Method for improving disease resistance of plants

Abstract

The invention discloses a method for improving plant disease resistance. The method comprises the following steps: improving the activity and / or content of RWR3 protein in a target plant to improve the disease resistance of the plant; the RWR3 protein is any one of the following A1)-A4): A1) a protein with an amino acid sequence as shown in a sequence 2; a2) a fusion protein related to plant disease resistance and obtained by connecting a tag to the N terminal and / or C terminal of the amino acid sequence shown in the sequence 2; a3) plant disease resistance related protein obtained by substituting and / or deleting and / or adding one or more amino acid residues to A1), and A4) plant disease resistance related protein having more than 75% of identity with A1). It is found for the first time that the RWR3 protein can improve the plant disease resistance, and a new target is provided for improvement of the rice disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for improving plant disease resistance. Background Technology

[0002] Rice occupies an irreplaceable and vital position in my country's agricultural production system, and its yield and quality directly affect the implementation of the national food security strategy and the development of the agricultural economy. However, for a long time, rice diseases caused by pathogenic microorganisms have been a major bottleneck restricting the high-quality development of the rice industry. For example, rice blast, a devastating disease of rice worldwide, is characterized by rapid spread, wide range of incidence, and severe damage. Once it breaks out, it can lead to a 10%-20% reduction in rice yield, or even more than 50% reduction, or even complete crop failure.

[0003] With the continuous advancement of modern biotechnology, researchers have increasingly delved into the molecular mechanisms of rice-pathogen interactions, moving from the macroscopic level to the gene level. They have clearly recognized that disease-resistance genes within rice play a crucial regulatory role in resisting pathogen invasion. Against this backdrop, utilizing disease-resistance genes to breed disease-resistant rice varieties has become the most effective, safest, and most economical way to reduce over-reliance on chemical pesticides, achieve green and sustainable rice production, and lower planting costs. It has also become a current research hotspot and core direction in the field of rice breeding. Summary of the Invention The technical problem to be solved by this invention is how to improve plant disease resistance.

[0004] To address the aforementioned technical problems, this invention first provides a method for improving plant disease resistance.

[0005] The method for improving plant disease resistance provided by this invention includes the following steps: increasing the activity and / or content of RWR3 protein in the target plant to improve plant disease resistance; The RWR3 protein is any one of the following (A1)-A4): A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) Proteins related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares more than 75% identity with A1 and is associated with plant disease resistance.

[0006] In the protein described in A2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0007] In the protein described in A3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0008] In the protein described in A4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained. The identity includes amino acid sequences having 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 2 of the present invention.

[0009] The proteins described in A1)-A4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] To address the aforementioned technical problems, this invention provides a method for cultivating transgenic plants with enhanced disease resistance.

[0011] The method for cultivating transgenic plants with enhanced disease resistance provided by this invention includes the following steps: increasing the activity and / or content of RWR3 protein in the target plant to obtain a transgenic plant; the transgenic plant exhibits higher disease resistance than the target plant. In any of the methods described above, the method for increasing the activity and / or content of RWR3 protein in the target plant is to overexpress RWR3 protein in the target plant.

[0012] Furthermore, the overexpression method involves introducing the gene encoding the RWR3 protein into the target plant.

[0013] Furthermore, the gene encoding the RWR3 protein is the DNA molecule shown in sequence 1 or sequence 3, positions 4811-8752.

[0014] In some embodiments, the gene encoding the RWR3 protein is introduced into the target plant via the recombinant vector pCAMBIA1300::RWR3. The recombinant vector pCAMBIA1300::RWR3 is obtained by replacing the DNA fragment between the Hind III and Kpn I restriction sites of the pCAMBIA1300-221-flag plasmid with the DNA molecule shown in sequence 3, while keeping the other sequences of the pCAMBIA1300-221-flag plasmid unchanged.

[0015] The application of any of the methods described above in plant breeding is also within the scope of protection of this invention.

[0016] To address the aforementioned technical problems, this invention also provides new uses for RWR3 protein or biomaterials related to RWR3 protein.

[0017] This invention provides the use of RWR3 protein or RWR3 protein-related biomaterials in any of the following C1)-C6): C1) Improve plant disease resistance; C2) Prepare products that enhance plant disease resistance; C3) Cultivate plants with enhanced disease resistance; C4) Prepare products from plants with enhanced disease resistance; C5) Plant breeding; C6) Prepare products for plant breeding.

[0018] In the above applications, the biological material is a nucleic acid molecule encoding the RWR3 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ.

[0019] The aforementioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0020] In this invention, the nucleic acid molecule is any one of D1) or D2): D1) The DNA molecule shown at positions 4811-8752 of sequence 1 or sequence 3; The DNA molecule that has 75% or more identity with the nucleotide sequence defined by D2) and D1) and encodes the RWR3 protein.

[0021] Those skilled in the art can readily mutate the nucleotide sequence encoding the RWR3 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity with the RWR3 protein nucleotide sequence isolated according to this invention, provided they encode the RWR3 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention. The identity refers to the sequence similarity to a natural nucleic acid sequence, including nucleotide sequences that have an identity of 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0022] The expression cassette described above may include a promoter, the aforementioned nucleic acid molecule, and a terminator. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters, and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0023] The aforementioned vectors refer to vectors capable of delivering the aforementioned nucleic acid molecules into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the aforementioned nucleic acid molecules with a plant expression vector. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). Nos The untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing recombinant vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer resistance to glyphosate) or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0024] The aforementioned microorganisms may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The recombinant microorganisms refer to those whose genes have been manipulated and modified to obtain recombinant microorganisms with altered functions. For example, recombinant microorganisms obtained by introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring do not necessarily need to be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0025] The aforementioned transgenic plant tissues can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0026] The organs of the aforementioned transgenic plants can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0027] The aforementioned transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0028] The aforementioned RWR3 protein or the aforementioned biomaterials related to the RWR3 protein are also within the scope of protection of this invention.

[0029] The indicators for plant breeding mentioned above include disease resistance (such as rice blast resistance).

[0030] The purpose of any of the above-mentioned plant breeding programs includes developing disease-resistant plant varieties (such as rice blast-resistant plant varieties).

[0031] The disease resistance mentioned above refers to rice blast resistance.

[0032] In some embodiments, the rice blast is rice blast caused by the rice blast fungus strain RB22.

[0033] The plant mentioned above is any one of the following B1)-B4): B1) Monocotyledonous or dicotyledonous plants; B2) Grasses; B3) Plants of the genus *Oryza*; B4) Rice.

[0034] This invention provides an RWR3 protein derived from rice, the amino acid sequence of which is shown in Sequence 2. This invention also constructs... RWR3 Transgenic rice lines NLR#1 and NLR#2 were used, and their resistance to rice blast was analyzed. The results showed that overexpression of NLR#1 and NLR#2... RWR3 It can enhance the rice blast resistance. This invention is the first to discover that the RWR3 protein can regulate plant rice blast resistance, providing a new target for improving rice disease resistance. Attached Figure Description

[0035] Picture 1 For T2 generation RWR3 Detection of the target gene RWR3 in the homozygous transgenic rice lines NLR#1 and NLR#2.

[0036] Picture 2 For T2 generation RWR3 The disease resistance phenotypes of transgenic homozygous rice lines NLR#1 and NLR#2 after inoculation with rice blast fungus strain RB22. RWR3 Two independent homozygous transgenic lines were obtained by transforming susceptible rice TP309 with the gene. A scratch inoculation experiment was conducted using the rice blast fungus strain RB22, and phenotypic data were recorded 6 days after inoculation. The scale bar is 1.5 cm.

[0037] Picture 3 For T2 generation RWR3 The length of lesions in transgenic homozygous rice lines NLR#1 and NLR#2 after inoculation with *Magnapordia oryzae* strain RB22 was statistically analyzed. A statistical test was conducted to determine whether there was a difference in lesion length between TP309 and the two independent transgenic homozygous lines. The statistical test method was one-way ANOVA, where **** represents... P The value is less than 0.0001. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] The rice variety Zhenshan 97 described in the following examples is described in the literature “Song, JM, Xie, WZ, Wang,S., Guo, YX, Koo, DH, Kudrna, D., Gong,C.,Huang, Y., Feng, JW,Zhang,W., Zhou, Y., Zuccolo,A., Long, E.,Lee,S., Talag, J.,Zhou,R., Zhu,XT,Yuan,D.,Udall, J., Xie,W., Wing, AR, Zhang, Q., Poland,J., Zhang,J.,Chen, LL(2021). Two gap-free reference genomes and a global view of the centromerearchitecture in rice. Molecular plant, 14(10), 1757-1767.”

[0041] The rice variety TP309 and the rice blast fungus strain RB22 described in the following examples are both described in the literature “Zhao, Y.,Shi, Y., Jiang, G., Wu, Y., Ma, M., Zhang, X., Liang, X., Zhou, JM (2022). Rice extra‐large G proteins play pivotal roles in controlling disease resistance and yield‐related traits. New Phytologist, 234(2), 607-617.”

[0042] The carrier pCAMBIA1300-221-flag used in the following embodiments is described in the literature “Liu, C., Ou, S., Mao, B., Tang, J., Wang, W., Wang, H., Cao, S., Schläppi, RM, Zhao, B., Xiao, G., Wang, X., Chu, C. (2018). Early selection of bZIP73 facilitatedaptation of japonica rice to cold climates. Nature communications, 9(1), 3302.”

[0043] Example 1 RWR3 Preparation of transgenic rice and analysis of its resistance to rice blast one, RWR3 Construction of gene expression vectors 1. RWR3 Obtaining gene fragments Extracting rice ( Oryza sativa DNA from the cultivated variety Zhenshan 97 was amplified by PCR using primers RWR3-F / RWR3-R. The PCR products were then sequenced. The primer sequences are as follows: RWR3-F: 5'-CGACGGCCAGTGCCAAGCTTCAGCGCATACAGTATGGGCTTAGATGG-3'.

[0044] RWR3-R: 5'-GATTTCGAACCCGGGGTACCGCAGATGATGAGATCGACAGATCGTTG-3'.

[0045] Sequencing results showed that PCR amplification yielded a PCR product of 10792 bp in size. This PCR product contained the DNA molecule shown in sequence 3, which included a promoter sequence of approximately 4 kb in size and a sequence of approximately 4 kb in size. RWR3 The gene sequence (supposedly shown at positions 4811-8752 of sequence 3) and the post-gene sequence, which is approximately 2 kb in size.

[0046] 2. RWR3 Obtaining gene expression vectors The pCAMBIA1300-221-flag plasmid was double-digested with Hind III and Kpn I to recover the large fragment and obtain the pCAMBIA1300-221-flag vector backbone. The PCR product obtained in step 1 was purified using a DNA purification kit. Homologous recombination technology was used to react the pCAMBIA1300-221-flag vector backbone with the purified PCR product to obtain the recombinant vector pCAMBIA1300::RWR3, which was then sequenced for verification.

[0047] Sequencing results show that the recombinant vector pCAMBIA1300::RWR3 is obtained by replacing the DNA fragment between the Hind III and Kpn I restriction sites of the pCAMBIA1300-221-flag plasmid with the DNA molecule shown in sequence 3, while keeping the other sequences of the pCAMBIA1300-221-flag plasmid unchanged.

[0048] two, RWR3 Genetically modified rice obtained 1. Obtaining recombinant Agrobacterium The recombinant vector pCAMBIA1300::RWR3 obtained in step one was transformed into Agrobacterium EHA105. Single colonies were picked and cultured overnight at 28°C with shaking in LB liquid medium containing kanamycin and rifampicin antibiotics to obtain transformants.

[0049] The transformants were identified by bacterial culture PCR (the primers for PCR identification were RWR3-F: 5′-GTGAATACGATCACCTTCGAG-3′ and Flag-R: 5′-CAAGACCGGCAACAGGATTC-3′, and the positive recombinant bacteria were obtained with a length of about 800 bp). The positive recombinant bacteria were named RWR3 / pCAMBIA1300::RWR3 and stored at -80℃ for later use.

[0050] 2. Obtaining genetically modified rice Rice was transformed using Agrobacterium tumefaciens-mediated transformation to obtain T0 generation transgenic rice. The specific steps are as follows: 1) Preparation of transformation receptors: Rice embryos (rice variety TP309) 14 days after pollination or naturally dispersed, bright yellow embryogenic callus (1 mm) derived from embryos were selected as receptors for Agrobacterium infection.

[0051] 2) Cultivation of Agrobacterium-mediated strains: Take a small amount of Agrobacterium stock solution (20% glycerol) and streak it onto YEB solid medium (containing 50 mg / L kanamycin and 50 mg / L rifamycin). Incubate at 28°C in the dark until single colonies with a diameter of 1 mm appear. Inoculate a single colony onto the same medium and incubate until the vigorous growth phase. Pick a small amount of cells and inoculate into 20 mL of YEB liquid medium (containing the corresponding antibiotics). Incubate overnight at 28°C in the dark with shaking at 220 rpm. The next day, transfer a 2% inoculum to 20 mL of YEB liquid medium containing 100 μM acetylsyl syringone. Incubate until mid-logarithmic growth, then dilute with at least three times the volume of liquid basal medium until slight turbidity is visible to the naked eye (OD). 600 Approximately 0.1), for use in conversion.

[0052] 3) Agrobacterium infection and co-culture: Take bacterial culture into a culture flask, add pre-cultured callus tissue, shake slightly and let stand for 10 minutes. After drying the callus tissue on sterile filter paper, place it on a culture medium supplemented with 100 μM acetylsyl syringone and incubate in the dark at 25°C for 3 days.

[0053] 4) Screening of resistant callus: The co-cultured callus tissue was transferred to a selection medium containing 25 mg / L hygromycin B and 500 mg / L cephalosporin. After screening 1 to 2 times, resistant callus tissue was observed to grow.

[0054] 5) Regeneration of resistant plants: Transfer resistant callus tissue to a medium containing 25 mg / L hygromycin, with the same light conditions as before. When the differentiated resistant shoots grow to 2-3 cm, transfer them to a regeneration seedling subculture medium containing 25 mg / L hygromycin. Once they have grown into complete plants, transfer them from the solid medium to an open culture medium containing nutrient solution. After new roots have formed, transfer the seedlings to a greenhouse.

[0055] 3. RWR3 Identification of genetically modified rice T0 generation transgenic rice plants that had grown for approximately two weeks were selected, and leaves approximately 3 cm long were cut and rapidly flash-frozen in liquid nitrogen. Total DNA was extracted, and PCR amplification was performed using wild-type rice TP309 as a control. The PCR amplification products were recovered and sequenced. The sequencing results showed that the transgenic rice with the target gene integrated into the genome was positive. RWR3 Genetically modified rice. Used for PCR detection. RWR3 The primer sequences successfully inserted into the rice genome are as follows (the amplified primers are located at...). RWR3 (3' end connection segment with the carrier skeleton) RWR3-F: 5′-GTGAATACGATCACCTTCGAG-3′.

[0056] Flag-R: 5′-CAAGACCGGCAACAGGATTC-3′.

[0057] 4. Homozygous RWR3 The acquisition of genetically modified rice Those identified as positive RWR3 Transgenic rice plants were propagated to the T2 generation by single-plant seed collection. 30-40 T2 generation seeds were soaked in water containing 25 mg / L hygromycin for germination. Homozygous plants were identified by the growth status of the plant roots and then transplanted. T2 generation plants were selected. RWR3 Transgenic rice lines NLR#1 and NLR#2 were used in the following rice blast resistance analysis experiment. The primers from step 3 were used to detect the T2 generation. RWR3 In the transgenic rice homozygous lines NLR#1 and NLR#2 RWR3 The target gene has been successfully transferred into the rice genome. Picture 1 ).

[0058] three, RWR3 Analysis of rice blast resistance in genetically modified rice Test materials: Wild-type rice TP309, T2 generation RWR3 Transgenic rice lines NLR#1 and NLR#2.

[0059] Experimental Methods: The test materials were cultivated using conventional methods and cultured under the following conditions: 14 hours of light, 10 hours of darkness, and a temperature of 28℃. When the rice reached the three-leaf stage (approximately 15 days), an in vitro inoculation experiment was conducted using scuffing. The specific steps were as follows: Rice leaves were cut into 4.5cm segments and placed in ddH2O. Each leaf was scuffed twice with a needle, with equal spacing between the scuffs. The leaves were then placed in large round petri dishes (labeled and placed in different positions within different petri dishes, and kept moist using ddH2O misting). The activated rice blast fungus strain RB22 was diluted with ddH2O to a concentration of 1.5 × 10⁻⁶. 5 A spore concentration of 1 spore / mL was determined, and the spore solution was dropped onto the wound. The large round dish was then transferred to an artificial climate incubator, where humidity was maintained as needed. After full disease development (approximately 5-7 days), the phenotype and lesion size were statistically analyzed based on the severity of the disease.

[0060] 1. Phenotypic observation Phenotypic results were recorded on day 6 post-RB22 dermatome inoculation. Results are as follows: Picture 2 As shown, the results indicate that compared to wild-type rice TP309, two... RWR3 The leaf lesions in genetically modified rice lines are visibly smaller.

[0061] 2. Statistics on the size of lesions On day 6 after RB22 inoculation via scratch, the length of lesions was recorded using a ruler. A one-way ANOVA test was used to examine whether there was a difference in lesion length between TP309 and two independent transgenic plants. Results are as follows: Picture 3 As shown, the results indicate that compared to wild-type rice TP309, two... RWR3 The size of lesions in transgenic rice lines was significantly reduced. Among them, wild-type rice TP309 and T2 generation showed significantly smaller lesions. RWR3 The lesion sizes of the transgenic rice lines NLR#1 and NLR#2 were 0.925 cm, 0.746 cm, and 0.748 cm, respectively.

[0062] The above experimental results show that, compared with wild-type rice TP309, RWR3 Transgenic rice showed significantly increased resistance to blast fungus RB22 after inoculation with RB22 spores, indicating that the RWR3 protein is involved in regulating rice blast resistance. The RWR3 protein and its encoding gene can be used for the breeding and identification of disease-resistant plant varieties in agricultural production, playing an important role in the field of plant disease resistance breeding.

[0063] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for improving plant disease resistance, comprising the following steps: increasing the activity and / or content of RWR3 protein in the target plant to improve plant disease resistance; The RWR3 protein is any one of the following (A1)-A4): A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) Proteins related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares more than 75% identity with A1 and is associated with plant disease resistance.

2. A method for cultivating transgenic plants with enhanced disease resistance, comprising the following steps: increasing the activity and / or content of RWR3 protein in a target plant to obtain a transgenic plant; wherein the transgenic plant exhibits higher disease resistance than the target plant; The RWR3 protein is any one of the following (A1)-A4): A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) Proteins related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares more than 75% identity with A1 and is associated with plant disease resistance.

3. The method according to claim 1 or 2, characterized in that: The disease resistance mentioned refers to resistance to rice blast.

4. The method according to any one of claims 1-3, characterized in that: The plant is any one of the following B1)-B4): B1) Monocotyledonous or dicotyledonous plants; B2) Grasses; B3) Plants of the genus *Oryza*; B4) Rice.

5. The application of the method according to any one of claims 1-4 in plant breeding.

6. Application of RWR3 protein or RWR3 protein-related biomaterials in any of the following C1)-C6): C1) Improve plant disease resistance; C2) Prepare products that enhance plant disease resistance; C3) Cultivate plants with enhanced disease resistance; C4) Prepare products from plants with enhanced disease resistance; C5) Plant breeding; C6) Preparation of plant breeding products; The RWR3 protein is any one of the following (A1)-A4): A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein associated with plant disease resistance obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; A3) Proteins related to plant disease resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of A1); A4) is a protein that shares more than 75% identity with A1 and is associated with plant disease resistance.

7. The application according to claim 6, characterized in that: The biological material is a nucleic acid molecule encoding the RWR3 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ.

8. The application according to claim 6 or 7, characterized in that: The nucleic acid molecule is either D1) or D2) as follows: D1) The DNA molecule shown at positions 4811-8752 of sequence 1 or sequence 3; The DNA molecule that has 75% or more identity with the nucleotide sequence defined by D2) and D1) and encodes the RWR3 protein.

9. The RWR3 protein as described in claim 6.

10. The biomaterial associated with the RWR3 protein as described in claim 6.

Images