A gene REL that enhances plant disease resistance and its application

Abstract

This invention provides a gene REL for enhancing plant disease resistance and its applications, belonging to the fields of plant molecular biology and plant genetic engineering. This invention relates to a plant-derived disease resistance gene REL, its recombinant expression vector, and its applications. The gene is derived from *Nicotiana benthamiana* and has the nucleotide sequence shown in SEQ ID NO.1, with the encoded product having the amino acid sequence shown in SEQ ID NO.3. This invention also provides a recombinant expression vector comprising the gene REL described herein. This gene plays a crucial role in tobacco resistance to various *Phytophthora* fungi. This invention can be applied to crop breeding for disease resistance improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering. Specifically, this invention relates to a gene REL that enhances plant disease resistance and its application. Background Technology

[0002] Crop diseases caused by Phytophthora fungi cause enormous economic losses to my country's agricultural production every year. Among them, potato late blight, caused by Phytophthora infestans, is one of the most devastating diseases to damage agricultural production. In the mid-19th century, this disease led to the Irish Famine, causing the deaths of millions of people in Ireland. Even now, late blight caused by Phytophthora infestans continues to restrict the development and production of potatoes and tomatoes. [1] Furthermore, *Phytophthora sojae* and *Phytophthora parasitica* are also *Phytophthora* fungi that cause huge economic losses to agricultural production, posing a serious threat to the production of important crops such as soybeans (*Phytophthora sojae*) and tobacco (*Phytophthora capsici*). [2] Phytophthora infestans causes rapid disease development, mutations quickly, and poses a serious threat in the field. Therefore, effective control requires improving crop resistance.

[0003] Plants can recognize pathogens in the environment through pattern recognition receptors (PRRs) on the cell membrane surface and intracellular immune recognition receptors (NLRs), thereby activating an immune response and resisting pathogen invasion. [3] Based on the "gene-to-gene" hypothesis, the protein encoded by the intracellular NLR gene has a one-to-one recognition relationship with the effector molecules secreted by pathogens. That is, the disease resistance protein encoded by the NLR can only specifically recognize effector molecules secreted by pathogens. However, pathogens can easily evade this recognition through their own mutations. After the effector molecules mutate, the disease resistance function of the NLR resistance protein in the plant is lost. [4] Microbe-associated molecular patterns (MAMPs) are a class of highly conserved immune response elicitors that are components of pathogens or play a functional role. Pattern recognition receptors (PRRs) on plant cell membranes can recognize pathogen model molecules (MAMPs), triggering different downstream signaling pathways, inducing reactive oxygen species (ROS) bursts, necrosis responses (HR), etc., thereby resisting pathogen invasion. [5-6]Currently identified cloned recognition receptors include FLS2, a recognition receptor for bacterial flagellin, and CERK1, a recognition receptor for chitin in fungal cell walls. [7-8] FLS2 and CERK1 activate plant immune responses by recognizing bacterial flagellin and chitin in fungal cell walls. Both bacterial flagellin and fungal cell wall chitin are crucial components for bacterial and fungal growth and pathogenicity, and are unlikely to mutate during evolution. Therefore, immune responses mediated by pattern recognition receptors on plant cell membranes are characterized by broad-spectrum and persistent immunity. [ 9 ] Therefore, screening for the recognition receptors of pathogenic MAMPs and modifying these receptors using disease-resistant genetic engineering technology to obtain more broad-spectrum and efficient disease-resistant varieties has become a more promising control method in plant disease resistance breeding.

[0004] [1] Fry WE, Birch PR, Judelson HS, Grünwald NJ, Danies G, Everts KL, GevensAJ, Gugino BK, Johnson DA, Johnson SB, McGrath MT, Myers KL, Ristaino JB, Roberts PD, Secor G, Smart CD. Five reasons to consider Phytophthora infestans are emerging pathogen.Phytopathology.2015Jul;105(7):966-81.doi:10.1094 / PHYTO-01-15-0005-FI.Epub 2015 Jun 26.PMID:25760519.

[0005] [2]Wang, W., Jiao, F. Effectors of Phytophthora pathogens are powerfulweapons for manipulating immunity host. Planta 250, 413–425 (2019).

[0006] [3]Vleeshouwers VG,Raffaele S,Vossen JH,Champouret N,Oliva R,SegretinME,Rietman H,Cano LM,Lokossou A,Kessel G,Pel MA,Kamoun S.Understanding andexploiting late blight resistance in the age of effectors.Annu RevPhytopathol.2011;49:507-31.doi:10.1146 / annurev-phyto-072910-095326.PMID:21663437.

[0007] [4]Dodds PN.Plant science.Genome evolution in plantpathogens.Science.2010 Dec 10;330(6010):1486-7.doi:10.1126 / science.1200245.PMID:21148378;PMCID:PMC3076603.

[0008] [5]Bigeard J,Colcombet J,Hirt H.Signaling mechanisms in pattern-triggered immunity (PTI).Mol Plant.2015Apr;8(4):521-39.doi:10.1016 / j.molp.2014.12.022.Epub 2015 Jan 9.PMID:25744358.

[0009] [6]Wu Y,Zhou JM.Receptor-like kinases in plant innate immunity.JIntegr Plant Biol.2013 Dec;55(12):1271-86.doi:10.1111 / jipb.12123.PMID:24308571.

[0010] [7]Gómez-Gómez L, Boller T. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. MolCell. 2000 Jun; 5(6):1003-11.doi:10.1016 / s1097-2765(00)80265-8.PMID:10911994.

[0011] [8]Miya A,Albert P,Shinya T,Desaki Y,Ichimura K,Shirasu K,Narusaka Y,Kawakami N,Kaku H,Shibuya N.CERK1, a LysM receptor kinase,is essential forchitin elicitor signaling in Arabidopsis.Proc Natl Acad Sci US A.2007Dec 4;104(49):19613-8.doi:10.1073 / pnas.0705147104.Epub 2007Nov 27.PMID:18042724;PMCID:PMC2148337.

[0012] [9] Tsuda K, Katagiri F. Comparing signaling mechanisms engaged inpattern-triggered and effector-triggered immunity. Curr Opin PlantBiol. 2010Aug; 13(4):459-65.doi:10.1016 / j.pbi.2010.04.006.Epub 2010 May12.PMID:20471306. Summary of the Invention

[0013] One of the objectives of this invention is to provide a gene REL.

[0014] The second objective of this invention is to provide a recombinant expression vector containing the REL gene.

[0015] The third objective of this invention is to provide applications for gene REL.

[0016] The specific details of this invention are as follows:

[0017] This invention provides a gene REL derived from tobacco, with a nucleotide sequence as shown in SEQ ID NO.1, or a tobacco-derived gene having more than 80% homology with sequence SEQ ID NO.1 that enhances plant disease resistance. In some embodiments, the nucleotide sequence is as shown in SEQ ID NO.1, or a tobacco-derived gene having more than 85% homology with sequence SEQ ID NO.1 that enhances plant disease resistance. In some more specific examples, the nucleotide sequence is as shown in SEQ ID NO.1, or a tobacco-derived gene having more than 90% homology with sequence SEQ ID NO.1 that enhances plant disease resistance, or even more specifically, a tobacco-derived gene having more than 95% homology with sequence SEQ ID NO.1 that enhances plant disease resistance.

[0018] The present invention also provides a fragment of the knockout gene REL, the sgRNA sequence of which is SEQ ID NO.2.

[0019] This invention also provides a protein encoded by a gene REL or a protein having an amino acid sequence with at least 50% similarity to a protein encoded by a gene REL. In a specific example, the amino acid sequence of the protein encoded by the gene REL of this invention is shown in SEQ ID NO.3, and the amino acid sequence of the protein having at least 50% similarity to the protein encoded by the gene REL is shown in SEQ ID NO.4. Alternatively, it can be a protein that enhances plant disease resistance with an amino acid sequence with at least 80% similarity; more preferably, it is a protein that enhances plant disease resistance with an amino acid sequence with at least 85% similarity, or a protein that enhances plant disease resistance with an amino acid sequence with at least 90% similarity, or a protein that enhances plant disease resistance with an amino acid sequence with at least 95% similarity.

[0020] Using the amino acid sequence encoded by the gene of this invention, signal peptide sequences can be designed and artificially added to facilitate expression in plants.

[0021] Using the gene-encoded amino acid sequence of the present invention, codon-optimized nucleic acid sequences that are beneficial for expression in plants can be designed and synthesized artificially.

[0022] The present invention also provides a recombinant expression vector comprising the aforementioned gene REL.

[0023] The expression vector is preferably a plant transformation plasmid, such as pBin::eGFP, pCambia, or pTF101.1.

[0024] Preferably, the recombinant expression vector is pBin::REL-eGFP, obtained by inserting the gene REL into the binary vector pBin::eGFP restriction site KpnI containing C-terminal eGFP.

[0025] A transformant obtained by introducing the recombinant expression vector into a host cell, wherein the host cell is preferably an Escherichia coli cell or an Agrobacterium cell.

[0026] Primer pairs that amplify the full length or any fragment of the gene REL are also within the scope of protection of this invention.

[0027] The present invention also provides the application of the gene REL or its encoded protein or recombinant expression vector or transformant in improving plant immune resistance or disease resistance.

[0028] The present invention also provides the application of the gene REL or its encoded protein or recombinant expression vector or transformant in enhancing the immune resistance of plants to pathogens or enhancing plant resistance to diseases caused by pathogens.

[0029] Furthermore, the pathogenic bacteria described in this invention are pathogenic bacteria capable of secreting elicitor proteins, such as Phytophthora or Pythium. Phytophthora can include pathogenic bacteria that can cause plant diseases, such as Phytophthora soybeani, Phytophthora pathogenicum, Phytophthora capsicum, and Phytophthora tobaccoi.

[0030] The present invention also provides the application of the gene REL or its encoded protein or recombinant expression vector or transformant in the breeding of plants with immune resistance to pathogenic bacteria.

[0031] The present invention also provides the application of the tobacco gene REL or its encoded protein or recombinant expression vector or transformant in varieties that acquire significant disease resistance after introduction into plants.

[0032] In some embodiments, the plant described in this invention is a solanaceous plant or a legume, such as, but not limited to, tobacco, chili pepper, tomato, potato, or soybean.

[0033] This study analyzed cell membrane receptors in tobacco and found that the immune receptor REL is involved in recognizing elicitors secreted by different *Phytophthora* and *Pythium* fungi, playing a decisive role in the immune responses induced by these elicitors in plants. The REL gene plays a crucial role in tobacco's recognition of different *Phytophthora* and *Pythium* fungi. Furthermore, as an important economic crop and a typical representative of the Solanaceae family, research on REL in tobacco can drive related research on cell membrane receptors in many other Solanaceae plants such as peppers, tomatoes, and potatoes. In addition, the elicitors are conserved among different *Phytophthora* and *Pythium* fungi, indicating that REL has the ability to recognize different *Phytophthora* and *Pythium* fungi. These studies will better elucidate the disease resistance functions of plants against *Phytophthora* and different pathogens, providing excellent disease resistance gene resources for disease resistance genetic engineering breeding.

[0034] The beneficial effects of this invention are:

[0035] The protein encoded by the REL gene described in this invention activates the plant's innate immunity by recognizing elicitors secreted by pathogens, thereby enhancing plant disease resistance. Overexpression in plants does not affect plant growth traits, and it exhibits broad-spectrum recognition of Phytophthora, significantly enhancing plant resistance to this fungus. This invention can be applied to crop breeding for disease resistance improvement, potentially increasing plant resistance to Phytophthora blight and thus achieving the goal of increasing yield and reducing pesticide use. Attached Figure Description

[0036] Figure 1 Screening and acquisition of REL genes. A. Detection of REL gene expression in TRV2::REL-silenced tobacco. Real-time quantitative PCR was used to detect the REL gene expression level in TRV2::REL-treated silent tobacco, where TRV::GFP was the control plant, and -1 and -2 were different plants with REL gene silencing. B. Cell necrosis induced by Phytophthora inducing INF1 and different exoside effectors in silent tobacco.

[0037] Figure 2 Identification of homozygous mutants of REL knockout tobacco. PCR cloning of REL gene sgRNA in knockout tobacco.

[0038] (Gene editing target) sequence, where WT is wild-type tobacco, and -1 and -2 are different REL knockout strains.

[0039] Figure 3 Cell necrosis symptoms induced by the expression of different Phytophthora and Pythium elicitors in REL gene knockout plants. Ps: Phytophthora soybeani, Pi: Phytophthora pathogenica, PPTG: Phytophthora tobaccoi, Pc: Phytophthora capsici, Py: Pythium oligomastii.

[0040] Figure 4Symptoms of disease in tobacco plants with WT and REL gene knockout after inoculation with *Phytophthora indicum*. A, Symptoms 3 days after inoculation with *Phytophthora indicum*. B, Symptoms 4 days after inoculation with pathogenic *Phytophthora indicum*.

[0041] Figure 5 Biomass assays of tobacco plants with WT and REL gene knockouts after inoculation with *Phytophthora indicum*. A, Biomass 3 days after inoculation with *Phytophthora indicum*. B, Biomass 4 days after inoculation with pathogenic *Phytophthora indicum*.

[0042] Figure 6 Disease symptoms and protein expression detection of pBin::eGFP and pBin::REL-eGFP transgenic tobacco after inoculation with *Phytophthora infestans*. Western blot was used to detect the expression levels of control eGFP and REL-eGFP, with anti-GFP as the detection antibody. A, Protein expression detection of pBin::eGFP and pBin::REL-eGFP in tobacco. B, Disease symptoms after inoculation with *Phytophthora infestans*.

[0043] Figure 7 Disease symptoms and protein expression detection of pBin::eGFP and pBin::REL-eGFP transgenic peppers after inoculation with Phytophthora capsici. Western blot was used to detect the expression levels of control eGFP and REL-eGFP, and the detection antibody was anti-GFP. A, Protein expression detection of pBin::eGFP and pBin::REL-eGFP in peppers. B, Disease symptoms 3 days after inoculation with Phytophthora capsici. Detailed Implementation

[0044] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The primers involved in the embodiments of the present invention were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0045] The Phytophthora tobaccoii, Phytophthora pathogenica, and Phytophthora capsici used in the following examples were all collected and isolated in the field in the laboratory and are now preserved in this laboratory. The inventors and the applicant promise to make them permanently available to the public.

[0046] Example 1. Screening and obtaining the REL gene

[0047] Using virus-induced gene silencing technology, cell membrane receptor genes were silenced in tobacco leaves. After treatment, tobacco seedlings were cultured in a greenhouse (21-23℃, 14 hours light / 10 hours dark) for four weeks. The silenced tobacco was then treated with the Phytophthora exciton INF1, yeast-purified INF1 protein, and different exotropic effectors XEG1 and NPP1. Based on the INF1-induced cell necrosis, the receptor protein REL, which recognizes the exciton, was screened. The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.1.

[0048] The specific steps are as follows:

[0049] 1) Culture of Agrobacterium containing silencing vector

[0050] Single colonies of Agrobacterium GV3101 transfected with the pTRV2::REL vector (REL gene silencing vector), single colonies of Agrobacterium GV3101 containing the pTRV2::GFP vector (control), and single colonies of Agrobacterium GV3101 containing the virus pTRV1 were picked from the plates and inoculated into 2 mL of LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and incubated overnight at 30°C and 200 rpm until the OD600 reached 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10 mM 2-[N-morpholino]ethanesulfonic acid, 10 mM MgCl2, 200 μM Macetosyringone, pH 5.6) and centrifuged again to collect the bacterial cells. The washing was repeated three times, and the bacterial suspension was diluted with buffer. pTRV1 was mixed 1:1 with Agrobacterium tumefaciens containing pTRV2::REL and pTRV2::GFP, respectively, and labeled as TRV::REL and TRV::GFP, with a final concentration of 1.0 for each.

[0051] 2) Gene silencing in tobacco

[0052] The prepared Agrobacterium tumefaciens solution was injected into four leaves of two-week-old tobacco seedlings using a syringe. After four weeks of cultivation in a greenhouse (21-23℃, 14 hours light / 10 hours dark), the gene silencing level was tested.

[0053] Reference method: Dong, Y., Burch-Smith, TM, Liu, Y., Mamillapalli, P., Dinesh-Kumar, SP2007. A ligation-independent cloning TRV vector for high-throughputvirus induced gene. Plant Physiology, 145, 1161-1170.

[0054] 3) REL gene silencing efficiency detection

[0055] Total RNA was extracted from tobacco leaves that had been silent for four weeks. The extraction of total RNA was performed using an Omega RNA extraction kit according to the instructions, and the RNA content and quality were determined using a spectrophotometer.

[0056] First-strand reverse transcription: Using 0.7 μg of RNA as a template, cDNA was synthesized according to the instructions for use of Takara's PrimeScript reverse transcriptase reagent, and the volume was adjusted to 20 μL. The reverse transcription product was diluted 10-fold with water for real-time quantitative PCR to detect gene silencing efficiency.

[0057] Real-time quantitative PCR reaction:

[0058] Pre-quantification primer: SEQ ID NO.5

[0059] 5'-GTCCACCATCGATCTCTCCA-3'

[0060] Primer after quantification: SEQ ID NO.6

[0061] 5'-GGCATAGTTGTAAGGGAATG-3'

[0062] The PCR reaction system contained 5 μL cDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4 μL pre- and post-primer primers, 0.4 μL ROX Reference Dye II, and 13.8 μL water. The reaction program was: I: 95°C for 30 seconds, II: 95°C for 5 seconds, 60°C for 34 seconds, with 40 cycles in step II. The melting curve analysis program was: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds. Data analysis was performed using the 2-ΔΔCT method, and the results are shown below. Figure 1Shown in A. Reference: Livak, KJ, and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402-408.

[0063] 4) Agrobacterium culture

[0064] Single colonies of GV3101 transfected with the pathogenic *Phytophthora indicum* INF1 vector, *Phytophthora sacchari* XEG1 vector, and *Phytophthora camphorata* NPP1 vector were picked from the plates and inoculated into 2 mL of LB broth (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and incubated overnight at 30°C and 200 rpm on a shaker until OD reached. 600 The concentration was 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10mM 2-[N-morpholino]ethanesulfonic acid, 10mM MgCl2, 200μM acetosyringone, pH 5.6) and then centrifuged again to collect the cells. After washing three times, the bacterial suspension was diluted with buffer to a final concentration of 0.2 for each solution.

[0065] 5) Silenced tobacco leaves were injected with purified INF1 protein from Agrobacterium and yeast encoding the XEG1, INF1, and NPP1 vectors. The tobacco was then cultured in a greenhouse (21-23℃, 14 hours light / 10 hours dark). Three days after injection, photographs were taken and cell necrosis was recorded. It was found that after injection of XEG1, INF1, NPP1, and purified INF1 protein, INF1-induced cell necrosis completely disappeared in tobacco leaves with the REL receptor gene silenced. Figure 1 As shown in Figure B, these results demonstrate that REL can specifically recognize the exciton INF1.

[0066] Example 2. Transient expression of different Phytophthora and Pythium elicitor proteins on REL-knockout tobacco.

[0067] 1) Obtaining homozygous REL gene knockout tobacco

[0068] The sgRNA used to knock out the REL gene has the sequence shown in SEQ ID NO.2 (5'-ACAGCTCCTTCAACGAATGTTGG-3'). REL-knockout tobacco plants were obtained and placed in a greenhouse (21-23℃, 14 hours light / 10 hours dark). Leaves were taken from each tobacco plant, flash-frozen in liquid nitrogen, ground, and the genome was extracted using the DNAsecure Plant Kit (TIANGEN). Using the extracted genome as a template, PCR was used to amplify approximately 150 bp fragments upstream and downstream of the sgRNA target sequence.

[0069] PCR amplification primer sequences:

[0070] Upstream primer: SEQ ID NO.7

[0071] (5'-TAGAAATCTAACGTCTCTTTCCG-3')

[0072] Downstream primer: SEQ ID NO.8

[0073] (5'-CCAATACTTGATGTAATGCTCCC-3')

[0074] The 50 μL reaction mixture consisted of 25 μL 2×PhantaMax Master Mix, 1 μL template gDNA, and water added to a final volume of 50 μL. The PCR amplification program was: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for 40 cycles, followed by a final extension at 72℃ for 10 minutes. The PCR products were sent to Nanjing Sangon Biotech Co., Ltd. for sequencing. The sequencing results of the homozygous REL gene knockout tobacco were as follows: Figure 2 As shown.

[0075] 2) Transient expression of different Phytophthora elicitor proteins on REL-knockout tobacco

[0076] Single colonies of Agrobacterium GV3101 transfected with the INF1 elicitor or a vector containing the INF1 homologous gene of Phytophthora soybeani, Phytophthora tobaccoi, Phytophthora pathogenica, Phytophthora capsici, and Pythium oligomaleum were picked from plates and inoculated into 2 mL of LB broth (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and incubated overnight at 30°C and 200 rpm on a shaker until OD reached. 600The concentration was 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10mM 2-[N-morpholino]ethanesulfonic acid, 10mM MgCl2, 200μM acetosyringone, pH 5.6) and then centrifuged again to collect the cells. After washing three times, the bacterial suspension was diluted with buffer to a final concentration of 0.2 for each solution.

[0077] 2) Agrobacterium-mediated transformation of different pathogen elicitor vectors was injected into 6-week-old tobacco leaves. The tobacco was then cultured in a greenhouse (21-23℃, 14 hours light / 10 hours dark). Three days after injection, photographs were taken and cell necrosis was recorded. It was found that the cell necrosis induced by injection of different Phytophthora elicitors completely disappeared in tobacco leaves with the receptor gene REL knocked out. Figure 3 As shown, these results demonstrate that REL has a broad spectrum of recognition for elicitors secreted by different Phytophthora species.

[0078] Example 3. Knockout of tobacco inoculated with Phytophthora nicotineans and pathogenic Phytophthora.

[0079] 1) Inoculation with Phytophthora indica

[0080] Place the knocked-out tobacco plants in a plastic incubator. Place a drop of 10 μL sterile water in the center of a flattened leaf, and inoculate with a 5 mm diameter fresh *Phytophthora indicum* fungus cake. Seal the plastic box to maintain humidity. Inoculated plants are first placed in darkness for 24 hours, then transferred to a greenhouse (21-23℃, 14 hours light / 10 hours darkness). Photographs are taken and leaf disease development is recorded three days after inoculation. Compared to the control WT plants, the leaves of the knocked-out REL plants inoculated with *Phytophthora indicum* showed significantly larger lesions. Figure 4 A). These results confirm that REL plays an important role in the fight against fungi in tobacco.

[0081] 2) Inoculation with pathogenic fungi

[0082] Place the knocked-out tobacco leaves in a plastic inoculation dish. Induce the production of zoospores of *Phytophthora infestans* using ddH2O. Inoculate the leaves with 10 μL of ddH2O (enough for 1000 zoospores), and seal the inoculation dish to maintain humidity. Incubate the inoculated plants in the dark for 96 hours. Photograph and record leaf disease development 4 days after inoculation. Compared to the control WT plants, the leaves with knocked-out RELs showed significantly larger lesions after inoculation with *Phytophthora infestans*. Figure 4 B). These results confirm that REL is essential for tobacco to combat antifungal fungi.

[0083] 3) Phytophthora biomass detection

[0084] Cut a piece of aluminum foil that can cover the lesions. Using the cut aluminum foil as a template, cut leaves of the same size from the infected leaves. Quick-freeze the leaves in liquid nitrogen, grind them, and extract the genome using the DNAsecure Plant Kit (TIANGEN). Use the extracted genome as a template to perform a real-time quantitative PCR reaction to detect biomass.

[0085] Real-time quantitative PCR reaction:

[0086] Pre-quantification primer for tobacco: SEQ ID NO.9

[0087] 5'-AGTATGCCTGGGTGCTTGAC-3'

[0088] Tobacco quantification primer: SEQ ID NO.10

[0089] 5'-ACAGGGACAGTTCCAATACCA-3'

[0090] Pre-quantitative primers for Phytophthora indicum: SEQ ID NO.11

[0091] 5'-ATGAACTTCCGCGCTCTGTT-3'

[0092] Primers for quantitative analysis of Phytophthora indicum: SEQ ID NO.12

[0093] 5'-CAGTGACGCGCACGTAGAC-3'

[0094] Pre-primer for quantitative analysis of Phytophthora infectivity: SEQ ID NO.13

[0095] 5'-GCATTGAGGGGGCGTATT-3'

[0096] Primer for quantitative analysis of pathogenic Phytophthora: SEQ ID NO.14

[0097] 5'-GCGGAAGAAGGAAGATTCGA-3'

[0098] The PCR reaction system contained 5 μL cDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4 μL pre- and post-primer primers, 0.4 μL ROX Reference Dye II, and 13.8 μL water. The reaction program was: Step I: 95°C for 30 seconds, Step II: 95°C for 5 seconds, 60°C for 34 seconds, with 40 cycles in Step II. The melting curve analysis program was: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds. Data analysis was performed using 2... -ΔΔCT Methods and test results are as follows Figure 5As shown, Figure A represents the biomass of *Phytophthora nicotinea* 3 days after inoculation. Figure B represents the biomass of *Phytophthora nicotinea* 4 days after inoculation with pathogenic *Phytophthora*. Reference: Livak, KJ, and Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCT method.Methods 25, 402-408.

[0099] The results showed that knocking out the REL gene significantly increased the biomass of Phytophthora tobacco colonization.

[0100] Example 4. Cloning and Sequence Structure Analysis of the REL Gene

[0101] Tobacco seeds were sown directly in pots filled with nutrient soil and cultured in a greenhouse (21-23℃, 14 hours of light / 10 hours of darkness). Six-week-old plants were used for RNA extraction.

[0102] Total RNA extraction: Using tobacco leaves as material, total RNA was extracted using the Omega RNA extraction kit according to the instructions, and the RNA content and quality were detected using a spectrophotometer.

[0103] First-strand reverse transcription: Using 0.7 μg of RNA as a template, cDNA synthesis was performed according to the instructions for use of Takara's PrimeScript reverse transcriptase reagent, and the volume was adjusted to 20 μL. An appropriate amount of the reverse transcription product was used for subsequent gene cloning PCR.

[0104] Using the first strand of cDNA as a template for RT-PCR, PCR is performed using standard methods to amplify the REL gene fragment or the full-length gene:

[0105] PCR amplification primer sequences:

[0106] Upstream primer: SEQ ID NO.15

[0107] (5'-ACGATAGCCGGTACCCCCGGGATGGAGTATCATAAATCCGTTTGTG-3')

[0108] Downstream primer: SEQ ID NO.16

[0109] (5'-GCCCTTGCTCACCATCCCGGGGCCTCTTCTTTGAAACTTAGC-3'),

[0110] The 50 μL reaction mixture consisted of 25 μL 2×Phanta Max Master Mix, 1 μL template cDNA, and water to a final volume of 50 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min 30 s, for 40 cycles, followed by a final extension at 72°C for 10 min. Separation was performed by electrophoresis on an agarose gel, followed by ethidium bromide (EB) staining and photographing. The results were recorded, and the REL PCR products were recovered by gel excision. The electrophoretic bands were recovered using the Agarose Gel DNA Purification Kit (TaKaRa). The PCR product of REL recovered from gel excision was ligated into the KpnI-digested pBin::eGFP vector using the ClonExpress II One Step Cloning Kit (Vazyme) according to the instructions to obtain the pBin::REL-eGFP plasmid. This plasmid was transformed into *E. coli* competent cells JM109, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated at 37°C for 16 hours. After colony PCR verification, three clones were selected, and the plasmid was extracted using a plasmid extraction kit (Takara). The plasmid was sent to Nanjing Sangon Biotech Co., Ltd. for sequencing; the sequence is shown in SEQ ID NO.1. The correctly sequenced plasmid was electroporated into *Agrobacterium* GV3101, plated on LB agar plates (containing 50 μg / mL kanamycin and 50 μg / mL rifampin), and incubated at 30°C for 48 hours. After colony PCR verification, the correct clones were selected for subsequent experiments.

[0111] Example 5. Transient expression of the REL gene in tobacco

[0112] 1) Agrobacterium culture

[0113] Single colonies of Agrobacterium GV3101 transfected with pBin::REL-eGFP vector (a binary expression vector for the REL gene, with a partial sequence as shown in SEQ ID NO.1) and pBin::eGFP vector (with a partial sequence as shown in SEQ ID NO.17), as well as the silencing repressor P19, were picked from the plates and inoculated into 2 mL of LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and incubated overnight at 30°C and 200 rpm on a shaker until OD. 600The concentration was 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10mM 2-[N-morpholino]ethanesulfonic acid, 10mM MgCl2, 200μM acetosyringone, pH 5.6) and then centrifuged again to collect the bacterial cells. After washing three times, the bacterial suspension was diluted with buffer. Agrobacterium P19 and pBin::REL-eGFP or pBin::eGFP were mixed 1:1 to a final concentration of 0.6 for each.

[0114] 2) Transient expression of REL in tobacco

[0115] The prepared Agrobacterium was injected into tobacco leaves using a syringe. After injection, the tobacco was cultured in a greenhouse (21-23℃, 14 hours of light / 10 hours of darkness).

[0116] 3) Detection of cumulative REL protein levels

[0117] Two days after injection, tobacco leaves were collected for protein accumulation detection. The collected tobacco leaves were flash-frozen in liquid nitrogen, ground, and added to protein extraction buffer (components: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM ethylenediaminetetraacetic acid, 1.0% (v / v) NP-40, 1 mM phenylmethylsulfonylfluoride, and 1.0% (v / v) protease inhibitor cocktail). The mixture was incubated on ice for 30 minutes. The supernatant was collected by centrifugation at 18000g, and 80 μL of the supernatant was added to 20 μL of 5-fold protein loading buffer. The mixture was then boiled in a water bath for 10 minutes. 20 μL of the sample was then electrophoretically separated on an SDS-PAGE gel at 120V for 1.5 hours. After the reaction, the protein sample was transferred to a PVDF membrane and incubated with 5% PBST milk for sealing. After incubating with 1:5000 diluted GFP primary antibody (Abmart) for 2 hours, wash the membrane three times for 5 minutes each time with PBST. Then, add 1:10000 diluted mouse antibody (LI-COR, irdye 800, 926-32210), incubate for 30 minutes, wash the membrane three times for 5 minutes each time with PBST, and scan the membrane for photographs. Figure 6 A).

[0118] 4) Overexpression of REL significantly enhances tobacco's resistance to Phytophthora.

[0119] Two days after REL expression in tobacco, leaves were inoculated with *Phytophthora capsici* and *Phytophthora nicotine*. Disease symptoms were observed 2 and 3 days after inoculation. Figure 6B) Record the results by photograph. Compared with the negative control, tobacco overexpressing REL showed a significant reduction in lesions after inoculation with two different Phytophthora fungi. These results demonstrate that REL overexpression significantly improves the resistance of tobacco to different Phytophthora fungi.

[0120] Example 6. Expression of the REL gene in peppers enhances resistance to Phytophthora capsici.

[0121] 1) Agrobacterium culture

[0122] Single colonies of Agrobacterium GV3101 transfected with pBin::REL-eGFP vector (a binary expression vector for the REL gene, with a partial sequence as shown in SEQ ID NO.1) and pBin::eGFP vector (with a partial sequence as shown in SEQ ID NO.17), as well as the silencing repressor P19, were picked from the plates and inoculated into 2 mL of LB liquid medium (Kan 50 μg / mL, Rif 50 μg / mL) and incubated overnight at 30°C and 200 rpm on a shaker until OD. 600 The concentration was 2.0. The overnight cultured Agrobacterium GV3101 was centrifuged at 3000g for 5 minutes to collect the bacterial cells. The bacterial suspension was resuspended in buffer (components: 10mM 2-[N-morpholino]ethanesulfonic acid, 10mM MgCl2, 200μL Macetosyringone, pH 5.6) and then centrifuged again to collect the bacterial cells. After washing three times, the bacterial suspension was diluted with buffer. Agrobacterium P19 and pBin::REL-eGFP or pBin::eGFP were mixed 1:1 to a final concentration of 0.6 for each.

[0123] 2) Transient expression of REL in chili peppers

[0124] Agrobacterium containing pBin::REL-eGFP or pBin::eGFP vector was injected into pepper leaves, and the peppers were then cultured in a greenhouse (21-23℃, 14 hours light / 10 hours dark).

[0125] 3) Detection of cumulative REL protein levels

[0126] Two days after injection, pepper leaves were collected for protein accumulation detection. The collected pepper leaves were flash-frozen in liquid nitrogen, ground, and added to a protein extraction buffer (components: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM ethylenediaminetetraacetic acid, 1.0% (v / v) NP-40, 1 mM phenylmethylsulfonylfluoride, and 1.0% (v / v) protease inhibitor cocktail). The mixture was incubated on ice for 30 minutes. The supernatant was collected by centrifugation at 18000g, and 80 μL of the supernatant was added to 20 μL of 5-fold protein loading buffer. The mixture was then boiled for 10 minutes. 20 μL of the sample was electrophoretically separated on an SDS-PAGE gel at 120V for 1.5 hours. After the reaction, the protein sample was transferred to a PVDF membrane, incubated with 5% PBST milk, and then sealed. After incubating with 1:5000 diluted GFP primary antibody (Abmart) for 2 hours, wash the membrane three times for 5 minutes each time with PBST. Then, add 1:10000 diluted mouse antibody (LI-COR, irdye 800, 926-32210), incubate for 30 minutes, wash the membrane three times for 5 minutes each time with PBST, and scan the membrane for photographs. Figure 7 A).

[0127] 4) Overexpression of REL significantly enhances the resistance of peppers to Phytophthora.

[0128] Two days after REL expression in pepper leaves, *Phytophthora capsici* was inoculated. Symptoms were observed three days after inoculation. Figure 7 B) Record the results by taking photos. Compared with the negative control, peppers overexpressing REL showed a significant reduction in lesions after inoculation with Phytophthora capsici. These results confirm that overexpression of REL significantly improves the resistance of peppers to Phytophthora capsici.

Claims

1. Genes as shown in SEQ ID NO.1 REL Contains the gene shown in SEQ ID NO.1 REL Recombinant expression vectors or those containing the gene shown in SEQ ID NO.1 REL The application of the transformant in improving the resistance of pepper to Phytophthora capsici specifically involves the use of the gene shown in SEQ ID NO.

1. REL It has been expressed in chili peppers.

2. Genes as shown in SEQ ID NO.1 REL Contains the gene shown in SEQ ID NO.1 REL Recombinant expression vectors or those containing the gene shown in SEQ ID NO.1 REL The application of the transformant in improving tobacco resistance to Phytophthora nicotineae specifically involves the use of the gene shown in SEQ ID NO.

1. REL It has been expressed in tobacco.

3. Genes as shown in SEQ ID NO.1 REL Contains the gene shown in SEQ ID NO.1 REL Recombinant expression vectors or those containing the gene shown in SEQ ID NO.1 REL The application of the transformant in breeding pepper varieties with immunity to Phytophthora capsici; specifically, the gene shown in SEQ ID NO.1 is used to transform the transformant. REL It has been expressed in chili peppers.

4. Genes as shown in SEQ ID NO.1 REL Contains the gene shown in SEQ ID NO.1 REL Recombinant expression vectors or those containing the gene shown in SEQ ID NO.1 REL The application of the transformant in breeding tobacco varieties resistant to Phytophthora indicum specifically involves using the gene shown in SEQ ID NO.

1. REL It has been expressed in tobacco.

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