Application of PIBP4 in disease resistance breeding

Abstract

The application relates to application of PIBP4 in disease-resistant breeding, and specifically provides use of PIBP4, Rab5a or OsRac1 protein or a coding sequence thereof in screening of a candidate substance capable of enhancing rice blast resistance of plants. The application provides a new target for cultivating broad-spectrum disease-resistant rice.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and botany, and more specifically, to a method for improving plant disease resistance. Background Technology

[0002] Rice blast is a disease of rice caused by the fungus *Magnaporthe oryzae*. This disease is widespread and occurs in all rice-producing regions of the world. Rice blast can occur at any stage of rice growth, primarily affecting the leaves, stems, and panicles. On leaves, it forms typical spindle-shaped lesions with a grayish-white center and brown edges. When the panicle is infected, it can lead to underdeveloped grains, and in severe cases, even total crop failure. Under normal circumstances, it reduces rice yield by 10%-30%, and in severe cases, by up to 40%-50%, making it one of the diseases with the greatest impact on rice yield and quality.

[0003] Some varieties contain disease resistance genes, such as PigmR, which can recognize effectors of rice blast fungus and initiate an immune response, thereby effectively resisting the disease. The mechanism of action of these disease resistance genes is still unclear, and further research is needed to elucidate the regulatory pathways of these genes, providing new targets for breeding broad-spectrum disease-resistant rice. Summary of the Invention

[0004] This invention provides the use of PIBP4, Rab5a, and OsRac1 genes in plant disease resistance.

[0005] The present invention first provides the use of PIBP4, Rab5a or OsRac1 protein, its coding sequence, nucleic acid construct containing said coding sequence, cell expressing said protein, or cell containing said coding sequence or nucleic acid construct in screening candidate substances that can enhance plant resistance to rice blast.

[0006] This invention also provides a method for screening candidate substances that can enhance plant resistance to rice blast, comprising:

[0007] (1) Contact the substance with a system containing PIBP4 and Rab5a, and

[0008] (2) Detect the interaction between PIBP4 and Rab5a and compare it with the control. If the interaction between PIBP4 and Rab5a is stronger than that of the control, then the substance is a candidate substance for enhancing rice blast resistance.

[0009] In one or more embodiments, the control is the same system that does not contain the substance.

[0010] In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0011] In one or more embodiments, the system is a cellular system, and the interaction occurs on the endoplasmic reticulum and / or cell membrane.

[0012] In one or more embodiments, the interaction is detected by a method selected from: yeast double hybridization, immunoprecipitation, and pull-down.

[0013] This invention also provides a method for screening candidate substances that can enhance plant resistance to rice blast, comprising:

[0014] (1) Contacting the substance with a system containing PIBP4, Rab5a, PigmR and a biological membrane (e.g., a cell membrane), and

[0015] (2) Detect the dot localization of PigmR on the biofilm and compare it with the control. If the dot localization of PigmR increases, the substance is a candidate substance for enhancing rice blast resistance.

[0016] In one or more embodiments, the control is the same system that does not contain the substance.

[0017] In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0018] In one or more embodiments, step (1) includes introducing a substance into cells expressing PIBP4, Rab5a, and PigmR, for example, introducing a nucleic acid construct expressing the substance into the cells.

[0019] In one or more embodiments, the point localization of PigmR on the biomembrane is detected by immunofluorescence assay.

[0020] This invention also provides a method for screening candidate substances that can enhance plant resistance to rice blast, comprising:

[0021] (1) Contact the substance with a system containing PigmR and OsRac1, and

[0022] (2) Detect the activation of OsRac1 and compare it with the control. If the activation of OsRac1 is enhanced, the substance is a candidate substance for enhancing rice blast resistance.

[0023] In one or more embodiments, the control is the same system that does not contain the substance.

[0024] In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0025] In one or more embodiments, activation of the OsRac1 is detected by the Raichu reporting system detection method.

[0026] This invention provides a nucleic acid construct comprising

[0027] (1) Nucleic acid sequences encoding PIBP4, Rab5a, and OsRac1 proteins;

[0028] (2) Nucleic acid sequences that specifically interfere with the transcription and / or expression of PIBP4, Rab5a, and OsRac1 genes.

[0029] In one or more embodiments, the amino acid sequence of the PIBP4 protein is as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it.

[0030] In one or more embodiments, the nucleic acid sequence of the PIBP4 protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

[0031] In one or more embodiments, the amino acid sequence of the Rab5a protein is as shown in SEQ ID NO:3 or a sequence having at least 80% identity with it.

[0032] In one or more embodiments, the nucleic acid sequence of the Rab5a protein is as shown in SEQ ID NO:4 or a sequence having at least 80% identity with it.

[0033] In one or more embodiments, the amino acid sequence of the OsRac1 protein is as shown in SEQ ID NO:5 or a sequence having at least 80% identity with it.

[0034] In one or more embodiments, the nucleic acid sequence of the OsRac1 protein is as shown in SEQ ID NO:6 or a sequence having at least 80% identity with it.

[0035] In one or more embodiments, the nucleic acid that specifically interferes with gene transcription and / or expression is sgRNA, wherein the sequence targeted by the sgRNA interfering with the PIBP4 gene comprises the sequence shown in SEQ ID NO:7 or 8, the sequence targeted by the sgRNA interfering with the Rab5a gene comprises the sequence shown in SEQ ID NO:9, and the sequence targeted by the sgRNA interfering with the OsRac1 gene comprises the sequence shown in SEQ ID NO:10. In one or more embodiments, the specific interference is performed using a CRISPR method based on the Cas9 enzyme.

[0036] In one or more embodiments, the nucleic acid construct is a vector.

[0037] This invention provides a genetically engineered host cell, which

[0038] (1) Expressing, containing, or secreting one or more proteins selected from the following: PIBP4, Rab5a, OsRac1, or

[0039] (2) Includes the nucleic acid constructs described in any of the embodiments described herein.

[0040] In one or more embodiments, the host cell is not a plant cell.

[0041] This invention provides a method for regulating plant resistance to rice blast, the method comprising: upregulating the expression or activity of one or more proteins selected from the group consisting of PIBP4, Rab5a, and OsRac1, thereby increasing plant resistance to rice blast; or downregulating the expression or activity of one or more proteins selected from the group consisting of PIBP4, Rab5a, and OsRac1, thereby decreasing plant resistance to rice blast. In one or more embodiments, the plant is a grass, preferably a plant of the genus Oryza, more preferably rice (Oryza sativa L.).

[0042] In one or more embodiments, the upregulation of protein expression in plants includes: transferring the coding sequence of a protein into a plant to obtain a transformed plant.

[0043] In one or more embodiments, the downregulation of protein expression in the plant includes:

[0044] (1) Specific interference is selected from the transcription and / or expression of one or more of the following genes: PIBP4, Rab5a, and OsRac1 genes.

[0045] (2) Downregulate the activity of one or more proteins selected from the following: PIBP4, Rab5a, and OsRac1 proteins.

[0046] (3) One or more of the following proteins whose activity is reduced in plants: PIBP4, Rab5a, OsRac1 protein.

[0047] In one or more embodiments, (1) the interference is to interfere with the transcription of a gene or the translation of its transcript.

[0048] In one or more embodiments, (2) the downregulation includes in plants:

[0049] (i) Expressing specific antibodies or ligands that can downregulate protein activity (e.g., inhibitory antibodies), or

[0050] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0051] In one or more embodiments, the downregulation of protein expression in the plant includes: transferring nucleic acids transcribed and / or expressed from a specific interfering protein encoding gene into the plant to obtain a transformed plant.

[0052] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the protein-encoding gene is selected from the group consisting of (1) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (2) nucleic acid constructs that can express or form (1).

[0053] In one or more embodiments, the nucleic acid used to specifically interfere with the transcription and / or expression of the protein-coding gene is sgRNA, wherein the sequence targeted by the sgRNA interfering with the PIBP4 gene comprises the sequence shown in SEQ ID NO:7 or 8, the sequence targeted by the sgRNA interfering with the Rab5a gene comprises the sequence shown in SEQ ID NO:9, and the sequence targeted by the sgRNA interfering with the OsRac1 gene comprises the sequence shown in SEQ ID NO:10. In one or more embodiments, the specific interference is performed using a CRISPR method based on the Cas9 enzyme.

[0054] This invention provides the use of substances that regulate the expression or activity of one or more proteins selected from the following in regulating plant resistance to rice blast: PIBP4, Rab5a, and OsRac1 proteins.

[0055] In one or more embodiments, the substance is a promoter of the expression or activity of PIBP4, Rab5a, or OsRac1 proteins, and the regulation is to upregulate the expression or activity of PIBP4, Rab5a, or OsRac1 proteins in plants, thereby improving plant resistance to rice blast.

[0056] In one or more embodiments, the promoter is a PIBP4, Rab5a, or OsRac1 protein or its coding sequence.

[0057] In one or more embodiments, the substance is an inhibitor of the expression or activity of PIBP4, Rab5a, or OsRac1 proteins, and the regulation is to downregulate the expression or activity of PIBP4, Rab5a, or OsRac1 proteins in plants, thereby reducing plant resistance to rice blast.

[0058] In one or more embodiments, the inhibitor is selected from:

[0059] (1) Inhibitors that specifically interfere with the transcription and / or expression of one or more of the PIBP4, Rab5a, and OsRac1 genes.

[0060] (2) Inhibitors that downregulate one or more activities of PIBP4, Rab5a, or OsRac1 proteins.

[0061] (3) PIBP4, Rab5a or OsRac1 protein variants or their coding sequences whose activity is downregulated.

[0062] In one or more embodiments, (1) the inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, small molecule compounds or combinations thereof, and (ii) nucleic acid constructs that can express or form (i).

[0063] In one or more embodiments, (1) the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of the PIBP4 gene.

[0064] In one or more embodiments, (2) the inhibitor is selected from the group consisting of (i) specific antibodies or ligands of the PIBP4 protein, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs capable of expressing (i).

[0065] In one or more embodiments, the nucleic acid used to specifically interfere with the transcription and / or expression of the protein-coding gene is sgRNA, wherein the sequence targeted by the sgRNA interfering with the PIBP4 gene comprises the sequence shown in SEQ ID NO:7 or 8, the sequence targeted by the sgRNA interfering with the Rab5a gene comprises the sequence shown in SEQ ID NO:9, and the sequence targeted by the sgRNA interfering with the OsRac1 gene comprises the sequence shown in SEQ ID NO:10. In one or more embodiments, the specific interference is performed using a CRISPR method based on the Cas9 enzyme.

[0066] The present invention also provides the use of the PIBP4, Rab5a or OsRac1 gene as a molecular marker for identifying plant resistance to rice blast.

[0067] In one or more embodiments, the identification includes identifying: comparing the expression or activity of the PIBP4, Rab5a, or OsRac1 genes in the plant with that in the wild-type plant; if the expression or activity of one or more of the PIBP4, Rab5a, or OsRac1 genes is upregulated, the plant has strong disease resistance; if the expression or activity of one or more of the PIBP4, Rab5a, or OsRac1 genes is downregulated, the plant has weak disease resistance.

[0068] The present invention also provides a method for regulating the accumulation of PigmR protein in cell membrane microdomains, the method comprising: (1) upregulating the protein expression or activity of PIBP4 and / or Rab5a in plants, thereby promoting the accumulation of PigmR protein in cell membrane microdomains; (2) downregulating the protein expression or activity of PIBP4 and / or Rab5a in plants, thereby reducing the accumulation of PigmR protein in cell membrane microdomains.

[0069] The present invention also provides a method for activating OsRac1, comprising contacting it with the PigmR protein. Attached Figure Description

[0070] Figure 1 PigmR and PIBP4 interact.

[0071] (A) Yeast double heterozygosity indicates that PIBP4 interacts with PigmR-CC and the full-length domain, but does not interact with the NBS and LRR domains.

[0072] (B) The tobacco luciferase complementation experiment showed that PIBP4 interacts with PigmR-CC and the full-length domain, but does not interact with the NBS and LRR domains.

[0073] (C)BiFC experiments demonstrated that the interaction between PIBP4 and PigmR-CC occurs in the cell membrane and endoplasmic reticulum, Bar: 5 μm.

[0074] Figure 2 PIBP4 knockout affects PigmR-mediated ETI immune responses.

[0075] (A) Spraying inoculation of the non-toxic rice blast race TH12 corresponding to PigmR significantly reduced the rice blast resistance of PIBP4 knockout materials. NIP materials served as susceptible controls for the TH12 rice blast race. Scale bar: 2cm.

[0076] (B) In vitro inoculation of PIBP4-related materials with TH12 showed the same phenotype as that of spray inoculation. Scale bar: 1 cm.

[0077] Figure 3 PIBP4 and Rab5a interaction

[0078] (A) Yeast double hybridization indicates that PIBP4 and OsRab5a, as well as the activated form OsRab5a (Q70L), interact.

[0079] (B)CoIP indicates that PIBP4 and OsRab5a, OsRab5a(Q70L) interact.

[0080] Figure 4 PigmR and Rab5a interact.

[0081] (A) The tobacco luciferase complementation experiment showed that Rab5a interacts with PigmR-CC and the full-length domain, but does not interact with the NBS and LRR domains.

[0082] (B)CoIP indicates that Rab5a and PigmR interact.

[0083] (C)BiFC experiments demonstrate that the interaction between Rab5a and PigmR-CC occurs in the cell membrane and endoplasmic reticulum, Bar: 5 μm.

[0084] (D) In ​​vitro inoculation of the virus-free rice blast race TH12 corresponding to PigmR showed a significant decrease in rice blast resistance in Rab5a knockout materials. NIP materials served as susceptible controls for the TH12 rice blast race. Scale bar: 1 cm.

[0085] (E) Rab5a-related materials were inoculated in vitro with TH12 and exhibited the same phenotype as those in spray inoculation. Scale bar: 2cm.

[0086] Figure 5 The PIBP4-Rab5a transporter is involved in the accumulation of PigmR protein microregions.

[0087] (A) PIBP4 and PigmR are colocalized in the cell membrane and endoplasmic reticulum.

[0088] (B) Rab5a and PigmR colocalize in the cell membrane and endoplasmic reticulum.

[0089] (C) Immunofluorescence experiments on root tip of PigmR-7Myc, PigmR-7Myc / рibр4, and PigmR-7Myc / osrab5a materials revealed that knockout of PIBP4 and Rab5a affected the localization of PigmR in the cell membrane microdomain. GSD1 is a remorin family protein that serves as a marker protein for localization in the cell membrane microdomain. Bar: 10 μm.

[0090] (D) TIRFM experiments showed that PIBP4 and Rab5a knockout affected the localization of PigmR-CC in the cell membrane microregion, Bar: 5 μm.

[0091] Figure 6 PigmR activates OsRac1 to enhance rice blast resistance.

[0092] (A)CoIP indicates that OsRac1 and PigmR interact.

[0093] (B) The tobacco luciferase complementation experiment showed that OsRac1 interacts with PigmR-CC and the full-length domain, but does not interact with the NBS and LRR domains.

[0094] (C) Spraying inoculation of the non-toxic rice blast race TH12 corresponding to PigmR significantly reduced the rice blast resistance of OsRac1 knockout materials. NIP materials served as susceptible controls for the TH12 rice blast race. Bar: 2cm.

[0095] (D) In ​​vitro inoculation of OsRac1-related materials with TH12 showed the same phenotype as that of spray inoculation, Bar: 1cm.

[0096] (E) The activation of OsRac1 was characterized using the Raichu-Rac1 reporter system. PigmR was found to activate OsRac1. Pish was used as a negative control. Bar: 5 μm.

[0097] (F) Statistical results after Raichu system quantization.

[0098] (G)OsRac1 enhances tobacco cell death induced by PigmR-CC.

[0099] (H) The conductivity of the dead tobacco cells was measured to characterize the degree of tobacco cell death. Detailed Implementation

[0100] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0101] The inventors have revealed for the first time that PIBP4 participates in regulating PigmR-mediated resistance to rice blast fungus by regulating OsRab5a. They discovered that the two interact to form a transport mechanism that participates in the accumulation of PigmR protein in cell membrane microdomains and further activates Rac1 protein located in cell membrane microdomains, thereby generating reactive oxygen species.

[0102] To elucidate the signal transduction mechanism of PigmR, we screened PigmR interacting proteins using yeast two-hybrid technology. Among them, an important protein, PIBP4 (PigmR Interacting and Blast resistance Protein 4, Os03g0741600 / NCBI: LOC4334064), belongs to the prenylated Rab GTPase receptor (PRA) family. This family plays an important role in regulating the activity of small G protein Rab GTPase and vesicle transport.

[0103] We demonstrated that PIBP4 does indeed interact with PigmR through Split-Luc experiments in tobacco, yeast two-hybrid experiments, and BiFC experiments. Using RNAi and gene editing technologies, we constructed a PIBP4 mutant with a NIL-Pigm background. Inoculation experiments with rice blast showed that the PIBP4 gene mutation partially reduced PigmR-mediated rice blast resistance. These data indicate that PIBP4 participates in regulating PigmR-mediated rice blast resistance. Subsequently, through interacting protein screening, we identified the PIBP4-regulated small G protein OsRab5a (Os12g0631100 / NCBI: LOC4352862). We found that the interaction between the two forms a transport mechanism involved in the accumulation of PigmR protein in cell membrane microdomains, further activating the Rac1 protein located in these microdomains, thereby generating reactive oxygen species and inducing disease resistance in rice.

[0104] This invention provides a method for regulating plant resistance to rice blast. The term "regulation" as used herein includes both "upregulation" and "downregulation." Therefore, the method described herein includes: upregulating the expression or activity of one or more proteins selected from the group consisting of PIBP4, Rab5a, and OsRac1, thereby enhancing plant resistance to rice blast. The plant described herein can be any plant, preferably a model plant (e.g., Arabidopsis thaliana, tobacco) or a grass species, such as rice, preferably rice (Oryza sativa L.).

[0105] In this study, the PIBP4, Rab5a, and OsRac1 genes can be derived from any plant, with rice (Oryza sativa L.) being the preferred source. Their amino acid sequences are shown in SEQ ID NO:1, 3, and 5, respectively, and their CDS are shown in SEQ ID NO:2, 4, and 6, respectively.

[0106] The term "rice blast resistance" as used in this article refers to a plant's ability to resist diseases caused by the rice blast fungus (Magnaporthe oryzae). Symptoms of rice blast include, but are not limited to: the formation of spindle-shaped lesions on leaves, with a grayish-white center and brown edges; and in the panicle, it leads to underdeveloped grains, and in severe cases, complete crop failure.

[0107] Any substance that can enhance the activity, stability, expression, duration of action, or transcription and translation of PIBP4, Rab5a, and OsRac1 genes can be used in this invention as a "promoter" for the PIBP4, Rab5a, and OsRac1 genes to regulate plant agronomic traits. For example, vectors that enhance the expression or activity of PIBP4, Rab5a, and OsRac1.

[0108] Alternatively, to upregulate the activity of PIBP4, Rab5a, and OsRac1, promoters of PIBP4, Rab5a, and OsRac1 can be introduced into the plant. Promoters of PIBP4, Rab5a, and OsRac1 include, but are not limited to, small molecule compounds, nucleic acid molecules, or combinations thereof. Preferably, the nucleic acid molecule is a nucleic acid construct containing the coding sequences of PIBP4, Rab5a, and OsRac1. The nucleic acid construct is an expression vector or an integration vector.

[0109] On the other hand, any substance that can reduce the activity, stability, expression, duration of action, or transcription and translation of PIBP4, Rab5a, and OsRac1 can be used in this invention as an inhibitor of PIBP4, Rab5a, and OsRac1. Such inhibitors can be used to regulate plant resistance to rice blast.

[0110] This invention also provides the use of PIBP4, Rab5a, and OsRac1 as targets in regulating plant resistance to rice blast or in screening for substances that can regulate plant resistance to rice blast.

[0111] For example, to downregulate the expression or activity of PIBP4, Rab5a, or OsRac1, repressive molecules that specifically interfere with the transcription and / or expression of PIBP4, Rab5a, or OsRac1, or downregulate the activity of PIBP4, Rab5a, or OsRac1 proteins, can be transferred into cells or plants, causing the cells or plants to not express or reduce the expression of the PIBP4, Rab5a, or OsRac1 genes. The repressive molecule targets the PIBP4, Rab5a, or OsRac1 genes or their transcripts or expressed proteins. Therefore, the repressive molecule can target the protein or its coding sequence shown in SEQ ID NO:1 or 2. The inhibitory molecule may be a small molecule compound known to inhibit the activity of PIBP4, Rab5a or OsRac1, an antibody or ligand of PIBP4, Rab5a or OsRac1 protein or a binding fragment thereof, or an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA or sgRNA that interferes with the expression of PIBP4, Rab5a or OsRac1 genes.

[0112] Furthermore, to downregulate the expression or activity of PIBP4, Rab5a, or OsRac1 genes, gene knockout vectors can be introduced into cells. Therefore, the inhibitor can be a reagent that knocks out or reduces the PIBP4, Rab5a, or OsRac1 gene using a technology selected from ZFN, TALEN, and CRISPR, such as sgRNA. ZFN, TALEN, and CRISPR / Cas9 technologies suitable for use in this invention are well known in the art. Each technology achieves the knockout of the target gene through the combined action of a DNA recognition domain and a nuclease. In these embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0113] The present invention also provides the use of substances in regulating plant resistance to rice blast, said substances being selected from the group consisting of PIBP4, Rab5a or OsRac1 genes or encoded proteins, or promoters or inhibitors thereof.

[0114] The present invention also provides polynucleotides expressing PIBP4, Rab5a or OsRac1 proteins or nucleic acid constructs containing said polynucleotides.

[0115] This invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes. "Polynucleotide encoding a polypeptide" can include a polynucleotide encoding the polypeptide, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0116] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:1 or 2.

[0117] It should be understood that although the genes provided in the examples of this invention are derived from rice, gene sequences derived from other similar plants (especially those belonging to the same family or genus as rice) that have a certain degree of homology (e.g., 70% or more, such as 80%, 85%, 90%, 95%, or even 98% sequence identity) with the sequences of this invention (preferably, as shown in SEQ ID NO: 1 or 2) are also included within the scope of this invention, provided that those skilled in the art can easily isolate such sequences from other plants based on the information provided in this application after reading it. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0118] The full-length nucleotide sequence or fragment thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available DNA library or a cDNA library prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, it is cloned into a vector, transformed into cells, and then the relevant sequence is isolated from the proliferated host cells using conventional methods. In addition, the relevant sequence can also be synthesized artificially. Currently, DNA sequences encoding the protein of the present invention (or its fragments, or derivatives) can be obtained entirely through chemical synthesis. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0119] This invention also provides a recombinant vector comprising the polynucleotides of this invention. As a preferred embodiment, the recombinant vector contains a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter (e.g., a 35S promoter), a target gene, and a terminator. If desired, the recombinant vector may further include elements selected from the group consisting of: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; an resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator. The vector can be an expression vector or an integration vector; the former is used to express genes, dsRNA, related enzymes, sgRNA, etc.; the latter is used to integrate the desired expressed nucleic acid sequence into the genome. Exemplary vectors include p1301s, pAbAi, pGADT7, p1300-GFP, pA7-GFP, pGreenII-62sk, and pGreenII-0800.

[0120] The methods used to prepare recombinant vectors are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0121] Those skilled in the art can use well-known methods to construct expression vectors containing the genes described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When constructing recombinant expression vectors using the genes of this invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0122] Vectors, including the genes, expression cassettes, or other components of this invention, can be used to transform suitable host cells to enable the host to express proteins. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Streptomyces*, or *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), CaCl2 treatment or electroporation can be used. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.) can be used. Transformation of plants can also be performed using methods such as *Agrobacterium* transformation or gene gun transformation, for example, spraying, leaf disc transformation, embryo transformation, flower bud soaking, etc. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants. When the polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0123] Those skilled in the art know how to select appropriate vectors, promoters, enhancers, and host cells.

[0124] The peptides described herein may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0125] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0126] Example

[0127] Experimental methods

[0128] Cloning steps

[0129] PIBP4 gene cDNA amplification

[0130] Obtain the target band using PCR method.

[0131] (1) Design primers

[0132] (2) Conventionally, genomic DNA, cDNA, or plasmids are used as templates. Due to the high GC content of the rice genome, we preferentially use Toyobo's high-fidelity DNA polymerase KOD-Fx for amplification of the target fragment. The PCR system (40 μl) is as follows:

[0133]

[0134] Reaction conditions: 95℃, 5min; 94℃, 30s; 55~65℃, 30s; 68℃, 1kb / min, 30~35Cycles; 68℃, 10min; 16℃, 5min.

[0135] Agarose gel DNA recovery

[0136] After PCR amplification or enzyme digestion, horizontal agarose gel electrophoresis is performed. After electrophoresis, DNA is recovered according to the instructions of the Biotech Agarose Gel DNA Recovery Kit. The simplified steps are as follows:

[0137] (1) Prepare an agarose gel of appropriate concentration according to the size of the required electrophoretic fragments. After it is completely solidified, perform horizontal electrophoresis in 1×TAE solution at 120V for 15-20 minutes.

[0138] (2) Cut the target strip in a UV gel cutter, add 300 μl of sol-binding solution to every 0.1 g of gel, place in a 65°C metal bath, and mix every 2-3 minutes until the gel block is completely melted.

[0139] (3) Add 150 μl of isopropanol to each 0.1 g gel, shake to mix, add the solution to the adsorption column, centrifuge at 12000 g for 1 min, and discard the centrifuged liquid; add 700 μl of wash buffer WB, centrifuge at 12000 g for 1 min, and discard the waste liquid; add another 500 μl of wash buffer WB, repeat the centrifugation once, and discard the waste liquid;

[0140] (4) Centrifuge the empty column at 12000g for 2 min, transfer the adsorption column to a new 1.5ml centrifuge tube, and place it in a 65℃ metal bath with the lid off for 2 min to allow the alcohol to evaporate completely.

[0141] (5) Add 50 μl of elution buffer preheated to 65 °C to the adsorption column, incubate in a 65 °C metal bath for 2 min, and centrifuge at 12000g for 2 min. Measure the concentration of the eluent using a NanoDrop 2000 (Thermo Fisher) and store at -20 °C.

[0142] Enzyme digestion system (50 μl)

[0143] The vector was subjected to an enzymatic digestion reaction, and the system is as follows:

[0144]

[0145]

[0146] Reconnection

[0147] Follow the instructions for the Vazyme ClonExpress II One Step Cloning Kit (C112).

[0148] Preparation of competent Escherichia coli

[0149] (1) Take a tube of competent cells frozen at -80℃, streak it on LB solid medium, and incubate it upside down at 37℃ overnight;

[0150] (2) Select single clones and culture them overnight in liquid LB medium at 37°C with shaking.

[0151] (3) Inoculate the small-scale shaken product into an appropriate amount of LB liquid medium at a ratio of 1–5:100, and incubate at 20°C for 200 minutes.

[0152] rpm, shake and incubate until OD600 is approximately 0.55;

[0153] (4) After placing the bacterial culture on ice for 10 minutes, centrifuge at 2500g at 4℃ for 10 minutes;

[0154] (5) Discard the supernatant, resuspend the precipitate at a ratio of 32ml Inoue buffer / 100ml bacterial culture, let it stand on ice for a period of time, and centrifuge again under the same conditions.

[0155] (6) Discard the supernatant, resuspend the precipitate at a ratio of 8ml Inoue buffer / 100ml bacterial solution, add 0.6ml of filtered and sterilized DMSO, mix well, dispense into approximately 100μl / tube, freeze quickly in liquid nitrogen and store at -80℃.

[0156] Preparation of relevant reagents

[0157]

[0158] When preparing the solution, first add PIPES, adjust the pH to 6.7 with solid KOH to dissolve the PIPES, then add other appropriate salt ions, and after making up the volume, filter it through a disposable 0.45μm filter (Millipore, Cat#SLHP033RB) for sterilization.

[0159] Escherichia coli competent transformation and identification

[0160] (1) Take the frozen competent cells out of the -80℃ freezer and place them on ice to thaw for 5 minutes;

[0161] (2) Add an appropriate amount of the ligation product, mix well and place on ice for 15-30 minutes;

[0162] (3) Heat shock in a 42℃ water bath or metal bath for 90 seconds, then quickly place on ice for 5 minutes.

[0163] (4) Add 100 μl of antibiotic-free LB to each tube, shake and incubate at 37°C for 30 min, take 100 μl and spread it on the corresponding LB solid plate, invert and incubate overnight.

[0164] (5) Pick a single colony and mix it with 30 μl of sterile water. Take 2 μl as a template and perform colony collection in a 20 μl system.

[0165] For PCR-positive samples, take 20 μl and incubate overnight at 37°C with shaking in LB liquid medium containing appropriate antibiotics.

[0166] (6) Take 1-2 ml of overnight culture product to extract plasmid (see 2.3.2.1), perform enzyme digestion and identification with appropriate enzymes, and send positive plasmids to the company for sequencing.

[0167] plasmid extraction

[0168] Small-scale plasmid extraction using alkaline lysis method

[0169] The following procedures can be performed at room temperature.

[0170] (1) Take 1-2 ml of overnight culture product, centrifuge at 12000g for 1 min, and discard the supernatant;

[0171] (2) Add 200 μl of pre-cooled solution I and shake thoroughly to mix;

[0172] (3) Add 200 μl of solution II, gently invert 6-8 times, avoiding vigorous shaking that could damage genomic DNA.

[0173] fracture;

[0174] (4) Add 400 μl of pre-cooled solution III, gently invert to mix, and let stand at room temperature for 5 min;

[0175] (5) Centrifuge at 12000g for 10 min, transfer the supernatant to a 1.5ml centrifuge tube, add an equal volume of isopropanol, shake well to mix, and let stand at room temperature for 5 min;

[0176] (6) Centrifuge at 12000g for 10 minutes, carefully discard the supernatant, and be careful not to let the precipitate flow away;

[0177] Add 1 ml of 70% ethanol and suspend the precipitate by tapping the bottom of the tube.

[0178] (7) Centrifuge at 7500g for 5 minutes and discard the supernatant;

[0179] (8) Repeat the washing process, aspirate the supernatant, place in a 65°C metal bath to dry for 2 min, and add 50 μl of water.

[0180] Dissolve the precipitate with ddH2O and store at -20℃.

[0181] Example 1. PigmR and PIBP4 have a direct interaction.

[0182] Through yeast double hybridization and SLC experiments, such as Figure 1 As shown in (A) and (B), PigmR interacts with PIBP4 through the CC domain. BIFC experiments revealed that this interaction mainly occurs in the cell membrane and endoplasmic reticulum. Figure 1 (C)).

[0183] Example 2. Positive regulation of PigmR-mediated rice blast resistance by PIBP4

[0184] In the context of Pigm near-isogeninized lines, materials were constructed that overexpressed and knocked out PIBP4 (using CRISPR-Cas9 technology for gene knockout, with target sequences being ggcgctcatcgcggcgagg and cctcaccctcctcgggagc). Inoculation of these materials with the virus-free rice blast race corresponding to PigmR revealed that PIBP4 knockout significantly reduced PigmR-mediated rice blast resistance. Figure 2 (A) and (B)) This result indicates that PIBP4 positively regulates PigmR-mediated rice blast resistance.

[0185] Example 3. PIBP4 interacts with Rab5a, and Rab5a participates in PigmR-mediated rice blast resistance.

[0186] PIBP4 belongs to the PRA family of proteins, which typically recruit isopentenylated Rab proteins to participate in intracellular transport processes. Through screening, we found an interaction between PIBP4 and OsRab5a. Figure 3 (A)), and mainly interacts with the activated form of OsRab5a, OsRab5a(Q70L). Figure 3 (B)).

[0187] like Figure 4As shown in (A) and (B), further research revealed that Rab5a also interacts with PigmR, and BiFC experiments showed that this interaction also occurs on the endoplasmic reticulum and cell membrane. Figure 4 (C)), Rab5a can also positively regulate PigmR-mediated rice blast resistance ( Figure 4 (D) and (E)). These results suggest that PIBP4 and Rab5a, as a transporter, jointly participate in PigmR-mediated rice blast resistance.

[0188] Example 4. PIBP4-Rab5a transporter involved in PigmR protein microregion accumulation

[0189] like Figure 5 As shown in (A) and (B), colocalization experiments revealed punctate colocalization of PigmR and PIBP4, and PigmR and Rab5a, on the cell membrane. This localization often suggests that the protein is localized in the microdomain formed by lipid rafts in the cell membrane. We subsequently verified whether PIBP4 and Rab5a are involved in the transport of PigmR to the cell membrane microdomains, as shown in... Figure 5 As shown in (C), immunofluorescence experiments on PigmR-Myc materials revealed a significant reduction in the punctate localization of PigmR on the cell membrane in PIBP4 and Rab5a mutants. Similarly, TIRFM assays were performed to observe the effects of transient expression of PigmR-CC protein in protoplasts. Figure 5 (D) It was also found that PIBP4 and Rab5a affect the accumulation of PigmR in cell membrane microregions. Rab5a was knocked out using CRISPR-Cas9 technology, with the target sequence being: atgtcgtagacaactatcg.

[0190] Example 5. PigmR activates OsRac1 to enhance rice blast resistance

[0191] like Figure 6 (A) and (B), CoIP, SLC experiments showed that PigmR can interact with OsRac1. Similarly, OsRac1 knockout materials in the NIL-Pigm background (gene knockout using CRISPR-Cas9 technology, target sequence: cctctgggacactgcaggt) showed significantly reduced resistance to PigmR-free rice blast races. Figure 6 (C) and (D) indicate that OsRac1 is involved in PigmR-mediated immune responses.

[0192] Next, we hypothesized whether the PIGmR protein in the microregion could activate the OsRac1 protein above it, and used the Raichu-Rac1 reporter system to characterize Rac1 activation. Figure 6(E)) It was found that co-expression of PigmR-Flag and Raichu-Rac1 in rice protoplasts significantly increased Rac1 activation compared to the control. Figure 6 (F)) Simultaneously, cell death induced by co-expression of PigmR-CC-Flag and Myc-Rac1 in tobacco was more severe than that induced by co-expression of Myc-Rac1 (DN) and Myc-EV (Disease-Induced Cell Death). Figure 6 (G) and (H)), these results indicate that PigmR activates OsRac1 to enhance rice blast resistance.

[0193] Partial sequence

[0194] SEQ ID NO:1 PIBP4 protein

[0195] MRNSTSSAAAQPAPASAAMYGSYASPSSGAGGYAKIPTYPPPPSAYPAAPPPPVMGQPVPPPPAQLHDPTAPPSPIAKAAELVTRFREQGQALIAARRPWGEVFRAPAFSRPPSVGEAVARARR NAAYFRANYALAVLAVVASLLWHPGTLFALLALCAAWFFLYFARPASSAGQPLRLLGMEFEDGTVLAALTGVTVIALLFTNVGWNVIGSVMIGAALVAAHATLRSTDDLFLTEQEAAGDGLVAAG MSAAGPILPT YVRIA

[0196] SEQ ID NO:2PIBP4 CDS

[0197] atgcgcaactccacctcctccgccgccgcccagcccgcgccggcctccgccgccatgtacggctcctacgcctcaccctcctcgggagcaggagggtacgccaagatccccacctacccgccccctccctccgcctaccccgccgccccgcctcctccggtgatgggccagcccgtgccgccgccgcccgcgcagctgcatgacccgacggcgccgccgtccccgatcgccaaggcggcggagctggtgacgaggttccgggagcaggggcaggcgctcatcgcggcgaggcggccgtggggggaggtgttccgggcgccggccttctccaggccgcccagcgtcggggaggcggtggccagggcgcggcggaacgctgcctacttccgggccaactacgcgctcgccgtgctcgccgtcgtcgcggcctcgctgctctggcaccccgggacgctcttcgcgctcctcgccctctgcgccgcctggttcttcctctacttcgcgcgccccgcgtcgtcggcggggcagccgctccgcctcctcgggatggagttcgaggacggcaccgtcctcgccgcgctcaccggggtcaccgtcatcgcgctgctcttcaccaacgtcggctggaacgtcatcggctcggtcatgatcggcgccgccctcgtcgccgcgcacgccacgctccgctccaccgacgacctcttcttgaccgagcaggaggccgccggcgacggcctggtggccgccggcatgagcgccgccggaccaatcttgcccacctacgttcgcatcgcttga

[0198] SEQ ID NO:3Rab5a protein

[0199] MAANPGNKIRNAKLVLLGDVGTGKSSLVLRFVKGQFVEFQESTIGAAFFSQTLAVNDETVKFEIWDTAGQERYHSLAPMYYRGAAAAIVVYDITNAASFTRAKKWVQELQAQGNPNTIMALAGNKADMVEARQVPAEEAKTYAQENGLFFMETSAKTAINVNDVFHEIAKRLLQGQQAQDTPAGMVLNQRPAERMVSSSSCCS*

[0200] SEQ ID NO:4Rab5a CDS

[0201] atggcggccaaccccggcaacaagatccgcaacgccaagctggttcttcttggagatgtgggcacgggcaagtcgagcctcgttctccggtttgtgaagggccagtttgttgagttccaggagtccaccatcggcgcggccttcttctcgcaaaccttggcggttaacgatgagacggtgaagttcgaaatctgggatactgcagggcaggagaggtatcatagcttggctccgatgtactatcggggtgcggctgccgcgatagttgtctacgacatcacaaatgcggcctctttcacacgtgcaaaaaaatgggttcaagaacttcaagcgcaaggaaacccaaacacgataatggctcttgctggtaacaaggctgatatggtagaggcgaggcaggtgccagcagaagaggcaaagacctacgcacaagagaatggccttttcttcatggaaacatctgcgaaaacggcgatcaatgtgaacgatgtattccacgagatcgcaaagagattgcttcaaggacagcaggctcaagacacaccggctggaatggttctcaaccagagaccagctgagaggatggtgagcagttcttcatgctgctcataa

[0202] SEQ ID NO:5OsRac1 protein

[0203] MSSAAAATRFIKCVTVGDGAVGKTCMLICYTCNKFPTDYIPTVFDNFSANVSVDGSVVNLGLWDTAGQEDYSRLRPLSYRGADVFILSFSLISRASYENVQKKWMPELRRFAPGVPVVLVGTKLDLREDRAYLADHPASSIITTEQGEELRKLIGAVAYIECSSKTQRNIKAVFDTAIKVVLQPPRHKDVTRKKLQSSSNRPVRRYFCGSACFA*

[0204] SEQ ID NO:6 OsRac1 CDS

[0205] atgagctcggcggcggcggcgacgaggttcatcaagtgcgtcaccgtgggggacggcgcggtggggaagacgtgcatgctcatctgctacacctgcaacaagttccccaccgattacatccccaccgtgttcgacaacttcagcgccaatgtctccgtggacgggagcgtcgtcaacctcggcctctgggacactgcaggtcaggaggattacagcaggttgaggcctctgagctacaggggagccgatgtgttcatcctgtccttctccctgataagcagggcgagctatgagaatgttcagaagaagtggatgccagagcttcgccggtttgcgcctggtgttcctgtagttcttgttggaaccaagttggatctccgtgaagatagggcctatcttgctgatcatccagcttcttccataataacaacggagcagggagaagaactgaggaagctaataggagcggtcgcctacatcgaatgcagctccaagacacagagaaacattaaagctgttttcgacactgccatcaaagtggtgcttcaacctccaagacataaggatgtaaccagaaagaaactccaatcaagctccaatcggccagtaaggcggtacttttgcggaagcgcttgtttcgcgtag

[0206] SEQ ID NO:7 PIBP4 knockout target sequence

[0207] ggcgctcatcgcggcgagg

[0208] SEQ ID NO:8PIBP4 knockout target sequence

[0209] cctcaccctcctcgggagc

[0210] SEQ ID NO:9Rab5a knockout target sequence

[0211] atgtcgtagacaactatcg

[0212] SEQ ID NO:10OsRac1 knockout target sequence

[0213] cctctgggacactgcaggt .

Claims

1. The use of PIBP4, Rab5a, or OsRac1 proteins or their coding sequences in screening candidate substances that can enhance plant resistance to rice blast.

2. A method for screening candidate substances that can enhance plant resistance to rice blast, comprising: (1) Contact the substance with a system containing PIBP4 and Rab5a, and (2) The interaction between PIBP4 and Rab5a was detected and compared with the control. If the interaction between PIBP4 and Rab5a was stronger than that of the control, then the substance was a candidate substance for enhancing rice blast resistance. The interaction was detected by any of the following methods: yeast double hybridization, immunoprecipitation, or pull-down.

3. A method for screening candidate substances that can enhance plant resistance to rice blast, comprising: (1) Contacting the substance with a system containing PIBP4, Rab5a, PigmR and a biological membrane (e.g., a cell membrane), and (2) The dotted localization of PigmR on the biofilm was detected and compared with the control. If the dotted localization of PigmR increased, the substance was a candidate substance for enhancing rice blast resistance. The point localization of PigmR on biomembranes was detected by immunofluorescence assay.

4. A method for screening candidate substances that can enhance plant resistance to rice blast, comprising: (1) Contact the substance with a system containing PigmR and OsRac1, and (2) Detect the activation of OsRac1 and compare it with the control. If the activation of OsRac1 is enhanced, the substance is a candidate substance for enhancing rice blast resistance.

5. A method for regulating plant resistance to rice blast, the method comprising: Upregulating the expression or activity of one or more proteins selected from the following in plants—PIBP4, Rab5a, and OsRac1—can enhance plant resistance to rice blast; or downregulating the expression or activity of one or more proteins selected from the following in plants—PIBP4, Rab5a, and OsRac1—can reduce plant resistance to rice blast. The upregulation of protein expression in plants includes: transferring the protein's coding sequence into plants to obtain transformed plants. The downregulation of protein expression in plants includes: (1) Specifically interferes with the transcription and / or expression of one or more of the PIBP4, Rab5a, and OsRac1 genes. (2) Downregulate the activity of one or more of the proteins PIBP4, Rab5a, and OsRac1. (3) Express one or more of the PIBP4, Rab5a, and OsRac1 proteins with reduced activity in plants.

6. The use of substances that regulate the expression or activity of one or more of the following proteins in regulating plant resistance to rice blast: PIBP4, Rab5a, and OsRac1 proteins, among which... The substance is a promoter of protein expression or activity, and the regulation is to upregulate protein expression or activity in plants, thereby improving plant resistance to rice blast, or The substance is an inhibitor of protein expression or activity, and the regulation is to downregulate the expression or activity of PIBP4 protein in plants, thereby reducing the plant's resistance to rice blast. Preferably, the promoter is a protein or its coding sequence; the inhibitor is selected from: (1) an inhibitor that specifically interferes with the transcription and / or expression of one or more of the PIBP4, Rab5a, and OsRac1 genes, (2) an inhibitor that downregulates the activity of one or more of the PIBP4, Rab5a, and OsRac1 proteins, and (3) a downregulated PIBP4, Rab5a, and OsRac1 protein variant or its coding sequence. More preferably, (1) the inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA or combinations thereof, and (ii) nucleic acid constructs that can express or form (i).

7. The uses of PIBP4, Rab5a, and OsRac1 genes: as molecular markers for identifying plant resistance to rice blast. Preferably, the identification includes: comparing the expression or activity of the plant's PIBP4, Rab5a, and OsRac1 genes with that of wild-type plants; if the expression or activity of one or more of the PIBP4, Rab5a, and OsRac1 genes is upregulated, the plant has strong disease resistance; if the expression or activity of one or more of the PIBP4, Rab5a, and OsRac1 genes is downregulated, the plant has weak disease resistance.

8. A method for activating OsRac1, comprising contacting it with the PigmR protein.

9. A nucleic acid construct comprising: (1) Encoding one or more of the following nucleic acid sequences: PIBP4, Rab5a, and OsRac1 proteins. (2) Specific interference with the transcription and / or expression of one or more of the PIBP4, Rab5a, and OsRac1 genes using nucleic acid sequences. The amino acid sequence of the PIBP4 protein is shown in SEQ ID NO:1 or a sequence having at least 80% identity with it. The amino acid sequence of the Rab5a protein is shown in SEQ ID NO:3 or a sequence having at least 80% identity with it. The amino acid sequence of the OsRac1 protein is shown in SEQ ID NO:5 or a sequence having at least 80% identity with it. Preferably, the nucleic acid construct is a vector.

10. A genetically engineered host cell, whose (1) Expressing, containing, or secreting one or more proteins selected from the following: PIBP4, Rab5a, OsRac1, or (2) Contains the nucleic acid construct according to claim 9 The host cell is not a plant cell.

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