Immunoregulatory protein GmNFR5a and application thereof in plant disease resistance

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly discloses immunoregulation GmNFR5a protein and application thereof in plant disease resistance. According to the invention, the function of cell membrane protein in soybean is analyzed, and the immunoregulation protein GmNFR5a is found to play a key role in identifying chitosan oligosaccharide or chitosan oligosaccharide secreted by phytophthora or fungi and play a decisive role in induced immunoreaction in plants. As important commercial crops and food crops, the research on GmNFR5a in soybean can promote the related research on cell membrane receptors of other plants such as pepper, tomato, potato, alfalfa and the like. The researches can more deeply reveal the disease-resistant mechanism of plants to phytophthora and other pathogenic bacteria, and provide high-quality disease-resistant gene resources for disease-resistant gene engineering breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and specifically discloses an immune regulatory protein GmNFR5a and an application thereof in plant disease resistance. Background Art

[0002] Plant diseases are a serious problem facing the world, posing a major threat to food security and production. Among them, crop diseases caused by oomycetes cause huge economic losses to my country's agricultural production every year. Phytophthora sojae ) in the field, the disease is fast, the scope is wide, and the mutation is fast, which seriously harms the yield and quality of soybeans. At present, the prevention and control of crop diseases in production is mainly based on chemical agents and the cultivation of disease-resistant varieties. However, the large-scale use of chemical agents not only causes the emergence of pests and diseases resistance, but also causes great harm to the environment and human health. And because it is difficult to organically combine the genetic disease resistance traits of plants with other agronomic traits, the effect of using traditional agronomic cultivation measures for disease prevention and control is very limited. In recent years, plant-induced disease resistance has been considered as a new technology and new approach for plant protection. Plant-induced disease resistance refers to the stimulation of plants to produce stress-resistant substances (plant leukothiocyanate, antibacterial protein, etc.) under the action of elicitors, thereby enhancing the resistance of plants to external adversity, also known as acquired immunity. Through elicitor induction, the damage caused to plants by adversity stress can be reduced and some physiological functions can be repaired. Therefore, it is particularly important to use the plant's own resistance genes to resist the invasion of pathogens from the aspects of pesticide use, environmental protection and drug resistance.

[0003] At the same time, in order to resist the invasion of pathogens, plants can activate the immune mechanism by identifying the pattern recognition receptors (PRRs) on the cell membrane surface of pathogens in the environment and the intracellular immune recognition receptors (NLRs). MAMPs from pathogens can be recognized by receptor kinases (RLKs) or receptor proteins (RLPs). According to their extracellular domains, they can be divided into LRR, LysM, Lectin and EGF (Macho & Zipfel, 2014). Among them, LysM mainly binds to carbohydrate PAMPs and participates in the immune process between plants and microorganisms.

[0004] Chitin is the main component of fungal cell walls, also known as chitin polysaccharide or chitin, which is a polysaccharide polymerized from β-1,4-N-acetyl-D-glucosamine. Plants can produce chitosan oligosaccharides by secreting chitinase. Chitosan oligosaccharides have a low molecular weight and induce plant resistance through good water solubility. In previous studies, it was known that OsCEBiP in rice recognizes chitosan oligosaccharides and forms a "sandwich" structure with OsCERK1, and OsCERK1 transmits immune signals to the cell; in Arabidopsis, AtLYK5 phosphorylates AtCREK1 and recognizes chitosan oligosaccharides, mediating immune responses, and chitosan oligosaccharides induce the interaction of two RLKs proteins. Pathogens can secrete chitin deacetylase to enzymatically hydrolyze the acetyl groups of chitosan oligosaccharides to produce chitosan oligosaccharides, thereby weakening the immunity induced by chitosan oligosaccharides. As an important plant immune activator, chitosan oligosaccharides are widely used in agricultural production. However, the plant immune signals induced by chitosan oligosaccharides are still unclear. Therefore, studying the immune signaling pathway of chitosan oligosaccharides in plants can better explain the immune mechanism induced by chitosan oligosaccharide elicitors and provide a new direction for the future development of plant immune-inducing agents.

[0005] Therefore, the use and modification of plant immune regulatory elements is a new way to achieve long-term and safe prevention and control of plant diseases, which is more in line with the requirements of green and sustainable development of agriculture today. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an immune regulatory protein GmNFR5a and its application in plant disease resistance.

[0007] On the one hand, the present invention provides immune regulatory genes GmNFR5a , the immune regulatory gene GmNFR5a The nucleotide sequence is any of the following, 1) The nucleotide sequence shown in SEQ ID NO.1 and its complementary sequence; or 2) A nucleotide sequence expressing a protein with the same function as the nucleotide sequence shown in SEQ ID NO.1 by replacing, deleting and / or adding one or more nucleotides; or 3) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions; or 4) A sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO.1, and the polypeptide encoded by the sequence has the function of improving plant disease resistance.

[0008] On the other hand, the present invention provides immune regulatory genes GmNFR5a The encoded polypeptide has an amino acid sequence as shown in any of the following: 1) the amino acid sequence shown in SEQ ID NO.2; or 2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and the polypeptide has the function of improving plant disease resistance; or 3) An amino acid sequence obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO. 2, and the amino acid sequence has the function of regulating plant disease resistance.

[0009] On the other hand, the present invention also provides a recombinant expression vector, the recombinant vector comprising the immune regulatory gene GmNFR5a .

[0010] The expression vector is preferably a plant transformation plasmid, which can be in various forms such as pBin::eGFP, pCambia or pTF101.1. More preferably, the recombinant expression vector is a gene GmNFR5a It was inserted into the restriction site SmaI of the binary vector pBin::eGFP containing C-terminal eGFP, and finally the vector pBin::GmNFR5a-eGFP was obtained.

[0011] On the other hand, the present invention also provides a host cell comprising the immune regulatory gene GmNFR5a , or comprising the recombinant vector. Preferably, the host cell is an Escherichia coli or Agrobacterium cell.

[0012] On the other hand, the present invention also provides the immune regulatory gene GmNFR5a , or the polypeptide, or the recombinant expression vector, or the host cell, characterized in that the application is any of the following: 1) Increase or decrease the immune-induced disease resistance of plants; 2) Enhance or weaken the ability of plants to recognize chitosan oligosaccharides or chitosan oligosaccharides secreted by pathogens; 3) Screening of disease-resistant / susceptible plant varieties.

[0013] Furthermore, the disease-resistant or disease-susceptible pathogenic bacteria include but are not limited to Phytophthora nicotianae, Phytophthora sojae, Phytophthora infestans and Phytophthora capsici.

[0014] Furthermore, the plant is a Solanaceae plant or a Leguminosae plant, preferably tobacco, tomato, potato or soybean.

[0015] Compared with the prior art, the present invention analyzes the function of cell membrane proteins in soybeans and finds that the immune regulatory protein GmNFR5a plays a key role in identifying chitosan oligosaccharides or chitosan oligosaccharides secreted by Phytophthora or fungi. These elicitors play a decisive role in the immune response induced in plants. At the same time, the elicitors are conserved in different Phytophthora or fungi, so GmNFR5a has the ability to recognize different Phytophthora and fungi. In addition, the gene of the present invention GmNFR5a The encoded protein activates plant innate immunity by regulating the elicitors secreted by pathogens, thereby enhancing plant disease resistance. GmNFR5a The gene does not affect the growth traits of plants, but significantly weakens the disease resistance of soybeans to Phytophthora. The invention can be used in improving the disease resistance of crop breeding to improve the disease resistance of plants to blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For ALSV:: GmNFR5a Silent Soybean GmNFR5a Gene expression detection and growth phenotype.

[0017] Figure 2 Immune responses induced by chitosan oligosaccharides (CSOS) and chitosan oligosaccharides (CTOS) in soybeans with silenced GmNFR5a.

[0018] Figure 3 The infection phenotype and biomass were shown 2 days after inoculation with Phytophthora sojae.

[0019] Figure 4 The in vivo interaction between pBin::GmNFR5a-eGFP and pBin::GmCERK1-HA proteins. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these examples are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the scope of protection of the present invention. The primers involved in the embodiments of the present invention are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0021] Example 1 Based on previous research results, all the cell membrane receptor protein LRR-RLPs genes of Nicotiana benthamiana were silenced (Wang, Y., Xu, Y., Sun, Y. et al. (2018). Leucine-rich repeat receptor-like genescreen reveals that Nicotiana RXEG1 regulates glycoside hydrolase 12 MAMP detection. Nat Commun 9, 594.). Soybean cell membrane receptor kinase was obtained through screening and sequencing. GmNFR5a The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0022] Example 2 Using virus-induced gene silencing technology, we first injected soybean cell membranes into Nicotiana benthamiana GmNFR5a Gene silencing vector. The treated tobacco seedlings were cultured in a greenhouse (21-23°C, 14 hours of light / 10 hours of darkness) for 17 days, and the virus particles were extracted. Then, the virus particles were rubbed on the leaves of 10-day-old soybean seedlings and cultured in a greenhouse (22°C, 8 hours of light / 16 hours of darkness) for 20 days. Then, the silenced seedlings were treated with chitosan oligosaccharides (CSOS) and chitosan oligosaccharides (CTOS), and the immune regulatory protein GmNFR5a that recognizes chitosan oligosaccharides and chitosan oligosaccharides was screened according to the immune response induced by different elicitors.

[0023] The specific steps are as follows: 1) Agrobacterium infiltration The vectors pALSV1, pALSV2, pALSV2-CH42, pALSV2-GmPDS, pALSV2-GmNFR5a and viral gene silencing suppressor P19 were transformed into Agrobacterium GV3101 by electroporation. -1 Rifampicin and 50 mg·L -1 After the single colony was picked, it was cultured in YEP liquid medium at 28°C overnight. Then, it was centrifuged at 5,000 r / min for 8 minutes, the supernatant was discarded, and an appropriate amount of Agrobacterium infiltration buffer (10 mmol·L -1 MES, 10 mmol·L -1 MgCl2 and 150 μmol·L -1Acetosyringone) was added to resuspend the bacteria. After being placed in a 28°C constant temperature incubator in the dark for 3 hours, the OD600 of the bacterial solution was adjusted to 1.0. Subsequently, Agrobacterium carrying pALSV1, pALSV2, pALSV2-GmPDS, pALSV2-CH42 and pALSV2-GmNFR5a were mixed in equal proportions, and an equal proportion of Agrobacterium containing P19 was added to each combination and injected into Nicotiana benthamiana plants at the 6-8 leaf stage.

[0024] Table 1 Primer sequences for constructing pALSV2-GmNFR5a

[0025] 2) Virus particle enrichment 17 days after Agrobacterium infiltration, 0.4 g of systemically infected leaves were weighed and ground into powder in a mortar with liquid nitrogen. Then, 800 μL of 0.01 mol·L -1 , pH=7.5 PB buffer, mix well and incubate on ice for 30 minutes. Next, centrifuge at 4°C and 8,500 r / min for 10 minutes, take the supernatant, add the corresponding volume of 40% PEG / NaCl solution (add 23 μL of 40% PEG / NaCl to every 100 μL supernatant), mix well and incubate on ice again for 1 hour. Then, centrifuge at 4°C and 13,000 r / min for 15 minutes, discard the supernatant, and use 100 μL of 0.01 mol·L -1 The precipitate was suspended in PB buffer with pH = 7.0 to obtain enriched virus particles. The concentration of virus particles was measured by OD260 and the final concentration was adjusted to 2 μg·μL -1 , store at 4℃ for future use.

[0026] 3) Virus particle friction inoculation of soybeans Select 8-10 day old soybean seedlings, sprinkle emery evenly on the soybean leaves, then use a pipette to absorb 20 μL of extracted virus particles, put on disposable gloves and evenly apply it on the true leaves of soybean, rubbing 40 μg of virus particles on each true leaf.

[0027] 4) Silencing efficiency detection When the soybean system leaves inoculated with ALSV:GmPDS showed an albino phenotype, the albino leaves at the top of the soybean were collected for total RNA extraction, and the soybean system leaves inoculated with the ALSV empty vector were used as controls. Then, cDNA was synthesized according to the rapid reverse transcription kit of Nanjing Novozyme Biotechnology Co., Ltd., and then quantitative PCR was performed using the ChamQ Universal SYBR qPCR MasterMix quantitative kit, using GmActin as an internal reference gene to detect the silencing efficiency of GmNFR5a. Primers for silencing efficiency detection: upstream primer: 5'-GGTGACAATGTTTCCCTTG-3' (SEQ ID NO. 5), downstream primer: 5'-AGAACCAGTAGCGTGCATC-3' (SEQ ID NO. 6).

[0028] Test results such as Figure 1 As shown, the silencing efficiency of soybean GmNFR5a gene is about 80%, and silencing GmNFR5a gene does not affect the normal growth of soybean.

[0029] 5) Soybeans with silenced GmNFR5a gene (ALSV-GmNFR5a) and controls (ALSV-GFP) were treated with CTOS (chitosan oligosaccharide) and CSOS (chitosan oligosaccharide), respectively. MAPK activity was detected by MAPK-specific antibody P44 / 42, as shown in Figure 2 As shown in Figure 2, CTOS and CSOS significantly weakened the MAPK in soybeans with GmNFR5a silenced. Then the changes of reactive oxygen species were detected by luminol / HRP method. Figure 2 As shown, the results showed that silence GmNFR5a After silencing the gene, the MAPK activity induced by CSOS and CTOS was significantly weakened by using MAPK-specific antibody P42 / 44. GmCERK1 The gene plant was used as a positive control. CSOS could not induce ROS production, and the silenced GmNFR5a After the gene was expressed, the ROS induced by CTOS was also significantly weakened, indicating that GmNFR5a is involved in the recognition of CTOS and CSOS immune signals.

[0030] Example 3 Cotyledons were removed from 10-day-old soybean seedlings. On the 7th day, the cotyledons were twisted off the hypocotyls and the intact cotyledons were harvested. Subsequently, the individual cotyledons were surface-sterilized with 70% ethanol. Next, a small circular incision with a diameter of approximately 0.4 cm was cut approximately 0.3 cm from the end of the petiole of the cotyledon to inoculate the K599 Agrobacterium tumefaciens cell suspension. Before inoculation, the K599 Agrobacterium was centrifuged at 2500×g in a tabletop centrifuge for 20 minutes. Subsequently, the K599 Agrobacterium was gently resuspended in 10 mM MgCl2 and adjusted to a final OD600 of approximately 0.4 for inoculation of cotyledons. The inoculated cotyledons were placed in a sterile Petri dish containing MS medium and incubated in a growth chamber at 22°C with a 16-hour photoperiod. During this period, the production of green fluorescence in the hairy roots was monitored, and qRT-PCR was used to measure the green fluorescent hairy roots after 4 weeks. GmNFR5a Finally, red fluorescent-labeled Phytophthora sojae was inoculated on the hairy roots and cultured in a dark environment at 25°C for 2 days. The length of the lesions was counted and the biomass was detected by qRT-PCR.

[0031] Table 2 Primer sequences for quantitative PCR reaction

[0032] The composition of the PCR reaction system is as follows: cDNA 5 μL, SYBR Premix Ex Taq II (Tli RNase HPlus) 10 μL, front and back primers 0.4 μL each, ROX Reference Dye II 0.4 μL, deionized water 13.8 μL. The reaction program was set as follows: Phase I: 95°C 30 seconds; Phase II: 95°C 5 seconds, 60°C 34 seconds, and 40 cycles were performed in Phase II. The program for melting curve analysis was: 95°C 15 seconds, 60°C 1 minute, 95°C 15 seconds.

[0033] Data analysis was performed using 2 -ΔΔCT Method, test results such as Figure 3 As shown, in soybean root hair silencing GmNFR5a Gene, chitosan oligosaccharide (CSOS) and chitosan oligosaccharide (CTOS) induced soybean resistance significantly weakened. 48 h after inoculation of soybean phytophthora, the infection was observed by fluorescence microscopy, and the soybean phytophthora biomass was detected by qRT-PCR. RFP-P6497: red fluorescent marker soybean phytophthora strain P6497. The results showed that both CSOS and CTOS could induce soybean resistance to phytophthora, and the soybean phytophthora biomass was significantly lower than that of the control (GFP-dH2O), and silencing GmNFR5aAfter silencing the GmCERK1 gene, the resistance induced by CSOS and CTOS was significantly weakened, and there was no significant difference in biomass compared with the control (GFP-dH2O); silencing GmCERK1 was used as a positive control. After silencing the GmCERK1 gene, the resistance induced by CSOS and CTOS was significantly weakened.

[0034] Example 4 Soybean ( Glycine max ) seeds were directly sown in pots filled with nutrient soil and cultured in a greenhouse (22°C, 8 h light / 16 h dark). Two-week-old plants were used for RNA extraction.

[0035] Total RNA extraction: Soybean leaves were used as materials, and total RNA was extracted using the RNA extraction kit provided by Omega according to the instructions, and the RNA content and quality were detected using a spectrophotometer.

[0036] Reverse transcription to generate the first strand: Take 0.7 μg RNA as a template, perform cDNA synthesis according to the instructions of Takara's PrimeScript reverse transcriptase reagent, and adjust the volume to 20 μL. Take an appropriate amount of reverse transcription product for subsequent gene cloning PCR.

[0037] The first strand of cDNA was used as a template for qRT-PCR and PCR amplification was performed using conventional methods to obtain GmNFR5a Gene fragment or full-length gene. PCR amplification primer sequence, , . A 50 μL PCR reaction system was set up, which contained 25 μL of 2 × Phanta Max Master Mix, 1 μL of template cDNA, and water was added to a total volume of 50 μL. The PCR amplification program was set as follows: pre-denaturation at 95°C for 5 minutes, followed by 40 cycles of amplification (denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds), and finally extension at 72°C for 10 minutes. After amplification, electrophoresis separation was performed on agarose gel, stained with ethidium bromide (EB), and the results were recorded by photography. Subsequently, the gel block containing the PCR product of GmNFR5a was cut out for recovery.

[0038] After the electrophoresis bands were recovered using Agarose Gel DNA Purification Kit (TaKaRa), GmNFR5aThe PCR product was ligated to the pBin::eGFP vector digested with SmaI according to the instructions of ClonExpress II One Step Cloning Kit (Vazyme) to obtain the pBin::GmNFR5a-eGFP plasmid. Next, this plasmid was transformed into E. coli competent cells JM109 and spread on LB plates containing kanamycin (50 μg / mL) and cultured at 37°C for 16 hours. After that, three clones were picked by colony PCR verification, and the plasmid was extracted using a plasmid extraction kit (Takara). The extracted plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with SEQ ID NO.1. For the correctly sequenced plasmid, it was transformed into Agrobacterium GV3101 by electroporation and spread on LB plates containing kanamycin (50 μg / mL) and rifampicin (50 μg / mL) and cultured at 30°C for 48 hours. Finally, through colony PCR verification, the correct clones were picked for subsequent experiments.

[0039] Example 5 1) Agrobacterium culture Pick the cells transformed with pBin::GmNFR5a-eGFP vector ( GmNFR5aGene binary expression vector) and detected using universal primers (SEQ ID NO.13 and SEQ ID NO.14) of pBin::eGFP vector. Subsequently, it was compared with SEQ ID NO.1 to confirm the positive transformed Agrobacterium GV3101 single colony. Silencing suppressor P19 was added and inoculated into 2 mL LB liquid medium containing 50 μg / mL kanamycin (Kan) and 50 μg / mL rifampicin (Rif), and cultured overnight on a constant temperature shaker at 30℃ and 200 rpm until OD600 reached 2.0. Then, the overnight cultured GV3101 Agrobacterium liquid was centrifuged at 3000 g for 5 minutes to collect the bacteria. After suspending the bacterial liquid with buffer (components: 10 mM 2-[N-morpholino]ethanesulfonic acid, 10 mM MgCl2, 200 μM acetosyringone, pH 5.6), the bacteria were collected by centrifugation again. After washing for 3 times, dilute the bacterial solution with buffer. Mix P19 and pBin::GmNFR5a-eGFP or pBin::eGFP Agrobacterium at a ratio of 1:1, and the final concentration is 0.6. The universal primer sequence of pBin::eGFP vector: upstream primer 5'-CATTTCATTTGGAGAGGACC-3' (SEQ ID NO.13), downstream primer 5'-TATCTGGGAACTACTCACAC-3' (SEQ ID NO.14).

[0040] 2) Transient expression of GmNFR5a in tobacco The prepared Agrobacterium was injected into tobacco leaves with a syringe, and the tobacco was cultured in a greenhouse (21-23°C, 14 h light / 10 h dark) after injection.

[0041] 3) Detection of GmNFR5a protein accumulation Tobacco leaves were collected two days after injection to detect protein accumulation. The collected tobacco leaves were quickly frozen with liquid nitrogen, then ground and added with protein extract (components include: 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM ethylenediaminetetraacetic acid, 1.0% (v / v) NP-40, 1 mM phenylmethylsulfonyl fluoride, 1.0% (v / v) protease inhibitor cocktail), and mixed on ice for 30 minutes. Then centrifuged at 18000 g for 5 minutes, the supernatant was collected, 80 μL of the supernatant was taken and 20 μL of 5-fold concentrated protein loading buffer was added, mixed evenly and treated in a boiling water bath for 10 minutes. 20 μL of the sample was taken for electrophoresis separation on SDS-PAGE gel, the voltage was set to 120 V, and the run was 1.5 hours. After the reaction was completed, the protein sample was transferred to a PVDF membrane and incubated with 5% PBST milk solution to seal the membrane. Then, 1:5000 diluted GFP primary antibody (Abmart) was added. After incubation for 2 hours, the membrane was washed 3 times with PBST, 5 minutes each time. Then, 1:10000 diluted mouse antibody (LI-COR, irdye 800, 926-32210) was added. After incubation for 30 minutes, the membrane was washed again with PBST 3 times, 5 minutes each time. Finally, the membrane was scanned and photographed. The results are shown in Figure 4 .

[0042] pBin::GmNFR5a-eGFP and pBin::GmCERK1-HA proteins were co-expressed in Nicotiana benthamiana. The expression levels of GmNFR5a-eGFP and GmCERK1-HA were detected by Western blot, and the detection antibodies were anti-GFP and anti-HA. The interaction between GmNFR5a and GmCERK1 was confirmed by in vivo Co-IP experiment (see Figure 4 ). After CTOS or CSOS treatment, the interaction between GmNFR5a and GmCERK1 was enhanced. This indicates that after GmNFR5a recognizes CTOS or CSOS, it transmits the immune signal of CTOS or CSOS by activating GmCERK1.

[0043] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values.

Claims

1. Immune regulatory genes GmNFR5a and the use of the polypeptide encoded by it, characterized in that, The application is any of the following, 1) Increase or decrease the immune-induced disease resistance of plants; 2) Enhance or weaken the ability of plants to recognize chitosan oligosaccharides or chitosan oligosaccharides secreted by pathogens; 3) Screening for disease-resistant / susceptible plant varieties; The immune regulatory gene GmNFR5a The nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence of the polypeptide is shown as SEQ ID NO.2, and the plant is tobacco or soybean.

2. The use according to claim 1, characterized in that: The disease-resistant or disease-susceptible pathogens are tobacco phytophthora, soybean phytophthora, phytophthora infestans and pepper phytophthora.

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