Nlr plug-in gene for improving disease resistance of plants and application thereof

Abstract

The application discloses an NLR plug-in gene for improving plant disease resistance and application thereof, and belongs to the field of plant molecular biology and biotechnology.The NLR plug-in gene StEM1 has a whole gene sequence as shown in SEQ ID NO.1 and a CDS sequence as shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is as shown in SEQ ID NO.3.The gene plays a key role in improving plant disease resistance, especially resistance to field late blight variant bacteria.In the NLR background, overexpression of the gene significantly promotes the disease resistance of tobacco and potato to pathogenic pythium, and the gene is a gene for enhancing plant disease resistance by improving ETI immunity.Overexpression of the gene in the NLR background is obtained by a genetic transformation method, and a strain line is obtained to resist field variant strains, so as to cope with field diseases of crops.The application provides an excellent gene resource for green prevention and control of late blight and disease-resistant genetic engineering breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant molecular biology and plant genetic engineering, and particularly relates to an NLR plug-in gene for improving plant disease resistance and application thereof. BACKGROUND

[0002] Late blight is one of the major diseases caused by the pathogenic fungus Phytophthora infestans, which causes serious harm to the potato industry in China. In recent years, potato has become the fourth major staple crop in China. However, the statistics of the National Agricultural Technology Extension Center show that the potato late blight in China has an annual incidence area of more than 30 million mu, causing economic losses of more than 8 billion yuan per year, which is the primary factor restricting the safe production of potato in China. Late blight has the characteristics of fast onset, rapid spread, and strong host adaptability. Under suitable environmental conditions, late blight spreads rapidly and causes large-scale death in a short period of time. Therefore, improving the resistance of crop varieties is of great significance for effectively preventing and controlling potato late blight.

[0003] The interaction between P. infestans and potato conforms to the "gene-for-gene" hypothesis (Mundt, 2014). P. infestans carries avirulence (AVR) genes, while potato has evolved disease resistance (R) receptors. If these plant R receptors can specifically recognize the products of AVR genes, they can provide potato with resistance to late blight (Dangl et al., 2013; Bradshaw et al., 2004). However, the large-scale use of potato disease-resistant varieties has exerted a huge selective pressure on P. infestans, driving genetic mutations at AVR gene loci of the fungus to escape the recognition of R genes, resulting in the loss of disease resistance of potato disease-resistant varieties in a short period of time (Vleeshouwers et al., 2011). Due to the high frequency of mutations in the AVR genes of P. infestans, most of the R genes of potato have lost their value in the field, and the breeding of potato for resistance to late blight is facing great challenges (Fry, 2008).

[0004] The mutation mode of AVR gene is the key to determine the broad-spectrum resistance of R gene. It has been reported that there are 10 Avr genes of P. infestans, and the coding products of these genes can be specifically recognized by the receptor proteins encoded by the resistance genes of Solanaceae plants (Vleeshouwers et al., 2011). In order to escape the recognition of R gene, P. infestans has undergone various heritable mutations at the AVR gene site. Currently, the three main mutation modes include gene loss, sequence substitution and gene silencing. The mutations of these avirulence genes lead to the loss of function of resistance genes (Zhang et al., 2025). At present, the good resistance genes in China have also evolved toxic strains in the field to overcome the resistance genes. Therefore, the development of NLR (containing nucleotide binding site (NBS) and leucine-rich repeat (LRR) receptor protein gene) plug-in genes to enhance NLR-mediated ETI (effector-triggered immunity) immunity can provide great potential for the reuse and improvement of NLR for crop resistance. SUMMARY

[0005] The purpose of the present application is to provide an NLR plug-in gene for improving plant disease resistance and its application.

[0006] Another purpose of the present application is to provide a method for improving the resistance of plants to late blight.

[0007] Still another purpose of the present application is to provide a method for breeding new germplasm of plants with improved resistance to late blight.

[0008] The purpose of the present application can be achieved by the following technical solutions:

[0009] In the first aspect, the present application claims the application of NLR plug-in gene protein for improving plant disease resistance in at least one of the following (c1)-(c2):

[0010] (c1) in improving the resistance of plants carrying NLR resistance genes to late blight;

[0011] (c2) in breeding new germplasm of plants carrying NLR resistance genes with improved resistance to late blight;

[0012] The amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO. 3.

[0013] In the second aspect, the present application claims the application of biological materials related to NLR plug-in gene protein for improving plant disease resistance in at least one of the following (c1)-(c2):

[0014] (c1) in improving the resistance of plants carrying NLR resistance genes to late blight;

[0015] (c2) use in breeding new germplasm of plants carrying NLR disease resistance genes for improving late blight resistance;

[0016] The amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO. 3; the biological material related to the NLR plug-in gene protein for improving plant disease resistance is at least one of the following (b1) - (b11):

[0017] (b1) a nucleic acid molecule encoding the NLR plug-in gene protein;

[0018] (b2) a primer pair for cloning the nucleic acid molecule of (b1);

[0019] (b3) an expression cassette containing the nucleic acid molecule of (b1);

[0020] (b4) a recombinant vector containing the nucleic acid molecule of (b1), or a recombinant vector containing the expression cassette of (b3);

[0021] (b5) a recombinant microorganism containing the nucleic acid molecule of (b1), or a recombinant microorganism containing the expression cassette of (b3), or a recombinant microorganism containing the recombinant vector of (b4);

[0022] (b6) a transgenic plant cell line containing the gene of (b1), or a transgenic plant cell line containing the expression cassette of (b3), or a transgenic plant cell line containing the recombinant vector of (b4);

[0023] (b7) a transgenic plant tissue containing the gene of (b1), or a transgenic plant tissue containing the expression cassette of (b3), or a transgenic plant tissue containing the recombinant vector of (b4);

[0024] (b8) a transgenic plant organ containing the gene of (b1), or a transgenic plant organ containing the expression cassette of (b3), or a transgenic plant organ containing the recombinant vector of (b4);

[0025] (b9) a transgenic plant containing the gene of (b1), or a transgenic plant containing the expression cassette of (b3), or a transgenic plant containing the recombinant vector of (b4);

[0026] (b10) tissue culture produced from regenerable cells of the transgenic plant of (b9);

[0027] (b11) protoplasts produced from the tissue culture of (b10).

[0028] Further, the nucleic acid molecule in (b1) is a CDS sequence (Coding sequence) or a genomic sequence encoding the NLR adaptor gene protein, the CDS sequence is shown as SEQ ID NO. 2, and the genomic sequence is shown as SEQ ID NO. 1.

[0029] Further, the nucleotide sequences of the primer pair in (b2) are shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0030] Further, the application is to improve the activity or / and content of the NLR adaptor gene protein in plants carrying NLR disease resistance genes (such as R8, Rpi-blb1, Rpi-blb2, Rpi-vnt1.1, Rpi-ber, etc.), or to overexpress the nucleic acid molecule encoding the NLR adaptor gene protein, which can improve the disease resistance function of the NLR disease resistance gene, and further improve the disease resistance of the plant to late blight or cultivate new germplasm of the plant with improved disease resistance to late blight and apply in production.

[0031] The NLR adaptor gene screened in the application is derived from potato and is relatively conserved in Solanaceae plants. The gene, as a plant-derived NLR (a receptor protein gene containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR)) adaptor gene, can improve the disease resistance function of the NLR disease resistance gene, enhance the disease resistance of the plant by improving the ETI immunity, and is named as gene StEM1.

[0032] The NLR disease resistance gene is a type of disease resistance protein gene containing NBS and LRR domains evolved in plants. In the prior art, the NLR disease resistance genes with clear disease resistance function verified by experiments include R8, Rpi-blb1, Rpi-blb2, Rpi-vnt1.1, Rpi-ber, etc.

[0033] The recombinant vector containing the NLR adaptor gene (gene StEM1) in the application is a recombinant expression vector obtained by inserting the gene StEM1 into an expression vector. The gene StEM1 can be the aforementioned nucleic acid molecule, or a codon-optimized nucleic acid sequence designed and artificially synthesized to facilitate expression in plants. The expression vector is preferably a plant transformation plasmid, which can be an expression vector pBin308 (NCBI ID: 2967819), pK7-GR-GFP (Catalog# 15534), or pK7WGF2 (VIB Vector Vault, ID: 1-50), etc.

[0034] In the specific embodiment of the present application, the genomic sequence (SEQ ID NO. 1) of the gene StEM1 is inserted into the restriction site SmaI of the binary vector pBin308 containing C-terminal MYC to obtain a recombinant expression vector pBin308::StEM1-MYC containing the gene StEM1.

[0035] The recombinant vector is introduced into host cells to obtain a recombinant microorganism, and the host cells are preferably E. coli cells or Agrobacterium cells.

[0036] In a third aspect, the present application claims a method for improving the resistance of plants to late blight, which comprises increasing the activity or / and content of NLR plug-in gene protein or overexpressing a nucleic acid molecule encoding the NLR plug-in gene protein in plants carrying NLR disease resistance genes; the amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO. 3.

[0037] In a fourth aspect, the present application claims a method for breeding new germplasm of plants with improved resistance to late blight, which comprises overexpressing a nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 in plants carrying NLR disease resistance genes, improving the resistance of plants to late blight, or breeding new germplasm of plants with improved resistance to late blight and applying it in production.

[0038] In the technical solution of the present application, the disease resistance is preferably the resistance to Oomycetes pathogenic bacteria (such as Phytophthora infestans) or diseases caused by Oomycetes pathogenic bacteria (such as late blight); further preferably, the resistance to late blight.

[0039] In the technical solution of the present application, the plant is a Solanaceae plant, preferably tobacco, tomato or potato.

[0040] The present application analyzes the potato infection sample carrying the disease resistance gene and finds that the gene StEM1 is significantly induced when ETI is activated, and the expression pattern shows that it may be involved in the regulation of ETI immunity. Overexpression of the gene in the background of NLR disease resistance gene can significantly improve the resistance of plants to field late blight variant strains. Sequence analysis of the gene shows that the gene is conserved in Solanaceae plants. Therefore, StEM1 gene is a specific regulator of ETI, which enhances the resistance of plants to late blight by improving ETI. This research can better cope with the field late blight variant strains, and provide excellent gene resources for green prevention and control of late blight and disease resistance genetic breeding.

[0041] The beneficial effects of the present application are:

[0042] The gene StEM1 improves the disease resistance function of the NLR disease resistance gene as a plug-in of the NLR, thereby enhancing the disease resistance of the plant. Overexpression of the gene StEM1 in potatoes can significantly enhance the resistance to the field variant strain of the pathogenic Phytophthora infestans under the premise of not affecting the agronomic traits by improving the function of the NLR disease resistance gene, and the technology can be applied to crop disease resistance breeding, and the resistance of crops to late blight is improved, the use of pesticides is reduced, and the yield is increased. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The results of detecting the resistance to the field pathogenic P. infestans strain of StEM1 (pBin308::StEM1-MYC) overexpressed in the NLR gene tobacco background are shown. The inoculated P. infestans strain is the field isolate JH19 which can overcome the NLR disease resistance gene R8, and EV-RFP is a negative control.

[0044] Figure 2 The results of detecting the protein accumulation of StEM1, StEM1 D259A mutant and negative control EV-RFP are shown. The detected antibody is MYC antibody.

[0045] Figure 3 The detection results of the potato transgenic positive seedlings are shown. NLR Desiree is a potato carrying NLR, which is used as a background plant receptor. Among them, A is the main cultivar Desiree (named Desiree NLR ) carrying NLR disease resistance gene R8, and B is the main cultivar Desiree carrying NLR disease resistance gene R8. D259A overexpressing StEM1 and StEM1 D259A mutant gene.

[0046] Figure 4 The results of detecting the resistance to the field pathogenic P. infestans of StEM1 (pBin308::StEM1-MYC) overexpressed in the potato background carrying NLR disease resistance gene R8 are shown. StEM1 D259A is a mutant of StEM1. DETAILED DESCRIPTION

[0047] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available from conventional biochemical reagent stores unless otherwise specified. The primers involved in the examples of the present application are synthesized by Shanghai Shengong Bioengineering Co., Ltd.

[0048] Example 1. Cloning, sequence analysis and expression vector construction of StEM1 gene

[0049] Potato (Solanum tuberosum) was grown in a 16 h light / 8 h dark cycle at 22°C, and 18°C, respectively. Six-week-old plants were used for leaf removal and inoculation with P. infestans. Two days later, the leaves were harvested for RNA extraction.

[0050] Total RNA extraction: The leaves of potato infected by P. infestans were used as the material, and the total RNA was extracted using the RNA extraction kit of Novagen (according to the instructions). The concentration and quality of the RNA were detected by spectrophotometer.

[0051] Synthesis of the first strand of reverse transcription: 1 pg - 1 μg of RNA was used as the template, and the cDNA was synthesized according to the reverse transcription kit of Novagen, and the volume was adjusted to 15 μL.

[0052] The primers were designed for PCR amplification with cDNA as the template. The PCR product was recovered by electrophoresis and gel cutting, and was ligated into the SmaI-digested pBin308 vector according to the ClonExpress II One Step Cloning Kit (Cat#C115) of Novagen to obtain the pBin308::StEM1-MYC recombinant expression vector. The Escherichia coli DH5a was transformed, and the single colonies were selected on the LB plate containing kanamycin and were sent to Shanghai Shengong Bioengineering Co., Ltd. for sequencing. The correct single colony was shaken, and the plasmid was extracted according to the plasmid extraction kit of Novagen. The Agrobacterium GV3101 was transformed by electroporation, and the glycerol bacteria were stored for subsequent experiments.

[0053] The designed primers for PCR amplification are as follows:

[0054] Upstream primer: 5'-CGAATTCTGCAGTCGACcccATGGGGAATAATTGTGTTCATGCAA

[0055] AGATA-3' (SEQ ID NO. 4, suitable for cloning of genomic and CDS sequences, the bold part is the specific primer of the gene, and the non-bold part is the homologous sequence of the pBin308 vector)

[0056] Downstream primer: 5'-TACAAGAAAGCTGGGTCcccACAAGCCACCATCGGCTCCCT-3' (SEQ ID NO. 5, suitable for cloning of genomic and CDS sequences, the bold part is the specific primer of the gene, and the non-bold part is the homologous sequence of the pBin308 vector).

[0057] Construction of expression vector of StEM1 kinase inactive mutant gene:

[0058] Two fragments of the gene were amplified using primer pair of SEQ ID NO. 4 / SEQ ID NO. 7 and primer pair of SEQ ID NO. 5 / SEQ ID NO. 6, respectively, with pBin308::StEM1-MYC as template, and the multi-fragment was ligated in the linearized pBin308 vector using In-Fusion cloning method, named as pBin308-StEM1 D259A -MYC, transform E. coli DH5a, select on LB plates containing kanamycin, pick single colonies for sequencing in Shanghai Sunbiotech Bioengineering Co., Ltd. Shake the correct single colony, extract the plasmid according to the Novagen plasmid extraction kit, and electroporate into Agrobacterium GV3101, and store the glycerol bacteria for subsequent experiments. The primers used are as follows:

[0059] Upstream primer: 5'-GCATCGTGACCTTAAGCCTGAA-3' (SEQ ID NO. 6, used for mutation of aspartic acid at position 259 of the StEM1 gene enzyme active site to alanine)

[0060] Downstream primer: 5'-CAGGCTTAAGGTCACGATGCATTA-3' (SEQ ID NO. 7, used for mutation of aspartic acid at position 259 of the StEM1 gene enzyme active site to alanine).

[0061] Example 2. Overexpression of StEM1 gene in transgenic tobacco carrying NLR (R8) significantly enhances the resistance of NLR to field variant strains

[0062] 1) Antibiotics and hormones used in tobacco transformation.

[0063] A. Timentin TMT (100 mg / ml): weigh 10 g of TMT (Sigma, Cat# T8660), dissolve in 100 mL of sterilized ddH2O, filter sterilization, and aliquot into 15 mL centrifuge tubes, store at -20°C for standby.

[0064] B. NAA stock solution (1 mg / mL): weigh 10 mg of NAA, dissolve with 1M NaOH, and add water to 10 mL. Filter sterilization, aliquot into sterilized 1.5 mL centrifuge tubes, and store at -20°C for standby.

[0065] C. Cell division factor 6-BA (2 mg / mL): weigh 20 mg of 6-BA, dissolve with alcohol to 10 mL. Filter sterilization, aliquot into sterilized 1.5 mL centrifuge tubes, and store at -20°C for standby.

[0066] D. MR medium: 4.43 g MS powder (PhytoTech LABS, cat# M516), sucrose 20 g, MES (2-morpholinoethanesulfonic acid, Cat# M7670) 0.5 g, 500 μL of 2 mg / mL of 6-BA, 100 μL of 1 mg / mL of NAA, agar 8 g, bring to 1 L.

[0067] E. MRTK medium: MR medium with final concentration of 50 μg / mL of timentin and 50 μg / mL of kanamycin.

[0068] 2) Construction of transgenic tobacco carrying NLR (R8).

[0069] A. R8 transgene vector construction. The vector used was pK7WGF2 (VIB Vector Vault, ID: 1-50) with EcoRV cloning site, the plasmid was linearized by restriction enzyme EcoRV digestion, the R8 gene sequence was amplified with primer pair SEQ ID NO. 8 / SEQ ID NO. 9 using the plasmid pBINPLUS-R8 (Vossen. et al, 2016) as template, and connected by In-Fusion cloning method (Novagen kit, Cat# C115), transformed into E. coli, picked single colonies, extracted plasmid, transformed into Agrobacterium GV3101, picked single colonies and used LB medium containing 50 μg / mL of rifampicin antibiotic for subsequent co-cultivation.

[0070] B. R8 transgenic line construction. N. benthamiana seeds were washed with 70% (v / v) ethanol for 30-60 seconds, then sterilized in 0.5% hypochlorite acid (volume ratio) for 15 minutes, washed with ultrapure water for 2-3 times, and then dried on filter paper. The seeds were sowed on MS (4.43 g MS powder plus 9 g agar, and then diluted to 1 L) solid medium. After the culture dish was sealed with parafilm, it was placed in a greenhouse (25-28°C) with 14 h light and 10 h dark alternation. After the mature leaves grew, the leaves were cut into 1 cm square leaf discs with a surgical knife or sterilized scissors. The leaf discs were soaked in the prepared Agrobacterium suspension for 30 minutes, dried with filter paper, and then laid on MR plates for 2-3 days for co-cultivation. The co-cultivated leaf discs were washed in sterilized water containing 50 μg / mL timentin for 10 minutes, and then laid on MRTK plates with the leaf surface facing up for screening for about one month. After the regenerated seedlings grew, the positive seedlings were verified by Agrobacterium-mediated transient expression system expressing the avirulence gene AVR8 (Vossen, Jack H., et al. The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties. Theoretical and Applied Genetics 129 (2016): 1785-1796.).

[0071] 3) Overexpression of StEM1 in NLR (R8) transgenic tobacco.

[0072] Recombinant expression plasmids pBin308::StEM1-MYC, pBin308-StEM1 D259A The Agrobacterium transformants of the -MYC and control plasmid EV-RFP were inoculated into 4-5 mL of LB liquid medium containing the corresponding antibiotics, and then cultured at 30°C with 220 rpm shaking for about 16 hours. The bacterial cells were collected, and the Agrobacterium suspension obtained by shaking culture was collected in a 2 mL EP tube. The tube was centrifuged at 4000-5000 rpm and room temperature (25±5°C) for 5 minutes, and the supernatant was discarded. The bacterial cells were suspended in 1 mL of MES Buffer or sterilized water, and then centrifuged and discarded. The bacterial cells were suspended again in 1 mL of MES Buffer. The OD600 of the bacterial cell suspension was measured to determine the concentration of the bacterial cell suspension. A small amount of the bacterial cell suspension was diluted 20 times, and the concentration of the bacterial cell suspension was determined by spectrophotometry. The concentration of the bacterial cell suspension was diluted to the desired concentration. The final concentration of the bacterial cell suspension was generally 0.4-0.6 OD600, and the final concentration of the bacterial cell suspension for resistance identification was preferably 0.1-0.2 OD600. The preparation of the MES Buffer is shown in Table 1.

[0073] Table 1. Recipe of MES Buffer

[0074]

[0075] The prepared Agrobacterium suspension was loaded into 1 mL sterile syringes (without needle) and injected evenly into the areas between the main veins of the NLR (R8) transgenic tobacco leaves from the back side of the leaves, avoiding the main leaf veins. After injection, the tobacco plants were transferred to a greenhouse (21-23°C, 16 h light / 8 h dark) for 2 days of culture.

[0076] 4) Detection of StEM1 protein accumulation in tobacco

[0077] The tobacco leaves after 2 days of injection were collected, 3-5 0.5 cm leaf discs were punched, frozen immediately in liquid nitrogen, and ground into powder using a grinder. Protein loading buffer (4x Laemmli sample buffer 25 μΐ, 1 M DTT 20 μΐ, ddH2O 55 μΐ) was added immediately, mixed well, and boiled in a water bath for 10 minutes. 10-20 μΐ of sample was taken for denaturing electrophoresis on an SDS-PAGE gel, running at 80 V for 20 minutes and 120 V for 1.5 hours. After the reaction, the membrane PVDF was transferred and blocked in 5% TBST skim milk for 1 hour. After incubation with 1:5000 diluted MYC primary antibody (anti-c-Myc antibody, Abmart, M20002L) for 2 hours, the membrane was washed with TBST for 5 minutes three times, followed by incubation with 1:20000 diluted mouse anti- (IRDye 800CW Goat anti-Mouse IgG Secondary Antibody, LICOR bio) for 1 hour, and then washed with TBST for 5 minutes three times. The membrane was scanned and photographed. The results of protein accumulation detection are shown in Figure 2

[0078] 5) Overexpression of StEM1 gene in NLR (R8) transgenic tobacco significantly enhances the resistance of NLR to field variant strains of late blight

[0079] ​The tobacco leaves injected 2 days were inoculated in vitro with the field-isolated variant strain JH19 (Zhang, X., Wu, J., Wang, X., Zhang, F., Yang, L., Wang, L., Wu, Y., et al. & (2025). Genotypic and virulence dynamics of Phytophthora infestans populations in southwestern China. Journal of Integrative Agriculture.) that overcomes the resistance mediated by NLR resistance gene R8, and the symptoms of disease were observed and photographed 4-5 days after inoculation. Compared with the negative control (EV-RFP), overexpression of the StEM1 gene significantly enhanced the resistance of NLR to the field variant strain of late blight, and overexpression in tobacco without NLR (e.g., R8) did not affect the disease resistance of tobacco to the field strain (e.g., JH19) (Fig. 1). Figure 1

[0080] Example 3. Overexpression of StEM1 gene in transgenic potato carrying NLR significantly enhances the resistance of NLR to field variant strains

[0081] 1) Reagents, antibiotics and medium formulations involved in the experiment process:

[0082] A. 1M (i.e., 1 mol / L) NaOH: weigh 2 g of NaOH and add water to 50 mL.

[0083] B. NAA mother liquor: (1) 10 mg / mL: weigh 100 mg of NAA, 1M NaOH to dissolve, add water to 10 mL. Filter sterilization and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C for standby use. (2) 0.1 mg / mL: take 100 μL of 10 mg / mL NAA mother liquor, dilute to 9.9 mL of water, filter sterilization and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C for standby use.

[0084] C. Acetyl-syringone AS (40 mg / mL): weigh 4 g of AS (Solabio Cat# IA2900), dissolve in 100 mL of methanol or DMSO (dimethyl sulfoxide), filter sterilization after dissolution, aliquot into 15 mL centrifuge tubes, wrap with tin foil to avoid light, and store at -20°C for standby use.

[0085] ​D. Zeatin ZT (1 mg / ml): Weigh 0.2 g ZT (Sigma Cat# T8110), dissolve with a small amount of 1 M NaOH, add 200 mL sterile ddH20, filter sterilize and aliquot into 15 mL centrifuge tubes, store at -20 °C.

[0086] E. Timentin TMT (100 mg / ml): Weigh 10 g TMT (Sigma, Cat# T8660), dissolve in 100 mL sterile ddH20, filter sterilize and aliquot into 15 mL centrifuge tubes, store at -20 °C.

[0087] F. 2,4-Dichlorophenoxyacetic acid 2,4-D (1 mg / mL): Weigh 0.01 g 2,4-D, dissolve in 10 mL sterile ddH20 with a small amount of NaOH to aid dissolution, filter sterilize and aliquot into 1.5 mL centrifuge tubes, store at -20 °C.

[0088] G. Rifampicin (25 mg / mL): 0.25 g rifampicin (Sigma, Cat# R8011) dissolved in 10 mL methanol or ethanol, filter sterilize and aliquot into sterile 1.5 mL centrifuge tubes.

[0089] H. G418 (5 mg / ml): Take 1 mL of G418 stock solution (50 mg / ml, regular supply), add 9 mL water, filter sterilize and aliquot.

[0090] I. MS20 liquid medium (1 L): Weigh 4.43 g MS powder (PhytoTech LABS, cat# M516), 20 g sucrose, bring to 1 L.

[0091] J. PACM: 0.44 g MS powder, 0.2 g caseine hydroiysate (regular reagent), 3 g sucrose, 100 μL 2,4-D (1 mg / mL), 50 μL kinetin (1 mg / mL), pH 6.5, bring to 100 mL.

[0092] K. MS20 solid medium: Add 9 g agar to MS20 liquid, bring to 1 L.

[0093] L. Z1N2: 2.215 g MS powder, 10 g sucrose, 1.55 g phytagel, 500 μL ZT, 100 μL NAA (10 mg / ml), bring to 500 mL.

[0094] M. Z1N2AS (1 L): 1 L MS20 with 1 mL ZT, 200 μL NAA (10 mg / ml), 1 ml AS.

[0095] N. Recovery medium (1 L): 1 L MS20 with 2 mL ZT, 100 μL NAA (0.1 mg / ml), 2 mL TMT.

[0096] O. Z2N0.01 (1 L): 1 L MS20 with 2 mL ZT, 100 μL NAA (0.1 mg / ml), 2 mL TMT, 2 mL, 3 mL Hygromycin.

[0097] P. Rooting medium (1 L): 1 L MS30, 2 mL TMT, 3 mL Hygromycin.

[0098] 2) Construction of stable transgenic potato StEM1 overexpression lines

[0099] A. Preparation of Agrobacterium: Agrobacterium containing transgenic plasmid pBin308-StEM1-MYC and pBin308-StEM1 D259A -MYC prepared in Example 1 were taken out from -80 °C, streaked on LB plates containing kanamycin, and allowed to grow into single colonies at 28-30 °C for 2 d with shaking. The buffer used in this process was MS20 containing 0.1 mM AS at final concentration. The bacterial cells were resuspended in MS20 liquid medium, and the concentration of Agrobacterium was adjusted to OD 600 = 0.5, and the volume was adjusted to 20 mL for infection of potato stem segments carrying NLR (e.g. R8) (Zhu, S., Vossen, J. H., Bergervoet, M., Nijenhuis, M., Kodde, L., Kessel, G. J.,... & Jacobsen, E. (2015). An updated conventional-and a novel GM potato late blight R gene differential set for virulence monitoring of Phytophthora infestans. Euphytica, 202, 219-234.).

[0100] B. Pre-culture of explants: Sterilized filter paper was laid on Z1N2 medium, 2 ml PACM was added, a certain number of R8-carrying potato stem segments were cut, about 20 explants per plate, and placed neatly, and cultured under light for 60-72 h.

[0101] C. Agrobacterium infection of potato explants: The pre-cultured stem segments were placed in bacterial solution with OD600 of 0.5, and infected for 10-15 min with constant shaking, 1 filter paper was laid on the Z1N2AS culture plate, and the infected stem segments were placed therein, and cultured in the dark at 24°C for 60-72 h.

[0102] D. Callus differentiation: The explants were cultured in recovery medium for one week, and then placed on differentiation medium Z2N0.01 to form callus, and the Z2N0.01 medium was replaced every two weeks until the callus differentiated into sprouts.

[0103] E. Screening of transformed lines on rooting medium: When the callus differentiated into sprouts, the sprouts were cut off and moved to rooting medium containing a higher concentration of antibiotic for screening, and the successfully transformed plants would grow roots from the cut, and 1-1.5 cm of the sprout tip was cut from the normally rooted plants and then inserted into the screening medium for secondary screening.

[0104] 3) Identification of potato transgenic positive seedlings

[0105] The leaves of the 4-5 week old regenerated seedlings obtained by transformation were quickly frozen in liquid nitrogen, and the sample was ground into powder in a grinder, 25 μl of 4x loading buffer, 20 μl of 1 M DTT, and 55 μl of ddH2O were added to the protein loading solution (Bio-RAD, Cat#161-0747) and immediately mixed, and boiled in a water bath for 10 min, 10-20 μl of the sample was subjected to denaturing electrophoresis on an SDS-PAGE gel, 80 V for 20 min, and 120 V for 1.5 h. After the reaction, the membrane PVDF was transferred, and 5% (g / 100 ml) TBST skimmed milk was added for incubation for 1 h. After incubation with 1:5000 diluted MYC primary antibody (anti-c-Myc antibody, Abmart, M20002L) for 2 h, the membrane was washed with TBST for 5 min three times, then 1:20000 diluted mouse anti- (IRDye 800CW Goat anti-Mouse IgG Secondary Antibody, LICORbio) was added for incubation for 1 h, and the membrane was washed with TBST for 5 min, repeated three times, scanned and photographed, and whether it was a transgenic positive seedling was judged by the presence or absence of a band (as shown in Figure 3

[0106] ​4) Overexpression of StEM1 gene in transgenic potato cultivar Desiree (designated Desiree NLR ) carrying NLR (R8) significantly enhanced NLR resistance to field variant strains

[0107] After 5-6 weeks of greenhouse culture, mature leaves with flat blades were selected, inoculated with zoospores of the late blight variant strain JH19 in the inoculation tray with the back facing up, and the resistance phenotype was observed after 4-5 days. Compared with the negative control and StEM1 mutant, the potato overexpressing StEM1 significantly enhanced NLR resistance to the field variant strain of late blight. Overexpression of the gene in potato Desiree without NLR did not affect the disease resistance of potato to the field strain (as shown in Figure 4 ).

[0108] SEQUENCE LISTING

[0109] SEQ ID NO. 1 (full gene sequence of gene StEM1)

[0110]

[0111] SEQ ID NO. 2 (CDS sequence of the gene StEM1)

[0112]

[0113] SEQ ID NO. 3 (amino acid sequence of protein StEM1)

[0114] MGNNCVHAKISKDGFFSSSWWSRSPEMITYEKKESSFQEGLNNVVQSNPPELAKIESRKSDVKGTDQVMIIVTDEKKDAWMMKQEEMITITVDLKQEKTNNAKPKKPHNVKRMASAGLQVDSVLKTRTGHLKEHYNLGEKLGHGQFGTTFLCIEKGTGKKYACKSIAKRKLLTDEDVDDVRREIQIMHHLSGHPTVISIKGAYEDAVAVHVVMELCTGGELFDRIIKRGHYSERQAAELARTILGVVEACHSLGVMHRDLKPENFLFVNAEEDSPLKTIDFGLSMFFKPGQLFDDVVGSPYYVAPEVLRKRYGPEADIWSAGVIIYILLSGVPPFWGESEEEIFDEVLHGDIDFELDPWPKISQGAKDLVRRMLIRDPKKRLTAHEVLCHPWVQIDGVAPDKPLDSAIFTRLTQFSAMNKLKKMAIRVIAERLSEEEIAGLKEMFKMIDTDNSGQITFDELKIGLKKFGTNLNESEIRDLMKAADIDNSGTIDYGEFVAAMLHANKIEKEDYLFAAFSYFDKDGSGYITADELQKACEEFGIEDVHLEEIIQEADQDNDGRIDYNEFVAMMHKGNADLGKKRLPNNFNIGYREPMVAC.

[0115] SEQ ID NO. 4 (suitable for cloning of genomic and CDS sequences, the bold part is the specific primer of the gene, and the non-bold part is the homologous sequence of the pBin308 vector)

[0116] 5'-CGAATTCTGCAGTCGACcccATGGGGAATAATTGTGTTCATGCAAAGATA-3'

[0117] SEQ ID NO. 5 (suitable for cloning of genomic and CDS sequences, the bold part is the specific primer of the gene, and the non-bold part is the homologous sequence of the pBin308 vector)

[0118] 5'-TACAAGAAAGCTGGGTCcccACAAGCCACCATCGGCTCCCT-3'

[0119] SEQ ID NO. 6

[0120] 5'-GCATCGTGACCTTAAGCCTGAA-3'

[0121] SEQ ID NO. 7

[0122] 5'-CAGGCTTAAGGTCACGATGCATTA-3'

[0123] SEQ ID NO. 8

[0124] 5'-CAGGCGGCCGCACTAGTGATATGAATGAAAATGAAATTGAGGAAATGTTAG-3'

[0125] SEQ ID NO. 9

[0126] 5'-GCAGATCCAGCAGATCCGATATCTCTTCGACTTCTTCTTACGAGGTCTATACGGT-3'

Claims

1. Use of an NLR plug-in gene protein for improving plant disease resistance in at least one of the following (c1)-(c2): (c1) in improving the late blight disease resistance of a plant carrying an NLR disease resistance gene; (c2) in breeding a new germplasm of a plant carrying an NLR disease resistance gene with improved late blight disease resistance; the amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO. 3; the plant is tobacco, tomato or potato.

2. Use of biological material related to an NLR plug-in gene protein for improving plant disease resistance in at least one of the following (c1)-(c2): (c1) in improving the late blight disease resistance of a plant carrying an NLR disease resistance gene; (c2) in breeding a new germplasm of a plant carrying an NLR disease resistance gene with improved late blight disease resistance; the amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO. 3; the biological material related to an NLR plug-in gene protein for improving plant disease resistance is at least one of the following (b1), (b3)-(b11): (b1) a nucleic acid molecule encoding the NLR plug-in gene protein; (b3) an expression cassette containing the nucleic acid molecule of (b1); (b4) a recombinant vector containing the nucleic acid molecule of (b1), or an expression cassette containing (b3); (b5) a recombinant microorganism containing the nucleic acid molecule of (b1), or an expression cassette containing (b3), or a recombinant vector containing (b4); (b6) a transgenic plant cell line containing the gene of (b1), or an expression cassette containing (b3), or a recombinant vector containing (b4); (b7) a transgenic plant tissue containing the gene of (b1), or an expression cassette containing (b3), or a recombinant vector containing (b4); (b8) a transgenic plant organ containing the gene of (b1), or an expression cassette containing (b3), or a recombinant vector containing (b4); (b9) a transgenic plant containing the gene of (b1), or an expression cassette containing (b3), or a recombinant vector containing (b4); (b10) a tissue culture produced from regenerative cells of the transgenic plant of (b9); (b11) a protoplast produced from the tissue culture of (b10); the plant is tobacco, tomato or potato.

3. Use according to claim 2, characterized in that, the nucleic acid molecule in (b1) is a CDS sequence encoding the NLR plug-in gene protein, or a genomic sequence, the CDS sequence is shown in SEQ ID NO. 2, and the genomic sequence is shown in SEQ ID NO.

1.

4. Use according to claim 1 or 2, characterized in that, Increasing the activity or / and content of the NLR insert gene protein or overexpressing the nucleic acid molecule encoding the NLR insert gene protein in the plant carrying the NLR disease resistance gene can improve the disease resistance function of the NLR disease resistance gene, and further improve the disease resistance of the plant to the late blight or cultivate new germplasm of the plant with improved disease resistance to the late blight and apply it in production.

5. A method for improving plant resistance to late blight, characterized in that, Increasing the activity or / and content of the NLR insert gene protein or overexpressing the nucleic acid molecule encoding the NLR insert gene protein in the plant carrying the NLR disease resistance gene; the amino acid sequence of the NLR insert gene protein is shown in SEQ ID NO. 3; and the plant is tobacco, tomato or potato.

6. A method of breeding a new germplasm of a plant with improved resistance to late blight, characterized in that, Overexpressing the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 in the plant carrying the NLR disease resistance gene can improve the disease resistance of the plant to the late blight or cultivate new germplasm of the plant with improved disease resistance to the late blight and apply it in production; and the plant is tobacco, tomato or potato.

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