NLR plug-in gene for improving plant disease resistance and application of NLR plug-in gene

Abstract

The invention discloses an NLR plug-in gene for improving plant disease resistance and application of the NLR plug-in gene, and belongs to the field of plant molecular biology and biotechnology. The NLR plug-in gene StEM1 has a complete 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 of the NLR plug-in gene StEM1 is as shown in SEQ ID NO.3. The gene plays a key role in improving the plant disease resistance, especially the resistance to field late blight variation flora. Under the background of NLR, the gene is overexpressed to remarkably promote the disease resistance of tobacco and potatoes to phytophthora infestans, and the gene is a gene for enhancing the plant disease resistance by improving ETI immunity. Through a genetic transformation method, a strain which overexpresses the gene under the background of NLR obtains resistance to field variant strains so as to cope with field diseases of crops. And excellent gene resources are provided for green prevention and control of late blight and breeding of disease-resistant gene engineering.

Description

Technical Field

[0001] The present invention 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 an application thereof. Background Art

[0002] Late blight, caused by the pathogenic fungus Phytophthora infestans, is a major disease that severely harms my country's potato industry. In recent years, potatoes have become my country's fourth-largest staple crop. However, statistics from the National Agricultural Technology Extension Center show that late blight regularly affects over 30 million mu (approximately 1.5 acres) of potato land, causing annual economic losses exceeding 8 billion yuan and becoming the primary constraint on safe potato production in my country. Late blight is characterized by rapid onset, rapid spread, and strong host adaptability. Under favorable environmental conditions, it spreads rapidly, causing widespread mortality in a short period of time. Therefore, improving crop resistance is crucial for the effective prevention and control of potato late blight.

[0003] The interaction between Phytophthora infestans and potato conforms to the "gene-for-gene" hypothesis (Mundt, 2014). P. infestans carries avirulence (AVR) genes, while potato has evolved resistance (R) receptors. If these plant R receptors can specifically recognize the AVR gene product, they can provide resistance to late blight in potato (Dangl et al., 2013; Bradshaw et al., 2004). However, the widespread use of disease-resistant potato varieties has exerted significant selective pressure on P. infestans, driving genetic mutations at the AVR loci in the fungus, allowing it to evade recognition by the R gene and quickly losing its resistance (Vleeshouwers et al., 2011). Due to the high frequency of mutations in the AVR gene in late blight, most potato R genes have lost their usefulness in the field, posing a significant challenge to breeding potato varieties for late blight resistance (Fry, 2008).

[0004] The pattern of AVR gene mutation is crucial for determining the broad-spectrum resistance of R genes. Ten AVR genes have been reported for Phytophthora infestans, each encoding a receptor protein specifically recognized by the same Solanaceae disease resistance gene (Vleeshouwers et al., 2011). To evade R gene recognition, P. infestans undergoes a variety of heritable mutations at the AVR locus. Three currently known mutation patterns include gene loss, sequence substitution, and gene silencing. These avirulent gene mutations result in functional loss of the resistance gene (Zhang et al., 2025). Currently, well-performing disease resistance genes in China have also evolved into virulent strains in the field, capable of overcoming the resistance genes. Therefore, developing plug-in genes for NLRs (receptor protein genes containing nucleotide binding sites (NBS) and leucine-rich repeats (LRRs)) to enhance NLR-mediated ETI (effector-triggered immunity) offers great potential for repurposing NLRs and improving crop resistance. Summary of the Invention

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

[0006] Another object of the present invention is to provide a method for improving plant resistance to late blight.

[0007] Another object of the present invention is to provide a method for cultivating new plant germplasm with improved resistance to late blight.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks to protect at least one use of an NLR plug-in gene protein for improving plant disease resistance in the following (c1)-(c2):

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

[0011] (c2) Application in breeding new plant germplasms carrying NLR disease 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 a second aspect, the present invention seeks to protect at least one application of a biomaterial related to an NLR plug-in gene protein that improves plant disease resistance in the following (c1)-(c2):

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

[0015] (c2) Application in breeding new plant germplasms carrying NLR disease resistance genes with improved resistance to late blight;

[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) to (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 described in (b1);

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

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

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

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

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

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

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

[0026] (b10) Tissue cultures produced from regenerable cells of the transgenic plant described in (b9);

[0027] (b11) Protoplasts produced from the tissue culture described in (b10).

[0028] Furthermore, the nucleic acid molecule described in (b1) is a CDS sequence (coding sequence) or a genomic sequence encoding the NLR plug-in gene protein, the CDS sequence is shown as SEQ ID NO.2, and the genomic sequence is shown as SEQ ID NO.1.

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

[0030] Furthermore, the above-mentioned application is: increasing the activity and / or content of the NLR plug-in gene protein in plants carrying NLR disease-resistant genes (such as R8, Rpi-blb1, Rpi-blb2, Rpi-vnt1.1, Rpi-ber, etc.), or overexpressing the nucleic acid molecule encoding the NLR plug-in gene protein, which can improve the disease resistance function of the NLR disease-resistant gene, thereby improving the plant's resistance to late blight or cultivating new plant germplasm with improved resistance to late blight and applying it in production.

[0031] The NLR plug-in gene screened by the present invention is derived from potato and is relatively conserved among Solanaceae species. As a plant-derived NLR (receptor protein gene containing a nucleotide binding site (NBS) and leucine-rich repeats (LRR)) plug-in gene, this gene can improve the disease resistance function of the NLR disease resistance gene and enhance plant disease resistance by improving plant ETI immunity. It is named gene StEM1.

[0032] The NLR disease resistance gene is a type of disease resistance protein gene that has evolved from plant species and contains NBS and LRR domains. In the prior art, NLR disease resistance genes that have been experimentally verified to have clear disease resistance functions include R8, Rpi-blb1, Rpi-blb2, Rpi-vnt1.1, Rpi-ber, etc.

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

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

[0035] The above-mentioned recombinant vector is introduced into a host cell to obtain a recombinant microorganism. The host cell is preferably an Escherichia coli cell or an Agrobacterium cell.

[0036] In the third aspect, the present invention claims protection for a method for improving the disease resistance of plants to late blight, which increases the activity and / or content of NLR plug-in gene protein in plants carrying NLR disease resistance genes, or overexpresses a nucleic acid molecule encoding the NLR plug-in gene protein; 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 invention seeks to protect a method for cultivating new plant germplasm with improved resistance to late blight, which comprises overexpressing a nucleic acid molecule with a nucleotide sequence such as SEQ ID NO.1 or SEQ ID NO.2 in a plant carrying an NLR disease-resistance gene, thereby improving the plant's resistance to late blight or cultivating new plant germplasm with improved resistance to late blight and applying it in production.

[0038] In the technical solution of the present invention, the disease resistance is preferably resistance to oomycete pathogens (such as Phytophthora infestans) or diseases caused by oomycete pathogens (such as late blight); more preferably, it is resistance to late blight;

[0039] The plant described in the technical solution of the present invention is a Solanaceae plant, preferably tobacco, tomato or potato.

[0040] Analysis of potato samples infected with disease-resistant genes revealed that the gene StEM1 is significantly induced to express upon ETI activation. This expression pattern suggests that it may be involved in regulating ETI immunity. Overexpressing this gene in the context of NLR disease-resistance genes significantly improved plant resistance to field-transmitted late blight variants. Sequence analysis revealed that this gene is conserved across Solanaceae plants. Therefore, StEM1 is a specific regulator of ETI, enhancing plant resistance to late blight by improving ETI. This research can better address late blight variants in the field and provide excellent genetic resources for both green late blight control and genetic engineering breeding for disease resistance.

[0041] Beneficial effects of the present invention:

[0042] The gene StEM1 described in this invention acts as an NLR plug-in to enhance the disease resistance function of NLR disease resistance genes, thereby enhancing plant disease resistance. Overexpressing the StEM1 gene in potatoes significantly improves resistance to field variants of Phytophthora infestans by enhancing the function of NLR disease resistance genes, without compromising agronomic traits. This technology can be applied to crop disease resistance breeding, reducing pesticide use and achieving yield increases by improving crop resistance to late blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 To test the resistance of StEM1 overexpressed in an NLR gene-enriched tobacco background (pBin308::StEM1-MYC) to field-isolated P. infestans strains, the inoculated strain was a field-isolated variant strain JH19 that overcomes the NLR resistance gene R8. EV-RFP served as a negative control.

[0044] Figure 2 To overexpress StEM1 and StEM1 in tobacco D259A Protein accumulation detection results of mutants and negative control EV-RFP, using MYC antibody.

[0045] Figure 3 This is the test result of the transgenic potato seedlings. NLR The potato carrying NLR is used as the background plant receptor. Among them, A is the main cultivated variety Desiree carrying the NLR disease resistance gene R8 (named Desiree NLR ) overexpression of StEM1 and mutant StEM1 in the background D259A Detection of positive seedlings obtained by gene expression; B. Overexpression of StEM1 and mutant StEM1 in the background of the main cultivar Desiree D259A Detection of positive seedlings obtained from the gene.

[0046] Figure 4 To test the resistance results of overexpressing StEM1 (pBin308::StEM1-MYC) in potato carrying NLR disease resistance gene R8 to field-induced Phytophthora infestans. D259A It is a mutant of StEM1. DETAILED DESCRIPTION

[0047] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The primers involved in the examples of the present invention were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.

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

[0049] Potatoes (Solanum tuberosum) were cultured at 22°C with 16 h of light and 18°C ​​with 8 h of darkness under alternating light and dark conditions. Sterile potato seedlings were grown in pots containing vermiculite and nutrient soil (mass ratio 1:1). Six-week-old plants were defoliated and inoculated in vitro with Phytophthora infestans. RNA was extracted 2 days later.

[0050] Extraction of total RNA: Total RNA was extracted from potato leaves infected with Phytophthora infestans using the RNA extraction kit from Novozymes (following the instructions). The RNA concentration and quality were measured using a spectrophotometer.

[0051] For the synthesis of the first strand of reverse transcription, 1 pg - 1 μg of RNA was used as a template and cDNA was synthesized using the reverse transcription kit of Novozymes, with the volume adjusted to 15 μL.

[0052] Primers were designed for PCR amplification using the cDNA as a template. PCR products were recovered by electrophoresis and excised from the gel. The product was then ligated into the pBin308 vector digested with SmaI using the ClonExpress II One-Step Cloning Kit (Cat#C115) from Novozymes to generate the pBin308::StEM1-MYC recombinant expression vector. This vector was then transformed into Escherichia coli DH5α and screened on LB plates containing kanamycin. Single colonies were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Correct single colonies were shaken, and the plasmid was extracted using the Novozymes plasmid extraction kit. The plasmid was then transformed into Agrobacterium tumefaciens GV3101 by electroporation, and the glycerol stock was stored for subsequent experiments.

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

[0054] Upstream primer: 5'-CGAATTCTGCAGTCGACcccATGGGGAATAATTGTGTTCATGCAA

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

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

[0058] The primer pairs SEQ ID NO.4 / SEQ ID NO.7 and SEQ ID NO.5 / SEQ ID NO.6 were used to amplify two fragments of the gene using pBin308::StEM1-MYC as templates. The fragments were then connected to the pBin308 linearized vector using the In-Fusion cloning method and named pBin308-StEM1. D259A -MYC, transform E. coli DH5α and screen on LB plates containing kanamycin. Single clones were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Correct clones were shaken, and the plasmid was extracted using the Novezan plasmid extraction kit. Transformed into Agrobacterium tumefaciens GV3101 by electroporation, the glycerol stock was preserved for subsequent experiments. The primers used were as follows:

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

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

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

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

[0063] A. Timentin TMT (100 mg / mL): Dissolve 10 g of Timentin TMT (Solarbol, Cat# T8660) in 100 mL of sterile ddH2O. Filter sterilize and aliquot into 15 mL centrifuge tubes. Store at -20°C until needed.

[0064] B. Naphthaleneacetic acid (NAA) stock solution (1 mg / mL): Weigh 10 mg of NAA, dissolve in 1M NaOH, and add water to 10 mL. Filter sterilize and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C until needed.

[0065] C. Cytokinin 6-BA (2 mg / mL): Weigh 20 mg of 6-BA and dissolve it in ethanol to 10 mL. Filter sterilize and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C until needed.

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

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

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

[0069] A. Construction of the R8 transgenic vector. The vector used was pK7WGF2 (VIB Vector Vault, ID: 1-50), with an EcoRV cloning site. This plasmid was linearized using the EcoRV restriction enzyme. The R8 gene sequence was amplified using the primer pair SEQ ID NO. 8 / SEQ ID NO. 9, using the R8-carrying plasmid pBINPLUS-R8 (Vossen et al., 2016) as a template. The R8 gene sequence was then ligated using In-Fusion cloning (Novagen kit, Cat# C115). Escherichia coli was transformed, and a single colony was picked. The plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101. A single colony was then shake-cultured in LB supplemented with 50 μg / mL rifampicin for subsequent co-cultivation.

[0070] B. Construction of R8 transgenic line. N. benthamiana seeds were washed with 70% (v / v) ethanol for 30–60 seconds, sterilized in 0.5% hypochlorous acid (v / v) for 15 minutes, washed 2–3 times with ultrapure water, dried on filter paper, and sown on MS solid medium (4.43 g MS powder plus 9 g agar, dilute to 1 L). The culture plates were sealed with parafilm and placed in a greenhouse (25–28°C) with alternating light cycles of 14 h and dark cycles of 10 h. After mature leaves emerged, they were cut into 1 cm square discs using a scalpel or sterilized scissors. The discs were immersed in the cultured Agrobacterium suspension for 30 minutes, dried on filter paper, and spread on MR plates for co-cultivation for 2–3 days. The co-cultivated leaves were removed and washed in sterile water containing 50 μg / mL timentin for 10 minutes. The discs were then spread with the leaf side facing up on MRTK plates for selection for about a month. After regenerated seedlings emerged, the avirulent gene AVR8 (Vossen, Jack et al.) was expressed using an Agrobacterium-mediated transient expression system. H., et al. The Solanum demissum R8 late blight resistancegene is an Sw-5 homologue that has been deployed worldwide in late blightresistant varieties. Theoretical and Applied Genetics 129 (2016): 1785-1796.) Verification of positive seedlings.

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

[0072] Recombinant expression plasmids pBin308::StEM1-MYC, pBin308-StEM1 D259A Inoculate a single colony of Agrobacterium transformants expressing -MYC and the control plasmid EV-RFP into 4-5 mL of LB liquid medium containing the corresponding antibiotics and culture at 30°C with shaking at 220 rpm for approximately 16 h. Collect the cells and place the shaken Agrobacterium suspension into a 2 mL EP tube. Centrifuge at 4000-5000 rpm at room temperature (25±5°C) for 5 min, discard the supernatant, and resuspend the cells in 1 mL of MES Buffer or sterile water. Centrifuge and discard the supernatant. Repeat the suspension once. Resuspend the cells in 1 mL of MES Buffer to the same volume. Measure the OD600 value to determine the concentration of the culture solution. Take a small amount of the culture solution and dilute it 20-fold. Detect the concentration of the Agrobacterium culture solution using a spectrophotometer. Dilute the cells to the desired concentration. The final injection concentration is generally 0.4-0.6, and the final concentration for resistance identification is preferably 0.1-0.2. The configuration of MES Buffer is shown in Table 1.

[0073] Table 1 MES Buffer recipe

[0074]

[0075] The prepared Agrobacterium suspension was placed in a 1 mL sterile syringe (with the needle removed). The suspension was injected evenly onto the underside of NLR (R8) transgenic tobacco leaves, avoiding the main veins and extending evenly along the midrib. After injection, the tobacco plants were transferred to a greenhouse (21-23°C, 16 h light / 8 h dark) and cultured for 2 days.

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

[0077] Two days after injection, tobacco leaves were harvested and cut into 3-5 0.5 cm leaf discs. These were quickly frozen in liquid nitrogen and ground into a powder using a grinder. Protein loading buffer (25 μl of 4× Laemmli sample buffer, 20 μl of 1 M DTT, and 55 μl of ddH₂O) was added and immediately mixed. The mixture was incubated in a boiling water bath for 10 minutes. A 10-20 μl sample was then run on a denaturing SDS-PAGE gel at 80 V for 20 minutes and 120 V for 1.5 hours. After the reaction, the membrane was transferred to a PVDF membrane and blocked with 5% skim milk powder in TBST for 1 hour. After adding 1:5000 diluted MYC primary antibody (anti-c-Myc antibody, Abmart, M20002L) and incubating for 2 hours, the membrane was washed three times with TBST for 5 minutes. Then, 1:20000 diluted mouse antibody (IRDye 800CW Goat anti-MouseIgG Secondary Antibody, LICORbio) was added and incubated for 1 hour, and the membrane was washed with TBST for 5 minutes. Repeat this three times and scan the membrane to take pictures. The protein accumulation test results are shown in Figure 2. Figure 2 shown.

[0078] 5) Overexpression of the StEM1 gene in NLR (R8) transgenic tobacco significantly enhanced NLR resistance to field variants of late blight.

[0079] Tobacco leaves injected 2 days ago 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 dynamicsof Phytophthora infestans populations in southwestern China. Journal of Integrative Agriculture.). Symptoms were observed and photographed 4-5 days after inoculation. Compared with the negative control (EV-RFP), overexpression of the gene StEM1 significantly enhanced NLR resistance to field variant strains of late blight. Overexpression in tobacco plants that do not carry NLR (such as R8) did not affect tobacco's resistance to field strains (such as Figure 1 shown).

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

[0081] 1) Reagents, antibiotics, and culture medium formulas involved in the experiment:

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

[0083] B. Naphthaleneacetic acid (NAA) stock solution: (1) 10 mg / mL: Weigh 100 mg of NAA, dissolve in 1M NaOH, and add water to 10 mL. Filter sterilize and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C until needed. (2) 0.1 mg / mL: Take 100 μL of the 10 mg / mL NAA stock solution, dilute it with 9.9 mL of water, filter sterilize and aliquot into sterile 1.5 mL centrifuge tubes. Store at -20°C until needed.

[0084] C. Acetosyringone (AS) (40 mg / mL): Weigh 4 g of AS (Solybo, Cat# IA2900) and dissolve it in 100 mL of methanol or DMSO (dimethyl sulfoxide). After dissolution, filter and sterilize. Aliquot into 15 mL centrifuge tubes, wrap in tin foil, and store at -20°C in the dark until needed.

[0085] D. Zeatin ZT (1 mg / ml): Weigh 0.2 g ZT (Solarbo Cat# T8110) and dissolve in a small amount of 1 M NaOH. Add 200 mL of sterile ddH2O, filter sterilize, and aliquot into 15 mL centrifuge tubes. Store at -20°C until needed.

[0086] E. Timentin TMT (100 mg / mL): Weigh 10 g of TMT (Solarbold, Cat# T8660) and dissolve it in 100 mL of sterile ddH2O. Filter-sterilize and aliquot into 15 mL centrifuge tubes. Store at -20°C until needed.

[0087] F. 2,4-Dichlorophenoxyacetic acid 2,4-D (1 mg / mL): Weigh 0.01 g of 2,4-D and dissolve it in 10 mL of sterile ddH2O with a small amount of NaOH to aid solubilization. Filter sterilize and aliquot into 1.5 mL centrifuge tubes. Store at -20°C until needed.

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

[0089] H. Hygromycin (5 mg / ml): Take 1 mL of hygromycin stock solution (50 mg / ml, commonly available), add 9 mL of water, filter sterilize, and aliquot.

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

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

[0092] K. MS20 solid medium: Add 9 g agar to the MS20 liquid and adjust the volume to 1 L.

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

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

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

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

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

[0098] 2) Construction of a stable transgenic potato line overexpressing StEM1

[0099] A. Agrobacterium preparation: Take out the transgenic plasmids pBin308-StEM1-MYC and pBin308-StEM1 prepared in Example 1 from -80°C. D259A -MYC Agrobacterium was streaked onto LB plates containing kanamycin to grow single colonies, cultured at 28-30°C for 2 days, and shaken. The buffer used in this process was MS20 containing a final concentration of 0.1 mM AS, so the bacteria were resuspended in MS20 liquid medium and the concentration of Agrobacterium was adjusted to OD 600 = 0.5, dilute to 20 mL and use to infect potato stem segments carrying NLR (e.g. R8) (Zhu, S., Vossen, JH, Bergervoet, M., Nijenhuis, M.,Kodde, L., Kessel, GJ, ... & Jacobsen, E. (2015). An updated conventional-and a novel GM potato late blight R gene differential set for virulencemonitoring of Phytophthora infestans. Euphytica, 202, 219-234.).

[0100] B. Explant pre-culture: Spread two sterilized filter papers on Z1N2 medium, add 2 ml of PACM, cut a certain number of potato stem segments carrying R8, about 20 explants per plate, arrange them neatly, and culture under light for 60-72 hours.

[0101] C. Agrobacterium infection of potato explants: Place the pre-cultured stem segment in a bacterial solution with an OD600 of 0.5 and infect for 10-15 min with constant shaking. Place the infected stem segment on a Z1N2AS culture plate with a piece of filter paper and incubate in the dark at 24°C for 60-72 h.

[0102] D. Callus Differentiation: Culture the explants in recovery medium for one week. Then, place the explants on differentiation medium Z2N0.01 to continue culturing callus. Change the Z2N0.01 medium every two weeks until callus differentiation and budding occur.

[0103] E. Screening of transformed strains on rooting medium: When callus tissue differentiates into buds, cut the buds and transfer them to rooting medium containing a high concentration of antibiotics for screening. Successfully transformed plants will grow roots from the incision. Bud tips of about 1-1.5 cm in diameter are cut from plants that have normal rooting and inserted into screening medium for secondary screening.

[0104] 3) Identification of transgenic potato seedlings

[0105] Leaves from 4-5-week-old regenerated seedlings obtained by transformation were snap-frozen in liquid nitrogen and ground into powder using a grinder. Protein loading buffer (25 μl of 4× loading buffer from Bio-RAD, Cat#161-0747, 20 μl of 1 M DTT, and 55 μl of ddH2O) was added and immediately mixed. The mixture was incubated in a boiling water bath for 10 minutes. A 10-20 μl sample was then run on a denaturing SDS-PAGE gel at 80 V for 20 minutes and 120 V for 1.5 hours. After the reaction, the membrane was transferred to a PVDF membrane and blocked with 5% (g / 100 ml) skim milk powder in TBST for 1 hour. After adding 1:5000 diluted MYC primary antibody (anti-c-Myc antibody, Abmart, M20002L) and incubating for 2 hours, the membrane was washed with TBST for 5 minutes three times. Then, 1:20000 diluted mouse antibody (IRDye 800CW Goatanti-Mouse IgG Secondary Antibody, LICORbio) was added and incubated for 1 hour, and the membrane was washed with TBST for 5 minutes. Repeat this three times, scan the membrane and take pictures. The presence of bands was used to determine whether the seedlings were transgenic positive (such as Figure 3 shown).

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

[0107] After culturing transgenic positive seedlings in the greenhouse for 5-6 weeks, mature leaves with flat leaves were selected and placed with the back side facing up in an inoculation tray. Zoospores of the late blight variant strain JH19 were inoculated and the resistance phenotype was observed 4-5 days later. Compared with the negative control and the StEM1 mutant, potatoes overexpressing StEM1 significantly enhanced NLR resistance to the field variant strain of late blight. Overexpressing this gene in Desiree, a potato that does not carry NLR, did not affect the potato's resistance to the field strain (e.g., Figure 4 shown).

[0108] Sequence Listing

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

[0110]

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

[0112]

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

[0114] .

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

[0116] 5'-CGAATTCTGCAGTCGACccATGGGGAATAATTGTGTTCATGCAAAGATA-3'

[0117] SEQ ID NO.5 (Suitable for cloning genomic and CDS sequences. The bold part is the specific primer for this 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. Application of NLR plug-in gene proteins for improving plant disease resistance in at least one of the following (c1)-(c2): (c1) Application in improving the resistance of plants carrying NLR disease resistance genes to late blight; (c2) Application in breeding new plant germplasms carrying NLR disease resistance genes with improved resistance to late blight; 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. Application of biomaterials related to NLR plug-in gene proteins that improve plant disease resistance in at least one of the following (c1)-(c2): (c1) Application in improving the resistance of plants carrying NLR disease resistance genes to late blight; (c2) Application in breeding new plant germplasms carrying NLR disease resistance genes with improved resistance to late blight; 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) to (b11): (b1) a nucleic acid molecule encoding the NLR plug-in gene protein; (b2) a primer pair for cloning the nucleic acid molecule described in (b1); (b3) an expression cassette containing the nucleic acid molecule described in (b1); (b4) a recombinant vector containing the nucleic acid molecule described in (b1), or a recombinant vector containing the expression cassette described in (b3); (b5) a recombinant microorganism containing the nucleic acid molecule described in (b1), or a recombinant microorganism containing the expression cassette described in (b3), or a recombinant microorganism containing the recombinant vector described in (b4); (b6) a transgenic plant cell line containing the gene described in (b1), or a transgenic plant cell line containing the expression cassette described in (b3), or a transgenic plant cell line containing the recombinant vector described in (b4); (b7) transgenic plant tissue containing the gene described in (b1), or transgenic plant tissue containing the expression cassette described in (b3), or transgenic plant tissue containing the recombinant vector described in (b4); (b8) A transgenic plant organ containing the gene described in (b1), or a transgenic plant organ containing the expression cassette described in (b3), or a transgenic plant organ containing the recombinant vector described in (b4); (b9) A transgenic plant containing the gene described in (b1), or a transgenic plant containing the expression cassette described in (b3), or a transgenic plant containing the recombinant vector described in (b4); (b10) Tissue cultures produced from regenerable cells of the transgenic plant described in (b9); (b11) protoplasts produced from the tissue culture described in (b10); The plant is tobacco, tomato or potato.

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

1.

4. The use according to claim 2, characterized in that The nucleotide sequences of the primer pair described in (b2) are shown in SEQ ID NO. 4 and SEQ ID NO.

5.

5. The use according to claim 1 or 2, characterized in that: Increasing the activity and / or content of the NLR plug-in gene protein in plants carrying the NLR disease-resistance gene, or overexpressing the nucleic acid molecule encoding the NLR plug-in gene protein, can enhance the disease resistance function of the NLR disease-resistance gene, thereby improving the plant's resistance to late blight or cultivating new plant germplasm with improved resistance to late blight and applying it in production.

6. A method for improving plant resistance to late blight, characterized in that: The activity and / or content of the NLR plug-in gene protein is increased in a plant carrying an NLR disease-resistant gene, or a nucleic acid molecule encoding the NLR plug-in gene protein is overexpressed; the amino acid sequence of the NLR plug-in gene protein is shown in SEQ ID NO.3; and the plant is tobacco, tomato or potato.

7. A method for cultivating new plant germplasm with improved resistance to late blight, characterized in that: Overexpressing a nucleic acid molecule with a nucleotide sequence such as SEQ ID NO.1 or SEQ ID NO.2 in a plant carrying an NLR disease-resistance gene improves the plant's disease resistance to late blight or cultivates new plant germplasm with improved late blight resistance and uses it in production; the plant is tobacco, tomato or potato.

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