Application of npr1 protein and related biological materials in regulating resistance of plants to root-knot nematode

Abstract

The application discloses application of NPR1 protein and related biological materials thereof in regulating resistance of plants to clubroot disease, and belongs to the technical field of genetic engineering. The technical problem to be solved by the application is how to regulate resistance of plants to Plasmodiophora brassicae. Specifically, the technical problem to be solved by the application is how to regulate resistance of cruciferous plants to Plasmodiophora brassicae. To solve the above technical problem, the application provides application of NPR1 protein or biological materials related to the NPR1 protein in regulating resistance of plants to clubroot disease. The application discloses, for the first time, application of NPR1 protein and a coding gene thereof in regulating resistance of cruciferous plants to clubroot disease and / or Plasmodiophora brassicae. Research on the function of NPR1 and application of the function in cruciferous plant breeding are of great significance to the mechanism of resistance of cruciferous plants to clubroot disease and / or Plasmodiophora brassicae and to resistance breeding.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically involving the application of NPR1 protein and related biomaterials in regulating plant clubroot resistance. Background Technology

[0002] Plasmodiophora brassicae Woronin is an organism belonging to the phylum Myxomycetes and class Plasmodiomycetes. It primarily lives parasitically on the roots of cruciferous plants, causing clubroot disease. This disease can severely impact the yield and quality of cruciferous crops, including common vegetables such as Chinese cabbage, bok choy, flowering cabbage, and mustard greens. Clubroot disease caused by Plasmodiophora brassicae leads to stunted seedlings and leaves that wilt easily in sunlight. Nodules appear on the roots of diseased plants, and in severe cases, the root system can completely rot. Control methods include avoiding the use of glyphosate herbicides, promptly removing and destroying diseased plants, treating the soil with chemical agents such as dichlorvos, adjusting soil pH, and strengthening cultivation management.

[0003] The pathogenic mechanism of *Platycodon brassicae* is complex, with its zoospores and dormant spores playing crucial roles. Recent research advances have included exploring the roles of plant hormones such as auxins, cytokinins, salicylic acid, jasmonic acid, and abscisic acid in the pathogenic process, as well as the effects of substances like oxidases, glucosinolates, and flavonoids. To better control clubroot disease, researchers have established a rapid detection and risk warning system for *Platycodon brassicae* in soil using quantitative real-time PCR (qPCR). This system helps identify the presence of the pathogen and its potential risks, providing technical support for early detection and control. *Platycodon brassicae* exists in different physiological races, and the pathogenicity of these races varies in different regions. For example, the pathogen is more virulent in Wenling and Shanghai, while the pathogen in cabbage from Wenzhou is less virulent. Furthermore, different host varieties show significant differences in resistance responses to the same or different pathogenic sources. Therefore, screening for resistant varieties and clarifying the pathogenic mechanism of the pathogen are crucial for the control of clubroot disease.

[0004] Cruciferous crops are not only important sources of oilseeds but also crucial vegetable crops. Currently, neither chemical nor agricultural control methods are effective in controlling clubroot disease, and these methods are costly and environmentally polluting. Therefore, identifying resistance genes for *Cladophora brassicae* and improving plant resistance to this fungus is of great significance for crop breeding and agricultural production.

[0005] NPR1 (non-expressor of PR genes) is a salicylic acid receptor protein that plays an important role in salicylic acid-mediated defense responses. Whether NPR1 is involved in regulating plant resistance to *Plasmodiophora brassicae* remains to be studied. Summary of the Invention

[0006] The technical problem this application aims to solve is: how to regulate the resistance of plants to *Cladosporium brassicae*. Specifically, the technical problem this application aims to solve is: how to regulate the resistance of cruciferous plants to *Cladosporium brassicae*.

[0007] To solve the above-mentioned technical problems, this application provides any of the following applications:

[0008] A1) The application of NPR1 protein or biomaterials related to said NPR1 protein in regulating plant clubroot resistance;

[0009] A2) The application of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of products that regulate plant clubroot resistance;

[0010] A3) The application of NPR1 protein or biomaterials related to said NPR1 protein in regulating resistance to *Cladosporium brassicae* in plants;

[0011] A4) The application of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of products that regulate resistance to *Cladosporium brassicae* in plants;

[0012] A5) The application of NPR1 protein or biological materials related to said NPR1 protein in plant breeding or plant-assisted breeding;

[0013] A6) The use of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of plant breeding or plant-assisted breeding products;

[0014] The NPR1 protein may be any of the following proteins:

[0015] a1) A protein whose amino acid sequence contains SEQ ID NO:2;

[0016] a2) Proteins obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in a1) that have more than 80% similarity to the amino acid sequence shown in a1) and are associated with resistance to *Plantago brassicae*.

[0017] a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

[0018] a1) The NPR1 protein may be a protein whose amino acid sequence is SEQ ID NO:2.

[0019] In this application, SEQ ID NO:2 consists of 593 amino acid residues.

[0020] In this application, the protein may be derived from Arabidopsis thaliana.

[0021] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0022] 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.

[0023] Further, the connection described in a3) may be a peptide bond formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in a1) or a2). Alternatively, the connection described in a3) may be a peptide bond formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in a1) or a2).

[0024] In this application, the regulation may be to increase, promote, or adjust.

[0025] In this application, the regulation may also be a reduction, suppression, or downregulation.

[0026] In this application, the indicators for plant breeding may include resistance to clubroot fungus.

[0027] In this application, the purpose of plant breeding may include cultivating plants with altered resistance to *Cladosporium brassicae*. Specifically, the purpose of plant breeding includes cultivating plants with reduced resistance to *Cladosporium brassicae* and / or cultivating plants with increased resistance to *Cladosporium brassicae*.

[0028] The plants with reduced resistance to *Plasmodiophora brassicae* obtained in this application can be used to study the resistance mechanism of *Plasmodiophora brassicae* in plants.

[0029] The plants obtained in this application with enhanced resistance to *Platycorrhizal* can be used for *Platycorrhizal* resistance breeding.

[0030] Furthermore, the biomaterial may be any of the following:

[0031] B1) Nucleic acid molecules that inhibit or reduce the expression of the NPR1 protein-encoding gene;

[0032] B2) Expression cassettes and / or constructs containing the nucleic acid molecules described in B1);

[0033] 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);

[0034] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette and / or construct described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0035] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette and / or construct described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0036] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette and / or construct described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0037] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette and / or construct described in B2), or transgenic plant organs containing the recombinant vector described in B3);

[0038] B8), the nucleic acid molecule encoding the NPR1 protein;

[0039] B9) Expression cassettes and / or constructs containing the nucleic acid molecules described in B8), recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues and / or transgenic plant organs.

[0040] Furthermore, the nucleic acid molecule described in B1) may be RNA encoding a gene that targets the NPR1 protein or DNA encoding the RNA.

[0041] Furthermore, the nucleotide at the target site of the nucleic acid molecule described in B1) may be positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0042] Furthermore, the nucleic acid molecule described in B8) can be any one of the following DNA molecules described in g1)-g3):

[0043] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1;

[0044] g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3;

[0045] g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates the resistance of plant *Plantochloa crus-galli*.

[0046] In some embodiments of this application, the expression cassette described in B2) may be an expression cassette for transcribing the sgRNA of the gene encoding the NPR1 protein described above.

[0047] The recombinant vector described in B3) may be pHEE401E-NPR1. The pHEE401E-NPR1 vector can transcribe sgRNA targeting the DNA molecule shown in SEQ ID NO:3 and encode the effector protein of the CRISPR / Cas9 system: the Cas9 protein.

[0048] Furthermore, in the aforementioned application, the expression cassette (B9) refers to a DNA sequence encoding the NPR1 protein that can express the NPR1 protein in a host cell. This DNA sequence may include not only a promoter that initiates transcription of the NPR1 protein encoding gene, but also a terminator and / or enhancer sequence that terminates transcription of the NPR1 protein encoding gene.

[0049] Furthermore, in the aforementioned applications, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0050] Furthermore, in the aforementioned applications, the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0051] Furthermore, in the aforementioned applications, the transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0052] Furthermore, in the aforementioned applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0053] This application also provides a method for regulating resistance to *Plantochloa crus-galli* in plants, the method comprising regulating resistance to *Plantochloa crus-galli* in recipient plants by regulating the expression level of the NPR1 gene encoding the NPR1 protein in the recipient plant and / or regulating the content of the NPR1 protein in the recipient plant.

[0054] Furthermore, the method includes reducing the resistance of the recipient plant to *Cladosporium brassicae* by reducing the expression level of the gene encoding the NPR1 protein and / or reducing the content of the NPR1 protein in the recipient plant, wherein the recipient plant contains the gene encoding the NPR1 protein.

[0055] This application also provides a method for obtaining a target plant with reduced resistance to *Plantochloa brassicae*, the method comprising obtaining a target plant with reduced resistance to *Plantochloa brassicae* by reducing the expression level of the gene encoding the NPR1 protein in the recipient plant and / or reducing the content of the NPR1 protein in the recipient plant, wherein the recipient plant contains the gene encoding the NPR1 protein.

[0056] Furthermore, the method can achieve the purpose of reducing the expression level of the gene encoding the NPR1 protein in the recipient plant and / or reducing the content of the NPR1 protein in the recipient plant through the manner described in C1) below.

[0057] C1) By knocking out the gene encoding the NPR1 protein in the recipient plant using a genome editing system, the expression level of the gene encoding the NPR1 protein and / or the content of the NPR1 protein in the recipient plant are reduced.

[0058] Furthermore, the genome editing system is a CRISPR / Cas system.

[0059] Furthermore, the CRISPR / Cas system can express gRNA that targets the gene encoding the NPR1 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein.

[0060] Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0061] Furthermore, the method can achieve the purpose of reducing the expression level of the gene encoding the NPR1 protein in the recipient plant and / or reducing the content of the NPR1 protein in the recipient plant through the manner described in C2) below.

[0062] C2) Perform any of the following mutations on the genome sequence of the recipient plant:

[0063] C2-1) The NPR1 gene with the nucleotide sequence SEQ ID NO:3 is deleted at positions 113-121, thereby knocking out the NPR1 gene;

[0064] C2-2) An adenine deoxyribonucleotide was inserted between positions 113 and 114 of the NPR1 gene with the nucleotide sequence SEQ ID NO:3, thereby knocking out the NPR1 gene.

[0065] Furthermore, in this application, the plant is a dicotyledonous plant.

[0066] In this application, the dicotyledonous plant may be selected from plants of the Brassicaceae family.

[0067] 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, lotus white), 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), European rapeseed (rapeseed);

[0068] and / or Radish genus: red-skinned radish, white-skinned radish, green-skinned radish, etc.;

[0069] and / or Capsella: Capsella bursa-pastoris;

[0070] And / or horseradish: horseradish (horse radish, wasabi);

[0071] And / or Arabidopsis: Arabidopsis.

[0072] This application also provides biological materials related to the NPR1 protein, which may be any one of B1) to B9) above.

[0073] Furthermore, the nucleic acid molecule described in B1) may be RNA encoding a gene that targets the NPR1 protein or DNA encoding the RNA.

[0074] Further, the nucleotides of the target site of the nucleic acid molecule described in B1) are positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0075] Furthermore, the nucleic acid molecule described in B8) can be any one of the following DNA molecules described in g1)-g3):

[0076] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1;

[0077] g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3;

[0078] g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates the resistance of plant *Plantochloa crus-galli*.

[0079] This application also provides a composition for genome editing, said composition being the genome editing system described above.

[0080] Furthermore, the genome editing system is a CRISPR / Cas system.

[0081] Furthermore, the CRISPR / Cas system can express gRNA that targets the gene encoding the NPR1 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein.

[0082] Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0083] The beneficial technical effects achieved by this application are as follows:

[0084] This application discloses for the first time the application of the NPR1 protein and its encoding gene in regulating clubroot resistance and / or clubroot resistance in cruciferous plants. Studying the function of NPR1 and applying it in cruciferous breeding is of great significance for understanding the mechanisms of clubroot resistance and / or clubroot resistance in cruciferous plants, as well as for resistance breeding. Attached Figure Description

[0085] Figure 1 Sequencing mutation sites for the NPR1 gene knockout mutants npr1-1 and npr1-2. The npr1-1 mutant has a deletion of bases 113-121 in SEQ ID NO:3 of the NPR1 gene, while the npr1-2 mutant has an insertion of an adenine deoxyribonucleotide (A) between positions 113 and 114 in SEQ ID NO:3 of the NPR1 gene.

[0086] Figure 2 This study analyzed the resistance of transgenic Arabidopsis thaliana plants to clubroot disease. Figure A shows wild-type Arabidopsis thaliana Est-1, NPR1 gene knockout (npr1-1 and npr1-2) mutant plants inoculated with *Plasmodiophora stylosa* collected from Dayi County, Sichuan Province. After 21 days of inoculation, the disease symptoms were observed, recorded, and photographed. Whole plant images were taken using a Canon EOS 6D Mark II SLR camera; the white bar indicates 1 cm. Root images were taken using a stereomicroscope; the red bar indicates 1 mm. Figures B and C show the disease incidence and disease index of wild-type Arabidopsis thaliana Est-1, NPR1 gene knockout (npr1-1 and npr1-2) mutant plants 21 days after inoculation with *Plasmodiophora stylosa*. The experiment was repeated in triplicate, with at least 12 Arabidopsis thaliana plants inoculated each time. Different letters in the figures indicate statistically significant differences (P < 0.05, mean ± SE, one-way ANOVA, Tukey's post-test). Detailed Implementation

[0087] Terminology in this application:

[0088] 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.

[0089] 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.

[0090] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows:

[0091] 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.

[0092] The aforementioned 80% or higher consistency can be 80%, 85%, 90%, or 95% or higher.

[0093] 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%.

[0094] 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.

[0095] As used in this article, "plant" includes explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0096] The term "cruciferous plants" as used in this article may be selected from, but is not limited to, any of the following:

[0097] Brassica genus: Chinese cabbage, purple rapeseed (red rapeseed) (Guangdong cabbage), cabbage (cabbage, lotus white), kohlrabi (cabbage variety), cauliflower (cauliflower), broccoli (cabbage variety), mustard greens (pickled mustard greens, pickled mustard greens, turnips (root mustard greens), yellow mustard (seed powder)), snow cabbage (mustard green variety), bok choy (small bok choy), Shanghai bok choy (small bok choy variety), European rapeseed (rapeseed);

[0098] and / or Radish genus: red-skinned radish, white-skinned radish, green-skinned radish, etc.;

[0099] and / or Capsella genus:: Capsella bursa-pastoris;

[0100] And / or horseradish: horseradish (horse radish, wasabi);

[0101] And / or Arabidopsis: Arabidopsis.

[0102] As used herein, "plant part" can refer to any organ or intact tissue of a plant, such as meristem, bud organ / structure (e.g., leaf, stem, or node), root, flower or floral organ / structure (e.g., flower, bract, sepal, petal, stamen, carpel, anther, and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., mature ovary), propagule, or other plant tissue (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof. The plant parts disclosed herein can be viable, non-viable, renewable, and / or non-renewable. "Propagule" can include any plant part that can grow into a whole plant.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0107] 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.

[0108] 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.

[0109] 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).

[0110] 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.

[0111] In this application, "editing" or "genome editing" means using targeted genome editing technology to produce a targeted mutation, deletion, inversion, or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, or at least 10,000 nucleotides of endogenous plant genome nucleic acid sequence.

[0112] In this application, “editing” or “genome editing” also covers the use of targeted genome editing technology to target or integrate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides into the endogenous genome of a plant.

[0113] In this application, a “target site” for genome editing refers to a location within a plant genome of a polynucleotide sequence that is targeted and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone and / or its complementary DNA strand. The site-specific nuclease may bind to the target site, for example, via a non-coding guide RNA (e.g., but not limited to CRISPR RNA (crRNA) or single-strand guide RNA (sgRNA)). The non-coding guide RNA provided herein may be complementary to the target site (e.g., complementary to the strand of a double-stranded nucleic acid molecule or the chromosome of the target site). A “target site” also refers to a location within the plant genome of a polynucleotide sequence that is bound and cleaved by another site-specific nuclease, which may not be guided by a non-coding RNA molecule, such as a broad-spectrum nuclease, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN), to introduce a double-strand break (or single-strand nick) into the polynucleotide sequence and / or its complementary DNA strand.

[0114] In this application, the term "guide RNA" or "gRNA" is a short RNA sequence comprising (1) a structural or scaffold RNA sequence required to bind or interact with RNA-guided nucleases and / or other RNA molecules (e.g., tracrRNA), and (2) an RNA sequence that is identical or complementary to the target sequence or target site (referred to herein as the "guide sequence"). A "single-stranded guide RNA" (or "sgRNA") is an RNA molecule comprising tracrRNA and crRNA covalently linked by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or target sequence ("guide sequence") that is identical or complementary to a target site within the plant genome, for example, at or near the GA oxidase gene. An interphase sequence adjacent motif (PAM) may be present immediately adjacent to the 5' end of a genomic target site sequence complementary to the target sequence of the guide RNA and upstream of it in the genome, i.e., downstream (3') of the sense (+) strand immediately adjacent to the genomic target site (relative to the target sequence of the guide RNA), as is known in the art. The genomic PAM sequence (relative to the target sequence of the guide RNA) on the sense (+) strand adjacent to the target site may contain 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') of the target sequence of the guide RNA) is typically not complementary to the genomic PAM sequence. The guide RNA can usually be a non-coding RNA molecule that does not encode a protein.

[0115] In this application, "RNA-directed nuclease" refers to RNA-directed DNA endonucleases associated with the CRISPR system. Unrestricted examples of RNA-directed nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs or modified forms thereof. In one aspect of the implementation, the RNA-directed nuclease is Cas9. In another aspect, the RNA-directed nuclease comprises N-terminal and C-terminal nuclear localization sequences (NLS).

[0116] In some embodiments of this application, the composition for genome editing may be co-delivered with a DNA molecule containing a selection or screening marker gene.

[0117] Furthermore, the Cas9 protein described in this application is not limited to a specific protein, as long as it can be used in conjunction with the sgRNA described in this application. Furthermore, the Cas9 protein described herein is selected from Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9HF (high fidelity), nicked Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9), and Treponema sp. Cas9, as well as other orthologs of Cas9 from other organisms, but not limited to these. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut). TM HiFiCas9 protein, etc.

[0118] NPR1 plays a crucial role in plant immune signaling networks. This invention uses Arabidopsis thaliana as a model plant to verify the contribution of the NPR1 gene to resistance to *Plasmodiophora brassicae*. The results showed that the NPR1 gene knockout mutant in Arabidopsis thaliana exhibited significantly reduced resistance to *Plasmodiophora brassicae* compared to wild-type Arabidopsis thaliana Est-1. This indicates that the NPR1 gene functions in plant resistance to *Plasmodiophora brassicae*, and the NPR1 protein and its encoding gene can provide new gene resources for breeding resistance to *Plasmodiophora brassicae* in cruciferous crops.

[0119] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and 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 invention in any way.

[0120] 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.

[0121] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0122] The following examples used GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used. Different letters indicate that there is a significant difference between the two groups of data (P < 0.05), and the same letters indicate that there is no significant difference between the two groups of data (P > 0.05).

[0123] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods and techniques used in the following embodiments are conventional techniques in the art. Unless otherwise specified, the experimental reagents and materials used in the following embodiments are commercially available conventional reagents and materials.

[0124] The pCBC-DT1T2 and pHEE401E vectors used in the following examples are disclosed in the literature “Ge, Z. et al. LLG2 / 3 areco-receptors in BUPS / ANX-RALF signaling to regulate Arabidopsis pollentubeintegrity. Current Biology 29, 3256-3265.e5 (2019)”. These materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The aforementioned biological materials are only for repeating the experiments of this application and may not be used for other purposes.

[0125] 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.

[0126] The Plasmodiophora brassicae fieldisolateDayi (PbDy) and Arabidopsis thaliana Est-1 from Dayi County, Sichuan Province, used 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 biological materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. These biological materials are only used to replicate the relevant experiments of this application and may not be used for other purposes.

[0127] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0128] The following examples used GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used. Different letters indicate that there is a significant difference between the two groups of data (P < 0.05), and the same letters indicate that there is no significant difference between the two groups of data (P > 0.05).

[0129] Example 1: Analysis of resistance to clubroot bacteria in NPR1 knockout Arabidopsis thaliana

[0130] The genomic sequence of the NPR1 gene in Arabidopsis thaliana is a DNA molecule with the nucleotide sequence of SEQ ID NO:3, and its coding sequence (CDS) is a DNA molecule with the nucleotide sequence of SEQ ID NO:1, encoding the NPR1 protein with the amino acid sequence of SEQ ID NO:2.

[0131] I. Obtaining NPR1 knockout in Arabidopsis thaliana

[0132] 1. Construction of NPR1 knockout vector

[0133] 1) Knockout target design

[0134] The target sites for knocking out the NPR1 gene were designed using the website http: / / skl.scau.edu.cn / . The target site sequences are as follows:

[0135] Target site 1: 5'-CGCCGTGAAGCTCGAGCTTAAGG-3' (SEQ ID NO:4), where positions 1 to 20 are the target site sequence and positions 21 to 23 are the PAM sequence;

[0136] Target site 2: 5'-GCCGCCGAACAAGTACTCACCGG-3' (SEQ ID NO: 5), where positions 1 to 20 are the target site sequence and positions 21 to 23 are the PAM sequence;

[0137] 2) Primer design

[0138] Primers were designed based on the target site sequence. The primer sequences are as follows (target site sequence in uppercase format):

[0139] Target1-BsF:atatatggtctcgattgCGCCGTGAAGCTCGAGCTTAgtt;

[0140] Target1-F0:tgCGCCGTGAAGCTCGAGCTTAgttttagagctagaaatagc;

[0141] Target2-R0:aacGTGAGTACTTGTTCGGCGGCcaatctcttagtcgactctac;

[0142] Target2-BsR:attattggtctcgaaacGTGAGTACTTGTTCGGGCGGCc.

[0143] 3) PCR amplification

[0144] Four-primer PCR amplification was performed using the pCBC-DT1T2 vector as a template. A 50 μL reaction mixture was prepared as follows: 25 μL 2×KOD Buffer, 10 μL 2 mM dNTPs, 1 μL KOD DNA Polymerase, 1.5 μL pCBC-DT1T2, 1.5 μL Target1-BsF, 1.5 μL Target2-BsR, 1.5 μL Target1-F0, 1.5 μL Target2-R0, and 6.5 μL ddH2O. The reaction conditions were: pre-denaturation at 94℃ for 2 min, denaturation at 98℃ for 2 min, annealing at 55℃ for 30 s, extension at 68℃ for 30 s / kbp, final extension at 68℃ for 10 min, and storage at 4℃ for 2 min. PCR products were recovered by agarose gel electrophoresis.

[0145] 4) Enzyme digestion and ligation

[0146] The PCR fragment was ligated into the pHEE401E vector using a cut-and-ligate method to obtain the recombinant vector pHEE401E-NPR1, i.e., the NPR1 knockout vector. A 15 μL reaction mixture was prepared as follows: 2 μL of the PCR product from step 3, 2 μL of the pHEE401E vector, 1.5 μL of 10×T4 DNA ligase buffer, 1.5 μL of 10×BSA, 1 μL of Bsa I, 1 μL of T4 DNA ligase, and 6 μL of ddH2O. The reaction conditions were: 37℃ for 5 h, 50℃ for 5 min, 80℃ for 10 min, and storage at 4℃ for 2 min.

[0147] 5) Reaction product conversion and coating

[0148] Add 15 μL of the reaction product to 100 μL of *E. coli* DH 5α competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, incubate on ice for 2 min, add 500 μL of LB medium, and incubate at 37°C in a shaker for 1 h. Spread the bacterial culture evenly on LB agar plates containing 50 mg / L kanamycin. Invert the plates and incubate overnight at 37°C.

[0149] 6) Cloning identification

[0150] Following colony PCR, PCR-positive single colonies were selected and sent to a sequencing company for routine Sanger sequencing. Sequencing results showed that the recombinant vector pHEE401E-NPR1 was obtained by replacing the fragment between the BsaI restriction sites of the pHEE401E vector with the DNA molecule shown in SEQ ID NO:6. The recombinant vector pHEE401E-NPR1 contains expression cassettes for two sgRNAs and one Cas9 protein. It can express sgRNA1 (target sites 1-20 of SEQ ID NO:4), sgRNA2 (target sites 1-20 of SEQ ID NO:5), and the CRISPR / Cas9 effector protein Cas9. After being introduced into the receptor, the two transcribed guide RNAs (sgRNA1 and sgRNA2) can target the target sequence near the PAM of the receptor genome, i.e., the NPR1 gene, through base complementarity pairing. The Cas9 protein causes a double-strand break in the DNA at the target site of the NPR1 gene. Through the organism's own DNA damage repair response mechanism, gene mutations occur in the cleaved region during the repair process, thereby knocking out the NPR1 gene.

[0151] 2. Obtaining transgenic Arabidopsis thaliana

[0152] 1) Agrobacterium-mediated transformation

[0153] The pHEE401E-NPR1 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 suspension was then evenly spread onto 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.

[0154] 2) Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0155] Agrobacterium GV3101 carrying the pHEE401E-NPR1 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 in a shaker for 12 h.

[0156] 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.

[0157] 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.

[0158] Est-1 Arabidopsis thaliana 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 16h light / 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.

[0159] After the seeds mature, they are surface disinfected with 30% 84 disinfectant for 10 minutes, sterilized by washing with 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 16h light / 8h dark). After 10-12 days, healthy seedlings are selected and transplanted into a plant culture room.

[0160] 3) Obtaining NPR1 knockout in Arabidopsis thaliana

[0161] Transgenic T0 generation Arabidopsis thaliana was continuously self-pollinated to obtain T2 generation transgenic lines. T2 generation transgenic Arabidopsis thaliana plants grown for approximately 4 weeks in a short-day plant culture chamber (temperature 23℃, 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. The target sequence was amplified using primers NPR1-crispr-F and NPR1-crispr-R. The PCR products were sent to a sequencing company for routine Sanger sequencing, identifying two different NPR1 knockout Arabidopsis thaliana mutants. Figure 1 The primers were named npr1-1 and npr1-2, respectively. The primer sequences are as follows:

[0162] NPR1-crispr-F:5'-CTTGGCTCTGGCTCGTCAATG-3'

[0163] NPR1-crispr-R:5'-GTGGCAGCAATTCTCGTCTG-3'

[0164] NPR1 knockout Arabidopsis npr1-1 is an Arabidopsis mutant with a homozygous mutation in the NPR1 gene (the same mutation occurred on both chromosomes). The difference in the genome sequence between it and wild-type Arabidopsis Est-1 is the deletion of bases 113-121 of the NPR1 gene in SEQ ID NO:3, which leads to the knockout of the NPR1 gene.

[0165] NPR1 knockout Arabidopsis npr1-2 is an Arabidopsis mutant with a homozygous mutation in the NPR1 gene (the same mutation occurred on both chromosomes). The difference between the NPR1 and wild-type Arabidopsis Est-1 genome sequence is that an adenine deoxyribonucleotide (A) was inserted between positions 113 and 114 of the NPR1 gene in SEQ ID NO:3, resulting in the knockout of the NPR1 gene.

[0166] II. Analysis of resistance to *Plasmodiophora stylosa* in NPR1 knockout Arabidopsis thaliana

[0167] 1. Inoculation with *Cladosporium brassicae*

[0168] Twenty-four wild-type Arabidopsis thaliana Est-1, twenty-four NPR1 knockout Arabidopsis thaliana npr1-1, and twenty-four npr1-2 mutant lines were taken and planted in holes, four plants per hole, in a short-day plant culture chamber (temperature 23℃, relative humidity 60%, photoperiod 10h light and 14h dark). After the plants emerged and grew for 20 days, a root drenching experiment was conducted to investigate the infection of *Platycodon brassicae*. The experiment was repeated three times, with at least 12 wild-type Arabidopsis thaliana Est-1, npr1-1, and npr1-2 mutant lines inoculated each time. The specific steps are as follows:

[0169] 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.

[0170] 2) Filter with 8 layers of gauze and transfer the filtrate into a clean Erlenmeyer flask.

[0171] 3) Measure the spore concentration of *Plasmodiophora brassicae* in the bacterial culture using a hemocytometer.

[0172] 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.

[0173] 2. Disease index and incidence rate statistics

[0174] 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:

[0175] Grade 0, no symptoms, intact root system;

[0176] Grade 1, with small nodules on the lateral roots, but no symptoms on the main root;

[0177] Grade 2, fewer lateral roots, small lumps on the main root;

[0178] Grade 3, with large lumps on the lateral roots and main root;

[0179] Grade 4, the main root is completely swollen and rotten.

[0180] The formulas for calculating incidence and disease index are as follows:

[0181] Incidence rate = (Number of diseased plants / Total number of inoculated plants) × 100%

[0182] Disease index = (Σ(Disease level × Number of diseased plants at each level)) / (Total number of inoculated plants × Highest disease level 4) × 100

[0183] Symptoms, incidence, and disease index of plants 21 days after inoculation with clubroot fungus are as follows: Figure 2 As shown in Figure AC, the results indicate that wild-type Arabidopsis thaliana Est-1, after inoculation with *Cladophora brassicae*, exhibited resistance, meaning root development was generally normal, with only a few plants showing mild symptoms. However, in the context of the resistant material Est-1, the NPR1 gene knockout mutants npr1-1 and npr1-2 showed susceptibility, characterized by root swelling and lateral root destruction. These results demonstrate that the NPR1 gene mediates resistance in Arabidopsis thaliana to *Cladophora brassicae*, providing a new gene resource for breeding plants with resistance to clubroot disease.

[0184] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application, characterized in that, The application is any one of the following: A1) The application of NPR1 protein or biomaterials related to said NPR1 protein in regulating plant clubroot resistance; A2) The application of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of products that regulate plant clubroot resistance; A3) The application of NPR1 protein or biomaterials related to said NPR1 protein in regulating resistance to *Cladosporium brassicae* in plants; A4) The application of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of products that regulate resistance to *Cladosporium brassicae* in plants; A5) The application of NPR1 protein or biological materials related to said NPR1 protein in plant breeding or plant-assisted breeding; A6) The use of NPR1 protein or biomaterials related to said NPR1 protein in the preparation of plant breeding or plant-assisted breeding products; The NPR1 protein is any of the following proteins: a1) A protein whose amino acid sequence contains SEQ ID NO:2; a2) Proteins obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in a1) that have more than 80% similarity to the amino acid sequence shown in a1) and are associated with resistance to *Plantago brassicae*. a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

2. The application according to claim 1, characterized in that, The biomaterial is any one of the following: B1) Nucleic acid molecules that inhibit or reduce the expression of the NPR1 protein-encoding gene; 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); B8), the nucleic acid molecule encoding the NPR1 protein; B9) Expression cassettes and / or constructs containing the nucleic acid molecules described in B8), recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues and / or transgenic plant organs.

3. The application according to claim 2, characterized in that, B1) The nucleic acid molecule is either RNA that targets the gene encoding the NPR1 protein or DNA that encodes the RNA; Further, the nucleotides of the target site of the nucleic acid molecule described in B1) are positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5; B8) The nucleic acid molecule is any one of the following (g1)-g3): g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1; g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3; g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates the resistance of plant *Plantochloa crus-galli*.

4. The application according to any one of claims 1-3, characterized in that, The plants were selected from dicotyledonous plants.

5. A method for regulating resistance to *Cladosporium brassicae* in plants, characterized in that, The method includes regulating the resistance of recipient plants to *Cladosporium brassicae* by regulating the expression level of the gene encoding the NPR1 protein of claim 1 in the recipient plant and / or regulating the content of the NPR1 protein in the recipient plant.

6. The method according to claim 5, characterized in that, The method includes reducing the resistance of recipient plants to *Cladosporium brassicae* by reducing the expression level of the gene encoding the NPR1 protein and / or reducing the content of the NPR1 protein in the recipient plants, wherein the recipient plants contain the gene encoding the NPR1 protein.

7. A method for obtaining a target plant with reduced resistance to *Cladosporium brassicum*, characterized in that, The method includes obtaining a target plant with reduced resistance to *Cladosporium brassicae* by reducing the expression level of the gene encoding the NPR1 protein of claim 1 and / or reducing the content of the NPR1 protein in the recipient plant, wherein the recipient plant contains the gene encoding the NPR1 protein.

8. The method according to any one of claims 5 to 7, characterized in that, The purpose of reducing the expression level of the gene encoding the NPR1 protein in the recipient plant and / or reducing the content of the NPR1 protein in the recipient plant is achieved by means of C1) or C2) described below. C1) By knocking out the gene encoding the NPR1 protein in the recipient plant using a genome editing system, the expression level of the gene encoding the NPR1 protein and / or the content of the NPR1 protein in the recipient plant are reduced; Furthermore, the genome editing system is a CRISPR / Cas system; Furthermore, the CRISPR / Cas system can express gRNA targeting the gene encoding the NPR1 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein; Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5; C2) Perform any of the following mutations on the genome sequence of the recipient plant: C2-1) The nucleotide sequence of the NPR1 gene, which is SEQ ID NO:3, is deleted at positions 113-121; C2-2) An adenine deoxyribonucleotide was inserted between positions 113 and 114 of the NPR1 gene with SEQ ID NO:

3.

9. A biological material associated with the NPR1 protein, said biological material being any one of B1) to B9) of claim 2 or 3; Furthermore, B1) the nucleic acid molecule is RNA that targets the gene encoding the NPR1 protein or DNA that encodes the RNA; Further, the nucleotides of the target site of the nucleic acid molecule described in B1) are positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5; B8) The nucleic acid molecule is any one of the following (g1)-g3): g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1; g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3; g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates the resistance of plant *Plantochloa crus-galli*.

10. A composition for genome editing, said composition being the genome editing system of claim 8; Furthermore, the genome editing system is a CRISPR / Cas system; Furthermore, the CRISPR / Cas system can express gRNA targeting the gene encoding the NPR1 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein; Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

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