Powdery mildew resistance gene in carrot

Abstract

The present invention relates to a protein capable of providing resistance to powdery mildew caused by the plant pathogen Erysiphe heraclei in carrot plants. The invention also provides nucleic acid, a genomic fragment, and a dominant carrot resistance gene. The invention further provides carrot plants resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei. The present invention further relates to methods for identifying powdery mildew-resistant carrot plants, methods for providing powdery mildew-resistant carrot plants and means for identifying powdery mildew-resistant carrot plants. The present invention also relates to seeds, plant parts, callus, suspension cultures, somatic embryos, clones, and progeny of the present plants.

Description

[0001] POWDERY MILDEW RESISTANCE GENE IN CARROT

[0002] Description

[0003] The present invention relates to a protein capable of providing resistance to powdery mildew caused by the plant pathogen Erysiphe heraclei in carrot plants. The invention also provides nucleic acid, a genomic fragment, and a dominant carrot resistance gene. The invention further provides carrot plants resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei. The present invention further relates to methods for identifying powdery mildew-resistant carrot plants, methods for providing powdery mildew-resistant carrot plants and means for identifying powdery mildew -resistant carrot plants. The present invention also relates to seeds, plant parts, callus, suspension cultures, somatic embryos, clones, and progeny of the present plants.

[0004] The carrot plant, or Daucus carota, is a cultivated plant from the Umhelliferae (or Apiaceae) family, which is common in many parts of the world. The Umhelliferae family encompasses more than 3,500 species, including other vegetables, for example, caraway, celery, coriander, dill, fennel, parsley, and parsnip.

[0005] The cultivated carrot, in particular Daucus carota subsp. sativa, is grown for its taproot that is usually orange, but may be purple, black, red, yellow, or white as well The root length of carrot plants varies from 5 up to 40 cm and the diameter can vary between 1 to 10 cm. Taproots can have various shapes. Round, conical, or more cylindrical shapes are preferred depending on the purpose.

[0006] Although carrot plants are thought to be native to Central Asia, they are cultivated globally. In moderate climate zones, carrot plants are generally biennial plants that grow vegetatively in the first year after sowing. After overwintering, the plant will flower in the second year of cultivation. In tropical and subtropical areas, the carrot plant has an annual life cycle, and the shift from vegetative to generative growth occurs without vernalization.

[0007] Male sterility in Daucus is very useful for producing hybrid seeds as it enables 100 % crosspollination. Moreover, heterosis, or hybrid vigor, can be strong in carrot plants. Two types of male sterility have been described for the genus Daucus. In the so-called brown anther type, anthers degenerate and shrivel before they can spread pollen. In the petaloid type, the stamens are replaced by petal-like structures. Both types of male sterility are generally due to cytoplasmic male sterility caused by mitochondrial defects. Since mitochondria are transferred to the offspring by egg cells only, this trait is maternally inherited.

[0008] Carrot production is an important agricultural activity with roughly 40 million tons of carrots produced globally each year. Key factors that influence crop yield are soil fertility, availability of water, climate, and diseases or pests.

[0009] A commonly occurring disease in carrots, which leads to economic losses, is the foliar disease powdery mildew caused by the fungus Erysiphe heraclei.

[0010] Carrot plants are infected by E. heraclei through airborne spores called ascospores, which are dispersed by wind or water. When they land on the leaves of a susceptible carrot plant, they germinate and enter the plant cell. After infection, initially, small white spots appear, which gradually enlarge to form a powdery mycelium. This mycelium produces more spores that spread the disease further. The infection may eventually lead to white dense mycelium on all above-ground parts of the plant. As the mildew spots enlarge, the leaves can become chlorotic. Under severe disease pressure, the leaves turn brown, twisted and brittle before shriveling and dying, which makes mechanical harvesting by pulling carrots from the ground difficult.

[0011] Plants are more susceptible under dry conditions and warm temperatures. Susceptibility also increases with plant age. Therefore, powdery mildew is most common and severe during dry and hot summers.

[0012] One approach to prevent infection by E. heraclei is the application of fungicides. However, the use of pesticides in general is becoming more restrained and the general public is in favor of avoiding the application of these compounds. Also, organic growers do not apply fungicides in their cultivation. Carrots that are resistant to powdery mildew, in particular powdery mildew caused by E. heraclei, are therefore preferred.

[0013] Carrot plants resistant to powdery mildew caused by E. heraclei, are known in the art. For example, WO2017 / 144077 discloses carrot plants resistant to powdery mildew caused by the plant pathogen E. heraclei and describes two resistance providing regions on chromosome 3. However, WO2017 / 144077 does not describe a gene providing resistance to powdery mildew.

[0014] Genes encoding proteins capable of providing resistance to powdery mildew caused by E. heraclei in carrot plants have not been described yet.

[0015] However, knowledge of genes that provide resistance to powdery mildew, in contrast to genomic fragments providing resistance, is preferred as it enables more efficient breeding and the development of powdery mildew-resistant carrot plants with improved agronomic quality. In addition, it enhances the ability of breeders to stack the resistance gene with other resistance genes to provide carrot plants with a more robust resistance to powdery mildew. Furthermore, knowledge of resistance genes allows directed mutagenesis of these genes and / or integration of the resistance gene into the genome of a carrot plant. Dominant resistances are particularly preferred as a single copy of such a resistance is sufficient to obtain a strong resistance against disease making it easier to introduce such resistances in combination with other resistances.

[0016] Hence, there is a need for new resistance genes that confer resistance to powdery mildew in carrots.

[0017] The present invention meets the above object, amongst other objects, as outlined in the appended claims.

[0018] Specifically, this object, amongst other objects, is achieved by providing a protein capable of providing resistance to powdery mildew caused by the plant pathogen Erysiphe heraclei in carrot plants, wherein the protein comprises the amino acid sequence represented by SEQ ID No. 3, or an amino acid sequence having at least 90% sequence identity with SEQ ID No. 3, preferably at least 95% sequence identity, more preferably at least 98%, most preferably at least 99% sequence identity.

[0019] The invention also provides nucleic acid capable of encoding a protein as defined above. A person skilled in the art is aware that there is redundancy of the genetic code. Therefore, nucleic acids capable of encoding the protein as defined above may comprise synonymous variations in their nucleic acid sequence. Synonymous variation means that a codon substitution in the nucleic acid sequence does not change the encoded amino acid. Preferably, the nucleic acid capable of encoding a protein as 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% sequence identity, more preferably at least 98% sequence identity, most preferably at least 99% sequence identity. Optionally, this nucleic acid is an isolated nucleic acid.

[0020] The invention further provides a genomic fragment capable of encoding a protein as defined above or a nucleic acid as defined above. Preferably, the present genomic fragment 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, most preferably at least 99% sequence identity.

[0021] The invention further relates to a dominant carrot resistance gene capable of translating into a protein as defined above, or capable of being transcribed into a nucleic acid as defined above, or comprising a genomic fragment as defined above.

[0022] The invention also relates to a carrot plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the carrot plant comprises a dominant carrot resistance gene as defined above. Preferably, the carrot plant comprises the dominant carrot resistance gene in homozygous form. The carrot plant may be a hybrid carrot plant, preferably a cytoplasmic male sterile hybrid carrot plant. The invention further relates to a carrot plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the carrot plant comprises a protein as defined above, or a nucleic acid as defined above, or a genomic fragment as defined above. Preferably, the carrot plant comprises a nucleic acid as defined above. The carrot plant may be a hybrid carrot plant, preferably a cytoplasmic male sterile hybrid carrot plant.

[0023] The invention also relates to carrot plants, wherein the dominant carrot resistance gene is obtained, is obtainable, is derived, or is from a carrot plant of which representative seeds are deposited under deposit number NCIMB 44149 (NCIMB Limited, Wellheads Place, Dyce, Aberdeen, AB21 7GB, United Kingdom) on 17 May 2023.

[0024] According to a preferred embodiment, the carrot plants detailed above are not plants exclusively obtained by means of an essentially biological process.

[0025] The invention further relates to seeds, plant parts, cells, callus, suspension culture, somatic embryos, clones, or progeny of a carrot plant as defined above, wherein the seeds, plant parts, cells, callus, suspension culture, somatic embryos, clones, or progeny comprise a protein as defined above, a nucleic acid as defined above, a genomic fragment as defined above or a dominant carrot resistance gene as defined above.

[0026] The invention further provides methods for identifying a powdery mildew-resistant carrot plant, wherein the method comprises the step of detecting the presence of the dominant carrot resistance gene. Preferably, the method comprises the steps of: isolating cellular material from the plant; and establishing the presence of a protein as defined above, a nucleic acid as defined above, a genomic fragment as defined above, or a dominant carrot resistance gene as defined above in the isolated cellular material.

[0027] Cellular material can be cells or parts of cells, such as protein, nucleic acid, in particular genomic DNA or mRNA, or a mixture thereof.

[0028] Preferably, establishing the presence comprises nucleic acid amplification and detection. A nucleic acid as defined above may be amplified using a forward primer which is a nucleic acid having a sequence represented by SEQ ID No. 4 and reverse primer which is a nucleic acid having a sequence represented by SEQ ID No. 5. A person skilled in the art can determine the appropriate conditions for nucleic acid amplification and detection in a routine manner.

[0029] The invention also provides methods for providing a powdery mildew-resistant carrot plant, wherein the method comprises the step of introducing a dominant carrot resistance gene as defined above in the genome of a susceptible carrot plant, preferably wherein the resulting powdery mildew -resistant carrot plant is not exclusively obtained by means of an essentially biological process. Mutagenesis, or transformation with Agrobacterium or CRISPR / Cas may be used to introduce a nucleic acid as defined above, a genomic fragment as defined above or a resistance gene as defined above by means of a non-essentially biological process into the genome a cell of a carrot plant that is not resistant to powdery mildew. Preferably, the method of providing further comprises the step of regenerating a carrot plant from the mutated or transformed cell to obtain a powdery mildew-resistant carrot plant that is not exclusively obtained by means of an essentially biological process. Methods for regenerating a carrot plant from a single cell are known in the prior art.

[0030] In an alternative embodiment, the present invention relates to a method for providing a carrot plant as defined above comprising the steps of introgression of a nucleic acid as defined above, a genomic fragment as defined above or a dominant carrot resistance gene as defined above into a carrot plant not comprising a nucleic acid as defined above, a genomic fragment as defined above or a dominant carrot resistance gene as defined above. Preferably, the method of providing further comprises the step of isolating cellular material from the obtained carrot plant and determining in the isolated cellular material the presence of a protein as defined above, a nucleic acid as defined above, a genomic fragment as defined above or a dominant carrot resistance gene as defined above.

[0031] The invention also relates to the use of a nucleic acid comprising a sequence selected from the group consisting of SEQ ID Nos. 1, 2, 4, 5, 6, 7, 8 and 9 for identifying, or providing, a powdery mildewresistant carrot plant.

[0032] The invention is further elucidated in the illustrative examples below.

[0033] EXAMPLES

[0034] Example 1 : Testing for resistance against Erysiphe heraclei in the glasshouse

[0035] The fungus Erysiphe heraclei was maintained on suitable susceptible carrot plants by placing infected leaves among the susceptible carrot plants. Infection was then spread among these plants by generating air currents which distributed the spores among the plants.

[0036] Plants to be evaluated for resistance were sown in soil in pots. When the plants were about 6 cm tall, inoculation took place by adding infected leaves, clearly containing fungal spores. Plants to be assayed for resistance were stroked first with these leaves, and then the inoculating leaves were placed between the young plants. Spores were spread further by blowing air. The temperature was 16 ± 2° C at night; 22 ± 2° C during daytime; min. 16 hours light (or more if day length was longer) and max. 8 hours dark. The relative humidity was kept high (95%) at night (8h). After 3 to 4 weeks the plants were evaluated; infected leaves were covered with a white powdery mycelium and spores. The severity of infection was reflected by scoring the symptoms: 0 (completely susceptible) and 9 (completely resistant). It was carefully checked that the susceptible control plants were indeed showing the symptoms of E. heraclei infection. Example 2: Identification of potential resistance genes

[0037] Gene mapping experiments were done to identify a resistance gene that confers powdery mildew resistance in carrot plants. Resistant carrot plants were initially identified by testing for resistance against Erysiphe heraclei in a glasshouse as described in Example 1.

[0038] After fine mapping in various populations, a resistance locus was identified comprising several putative disease resistance genes. One of these putative 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 an NB-ARC-LRR protein. The coding sequence of gene 8803 is present on a genomic DNA fragment represented by SEQ ID No. 1. Table 1. Sequence information

[0039] Example 3: Design of gRNAs and generation of CRISPR mutants

[0040] To determine which of these genes is needed to confer powdery mildew resistance, CRISPR / Cas was used to knockout each of the resistance genes in a resistant carrot plant. For this purpose, guide RNAs (gRNAs) were developed for each putative disease resistance gene.

[0041] Table 2. Guide RNAs used to generate CRISPR mutants of gene 8803.

[0042] CRISPR mutants comprising mutations in each putative powdery -mildew-resistance gene were generated by performing a protoplast PEG mediated transfection. An F 1 hybrid line was used, with a susceptible parent and a resistant parent. This means that only a single resistance allele needs to be mutated and the phenotype of a plant with a mutation can be directly assessed in a disease trail (Example 1).

[0043] The gRNAs and Cas9 proteins were mixed to form a ribonucleoprotein. Protoplasts were isolated from young leaves of resistant plants. The RNPs were delivered to protoplast by polyethylene glycol (PEG)-mediated transfection. Direct somatic embryos were subsequently induced. From these somatic embryos plants were derived to obtain regenerated mutated plants.

[0044] Example 5: Identification of mutations in CRISPR mutants

[0045] A small leaf tissue was taken from four regenerated mutated plants. DNA was isolated and mutations were identified in gene 8803 by long read sequencing.

[0046] Table 3. Primers forPCR amplification of gene 8803 and RT-PCR.

[0047] Subsequently a RT-PCR was conducted of gene 8803 using primers SEQ ID No. 4 (forward primer) and SEQ ID No. 5 (reverse primer). This should give a 3225 bp fragment. All samples were assessed by electrophoresis. No, or a low intensity DNA band was obtained for two mutants. This indicates no or extremely low expression of gene 8803. All other plants, including a positive control, show expression of gene 8803.

[0048] Example 5: Performing disease test with CRISPR mutants

[0049] The generated CRISPR mutants were assessed for resistance against Erysiphe heraclei as described in Example 1. The results showed that all four mutants comprising a mutation in gene 8803 were susceptible to Erysiphe heraclei. In contrast, plants without a mutation in gene 8803 remained resistant to powdery mildew caused by Erysiphe heraclei.

[0050] Example 6: Construction of resistance gene construct and transformation into carrot plants

[0051] An expression cassette for expression of gene 8803 in a carrot plant was designed. The construct comprises gene 8803 placed downstream of a strong constitutive promoter (i.e., the Cauliflower mosaic virus (CaMV) 35S promoter) and upstream of a terminator (i.e., the Agrobacterium nopaline synthase (NOS) terminator). This cassette may be introduced into a cell of a carrot plant 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 may be cloned into a disarmed Ti plasmid suitable for replication in Agrobacterium, e.g., a pPZP Agrobacterium binary vector. The cassette can then be delivered into the plant cell by Agrob acterium-mc&wAe transformation using methods known in the art. Alternatively, an expression cassette containing gene 8803 can be cloned into a vector system enabling integration of the cassette into the genome of a carrot plant cell using CRISPR-mediated homologous recombination.

Claims

CLAIMS1. Protein capable of providing resistance to powdery mildew caused by the plant pathogen Erysiphe heraclei in carrot plants, wherein the protein comprises the amino acid sequence represented by SEQ ID No. 3, or an amino acid sequence having at least 90% sequence identity with SEQ ID No. 3, preferably at least 95% sequence identity, more preferably at least 98%, most preferably at least 99% sequence identity.

2. Nucleic acid capable of encoding a protein according to claim 1.

3. Nucleic acid of claim 2, wherein the nucleic acid comprises a 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% sequence identity, more preferably at least 98% sequence identity, most preferably at least 99% sequence identity.

4. Genomic fragment capable of encoding a protein according to claim 1 or a nucleic acid according to claim 2 or claim 3.

5. Genomic fragment of claim 4, wherein the genomic fragment 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, most preferably at least 99% sequence identity.

6. Dominant carrot resistance gene capable of translating into a protein according to claim 1, or capable of being transcribed into a nucleic acid according to claim 2 or claim 3, or comprising a genomic fragment according to claim 4 or claim 5.

7. Carrot plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the carrot plant comprises a dominant carrot resistance gene according to claim 6.

8. Carrot plant according to claim 7, wherein the carrot plant is a hybrid carrot plant, preferably a cytoplasmic sterile hybrid carrot plant.

9. Carrot plant according to claim 7 or claim 8, wherein the dominant carrot resistance gene is obtained, is obtainable, is derived, or is from a carrot plant of which representative seeds are deposited under deposit number NCIMB 44149.

10. Seeds, plant parts, cells, callus, suspension cultures, somatic embryos, clones, or progeny of a carrot plant according to any one of claims 7 to 9, wherein the seeds, plant parts, cells, callus, suspension culture, somatic embryos, clones, or progeny comprise a dominant carrot resistance gene according to claim 6.

11. Method for identifying a powdery mildew-resistant carrot plant, wherein the method comprises the step of detecting the presence of a dominant carrot resistance gene according to claim 6 in the genome of the carrot plant.

12. Method for identifying according to claim 11, wherein the method comprises the steps of: isolating cellular material from the plant; and establishing the presence of a protein according to claim 1, a nucleic acid according to claim 2 or claim 3, a genomic fragment according to claim 4 or claim 5, or a dominant carrot resistance gene according to claim 6 in the isolated cellular material.

13. Method for identifying according to claim 11 or claim 12, wherein establishing the presence comprises nucleic acid amplification and detection.

14. Method for providing a powdery mildew-resistant carrot plant, wherein the method comprises the step of introducing a dominant carrot resistance gene according to claim 6 in the genome of a susceptible carrot plant, preferably wherein the resulting powdery mildew-resistant carrot plant is not exclusively obtained by means of an essentially biological process.

15. Use of a nucleic acid comprising a sequence selected from the group consisting of SEQ ID Nos. 1, 2, 4, 5, 6, 7, 8 and 9 for identifying, or providing, a powdery mildew-resistant carrot plant.

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