Methods for regulating plant disease resistance and related proteins and biomaterials

Abstract

This application discloses methods for regulating plant disease resistance, as well as related proteins and biological materials, belonging to the field of genetic engineering technology. The technical problem this application aims to solve is how to regulate plant disease resistance, such as how to improve the clubroot resistance and / or Brassica clubroot resistance of cruciferous plants. To solve the above technical problem, this application provides a method for regulating plant clubroot resistance and / or Brassica clubroot resistance. The method may include: regulating the expression level of the gene encoding the Xa10 protein in the recipient plant and / or regulating the content of the Xa10 protein in the recipient plant to regulate the clubroot resistance and / or Brassica clubroot resistance of the recipient plant. The Xa10 protein may be a protein with the amino acid sequence SEQ ID NO:2. Studying the function of Xa10 and applying it in cruciferous breeding is of great significance for improving the clubroot resistance and / or Brassica clubroot resistance of cruciferous plants.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically involving methods for regulating plant disease resistance and related proteins and biomaterials. Background Technology

[0002] Cruciferous crops are not only important sources of oilseeds but also crucial vegetable crops. Clubroot is a significant disease of cruciferous plants, a soil-borne root disease caused by *Platycodon brassicae*, which severely impacts cruciferous crop production. Currently, control measures for cruciferous plants primarily rely on agricultural and chemical control. Agricultural control includes selecting resistant varieties, optimizing planting structures, and implementing agricultural control measures, as well as chemical control methods. For example, effective control of clubroot can be achieved by selecting resistant (tolerant) varieties, adjusting sowing dates, using biocontrol agents for seed coating, and using disease-free soil and substrate-based seedling trays. Chemical control methods include seed treatment with seed coating agents containing cyazofamid as the main component, and implementing appropriate control techniques in rapeseed transplanting and direct-seeding areas, such as seed treatment and pesticide application. Environmental factors also play a role: the occurrence of clubroot is closely related to environmental factors such as soil pH, humidity, and temperature. Therefore, improving the soil environment, such as adjusting soil pH and managing fertilizer and water properly, can effectively reduce the incidence of clubroot.

[0003] Despite some progress in clubroot disease control, several challenges remain. For example, the pathogenic mechanism of clubroot is complex, and the resistance of resistant varieties is unstable, increasing the difficulty of control. Currently, chemical control is only partially effective against clubroot, and is costly and environmentally unfriendly. Therefore, identifying clubroot resistance genes and cultivating resistant materials is crucial for the control of clubroot.

[0004] Executor (E) genes, as a novel class of plant disease resistance genes, possess unique disease resistance characteristics and are also an important resource of disease resistance genes, thus becoming a research hotspot in the field of plant immunity. Rice Xa10 encodes an Executor protein, but its relationship with clubroot disease has not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by this application is how to regulate the disease resistance of plants, such as how to improve the resistance of cruciferous plants to clubroot disease and / or resistance to clubroot fungus.

[0006] To address the aforementioned technical problems, this application provides a method for regulating clubroot resistance and / or Brassica clubroot resistance in plants. The method may include: regulating the expression level of the gene encoding the Xa10 protein in the recipient plant and / or regulating the content of the Xa10 protein in the recipient plant to regulate the clubroot resistance and / or Brassica clubroot resistance in the recipient plant.

[0007] The Xa10 protein may be any of the following:

[0008] a1) The amino acid sequence of the protein is SEQ ID NO:2;

[0009] a2) Proteins obtained by substituting, deleting and / or adding amino acids to the amino acid sequence of the protein shown in a1), which have more than 80% identity with the amino acid sequence shown in a1), and are associated with plant clubroot resistance and / or Brassica clubroot resistance.

[0010] a3) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

[0011] Furthermore, the regulation can be to increase, promote, or upregulate.

[0012] Furthermore, the regulation can also be reduced, suppressed, or downregulated.

[0013] Furthermore, the protein may be derived from rice.

[0014] In this application, SEQ ID NO:2 consists of 126 amino acid residues.

[0015] a3) The connection can be made via peptide bonds. Specifically, the C-terminus of the tag is dehydrated and condensed with the N-terminus of the protein in a1) or a2) to form a peptide bond.

[0016] Alternatively, the N-terminus of the tag may be linked to a peptide bond formed by the dehydration condensation of an amino acid at the C-terminus of the protein (a1) or (a2).

[0017] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0018] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0019] Furthermore, the method may include increasing the clubroot resistance and / or clubroot resistance of the recipient plant by increasing the expression level of the gene encoding the Xa10 protein and / or the content of the Xa10 protein in the recipient plant.

[0020] This application also provides a method for preparing a target plant with increased resistance to clubroot disease and / or resistance to *Plasmodiophora brassicae*. The method may include increasing the expression level of the gene encoding the Xa10 protein and / or the content of the Xa10 protein in the recipient plant to obtain a target plant with greater resistance to clubroot disease and / or resistance to *Plasmodiophora brassicae* than the recipient plant.

[0021] Furthermore, in the method, the expression level of the Xa10 protein encoding gene and / or the content of the Xa10 protein in the recipient plant can be increased by introducing the Xa10 protein encoding gene into the recipient plant.

[0022] Furthermore, in the method, the encoding gene may be a DNA molecule as described in any of the following:

[0023] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2;

[0024] g2) DNA molecules that have more than 80% identity with the DNA molecules described in g1 and that regulate plant resistance to clubroot disease and / or resistance to *Platycodon brassicae*.

[0025] Furthermore, in the method described, the plant may be a dicotyledonous plant.

[0026] Furthermore, in the method, the gene encoding the Xa10 protein can be introduced into the recipient plant in plasmid form.

[0027] In some embodiments of this application, the plasmid is the pGreenHygromycin-p3.6k-Xa10 plasmid, which contains a DNA molecule with the nucleotide sequence of SEQ ID NO:3. Specifically, positions 1 to 3731 of SEQ ID NO:3 represent the nucleotide sequence of the p3.6k promoter, positions 3732 to 4112 represent the coding sequence of the Xa10 protein, and positions 4113 to 4507 represent the 3'UTR region of the Xa10 protein. The pGreenHygromycin-p3.6k-Xa10 plasmid can express the Xa10 protein with the amino acid sequence of SEQ ID NO:2.

[0028] In some embodiments of this application, the pGreenHygromycin-p3.6k-Xa10 plasmid is transformed into recipient plants according to conventional Agrobacterium-mediated transformation methods in the art.

[0029] This application also provides the use of proteins and / or biomaterials associated with said proteins in any of the following, A1), in regulating plant clubroot resistance;

[0030] A2) Application in the preparation of products that regulate plant clubroot resistance;

[0031] A3) Application in regulating resistance to *Plantema rubrum* in plants;

[0032] A4) Application in the preparation of products that regulate resistance to *Cladosporium brassicae* in plants;

[0033] A5) Applications in plant breeding or plant-assisted breeding;

[0034] A6) Application in the preparation of plant breeding or plant-assisted breeding products;

[0035] The protein may be the Xa10 protein mentioned above.

[0036] In the application described, the regulation may be to increase, promote, or upregulate.

[0037] In the application described, the regulation may also be a reduction, suppression, or downregulation.

[0038] In the aforementioned application, the purpose of plant breeding may be to obtain plants with higher resistance to clubroot disease and / or resistance to clubroot fungus than the parent plants.

[0039] In the aforementioned application, the evaluation indicators for plant breeding include resistance to clubroot disease and / or resistance to clubroot fungus.

[0040] Furthermore, in the aforementioned applications, the biomaterial may be any of the following:

[0041] B1) The nucleic acid molecule encoding the Xa10 protein;

[0042] B2) Expression cassettes and / or constructs containing the nucleic acid molecules described in B1);

[0043] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette and / or construct described in B2);

[0044] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette and / or construct described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0045] B5) Transgenic plant cell lines containing the nucleic acid molecules described in B1), transgenic plant cell lines containing the expression cassette and / or construct described in B2), or transgenic plant cell lines containing the recombinant vector described in B3);

[0046] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), transgenic plant tissue containing the expression cassette and / or construct described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0047] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), transgenic plant organs containing the expression cassette and / or construct described in B2), or transgenic plant organs containing the recombinant vector described in B3).

[0048] Furthermore, in the aforementioned application, the expression cassette described in B2) refers to DNA capable of expressing the Xa10 protein in a host cell, which may include not only promoters that initiate transcription of protein-coding genes, but also terminators and / or enhancer sequences that terminate transcription of protein-coding genes.

[0049] Further, in the aforementioned application, specifically, B2) the expression cassette is a DNA molecule whose nucleotide sequence is the sequence of SEQ ID NO:3 from position 1 to position 4507. Specifically, positions 1 to 3731 of SEQ ID NO:3 are the nucleotide sequence of the p3.6k promoter, positions 3732 to 4112 are the coding sequence for the Xa10 protein, and positions 4113 to 4507 are the 3'UTR region of the Xa10 protein.

[0050] Furthermore, in the aforementioned application, the recombinant expression vector (B3) may contain the encoding gene of the Xa10 protein (a DNA molecule with the nucleotide sequence of SEQ ID NO:1) and may express the Xa10 protein with the amino acid sequence of SEQ ID NO:2.

[0051] In some embodiments of this application, the recombinant expression vector is named pGreenHygromycin-p3.6k-Xa10 plasmid.

[0052] Furthermore, in the aforementioned applications, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0053] Furthermore, in the aforementioned applications, the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0054] Furthermore, in the aforementioned applications, the transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0055] Furthermore, in the aforementioned applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0056] Further, the nucleic acid molecule described in B1) may be any of the following: a DNA molecule and / or an RNA molecule transcribed from the DNA molecule:

[0057] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2;

[0058] g2) DNA molecules that have more than 80% identity with the DNA molecules described in g1 and that regulate plant resistance to clubroot disease and / or resistance to *Platycodon brassicae*.

[0059] Furthermore, in the aforementioned application, the plant is a dicotyledonous plant.

[0060] In this application, the dicotyledonous plant may be selected from plants of the Brassicaceae family.

[0061] In this application, the cruciferous plants may be selected from, but are not limited to, the Brassica genus: Chinese cabbage, purple cabbage (red rapeseed) (Guangdong cabbage), cabbage (cabbage), kohlrabi (cabbage variety), cauliflower (cauliflower, cabbage variety), broccoli (cabbage variety), mustard greens (pickled mustard greens, pickled mustard greens, turnip mustard greens (root mustard greens), yellow mustard (seed powder)), snow cabbage (mustard green variety), bok choy (small bok choy), Shanghai bok choy (small bok choy variety), and European rapeseed (rapeseed).

[0062] and / or Radish genus: red-skinned radish, white-skinned radish, green-skinned radish, etc.;

[0063] and / or Capsella: Capsella bursa-pastoris;

[0064] And / or horseradish: horseradish (horse radish, wasabi);

[0065] And / or Arabidopsis: Arabidopsis.

[0066] The beneficial technical effects achieved by this application are as follows:

[0067] This application discloses for the first time the application of the Xa10 protein and its encoding gene in regulating clubroot resistance in cruciferous plants and / or resistance to *Cladophora brassicae*. Studying the function of Xa10 and applying it in cruciferous breeding is of great significance for improving the resistance of cruciferous plants to clubroot and / or *Cladophora brassicae*.

[0068] Experiments have shown that the protein and its encoding gene provided in this application can regulate clubroot resistance and / or Brassica rapa resistance in plants: the clubroot resistance and / or Brassica rapa resistance of overexpressed lines were significantly higher than those of wild-type recipients. Therefore, the protein and its encoding gene provided in this application have important theoretical significance and practical value for cultivating plants with enhanced clubroot resistance and / or Brassica rapa resistance. Attached Figure Description

[0069] Figure 1This study analyzed the resistance of transgenic Arabidopsis thaliana plants to clubroot disease. A shows wild-type Arabidopsis thaliana Col-0 and p3.6k-Xa10_Col-0 transgenic plants inoculated with *Plasmodiophora stylosa* collected from Dayi County, Sichuan Province. After 21 days of inoculation, the disease symptoms were observed, recorded, and photographed. The entire plant was photographed using a Canon EOS 6D Mark II SLR camera; the white scale bar in the image indicates 0.5 cm. B shows the disease incidence and disease index of wild-type Arabidopsis thaliana Col-0 and p3.6k-Xa10_Col-0 transgenic plants 21 days after inoculation with *Plasmodiophora stylosa*. The experiment was conducted in triplicate, with at least 24 Arabidopsis thaliana plants inoculated each time. Detailed Implementation

[0070] Terminology in this application:

[0071] Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0072] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0073] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows:

[0074] In this application, "identity" refers to the similarity of amino acid sequences or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0075] The aforementioned 80% or higher consistency can be 80%, 85%, 90%, or 95% or higher.

[0076] The aforementioned 80% or higher level of consistency can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 85% or higher level of consistency can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 90% or higher level of consistency can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 95% or higher level of consistency can be at least 95%, 96%, 97%, 98%, or 99%.

[0077] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0078] As used in this article, "plant" includes explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0079] Plant cells are biological cells of plants, derived from plants or derived from cultures obtained by culturing cells taken from plants. As used herein, “transgenic plant cell” means any plant cell transformed with a stably integrated recombinant DNA molecule, construct, expression cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0080] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0081] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0082] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0083] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0084] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.

[0085] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0086] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0087] Rice Xa10 encodes an executor protein that plays a crucial role in plant disease resistance. Previous studies identified a highly clubroot-resistant gene, WTS (WeiTsing), which is specifically expressed on the periphery of the stele after induction by clubroot fungus, preventing pathogen invasion or colonization of the stele. This application constructed a Xa10 gene expression vector driven by the 3.6K promoter upstream of WTS, and obtained p3.6k-Xa10_Col-0 transgenic Arabidopsis thaliana in the susceptible material Col-0. The results showed that the Xa10-transgenic Arabidopsis thaliana exhibited significantly enhanced resistance to clubroot fungus infection compared to wild-type Arabidopsis thaliana Col-0. This indicates that the Xa10 gene functions in plant resistance to clubroot fungus, and the Xa10 protein and its encoding gene can provide new gene resources for clubroot resistance breeding in cruciferous crops.

[0088] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0089] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0090] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0091] The plant binary expression vector pGreenHygromycin in the following examples is the vector backbone obtained by deleting NLS-3×VENUS from pGreenHygromycinNLS-3×VENUS. pGreenHygromycinNLS-3×VENUS is described in the literature "Zhou, F. et al. Co-incidence of damage and microbial patterns controls localized immune responses in roots. Cell 180, 440-453 (2020)". It is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to replicate the relevant experiments of this application and may not be used for other purposes.

[0092] The Agrobacterium GV3101 used in the following examples is owned by the applicant and is described in the literature "Liang, X. et al. Ligand-triggered de-repression of Arabidopsis heterotrimeric G proteins coupled to immune receptor kinases. Cell Research 28, 529-543 (2019)". It is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this application and may not be used for other purposes.

[0093] The *Plasmodiophora brassicaefield isolate Dayi (PbDy)*, ​​*Arabidopsis thaliana* ecotype Est-1, and *Arabidopsis thaliana* ecotype Col-0 from Dayi County, Sichuan Province, in the following examples are owned by the applicant and described in the literature “Wang W. et al. WeiTsing, a pericycle-expressed ion channel, safeguards the stele to confer clubroot resistance. Cell 186, 2656-2671 (2023).” These materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. These biological materials are only for repeating the relevant experiments of this application and may not be used for other purposes.

[0094] The rice IRBB10 described in the following examples is described in the literature "The rice TAL effector-dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum. Plant Cell 26, 497-515 (2014)." It is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to replicate the relevant experiments of this application and may not be used for other purposes.

[0095] Unless otherwise specified, the cultivation conditions for the long-day plant culture chamber in the following examples are: temperature 22°C, relative humidity 60%, and a photoperiod of 16 hours of light and 8 hours of darkness. The cultivation conditions for the short-day plant culture chamber are: temperature 22°C, relative humidity 60%, and a photoperiod of 10 hours of light and 14 hours of darkness.

[0096] The amino acid sequence of the Xa10 protein in the following examples is SEQ ID NO:2, and the nucleotide sequence of its encoding sequence is SEQ ID NO:1.

[0097] Example 1: Analysis of resistance of Xa10 transgenic Arabidopsis thaliana to clubroot bacteria

[0098] 1. Construction of a plant binary expression vector for the Xa10 gene

[0099] 1) Obtaining the WTS starter sequence

[0100] DNA was extracted from the Arabidopsis ecotype Est-1, and the following primers were designed to amplify the 3.6kb promoter sequence upstream of the WTS gene. This promoter was named p3.6k, and its nucleotide sequence is the DNA molecule at positions 1 to 3731 of SEQ ID NO:3. The PCR fragment containing the p3.6k promoter was ligated between the SalI restriction sites of the pGreenHygromycin vector using SalI digestion to obtain the recombinant vector pGreenHygromycin-p3.6k.

[0101] p-GREEN-SalI-EP-F:gtaccgggccccccctcgagAGCCAGACAAACTCCCATCT;

[0102] p-GREEN-SalI-EP-R:agcttatcgataccgtcgacAATTTGTATCTTTGCAGTT.

[0103] 2) Obtaining the Xa10 gene fragment

[0104] RNA was extracted from rice IRBB10 and reverse transcribed to obtain the CDS. Based on the CDS and 3' UTR sequence of the rice Xa10 gene, the following amplification primers were designed for PCR amplification, and the PCR products were recovered by agarose gel electrophoresis.

[0105] Xa10-SalI-F: aactgcaaagatacaaattatgcagctgATGCTCACATT

[0106] Xa10-XbalI-R: cattaaagcaggactctagaTGACCACCCGGGGATCCTTT

[0107] 3) Obtaining the pGreenHygromycin-p3.6k-Xa10 expression vector

[0108] The DNA fragment between SalI and XbaI in the pGreenHygromycin-p3.6k vector was replaced with a Xa10 gene fragment (positions 3732 to 4112 of SEQ ID NO:3) to obtain the recombinant vector pGreenHygromycin-p3.6k-Xa10. Sequencing results showed that the recombinant vector pGreenHygromycin-p3.6k-Xa10 contained a DNA molecule with the nucleotide sequence of SEQ ID NO:3, where positions 1 to 3731 of SEQ ID NO:3 are the nucleotide sequence of the p3.6k promoter, positions 3732 to 4112 are the coding sequence of the rice-derived Xa10 gene, and positions 4113 to 4507 are the 3'UTR region of the Xa10 protein. The recombinant vector pGreenHygromycin-p3.6k-Xa10 is a WTS promoter-driven Xa10 gene expression vector that can express the Xa10 protein with the amino acid sequence of SEQ ID NO:2.

[0109] 2. Obtaining transgenic Arabidopsis thaliana

[0110] 1) Agrobacterium-mediated transformation

[0111] The pGreenHygromycin-p3.6k-Xa10 plasmid was introduced into Agrobacterium GV3101 strain via electroporation. The specific steps are as follows: 1 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent cells, transferred to an electroporation cuvette, and electroporated at 2500V / 6ms. Then, 500 μL of LB medium was added, and the mixture was incubated at 28℃ in a shaker for 1 h. The bacterial culture was then evenly spread on LB agar plates containing 50 mg / L kanamycin and 50 mg / L gentamicin. The plates were inverted and incubated at 28℃ for 48 h to screen for positive colonies.

[0112] 2) Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0113] Agrobacterium GV3101 carrying the pGreenHygromycin-p3.6k-Xa10 plasmid was streaked onto LB agar plates containing 50 mg / L kanamycin and 50 mg / L gentamicin, and incubated at 28°C for 48 h. Single colonies were picked and transferred to 2 mL of LB liquid medium containing 50 mg / L kanamycin and 50 mg / L gentamicin, and incubated overnight at 28°C for 12 h in a shaker.

[0114] Inoculate 2 mL of bacterial culture into 300 mL of LB liquid medium containing the corresponding antibiotic and incubate in a shaker at 28 °C for 12 h.

[0115] Collect bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, resuspend in 5% sucrose aqueous solution, and adjust OD. 600 Add 0.017% Silwet L-77 solution to a concentration between 0.8 and 1.0, and mix thoroughly.

[0116] Arabidopsis ecotype Col-0 plants with good growth were selected after about 8 weeks of growth in a long-day plant culture room (temperature 23℃, relative humidity 60%, light cycle of 16h light and 8h darkness). Mature pods were cut off, and the inflorescences were soaked in bacterial solution for 5 minutes for transformation. After 24 hours of cultivation in the dark, they were cultured under normal light.

[0117] After the seeds mature, they are surface disinfected with 30% 84 disinfectant for 10 minutes, sterilized by washing ddH2O 5 times, and spread evenly on 1 / 2 MS plates containing 25 mg / L hygromycin and 50 mg / L carbenicillin. They are vernalized at 4℃ for 48 hours. The plates are then transferred to a plant culture chamber (temperature 22℃, relative humidity 60%, photoperiod of 16 hours light and 8 hours dark). After 10 to 12 days, healthy seedlings (T0 generation) are selected and transplanted to a short-day plant culture room for conventional culture (temperature 22℃, relative humidity 60%, photoperiod of 10 hours light and 14 hours dark).

[0118] 3) Obtaining Xa10 transgenic Arabidopsis thaliana

[0119] Transgenic T0 generation Arabidopsis thaliana was self-pollinated to obtain T1 generation transgenic lines. T1 generation transgenic Arabidopsis thaliana plants grown for approximately 4 weeks in a short-day plant culture chamber (temperature 22℃, relative humidity 60%, photoperiod of 10h light and 14h darkness) were selected. One to two leaves were taken from each plant, and genomic DNA was extracted using the CTAB method for hygromycin resistance gene (HYG) detection. Plants testing positive by PCR were individually harvested for seeds and designated as T2 generation. This process was repeated, using a combination of hygromycin resistance screening and PCR detection to obtain homozygous T3 generation p3.6k-Xa10_Col-0 transgenic Arabidopsis thaliana plants.

[0120] Hyg-F: AAGCCTGAACTCACCGCGA;

[0121] Hyg-R:GTTTCCACTATCGGCGAGTAC.

[0122] II. Analysis of resistance of Col-0 and p3.6k-Xa10_Col-0 transgenic Arabidopsis to *Platycorrhizal*.

[0123] 1. Inoculation with *Cladosporium brassicae*

[0124] Wild-type Arabidopsis thaliana Col-0 and p3.6k-Xa10_Col-0 transgenic Arabidopsis thaliana plants were taken and cultured in a short-day plant culture room. After the plants emerged from the soil and grew for about 20 days, a root-drenching experiment was conducted to investigate the infection of *Plasmodiophora stylosa*. The experiment was repeated three times, with at least 24 wild-type Arabidopsis thaliana Col-0 and p3.6k-Xa10_Col-0 transgenic Arabidopsis thaliana plants inoculated each time. The specific steps are as follows:

[0125] 1) Chop the root tissue of Chinese cabbage containing *Cladosporium brassicae* from Dayi County, Sichuan Province, add an appropriate amount of sterilized ddH2O, and grind it with a tissue homogenizer.

[0126] 2) Filter with 8 layers of gauze and transfer the filtrate into a clean Erlenmeyer flask.

[0127] 3) Measure the spore concentration of *Plasmodiophora brassicae* in the bacterial culture using a hemocytometer.

[0128] 4) Dilute the concentration of *Plantochoria brassicae* to 1×10⁻⁶. 7 The bacterial culture was diluted to 1 mL using a pipette and then applied to the roots of Arabidopsis thaliana for inoculation.

[0129] 2. Disease index and incidence rate statistics

[0130] On day 21 after Arabidopsis thaliana root inoculation with *Plasmodiophora brassicae*, the incidence rate and disease index were statistically analyzed using the *Plasmodiophora brassicae* grading system. The statistical criteria for the *Plasmodiophora brassicae* grading system in Arabidopsis thaliana are as follows:

[0131] Grade 0, no symptoms, intact root system;

[0132] Grade 1, with small nodules on the lateral roots, but no symptoms on the main root;

[0133] Grade 2, fewer lateral roots, small lumps on the main root;

[0134] Grade 3, with large lumps on the lateral roots and main root;

[0135] Grade 4, the main root is completely swollen and rotten.

[0136] The formulas for calculating incidence and disease index are as follows:

[0137] Incidence rate = (Number of diseased plants / Total number of inoculated plants) × 100%;

[0138] Disease index = (Σ(disease level × number of diseased plants at each level)) / (total number of inoculated plants × highest disease level 4) × 100.

[0139] Symptoms, incidence, and disease index of plants 21 days after inoculation with clubroot fungus are as follows: Figure 1 As shown in Figures A and B, the results indicate that wild-type Arabidopsis thaliana Col-0, after inoculation with clubroot fungus, exhibited susceptibility, characterized by swollen taproots and destroyed lateral roots. However, against the background of the susceptible Col-0 material, the p3.6k-Xa10_Col-0 transgenic Arabidopsis plants showed resistance, with relatively normal root development and only a few plants exhibiting mild symptoms. These results demonstrate that the Xa10 gene can enhance Arabidopsis resistance to clubroot fungus, providing a new gene resource for clubroot resistance breeding in plants.

[0140] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for regulating plant resistance to clubroot disease and / or resistance to *Cladophora brassicae*, characterized in that, The method includes: regulating the clubroot resistance and / or Brassica clubroot resistance of recipient plants by regulating the expression level of the gene encoding the Xa10 protein in the recipient plant and / or regulating the content of the Xa10 protein in the recipient plant. The Xa10 protein is any of the following: a1) The amino acid sequence of the protein is SEQ ID NO:2; a2) Proteins obtained by substituting, deleting and / or adding amino acids to the amino acid sequence of the protein shown in a1), which have more than 80% identity with the amino acid sequence shown in a1), and are associated with plant clubroot resistance and / or Brassica clubroot resistance. a3) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

2. The method according to claim 1, characterized in that, The method includes increasing the resistance of recipient plants to clubroot disease and / or resistance to *Cladophora brassicae* by increasing the expression level of the gene encoding the Xa10 protein and / or the content of the Xa10 protein in the recipient plant.

3. A method for preparing target plants with increased resistance to clubroot disease and / or *Cladosporium brassicum*, characterized in that, The method includes obtaining a target plant with greater resistance to clubroot and / or resistance to *Platycodon brassicae* than the recipient plant by increasing the expression level of the gene encoding the Xa10 protein as described in claim 1 and / or increasing the content of the Xa10 protein in the recipient plant.

4. The method according to claim 2 or 3, characterized in that, The expression level of the Xa10 protein encoding gene and / or the content of the Xa10 protein in the recipient plant are increased by introducing the Xa10 protein encoding gene into the recipient plant.

5. The method according to any one of claims 2-4, characterized in that, The encoding gene is a DNA molecule that is any of the following: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2; g2) DNA molecules that have more than 80% identity with the DNA molecules described in g1 and that regulate plant resistance to clubroot disease and / or resistance to *Platycodon brassicae*.

6. The method according to claim 1 or 3, characterized in that, The plant in question is a dicotyledonous plant.

7. The use of proteins and / or biological materials associated with said proteins in any of the following: A1) Application in regulating plant resistance to clubroot disease; A2) Application in the preparation of products that regulate plant clubroot resistance; A3) Application in regulating resistance to *Plantema rubrum* in plants; A4) Application in the preparation of products that regulate resistance to *Cladosporium brassicae* in plants; A5) Applications in plant breeding or plant-assisted breeding; A6) Application in the preparation of plant breeding or plant-assisted breeding products; The protein is the Xa10 protein as described in claim 1.

8. The application according to claim 7, characterized in that, The biomaterial is any one of the following: B1) A nucleic acid molecule encoding the Xa10 protein as described in claim 1; B2) Expression cassettes and / or constructs containing the nucleic acid molecules described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette and / or construct described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette and / or construct described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Transgenic plant cell lines containing the nucleic acid molecules described in B1), transgenic plant cell lines containing the expression cassette and / or construct described in B2), or transgenic plant cell lines containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), transgenic plant tissue containing the expression cassette and / or construct described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), transgenic plant organs containing the expression cassette and / or construct described in B2), or transgenic plant organs containing the recombinant vector described in B3).

9. The application according to claim 8, characterized in that, The nucleic acid molecule described in B1) is any of the following DNA molecules and / or RNA molecules transcribed from the DNA molecule: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2; g2) DNA molecules that have more than 80% identity with the DNA molecules described in g1 and that regulate plant resistance to clubroot disease and / or resistance to *Platycodon brassicae*.

10. The application according to any one of claims 7-9, characterized in that, The plant in question is a dicotyledonous plant.

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