Patent Application
20240294935
A Sequence Listing in the form of an XML file (entitled “EAB-29201_SL.xml”, created on Apr. 30, 2024, and having a size of 18,522 bytes) is hereby incorporated by reference in its entirety.
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 oomycetes 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.
The present invention will be further detailed in the following examples and figures wherein:
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 MACPF1 gene, wherein said MACPF1 gene encodes for a protein sequence having at least 90% sequence identity with SEQ ID NO: 2, preferably at least 95%, more preferably at least 98%, most preferably at least 99%.
Research on the MACPF (Membrane Attack Complex/Perforin) superfamily is mainly focused on its function in humans where it plays a role in the immune system in defeating virus and bacteria related diseases. 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.
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. The MACPF1R gene is involved in a completely different mechanism than the known NBS-LRR mechanism (R genes) in the plant. Therefore, the presence of the MACPF1R 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.
The identification of a novel candidate dominant resistance gene, indicated here as the MACPF1R gene is obtained by gene mapping of multiple independent downy mildew resistance genes in Lettuce. For the first time a MACPF gene has been found in plants that can be linked to plant disease resistance. Using gene mapping methods a gene region was disclosed in lettuce which hosts a number of novel annotated genes that are associated with pathogen resistance, called the membrane attack complex and Perforin (MACPF) gene. In lettuce there are five MACPF homologs present in the lettuce genome, 4 are clustered on chromosome 9 and one is present on chromosome 7. Only one of them is involved with this Bremia resistance phenotype, MACPF1R. This MACPF1R gene of present invention gives resistance to all Bremia races B1 1 to 33, preferably BI 1 to 35, more preferably a broad spectrum Bremia lactucae resistance.
To demonstrate that the MACPF1 gene family is related to Bremia resistance, the putative resistance genes (MACPF1R) have been silenced by tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to induce susceptibility to B. lactucae infection in resistant L. serriola lettuce lines containing the MACPFR1 resistance gene. With VIGS it was demonstrated that the MACPF1R 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 a MACPF1R silencing construct. With VIGS, resistant lettuce lines (L. serriola) were made susceptible by removing the MACPF1R gene via virus induced gene silencing, thereby silencing the MACPF1R gene.
Furthermore it was shown by stable transformation of MACPF1R gene in the susceptible parent (L. sativa) that when MACPF1R was segregating in the next generation it resulted in resistant plants when MACPF1R was present.
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the mutations in the MACPF1 gene result in amino acid changes comprised of amino acid substitutions on the amino acid positions 25, 84, 178, 181, 204, 235, 236, 329, 450, 586, 588, and 589 in the MACPF1 protein represented by SEQ ID NO: 2.
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the mutations in the MACPF1 gene result in amino acid changes further comprised of an addition on amino acid position 590 in the MACPF protein represented by SEQ ID NO: 2.
According to yet another preferred embodiment, the present invention relates to the Lettuce plant, wherein the mutations in the MACPF1 gene result in amino acid changes are further comprised of an insertion of four amino acids on the positions 255 to 258 in the MACPF1 protein represented by SEQ ID NO: 2.
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the MACPF1 gene that comprises one or more mutations encodes for the protein sequence represented by SEQ ID NO: 4. Sequencing experiments showed that the protein encoded by the MACPF1R gene from the resistant plant compared with the protein encoded by the MACPF gene of a plant that is susceptible differs in 12 amino acid substitutions, one amino acid addition and an insertion of 4 amino acids; the gene (MACPF1R) that encodes for the protein that comprises all the above mutations is represented by SEQ ID NO: 3. The mutated protein is represented by SEQ ID NO: 4.
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 MACPF1 gene are obtained by gene editing techniques, preferably by mutagenesis and/or CRISPR/Cas.
According to another preferred embodiment, the present invention relates to the Lettuce plant, wherein the downy mildew is caused by an oomycete, more preferably Bremia lactucae.
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 B11 to B133. The resistant lettuce plant of present invention is resistance to all Bremia races B11 to B133, preferably B11 to B135, more preferably broad spectrum Bremia lactucae resistant.
According to yet another preferred embodiment, the present invention relates to the lettuce plant, wherein the resistance gene MACPF1R is obtainable from deposit number NCIMB 42435.
The present invention, according to a second aspect, relates to seed produced by the lettuce plant of present invention.
The present invention, according to a third aspect, relates to a resistance gene MACPF1R that confers broad spectrum resistance to oomycetes in lettuce plants, wherein the gene comprises a coding sequence that has 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%. SEQ ID NO: 3 represents the coding nucleotide sequence of MACPF1R resistance gene of Lactuca serriola and encodes for the MACPF1R protein sequence represented by SEQ ID NO: 4. SEQ ID NO: 4 represents the MACPF1R protein sequence of Lactuca serriola 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 MACPF1R, wherein the gene encodes for a MACPF1R 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 MACPF1R, wherein broad spectrum resistance to oomycetes in lettuce comprises resistance to Bremia lactucae of race B11 to B133.
According to yet another preferred embodiment, the present invention relates to the resistance gene MACPF1R, 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 seed produced by a lettuce plant of present invention.
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 MACPF1 gene of a lettuce plant, resulting in a MACPF1R resistance gene of present invention. The MACPF1 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 MACPF1 gene of Lactuca sativa. This sequence is the wild type sequence and does not contain the mutations as compared to the resistance (MACPF1R) gene of present invention.
According to another preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene result in amino acid changes comprised of amino acid substitutions on the amino acid positions 25, 84, 178, 181, 204, 235, 236, 329, 450, 586, 588, and 589 in the MACPF1 protein represented by SEQ ID NO: 2. SEQ ID NO: 2 represents the MACPF1 protein sequence of Lactuca sativa. This protein sequence does not comprise the mutations as compared to the MACPF1R protein of present invention. Therefore, L. sativa that express the protein of SEQ ID NO: 2 is susceptible to downy Mildew. SEQ ID NO: 2 represent the wild type protein sequence as found in lettuce (Lactuca sativa) that does not contain the mutations that result into the MACPF1R protein (SEQ ID NO: 4). Preferably the amino acid substitutions are S->A, H->Y, I->M, Y->F, T->A, K->T, Y->F, D->E, T->S, M->I, T->I, R->D, respectively on the amino acid position 25, 84, 178, 181, 204, 235, 236, 329, 450, 586, 588, and 589 in the MACPF1 protein. Table 8 shows an overview of the mutations in the MACPF1 protein in their respective positions. The mutated MACPF1 protein (MACPF1R) is represented by SEQ ID NO: 4.
According to yet another preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene result in amino acid changes further comprised of an addition, preferably of Aspartic Acid (D), on amino acid position 590 in the MACPF1 protein represented by SEQ ID NO: 2.
According to a preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene result in amino acid changes further comprised of an insertion of four amino acids on the positions 255 to 258 in the MACPF1 protein represented by SEQ ID NO: 2. The insertion of four amino acids is preferably TKND (SEQ ID NO: 13).
According to a preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene results in a protein represented by SEQ ID NO: 4.
According to another preferred embodiment, the present invention relates to the method, wherein the lettuce 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.
A lettuce plant comprised of the insertion of 4 amino acids in combination with an addition, in combination with the amino acid substitutions 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 MACPF1 gene to generate disease resistant crops.
According to yet another preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene are obtained by gene editing techniques, preferably by mutagenesis and/or CRISPR/Cas. Alternatively, a MACPF1R gene can be brought into the plant by means of transgenic techniques or by introgression.
According to another preferred embodiment, the present invention relates to the method, wherein the mutations in the MACPF1 gene are non-natural mutations. Mutations induced by gene editing techniques such as mutagenesis, CRISPR/Cas, transgenic techniques, or others can be regarded as non-natural mutations.
The present invention, according to a further aspect, relates to the use of a plasmid for introducing a resistance gene into the genome of a plant or plant cell, wherein the plasmid comprises the resistance gene MACPF1R of present invention. The resistance gene of present invention may be transferred (e.g. by transformation or transfection) into plants, such as lettuce plants, using a plasmid that comprises the the MACPF1R resistance gene of present invention wherein the gene comprises a coding sequence that has at least 90% sequence identity with SEQ ID NO: 3. The resistance gene MACPF1R encodes for a MACPF1R protein that has at least 85% sequence identity with SEQ ID NO: 4. The Resistance gene MACPF1R , after being transferred into the plant would provide broad spectrum resistance to oomycetes, i.e. resistance to Bremia lactucae of race B11 to B133.
In order to study the function of the MACPF1R gene and more specifically if the amino acid substitutions, additions and/or the amino acid insertion are causing the resistance, the MACPF1R construct has been developed. To study if the insertion or the amino acid substitution +addition is effecting resistance, three constructs were made: one with the resistance gene of present invention, one construct with the insertion and no substitutions or additions called LsMACPF1Ins and one construct with the substitution +addition and not the insertion called LsMACPF1Sub. Synthetically constructs with gateway sites were made by Gen9. These fragments were cloned into the vector pK7WG2,0 and transformed to A.tum GV2260. Finally those constructs were stably transformed into L. sativa cultivars Cobham Green and Wendel. The differences between the MACPF1 and MACPF1R protein are 12 amino acid substitutions, an amino acid addition and an insertion of 4 amino acids, see
The multiple constructs of above of the MACPF1 gene were transformed into lettuce (L. sativa) using co-cultivation with agrobacterium. The following construct were used:
Furthermore, it was shown by stable transformation of MACPF1R gene in the susceptible parent (L. sativa) that when MACPF1R was segregating in the next generation it resulted in resistant plants when the MACPF1R resistance gene was present. This was followed by primers specific for the MACPF1R gene. Sequences are present in table 1. Plants were selected based on the primers below in table 1 (SEQ ID NO: 5 and SEQ ID NO: 6, respectively).
Tobacco rattle virus (TRV)-derived VIGS vectors have been abundantly described to study gene function in Arabidopsis thaliana, Nicotiana benthamiana, Lycopersicon 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, lines containing MACPF1R were silenced for MACPF1 by VIGS. Independent of MACPF1R silencing the PDS gene is silenced as well that serves as positive control to indicate if VIGS is working. 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. Silencing of MACPF1R did not result in a visual phenotype. Therefore, all plants that were MACPF1-VIGS inoculated were harvested and put in a tray and sprayed with Bremia B130. This resulted in susceptible leaves while non-silenced MACPF1 plants stayed resistant.
The MACPF1 constructs (LsMACPF1R,-Ins and-Sub) were introduced in lettuce lines using co-cultivation with agrobacterium to get stable transformants. Introducing of the MACPF1R consensus sequence stable transformed in Bremia susceptible lettuce lines (Cobham Green and Wendel) result in Bremia resistant lines in T0, T1 and T2 generation. Outcome of the results are lettuce T1 plants containing the different constructs which are tested for resistance to the oomycete Bremia.
For LsMACPF1 Ins, 47 independent lines in the Cobham Green background and 9 independent lines in the Wendel background were made. In the case of LsMACPF1Sub 57 independent lines in Cobham Green background and 8 independent lines in the Wendel background were made. The seeds of those independent lines were tested in a Bremia seedling test in which 50 seeds per transformant were inoculated with Bremia. The results are that all plants (Wendel and Cobham Green) were susceptible for Bremia (B124 Bremia tested in Wendel, B124 and BL32 Bremia tested in Cobham Green).
The above experiments indicate that both substitutions, addition and the insertion in the MACPF1 gene/protein are necessary to provide the full resistant phenotype to Bremia. If we isolate only the insertion or only the substitution+addition from the resistant source, the Bremia resistance is lost. Therefore the substitutions, addition and insertion are needed to be present in the MACPF1 protein to make an active MACPF1R protein to form pores which could give resistance to Bremia in lettuce.
Leaves of resistant plants stably transformed with or without VIGS MACPF1, were put in trays with moistened paperboard. 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 this B. lactucae suspension. The trays are covered with a glass plate and stored in a climate chamber at 15° C. (12 hours of light). A black, opaque foil is placed over the trays for one day to improve growth of B. lactucae. After one day, the foil is removed. Eight to ten days after infection leaves are phenotypically scored by eye on the presence of Bremia and qPCR was performed to determine MACPF1R expression.
A number of gene expression experiments were conducted in lettuce tissues obtained form the VIGS experiment as outlined above, to determine MACPF1R expression. The response of lettuce leaf discs to Bremia lactucae infection was examined and gene expression studies were used to assess VIGS analysis.
To create more insight in the response of lettuce to infection with Bremia (Bremia lactucae), leaves of resistant and susceptible plants were harvested. cDNA was synthesized from RNA that had been isolated from infected leaf discs. The expression of MACPF1 was assessed in lettuce by conducting qPCR. Expression of Bremia lactucae actin and expression of MACPF1 were analyzed by qPCR using the primers as set out in Table 2 (SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively). This experiment was performed independent in duplo.
B. lactucae
B. lactucae
Results (
This application is a Divisional of U.S. patent application Ser. No. 17/268,540, filed Feb. 15, 2021, which is a U.S. National Stage Application of International Application No. PCT/EP2018/072249, filed Aug. 16, 2018, each of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17268540 | Feb 2021 | US |
| Child | 18427141 | US |
