosome 9. RCB1 and RBC2 originate from L. serriola and are located on Chromosome 9. SE20, SE28, SE29 and SE30 originate from L. serriola and are located on Chromosome 4.

[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 95% of the sequence length of the gene or protein sequence as claimed herein, preferably at least 96% sequence length, more preferably at least 97% sequence length, even more preferably at least 98% sequence length, even more preferably at least 99% sequence length, most preferably at least 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] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are located on at least two different chromosomes. Combining resistance genes located on different chromosomes decreases the chance of downy mildew breaking resistance and so allows for a lettuce plant with broader spectrum, more durable B. lactucae resistance compared to a lettuce plant with only a single resistance gene, or resistance genes all on one chromosome.

[0014] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are three, preferably four, more preferably five, most preferably six downy mildew resistance genes. Increasing the number of resistance genes decreases the chance of downy mildew breaking resistance and so allows for a lettuce plant with broader spectrum, more durable B. lactucae resistance compared to a lettuce plant with a lower number of resistance genes.

[0015] According to another preferred embodiment, the present invention relates to the lettuce plant, wherein the lettuce plant is resistant to at least one or more, preferably at least 3 or more, more preferably at least 5 or more, even more preferably at least 10 or more, most preferably all Bremia lactucae races selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU. Experiments have shown that a lettuce plant with two or more downy mildew resistance genes with different mechanisms of action or that are derived from at least two different wild-type lettuce sources is resistant to many races of B. lactucae, with certain combinations providing resistance to all B. lactucae races Bl:29 - Bl:41.

[0016] According to a preferred embodiment, the present invention relates to the lettuce plant wherein the two or more downy mildew resistance genes that have at least two different mechanisms of action for providing the downy mildew resistance are selected from the group consisting of an R gene encoding LRR, TIR, CC, NBS, Zinc Finger, and NB (-Arc) domains or combinations thereof, a FER gene encoding for (pseudo)kinase, and an MACPF gene encoding for a pore former. A lettuce plant according to a preferred embodiment comprises two or more resistance genes that have different mechanisms or modes of action for providing downy mildew resistance to said lettuce plant and preferably comprises (1) a combination of at least two R-genes of different types, wherein the types are selected from the group consisting of the LRR, TIR, CC or NB type, (2) a combination of a FER gene and an R-gene which is preferably of the LRR, TIR, CC or NB type, (3) a combination of an MACPF gene and an R-gene which is preferably of the LRR, TIR, CC or NB type, or (4) a combination of a FER gene, a MACPF gene and an R-gene which is preferably of the LRR, TIR, CC or NB type. Experiments have shown that a lettuce plant with the downy mildew resistance genes FER on chromosome 1 (a (pseudo)kinase), MACPF (pore former) on chromosome 9 and one selected from V04, Dm3 and SE17 on chromosome 2 (R gene), has a wide spectrum B. lactucae resistance as well as improved agronomical value compared to a lettuce plant with only one of those downy mildew resistance genes.

[0017] According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are:

[0018] - FER having 99% sequence identity with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, preferably SEQ ID No. 6,

[0019] - MACPF having 99% sequence identity with SEQ ID No. 18, and optionally

[0020] - one selected from V04 having 99% sequence identity with SEQ ID No. 10, SE17 having 99% sequence identity with SEQ ID No. 12, and DM3 comprising SEQ ID No. 31, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU.

[0021] The FER gene, identified in L. serriola and L. saligna, is a resistance gene in lettuce and provides resistance against Bremia disease, B. lactucae in yet another manner distinct from R genes of the MACPF gene. FER is a receptor like kinases (RLK) and member of the Catharanthus roseus receptor-like kinase 1-like (CrRLKIL) protein kinase subfamily. FER consists of four putative kinase domains, and FER acts like a sensor that detects if the plant is under attack by a pathogen, FER will subsequently activate, by phosphorylation, the plants downstream immune response in the lettuce, presumably via one or more pathways including the H+-exchange pathway, immune receptor complex pathway, and / or JA-pathway of the plants immune system, or a combination of these. The MACPF gene is involved in a completely different mechanism than the known NBS-LRR mechanism (R genes) in the plant. The MACPF superfamily is named after a domain that is common to the membrane attack complex (MAC) proteins of complement and Perforin. Many members are important pore-forming toxins in eukaryotes. The archetypal members of the family are complement C9 and Perforin, both of which function in human immunity. C9 functions by punching holes in the membranes of Gram-negative bacteria. Perforin is released by cytotoxic T cells and lyses virally infected and transformed cells. In addition, Perforin permits delivery of cytotoxic proteases called granzymes that cause cell death.

[0022] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are:

[0023] - MACPF having 99% sequence identity with SEQ ID No. 18,

[0024] - RCB1 having 99% sequence identity with SEQ ID No. 20,

[0025] - RCB2 having 99% sequence identity with SEQ ID No. 22, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU. The RCB1 gene is not a classical NBS-LRR gene, it is an RLK-LRR protein. The LRR motif of RCB1 contains leucine’s in a beta-sheet and the domain forms a horseshoe structure in total. Furthermore, the RCB1 gene contains a (pseudo)kinase domain, in which the RCB1 gene differs from other cases of R genes where multiple LRR regions and the NBS domain are present together with TIR or CC domain. It is thought that those domains determine effector recognition and therefore disease susceptibility / resistance. Experiments have shown that a lettuce plant with the downy mildew resistance genes MACPF, having the MACPF mechanism of action, and RCB 1 and RCB2, having an R gene mechanism of action has a broad spectrum and durable B. lactucae resistance.

[0026] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are:

[0027] - V04 having 99% sequence identity with SEQ ID No. 10,

[0028] - SA12 comprising SEQ ID No. 32, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29, BL:30, Bl:31, Bl:32, Bl:34, Bl:36, Bl:39, Bl:40 and Bl:41. The downy mildew resistance genes SA12 and V04 can be obtained, originate or derived from a plant or plant seeds as represented by deposit NCIMB44202. The deposits were made on 4thof August 2023 at NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland. Experiments have shown that a lettuce plant with the downy mildew resistance genes V04, originally derived from Lactuca virosa lettuce source, and SA12, originally derived from Lactuca serriola lettuce source, has a broad spectrum and durable B. lactucae resistance.

[0029] According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the two or more downy mildew resistance genes are: - V04 having 99% sequence identity with SEQ ID No. 10,

[0030] - SE17 having 99% sequence identity with SEQ ID No. 12, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU.

[0031] Experiments have shown that a lettuce plant with the downy mildew resistance genes V04, originally derived from Lactuca virosa Lettuce source, and SE17, originally derived from Lactuca serriola lettuce source, has a broad spectrum and durable B. lactucae resistance.

[0032] The present invention, according to a second aspect, relates to a seed, plant tissue, plant cell or plant part of the lettuce plant as disclosed herein comprising the two or more downy mildew resistance genes encoding downy mildew resistance proteins.

[0033] The present invention, according to a further aspect, relates to a combination of two or more downy mildew resistance genes for conferring resistance to downy mildew in a lettuce plant, wherein each of said two or more downy mildew resistance genes encodes for a downy mildew resistance protein having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with an amino acid sequence, respectively; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or in case of DM3, SA12 or SA07 comprising a marker sequence respectively; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35, preferably wherein the two or more downy mildew resistance genes encodes for at least FER and MACPF. Experiments have shown that a lettuce plant with the downy mildew resistance genes FER on chromosome 1, MACPF on chromosome 9 is resistant to B. lactucae races Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:40 and Bl:41 EU and has improved agronomical value compared to a lettuce plant with only one of those downy mildew resistance genes.

[0034] According to another preferred embodiment, the present invention relates to the combination of two or more downy mildew resistance genes providing resistance to at least one or more, preferably at least 3 or more, more preferably at least 5 or more, even more preferably at least 10 or more, most preferably all Bremia lactucae races selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU. Experiments have shown that a lettuce plant with two or more downy mildew resistance genes located on at least two different chromosomes has wide spectrum resistance to B. lactucae, and is resistant to many races of B. lactucae, with certain combinations providing resistance to all B. lactucae races Bl: 29 - Bl: 41. The present invention, according to a further aspect, relates to a method for identifying and / or selecting (i) a downy mildew resistant lettuce plant or (ii) a seed of the lettuce plant, the method comprises the step of establishing, in the genome of the lettuce plant or seed the presence of two or more downy mildew resistance genes encoding a downy mildew resistance protein as disclosed herein. The step of establishing, in the genome of the seed, the presence of any genetic information, including the presence of the downy mildew resistance genes encoding downy mildew resistance proteins - such as MACPF and FER - 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, wherein the step of establishing, comprises establishing the presence of the two or more downy mildew resistance genes, wherein each of the two or more downy mildew resistance genes encodes for a corresponding downy mildew resistance protein having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with an amino acid sequence as follows; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or wherein each of the two or more downy mildew resistance genes comprises a marker sequence as follows; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35, preferably wherein the two or more downy mildew resistance genes encodes for the corresponding downy mildew resistance proteins FER and MACPF. This method will provide the B. lactucae resistant lettuce plant with two or more downy mildew resistance genes of the present invention.

[0036] The present invention, according to a further aspect, relates to a 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 the combination of two or more downy mildew resistance genes as disclosed herein with a lettuce plant that is susceptible to downy mildew and does not comprise the combination of the two or more downy mildew resistance genes, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to downy mildew, preferably resistant to one or more B. lactucae races selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 EU, and Bl:41 EU, preferably resistant to all of said races. Preferably the selection of the Bremia resistant plants is performed as indicated above using the gene and / or protein sequences and / or marker sequences.

[0037] The present invention, according to a further aspect, relates to the use of a gene construct or plasmid for introducing two or more downy mildew resistance genes into the genome of a lettuce plant or lettuce plant cell and providing broad spectrum resistance to downy mildew caused by one or more of B. lactucae selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39 and Bl:40 EU, preferably resistant to all of said races, wherein the gene constructs are comprised of a combination of two or more downy mildew resistance genes operably linked to expression providing sequences in the lettuce 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, or two or more plasmids or vectors or linear gene constructs, that comprise the two or more downy resistance genes of present invention. The two or more downy mildew resistance genes will provide resistance to B. lactucae after being transferred into the lettuce plant.

[0038] The present invention will be further detailed in the following examples and figure wherein:

[0039] Figure 1: Shows an overview of the Bremia resistance genes that may be combined in the lettuce plant according to the present invention and their Bremia (B. lactucae ) resistance spectrum. Lettuce plants (Lactuca sativa) comprising the indicated resistance gene(s) were tested in a disease resistance test against the different bremia races, wherein (-) is resistant, (+) is susceptible. L. sativa lines Cobham Green was used as positive control and does not include any of the indicated resistance genes.

[0040] Figure 2: shows a leaf of a lettuce plant with the V04 resistance gene, a leaf of lettuce plant with the SA12 resistance gene and a leaf of a lettuce plant with the combination of the two resistance genes V04 + SA12 after 10 days of incubation with Bl:41. Signs of infection with Bremia are visibly on the lettuce plants comprising the single V04 or SA12 resistance gene, as observed as discoloured areas on upper leaf surfaces in combination with white, grey mould located on the lower side of the leaf. However, when combining the two resistance genes V04 + SA12 in the lettuce plant, no such disease symptoms were observed, indicating a synergistic effect of both gene against Bremia. Examples

[0041] Example 1 - Generation of Bremia resistant lettuce plants

[0042] Three different lettuce plants (L. sativa) were generated via marker assisted breeding comprising the V04 resistance gene, or the SA12 gene, or a plant comprising both resistance genes. The resistance genes were originally isolated from Lactuca species consisting of Lactuca seriola, Lactuca virosa or Lactuca saligna. In this case, the downy mildew resistance genes V04 was originally derived from Lactuca virosa, and the SA12, was originally derived from Lactuca serriola. The resistance genes V04 and SA12 were identified and screened using marker V04 and marker SA12 of Table 1 and subsequently used to combine the genes into a lettuce plant. The plants were subsequently tested in disease assays for Bremia resistance. The lettuce plant with the combination of V04 + SA12 had improved Bremia lactucae resistance properties compared to plants only containing one resistance gene of either V04 or SA12 but not the other (see example 2).

[0043] Table 1. Marker sequences for combining genes for Bremia lactucae resistance in lettuce (L. sativa).

[0044] Example 2 - Disease test and biotest for downy mildew in Lettuce (L. Sativa)

[0045] Leaves of the resistant plants obtained through marker assisted breeding were put in trays with moistened paperboard and infected with Bremia lactucae (races Bl;29 to Bl:41 EU). Infected seedlings were suspended in 20 ml water, were filtered by cheesecloth and the flow-through was collected in a spray flask. The trays were spray-inoculated with the Bremia lactucae suspension. The trays were covered with a glass plate and stored in a climate chamber at 15°C (12 hours of light). A black, opaque foil was placed over the trays for one day to improve growth of Bremia lactucae. After one day, the foil was removed. Experiments were performed in triple, and eight to ten days after infection leaves were phenotypically scored by eye on the presence of Bremia lactucae, i.e. being susceptible or resistant. Results of the disease tests are summarized in Figure 1 and provide an overview of the Bremia resistance genes that may be combined in the lettuce plant according to the present invention and their Bremia (B. lactucae) resistance spectrum, wherein (-) is resistant, (+) is susceptible. The L. sativa lines Cobham Green R273 was used as positive control and does not include any of the indicated resistance genes. Surprisingly, result show that when combining different resistance genes from at least two different lettuce plants from the group consisting of Lactuca seriola, Lactuca virosa and Lactuca saligna, improved or even synergistically improved disease resistance may be obtained. For example, the single V04 or SA12 resistance gene do not provide resistance to Bl:41, whereas when combining the two resistance genes V04 + SA12 in the lettuce plant, the plant becomes resistant to Bl:41, indicating a synergistic effect of both genes against Bremia in Lettuce. See also Figure 2, where signs of Bremia infection are clear on the lettuce plants comprising the single V04 or SA12 resistance gene, observed as discoloured areas on upper leaf surfaces in combination with white, grey mould located on the lower side of the leaf. When combining the two resistance genes V04 + SA12 in the lettuce plant, no such disease symptoms are observed.

Claims

Claims1. A downy mildew resistant lettuce plant of Lactuca Sativa, wherein the lettuce plant comprises two or more downy mildew resistance genes that have at least two different mechanisms of action for providing said downy mildew resistance, and / or wherein the two or more downy mildew resistance genes originate from at least two different lettuce plants selected from the group consisting of Lactuca serriola, Lactuca virosa and Lactuca saligna, wherein the two or more downy mildew resistance genes are selected from the group consisting of FER, V10, V04, SA07, Dm3, SE17, SA12, SL7, V06, MACPF, SA23, RCB1, RCB2, SA17, SE20, SE28, SE29 and SE30, wherein each of said two or more downy mildew resistance genes encodes for a downy mildew resistance protein having at least 90% sequence identity with an amino acid sequence, respectively; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or in case of DM3, SA12 or SA07 comprising a marker sequence respectively; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35.

2. Lettuce plant according to claim 1, wherein the two or more downy mildew resistance genes are located on at least two different chromosomes in said plant.

3. Lettuce plant according to claim 1 or 2, wherein the two or more downy mildew resistance genes are three, preferably four, more preferably five, most preferably six downy mildew resistance genes.

4. Lettuce plant according to any one of the claims 1 to 3, wherein the lettuce plant is resistant to at least one or more Bremia lactucae races selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU.

5. Lettuce plant according to any one of the claims 1 to 4, wherein the two or more downy mildew resistance genes that have at least two different mechanisms of action for providing the downy mildew resistance are selected from the group consisting of an R gene encoding LRR, TIR, CC, NBS, Zinc Finger, and NB (-Arc) domains or combinations thereof, a FER gene encoding for (pseudo)kinase, and an MACPF gene encoding for a pore former.

6. Lettuce plant according to any one of the claims 1 to 5, wherein the two or more downy mildew resistance genes are:- FER having 99% sequence identity with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, preferably SEQ ID No. 6,- MACPF having 99% sequence identity with SEQ ID No. 18, and optionally- one selected from V04 having 99% sequence identity with SEQ ID No. 10, SE17 having 99% sequence identity with SEQ ID No. 12, and DM3 comprising SEQ ID No. 31, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU.

7. Lettuce plant according to any one of the claims 1 to 5, wherein the two or more downy mildew resistance genes are:- MACPF having 99% sequence identity with SEQ ID No. 18,- RCB 1 having 99% sequence identity with SEQ ID No. 20,- RCB2 having 99% sequence identity with SEQ ID No. 22, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU.

8. Lettuce plant according to any one of the claims 1 to 5, wherein the two or more downy mildew resistance genes are:- V04 having 99% sequence identity with SEQ ID No. 10,- SA12 comprising SEQ ID No. 32, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29, BL:30, Bl:31, Bl:32, Bl:34, Bl:36, Bl:39, Bl:40 and Bl:41 EU.

9. Lettuce plant according to any one of the claims 1 to 5, wherein the two or more downy mildew resistance genes are:- V04 having 99% sequence identity with SEQ ID No. 10,- SE17 having 99% sequence identity with SEQ ID No. 12, wherein the lettuce plant is resistant to Bremia lactucae races Bl:29 - Bl:41 EU.

10. Seed, plant tissue, plant cell or plant part of the lettuce plant according to any one of the claims 1 to 9, comprising the two or more downy mildew resistance genes encoding downy mildew resistance proteins as defined in any one of the claims 1 to 9.

11. A combination of two or more downy mildew resistance genes for conferring resistance to downy mildew in a lettuce plant (Lactuca Sativd), wherein each of said two or more downy mildew resistance genes encodes for a downy mildew resistance proteinhaving at least 90% sequence identity with an amino acid sequence, respectively; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or in case of DM3, SA12 or SA07 comprising a marker sequence respectively; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35, preferably wherein the two or more downy mildew resistance genes encodes for at least FER and MACPF.

12. The combination of two or more downy mildew resistance genes according to claim 11, wherein the combination provides resistance to one or more Bremia lactucae races selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU.

13. Method for identifying and / or selecting (i) a downy mildew resistant lettuce plant or (ii) a seed of the lettuce plant according to any one of the claims 1 to 9, the method comprises the step of establishing, in the genome of the lettuce plant or seed the presence of two or more downy mildew resistance genes encoding a downy mildew resistance protein.

14. Method according to claim 13, wherein each of the two or more downy mildew resistance genes encodes for a corresponding downy mildew resistance protein having at least 95% sequence identity with an amino acid sequence as follows; FER with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6, V10 with SEQ ID No. 8, V04 with SEQ ID No. 10, SE17 with SEQ ID No. 12, SL7 with SEQ ID No. 14, V06 with SEQ ID No. 16, MACPF with SEQ ID No. 18, SA23 with SEQ ID No. 34, RCB1 with SEQ ID No. 20, RCB2 with SEQ ID No. 22, SE20 with SEQ ID No. 24, SE28 with SEQ ID No. 26, SE29 with SEQ ID No. 28 and SE30 with SEQ ID No. 30, and / or wherein each of the two or more downy mildew resistance genes comprises a marker sequence as follows; DM3 comprises SEQ ID No. 31, SA12 comprises SEQ ID No. 32, SA07 comprises SEQ ID No. 35, preferably wherein the two or more downy mildew resistance genes encodes for the corresponding downy mildew resistance proteins FER and MACPF.

15. 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 the combination of two or more downy mildew resistance genes according to claim 11 or 12 with a lettuce plantthat is susceptible to downy mildew and does not comprise said combination of two or more downy mildew resistance genes, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to downy mildew, preferably resistant to one or more Bremia lactucae races selected from the group consisting of Bl:29, Bl:30,Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU, preferably resistant to all of said races, preferably wherein the selection of plants is done according to the method of claim 13 or 14.

16. Use of a gene construct or plasmid for introducing two or more downy mildew resistance genes into the genome of a lettuce plant or lettuce plant cell and providing broad spectrum resistance to downy mildew caused by one or more of B. lactucae selected from the group consisting of Bl:29, Bl:30, Bl:31, Bl:32, Bl:33, Bl:34, Bl:35, Bl:36, Bl:37, Bl:38, Bl:39, Bl:40 and Bl:41 EU, wherein the gene constructs are comprised of a combination of two or more downy mildew resistance genes according to claim 11 or 12 operably linked to expression providing sequences in the lettuce plant or plant cell.

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