Powdery mildew resistance genes in carrots
The identification of a protein and nucleic acids conferring powdery mildew resistance in carrots addresses the lack of such genes, enabling effective breeding and genetic modification for enhanced resistance.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- ベジョー·ザデン·ベー·フェー
- Filing Date
- 2023-06-08
- Publication Date
- 2026-07-23
AI Technical Summary
Existing technologies lack specific genes that confer resistance to powdery mildew in carrot plants caused by Erysiphe heraclei, limiting efficient breeding and development of resistant varieties.
Identification and utilization of a protein (Sequence ID No. 3) and its encoding nucleic acids (SEQ ID NO: 2) that provide resistance to powdery mildew, along with a dominant carrot resistance gene (SEQ ID NO: 1) for introducing resistance into carrot plants, using methods like CRISPR/Cas and Agrobacterium transformation.
Enables the development of powdery mildew-resistant carrot plants through efficient breeding and genetic modification, enhancing resistance and facilitating integration with other resistance genes.
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Abstract
Description
Technical Field
[0001] The present invention relates to a protein capable of conferring resistance to powdery mildew caused by the plant pathogen Erysiphe heraclei in carrot plants. The present invention also provides a nucleic acid, a genomic fragment, and a dominant carrot resistance gene. The present invention further provides a carrot plant resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei. The present invention further relates to a method for identifying a powdery mildew-resistant carrot plant, a method for providing a powdery mildew-resistant carrot plant, and a means for identifying a powdery mildew-resistant carrot plant. The present invention also relates to seeds, plant parts, callus, suspension cultures, somatic embryos, clones, and progeny of the plants of the present invention.
Background Art
[0002] Carrot plants, or wild carrots (Daucus carota), are cultivated plants of the Umbelliferae (or Apiaceae) family, which are common in many regions of the world. The Umbelliferae family includes over 3,500 species, including other vegetables such as caraway, celery, coriander, dill, fennel, parsley, and parsnip.
[0003] Cultivated carrots, particularly Daucus carota subsp. sativa, are usually orange, but are cultivated for their taproots, which may also be purple, black, red, yellow, or white. The length of the roots of carrot plants can vary from 5 to 40 cm, and the diameter can vary from 1 to 10 cm. The taproot can have various shapes. Depending on the purpose, a circular, conical, or more cylindrical shape is preferred.
[0004] Carrots are thought to be native to Central Asia, but are cultivated worldwide. In regions with moderate climates, carrots are generally biennial plants that undergo vegetative growth in their first year after sowing. After overwintering, the plants flower in their second year of cultivation. In tropical and subtropical regions, carrots have an annual life cycle, and the transition from vegetative to reproductive growth occurs without vernalization.
[0005] Male sterility in the genus Daucus is very useful for producing hybrid seeds because it allows for 100% cross-pollination. Furthermore, heterosis, or hybrid vigor, can be strongly expressed in Daucus plants. Two types of male sterility have been described for Daucus. In the so-called brown anther type, the anthers degenerate and become shriveled before they can spread pollen. In the petal-like type, the stamens are replaced by petal-like structures. Both types of male sterility generally result from cytoplasmic male sterility caused by mitochondrial deficiency. Since mitochondria are transmitted to offspring only by the egg cell, this trait is maternal.
[0006] Carrot production is an important agricultural activity, with approximately 40 million tons of carrots produced worldwide every year.
[0007] Important factors influencing crop yield include soil fertility, water availability, climate, and diseases or pests.
[0008] A common disease affecting carrots that causes economic losses is powdery mildew, a leaf disease caused by the fungus Elisifé heracray.
[0009] Carrot plants are infected with E. heraclei via airborne spores called ascospores, which are dispersed by wind or water. Once the spores land on the leaves of a susceptible carrot plant, they germinate and invade the plant cells. After infection, small white spots first appear, which gradually enlarge to form a powdery mycelium. This mycelium produces more spores, further spreading the disease. The infection can eventually form a dense white mycelium on the entire above-ground part of the plant. As the powdery mildew spots enlarge, the leaves turn white. Under severe disease pressure, the leaves turn brown, twist, become brittle, and then wither and die, making mechanical harvesting by pulling the carrots from the ground difficult.
[0010] Plants are more susceptible to dry conditions and warm temperatures. Susceptibility also increases with the age of the plant. Therefore, powdery mildew is most common and severe during dry and hot summers.
[0011] One approach to prevent infection by E. heraclay is the application of fungicides. However, the use of pesticides is generally becoming more restricted, and the general public prefers to avoid applying these compounds. Organic farmers also do not apply fungicides in their cultivation. Therefore, carrots that are resistant to powdery mildew, especially powdery mildew caused by E. heraclay, are preferable.
[0012] Carrot plants resistant to powdery mildew caused by E. heracrayi are known in the art. For example, WO2017 / 144077 discloses carrot plants resistant to powdery mildew caused by the plant pathogen E. heracrayi and describes two resistance-contributing regions on chromosome 3. However, WO2017 / 144077 does not describe the genes that confer resistance to powdery mildew.
[0013] The gene encoding the protein that can confer resistance to powdery mildew caused by E. heracray in carrot plants has not yet been described.
[0014] However, knowledge of the genes that confer resistance to powdery mildew is desirable because, in contrast to resistance-contributing genome fragments, it enables more efficient breeding and the development of powdery mildew-resistant carrot plants with improved agricultural quality. In addition, it allows breeders to stack resistance genes with other resistance genes to enhance the ability of carrot plants to confer stronger resistance to powdery mildew. Furthermore, knowledge of resistance genes enables directional mutagenesis of these genes and / or integration of resistance genes into the carrot plant genome.
[0015] Overt resistance is particularly preferred because a single copy of such resistance is sufficient to obtain strong resistance to the disease, and it facilitates the introduction of such resistance in combination with other resistances. [Prior art documents] [Patent Documents]
[0016] [Patent Document 1] WO2017 / 144077 [Overview of the project] [Problems that the invention aims to solve]
[0017] Therefore, there is a need for novel resistance genes to confer powdery mildew resistance to carrots. [Means for solving the problem]
[0018] The present invention satisfies, among other purposes, the above-mentioned purposes, as outlined in the appended claims. [Modes for carrying out the invention]
[0019] Specifically, this objective is achieved, among other objectives, by providing a protein that can confer resistance to powdery mildew caused by the plant pathogenic fungus Elisife heracley in carrot plants, and which comprises the amino acid sequence represented by Sequence ID No. 3, or an amino acid sequence having at least 90% sequence identity with Sequence ID No. 3, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
[0020] The present invention also provides nucleic acids capable of encoding the proteins defined above. Those skilled in the art will recognize the existence of genetic coding redundancy. Therefore, nucleic acids capable of encoding the proteins defined above may contain synonymous mutations in their nucleic acid sequences. A synonymous mutation means that a codon substitution in the nucleic acid sequence does not alter the encoded amino acid. Preferably, the nucleic acid capable of encoding the proteins defined above comprises the sequence represented by SEQ ID NO: 2, or a sequence having at least 90% sequence identity with SEQ ID NO: 2, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%. Optionally, this nucleic acid is an isolated nucleic acid.
[0021] The present invention further provides a genome fragment capable of encoding the protein or nucleic acid defined above. Preferably, the genome fragment of the present invention comprises the sequence represented by SEQ ID NO: 1, or a sequence having at least 90% sequence identity with SEQ ID NO: 1, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.
[0022] The present invention further relates to a dominant carrot resistance gene that can be translated into the protein defined above, or transcribed into the nucleic acid defined above, or comprises the genomic fragment defined above.
[0023] The present invention also relates to a carrot plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei and contains the dominant carrot resistance gene as defined above. Preferably, the carrot plant contains the dominant carrot resistance gene in a homozygous form. The carrot plant may be a hybrid carrot plant, preferably a cytoplasmic male sterile hybrid carrot plant.
[0024] The present invention further relates to a carrot plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei and contains the protein as defined above, or the nucleic acid as defined above, or the genomic fragment as defined above. Preferably, the carrot plant contains the nucleic acid as defined above. The carrot plant may be a hybrid carrot plant, preferably a cytoplasmic male sterile hybrid carrot plant.
[0025] The present invention also relates to a carrot plant that can be obtained, derived, induced, or is derived from a carrot plant in which the dominant carrot resistance gene is deposited under the deposit number NCIMB 44149 (NCIMB Limited, Wellheads Place, Dyce, Aberdeen, AB21 7GB, United Kingdom) on May 17, 2023, with its representative seeds.
[0026] According to a preferred embodiment, the carrot plant detailed above is not a plant obtained only by essentially biological methods.
[0027] The present invention further relates to seeds, plant parts, cells, callus, suspension cultures, somatic embryos, clones, or progeny of the carrot plant as defined above, which contain the protein as defined above, the nucleic acid as defined above, the genomic fragment as defined above, or the dominant carrot resistance gene as defined above.
[0028] The present invention further provides a method for identifying a powdery mildew-resistant carrot plant, which includes the step of detecting the presence of the dominant carrot resistance gene. Preferably, the method - The process of isolating cellular material from plants, - A step of establishing the presence of the above-defined protein, above-defined nucleic acid, above-defined genome fragment, or above-defined dominant carrot resistance gene in the isolated cell material, Includes.
[0029] The cellular material may be a cell or part of a cell, such as a protein, nucleic acid, especially genomic DNA or mRNA, or a mixture thereof.
[0030] Preferably, the step of establishing presence includes amplification and detection of nucleic acids. The nucleic acids defined above can be amplified using a forward primer which is a nucleic acid having the sequence represented by SEQ ID NO: 4 and a reverse primer which is a nucleic acid having the sequence represented by SEQ ID NO: 5. Those skilled in the art can determine suitable conditions for nucleic acid amplification and detection in a conventional manner.
[0031] The present invention also provides a method for providing powdery mildew-resistant carrot plants, which includes the step of introducing the dominant carrot resistance gene defined above into the genome of a susceptible carrot plant, preferably such that the resulting powdery mildew-resistant carrot plant is not obtained solely by essentially biological methods.
[0032] Mutagenesis or transformation using Agrobacterium or CRISPR / Cas can be used to introduce the nucleic acids, genome fragments, or resistance genes defined above into the genome of carrot plant cells that are not resistant to powdery mildew by essentially non-biological means. Preferably, the method provided further includes the step of regenerating carrot plants from mutant or transformed cells to obtain powdery mildew-resistant carrot plants that are not obtained solely by essentially biological means. Methods for regenerating carrot plants from single cells are known in the prior art.
[0033] In an alternative embodiment, the present invention relates to a method for providing a carrot plant as defined above, comprising the step of transferring the nucleic acid, genome fragment, or dominant carrot resistance gene defined above into a carrot plant that does not contain the nucleic acid, genome fragment, or dominant carrot resistance gene defined above. Preferably, the method further comprises the steps of isolating cell material from the obtained carrot plant and determining the presence of the protein, nucleic acid, genome fragment, or dominant carrot resistance gene defined above in the isolated cell material.
[0034] The present invention also relates to the use of nucleic acids comprising sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, and 9 for identifying or providing powdery mildew-resistant carrot plants.
[0035] The present invention is further illustrated in the following exemplary embodiments. [Examples]
[0036] (Example 1: Testing of resistance to Elisifé heraclay in a greenhouse) The fungus Elisifé heraclay was maintained on suitable susceptible carrot plants by placing infected leaves among them. The infection was then spread among these plants by generating airflow to disperse the spores between them.
[0037] Plants to be evaluated for resistance were sown in soil in pots. When the plants reached a height of approximately 6 cm, inoculation was performed by adding infected leaves that clearly contained fungal spores. The plants to be assayed for resistance were first stroked with these leaves, and then the inoculated leaves were placed among the young plants. Spores were further dispersed by blowing air. The temperature was 16±2°C at night and 22±2°C during the day, with a minimum of 16 hours of light (or longer if the day length was longer) and a maximum of 8 hours of darkness. Relative humidity was maintained high (95%) at night (8 hours). After 3-4 weeks, the plants were evaluated, and the infected leaves were covered with white powdery mycelium and spores.
[0038] The severity of the infection was reflected by scoring the symptoms: 0 (fully susceptible) and 9 (fully resistant). We carefully checked that susceptible control plants actually showed symptoms of E. heracray infection.
[0039] (Example 2: Identification of potential resistance genes) Gene mapping experiments were performed to identify resistance genes that confer powdery mildew resistance in carrot plants. As described in Example 1, resistant carrot plants were first identified by testing their resistance to Elisifé Heraclay in a greenhouse.
[0040] After precise mapping across various populations, several resistance loci, including several presumed disease resistance genes, were identified. One of these presumed resistance genes was gene 8803. Gene 8803 has a coding sequence represented by SEQ ID NO: 2 and encodes a protein with an amino acid sequence represented by SEQ ID NO: 3. The protein encoded by gene 8803 is the NB-ARC-LRR protein. The coding sequence of gene 8803 is located on the genomic DNA fragment represented by SEQ ID NO: 1.
[0041] [Table 1A]
[0042] [Table 1B]
[0043] [Table 1C]
[0044] [Table 1D]
[0045] [Table 1E]
[0046] [Table 1F]
[0047] (Example 3: gRNA design and CRISPR mutant generation) To determine which of these genes is necessary to confer powdery mildew resistance, we used CRISPR / Cas to knock out each of the resistance genes in resistant carrot plants. For this purpose, guide RNAs (gRNAs) were developed for each putative disease resistance gene.
[0048] [Table 2]
[0049] CRISPR mutants containing mutations in each of the presumed powdery mildew resistance genes were generated by protoplast PEG-mediated transfection. F1 hybrid lines with both susceptible and resistant parents were used. This means that only a single resistance allele needed to be mutated, and the phenotype of the mutated plant could be directly evaluated in terms of disease traces (Example 1).
[0050] gRNA and Cas9 protein were mixed to form a ribonucleoprotein. Protoplasts were isolated from young leaves of resistant plants. RNPs were delivered to the protoplasts by polyethylene glycol (PEG) transfection. Subsequently, somatic cell embryos were induced directly. Plants were induced from these somatic cell embryos to obtain regenerated mutant plants.
[0051] (Example 4: Identification of mutations in CRISPR variants) Small leaf tissue samples were collected from four regenerated mutant plants. DNA was isolated, and the mutation in gene 8803 was identified by long-read sequencing.
[0052] [Table 3]
[0053] Next, RT-PCR of gene 8803 was performed using primers SEQ ID NO: 4 (forward primer) and SEQ ID NO: 5 (reverse primer). This should yield a 3225 bp fragment. All samples were evaluated by electrophoresis. For the two mutants, no DNA band was obtained, or a very weak DNA band was obtained. This indicates that gene 8803 is not expressed or is extremely low. All other plants, including the positive control, show expression of gene 8803.
[0054] (Example 5: Conducting disease tests using CRISPR mutants) As described in Example 1, the CRISPR mutants generated were evaluated for resistance to Elisifé heracray. The results showed that all four mutants containing mutations in gene 8803 were susceptible to Elisifé heracray. In contrast, plants without the mutation in gene 8803 remained resistant to powdery mildew caused by Elisifé heracray.
[0055] (Example 6: Construction of resistance gene constructs and transformation into carrot plants) An expression cassette for expressing gene 8803 in carrot plants was designed. This construct contains gene 8803 positioned downstream of a strong constitutive promoter (i.e., cauliflower mosaic virus (CaMV) 35S promoter) and upstream of a terminator (i.e., Agrobacterium nopaline synthase (NOS) terminator). This cassette can be introduced into carrot plant cells for transient or stable expression of gene 8803. Methods for transient or stable expression of heterologous genes in carrot plants are known. For example, an expression cassette containing gene 8803 can be cloned into a detoxified Ti plasmid suitable for replication in Agrobacterium (e.g., pPZP Agrobacterium binary vector). The cassette can then be delivered to plant cells by Agrobacterium-mediated transformation using methods known in the art. Alternatively, the expression cassette containing gene 8803 can be cloned into a vector system, and the cassette can be incorporated into the genome of carrot plant cells using CRISPR-mediated homologous recombination.
Claims
1. A protein capable of conferring resistance to powdery mildew caused by the plant pathogenic fungus Elisifé heracray in carrot plants, comprising an amino acid sequence represented by Sequence ID No. 3, or an amino acid sequence having at least 90% sequence identity with Sequence ID No. 3, preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
2. A nucleic acid capable of encoding the protein described in claim 1.
3. The nucleic acid according to claim 2, comprising the sequence represented by Sequence ID No. 2, or a sequence having at least 90% sequence identity with Sequence ID No. 2, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.
4. A genome fragment capable of encoding the protein described in claim 1 or the nucleic acid described in claim 2 or 3.
5. The genome fragment according to claim 4, comprising the sequence represented by Sequence ID No. 1, or a sequence having at least 90% sequence identity with Sequence ID No. 1, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.
6. A dominant carrot resistance gene comprising a genome fragment that can be translated into the protein described in claim 1, or transcribed into the nucleic acid described in claim 2 or 3, or described in claim 4 or 5.
7. A carrot plant that is resistant to powdery mildew caused by the plant pathogenic fungus Elisifé heracray, comprising the dominant carrot resistance gene described in claim 6.
8. The carrot plant according to claim 7, which is a hybrid carrot plant, preferably a cytoplasmically sterile hybrid carrot plant.
9. The carrot plant according to claim 7 or 8, wherein the dominant carrot resistance gene is obtained, can be obtained, induced, or derived from a carrot plant whose representative seeds are deposited under deposit number NCIMB 44149.
10. Seeds, plant parts, cells, callus, suspension cultures, somatic cell embryos, clones, or progeny of a carrot plant according to any one of claims 7 to 9, comprising the dominant carrot resistance gene described in claim 6.
11. A method for identifying powdery mildew-resistant carrot plants, comprising the step of detecting the presence of the dominant carrot resistance gene described in claim 6 in the genome of the carrot plant.
12. - A step of isolating cellular material from the aforementioned plant, - A step of establishing the presence of the protein described in claim 1, the nucleic acid described in claim 2 or 3, the genome fragment described in claim 4 or 5, or the dominant carrot resistance gene described in claim 6 in the isolated cell material, The identification method according to claim 11, including the method described in claim 11.
13. The identification method according to claim 11 or 12, wherein the step of establishing the presence includes amplification and detection of nucleic acids.
14. A method for providing a powdery mildew-resistant carrot plant, comprising the step of introducing the dominant carrot resistance gene described in claim 6 into the genome of a susceptible carrot plant, wherein the resulting powdery mildew-resistant carrot plant is not obtained solely by essentially biological means.
15. Use of nucleic acids containing sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, and 9 for the identification or provision of powdery mildew-resistant carrot plants.