CaSNAPIN gene and its applications

Abstract

This invention discloses the CaSNAPIN gene and its applications. Firstly, this invention investigates the function of RxLR126 in plant immunity and verifies the function of its interacting protein, CaSNAPIN. The study found that overexpression of CaSNAPIN does not induce necrosis in tobacco leaves, nor does it affect the infection of *Phytophthora capsici*, and it can inhibit necrosis induced by the death elicitor INF1, indicating that CaSNAPIN plays a negative regulatory role in plant immunity. This invention provides data support for exploring the pathogenic mechanism of *Phytophthora capsici*.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the CaSNAPIN gene and its applications. Background Technology

[0002] Phytophthora capsici, a destructive oomycete pathogen, has spread widely globally since its discovery in 1922, causing incalculable losses. It infects a wide range of hosts and possesses a strong survival ability. Its high genetic diversity allows it to evade host recognition immunity and also contributes to resistance to relevant pesticides.

[0003] RxLR effectors are an important class of intracellular effectors secreted by *Phytophthora capsici*, playing a crucial role in host infection. Previous studies cloned the effector RxLR126 (PHYCAscaffold_61:120642-121259) from the standard *Phytophthora capsici* strain LT1534, and investigated its function in *Phytophthora capsici* infection through molecular biology experiments. Key functional sites were identified, and a suspected interacting protein of RxLR126 was found in pepper (*Capsicum annuum*), providing data support for exploring the pathogenic mechanism of *Phytophthora capsici*. Summary of the Invention

[0004] The purpose of this invention is to provide the CaSNAPIN gene and its applications.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides the CaSNAPIN gene, which is a gene encoding the following protein:

[0006] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2;

[0007] (b) Proteins derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0008] Secondly, the present invention provides biological materials containing the CaSNAPIN gene, wherein the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.

[0009] Thirdly, this invention provides the application of the CaSNAPIN gene in the negative regulation of plant innate immunity.

[0010] Fourthly, this invention provides the application of the CaSNAPIN gene in suppressing pepper leaf necrosis induced by the death elicitor INF1;

[0011] The death exciton INF1 is:

[0012] (A) A protein consisting of the amino acid sequence shown in SEQ ID NO:3;

[0013] (B) A protein derived from (A) with the sequence shown in SEQ ID NO:3 replaced, deleted or added with one or more amino acids and having the same function.

[0014] Fifthly, the present invention provides a method for mitigating plant leaf necrosis caused by the death elicitor INF1, the method comprising: overexpressing the CaSNAPIN gene in a plant.

[0015] The overexpression method can be selected from the following 1) to 5), or any combination thereof:

[0016] 1) By importing a plasmid containing the gene;

[0017] 2) By increasing the copy number of the aforementioned genes on plant chromosomes;

[0018] 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes;

[0019] 4) By operatively linking a strong promoter to the gene;

[0020] 5) By importing enhancers.

[0021] The plants mentioned include, but are not limited to, chili peppers and tobacco.

[0022] In a sixth aspect, the present invention provides a method for enhancing pepper leaf necrosis induced by the death elicitor INF1, the method comprising: silencing the CaSNAPIN gene in the pepper.

[0023] Furthermore, a DNA fragment for silencing the CaSNAPIN gene was designed and synthesized, and the DNA fragment was ligated into the pTRV2 vector to obtain a recombinant vector; then, pTRV1 and the recombinant vector were transformed into Agrobacterium, and the resulting transformants co-infected peppers.

[0024] Preferably, the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO:5.

[0025] In a seventh aspect, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0026] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0027] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] This invention first verifies the functional relationship between the effector factor RxLR126 and the interacting protein CaSNAPIN. Simultaneously, the VIGS silencing technique is used to silence the interacting protein, verifying the effect of CaSNAPIN on the function of RxLR126. Specific results are as follows:

[0029] (1) RxLR126 inhibits plant PTI response induced by death elicitor: The pBIN-RxLR126-GFP vector was constructed using homologous recombination technology. RxLR126-GFP and GFP were transiently expressed in Nicotiana benthamiana using Agrobacterium transient transformation technology, and INF1 was transiently expressed 24 h later. The results were verified by classic experiments on plant PTI immunization at different time points. It was found that RxLR126 could inhibit the reactive oxygen species burst induced by death elicitor INF1, inhibit the upregulation of PTI-related resistance genes, inhibit the deposition of callose induced by death elicitor INF1, and weaken the MAPK phosphorylation cascade induced by death elicitor INF1.

[0030] (2) RxLR126 interacts with SNAPIN protein in chili pepper: Through three classic interaction verification experiments, namely co-immunoprecipitation (CO-IP), luciferase complementation assay (LUC), and bimolecular fluorescence complementation (Bifc), it was determined that the effector RxLR126 interacts with SNAPIN protein in chili pepper.

[0031] (3) RxLR126 and CaSNAPIN are located on the cell nucleus and cell membrane: By transiently expressing RxLR126-GFP and CaSNAPIN-GFP on Nitralium benzoate, fluorescence was observed on the cell nucleus and cell membrane using two-photon confocal microscopy, which preliminarily confirmed that RxLR126 and CaSNAPIN are located on the cell nucleus and cell membrane. By transiently expressing RxLR126-GFP and CaSNAPIN-RFP together on Nitralium benzoate, co-localization was observed using two-photon confocal microscopy.

[0032] (4) CaSNAPIN inhibits INF1-induced necrosis: The function of CaSNAPIN was verified in Nicotiana benthamiana using Agrobacterium-mediated transient transformation. The study found that overexpression of CaSNAPIN did not induce necrosis in Nicotiana benthamiana leaves, nor did it affect the infection by Phytophthora capsici, and it could inhibit necrosis induced by the death elicitor INF1. Overall, CaSNAPIN plays a negative regulatory role in plant immunity.

[0033] (5) Silencing CaSNAPIN in chili peppers: Bioinformatics analysis revealed that there are no homologous genes for CaSNAPIN in chili peppers. A specific gene fragment of 300 bp in the CaSNAPIN sequence was selected, and the CaSNAPIN protein was silenced in chili peppers using VIGS (Virus Induced Gene Silencing) technology. The silencing efficiency of CaSnapim was found to be above 75% by qRT-PCR technology.

[0034] (6) The function of RxLR126 in inhibiting the death elicitor INF1-induced necrosis in CaSNAPIN-silenced plants was weakened: RxLR126 was transiently expressed in CaSNAPIN-silenced pepper plants by Agrobacterium transient transformation, and INF1 was transiently expressed in the same position 24 h later. Mild leaf necrosis was found in CaSNAPIN-silenced plants 48 h later.

[0035] (7) Further screening of RxLR126 interacting proteins was performed using immunoprecipitation-mass spectrometry (IP-MS): RxLR126 was transiently expressed on Nitraria tangents, and after 48 h, RxLR126-GFP protein was enriched using anti-GFP agarose beads, with GFP-treated samples used as controls. IP-MS analysis was performed on the experimental and control groups to identify proteins suspected of interacting with RxLR126, and preliminary screening was conducted using a luciferase complementation assay.

[0036] The preliminary studies above confirmed that RxLR126 negatively regulates plant PTI immunity, indicating that this effector plays a role in evading plant immunity during *Phytophthora capsici* infection. The interaction between CaSNAPIN and RxLR126 was confirmed, preliminarily verifying the function of CaSNAPIN. Silencing CaSNAPIN in pepper weakened the inhibitory effect of RxLR126 on leaf necrosis induced by the death elicitor INF1. These findings lay a theoretical foundation for further research into the mechanism of RxLR126 function and the screening of downstream interacting proteins, while also providing data support for exploring the pathogenic mechanism of *Phytophthora capsici*. Attached Figure Description

[0037] Figure 1 To verify the function of the effector factor RxLR126 in inhibiting reactive oxygen species bursts in the preferred embodiment of the present invention; A: DAB staining experiment; B: quantitative detection of hydrogen peroxide (H2O2); C: expression level of the RBOHB gene in Nicotiana benthamiana under different treatment conditions.

[0038] Figure 2 The following are the expression of PTI pathway marker genes in a preferred embodiment of the present invention: A: NbWRKY7 expression in Nicotiana benthamiana; B: NbWrky8 expression in Nicotiana benthamiana; C: NbPTI5 expression in Nicotiana benthamiana.

[0039] Figure 3 The results of callosity deposition detection are shown in a preferred embodiment of the present invention.

[0040] Figure 4 The results of the MAPK phosphorylation cascade reaction detection experiment are shown in the preferred embodiment of the present invention.

[0041] Figure 5 For verification of the interaction between effector RxLR126 and CaSNAPIN in a preferred embodiment of the present invention: A: Verification of RxLR126 and CaSNAPIN through immunoprecipitation; B: Complementary interaction experiment of RxLR126 and CaSNAPIN luciferase.

[0042] Figure 6 The experimental results of two-photon fluorescence complementary interaction between RxLR126 and CaSNAPIN are shown in the preferred embodiment of the present invention.

[0043] Figure 7 The following is a preferred embodiment of the present invention: CaSNAPIN functional analysis; A: Schematic diagram of inoculation of *N. benthamiana* and trypan blue staining results; B: Western blotting detection, antibody is anti-GFP, and the bottom shows the results of Ponceau sulphureus staining; C: Schematic diagram of inoculation of *Agrobacterium benthamiana* leaves and *Phytophthora capsici* lesions; D: Biomass analysis; E: Bar chart of *Phytophthora capsici* lesion area; F: Western blotting detection, antibody is anti-GFP, and the bottom shows the results of Ponceau sulphureus staining.

[0044] Figure 8 This invention provides a preferred embodiment of the functional verification of CaSNAPIN inhibiting leaf necrosis induced by INF1; A: Schematic diagram of Nicotiana benthamiana inoculation and trypan blue staining results; B: Conductivity bar chart; C: Post-inoculation necrosis rate bar chart; D: Western Blot detection, antibody is anti-GFP, and the bottom is the Ponceau S staining result.

[0045] Figure 9In a preferred embodiment of the present invention, RxLR126-GFP, CaSNAPIN-GFP, and GFP are subcellularly localized; (a) subcellular localization; (b) plasmolysis subcellular localization.

[0046] Figure 10 In a preferred embodiment of the present invention, RxLR126-GFP and CaSNAPIN-RFP are co-localized in subcellular structures.

[0047] Figure 11 To verify the effect of RxLR126 on CaSNAPIN in a preferred embodiment of the present invention; A: Quantitative Western Blot, antibody is anti-GFP; B: SNAPIN expression level in chili pepper.

[0048] Figure 12 To verify the effect of CaSNAPIN on RxLR126 in a preferred embodiment of the present invention; A: Schematic diagram of inoculation with Nicotiana benthamiana and results of trypan blue staining; B: Conductivity bar chart; C: Statistical bar chart of necrosis rate after inoculation; D: Western Blot detection, the antibody is anti-GFP, and the bottom is the results of Ponceau S staining.

[0049] Figure 13 This is a preferred embodiment of the SNAPIN silencing sequence in chili peppers.

[0050] Figure 14 In a preferred embodiment of the present invention, the SNAPIN silencing phenotype and silencing efficiency in chili peppers were verified; A: chili pepper growth status after silencing; B: silencing efficiency detection, with the chili pepper housekeeping gene Actin as an endogenous reference.

[0051] Figure 15 The following is a functional verification of silent pepper plants in a preferred embodiment of the present invention: A: Effect of silencing CaSNAPIN on RxLR126 function; B: Statistics on pepper leaf necrosis rate; D: Western Blot detection, antibody is anti-GFP, and the bottom shows the results of Ponceau S staining.

[0052] Figure 16 The following is a network diagram of key interacting proteins in a preferred embodiment of the present invention: A: MCC algorithm subnetwork; B: DMNC algorithm subnetwork; C: degree algorithm subnetwork.

[0053] Figure 17 For verification of suspected interacting protein interactions in a preferred embodiment of the present invention; A: Schematic diagram of inoculation with Nicotiana benthamiana; B: Inoculation sequence of the experimental group.

[0054] Figure 18 This is the gene and amino acid sequence of the effector RxLR126 of this invention.

[0055] Figure 19 The gene and amino acid sequence of the interacting protein CaSnapin are shown in the preferred embodiment of the present invention.

[0056] Figure 20 The present invention provides the INF1 gene and amino acid sequence of the death elicitor in a preferred embodiment of the present invention.

[0057] Figure 21 The Avr3a gene and amino acid sequence are shown in a preferred embodiment of the present invention. Detailed Implementation

[0058] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0059] Example 1: Study on the mechanism by which the effector factor RxLR126 of Phytophthora capsici inhibits plant immunity and the interaction protein CaSNAPIN and its function.

[0060] 1. Experimental Materials

[0061] 1.1 Test strains and plant materials

[0062] The strains used in the experiment were all Phytophthora capsicum standard strain LT1534.

[0063] The model plant used in the experiment, Nicotiana benthamiana, is a variety preserved in our laboratory. The cultivation temperature was 22℃, the humidity was about 65-70%, the white light was 14h, the photoperiod was 10h, and the plant was grown in a greenhouse with alternating light and dark conditions.

[0064] The chili pepper (Capsicum annuum L.) was an inbred line variety preserved in our laboratory. It was cultivated at a temperature of 22℃ and a humidity of 65-70%, with 14 hours of white light and a photoperiod of 10 hours. It was grown in a greenhouse with alternating light and dark conditions.

[0065] The INF1 Agrobacterium strain was kindly provided by Professor Dou Daolong of Nanjing Agricultural University.

[0066] 1.2 Test medium

[0067] The pBIN-GFP2 vector was kindly donated by Professor Dou Daolong of Nanjing Agricultural University. The TRV silencing vector was kindly donated by Professor Li Feng of Huazhong Agricultural University. The pSPnYFP and pSPcYFP vectors were kindly donated by Professor Qiao Yongli of Shanghai Normal University. The nLUC and cLUC vectors were kindly donated by the College of Plant Protection of China Agricultural University.

[0068] The pSPYFP vector can be found in Walter, Michael, et al. "Visualization of protein interaction in living plant cells using bimolecular fluorescence complementation." The Plant Journal 40.3 (2004): 428-438.

[0069] The pBIN-GFP2 vector can be found in Daolong Dou, et al. RXLR-Mediated EntryofPhytophthora sojae Effector Avr1b into Soybean Cells Does Not RequirePathogen-Encoded Machinery. Plant Cell. 200820(7):1930-1947.

[0070] For nLUC and cLUC vectors, see Dongqin Chen*, FengshengHao, Huiqi Mu, NagibAhsan, JayJ.Thelen&Gary Stacey*. S-acylation of P2K1 mediates extracellularATP-inducedimmune signaling in Arabidopsis. Nature communications, 12: 2750, 2021.

[0071] The names and sequences of the primers involved are shown in Tables 1 and 2.

[0072] Table 1 Primer names and sequences

[0073]

[0074]

[0075] Table 2 Primer names and sequences

[0076]

[0077]

[0078] 1.3 Experimental Instruments, Equipment and Consumables

[0079] 1.3.1 The experimental kits are shown in Table 3.

[0080] Table 3 Experimental Reagent Kits

[0081]

[0082]

[0083] 1.3.2 Preparation of Commonly Used Reagents and Culture Media

[0084] (1) LB liquid culture medium: accurately weigh 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, add 1L of deionized water, stir on a magnetic stirrer until completely dissolved, dispense into 250mL Erlenmeyer flasks, sterilize and use.

[0085] (2) LB solid medium: Accurately weigh 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride, add 1L of deionized water, and stir on a magnetic stirrer until completely dissolved. Weigh 3g of agar powder and add it to a 250mL Erlenmeyer flask, then add 200mL of LB liquid medium, shake gently to mix, and sterilize before use.

[0086] (3) Oat culture medium: Weigh approximately 25g of oats, add 1L of deionized water, place on an induction cooker and heat, stirring continuously, maintaining a boiling state for 30 minutes. After boiling, filter out the oat residue using four layers of clean gauze. Recycle the oat liquid and add deionized water to bring the volume to 1L. Weigh 3g of agar powder and add it to a 250mL Erlenmeyer flask, then add 200mL of the oat liquid, gently shake to mix, and sterilize before use.

[0087] (4) 5×TBE buffer: Weigh 54g of tris(hydroxymethyl)aminomethane and 27.5g of boric acid, place them in a 1L beaker, add 20mL of 0.5M ethylenediaminetetraacetic acid (pH=8.0), add deionized water to bring the volume to 1L, and stir on a magnetic stirrer until the drug dissolves. Store at room temperature. When using, dilute with deionized water to prepare 0.5×TBE.

[0088] (5) 25 mg / mL rifampicin resistance: Weigh 1 g of rifampicin powder and pour it into a 50 mL Erlenmeyer flask wrapped with aluminum foil. Add 40 mL of dimethyl sulfoxide and stir on a magnetic stirrer until the rifampicin is completely dissolved. Filter the solution through a 0.22 μm bacterial filter in a clean bench to sterilize it, then aliquot it into 1.5 mL brown EP tubes and store at -20 °C.

[0089] (6) 50 mg / mL kanamycin sulfate resistance: Weigh 1 g of kanamycin sulfate powder and pour it into a 50 mL Erlenmeyer flask. Add 20 mL of deionized water and stir on a magnetic stirrer until the kanamycin sulfate is completely dissolved. After sterilization by filtration through a 0.22 μm bacterial filter in a clean bench, dispense into 1.5 mL EP tubes and store at -20 °C.

[0090] (7) 20 mg / mL acetosyringone solution: Weigh 1 g of acetosyringone powder and add it to a 50 mL BD tube. Add 50 mL of dimethyl sulfoxide and mix by inverting. After sterilization by filtration through a 0.22 μm bacterial filter in a clean bench, dispense and store at -20 °C.

[0091] (8) 0.5M MES: Weigh 24.4g of morpholinoethanesulfonic acid and place it in a clean 250mL beaker. Add 100mL of deionized water and stir continuously to dissolve the drug. Adjust the pH to 5.6 with 1M potassium hydroxide, and then add deionized water to bring the volume to 200mL. After sterilization by filtering through a 0.22μm bacterial filter in a clean bench, store in a sterile blue wide-mouth bottle at 4℃.

[0092] (9) 10mM MgCl2 buffer: Weigh 0.22g of magnesium chloride powder and place it in a 1L blue-mouth bottle. Add 1L of deionized water. Mix by inverting the bottle to dissolve the drug. Sterilize before use.

[0093] (10) 2× Loading Buffer: Weigh 3g of tris(hydroxymethyl)aminomethane hydrochloride, dissolve it, adjust the pH to 6.8, and then bring the volume to 50mL with deionized water. Stir well with a magnetic stirrer to prepare 4× tris(hydroxymethyl)aminomethane hydrochloride buffer for later use. Accurately weigh 4g of sodium dodecyl sulfate, 0.02g of Coomassie brilliant blue, and 3.1g of dithiothreitol, place them in a 100mL beaker, add 20mL of 4× tris(hydroxymethyl)aminomethane hydrochloride buffer and 24mL of glycerol, and finally add deionized water to bring the volume to 100mL. Stir well with a magnetic stirrer. After sterilization by filtration through a 0.22μm bacterial filter in a clean bench, transfer to a sterile blue-mouth bottle and store at room temperature.

[0094] (11) Electrophoresis solution: Weigh 14.4g glycine, 3.1g tris(hydroxymethyl)aminomethane and 1g sodium dodecyl sulfate, pour them into a 1L beaker, add deionized water to make up to 1L, place on a magnetic stirrer and mix thoroughly. After the drug is completely dissolved, transfer to a blue bottle and store at room temperature.

[0095] (12) Transfer solution: Weigh 14.4g glycine and 3g tris(hydroxymethyl)aminomethane, add 800mL deionized water, place on a magnetic stirrer and mix thoroughly. After complete dissolution, add 200mL methanol and transfer to a blue bottle for storage at 4℃.

[0096] (13) 10×TBST-Buffer: Weigh 24.2g of tris(hydroxymethyl)aminomethane and 80g of sodium chloride, add deionized water to make up to 1L, mix thoroughly on a magnetic stirrer, adjust the pH to 7.6 with 1M hydrochloric acid, transfer to a blue-mouth bottle and store at room temperature. When using, measure 100mL of 10×TBST-Buffer, add 900mL of deionized water, add 1mL of Tween-20, shake to mix and prepare 1×TBST-Buffer, and store at room temperature.

[0097] (14) Milk powder sealing solution: Weigh 7.5g of skim milk powder, pour it into a 250mL Erlenmeyer flask, add 150mL of 1×TBST-Buffer, and stir on a magnetic stirrer for at least 3 hours. After stirring, dispense into 50mL BD tubes and store at -20℃.

[0098] (15) DAB staining solution: Take a 250mL Erlenmeyer flask wrapped in aluminum foil, weigh 0.1g of 3,3-diaminobenzidine powder and dissolve it in 100mL of deionized water. Place the Erlenmeyer flask on a magnetic stirrer and mix for about 15min. After the powder is completely dissolved, adjust the pH of the solution to 5.8-6.0 with 1M sodium hydroxide solution. Prepare fresh before use.

[0099] (16) Trypan blue staining solution: Accurately weigh 0.4g of trypan blue powder and pour it into a 1L blue-necked bottle. Measure and add 200mL of glycerol, 200mL of lactic acid, 200mL of deionized water, and 200mL of phenol. Shake upside down for 1 minute to mix and dissolve the liquid and chemicals, thus preparing the trypan blue staining solution stock solution. When using, mix the trypan blue stock solution with 95% ethanol at a volume ratio of 1:2 before use.

[0100] (17) Chloral hydrate solution: Weigh 7 kg of chloral hydrate granules, put them into a 10 L capacity container, add 4 L of deionized water, and shake continuously to completely dissolve the chloral hydrate to obtain a chloral hydrate solution.

[0101] (18) Lysis Buffer: Weigh 1.6g of tris(hydroxymethyl)aminomethane hydrochloride, 8.8g of sodium chloride, and 0.17g of ethylenediaminetetraacetic acid, place them in a 1L beaker, add 500mL of deionized water, and stir on a magnetic stirrer to dissolve the drug. After the drug is completely dissolved, adjust the pH to 7.5, sterilize by vacuum filtration, and store at 4℃.

[0102] 2. Experimental Methods

[0103] 2.1 Primer Design

[0104] All primers used in this experiment were designed using DNAMAN software and the CE Design primer design website (https: / / crm.vazyme.com / cetool / simple.html) on the Nanjing Novizan website.

[0105] 2.2 Total DNA extraction, using standard methods.

[0106] 2.3 Total RNA extraction, using conventional methods.

[0107] 2.4 RNA is reverse transcribed into cDNA using conventional methods.

[0108] 2.5 Gene amplification, using conventional methods.

[0109] 2.6 Nucleic acid gel recovery, conventional method.

[0110] 2.7 Construction of recombinant plasmids, using conventional methods.

[0111] 2.8 Transformation of Escherichia coli with recombinant plasmids using conventional methods.

[0112] 2.9 Recombinant plasmid extraction, using conventional methods.

[0113] 2.10 Recombinant plasmids were transformed into Agrobacterium, using conventional methods.

[0114] 2.11 Agrobacterium-mediated transient expression technology, conventional method.

[0115] 2.12 Western blot, a conventional method.

[0116] 2.13 Callose Deposition

[0117] (1) Place the phenol in a 65°C oven and heat it to melt.

[0118] (2) Prepare lactophenol solution: Mix deionized water, lactic acid, phenol and 20% glycerol in a volume ratio of 1:1:1:2, and store in the dark for later use.

[0119] (3) Prepare mixture A: Estimate the volume of mixture A based on the volume of the sample to be tested, and mix anhydrous ethanol and lactol in a volume ratio of 2:1 to prepare mixture A.

[0120] (4) Place the sample to be tested into mixture A and vacuum it for 30 minutes until bubbles appear on the surface of the blade.

[0121] (5) Place the sample to be tested and mixture A in a water bath at 60°C for 30 minutes, and mix them every 10 minutes until the sample to be tested is transparent and colorless.

[0122] (6) Wash three times with deionized water, then add aniline blue staining solution. Place on a decolorizing shaker and stain overnight at room temperature in the dark. After washing the sample with deionized water, store it in 50% glycerol.

[0123] (7) Observe and photograph under a fluorescence microscope, and use ImageJ software to process and calculate the fluorescence intensity of callosity deposition.

[0124] 2.14 DAB staining

[0125] (1) Weigh 0.2g of diaminobenzidine powder and place it in a 250mL Erlenmeyer flask wrapped in tin foil. Add 200mL of deionized water and stir on a magnetic stirrer for 15min. After the diaminobenzidine powder dissolves, adjust the pH to 5.8 with sodium hydroxide solution.

[0126] (2) Place no more than ten leaves to be tested in a 10cm petri dish, add the prepared diaminobenzidine staining solution, wrap with tin foil, place on a decolorizing shaker and shake for 12h to 24h depending on the staining situation.

[0127] (3) After staining, discard the diaminobenzidine staining solution, add 95% alcohol, place in a 65℃ oven, and change the alcohol every 12 hours until decolorization is complete.

[0128] (4) Observe whether brown precipitate can be produced in the stained area, record the size of the stained area and take a picture of the leaf.

[0129] 2.5.15 Determination of H2O2 content

[0130] 1) Prepare a 10 mM H2O2 standard solution, and dilute it sequentially according to Table 4:

[0131] Table 4 Standard Solution Preparation System

[0132]

[0133] 2) 5% titanium sulfate: Accurately weigh 0.3g of titanium sulfate and add it to 6mL of distilled water. After dissolving, it becomes a 5% titanium sulfate solution. Store at 4℃.

[0134] 3) Take the processed fresh plant tissue, wash it clean, dry it, weigh 5g, and put it into a pre-cooled mortar.

[0135] 4) Add 5 mL of pre-cooled acetone at 4°C and grind rapidly under ice bath conditions. Then transfer the grinding liquid to a pre-cooled centrifuge tube, centrifuge at 12000 r / min at 4°C for 20 min, collect the supernatant, measure the total volume of the extract, and store at 4°C for later use.

[0136] 5) Set up blank tubes, standard tubes, and experimental tubes according to Table 5, and add them in sequence.

[0137] Table 5. Reaction Solution Preparation System

[0138]

[0139] 6) Mix the above solution well, let it stand at room temperature for 5 min, centrifuge at 12000 r / min for 15 min, discard the supernatant, keep the precipitate, wash the precipitate once with pre-cooled acetone, add 2 mL of acidic base solution to the precipitate in each tube, shake to completely dissolve the precipitate.

[0140] 7) Using a glass cuvette with a light path of 1 cm, zero the blank tube, measure the absorbance at 412 nm using a spectrophotometer, and record the result.

[0141] 8) Using the gradient acetone-H2O2 standard solutions (0.3mM, 0.5mM, 0.8mM, 1mM, 3mM, 5mM, 8mM) as the abscissa and the corresponding absorbance values ​​as the ordinate, calculate the standard curve and obtain the regression curve. Substitute the absorbance value of the sample to be tested into the regression curve equation to obtain the concentration of H2O2 in the sample.

[0142] 2.16 Trypan blue staining

[0143] (1) Place the phenol in a 65°C oven to melt it beforehand.

[0144] (2) Trypan blue staining solution: Weigh 0.4g of trypan blue powder using an analytical balance and pour it into a 1L blue-mouth bottle. Measure 200mL of glycerol, lactic acid, deionized water, and phenol, respectively, and pour them into the blue-mouth bottle. Tighten the cap and mix by inverting the bottle for 1-2 minutes to dissolve and mix the reagents, thus preparing the trypan blue stock solution.

[0145] (3) Chloral hydrate solution: Weigh about 7 kg of chloral hydrate and measure about 4 L of deionized water. Shake continuously to dissolve the chloral hydrate.

[0146] (4) When using, mix trypan blue stock solution with 95% ethanol at a volume ratio of 1:2 to prepare trypan blue working solution.

[0147] (5) Place the 1L beaker containing 100mL trypan blue working solution into a pot of boiling water and preheat it in the pot of boiling water for 1 minute.

[0148] (6) Take ten leaves to be stained and transfer them to the preheated working solution. Continue boiling in the water bath for 1 min to 2 min, and keep shaking the beaker.

[0149] (7) After the water bath, transfer all the working solution and leaves from the beaker to a 15cm glass petri dish. Lay the leaves flat without folding them, and immerse them completely in the trypan blue staining solution overnight for 12 hours.

[0150] (8) Decolorization: Remove the stained leaves and lay them flat in another 15cm glass petri dish. Pour in chloral hydrate solution.

[0151] (9) Submerge all leaves and soak for 12 hours. Replace with chloral hydrate and continue to replace until the leaves are completely decolorized.

[0152] (10) After decolorization, discard the chloral hydrate solution and replace it with a 95% ethanol solution. Ensure that each leaf is completely submerged in alcohol. After soaking for 1 day, remove the leaves and spread them out as much as possible, avoiding any wrinkles. Once the leaves are completely dry and free of water stains, quickly take a photo to record the process.

[0153] 2.17 Subcellular localization

[0154] (1) The CDS sequence of RxLR126174 was inserted into the pBIN-GFP vector via Kpn1 and Sal1 restriction enzyme sites. The CDS sequence of SNAPIN was inserted into pBIN-RFP using Xho1 and Kpn1 restriction enzyme sites.

[0155] (2) The Agrobacterium strain GV3101 carrying the above-described plasmid was cultured to OD200. 600 0.5~1.0 (single-connection positioning OD) 600 =0.5, common positioning OD 600 =1.0).

[0156] (3) Inoculate the Agrobacterium tumefaciens solution onto tobacco plants with leaves that are about 5 weeks old. After 36 to 48 hours, cut a 1 cm section of the leaf. 2 Select the leaf from the left and right sides, avoiding pinholes and flat areas with veins. With the underside of the leaf facing upwards, use a micropipette to add an appropriate amount of sterile water to the underside of the leaf. After covering with a coverslip, use a two-photon confocal microscope to observe the subcellular localization.

[0157] 2.18 Co-immunoprecipitation (CO-IP)

[0158] (1) Six vigorous, uniformly growing six-week-old Nicotiana benthamiana plants were selected, and two plants were placed in each group. The experimental group and the control group were inoculated using Agrobacterium-mediated transient expression technology. After 48 hours, 5g of leaves were weighed, wrapped in aluminum foil, and quick-frozen in liquid nitrogen.

[0159] (2) Place six 50mL BD tubes, several 1.5mL EP tubes, 1mL pipette tips, and 200μL pipette tips in a -20℃ refrigerator for pre-cooling. Pre-cool the centrifuge at 4℃.

[0160] (3) After the cryogenic high-throughput tissue homogenizer is pre-cooled to -50℃, the sample is transferred to the pre-cooled sample tube, placed in the homogenizer, fixed, and homogenized at 50Hz for 60s. After completion, it is placed in liquid nitrogen to cool down and homogenized again. After homogenization, about 400μL of sample is quickly added to a pre-cooled 1.5mL EP tube, and an equal volume of 2× loading buffer is added. The tube is then vortexed and mixed thoroughly. After complete mixing, it is placed in a boiling water bath for 10min and stored at -20℃. The tube is named Western and kept for later use.

[0161] (4) Transfer the remaining sample powder to a pre-cooled 50mL BD tube, add lysis buffer and benzyl sulfonyl fluoride, mix quickly and thoroughly, and place on a 4℃ shaker for at least 4h for lysis.

[0162] (5) The pyrolysis mixture was placed in a centrifuge and centrifuged at 4°C and 4000 r / min for 10 min.

[0163] (6) During centrifugation, prepare three 1.5 mL pre-chilled EP tubes. Use a pre-chilled and cut 200 μL pipette tip to gently aspirate and mix the anti-FLAG agarose strain. Add 15 μL of beads to each tube. Add 500 μL of washing buffer, gently invert the tube, centrifuge at 1000 r / min at 4℃ for 1 min, discard the supernatant, and repeat three times. After washing, place the tubes on ice for later use.

[0164] (7) After centrifugation, transfer the supernatant to a new 1.5 mL pre-cooled EP tube and centrifuge at 4°C, 13000 r / min for 10 min.

[0165] (8) After the second centrifugation, transfer the supernatant to a new 50 mL pre-cooled BD tube. Take 70 μL of the supernatant, add 70 μL of 2× loading buffer, mix well, boil in a water bath for 10 min, and place at -20℃. Name it Input for later use.

[0166] (9) Slowly aspirate the previously washed FLAG-tagged agarose strain into a 50 mL pre-cooled BD tube containing the supernatant and incubate it on a 4°C shaker for 4 h.

[0167] (10) After incubation, insert the BD tubes onto ice and prepare three corresponding numbered 1.5 mL pre-chilled EP tubes. Using a cut, pre-chilled 1 mL pipette tip, add 1-1.5 mL of liquid from the BD tubes to the corresponding 1.5 mL pre-chilled EP tubes each time. Slowly invert the tubes and centrifuge at 4°C, 1000 rpm for 1 min, discarding the supernatant. Repeat this step until all liquid in the BD tubes has been centrifuged. The precipitate in the tubes is the agarose gel containing the tag protein.

[0168] (11) Take 500 μL of washing buffer and slowly rinse the cap of the BD tube, then transfer it into the BD tube to rinse the tube wall. After slowly inverting the tube, centrifuge at 1000 r / min at 4℃ for 1 min and discard the supernatant. Repeat this step 5-6 times.

[0169] (12) Add 70 μL of 2× loading buffer, mix gently, boil in water for 10 min, and place at -20℃. Name it IP for later use.

[0170] (13) Centrifuge the Western, Input, and IP samples at 13000 r / min for 10 min. Collect the supernatant for SDS-PAGE electrophoresis.

[0171] 2.19 Bimolecular Fluorescence Complementation Experiment

[0172] (1) The CDS sequence of RxLR126174 was inserted into the pSPYNE-35S vector via BamH1 and Xho1 restriction enzyme sites. The CDS sequence of SNAPIN was inserted into the pSPYCE-35S vector using BamH1 and Xho1 restriction enzyme sites.

[0173] pSPYNE-35s and pSPYCE-35s vectors can be found in Walter Michael, Christina Chaban, Katia Schütze, et al.Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.Plant Journal.200440(3):428-38.

[0174] (2) The Agrobacterium strain GV3101 carrying the above-described plasmid was cultured to OD200. 600 It is 1.0.

[0175] (3) Mix the ingredients according to Table 6 and apply them to tobacco plants with leaves around 5 weeks old. The experimental group and the control group were injected into different positions on one leaf, and the injection was repeated on more than 10 leaves.

[0176] Table 6 Experimental Control Table

[0177]

[0178] (4) 48 hours after inoculation, select areas without pinholes or veins and with flat leaves to prepare slides and observe them using a two-photon confocal microscope.

[0179] 2.20 Luciferase Complementation Assay

[0180] (1) The CDS sequence of RxLR126174 was inserted into the pNLUC vector via Kpn1 and Xho1 restriction enzyme sites. The CDS sequence of SNAPIN was inserted into the pCLUC vector using Kpn1 and Sal1 restriction enzyme sites. For pNLUC and pCLUC vectors, please refer to Dongqin Chen*, Fengsheng Hao, Huiqi Mu, Nagib Ahsan, Jay J. Thelen & Gary Stacey*. S-acylation of P2K1 mediates extracellular ATP-induced immunesignaling in Arabidopsis. Nature Communications, 12:2750, 2021.

[0181] (2) The Agrobacterium strain GV3101 carrying the above-described plasmid was cultured to OD200. 600 It is 1.0.

[0182] (3) Mix the ingredients according to Table 7 and apply them to tobacco plants with leaves that are about 5 weeks old. The experimental group and the control group were injected into different positions on one leaf, and more than 10 leaves were injected repeatedly.

[0183] Table 7 Experimental Control Table

[0184]

[0185] (4) 48 hours after inoculation, place the leaves face down into sterilization bags, add 1 mL to 2 mL of 1×D-fluorescein potassium salt working solution, gently press with your hand to spread the working solution evenly on the whole leaf, and place the leaves in the dark at room temperature for 5 min.

[0186] (5) Use a plant in vivo optical imaging system to observe the luminescence of luciferase.

[0187] 2.21 Electrolyte Leakage

[0188] (1) Use a 9mm diameter punch to punch holes in the leaf to be tested. Take 15 small leaf discs, avoiding the inoculation hole and other mechanically damaged areas.

[0189] (2) Transfer the small leaf disc to a 90 mm culture dish, add 15 mL of deionized water, place it on a decolorizing shaker and shake it at room temperature for 3 h.

[0190] (3) After incubation, transfer the deionized water in the petri dish to a 50 mL BD tube, and use a calibrated conductivity meter to measure the electrolyte concentration of the deionized water in the BD tube, which is recorded as n.

[0191] (4) Transfer the small leaf disc to the BD tube and place it in a boiling water bath for 25 minutes. After the deionized water in the BD tube cools to room temperature, use a conductivity meter to measure the electrolyte concentration in the BD tube and record it as N.

[0192] (5) Calculate the electrolyte leakage rate: (n / N)×100%.

[0193] 2.22 Virus-induced gene silencing

[0194] (1) Using the VIGS Tool silencing tool in the online website of Sol Genomics Network, a specific fragment of about 300 bp in the CDS sequence of SNAPIN protein was selected, and the fragment was constructed into the pTRV2-c2b viral silencing vector using the specific restriction enzyme site Sma1.

[0195] (2) The recombinant plasmid was transformed into Agrobacterium GV3101.

[0196] (3) Culture Agrobacterium and collect it, and adjust the OD600 of the bacterial solution to 1.0-1.2. Mix pTRV1 with pTRV2-c2b-SNAPIN, pTRV2-c2b-PDS (phytoene dehydrogenase), and pTRV2-c2b in a volume ratio of 1:1.

[0197] (4) Select chili seedlings that are about 2-3 weeks old, inoculate the mixed Agrobacterium tumefaciens solution onto the chili leaves, and culture for about 1 month.

[0198] (5) RNA was extracted from the leaves of the silenced plants, reverse transcribed into cDNA, and subjected to qRT-PCR. The expression level of SNAPIN was detected using the TRV2 empty vector as a blank to determine the silencing efficiency of the system. When the expression level of the target gene was reduced by more than 65% compared with that of the blank plant, the silencing was considered successful, and the next step of the experiment could be carried out on the successfully silenced plants.

[0199] 2.23 Immunoprecipitation coupled with mass spectrometry (IP-MS)

[0200] (1) Six vigorous, uniformly growing six-week-old Nicotiana benthamiana plants were selected, with each plant forming a group. The experimental group and the control group were inoculated using Agrobacterium-mediated transient expression technology. After 48 hours, 5g of leaves were weighed, wrapped in aluminum foil, and flash-frozen in liquid nitrogen.

[0201] (2) Place 12 50mL BD tubes, several 1.5mL EP tubes, 1mL pipette tips, and 200μL pipette tips in a -20℃ refrigerator for pre-cooling. Pre-cool the centrifuge at 4℃.

[0202] (3) After the cryogenic high-throughput tissue homogenizer is pre-cooled to -50℃, the sample is transferred to the pre-cooled sample tube, placed in the homogenizer, fixed, and homogenized at 50Hz for 60s. After completion, it is placed in liquid nitrogen to cool down and homogenized again. After homogenization, about 400μL of sample is quickly added to a pre-cooled 1.5mL EP tube, and an equal volume of 2× loading buffer is added. The tube is then vortexed and mixed thoroughly. After complete mixing, it is placed in a boiling water bath for 10min and stored at -20℃. The tube is named Western and kept for later use.

[0203] (4) Transfer the remaining sample powder to a pre-cooled 50mL BD tube, add lysis buffer and benzyl sulfonyl fluoride, mix quickly and thoroughly, and place on a 4℃ shaker for at least 4h for lysis.

[0204] (5) The pyrolysis mixture was placed in a centrifuge and centrifuged at 4°C and 4000 r / min for 10 min.

[0205] (6) During centrifugation, prepare six 1.5 mL pre-chilled EP tubes. Using a pre-chilled and cut 200 μL pipette tip, gently aspirate and mix the anti-FLAG agarose culture. Add 15 μL of agarose culture to each tube. Add 500 μL of washing buffer, gently invert the tube, centrifuge at 1000 rpm for 1 min at 4 °C, discard the supernatant, and repeat three times. After washing, place the tubes on ice for later use.

[0206] (7) After centrifugation, transfer the supernatant to a new 1.5 mL pre-cooled EP tube and centrifuge at 4°C, 13000 r / min for 10 min.

[0207] (8) After the second centrifugation, transfer the supernatant to a new 50 mL pre-cooled BD tube. Take 70 μL of the supernatant, add 70 μL of 2× loading buffer, mix well, boil in a water bath for 10 min, and place at -20℃. Name it Input for later use.

[0208] (9) Slowly aspirate the previously washed FLAG-tagged agarose strain into a 50 mL pre-cooled BD tube containing the supernatant and incubate it on a 4°C shaker for 4 h.

[0209] (10) After incubation, insert the BD tubes onto ice and prepare six corresponding numbered 1.5 mL pre-chilled EP tubes. Using a cut, pre-chilled 1 mL pipette tip, add 1-1.5 mL of liquid from the BD tubes to the corresponding 1.5 mL pre-chilled EP tubes each time. Slowly invert the tubes and centrifuge at 4°C, 1000 rpm for 1 min, discarding the supernatant. Repeat this step until all liquid in the BD tubes has been centrifuged. The precipitate in the tubes is the agarose gel containing the tag protein.

[0210] (11) Take 500 μL of washing buffer and slowly rinse the cap of the BD tube, then transfer it into the BD tube to rinse the tube wall. After slowly inverting the tube, centrifuge at 1000 r / min at 4℃ for 1 min and discard the supernatant. Repeat this step 5-6 times.

[0211] (12) Take 500 μL of phosphate buffer and slowly rinse the cap of the BD tube, then transfer it to the BD tube to rinse the tube wall. After slowly inverting the tube, centrifuge at 1000 r / min at 4℃ for 1 min and discard the supernatant. Repeat this step 2-3 times.

[0212] (13) Take 500 μL of PBS buffer to resuspend the beads, take 100 μL of the resuspended solution and add 70 μL of 2× loading buffer, mix gently, boil in a water bath for 10 min, and place at -20℃ and name it IP for later use.

[0213] (14) Centrifuge the remaining resuspension at 1000 r / min for 1 min at 4℃. Discard as much phosphate buffer as possible and store the dried agarose strain at -20℃.

[0214] (15) Centrifuge the Western, Input, and IP samples at 13000 r / min for 10 min. Collect the supernatant and perform SDS-PAGE electrophoresis to verify protein expression.

[0215] (16) If the target protein band is correct and there are no other bands, send the sample to Spectro-Zhonghe (Wuhan) Life Science Technology Co., Ltd. for mass spectrometry experiments.

[0216] 3. Results and Analysis

[0217] 3.1 Validation of the inhibitory function of the effector factor RxLR126 on PTI

[0218] 3.1.1 Effects of the effector factor RxLR126 on reactive oxygen species in plants

[0219] To verify the effect of the effector factor RxLR126 on reactive oxygen species (ROS) production in plants, diaminobenzidine (DAB) staining was used to measure ROS in plants. Peroxidase in plant cells reacts with hydrogen peroxide to produce free hydrogen, which binds to DAB, oxidizing DAB into a brown precipitate located at the site of hydrogen peroxide. RxLR126-GFP was transiently expressed on the left side of *Nicotiana benthamiana* leaves, while GFP was transiently expressed on the right side as a control. After 24 hours, INF1 was transiently expressed at the same location. Leaves were stained with DAB solution 24, 36, and 48 hours after inoculation with INF1. Simultaneously, to quantitatively determine the effect of the effector factor RxLR126 on ROS in plants, RxLR126-GFP was transiently expressed on the left side of *Nicotiana benthamiana* leaves, while GFP was transiently expressed on the right side as a control. After 24 hours, INF1 was transiently expressed at the same location. Two g of treated leaves were weighed at 24, 36, and 48 hours after inoculation with INF1, and the hydrogen peroxide content was determined using a kit. NbRBOHB is an NADPH oxidase, and its expression is upregulated when the elicitor INF1 excites reactive oxygen species in plants. In *Nicotiana benthamiana*, RxLR126-GFP was transiently expressed, followed by INF1 expression 24 hours later. Samples were collected 24 hours after inoculation, and total RNA was extracted from the leaves. This RNA was reverse transcribed into cDNA, and the expression level of NbRBOHB was detected using qRT-PCR.

[0220] Figure 1 Results A showed that after treatment with empty vector GFP and effector factor RxLR126-GFP, followed by inoculation with INF1 Agrobacterium, leaves of both treatments exhibited varying degrees of reactive oxygen species (ROS) bursts. Leaves treated with empty vector GFP served as a control, while leaves treated with effector factor RxLR126 showed significantly lower ROS burst intensity. Figure 1 Results B showed that, under the same conditions, after inoculation with INF1 Agrobacterium, the reactive oxygen species content in leaves treated with the empty vector GFP was higher than that in samples treated with the effector factor RxLR126. Figure 1 The results showed that RxLR126-GFP treatment downregulated the RBOHB gene in Nicotiana benthamiana, indicating that the ROS burst induced by INF1 was suppressed. These results suggest that the effector RxLR126 can inhibit the reactive oxygen species (ROS) burst induced by INF1.

[0221] 3.1.2 Effects of the effector factor RxLR126 on PTI-related resistance genes in plants

[0222] The induced expression of characteristic immune response genes in plants is also a response that plants produce when facing infection by external pathogens. To verify whether the effector factor RxLR126 affects PTI-related resistance genes in plants, RxLR126 and the control GFP were transiently expressed. 24 h later, INF1 was transiently expressed at the same location. Samples were collected quantitatively at 0 h, 6 h, 12 h, 24 h, and 48 h after INF1 inoculation. Total RNA was extracted from the samples and reversed to cDNA. Five PTI-related resistance genes, NbWRKY7, NbWRKY8, NbPTI5, NbACRE31, and NbCYP71D20, were selected for qRT-PCR detection.

[0223] Figure 2 The results showed that, compared with samples treated with empty vector GFP as a control, the effector RxLR126-GFP could inhibit the PTI pathway marker gene NbWRKY7 induced by INF1. Figure 2 A), NbWRKY8 Figure 2 B) and NbPTI5 Figure 2 C) upregulation. The results showed that the effector RxLR126 could inhibit the expression of plant PTI-related resistance genes induced by INF1.

[0224] 3.1.3 Effects of the effector factor RxLR126 on callose deposition in plants

[0225] Callose is a β-(1,3)-D-glucan widely found in plants. Callose not only participates in normal plant growth and development but also plays an important role in plant defense. Aniline blue is an acidic staining agent, poorly soluble in alcohol and water, but it specifically binds to plant callose and emits fluorescence under specific wavelength excitation. RxLR126-GFP and control GFP were transiently expressed on leaves of *Nicotiana benthamiana*, and INF1 was transiently expressed at the same location 24 h later. Leaves were harvested at 0 h, 36 h, and 48 h after inoculation with INF1 for aniline blue staining. Twelve h later, the stained leaves were rinsed with alcohol, and the callose staining was observed under a fluorescence microscope.

[0226] After aniline blue staining, it can be observed that ( Figure 3 After treatment with the empty vector GFP, inoculation with Agrobacterium tumefaciens INF1 induced callose deposition in tobacco leaves. Furthermore, the amount of callose deposition increased continuously over time. In contrast, inoculation with INF1 on tobacco leaves treated with the effector factor RxLR126-GFP did not significantly increase callose deposition. These results indicate that the effector factor RxLR126 can inhibit INF1-induced callose deposition in leaves.

[0227] 3.1.4 Effects of the effector RxLR126 on the phosphorylation cascade of MAPK in plants

[0228] MAPK cascade signaling is a common mechanism for regulating growth and development and responding to environmental changes in all cellular organisms, and it also plays an important role in plant immunity. PTI immunization can induce rapid transient activation of MAPKs, enhancing local immune responses without triggering plant cell death. RxLR126-GFP and control GFP were transiently expressed on Nicotiana benthamiana, and INF1 was inoculated at the same location 24 h later. Samples were quantitatively collected at 0 h, 0.5 h, 1 h, 6 h, and 12 h after INF1 inoculation, and quantitative Western blotting was performed to detect the degree of phosphorylation using the corresponding antibodies.

[0229] according to Figure 4 The experimental results show that after treatment with the empty vector GFP and inoculation with INF1, the degree of MAPK phosphorylation in plants gradually increased. However, after inoculation with INF1 on Nicotiana benthamiana expressing RxLR126-GFP transiently, the intensity of MAPK phosphorylation was weaker compared to the control group. Quantitative analysis of the bands using ImageJ software also showed changes in the band values, reflecting changes in the degree of MAPK phosphorylation. In summary, the effector RxLR126 can inhibit the MAPK phosphorylation cascade induced by INF1 to a certain extent.

[0230] 3.2 Verification of the interaction between the effect factor RxLR126 and CaSNAPIN

[0231] 3.2.1 Immunoprecipitation assay to verify the interaction between effector factor RxLR126 and CaSNAPIN.

[0232] First, RxLR126-GFP co-inoculated with CaSNAPIN-FLAG served as the experimental group, while RxLR126-GFP and FLAG, and GFP and CaSNAPIN-FLAG co-inoculated as the control groups, were transiently expressed in Nicotiana benthamiana. Two days after inoculation, 5g of leaves were weighed for immunoprecipitation experiments, using anti-FLAG agarose beads for protein extraction. After enrichment, washing, and elution, Western blotting was used for experimental verification.

[0233] The results of the immunoprecipitation showed that ( Figure 5A) The INPUT section shows that both the effector proteins RxLR126-GFP and CaSNAPIN-FLAG are normally expressed and can be bound and colored by their corresponding protein antibodies. The effector RxLR126-GFP can be bound and colored by the GFP antibody, with a protein size of 46 kDa; CaSNAPIN-FLAG can be correctly bound and colored by the FLAG antibody, with a protein size of 20 kDa. The IP section bands show that CaSNAPIN-FLAG can be extracted after treatment with anti-FLAG agarose beads, and the protein size at the band position is the same as in the INPUT section. After GFP antibody treatment, a 46 kDa protein band can only be detected in the experimental group where RxLR126-GFP and CaSNAPIN-FLAG are co-inoculated, indicating that the anti-FLAG agarose beads enrich RxLR126-GFP along with CaSNAPIN-FLAG. Experimental results show that there is an interaction between RxLR126 and CaSNAPIN.

[0234] 3.2.2 Luciferase complementation assay to verify the interaction between effector factor RxLR126 and CaSNAPIN.

[0235] Agrobacterium expression vectors RxLR126-nLUC and CaSNAPIN-cLUC were constructed. After expression validation, RxLR126-nLUC and CaSNAPIN-cLUC were co-inoculated as the experimental group, while RxLR126-nLUC and cLUC, nLUC and CaSNAPIN-cLUC, and nLUC and cLUC were co-inoculated as the control group. Transient expression was performed on tobacco leaves. Two days after inoculation, potassium fluorescein was evenly added to the leaves, and after incubation in the dark for 5 minutes, fluorescence was observed using a chemiluminescence analyzer.

[0236] Figure 5 Experiment B showed that no significant fluorescence was observed in the control group's nLUC and cLUC, RxLR126-nLUC and cLUC, and the co-connected regions of nLUC and CaSNAPIN-cLUC. Conversely, significant fluorescence was observed in the co-connected regions of RxLR126-nLUC and CaSNAPIN-cLUC, indicating an interaction between the effector factors RxLR126 and CaSNAPIN.

[0237] 3.2.3 Bimolecular fluorescence complementation experiment to verify the interaction between the effector factor RxLR126 and CaSNAPIN.

[0238] Agrobacterium expression vectors RxLR126-nYFP and CaSNAPIN-cYFP were constructed. After expression validation, RxLR126-nYFP and CaSNAPIN-cYFP were co-inoculated as the experimental group, while RxLR126-nYFP and cYFP, nYFP and CaSNAPIN-cYFP, and nYFP and cYFP were co-inoculated as the control group. Transient expression was performed in Nigeriensis. Two days after inoculation, the luminescence of plant cells was observed using two-photon confocal microscopy at an excitation wavelength of 514 nm and an emission wavelength of 527 nm.

[0239] Figure 6 Experimental results showed that no significant fluorescence was observed in the control group regions of nYFP and cYFP, RxLR126-nYFP and cYFP, and the co-connected regions of nYFP and CaSNAPIN-cYFP. Conversely, significant fluorescence was observed in the co-connected regions of RxLR126-nYFP and CaSNAPIN-cYFP, indicating an interaction between the effector factors RxLR126 and CaSNAPIN.

[0240] 3.3 CaSNAPIN Function Verification

[0241] 3.3.1 Pathogenicity verification of CaSNAPIN

[0242] A CaSNAPIN-GFP Agrobacterium expression vector was constructed. After Western blotting validation, CaSNAPIN-GFP, GFP, and INF1 Agrobacterium cultures were cultivated, and the OD600 of the culture was adjusted to approximately 0.5–0.6. Transient expression was then performed on *N. benthamiana* leaves. The transient expression of CaSNAPIN-GFP served as the experimental group, with GFP and buffer as negative controls and the death elicitor INF1 as a positive control. Fifteen *N. benthamiana* leaves were inoculated at a time, with three replicates. Leaves were harvested based on the degree of necrosis, photographed, and then stained with trypan blue and destained with chloral hydrate. The photographs were then archived.

[0243] Figure 7 The results in (A and B) showed that no changes occurred in the negative control sites of transient expression of the effector RxLR126 and transient expression of empty vector GFP and buffer in *N. benthamiana* leaves. Simultaneously, immunoblotting analysis also indicated normal expression of RxLR126-GFP and GFP in *N. benthamiana* leaves. Conversely, the areas inoculated with the positive control INF1 showed significant necrosis. This indicates that the effector RxLR126 itself does not induce necrosis in *N. benthamiana* leaves.

[0244] 3.3.2 Functional validation of CaSNAPIN against infection by Phytophthora capsici.

[0245] To verify the effect of CaSNAPIN on the infection of *Phytophthora capsici* clumps, CaSNAPIN-GFP was transiently expressed on the left side of *Nicotiana benthamiana* leaves, and GFP was transiently expressed on the right side. After 24 hours, the leaves were removed and laid flat in a tray. *Phytophthora capsici* clumps were inoculated at the injection sites on both sides of the leaves, sealed with sealing film, and incubated in the dark at 28°C. The results were observed under UV light after 48 hours of incubation. Each treatment involved at least 30 leaves, and the treatment was repeated three times.

[0246] Experimental results ( Figure 7 C) indicates that, compared to the side treated with empty vector GFP and inoculated with *Phytophthora capsici*, the area of ​​lesions caused by transient expression of RxLR126-GFP was not significantly different after inoculation with *Phytophthora capsici*. This suggests that the effector factor RxLR126 had no effect on *Phytophthora capsici* infection. The area of ​​*Phytophthora capsici* infection was also statistically analyzed. Figure 7 E) and extracted DNA from the infected leaves for biomass analysis. Figure 7 F), the results were consistent with the infection results. Western blotting confirmed that both RxLR126-GFP and GFP were normally expressed in tobacco.

[0247] 3.3.3 Functional Verification of CaSNAPIN in Inhibiting INF1 Necrosis

[0248] To verify whether the effector factor RxLR126 could inhibit cell death induced by INF1 (the amino acid sequence of INF1 is shown in EQ ID NO:3), Agrobacterium transient expression technology was used to transiently express CaSNAPIN-GFP, GFP, buffer, and Avr3a (the amino acid sequence of Avr3a is shown in EQ ID NO:4) on Nicotiana benthamiana leaves. CaSNAPIN-GFP was used as the experimental group, GFP and buffer were used as negative controls, and the effector protein Avr3a was used as a positive control. After 24 hours of transient expression, INF1 was injected into the same site. Leaf necrosis was observed regularly, and leaves were stained with trypan blue. At least 15 Nicotiana benthamiana leaves were treated each time, and the treatment was repeated 3 times.

[0249] Figure 8 Results showed that transient expression of the effector factor RxLR126-GFP and the positive control Avr3a followed by inoculation with INF1 did not result in cell death at the inoculation site. Conversely, inoculation with GFP and buffer resulted in cell death at the INF1 inoculation site. These results indicate that the effector factor RxLR126 can inhibit INF1-induced cell death. Figure 8 C represents the area statistics of the necrotic region in Benedict's tobacco leaves. Figure 8B is a bar chart of conductivity from electrolyte leakage due to leaf necrosis; the results of these two experiments are the same as those of the inoculation experiment. Immunoblotting results ( Figure 8 D) This demonstrates that RxLR126-GFP, GFP, and Avr3a are all normally expressed in tobacco.

[0250] 3.4 Subcellular localization of effector factors RxLR126 and CaSNAPIN

[0251] 3.4.1 Subcellular localization of effector factors RxLR126 and CaSNAPIN

[0252] To determine the cellular localization of the effector factors RxLR126 and CaSNAPIN, RxLR126-GFP, CaSNAPIN-GFP, and GFP were inoculated into leaves of Nicotiana benthamiana. After transient expression for 48 h, the fluorescence signals were detected using two-photon confocal microscopy at an excitation wavelength of 384 nm and an emission wavelength of 507 nm. Simultaneously, mannitol was injected into the plant cells before slide preparation to induce plasmolysis, further allowing for observation of the cellular localization of the effector factors RxLR126 and CaSNAPIN.

[0253] according to Figure 9 (a) Localization results showed that RxLR126-GFP was located in the cell nucleus; CaSNAPIN-GFP was located in the cell nucleus. Based on... Figure 9 (b) The results showed that after plasmolysis, fluorescence was still clearly visible on the plant cell membrane, indicating that RxLR126-GFP and CaSNAPIN-GFP were also localized on the cell membrane.

[0254] 3.4.2 Subcellular co-localization of effector factors RxLR126 and CaSNAPIN

[0255] First, the Agrobacterium expression vector pBIN-CaSNAPIN-RFP was constructed. After validating the expression, RxLR126-GFP and CaSNAPIN-RFP were used as experimental groups, while RxLR126-GFP and RFP, GFP and CaSNAPIN-RFP, and GFP and RFP were used as control groups for transient co-inoculation. 48 hours after inoculation, the co-localization of RxLR126-GFP and CaSNAPIN-RFP was observed using two-photon confocal microscopy at excitation wavelengths of 384 nm and 507 nm, and at excitation wavelengths of 532 nm and 588 nm.

[0256] Figure 10Experimental results show that RxLR126-GFP and CaSNAPIN-RFP are colocalized in the cell nucleus and cell membrane, indicating that their interaction sites are in the cell nucleus and cell membrane.

[0257] 3.5 Effect of CaSNAPIN on the effect factor RxLR126

[0258] 3.5.1 Effect of the effector factor RxLR126 on CaSNAPIN expression

[0259] To investigate whether RxLR126 affects CaSNAPIN expression, RxLR126-GFP was divided according to OD... 600 RxLR126 and GFP were collected at concentrations of 0.5, 1.0, 1.5, 20, and 2.5, and CaSNAPIN-FLAG was diluted to an OD600 of 1.0. Different concentrations of RxLR126 and CaSNAPIN were mixed at a 1:1 volume ratio and inoculated into whole leaves of Nicotiana benthamiana. Quantitative sampling was performed 48 h after inoculation, and quantitative Western blotting was conducted to observe band changes. RxLR126 and GFP were inoculated into peppers, and samples were collected at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h. Total RNA was extracted from pepper leaves, reverse transcribed into cDNA, and the expression level of SNAPIN was detected using qRT-PCR.

[0260] Figure 11 A indicates that as the concentrations of RxLR126 and GFP continue to increase, the corresponding expression level of CaSNAPIN does not change significantly, suggesting that RxLR126 has no effect on CaSNAPIN expression. Figure 11 B indicates that transient expression of RxLR126 in chili peppers did not result in significant up- or down-regulation of SNAPIN protein expression. These results combined suggest that RxLR126 does not affect SNAPIN protein expression.

[0261] 3.5.2 Effects of CaSNAPIN on the function of the effector factor RxLR126

[0262] To investigate the effect of CaSNAPIN on the inhibition of necrosis induced by the death elicitor INF1 in RxLR126, CaSNAPIN-GFP and RxLR126-GFP were co-transiently expressed in *Nicotiana benthamiana* as the experimental group. Avr3a was inoculated as a positive control, and GFP and buffer were inoculated as negative controls. INF1 was transiently expressed 24 h post-inoculation. The necrosis induced by INF1 was observed periodically and stained with trypan blue. At least 15 leaves were treated each time, with three replicates.

[0263] Figure 12A showed that no leaf necrosis occurred at the co-inoculation sites of CaSNAPIN-GFP and RxLR126-GFP, and no leaf necrosis occurred at the inoculation sites of the positive control Avr3a. As a negative control, cell death occurred at the inoculation sites of INF1 after inoculation with GFP and buffer. The results indicate that CaSNAPIN does not affect the function of the effector RxLR126 in inhibiting INF1-induced cell death. Figure 12 B is a bar chart of conductivity from electrolyte leakage due to blade necrosis. Figure 12 C represents the area of ​​necrotic regions in the tobacco leaf. The results of these two experiments were consistent with those of the inoculation experiment. Immunoblotting results ( Figure 12 D) This demonstrates that RxLR126-GFP, CaSNAPIN-GFP, GFP, and Avr3a are all normally expressed in tobacco.

[0264] 3.6 Silencing CaSNAPIN in Chili Peppers Using VIGS

[0265] 3.6.1 Construction of Silent Carrier

[0266] Bioinformatics analysis revealed that CaSNAPIN does not have a homologous gene in chili peppers. Using the VIGS tool from the Sol Genomics Network website, a suitable gene fragment for silencing was selected, approximately 300 bp in length. Figure 13 The identified 300bp fragment was BLASTed on the NCBI website to confirm that the sequence's reproducibility met the requirements for silencing experiments. The selected fragment was amplified and constructed into the pTRV2-C2b silencing vector. The recombinant plasmid was transformed with Agrobacterium, colonies were selected, and PCR verification was performed using vector primers to screen for positive colonies.

[0267] 3.6.2 Verification of Silent Efficiency

[0268] TRV1 and TRV2-CaSNAPIN were used as experimental groups, TRV1 and TRV2-GFP as negative controls, and TRV1 and TRV2-PDS as positive controls. Transient expression was inoculated into the leaves of pepper seedlings with four to six leaves. About 8 peppers were inoculated in each of the experimental and negative control groups, and 2-3 peppers were used as positive controls.

[0269] Phytoene desaturase (PDS) is the rate-limiting enzyme in the carotenoid biosynthesis pathway, catalyzing the conversion of colorless phytoene into carotene, and is a crucial component of biological carotenoid biosynthesis. When PDS expression in plants is affected, the plants are influenced by carotenoid expression, leading to leaf whitening. When transient co-expression of TRV1 and TRV2-PDS in pepper leaves resulted in leaf whitening, it indicates that the silencing system was functioning normally.

[0270] Approximately four weeks after inoculation, albinism was observed in plants inoculated with TRV2-PDS, confirming the normal functioning of the silencing system. For plants inoculated with TRV2-CaSNAPIN, RNA was extracted from the topmost leaves and reverse transcribed. The expression level of CaSNAPIN in the leaves was then detected using qRT-PCR. CaSNAPIN-specific primers were designed using the NCBI website, with CaActin as an internal reference gene, to detect its expression level.

[0271] Figure 14 As shown in Figure A, pepper plants inoculated with TRV2:PDS exhibited an albino phenotype. This indicates that the pepper VIGS silencing system functioned, and the PDS gene in the pepper was silenced. Figure 14 B shows that, after qRT-PCR experiments, the SNAPIN expression level in TRV2:CaSNAPIN plants was reduced by more than 75% compared to TRV2:GFP plants. This indicates that SNAPIN was successfully silenced in TRV2:CaSNAPIN plants.

[0272] 3.6.3 Validation of the function of the effector RxLR126 in CaSNAPIN-silenced plants

[0273] Based on the aforementioned experiments, the effector RxLR126 can inhibit INF1-induced necrosis in pepper. To determine whether CaSNAPIN is involved in the function of the effector RxLR126, the effectors RxLR126-GFP, Avr3a, GFP, and buffer were transiently expressed in TRV2-GFP and TRV2-CaSNAPIN-silenced plants, respectively. RxLR126-GFP served as the experimental group, Avr3a as the positive control, and GFP and buffer as negative controls. Twenty-four hours after transient expression, INF1 was inoculated at the same location, and necrosis was observed periodically.

[0274] Figure 15A showed that transient expression of RxLR126 in TRV2:GFP-silenced plants did not induce leaf necrosis after inoculation with INF1. However, transient expression of RxLR126 in TRV2:CaSNAPIN-silenced plants resulted in some degree of leaf necrosis after inoculation with INF1. This indicates that CaSNAPIN silencing led to a partial loss of the function of RxLR126 in inhibiting INF1-induced leaf death. Figure 15 B shows that, statistical analysis of leaf necrosis rates in TRV2:GFP and TRV2:CaSNAPIN treatments revealed a higher necrosis rate in plants silenced by TRV2:CaSNAPIN compared to those treated by TRV2:GFP, confirming the above conclusion. (Immunoblotting results) Figure 15 C) This demonstrates that both RxLR126-GFP and GFP are normally expressed in chili peppers.

[0275] 3.7 Screening for other interacting proteins of effector factor RxLR126 using IP-MS

[0276] To screen downstream interacting proteins of the effector factor RxLR126, IP-MS was used. Immunoprecipitation tandem mass spectrometry (IP-MS) is a method for studying protein interactions based on the use of specific antibodies to extract the interacting proteins of the target protein from complex samples, followed by mass spectrometry detection to identify these interacting proteins. The effector factors RxLR126-GFP and GFP were transiently expressed on whole Nicotiana benthamiana plants, with three plants inoculated for each experimental group. After 48 hours of transient expression, an immunoprecipitation experiment was performed, and anti-GFP beads were used to enrich the effector factors RxLR126 and GFP. After enrichment and washing, the three replicate beads from each of the two experimental groups were analyzed by mass spectrometry to identify proteins that may interact with the effector factor RxLR126.

[0277] Mass spectrometry results as follows Figure 16 As shown, the experimental results are based on the CytoHubba interaction network node scoring algorithm to score interacting proteins. Three different scoring algorithms were used: the MCC algorithm (…). Figure 16 A) DMNC algorithm ( Figure 16 B) degree Figure 16 C). Based on the algorithm abundance, proteins with higher scores were selected for preliminary protein-protein interaction verification (Table 8).

[0278] Table 8 Candidate Interacting Proteins

[0279]

[0280] In this experiment, the luciferase complementation method was used to conduct preliminary interaction verification of candidate proteins. RxLR126-Nluc and CaSNAPIN-Cluc were co-coupled as a positive control, and RxLR126-Nluc and the suspected interacting protein-Cluc were co-coupled as the experimental group. Figure 17 Significant fluorescence was observed in the co-inoculation region of RxLR126-Nluc and CaSNAPIN-Cluc in the positive control group, but no fluorescence was observed in the inoculation regions of other experimental groups. These results indicate that none of the ten initially selected interacting proteins interact with RxLR126.

[0281] In this invention, the amino acid sequences of RxLR126, CaSNAPIN, INF1, and Avr3a and their coding gene sequences are shown below. Figures 18-21 .

[0282] In nature, plants are constantly attacked by various microorganisms. Different types of microorganisms establish different types of interactions with plants, either killing plants to obtain nutrients from dead tissues, relying on the metabolism of living plants to obtain nutrients, or infecting established plants before killing them. *Phytophthora capsici*, as a semi-biotrophic fungus, has a wide range of infecting hosts and a strong ability to evade plant immune recognition, causing serious damage to crops. Therefore, elucidating the molecular interaction mechanisms between pathogens and plants is particularly important.

[0283] Effector factors are powerful tools that pathogens and plants have developed through continuous struggle and evolution, but they are also a double-edged sword. On the one hand, they play a crucial role in the infection of plants; pathogens can use effector factors to evade plant immune recognition and suppress the plant's immune response, thereby achieving infection. On the other hand, effector factors are targets for plant recognition of pathogens, and plants initiate immune suppression against these effector factors to protect themselves. Currently, the most studied effector factors are the RxLR effectors. This large class of effectors plays a significant role in pathogen infection and is key to the pathogenicity of *Phytophthora capsici*.

[0284] In previous studies, the effector factor RxLR126 of *Phytophthora capsici* was selected and found to be induced in the early stages of *Phytophthora capsici* infection. *Phytophthora capsici* is in a biotrophic stage during the early infection phase; therefore, RxLR126 may play a role in suppressing plant immunity to aid infection. Furthermore, previous experiments demonstrated that RxLR126 can inhibit leaf necrosis induced by the death elicitor INF1, indicating that RxLR126 can suppress plant immunity. PTI signaling can induce a series of defensive responses, including ROS bursts, callose deposition in the cell walls of infected sites, upregulation of immune gene expression, and activation of the MAPK cascade. According to related research, some RxLR effectors can influence ROS production pathways and interfere with plant signal transduction to suppress plant immunity. This study further investigated the function of RxLR126, exploring its effects on plant ROS outbreaks, detection of related immune pathway marker genes, callose deposition, and the role of classic PTI-related reactions such as the MAPK phosphorylation cascade. This further confirmed that RxLR126 has the function of inhibiting plant PTI immunity.

[0285] Effector factors that suppress plant immunity during infection must have target proteins in the plant host, achieving their purpose through protein-protein interactions. Dou et al. discovered that RxLR242 can target plant RAB proteins to suppress plant immunity. Previously, a yeast screening library identified host proteins that might interact with the effector factor RxLR126, but further interaction verification was not conducted. This study verified the interaction with the SNAPIN protein in pepper. Using immunoprecipitation, luciferase complementation, and bimolecular fluorescence complementation, it was determined that RxLR126 can interact with the pepper-derived SNAPIN protein. SNAPIN participates in intracellular substance transport, signal transduction, and assists SNARE proteins in vesicle transport. Using Agrobacterium-mediated transient transformation, the function of SNAPIN was verified, revealing that SNAPIN also inhibits the death elicitor INF1, thus acting as a negative regulator of immunity.

[0286] To confirm the role of SNAPIN in the function of RxLR126, this study used VIGS technology to silence the SNAPIN protein in peppers, successfully obtaining silenced pepper plants. Subsequently, the function of RxLR126 in inhibiting necrosis induced by the death elicitor INF1 was verified in the silenced pepper plants. Compared with pepper plants without SNAPIN silence, the silenced plants exhibited a certain degree of leaf necrosis phenotype. This indicates that the function of RxLR126 in inhibiting INF1-induced necrosis was weakened, confirming that SNAPIN plays a role in the function of RxLR126. However, even after silencing SNAPIN, RxLR126 still retains its inhibitory effect on necrosis, suggesting that other proteins may be involved in the function of RxLR126. Furthermore, whether CaSNAPIN is involved in the function of RxLR126 in inhibiting PTI has not yet been verified, and further research is needed to supplement this work with related experiments.

[0287] Meanwhile, to confirm whether other plant-derived proteins mediate the function of RxLR126, this study screened potential interacting proteins for RxLR126 using IP-MS, resulting in a series of potentially interacting protein families. Among the candidate proteins are those closely related to plant immunity, which may interact with RxLR126 to mediate its function. These will be verified using classic protein-protein interaction experiments.

[0288] Current research confirms the function of RxLR126 in inhibiting plant PTIs and demonstrates its interaction and functional relationship with CaSNAPIN proteins. However, the deeper interaction mechanisms between the two require further detailed investigation to provide more theoretical support for studying the pathogenesis and virulence mechanisms of Phytophthora capsici.

[0289] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of the CaSNAPIN gene in suppressing tobacco leaf necrosis induced by the death elicitor INF1, among which, The death elicitor INF1 is a protein consisting of the amino acid sequence shown in SEQ ID NO:3, and the CaSNAPIN gene therein encodes a protein consisting of the amino acid sequence shown in SEQ ID NO:

2.

2. Application of the CaSNAPIN gene in suppressing pepper leaf necrosis induced by the death elicitor INF1. in, The death elicitor INF1 is a protein consisting of the amino acid sequence shown in SEQ ID NO:3, and the CaSNAPIN gene therein encodes a protein consisting of the amino acid sequence shown in SEQ ID NO:

2.

3. A method for mitigating plant leaf necrosis caused by the death elicitor INF1, characterized in that, The method includes: overexpressing the CaSNAPIN gene in a plant, wherein the death elicitor INF1 is a protein consisting of the amino acid sequence shown in SEQ ID NO:3, the CaSNAPIN gene encodes a protein consisting of the amino acid sequence shown in SEQ ID NO:2, and wherein the plant is tobacco and pepper.

4. The method according to claim 3, characterized in that The overexpression mode is selected from the following 1) to 2), or a combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the genes described on the plant chromosomes.

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