Downy mildew resistant plants having enhanced root hair formation

Abstract

The present invention relates to a plant that is resistant to downy mildew having enhanced root hair formation and to a combination of genes comprised of a mutated root hair gene and a mutant DMR6 gene for providing a downy mildew resistant plant. Furthermore, the present invention relates to a method for providing a plant that is resistant to downy mildew, mutating DMR6 and root hair gene(s), and use of one or more mutated root hair gene sequence(s), or the cDNA sequence(s) thereof, for providing an agronomically improved plant.

Description

[0001] Disease resistant plants having enhanced root hair formation

[0002] Description

[0003] The present invention relates to a plant that is resistant to downy mildew having enhanced root hair formation and to a combination of genes comprised of a mutated root hair gene and a mutant DMR6 gene for providing a downy mildew resistant plant. Furthermore, the present invention relates to a method for providing a plant that is resistant to downy mildew, mutating DMR6 and root hair gene(s), and use of one or more mutated root hair gene sequence(s), or the cDNA sequence(s) thereof, for providing an agronomically improved plant.

[0004] Downregulation or deactivation of DMR6 via mutations in the gene, which encodes an oxidoreductase, helps a plant to gain resistance against pathogens. For example, W02008 / 092505 discloses disease resistant plants and a method for improving plant resistance to pathogens, especially focusing on oomycetes and fungi, by targeting the DMR6 gene in plants.

[0005] Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule, the oxidant, to another, the reductant. It was found that lack of a functional DMR6 protein results in improved disease resistance. This approach is applied to various crops susceptible to infections, including lettuce, spinach, pepper, tomato and brassica aiming for broad-spectrum, durable resistance. This form of resistance is in particular effective against pathogens of the phylum Oomycota, such as Albugo, Aphanomyces, Basidiophora, Bremia, Hyaloperonospora, Pachymetra, Paraperonospora, Perofascia, Peronophythora, Peronospora, Peronosclerospora, Phytium, Phytophthora, Plasmopara, Protobremia, Pseudoperonospora, Sclerospora, Viennotia species, as well as to pathogens belonging to the Fungi.

[0006] Furthermore, downregulation or deactivation of DMR6 is suitable for a large number of plant diseases caused by oomycetes such as Bremia lactucae on lettuce, Peronospora effusa on spinach, Pseudoperonospora cubensis on members of the Cucurbitaceae family, e.g. cucumber and melon, Peronospora destructor on onion, Plyaloperonospora parasitica on members of the Brasicaceae family, e.g. cabbage, Plasmopara viticola on grape, Phytophthora infestans on tomato and potato, and Phytophthora sojae on soybean.

[0007] A disadvantage of downregulating or deactivating DMR6 or other so-called susceptibility genes is that under field conditions, improved pathogen resistance can be accompanied by undesired phenotypes such as stunted growth or spontaneous occurrence cell death. Without being bound to theory, it is believed that downregulation or deactivation of susceptibility genes is often associated with pleiotropy, that could lead to said undesired phenotypes. Therefore, the use of such downregulated or deactivated susceptibility genes would generally require the skilled person to contemplate mitigating the undesirable phenotypes associated with the resistance against Downy Mildew pathogens. More specifically, a mutated DMR6 in lettuce (Lactuca sativa) confers highresistance to Bremia lactucae (B. lactucae) in a non-race specific manner. However, under field conditions growth reduction is observed in lettuce and based on multiple field trials performed in The Netherlands (Voorst), France (Allonnes) and Spain (Murcia), it was shown that nutrient availability seems to play a crucial role in the observed reduction of the growth. Phenotyping the roots of the DMR6 mutant plants on agar plates showed a drastic reduction of root hairs and root hair development in comparison to the non-mutated wild type DMR6 plants.

[0008] Considering the above, there is a need in the art for disease resistant plants comprising one or more DMR6 gene(s) which do not show an undesirable phenotype. In addition, there is a need in the art for novel genetics and methods for providing plants having improved disease resistance and avoiding or mitigating the undesirable phenotype that can be associated with a downregulated or deactivated DMR6 gene.

[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 plant that is resistant or has enhanced resistance to downy mildew, wherein the plant comprises at least one DMR6 gene encoding a DMR6 protein, wherein the at least one DMR6 gene is mutated and the mutation results in a reduced level, reduced activity or absence of the DMR6 protein as compared to a plant that is not resistant to said downy mildew, wherein the plant further comprises a mutation in one or more root hair genes selected from the group consisting of TTG1, GL2, GL3, encoding a root hair protein TTG1, GL2 and GL3, respectively, wherein the mutation in said one or more root hair genes result in a reduced level, reduced activity or absence of said one or more root hair proteins, as compared to a plant that does not comprises said mutations in said root hair genes, wherein preferably

[0011] the plant is a lettuce plant (Lactuca sativa) and the at least one DMR6 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 2 and / or the TTG1 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 16 or

[0012] the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 18 and / or theTTG1 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 24,

[0013] wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

[0014] In lettuce, the at least one DMR6 gene comprises a DMR6 gene coding sequence of SEQ ID No.l encoding for a protein of SEQ ID No. 2, the TTG1 gene comprises a TTG1 gene coding sequence of SEQ ID No.5 encoding for a protein of SEQ ID No. 6, the GL2 gene comprises a GL2 gene coding sequence of SEQ ID No.l 1 encoding for a protein of SEQ ID No. 12, the GL3 gene comprises a GL3 gene coding sequence of SEQ ID No.15 encoding for a protein of SEQ ID No.

[0015] 16.

[0016] In spinach, the at least one DMR6 gene comprises a DMR6 gene coding sequence of SEQ ID No. 17 encoding for a protein of SEQ ID No. 18, the TTG1 gene comprises a TTG1 gene coding sequence of SEQ ID No. 21 encoding for a of SEQ ID No. 22, the GL2 gene comprises a GL2 gene coding sequence of SEQ ID No. 23 encoding for a protein of SEQ ID No. 24.

[0017] Surprisingly we have found that when mutating TTG1 in DMR6 mutant plants, more specifically lettuce plants, these TTG1 mutants display an altered root hair patterning, and an enhanced or increased root hair and / or restoration of root hair formation and restoring plant growth (reducing / removing the pleo tropic effects) in comparison to DMR6 mutant plants. The plant of present invention is resistant or has enhanced resistance to downy mildew in comparison to a plant which does not comprise the mutations in DMR6 gene. Furthermore, mutating any one of the GL2, GL3 genes, which are in close interaction with the TTG1 gene, seem to also provide the altered root hair patterning, and an enhanced or increased root hair and / or restoration of root hair formation in comparison to DMR6 mutant plants and restoring plant growth (reducing / removing the pleotropic effects). TTG1 partners with other proteins, such as Glabra3 (GL3), to form a complex that regulates the fate of root epidermal cells. GL3 is a key regulator of trichome and root hair initiation and patterning and plays a crucial role in coordinating the spatial and temporal patterning of trichomes and root hairs on the plant surface. Furthermore, TTG1 interacts closely with Glabra2 (GL2) that directs specific cell fates in the root and shoot epidermis, helping to define which cells will form root hairs and which will remain hairless and to ensure the precise patterning of root hairs and trichomes. GL2 acts downstream of GL3. TTG1, through its action in this protein complex, suppresses the formation of root hairs in certain epidermal cells. In this way, it contributes to the patterned development of root hairs, ensuring that root hairs form in a precise spatial arrangement along the root surface. Root hair development is regulated by signallingpathways, including auxin, ethylene, and nutrient-specific pathways and a complex network of genes that control their initiation, elongation, and morphology, including RHD6 (Root Hair Defective 6), RSL (Root Hair Specific-Like), including RSL4 and RSL2, which are essential for root hair elongation, TTG1 (Transparent Testa Glabral) to regulate which cells will become trichoblasts (root hair cells) or atrichoblasts (non-root hair cells), and Auxin-related genes (e.g., ARF7, ARF19) involved in root hair elongation. Therefore, mutating any one of the GL2, GL3 genes, and / or mutating a combination of these may very likely have the same effect on a DMR6 mutant plant with reduced root hair density, as mutating TTG1.

[0018] 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 98% of the sequence length of the gene or protein sequence as claimed herein, preferably at least 99%, more preferably 100%, most preferably 100% sequence length. Alternatively, or additionally, the gene or protein sequence alignment is performed from start(codon) to stop(codon) of the coding sequence or protein sequence. For example, Clustal omega, Clustal W, or Geneious Prime (Clustal Omega algorithm) can be used to align sequences and calculate the % sequence identity.

[0019] Root hair formation is crucial for plant development because root hairs significantly increase the root surface area, enhancing the plant's ability to absorb water and essential nutrients. They are specialized tubular outgrowths of epidermal cells, and their formation and growth are tightly regulated by genetic, hormonal, and environmental factors. Root hairs develop from specialized cells in the root epidermis known as trichoblasts and root hairs facilitate nutrient acquisition by increasing the root’s absorptive surface. The root hairs grow at the root surface, penetrating the soil and creating close contact with the rhizosphere. This close interaction helps plants absorb minerals and nutrients that are essential for growth and development, such as nitrogen (N), phosphorus (P), and potassium (K).

[0020] According to a preferred embodiment, the present invention relates to the plant, wherein the mutation in the one or more root hair genes is an early stop mutation resulting in a full knockout of the one or more root hair genes. Experiments have shown that for the DMR6 / TTG1 double mutant lettuce, the mutations in the DMR6 gene and TTG1 gene results in truncated nonfunctional proteins.

[0021] According to another preferred embodiment, the present invention relates to the plant wherein the mutation in the one or more root hair genes results in a non-functional or truncated protein being encoded by the one or more root hair genes.

[0022] According to yet another preferred embodiment, the present invention relates to the plant, wherein said reduced level, reduced activity or absence of the DMR6 or of said one or more root hair genes is provided by one or more mutations in the regulatory regions or non-coding sequences of said gene. Mutations in the regulatory sequences of the genes can provide an identical result, i.e.wherein the mutation in said one or more root hair genes or DMR6 gene(s) result in a reduced level, reduced activity or absence of the proteins encoded by said genes, as compared to mutations in the gene sequences itself encoding the protein.

[0023] According to a preferred embodiment, the present invention relates to the plant wherein the plant is a lettuce plant and the mutation in the DMR6 genes comprises a G368A mutation in the DMR6 gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No.3. The mutated DMR6 gene comprises the gene coding sequence of SEQ ID No.3 comprising the G368A mutation and encodes for the mutated DMR6 protein of SEQ ID No.4.

[0024] According to a preferred embodiment, the present invention relates to the plant, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C337T or G531A mutation in the TTG1 gene coding sequence, preferably wherein the mutated TTG1 gene coding sequence comprises the sequence of SEQ ID No.7 or SEQ ID No.9, respectively, or wherein the plant is a spinach plant and the mutation in the DMR6 genes comprises a G396A mutation in the DMR6 gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No. 19.

[0025] Experiments show that both the indicated mutations in TTG1 in a lettuce plant comprising a mutated DMR6 in its genome, provides a downy mildew resistant lettuce plant having agronomically improved phenotype, i.e. was not affected in growth or lettuce head size, in contrast to a lettuce comprising a mutated DMR6 in its genome but no mutated TTG1 gene. The double mutant DMR6 / TTG1 shows enhanced plant growth which can be determined by measuring the fresh weight of the crop and quantifying the root system architecture, in comparison to the single DMR6 mutants.

[0026] According to another preferred embodiment, the present invention relates to the plant, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C862T mutation in the GL2 gene coding sequence, preferably wherein the mutated GL2 gene coding sequence comprises the sequence of SEQ ID No.13. The mutated GL2 gene comprises the coding sequence of SEQ ID No.13 comprising the C862T mutation and encodes for the mutated GL2 protein of SEQ ID No.14. Same as for TTG1, a mutation in GL2 in a plant having a mutated DMR6, provides a downy mildew resistant lettuce plant having agronomically improved phenotype.

[0027] According to yet another preferred embodiment, the present invention relates to the plant that is resistant to a pathogen, wherein the plant is one or more selected from the group consisting of Lactuca sativa, Spinacia oleracea, preferably Lactuca sativa.

[0028] Examples of lettuce plants according to present invention are for example wherein in said plant for TTG1 a stretch of 7 or 8 nt has been deleted counted from position 839 of SEQ ID No.5,and for example for DMR6 where a stretch of 7 to 8 nucleotides has been deleted counted from position 115 of SEQ ID No.l, wherein the DMR6 gene and TTG1 are mutated and these mutations results in a reduced level, reduced activity or absence of the DMR6 and TTG1 protein as compared to a plant that is not resistant to said downy mildew, and the plant has an agronomical elite phenotype wherein no negative or impaired growth or pleotropic effects were observed. A further example according to present invention could be that instead or in addition to mutating TTG1, the GL2 gene is mutated by deletion of a 4 to 7 nucleotide stretch counted from position 109 of SEQ ID No.l 1.

[0029] According to a preferred embodiment, the present invention relates to the plant, wherein the mutation in the DMR6 gene and / or mutation in said one or more root hair genes is via random mutagenesis, preferably using a chemical mutagen such as EMS, or targeted mutagenesis such as Zinc-Finger Nuclease, TALEN or CRISPR / CAS, preferably via EMS. The present plants can be obtained by mutagenesis. For example, mutations, either at the expression level or the protein level, can be introduced in these plants by using mutagenic chemicals such as ethyl methane sulfonate (EMS) or by irradiation of plant material with gamma rays or fast neutrons. The resulting mutations can be directed or random. In the latter case, mutagenized plants carrying mutations in the present gene can be readily identified by using the TILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum et al. (2000) Targeted screening for induced mutations. Nat. Biotechnol. 18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135, 630-636). Briefly, this method is based on the PCR amplification of a gene of interest from genomic DNA of a large collection of mutagenized plants in the M2 generation. By DNA sequencing or by scanning for point mutations using a single-strand specific nuclease, such as the CEL-I nuclease (Till et al. (2004) Mismatch cleavage by singlestrand specific nucleases. Nucleic Acids Res. 32, 2632-2641) individual plants having a mutation in the present genes are identified.

[0030] According to another preferred embodiment, the present invention relates to the plant, wherein the mutated DMR6 gene and / or mutated one or more root hair genes are heterozygous, preferably homozygous present in the genome of said plant.

[0031] According to yet another preferred embodiment, the present invention relates to the plant, wherein the downy mildew is caused by a pathogen are selected from Bremia lactucae on lettuce, Peronospora effusa on spinach, preferably Bremia lactucae on lettuce.

[0032] According to another preferred embodiment, the present invention relates to the plant, wherein the mutated DMR6 gene and / or mutated one or more root hair genes, preferably the mutated TTG1 gene, are obtainable from the deposit NCIMB 44395. Seeds are deposited atNCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland on 13 June 2024 under the number NCIMB 44395.

[0033] The present invention, according to a second aspect, relates to seeds, plant cell, plant tissue or plants parts of a plant as defined herein.

[0034] The present invention, according to a third aspect, relates to a combination of genes comprised of a mutated root hair gene and at least one mutated DMR6 gene for providing a downy mildew resistant plant according to any one of the claims 1 to 13, wherein the root hair gene is selected from the group consisting of TTG1, GL2, GL3, encoding a root hair protein TTG1, GL2, GL3 , respectively,

[0035] preferably wherein the plant is a lettuce plant (Lactucci sativa) and the at least one DMR6 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.

[0036] 2 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 16, or

[0037] preferably wherein the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% with SEQ ID No. 18 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% sequence identity preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% with SEQ ID No. 24,

[0038] wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

[0039] The present invention, according to a further aspect, relates to a method for providing a plant that is resistant to a pathogen, said method comprises the step of introducing in said plant a mutation in at least one DMR6 gene encoding a DMR6 protein, wherein the mutation results in a reduced level, reduced activity or absence of DMR6 protein as compared to a plant that is not resistant to said pathogen, wherein the method further comprises the step of introducing in said plant a mutation in one or more root hair genes selected from the group consisting of TTG1, GL2, GL3, preferably GL2, more preferably TTG1, wherein the mutation in said root hair genes result ina reduced level, reduced activity or absence of said one or more root hair genes, as compared to a plant that does not comprises said mutations in said root hair genes, and

[0040] wherein the plant is a lettuce plant (Lactucci sativa) and the at least one DMR6 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 2 and / or the TTG1 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% sequence identity with SEQ ID No. 16, or

[0041] wherein the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 18 and / or the TTG1 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% preferably at least 96%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 24,

[0042] wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

[0043] Similarly, or alternatively, it can be understood that the method also relates to methods using a DMR6 mutant plant as starting point (i.e. the DMR6 gene was already mutated) and wherein then the root hair genes need to be mutated to obtain the plant that is resistant to downy mildew. In these methods, the resulting plant is resistant to downy mildew and has an improved agronomical phenotype in comparison to a DMR6 mutant plant that does not comprise the mutated root hair gene.

[0044] According to a preferred embodiment, the present invention relates to the method for providing a plant that is resistant to downy mildew, wherein the plant is as defined herein.

[0045] According to another preferred embodiment, the present invention relates to the method for providing a plant that is resistant to a pathogen wherein the mutation in the one or more root hair genes is an early stop mutation resulting in a full knockout of the one or more root hair genes, preferably wherein the mutation in the one or more root hair genes results in a non-functional or truncated protein being encoded by the one or more root hair genes.

[0046] According to yet another preferred embodiment, the present invention relates to the method for providing a plant that is resistant to a pathogen wherein said reduced level, reducedactivity or absence of the at least one DMR6 gene(s) or of said one or more root hair genes is provided by one or more mutations in the regulatory regions or non-coding sequences of said genes.

[0047] According to a preferred embodiment, the present invention relates to the method wherein the plant is a lettuce plant and the mutation in the at least one DMR6 gene comprises a G368A mutation in the gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No.3. The mutated DMR6 gene comprises the coding sequence of SEQ ID No.3 comprising said mutation and encodes for the mutated DMR6 protein of SEQ ID No.4.

[0048] According to another preferred embodiment, the present invention relates to the method for providing a plant that is resistant to downy mildew, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C337T or G531A mutation in the TTG1 gene coding sequence, preferably wherein the mutated TTG1 gene coding sequence comprises the sequence of SEQ ID No.7 or SEQ ID No.9, respectively. The mutated TTG1 gene in lettuce comprises the coding sequence of SEQ ID No.7 and encodes for the mutated TTG1 protein of SEQ ID No.8, or the mutated TTG1 gene in lettuce comprises the coding sequence of SEQ ID No.9 and encodes for the mutated TTG1 protein of SEQ ID No.10.

[0049] According to another preferred embodiment, the present invention relates to the method for providing a plant that is resistant to downy mildew, wherein the plant is selected from the group consisting of Lactuca sativa, Spinacia oleracea, preferably Lactuca sativa.

[0050] According to yet another preferred embodiment, the present invention relates to the method for providing a plant that is resistant to a pathogen wherein the mutation in the at least one DMR6 gene and / or mutation in said one or more root hair genes is via random mutagenesis, preferably using a chemical mutagen such as EMS, ENU, MNU, or targeted mutagenesis such as Zinc-Finger Nuclease, TALEN or CRISPR / CAS, preferably via EMS.

[0051] The present invention, according to a further aspect, relates to a nucleic acid sequence comprising a mutated DMR6 gene comprising a mutated DMR6 gene coding sequence comprising SEQ ID No. 3, and / or a mutated TTG1 gene comprising a mutated TTG1 coding sequence comprising SEQ ID No.7 or SEQ ID No. 9 for providing a lettuce plant that is resistant to a pathogen and comprises agronomical elite phenotype in comparison to a lettuce plant not comprised said nucleic acid sequence. The double mutant DMR6 / TTG1 shows enhanced plant growth which can be determined by measuring the fresh weight of the crop and quantifying the root system architecture, in comparison to the single DMR6 mutants.

[0052] The present invention, according to a further aspect, relates to use of one or more mutated root hair gene sequence(s), or the cDNA sequence(s) thereof, for providing an agronomically improved plant, preferably a lettuce plant, wherein said plant comprises a mutated DMR6 gene thatprovides said plant resistance or enhanced resistance to downy mildew, wherein the mutated DMR6 gene and the one or more mutated root hair gene(s) are as disclosed herein. The agronomically improved plant is in comparison to a plant comprising the mutated DMR6 but not comprised of the one or more mutated root hair gene sequence(s), or the cDNA sequence(s) thereof.

[0053] The present invention, according to a further aspect, relates to use of one or more mutated root hair gene sequence(s), or the cDNA sequence(s) thereof, for improving tip burn resistance of a plant, preferably a lettuce plant, wherein the one or more mutated root hair gene(s) are as disclosed herein. Mutating the root hair genes showed to improve the tip burn resistance of especially leafy crops, more specifically a plant selected from the group consisting of Lactuca sativa, Spinacia oleracea, preferably Lactuca sativa. Preferably the one or more mutated root hair gene(s) are provided in said plant via EMS mutagenesis, CRISPR / Cas or gene editing techniques, or via gene constructs, Agrobacterium-mediated genetic transformation, preferably via EMS. Tip burn is a physiological disorder commonly observed in leafy vegetables such as lettuce, cabbage, spinach, and other crops. It is characterized by the browning or necrosis (death) of the leaf edges or tips, typically on the younger, developing leaves. This condition is not caused by pathogens but rather by environmental and nutritional imbalances. Tip burn is associated with browning or blackening of leaf margins or tips, starting on the inner leaves of a plant. Tissue may appear dry and papery or soft and mushy, depending on the plant and environmental conditions. Affected areas do not recover but may spread to other parts if growth conditions remain unfavorable. By managing growth conditions and ensuring consistent nutrient availability via optimal root development of the plant, tip burn can often be mitigated or prevented, preserving the quality and marketability of leafy crops.

[0054] The present invention, according to a further aspect, relates to a method for identifying or screening of (i) a plant that is resistant to a pathogen as defined herein or (ii) a seed of said plant, the method comprises the step of establishing, in the genome of the plant or seed the presence of a mutated DMR6 gene and one or more mutated root hair gene(s) as disclosed herein.

[0055] The present invention, according to a further aspect, relates to a method for producing (i) a plant according the invention or (ii) a seed of said plant, preferably a lettuce plant, the method comprises the step of introgressing in the genome of the plant or seed at least one mutated DMR6 gene and one or more mutated root hair gene(s) as disclosed herein.

[0056] The present invention will be further detailed in the following examples and figures wherein:

[0057] Figure 1: Shows root hairs on \ / i MS agar plates from non-mutated, wild type lettuce (upper panel, (wt), Corbana RP variety), a DMR6 single mutant lettuce (middle panel,(DMR6 mutant)), and a DMR6 + TTG1 double mutant lettuce plants (lower panel, (DMR6 + TTG1 mutant)). Four different roots from four separate plants are shown for each type of lettuce.

[0058] Figure 2: Shows the rhizotron of lettuce comprising the non-mutated wild type lettuce (Corbana RP, left), the single DMR6 mutant (Corbana DMR6 single (SEQ ID No.

[0059] 3), middle plant), and the DMR6 + TTG1 double mutant (Corbana DMR6 / TTG1 double mutant (SEQ ID No.3 and SEQ ID No. 9), right plant).

[0060] Figure 3: Shows the lettuce crops of a non-mutated wild type lettuce (Corbana RP), a lettuce plant of present invention (DMR6 + TTG1 double mutant) and a single DMR6 mutant lettuce plant (DMR6). It shows that the plant of present invention is comparable in size in respect to the wild type lettuce plant and illustrates the data about weight fresh shown in Table 1 of example 2. The single DMR6 mutant is strongly affected in view of fresh weight and lettuce head density or filling. The lettuce DMR6 single mutant showed reduced biomass especially observed in strongly reduced filling and compactness of the lettuce head. The lettuce of present invention (DMR6 + TTG1 double mutant) showed comparable filling and compactness of the lettuce head in respect with the wild type lettuce (Corbana RP).

[0061] Figure 4: Shows lettuce plants in the field and being infected with the B. Lactuca race BL41EU. A non-mutated wild type lettuce (Corbana RP) shows heavy signs of infection resulting in crops that are commercial unsuitable. In contrast, the lettuce plant of present invention (DMR6 + TTG1 double mutant) is free of symptoms and is comparable in size and weight in respect to the wild type lettuce plant. The single DMR6 mutant showed also fully resistance but was significantly affected in its growth and development as show in Figure 3.

[0062] Examples

[0063] Example 1 — Generation ofDMR6 + TTG1 double mutants in lettuce

[0064] TTG1 was identified as a candidate gene to enhance root hair formation in lettuce by creating a full knock out. Only one homolog of TTG1 gene was found in the lettuce genome encoding the TTG1 protein.Lettuce plants comprising the mutated DMR6 (for example plants comprising SEQ ID No.

[0065] 3) where subjected to ethyl methane sulfonate (EMS) treatment to induce mutations in the TTG1 gene. The EMS treated lettuce was subsequently screened for TTG1 mutants, more specifically screened for early stop mutations in the EMS M2 population fixed for the DMR6 mutation. The TTG1 gene coding sequence of SEQ ID No.5 encodes for the TTG1 protein comprising the amino acid sequence of SEQ ID No. 6. Two mutations were identified in TTG1, which provide for a stop mutation in TTG1, C337T and G531A, respectively in view of SEQ ID No.5, resulting in mutant TTG1 sequences of SEQ ID No. 7 and 9, respectively. Furthermore, via gene editing, for example via CRISPR / Cas, such mutant TTG1 plants may be generated, i.e. double mutants in view of DMR6 and TTG1. Examples of such mutants are for example for TTG1 where a stretch of 7 or 8 nt has been deleted counted from position 839 of SEQ ID No.5, and for example for DMR6 where a stretch of 7 to 8 nucleotides has been deleted counted from position 115 of SEQ ID No.l, wherein the DMR6 gene and TTG1 are mutated and these mutations may result in a reduced level, reduced activity or absence of the DMR6 and TTG1 protein as compared to a plant that is not resistant to said downy mildew, resulting in a plant according to present invention (results not shown).

[0066] Example 2 — root hair and disease resistance analysis of mutant DMR6, and TTG1 + DMR6 double mutants and wild type lettuce

[0067] DMR6 mutant lettuce plants, and DMR6 lettuce plants homozygous for the TTG1 mutation C337T or G531A, or the wild type lettuce (no mutations in TTG1 or DMR6) were phenotyped for:

[0068] (1) root hairs on agar plates

[0069] (2) root system architecture on rhizotrons with soil

[0070] (3) fresh weight on rhizotrons with soil

[0071] (4) Bremia resistance under field conditions

[0072] (1) Root hair on agar plates

[0073] When grown on Vi MS agar plates to be able to observe the root hairs, the DMR6 KO mutants (Figure 1 middle panel) show fewer and shorter root hairs compared to the wild type plants (figure 1, upper panel). The dmr6 / ttgl double mutant (Figure 1, lower panel) shows recovery of both root hair number and length.(2) root system architecture on rhizotrons

[0074] On the rhizotrons, the DMR6 single mutant lettuce displayed a less complex root system (Figure 2, middle plant), observed as a reduced number of lateral roots, and reduced total root length, as compared to the non-mutated lettuce (Corbana RP variety, Figure 2, left plant). The root system of the DMR6 single mutant thus explores less of the soil for the uptake of nutrients and water. Surprisingly the lettuce DMR6 + TTG1 double mutant (right plant in Figure 2) showed a significant recovery of the root system size and complexity. Also, the total root length of the double mutant was comparable to the total root length of the non-mutated lettuce, the non-mutated lettuce having only a slight increased total length, whereas the DMR6 single mutant plant showed a significant decrease in total root length (data not shown).

[0075] (3) fresh weight on rhizotrons

[0076] The fresh weight of the rhizotrons (roots) and the fresh weight of the lettuce head of the different lettuce plants is investigated under field conditions, a spring trial, Netherlands, Voorst; plants were planted week 142023, start evaluation in week 242023, final evaluation: week 26 2023. Under field conditions the double mutant DMR6 + TTG1 lettuce was found to be the only lettuce that was not significantly lower in rhizotron biomass than the non-mutated lettuce (see Tabel 1). The fresh weight of the double mutant plant followed the same trend as the root system of the previous experiment (2), having a significantly higher head fresh weight than the DMR6 single mutant lettuce. Furthermore, the performance of the mutant plants (single DMR6, double mutant DMR6 + TTG1) was also assessed under field conditions for two different seasons, spring and autumn (not shown). Results were relatively comparable between spring and autumn.

[0077] In a further experiment the lettuce plants (i.e. the mutant plants (single DMR6 plant and a double mutant DMR6 + TTG1 plant) and the wild type plant (Corbana RP) not comprising any mutations in the DMR6 or TTG1 gene) were tested under greenhouse conditions and a comparison was made in view of the number (#) of lateral roots and the total root length. Plants were grown (April-May-June 2023) on rhizotrons in a heated greenhouse (20 °C continuously) with shading cloths during excess warmth. The plants were watered by flooding of the tables. Light settings: 18 hours of light: off around sundown (around 21:00), on 6 hours later for the first 3 weeks. Then 16 hours of light off around sundown (around 21 :00), on 8 hours later.

[0078] Under greenhouse conditions a similar trend was observed as seen under field conditions, wherein the double mutant was not significantly lower in rhizotron biomass than the non-mutated, wild type lettuce, wherein the single mutant DMR6 showed reduced biomass as also observed in Figure 3. The lettuce DMR6 single mutant showed reduced biomass, especially observed instrongly reduced filling and compactness of the lettuce head. The lettuce of present invention (DMR6 + TTG1 double mutant) showed comparable filling and compactness of the lettuce head in respect to the wild type lettuce (Corbana RP).

[0079] Table 1. Average Fresh Weight (FW) of the lettuce head and root, and number of roots and total root length of the various lettuce plants, including a plant according to present invention. The results are the average of 10 plants.

[0080]

[0081] (4) Bremia resistance under field conditions

[0082] The disease resistance of various lettuce, i.e. a non-mutated, a single DMR6 mutant, and a double DMR6 + TTG1 mutant lettuce were assessed under field conditions in a spring trial, Netherlands, Voorst; plants were planted week 142023, start evaluation in week 242023, final evaluation: week 262023. A scale was used to score was from 0 to 4 being the material either resistant or susceptible, respectively. More specifically, under field condition a score of 0 = resistant, 1 = few Bremia spots on the older leaves, 2 = 50% of the leaves are covered with Bremia, 3 = 75% of the leaves are covered with Bremia and 4 = 100% of the leaves are covered with Bremia.

[0083] Table 2. Average disease score of the different lettuce plants suffering from Bremia. The results are the average of 10 plants.

[0084]

[0085]

[0086] Table 2 shows the resistance scoring for the different lettuce plants, see also Figure 4 showing the lettuce plants. The DMR6 mutant plants were fully resistant against Bremia lactucaea in the field (later collected and confirmed to be race, BL41EU), and served as control. In comparison with the control, the lettuce plant comprising the non-mutated DMR6 (wild type) was fully susceptible. Therefore, DMR6 mutant lettuce plant remained resistant during the field test. In view of the DMR6 + TTG1 double mutant lettuce, the performance of mutation G531A was tested in the field and was also fully resistant. No significant negative effects of the TTG1 in view of disease resistance of the double mutant lettuce was observed.

[0087] Example 3 - Sequence homology root development genes

[0088] The sequence homology among TTG1 proteins of lettuce (L.sativa) and spinach (S. oleracea), plants were analyzed using multiple alignment software. Sequences share a sequence homology of at least 75% sequence identity between plants.

Claims

Claims1. A plant that is resistant to downy mildew, wherein the plant comprises at least one DMR6 gene encoding a DMR6 protein, wherein the at least one DMR6 gene is mutated and the mutation results in a reduced level, reduced activity or absence of the DMR6 protein as compared to a plant that is not resistant to said downy mildew, wherein the plant further comprises a mutation in one or more root hair genes selected from the group consisting of TTG1, GL2, GL3, encoding a root hair protein TTG1, GL2 and GL3, respectively, wherein the mutation in said one or more root hair genes result in a reduced level, reduced activity or absence of said one or more root hair proteins, as compared to a plant that does not comprises said mutations in said root hair genes, and wherein the plant is a lettuce plant (Lactucci sativa) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 2 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% sequence identity with SEQ ID No. 16 orwherein the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 18 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 24, wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

2. Plant according to claim 1, wherein the mutation in the one or more root hair genes is an early stop mutation resulting in a full knockout of the one or more root hair genes.

3. Plant according to claim 1 or 2 wherein the mutation in the one or more root hair genes results in a non-functional or truncated protein being encoded by the one or more root hair genes.

4. Plant according to any one of the claims 1 to 3, wherein said reduced level, reduced activity or absence of the DMR6 protein and / or of said one or more root hair genes is provided by one or more mutations in the regulatory regions or non-coding sequences of said gene.

5. Plant according to any one of the claims 1 to 4, wherein the plant is a lettuce plant and the mutation in the DMR6 genes comprises a G368A mutation in the DMR6 gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No.3.

6. Plant according to any one of the claims 1 to 5, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C337T or G531 A mutation in the TTG1 gene coding sequence, preferably wherein the mutated TTG1 gene coding sequence comprises the sequence of SEQ ID No.7 or SEQ ID No.9, respectively.

7. Plant according to any one of the claims 1 to 6, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C862T mutation in the GL2 gene coding sequence, preferably wherein the mutated GL2 gene coding sequence comprises the sequence of SEQ ID No.13.

8. Plant according to any one of the claims 1 to 7, wherein the plant is a spinach plant and the mutation in the DMR6 genes comprises a G396A mutation in the DMR6 gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No. 19.

9. Plant according to any one of the claims 1 to 8, wherein the mutation in the DMR6 gene and / or mutation in said one or more root hair genes is via random mutagenesis, preferably using a chemical mutagen such as EMS, or targeted mutagenesis such as Zinc-Finger Nuclease, TALEN or CRISPR / CAS.

10. Plant according to any one of the claims 1 to 9, wherein the mutated DMR6 gene and / or mutated one or more root hair genes are heterozygous, preferably homozygous present in the genome of said plant.

11. Plant according to any one of the claims 1 to 10, wherein the downy mildew is caused by a pathogen are selected from Bremia lactucae on lettuce, Peronospora effusa on spinach.

12. Plant according to any one of the claims 1 to 11, wherein the plant is lettuce, wherein the mutated DMR6 gene and / or mutated one or more root hair genes, preferably the mutated TTG1 gene are as present in or obtainable from the deposit NCIMB 44395.

13. Seed, plant cell, plant tissue or plants parts of a plant as defined in any of the claims 1 to14. A combination of genes comprised of a mutated root hair gene and at least one mutated DMR6 gene for providing a downy mildew resistant plant according to any one of the claims 1 to 13, wherein the root hair gene is selected from the group consisting of TTG1, GL2, GL3, encoding a root hair protein TTG1, GL2, GL3 , respectively,preferably wherein the plant is a lettuce plant (Lactuca sativa) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 2 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% sequence identity with SEQ ID No. 16, or preferably wherein the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 18 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 24,wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

15. Combination of genes according to claim 14, wherein the mutated root hair gene and the mutated DMR6 gene are according to any one of the claims 1 to 13.

16. A method for providing a plant that is resistant to a pathogen, said method comprises the step of introducing in said plant a mutation in at least one DMR6 gene encoding a DMR6 protein, wherein the mutation results in a reduced level, reduced activity or absence of DMR6 protein as compared to a plant that is not resistant to said pathogen, wherein the method further comprises the step of introducing in said plant a mutation in one or more root hair genes selected from the group consisting of TTG1, GL2, GL3, preferably GL2, more preferably TTG1, wherein the mutation in said root hair genes result in a reduced level, reduced activity or absence of said one or more root hair genes, as compared to a plant that does not comprises said mutations in said root hair genes, andwherein the plant is a lettuce plant (Lactucci sativa) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 2 and / or the TTG1 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 6 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 12 and / or GL3 encodes for a protein having at least 95% sequence identity with SEQ ID No. 16, or wherein the plant is a spinach plant (Spinacia oleracea) and the at least one DMR6 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 18 and / or the TTG1gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 22 and / or GL2 gene encodes for a protein having at least 95% sequence identity with SEQ ID No. 24, wherein the mutation in said one or more root hair genes is preferably in the TTG1 gene and / or in the GL2 gene.

17. Method according to claim 16, wherein the plant is according to any one of the claims 1 to 12.

18. Method according to claim 16 or 17, wherein the mutation in the one or more root hair genes is an early stop mutation resulting in a full knockout of the one or more root hair genes, preferably wherein the mutation in the one or more root hair genes results in a non-functional or truncated protein being encoded by the one or more root hair genes.

19. Method according to any one of the claims 16 to 18, wherein said reduced level, reduced activity or absence of the at least one DMR6 or of said one or more root hair genes is provided by one or more mutations in the regulatory regions or non-coding sequences of said gene.

20. Method according to any one of the claims 16 to 19, wherein the plant is a lettuce plant and the mutation in the at least one DMR6 gene comprises a G368A mutation in the gene coding sequence, preferably wherein the mutated DMR6 gene coding sequence comprises the sequence of SEQ ID No.3.

21. Method according to any one of the claims 16 to 20, wherein the plant is a lettuce plant and the mutation in the one or more root hair genes comprises a C337T or G531A mutation in the TTG1 gene coding sequence, preferably wherein the mutated TTG1 gene coding sequence comprises the sequence of SEQ ID No.7 or SEQ ID No.9, respectively.

22. Method according to any one of the claims 16 to 21, wherein the plant is Lactuca sativa23. Method according to any one of the claims 16 to 22, wherein the mutation in the at least one DMR6 gene and / or mutation in said one or more root hair genes is via random mutagenesis, preferably using a chemical mutagen such as EMS, or targeted mutagenesis such as Zinc-Finger Nuclease, TALEN or CRISPR / CAS.

24. A nucleic acid sequence comprising a mutated DMR6 gene comprising a mutated DMR6 gene coding sequence comprising SEQ ID No. 3, and / or a mutated TTG1 gene comprising a mutated TTG1 coding sequence comprising SEQ ID No.7 or SEQ ID No. 9 for providing a lettuce plant that is resistant to a pathogen and comprises agronomical elite phenotype in comparison to a lettuce plant not comprised said nucleic acid sequence.

25. Use of one or more mutated root hair gene sequence(s), or the coding sequences thereof, for providing an agronomically improved plant or for improving the agronomic quality of a plant, preferably a lettuce plant, wherein said plant comprises at least one mutated DMR6 gene that provides said plant resistance to downy mildew.

26. Use according to claim 25, wherein the mutated DMR6 gene is according to any one of the claims 1 to 12.

27. Use according to claim 25 or 26, wherein the one or more mutated root hair gene(s) are according to any one of the claims 1 to 12.

28. Use of one or more mutated root hair gene sequence(s), or the coding sequence(s) thereof, for improving tip burn resistance of a plant, preferably a lettuce plant, wherein the one or more mutated root hair gene(s) are according to any one of the claims 1 to 12.

29. Use according to any one of the claims 21 to 27 and 28, wherein the one or more mutated root hair gene(s) are provided in said plant via random mutagenesis, preferably using a chemical mutagen such as EMS, or targeted mutagenesis such as Zinc-Finger Nuclease, TALEN or CRISPR / CAS.

30. Method for identifying or screening of (i) a plant according to any one of the claims 1 to 12 or (ii) a seed of said plant, preferably a lettuce plant, the method comprises the step of establishing, in the genome of the plant or seed the presence of at least one mutated DMR6 gene and one or more mutated root hair gene(s) according to any one of the claims 1 to 12.

31. Method for producing (i) a plant according to any one of the claims 1 to 12 or (ii) a seed of said plant, preferably a lettuce plant, the method comprises the step of introgressing in the genome of the plant or seed at least one mutated DMR6 gene and one or more mutated root hair gene(s) according to any one of the claims 1 to 12.

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