Patent Application
20220090119
The present invention relates to a lettuce plant that is resistant to downy mildew, more specifically to a lettuce plant that comprises a mutated gene that confers broad spectrum resistance to Bremia lactucae in lettuce. Furthermore the present invention relates a resistance gene and a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the step of mutating a gene.
Downy mildew refers to several types of oomycete microbes that are parasites of plants. Downy mildew can originate from various species, but mainly of Peronospora, Plasmopara and Bremia. Downy mildew is a problem in many food crops, in for example in lettuce caused by Bremia lactucae, affecting the production of this crop worldwide. Plants that are being affected include food crops such as brassicas (e.g. cabbage), potatoes, grape, spinach, lettuce, onion, tomato, cucumber plants. Downy mildew infection show symptoms of discoloured areas on upper leaf surfaces in combination with white, grey or purple mould located on the other side of the leaf surface below. Disease is spread from plant to plant by airborne spores.
Lettuce, mostly known as Lactuca sativa, but also including Lactuca species such as L. serriola, L. saligna or L. virosa, is a very important crop worldwide. Some of the most popular varieties available are Iceberg, Romaine, Butterhead, Batavia and Oakleaf. There are many plant pathogens that affect L. sativa, and some of the diseases caused by these pathogens are downy mildew, Sclerotinia rot, powdery mildew, fusarium wilt of which the most important disease is lettuce downy mildew, which is caused by the B. lactucae, an oomycete pathogen that belong to Peronosporaceae.
For some vegetable crops, such as lettuce, cultivars with resistance to downy mildew are available. However, the pathogen under pressure will mutate to break down the disease resistance and new disease resistance in crops is needed to control infection. Especially in lettuce the occurrence of resistant downy mildew is particularly complex as there are many different races, and new resistant downy mildew species emerging all the time.
In lettuce, infection of B. lactucae result in yellow to pale green lesions that eventually become necrotic due to secondary pathogens leading to major crop losses. Fungicides can be used to control B. lactucae, but eventually B. lactucae becomes immune to these chemicals, because over time the pathogen also acquires resistance to fungicides. Furthermore, there are multiple lettuce varieties available that are resistant to B. lactucae but resistance is quickly overcome because new Bremia races develop rapidly. Therefore, it is of the utmost importance to find other methods to control B. lactucae infection. Most preferably is to identify a resistance gene that gives broad resistance against B. lactucae and to provide for lettuce plants that are resistant to downy mildew. Therefore, identification of resistance genes is a promising alternative.
Considering the above, there is a need in the art for to provide plants that are resistant to downy mildew and wherein plants have a broad spectrum resistance against this pathogen. Furthermore, it is an object of present invention to provide a method to obtain such downy mildew resistant plants.
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.
Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a lettuce plant that is resistant to downy mildew, wherein said plant comprises one or more mutations in a SL7 gene, wherein said SL7 gene encodes for a protein sequence of SEQ ID No. 2 or having at least 90% sequence identity with SEQ ID No. 2, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%, wherein the one or more mutations in the SL7 gene result in amino acid substitutions on position 38 in the SL7 protein represented by SEQ ID No. 2. Preferably the serine (S) at position 38 in SEQ ID No. 2 is mutated to asparagine (N). The mutated SL7 gene is a dominant resistance gene, and may be homozygous or heterozygous present in a downy mildew resistant lettuce plant.
The majority of disease resistance genes in plants encode nucleotide-binding site leucine-rich repeat proteins, also known as NBS-LRR proteins (encoded by R genes). These proteins are characterized by nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains as well as variable amino- and carboxy-terminal domains and are involved in the detection of diverse pathogens, including bacteria, viruses, fungi, nematodes, insects and oomycetes. There are 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.
A leucine-rich repeat (LRR) is a protein structural domain composed of repeating 20 to 30 amino acid stretches that forms an a/I3 horseshoe fold. The domain is rich in the hydrophobic amino acid leucine. The region between the helices and sheets is the protein's hydrophobic core and is tightly sterically packed with leucine residues. On average classical NBS-LLR genes comprise six LRR regions. The SL7 gene is not a classical NBS-LRR gene, since the SL7 protein does not comprise multiple LRR regions, and it does not comprise the NBS domain. The SL7 gene contains only one LRR region, in which the SL7 gene differs from other cases of R genes where multiple LRR regions and the NBS domain are present. It is thought that those domains determine effector recognition and therefore disease susceptibility/resistance. The presence of the SL7 resistance gene will decrease the chances of the pathogen overcoming the resistance, as often seen with the R genes. Even so, combined with R genes, disease resistance (e.g. against downy mildew) may even be further improved.
For the first time a resistance gene has been found in a lettuce plant that is located on chromosome 3 and that can be linked to plant disease resistance. This SL7 gene of present invention gives resistance to Bremia lactucae races Bl17 to Bl35, with the exception of Bl16, Bl20, Bl21, Bl23, and Bl27. Preferably, spectrum resistance to Bremia lactucae in lettuce comprises resistance to Bremia lactucae of at least races Bl17, Bl18, Bl22, Bl24 to Bl26, and Bl28 to Bl35.
To demonstrate that the SL7 gene is related to Bremia resistance, this putative resistance gene has been silenced by tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to induce susceptibility to B. lactucae infection in resistant L. saligna lettuce lines containing the SL7 resistance gene. With VIGS it was demonstrated that the SL7 gene was associated with downy mildew resistance, VIGS gene silencing was used to create Bremia-susceptibility in resistant Lactuca species. Resistant lettuce plants were transient transformed with an SL7 silencing construct and made susceptible to B. lactucae infection, thus by removing the SL7 gene via virus induced gene silencing. According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene result in amino acid substitutions in the region comprised of amino acid positions 6 to 147 in the SL7 protein represented by SEQ ID No.2. The region comprised of amino acids 6 to 147 is an LRR region of SL7, comprising at least one or more LLR domains, preferably at least two, more preferably at least three, most preferably four LLR domains.
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 11 in the SL7 protein represented by SEQ ID No.2. Preferably the threonine (T) at position 11 in SEQ ID No. 2 is mutated to arginine (R).
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene result in amino acid substitutions in the region comprised of amino acid positions 26 to 72 in the SL7 protein represented by SEQ ID No.2. The region comprised of amino acids 26 to 72 is a single LRR domain of SL7.
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 40 in the SL7 protein represented by SEQ ID No.2. Preferably the lysine (K) at position 40 in SEQ ID No. 2 is mutated to threonine (T).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 48 in the SL7 protein represented by SEQ ID No.2. Preferably the serine (S) at position 48 in SEQ ID No. 2 is mutated to asparagine (N).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 61 in the SL7 protein represented by SEQ ID No.2. Preferably the threonine (T) at position 61 in SEQ ID No. 2 is mutated to serine (S).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 69 in the SL7 protein represented by SEQ ID No.2. Preferably the isoleucine (I) at position 69 in SEQ ID No. 2 is mutated to valine (V).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 84 in the SL7 protein represented by SEQ ID No.2. Preferably the cysteine (C) at position 84 in SEQ ID No. 2 is mutated to arginine (R).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 91 in the SL7 protein represented by SEQ ID No.2. Preferably the aspartic acid (D) at position 91 in SEQ ID No. 2 is mutated to asparagine (N).
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the one or more mutations in the SL7 gene further result in amino acid substitutions on position 129 in the SL7 protein represented by SEQ ID No.2. Preferably the valine (V) at position 129 in SEQ ID No. 2 is mutated to isoleucine (I).
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the mutations in the SL7 gene result in amino acid substitutions at position 11, 38, 40, 48, 61, 69, 84, 91 and 129 in the SL7 protein represented by SEQ ID No.2. Preferably the mutations are T11R, S38N, K40T, S48N, T61S, I69V, C84R, D91N, and V129I respectively. An SL7 resistance gene of present invention that encodes for the protein that comprises all the above mutations is represented by SEQ ID No. 3 and encodes for the SL7 protein is represented by SEQ ID No. 4.
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the mutations in the SL7 gene further result in amino acid substitutions at position 11, 40, and 84 in the SL7 protein represented by SEQ ID No.2. Preferably the mutations are T11R, S38N, K40T, and C84R.
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the SL7 gene that comprises one or more mutations and encodes for the protein sequence represented by SEQ ID No. 4. The mutated SL7 gene, being the SL7R gene, is represented by SEQ ID No. 3. Sequencing experiments showed that the protein encoded by the SL7R gene from the resistant plant compared with the protein encoded by the SL7 gene of a plant that is susceptible differs in several amino acids that have been mutated. In particular mutations in the LRR region of the SL7 protein in the amino acid 6 to 147 are of importance to provide resistance to downy mildew, more specifically in the LLR domain of the SL7 protein in the amino acid region of 26 to 72. The downy mildew is caused in the Lettuce plant by an oomycete, more preferably Bremia lactucae.
According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.
According to a preferred embodiment, the present invention relates to the lettuce plant, wherein the mutations in the SL7 gene are obtainable by gene editing techniques, preferably by mutagenesis (e.g. EMS) and/or CRISPR/Cas.
According to another preferred embodiment, the present invention relates to the lettuce plant, wherein the lettuce plant is resistant to downy mildew caused by one or more of Bremia lactucae selected from the group of race Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35. A lettuce plant of present invention comprising the SL7 resistance gene is susceptible to downy mildew caused by Bremia lactucae Bl16, Bl20, Bl21, Bl23, and Bl27. Preferably, spectrum resistance to Bremia lactucae in the lettuce of present invention comprises resistance to Bremia lactucae of at least races Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.
According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the resistance gene SL7R of SEQ ID No. 3 is obtainable from deposit number NCIMB 42785.
The present invention, according to a second aspect, relates to seeds produced by the lettuce plant of present invention. The seed comprises the SL7R gene as described above.
The present invention, according to a third aspect, relates to a resistance gene SL7R that confers resistance to Bremia lactucae in lettuce plants, wherein the gene comprises a coding sequence of SEQ ID No. 3 or having at least 90% sequence identity with SEQ ID No. 3, preferably at least 95%, more preferably at least 98%, most preferably at least 99%, most preferably 100%. The SL7R gene is a dominant gene. SEQ ID No.3 represents the coding nucleotide sequence of SL7R gene of Lactuca saligna and encodes for the SL7R protein sequence represented by SEQ ID No.4. SEQ ID No.4 represents the SL7R protein sequence of Lactuca saligna and lettuce plants that express this protein show complete resistance to downy mildew.
According to a preferred embodiment, the present invention relates to resistance gene SL7R, wherein the gene encodes for a SL7R protein that has at least 85% sequence identity with SEQ ID No. 4, preferably at least 90%, more preferably at least 95%, most preferably at least 98%, most preferably 100%.
According to another preferred embodiment, the present invention relates to the resistance gene SL7R, wherein resistance to Bremia lactucae in lettuce comprises resistance to Bremia lactucae of race Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35. Preferably, spectrum resistance to Bremia lactucae in lettuce comprises resistance to Bremia lactucae of at least races Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.
According to yet another preferred embodiment, the present invention relates to the resistance gene SL7R, wherein the plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.
The present invention, according to a further aspect, relates to a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the steps of,
The present invention, according to a further aspect, relates to a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method comprises the step of providing one or more mutations in a SL7 gene of a lettuce plant, resulting in a SL7R resistance gene of present invention. The SL7 gene comprises a coding sequence that has at least 90% sequence identity with SEQ ID No. 1, preferably at least 95%, more preferably at least 98%, most preferably at least 99%, most preferably 100%. SEQ ID No.1 represents the coding nucleotide sequence of the SL7 gene of Lactuca sativa. This sequence is the wild type sequence and does not contain the mutations as compared to the resistance gene of present invention.
According to another preferred embodiment, the present invention relates to the method, wherein the one or more mutations in the SL7 gene result in amino acid substitutions in the region comprised of amino acid positions 6 to 147, preferably comprised of amino acid positions 26 to 72, in the SL7 protein represented by SEQ ID No.2. Mutations are located in the LLR region of amino acid positions 6 to 147, preferably in the single LRR domain that is located from amino acid 26 to 72 of the SL7 protein.
According to yet another preferred embodiment, the present invention relates to the method, wherein the one or more mutations in the SL7 gene comprise amino acid substitutions at position 11, 38, 40, and 84 in the SL7 protein represented by SEQ ID No.2.
According to yet another preferred embodiment, the present invention relates to the method, wherein the one or more mutations in the SL7 gene further comprises amino acid substitutions at position 48, 61, 69, 91 and 129 in the SL7 protein represented by SEQ ID No.2.
According to a preferred embodiment, the present invention relates to the method, wherein the SL7 gene that comprises one or more mutations, being the SL7R gene, is represented by SEQ ID No. 3 and encodes for the protein sequence represented by SEQ ID No. 4. SEQ ID No.4 represents the SL7R protein sequence of Lactuca saligna. Lettuce, such as L. sativa that express the protein of SEQ ID No.4 is resistant to downy mildew.
According to a preferred embodiment, the present invention relates to the method, wherein the mutations in the SL7 gene are obtained by gene editing techniques, preferably by mutagenesis and/or CRISPR/Cas. The lettuce plant comprising the mutations in the SL7 gene is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa. A lettuce plant comprised of the mutations in the SL7 gene gives a high downy mildew resistance phenotype. A plant having this resistant phenotype can be obtained via use of gene editing and/or mutation techniques, such as EMS mutagenesis or CRISPR/Cas in concert with cloning techniques on the SL7 gene to generate disease resistant crops. Mutations induced by gene editing techniques such as mutagenesis, CRISPR/Cas, transgenic techniques, or others can be regarded as non-natural mutations. Alternatively, a SL7R gene can be brought into the plant by means of transgenic techniques or by introgression.
The present invention, according to a further aspect, relates to the use of a gene construct for introducing a resistance gene into the genome of a plant or plant cell, wherein the gene construct is comprised of the resistance gene SL7R of present invention which is operably linked to expression providing sequences in said plant. The resistance gene of present invention may be transferred (e.g. by transformation or transfection) into plants, such as lettuce plants, using a plasmid of vector or linear gene construct that comprises the SL7R resistance gene of present invention or wherein the gene comprises a coding sequence that has at least 90% sequence identity with SEQ ID No. 3. The resistance gene SL7R encodes for a SL7R protein that has at least 85% sequence identity with SEQ ID No. 4. The Resistance gene SL7R, after being transferred into the lettuce plant would provide resistance to Bremia lactucae i.e. resistance to Bremia lactucae of race Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.
The present invention will be further detailed in the following examples and figures wherein:
Gene Mapping SL7 Resistance Gene in L. saligna
Gene mapping experiments were done to identify the of the Bremia (Bremia lactucae) resistance gene SL7R from Lactuca saligna. SL7R was originally isolated from L. saligna lettuce accession LAC0364. A dominant resistance gene was mapped on chromosome 3, Lettuce genome V8. The SL7R resistance gene is the first Bremia resistance gene described and mapped on chromosome 3 in Lettuce.
After fine mapping 12000 plants two putative genes were present. The SL7R region is currently flanked by two markers based on a SNP at position V8_3_200695773 and a SNP at position V8_3_200770104. The two candidate genes are designates as Lsa011563 and Lsa042767. VIGS silencing was used to silence both genes independently in a resistant source (L. saligna), see below. These experiments indicated that when resistance gene Lsa011563 was silenced the plants became susceptible after Bremia infection, whereas when gene Lsa042767 was silenced, the plants remained resistant. This confirms that the Lsa011563 gene provide the plant resistance against Bremia. This resistance gene is renamed to the resistance gene SL7R of present invention.
Construction of SL7 Construct and Transformation into Lettuce (L. saligna).
After gene mapping two candidate resistance genes were found and designated as Lsa011563 (=SL7R) and Lsa042767. To identify which gene (or both) are responsible for the observed resistance, VIGS silencing can be used to silence both genes independently in the resistant source L. saligna. Therefore, two VIGS-constructs were made per gene, for Lsa011563 (=SL7) (1A and 1B) and for Lsa042767 (2A and 2B) and cloned in the K20 vector (See Table 1 for sequences, respectively SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8). Thus two SL7R specific VIGS constructs were made (1A and 1B) and two construct that target a different gene Lsa042767 (2A, 2B) can be used as a negative control. The multiple constructs of above were transformed into lettuce (L. sativa) using co-cultivation with agrobacterium (GV3101) to study the SL7R function. The two resistance candidate genes are individual silenced in VIGS independent experiments. With the leaves of VIGS-experiments independent disease tests (see below) were performed to observe that when SL7R was silenced, plants became susceptible to Bremia.
Tobacco rattle virus (TRV)-derived VIGS vectors have been abundantly described to study gene function in Arabidopsis thaliana, Nicotiana benthamiana, Solanum esculentum and other plants (see for example Huang C, Qian Y, Li Z, Zhou X.: Virus-induced gene silencing and its application in plant functional genomics. Sci China Life Sci. 2012; 55(2):99-108).
Briefly, Lettuce containing SL7 were silenced for SL7R by VIGS. Independent of SL7R silencing the PDS gene is silenced as well that serves as positive control to indicate if VIGS is working and to determine the efficiency. PDS is involved in carotenoid biosynthesis and is the first step in lycopene biosynthesis. This step is catalyzed by phytoene desaturase (PDS). When silencing of the PDS gene is achieved, this results in bleached leaves.
Furthermore, all plants that were SL7R-VIGS inoculated were harvested and put in a tray and sprayed with Bremia to test the effect of the gene silencing on disease resistance.
Leaves of resistant plants transiently transformed with the above described VIGS constructs (1A, 1B, 2A, 2B and PDS), were put in trays with moistened paperboard and infected with Bremia race 30. The infected seedlings are suspended in 20 mL water, filtered by cheesecloth and the flow-through is collected in a spray flask. One tray is spray-inoculated with the Bremia lactucae suspension. The trays are covered with a glass plate and stored in a climate chamber at 15° C. (12 hours of light). A black, opaque foil is placed over the trays for one day to improve growth of B. lactucae. After one day, the foil is removed. Experiments were performed in triple, and eight to ten days after infection leaves are phenotypically scored by eye on the presence of Bremia, i.e. being susceptible or resistant (
Disease resistance tests show that resistance gene SL7R provides resistance to most Bremia races from Bl:15 to Bl:35, with the exceptions being Bl:16, BL20, Bl21, Bl23, and Bl:27. Furthermore a qPCR was performed to determine SL7R expression.
A single gene line comprising the SL7R gene used internally to test Bremia diagnostic samples is R290. Seeds of this line are deposited at NCIMB (NCIMB Limited, Ferguson Building; Craibstone Estate, Bucksburn ABERDEEN, Scotland, AB21 9YA United Kingdom) on 12 Jul. 2017 under the number NCIMB 42785.
A number of gene expression experiments were conducted in lettuce tissues obtained from the VIGS experiment as outlined above, to determine SL7R expression. The response of lettuce leaves to Bremia lactucae infection was examined and gene expression studies were used to assess VIGS analysis.
To obtain more insight in the response of lettuce to infection with Bremia, leaves of resistant and susceptible plants were harvested. cDNA was synthesized from RNA that had been isolated from infected leaves. The expression of SL7R was assessed in lettuce by conducting qPCR. Expression of Bremia lactucae actin and expression of SL7R were analyzed by qPCR using the primers as set out in Table 2. (SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14, respectively).
B. lactucae
B. lactucae
In addition,
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/085244 | Dec 2018 | EP | regional |
This application is the United States national phase of International Application No. PCT/EP2019/083651 filed Dec. 4, 2019, and claims priority to International Application No. PCT/EP2018/085244 filed Dec. 17, 2018, the disclosures of which are hereby incorporated by reference in their entirety. The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 2102739_ST25.txt. The size of the text file is 21,911 bytes, and the text file was created on May 19, 2021.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/083651 | 12/4/2019 | WO | 00 |
