Application of immune negative regulator nbelp1 in improving plant disease resistance

Abstract

The application belongs to the technical field of genetic engineering, and relates to application of an immune negative regulatory factor NbELP1 in improving plant disease resistance. NbELP1 The gene is used as a plant immune negative regulatory factor, and negatively regulates early immune response induced by PAMP. NbELP1 Knocking out the gene can effectively improve the disease resistance of plants in response to pathogenic bacteria infection, and does not affect the normal growth of plants. NbELP1 The immune negative regulatory factor gene or its orthologous gene widely exists in various plants and is functionally conservative, is a high-quality gene resource with potential for genetic improvement of disease resistance of various plants, and has great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of the immune negative regulatory factor NbELP1 in improving plant disease resistance. Background Technology

[0002] Pattern-triggered immunity (PTI) is the first line of defense for plants against pathogen infection. Plants primarily detect pathogen-associated molecular patterns (PAMPs) of different pathogens through pattern recognition receptors (PRRs) located on the plasma membrane, thereby activating a series of downstream immune responses. PRRs mainly include two classes: receptor-like proteins (RLPs) and receptor-like kinases (RLKs). Both share the characteristic of containing an extracellular domain and a transmembrane structure, and rely on the extracellular domain to recognize specific ligands. The extracellular domains of RLKs and RLPs are mostly composed of a certain number of leucine-rich repeats (LRRs). Numerous studies have shown that LRR motifs play an important role in plant receptor ligand recognition and immune signal transduction. Unlike typical LRR-RLKs and LRR-RLPs, plant genomes also encode a class of extracellular LRR-only proteins (ELPs) that contain only extracellular LRR structures and lack transmembrane and intracellular kinase domains. Whether these proteins can serve as disease resistance gene resources has not yet been clearly reported in research.

[0003] Currently, negative immune regulators have become a valuable resource of disease-resistant genes. Creating durable disease-resistant plant materials by disrupting these negative regulators has become a novel disease control strategy. For example, the MLO gene is a classic example of an early and widely studied negative regulator in wheat. Loss-of-function mutants of MLO can confer durable, broad-spectrum resistance to powdery mildew in various plants. However, mutations in this gene usually lead to severe growth defects, limiting its widespread application in agricultural production. Subsequent long-term research has shown that genetic modifications that can salvage growth defects can compensate for the growth barriers caused by MLO mutations, ultimately leading to the development of new wheat germplasm with broad-spectrum resistance to powdery mildew that does not affect yield or quality. However, this process is time-consuming and resource-intensive, highlighting the significant value and potential of identifying plant disease-resistant negative regulators that do not affect agronomic traits in crop disease-resistant breeding. However, such negative regulators are currently extremely rare. Summary of the Invention

[0004] This invention verifies that NbELP1 in Solanaceae plants is an important negative regulator of immunity. It was generated through CRISPR / Cas9-mediated genome editing. NbELP1 The mutant showed no significant difference in growth and development compared to the wild type, but exhibited resistance to *Sclerotinia sclerotiorum* (…). Sclerotinia sclerotiorum The resistance to ) was significantly enhanced, and immune marker genes NbPR1 and NbPR4 All of these proteins showed significantly upregulated expression. Further comparative analysis revealed that NbELP1 homologs are highly conserved in various important food crops such as rice and wheat; economic crops such as apples and cotton; and forest trees such as poplar. NbELP1 Heterologous reintroduction of these homologous proteins into the mutants all exhibited similar negative immunomodulatory functions to NbELP1, indicating that the negative immunomodulatory function of this protein is conserved across different plants. Therefore, it has great potential for application in the genetic improvement of crop disease resistance.

[0005] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the NbELP1 protein described in this invention is represented by "NbELP1" in non-italicized font. NbELP1 Genes in italic font NbELP1 This indicates that, of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.

[0006] On one hand, this invention relates to the application of the immunomodulatory negative regulator NbELP1 in improving plant disease resistance, wherein the plant contains NbELP1 Genes or their orthologs, including: knockout genes in plants NbELP1 Genes or their orthologous genes can enhance the disease resistance of corresponding plants;

[0007] The disease resistance is activated by PAMP;

[0008] The NbELP1 The gene encodes the NbELP1 protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] SEQ ID NO: 1 is as follows:

[0010] MAAVIETQSLGFLVIVLALAASVRGNSEGDALYALRRSLSDPDNVLQSWDPNLVNPCTWFHVTCNGDNHVTRVDLGNSKLSGHLVPELGKLEHLQYLELYKNNIQGTI PKELGNLKSLISLDLYNNNISGTIPPSLGKLKNLVFLRLNDNQLTGPIPREFSSVSSLKVVDVSNNNLCGTIPTSGPFEHIPLNNFEHNPRLEGPELLGLASYDTNCS.

[0011] Furthermore, in the application of the immune negative regulator NbELP1 provided by this invention in improving plant disease resistance, the... NbELP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2.

[0012] SEQ ID NO: 2 is as follows:

[0013] .

[0014] Furthermore, in the application of the immune negative regulatory factor NbELP1 provided by the present invention in improving plant disease resistance, the plant is selected from one of the following: Solanaceae plants, corn, rice, wheat, potato, apple, pear, peach, peanut, soybean, rapeseed, cotton, grape, cucumber, poplar, Artemisia annua, and wolfberry.

[0015] Generally, the NbELP1 gene or its orthologs can be obtained from public databases.

[0016] Exemplarily, in the application of the immune negative regulator NbELP1 provided by the present invention in improving plant disease resistance, the... NbELP1 The corresponding plant for this gene is a member of the Solanaceae family;

[0017] The NbELP1 Orthologous genes of genes TaELP1The plant corresponding to this gene is wheat;

[0018] The NbELP1 Orthologous genes of genes OsELP1 The plant corresponding to this gene is rice;

[0019] The NbELP1 Orthologous genes of genes MdELP1 The plant corresponding to this gene is the apple.

[0020] The NbELP1 Orthologous genes of genes PaELP1 The plant corresponding to this gene is the silver poplar.

[0021] The NbELP1 Orthologous genes of genes AtELP1 The gene corresponds to the plant Arabidopsis thaliana.

[0022] Furthermore, in the application of the immune negative regulator NbELP1 provided by this invention in improving plant disease resistance, the disease resistance refers to the plant's resistance to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance to infection.

[0023] On the other hand, the present invention relates to a method for improving the disease resistance of plants, wherein the plant comprises NbELP1 Genes or their orthologs, including: the introduction of knockout genes into plants NbELP1 A gene or a vector of its orthologous gene can enhance the corresponding plant's resistance to pathogen infection.

[0024] The pathogen infection resistance is activated by PAMP;

[0025] The NbELP1 The gene encodes the NbELP1 protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0026] Furthermore, in the method for improving plant disease resistance provided by the present invention, the... NbELP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2.

[0027] As verified by this invention,

[0028] Furthermore, in the method for improving plant disease resistance provided by the present invention, the plant is selected from one of the following: Solanaceae plants, corn, rice, wheat, potato, apple, pear, peach, peanut, soybean, rapeseed, cotton, grape, cucumber, poplar, Artemisia annua, and wolfberry.

[0029] For example, the Solanaceae plants include Nicotiana benthamiana (… Nicotiana benthamiana ).

[0030] Furthermore, in the method for improving plant disease resistance provided by the present invention, the... NbELP1 The corresponding plant for this gene is a member of the Solanaceae family;

[0031] The NbELP1 Orthologous genes of genes TaELP1 The plant corresponding to this gene is wheat;

[0032] The NbELP1 Orthologous genes of genes OsELP1 The plant corresponding to this gene is rice;

[0033] The NbELP1 Orthologous genes of genes MdELP1 The plant corresponding to this gene is the apple.

[0034] The NbELP1 Orthologous genes of genes PaELP1 The plant corresponding to this gene is the silver poplar.

[0035] The NbELP1 Orthologous genes of genes AtELP1 The gene corresponds to the plant Arabidopsis thaliana.

[0036] Furthermore, in the method for improving plant disease resistance provided by the present invention, the pathogen includes *Sclerotinia sclerotiorum*. Sclerotinia sclerotiorum .

[0037] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0038] This invention is the first to verify the encoding gene of plant extracellular LRR-only protein. NbELP1 It is a key negative regulator of immunity. Knocking out this gene can significantly enhance plant resistance to PAMP-activated pathogens (such as Sclerotinia sclerotiorum) without affecting normal plant growth and development, thus solving the common problem of negative correlation between resistance and growth in disease resistance breeding. Further research shows that... NbELP1 This invention contains highly conserved orthologous genes in various important food, economic, and forest crops, all of which perform similar negative immunomodulatory functions. This breaks through species limitations and has the potential for widespread application in the genetic improvement of disease resistance in various plants. In summary, this invention provides a high-efficiency, safe, and widely applicable high-quality gene target and operational scheme for crop disease resistance breeding, possessing significant theoretical value and enormous application prospects. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 yes NbELP1 A graph showing the expression patterns of genes in response to Sclerotinia sclerotiorum infection at different time points.

[0041] Figure 2 This is an evolutionary analysis diagram of NbELP1 homologous proteins in 37 plant species.

[0042] Figure 3 These two were obtained using CRISPR-Cas9-mediated gene editing technology. NbELP1 Editing plants Nbelp1- 3 and Nbelp1-5 A schematic diagram of a genome editing event.

[0043] Figure 4 yes Nbelp1-3 and Nbelp1-5 Phenotypic diagram of plant growth.

[0044] Figure 5 yes Nbelp1-3 and Nbelp1-5 Phenotypic representation of plant disease resistance. In this figure, A shows infected photographs of different plants; B shows statistics on lesion area.

[0045] Figure 6 These are two defense-related genes in tobacco Benzoenterae. NbPR1 and NbPR4 exist Nbelp1-3 and Nbelp1-5 Analysis of expression patterns in plants.

[0046] Figure 7 yes NbELP1 A diagram illustrating the negative regulation of PAMP-induced early immune responses and the immunosuppressive function of orthologs of this gene in various plants. WT represents wild-type Nicotiana benthamiana plants. Nbelp1 express NbELP1 Gene-edited plants, EV indicates empty vector, OE indicates overexpression; WT / OE:EV indicates empty vector expression control in wild-type Nicotiana benthamiana leaves; Nbelp1 / OE:EV indicates in Nbelp1 Empty vectors are expressed in the leaves of the plant; Nbelp1 / OE: NbELP1 Indicates in Nbelp1 Expression in the leaves of the plant NbELP1 ; Nbelp1 / OE: MdELP1 , Nbelp1 / OE: PaELP1 , Nbelp1 / OE: AtELP1 , Nbelp1 / OE: TaELP1 , Nbelp1 / OE: OsELP1 They represent in Nbelp1 Heterologous expression in the leaves of the plant MdELP1 , PaELP1 , AtELP1 , TaELP1 and OsELP1 . Detailed Implementation

[0047] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0048] The primer information involved in the following examples is as follows:

[0049] Table 1: Primers used in this invention

[0050]

[0051] Other undocumented sequences can be obtained by those skilled in the art through public databases based on the information provided in this invention.

[0052] Example 1

[0053] This embodiment provides negative regulatory factors of plant immunity. NbELP1 Methods for obtaining gene sequences.

[0054] The present invention provides NbELP1 The gene was obtained from the Solanaceae Genome Database (https: / / solgenomics.net / ), with the gene number Niben101Scf02230g01003.1. Its coding region has a full-length nucleotide sequence of 648 bp, encoding 216 amino acids.

[0055] Example 2

[0056] This embodiment is... NbELP1 The expression patterns of genes in response to Sclerotinia sclerotiorum infection were analyzed.

[0057] Nicotiana benthamiana WT plants were cultured in a greenhouse (25℃, 16h / 8h light / dark alternation) for 4 weeks, and then used for various experiments. Sclerotinia sclerotiorum S. sclerotiorum The strain was inoculated onto PDA medium and incubated at 25°C for 1-2 days. Artificial wounds were created by puncturing the center of young *N. benthamiana* leaves using a disposable sterile syringe needle. Then, activated *Sclerotinia sclerotiorum* was inoculated into the wounds at the edges of the colonies using a 5 mm diameter punch, and the mycelial discs were placed into the wounds. The mixture was incubated at 25°C. Plant tissues from the boundary between diseased and healthy plants were collected at 0, 2, 4, 8, 16, and 24 hours after inoculation. Total RNA was extracted from the plant tissues using an RNA extraction kit, and cDNA was obtained by reverse transcription using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number R433-01). Using the cDNA obtained from different infection time stages as templates, *N. benthamiana*... Actin The gene is an internal reference gene, and qPCR analysis was performed. NbELP1 Expression patterns at different time points in the interaction between plants and pathogens. First, a 20 μL qPCR system was established: 10 μL of 2×ChamQ SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number Q311); 0.5 μL of qRT-NbELP1-F and qRT-NbELP1-R; 1 μL of cDNA; and 8 μL of ddH2O were added sequentially to ice. The qPCR reaction was performed according to the following system and procedure: pre-deformation: 95℃ for 30 seconds; cycling reaction: 95℃ for 10 seconds, 60℃ for 30 seconds, 40 cycles. Three biological replicates and three experimental replicates were performed for each treatment. Through 2... –ΔΔCT The method was used to analyze the qPCR results, and the results showed that... NbELP1 The transcriptional level of the gene began to rise 4 hours after inoculation with Sclerotinia sclerotiorum and peaked at 8-16 hours. Figure 1 ).

[0058] Example 3

[0059] This embodiment provides NbELP1 Homologous evolution analysis of gene-encoded proteins.

[0060] The search was conducted in the NCBI database to find homologous genes of this gene in different plants, primarily including the food crop maize (…). Zea mays ), rice ( Oryza sativum ),wheat( Wheat ),potato( Nightshade tuberous ) etc.; cash crop apples ( Domestic apple ),pear( Pyrus bretschneideri ),Peach( Prunus peach),peanut( Arachis hypogaea ), soybeans Glycine max ),rape( Brassica napus ),cotton( Gossypium hirsutum ),Grape( Vitis vinifera ),cucumber( Cucumis sativus ) etc.; silver poplar ( Populus alba ) etc.; and the medicinal plant Artemisia annua ( Artemisia annua ) and goji berries ( Lycium barbarum ), etc. A phylogenetic tree of NbELP1 was constructed using MEGA11 software, revealing a close evolutionary relationship between this gene and the XP_016543604.1 protein of the nightshade plant pepper (Pepper). Figure 2 Currently, no reports have been made regarding the negative regulatory functions of this protein on plant immunity and disease resistance in different plants.

[0061] Example 4

[0062] This embodiment provides NbELP1 Obtaining gene-edited plants.

[0063] (1) First construct NbELP1 Gene editing vectors.

[0064] A design was created using the online tool E-CRISP (http: / / www.e-crisp.org / E-CRISP / ) targeting... NbELP1 Two sgRNAs of the gene: sg-NbELP1-1 (CTCTAGCTGCTAGTGTTAGGGGG) and sg-NbELP1-2 (TATCCGGGTCGGATAAGCTACGG). Primers for the corresponding target sites were designed using Primer Premier 6.0 primer design software (see Table 1 for details). Conventional gene cloning methods were then used to... NbELP1 The sgRNA fragment was cloned into the intermediate vector AtU6-26-sgRNA-SK. This intermediate vector was then used to construct the final vector CAM-GFP-35SCAS for plant transformation. Positive clones were further identified using colony PCR; the primers are detailed in Table 1. Finally, three positive strains were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing comparison. The correctly sequenced strains were used to extract the plasmid (CAM-GFP-35SCAS+NbLRR-Cas9-5) using the Plasmid Mini Kit I (OMEGA, catalog number: D6943-01) and transformed into Agrobacterium GV3101 competent cells for subsequent genetic transformation.

[0065] (2) Genetic transformation of Tobacco Benedict.

[0066] Select plump, mature seeds of *Nicotiana benthamiana*, disinfect with 75% alcohol for 1 min, then with sodium hypochlorite solution for 10-20 min, rinse four times with sterile water, and inoculate onto 1 / 2 MS solid medium. Incubate in the dark at 25℃ for 3 days, then under light for one week. Continue incubation for 1-2 months, then harvest sterile, tender leaves (explants) and cut them into small pieces. Place the seedlings constructed in section 4.1 into the culture medium. NbELP1 Agrobacterium was used to propagate the gene-editing vector. Prepared explants were incubated in resuspended Agrobacterium culture for 8-10 min, then placed on MS medium and cultured in the dark at 25 °C for 2-4 days. The explants were then transferred to MS medium containing resistance to induce shoot formation, with the medium changed every 15 days. Finally, the differentiated resistant shoots were inoculated onto 1 / 2 MS medium containing resistance to allow them to form complete plantlets. Genomic DNA was extracted from these plantlets using the cetyltrimethylammonium bromide (CTAB) method and analyzed by PCR (primers are detailed in Table 1). Positive T0 generation seedlings were then subcultured. Genomic DNA was extracted from subsequent T1, T2, and T3 generations using the CTAB method, and PCR amplification was performed using gene-specific primers (see Table 1). The PCR products were sequenced to verify the genome editing event. Sequencing revealed two plants with different editing events. NbELP1 Gene-edited plants ( Figure 3 ).

[0067] Example 5

[0068] This embodiment provides NbELP1 Phenotypic analysis of gene-edited plants.

[0069] Two plants obtained from the T3 generation screening with different edit events were selected. NbELP1 Gene-edited plants ( Nbelp1-3 , Nbelp1-5 ) and Nicotiana benthamiana WT plants were cultured in a greenhouse (22℃, 16h / 8h light and dark alternation) and observed after 4 weeks of growth. Nbelp1-3 and Nbelp1-5 The growth status of the plants and WT. The results showed that... Nbelp1-3 and Nbelp1-5 The plants showed no significant difference in growth compared to WT, indicating that... NbELP1 Gene knockout does not affect the growth of Nicotiana benthamiana. Figure 4 ).

[0070] Example 6

[0071] This embodiment provides NbELP1 Disease resistance phenotype analysis of gene-edited plants.

[0072] After observing the growth phenotype in Example 5 above NbELP1Gene-edited plants were further used for disease resistance phenotype analysis. *Sclerotinia sclerotiorum* was inoculated onto PDA medium and incubated at 25°C for 1-2 days for later use. Nbelp1-3 , Nbelp1-5 Leaves from both the plant and WT plants were cut from the petiole and placed in a tray. Absorbent cotton soaked in sterile water was wrapped around the petiole of the leaves to maintain moisture. A hole was made in the center of the leaf using a disposable sterile syringe needle to create an artificial wound. Then, activated *Sclerotinia sclerotiorum* was used to make holes at the edge of the colony using a 5 mm diameter punch, and the mycelial cake was placed on the wound site. Water was sprayed and the area kept moist. Leaf disease was observed and photographed 24 hours after inoculation. The area of ​​lesions was statistically analyzed using ImageJ software. Each treatment had at least six biological replicates, and the experiment was repeated three times. Results showed that *Sclerotinia sclerotiorum* infection... Nbelp1- 3 , Nbelp1-5 The incidence of disease after leaf fall was significantly reduced compared to WT. Figure 5 In the A category, the lesion area was significantly smaller compared to the WT category. Figure 5 (B) indicates NbELP1 Negative regulation of resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum.

[0073] Example 7

[0074] This embodiment provides NbELP1 Expression analysis of immune marker genes in gene-edited plants.

[0075] by NbELP1 Gene-edited plants Nbelp1-3 and Nbelp1-5 Using leaf cDNA as a template, RT-qPCR was used to detect salicylic acid pathway defense-related genes in Nicotiana benthamiana. NbPR1 Genes related to jasmonic acid pathway defense NbPR4 The expression status. The results showed that, compared with the control WT, Nbelp1-3 and Nbelp1-5 Two defense-related genes in the plant NbPR1 and NbPR4 Both significantly upregulated expression ( Figure 6 ).show NbELP1 It negatively regulates the immune response to Benignto's tobacco.

[0076] Example 8

[0077] This embodiment provides NbELP1 Analysis of the conservation of immunosuppressive function of homologous genes.

[0078] (1) Determine the effect of NbELP1 on PAMP-triggered immune response.

[0079] First, WT and [other bacteria] were treated with the bacterial elicitor flg22. Nbelp1-3 , Nbelp1-5 Plants were further analyzed to determine ROS bursts in order to assess the effect of NbELP1 on PAMP-triggered immune responses. ROS bursts were measured from four-week-old WT and [unclear - likely referring to plant growth rates or morphology]. Nbelp1- 3 , Nbelp1-5 0.5 cm diameter circular leaflets were cut from the plant leaves and placed in 96-well microplates. 200 µL of distilled water was added to each well for overnight incubation. The next day, a reaction mixture containing 100 µM luminol (Solarbio), 20 µg / mL horseradish peroxidase (Solarbio), and 1 µM flg22 was prepared. The water in the 96-well plate was then removed using a pipette, and the reaction mixture was quickly added to the microplate. The plate was then placed in a Varioskan LUX multi-functional microplate reader, the kinetic cycle was set, and fluorescence values ​​were detected. Data for each sample are expressed as relative luminescence units (RLU) and cumulative luminescence values. Each treatment was performed in 6 biological replicates, and the experiment was repeated 3 times. Results showed that compared to WT, Nbelp1-3 and Nbelp1-5 Plant leaves treated with 1 µMflg22 produced a greater total amount of ROS. Figure 7 This indicates that in Benedict's tobacco, NbELP1 Negative regulation of PAMP-induced early immune responses.

[0080] (2) Measurement NbELP1 Conservation of the immunosuppressive function of homologous genes.

[0081] Given the significant potential of widely conserved immunomodulatory factors in the genetic improvement of crop disease resistance, this invention further evaluates the conservation of the immunosuppressive function of NbELP1 orthologs in different plants. This invention cloned NbELP1 from grain crops wheat and rice; economic crop apple; forest tree *Populus tomentosa*; and model plant *Arabidopsis thaliana*. NbELP1 orthologous genes TaELP1 , OsELP1 , MdELP1 , PaELP1 , AtELP1 Subsequently, the present invention in Nbelp1 Heterologous expression in the leaves of the plant TaELP1 , OsELP1 , MdELP1 , PaELP1 and AtELP1 The leaves were treated with 1 µM flg22, and the cumulative luminescence value of reactive oxygen species (ROS) emission was measured. The results showed that, compared with the expression... NbELP1 The results were consistent with those of the control group, showing that the expression of the above five genes... Nbelp1 In leaves, the excess ROS accumulation induced by flg22 was significantly reduced. Figure 7 This indicates TaELP1 , OsELP1 , MdELP1 , PaELP1 , AtELP1 Effectively inhibited by NbELP1 Knockout induces an enhanced immune phenotype.

[0082] Example 1 clarified the negative regulatory factors of plant immunity. NbELP1 The origin of the gene. Example 2 clarified that the gene was upregulated during the response of Nicotiana benthamiana to Sclerotinia sclerotiorum infection. Example 3, through phylogenetic analysis with 37 plants, including food crops, economic crops, forest trees, model plants, and medicinal plants, found that the gene is closely related to the evolutionary relationship of the XP_016543604.1 protein in the Solanaceae plant pepper. However, the function of this protein in negatively regulating plant immunity and disease resistance in different plants has not been reported. Example 4 constructed... NbELP1 Gene editing vectors were used, and two strains with different editing events were obtained through genetic transformation. NbELP1 Gene-edited plants. Example 5 clarifies... NbELP1 Gene knockout does not affect the growth of Nicotiana benthamiana. Example 6 clarifies that... NbELP1 Negative regulation of resistance to *Nicotinus benthamiana* to *Sclerotinia sclerotiorum*. Example 7 shows that knockout... NbELP1 Following the genes, two defense-related genes in *Smokebium benthamianum* NbPR1 and NbPR4 Both genes showed significant upregulation, indicating that this gene negatively regulates plant immunity. Example 8 clarified that in Nicotiana benthamiana, NbELP1 The negative regulation of PAMP-induced early immune responses further demonstrates that the immunosuppressive function of this gene's ortholog in various plants is conserved.

[0083] In summary, the gene encoding extracellular LRR-only proteins in plants NbELP1 This protein is upregulated during the response of *N. benthamiana* to *Sclerotinia sclerotiorum* infection and negatively regulates the immune response of *N. benthamiana* and its resistance to *Sclerotinia sclerotiorum*. This protein is widely distributed in various plants and its immunosuppressive function is conserved, making it a high-quality gene resource with potential for genetic improvement in various plants.

[0084] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate preferred embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of the immune negative regulator NbELP1 in improving plant disease resistance, characterized in that, including: Knock out plants NbELP1 Genes that enhance the disease resistance of corresponding plants; The disease resistance refers to the plant's resistance to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance to infection; The NbELP1 The gene encodes the NbELP1 protein, the amino acid sequence of which is shown in SEQ ID NO: 1; The NbELP1 The plant corresponding to the gene is tobacco.

2. The application of the immune negative regulator NbELP1 according to claim 1 in improving plant disease resistance, characterized in that, The NbELP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO:

2.

3. A method for improving plant disease resistance, characterized in that, including: Introducing knockout into plants NbELP1 Gene vectors enhance the corresponding plant's resistance to pathogen infection; The disease resistance refers to the plant's resistance to Sclerotinia sclerotiorum. Sclerotinia sclerotiorum Resistance to infection; The NbELP1 The gene encodes the NbELP1 protein, the amino acid sequence of which is shown in SEQ ID NO: 1; The NbELP1 The plant corresponding to the gene is tobacco.

4. The method for improving plant disease resistance according to claim 3, characterized in that, The NbELP1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2.

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