Osago12 protein and its coding gene in regulating plant resistance to rice gall dwarf virus

Abstract

The present invention relates to the field of biotechnology, and in particular to the application of OsAGO12 protein and its encoding gene expression in regulating plant resistance to rice gall stunt virus. The amino acid sequence of OsAGO12 protein is shown in SEQ ID NO.2, and its encoding gene OsAGO12 The nucleotide sequence of is shown in SEQ ID NO.1. The present invention uses overexpression and CRISPR / Cas9 technology to OsAGO12 The gene was overexpressed or knocked out in rice. OsAGO12 The gene can significantly enhance the resistance of rice to rice gall dwarf virus. The invention provides a new gene resource for rice improvement and breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of OsAGO12 protein and expression of its encoding gene in regulating plant resistance to rice gall dwarf virus. Background Art

[0002] Rice viral diseases are catastrophic and lack effective control measures. Research on virus-host interactions provides fundamental knowledge for understanding the mechanisms of crop viral disease outbreaks. As one of the world's most important food crops, rice feeds nearly half of the world's population. Global rice yield losses due to pests and diseases range from 24.6% to 40.9%. Rice viral diseases, in particular, transmitted by insect vectors, are characterized by their intermittent, migratory, and explosive nature, threatening the safety of most grasses and some dicots. Currently, we lack effective prevention and control measures for the catastrophic outbreaks of rice viral diseases. Once an epidemic occurs, the losses caused by the diseases are irreversible. Furthermore, genetic resources for virus-resistant rice breeding are extremely limited. Therefore, identifying rice antiviral genes and understanding their mechanisms of action are of paramount importance.

[0003] Rice gall dwarf virus (RGDV) is a species of the genus Phytoreovirus in the family Reoviridae. In China, it is primarily transmitted by the insect vector, the electric leafhopper. Rice gall dwarf disease exhibits fluctuating growth and decline. The disease causes severe dwarfing in rice plants, with leaves becoming shorter, darker, and stiffer, and with the appearance of tumor-like projections of varying sizes on the leaf dorsal surfaces and sheaths. The RGDV genome consists of 12 double-stranded RNAs encoding six structural proteins (P1, P2, P3, P5, P6, and P8) and six nonstructural proteins (Pns4, Pns9, Pns10, Pns11, and Pns12). Our understanding of RGDV's pathogenicity and host interactions remains limited, hindering the development of genetic resources for breeding rice gall dwarf virus resistance. However, research based on model organisms such as Arabidopsis thaliana has revealed a variety of antiviral mechanisms in plants, which provides a theoretical basis and research direction for the discovery of crop antiviral genes.

[0004] RNA silencing is a conserved form of viral resistance in plants. During the long-standing interactions between viruses and plants, plants do not sit idly by, but instead have evolved a complex array of defense mechanisms to combat viral infection, including RNA silencing, R genes, recessive resistance, phytohormone signaling, autophagy, and WUS (WUSCHEL)-mediated antiviral immunity. RNA silencing, a ubiquitous gene expression regulation mechanism in eukaryotes, is crucial for defense against pathogens, particularly viruses. Key proteins involved in this mechanism include Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR), which play roles in the initiation, execution, and signal amplification stages of the RNA silencing process, respectively. Downstream of RNA silencing, small RNAs (sRNAs) bind to AGO proteins to form RNA-induced silencing complexes (RISCs), which, based on sequence complementarity, target target genes. RNA silencing can inhibit the transcription of homologous DNA through transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM). It can also achieve posttranscriptional gene silencing (PTGS) by cleaving and degrading or inhibiting translation of target RNA. Viral infection leads to the production of large amounts of viral-derived small RNAs (vsiRNAs) in the host, which can be loaded into the AGO protein to form RISC targeting the viral genome, effectively inhibiting viral replication and spread.

[0005] AGO proteins play an important role in many areas of life, especially in the immune response, where they are a key element in defending against viral invasion. In the antiviral defense mechanism of plants, AGO proteins demonstrate their core function in resisting viruses by participating in RNA silencing. As core members and main executors of the RNA silencing pathway, AGO proteins frequently become targets of attack by pathogens, especially viruses that require a host system to replicate and spread. Many plant viruses produce RNA silencing suppressors (VSRs), which use different mechanisms to interfere with the host's RNA silencing process. Among them, the RISC complex centered on AGO is often the main target of VSR attack. For example, some virus-encoded silencing suppressors bind to AGO proteins through their GW / WG (glycine-tryptophan) motifs and further prevent AGO proteins from exercising their functions. For example, the p38 protein encoded by Turnip crinkle virus (TCV) has been shown to inhibit the RNA silencing pathway through multiple pathways. p38 not only binds to dsRNA but also to AGO1 and AGO12 proteins, disrupting the DCL4 enzyme, thereby reducing siRNA production and inhibiting RNA silencing. The silencing suppressor P1 encoded by sweetpotato mild mottle virus (SPMMV) contains three GW / WG repeats and may inhibit RISC formation through a strong interaction with Arabidopsis AGO1. Other viral-encoded silencing suppressors degrade AGO proteins, thereby weakening AGO-mediated antiviral immunity. For example, the silencing suppressor p25 encoded by Potato virus X (PVX) interacts with Arabidopsis AGO1, 2, 3, and 4, leading to their degradation via the 26S proteasome. The coat protein encoded by Tomato ringspot virus (ToRSV) interacts with tomato AGO1 via a GW / WG motif, inhibiting its translational repression activity and promoting AGO1 degradation through autophagy. In addition, during the RISC assembly process, the P0 protein encoded by Beet western yellows polerovirus (BWYV) binds to the components of RISC to further prevent sRNA from being loaded into AGO1, and AGO1 that is not assembled into RISC is easily degraded. In response, plants have evolved a variety of AGO proteins, and the functions of some AGO proteins are redundant; the diversity and redundancy of AGO proteins play an important role in maintaining the function of the RNA silencing pathway and inhibiting viral invasion. In addition, plant AGO proteins have also evolved multiple pathways to cope with the interference of VSR. For example, rice AGO1 can bind to miR168 to target and cut its own mRNA, forming a negative feedback regulation model.When AGO1 function is disrupted by VSR, miR168's inhibition of AGO1 is released, thereby increasing AGO1 expression and enhancing its inhibitory effect on viruses. This ingenious negative feedback regulatory pathway stabilizes AGO1's regulation of plant growth and development and resistance to viral infection. Rice AGO18, in turn, regulates miR168's inhibition of AGO1 through a "molecular lock" mechanism, further increasing the complexity of this pathway to respond to environmental changes and interference from pathogenic microorganisms. In the rice RNA silencing system, the genome encodes eight DCL proteins, 19 AGO proteins, and five RDR proteins. Although these genes play a role in rice's viral defense, detailed information on their specific functions awaits further study.

[0006] Thanks to the rapid advancements in gene editing technology, scientists can directly locate and modify specific genes within crop genomes, thereby improving disease resistance. Compared to traditional breeding methods, gene editing is more efficient and can introduce and improve disease resistance traits in a shorter timeframe. Furthermore, gene editing avoids the introduction of foreign DNA, facilitating public acceptance and regulatory approval. Using gene editing, we can test and evaluate the effects of potential antiviral genes, thereby accelerating the breeding process for antiviral resistance. Summary of the Invention

[0007] The object of the present invention is to provide an application of OsAGO12 protein and the expression of its encoding gene in regulating plant resistance to rice gall stunt virus.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The amino acid sequence of OsAGO12 protein is shown in SEQ ID NO.2.

[0010] The nucleotide sequence of the gene encoding the above-mentioned OsAGO12 protein is shown in SEQ ID NO.1.

[0011] Use of the above-mentioned OsAGO12 protein or encoding gene in regulating plant resistance to rice gall dwarf virus;

[0012] Among them, overexpression of OsAGO12 protein or increasing the expression of the gene encoding OsAGO12 protein can improve plant resistance to rice gall stunt virus;

[0013] Among them, the loss of OsAGO12 protein function or downregulation of the expression of the gene encoding OsAGO12 protein can reduce the plant's resistance to rice gall stunt virus;

[0014] Wherein, the plant is rice.

[0015] A method for improving plant resistance to rice gall stunt virus, comprising the steps of overexpressing the OsAGO12 protein encoding gene in a plant to obtain a transgenic plant;

[0016] Wherein, the plant is rice.

[0017] Furthermore, the above-mentioned method for improving plant resistance to rice gall stunt virus specifically includes: constructing a recombinant plant expression vector containing the OsAGO12 protein encoding gene, transforming the constructed recombinant plant expression vector into a recipient plant, and cultivating and screening to obtain transgenic plants with improved resistance to rice gall stunt virus.

[0018] A method for reducing plant resistance to rice gall stunt virus, comprising the steps of causing the OsAGO12 protein encoding gene to be functionally deleted or down-regulated to obtain a transgenic plant;

[0019] Wherein, the plant is rice.

[0020] Furthermore, the above-mentioned method for reducing plant resistance to rice gall stunt virus specifically includes: designing a CRISPR / Cas9-based sgRNA sequence with the above-mentioned OsAGO12 protein-encoding gene as the target, connecting a DNA fragment containing the sgRNA sequence to a CRISPR / Cas vector, transforming a recipient plant, and thereby obtaining a transgenic plant with a functional deficiency of the OsAGO12 protein-encoding gene; the transgenic plant has reduced resistance to rice gall stunt virus compared to the recipient plant.

[0021] A method for cultivating transgenic plants, comprising: introducing the above-mentioned OsAGO12 protein encoding gene into a recipient plant to obtain a transgenic plant; the transgenic plant has improved resistance to rice gall stunt virus compared to the recipient plant;

[0022] Wherein, the plant is rice.

[0023] The significant advantages of the present invention are:

[0024] This study used overexpression and CRISPR / Cas9 technology to overexpress or knockout the OsAGO12 gene in rice. The results showed that overexpressing the OsAGO12 gene significantly enhanced resistance to rice gall dwarf virus. This study provides a new gene resource for rice breeding and improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Western identification of OsAGO12 overexpressing rice.

[0026] Figure 2 Identification of OsAGO12 loss-of-function mutants in rice.

[0027] Figure 3 qRT-PCR detection of RGDV virus-related genes in OsAGO12-overexpressing rice, OsAGO12 loss-of-function mutant rice and wild-type rice (Zhonghua11) after RGDV infection.

[0028] Figure 4 Symptoms of different rice strains infected with RGDV. DETAILED DESCRIPTION

[0029] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The amino acid sequence of the OsAGO12 protein described in the following examples is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding the OsAGO12 is shown in SEQ ID NO. 1.

[0033] Example 1: Obtaining OsAGO12 protein and its encoding gene

[0034] Primers were designed based on the sequence shown in SEQ ID NO.2. The primer sequences are as follows:

[0035] AGO12-F: 5'-ATGTCTTCGCGCGGCGG-3',

[0036] AGO12-R: 5'-TCAGCAGTAGAACATGAACCGCT-3'.

[0037] Total RNA from Oryza sativa L. japonica cv. Zhonghua 11 (Xu Yu et al., "Cloning of the 'Zhonghua 11' Rice Glutenin Gt1 Gene and Construction of a Waxy Gene Promoter-Directed Gt1 Gene Expression Vector," Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204) was extracted using Invitrogen's TRIzol Reagent according to the manufacturer's instructions. Reverse transcription was performed using the company's SuperScript II reverse transcriptase to generate cDNA. The primer used for reverse transcription was a 16-nucleotide Oligod(T) primer.

[0038] The cDNA obtained by reverse transcription was used as a template and the primers AGO12-F and AGO12-R were used to perform a PCR (Polymerase Chain Reaction) reaction to obtain a 3177 bp PCR product having the nucleotide sequence shown in SEQ ID NO.1.

[0039] The PCR product was recovered and ligated with the pEASY-Blunt Zero Cloning Kit (Quanshijin, catalog number CB501-01) and transformed into E. coli DH5α to obtain transformants. The plasmids from the transformants were extracted and sent for sequencing. The plasmid that was sequenced correctly was named pEASY-Blunt-AGO12.

[0040] Example 2: Obtaining OsAGO12-overexpressing rice

[0041] I. Construction of the Overexpression Vector: Using the pEASY-Blunt-AGO12 plasmid as a template, PCR amplification was performed with primers AGO12-5'SalI and AGO12-3'PstI to generate a 3177-bp PCR product containing the target gene fragment. This PCR product was purified and recovered, and then ligated with the pCambia2300-Actin-Flag plasmid backbone digested with SalI and PstI via seamless cloning (see Novizan, catalog number C112-01) to generate the recombinant plasmid pCambia2300-Actin-FLAGAGO12. Sequencing confirmed that the recombinant plasmid pCambia2300-Actin-FLAGAGO12 was constructed by inserting the OsAGO12 gene between the SalI and PstI restriction sites of the pCambia2300-Actin-Flag plasmid, thus forming the overexpression vector.

[0042] AGO12-5'SalI:5'-aggggatcctctagagtcgacATGTCTTCGCGCGGCGG-3',

[0043] AGO12-3'PstI:5'-taaagcagggcatgcctgcagTCAGCAGTAGAACATGAACCGCT-3'.

[0044] 2. Obtaining transgenic rice overexpressing OsAGO12

[0045] 1) Callus induction culture

[0046] The seeds of Zhonghua 11 rice (hereinafter also referred to as wild-type rice) are shelled, first soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes; the surface is sterilized. The solution on the surface of the seeds is washed off with plenty of sterile water, and the moisture on the surface of the seeds is absorbed with sterile filter paper. The seeds are placed on a plate of mature embryo callus induction medium, the edge of the plate is sealed with Parafilm film, and cultured in a 26°C incubator in the dark. After about 15 days, the grown callus tissue is carefully removed and transferred to the mature embryo subculture medium, and culture is continued under the same conditions. Subculture is required every two weeks. When used for transformation, it is necessary to select granular callus tissue that has been subcultured for about 5 days and is light yellow.

[0047] 2) Cultivation of Agrobacterium

[0048] The overexpression vector pCambia2300-Actin-FLAGAGO12 was electroporated into Agrobacterium EHA105 to obtain the recombinant bacteria EHA105 / pCambia2300-Actin-FLAGAGO12.

[0049] Streak the recombinant strain EHA105 / pCambia2300-Actin-FLAGAGO12 on an LB plate containing antibiotics (50 mg / L Kanamycin, 50 mg / L Rifampicin) and culture at 28°C for 2 days. Pick a single colony and inoculate it into liquid LB medium and culture it at 28°C with shaking until the OD 600 The concentration of acetosyringone was about 0.5, and acetosyringone was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus tissue.

[0050] 3) Co-culture of rice callus and Agrobacterium

[0051] Place the subcultured callus into a sterile Erlenmeyer flask and pour the Agrobacterium suspension into the flask until it is submerged. Incubate at room temperature for 20 minutes, gently shaking the flask occasionally to ensure full contact between the callus and the bacterial suspension. Gently remove the callus with sterile tweezers, place it on sterile filter paper to absorb excess bacterial suspension, and transfer it to a co-cultivation medium plate lined with sterile filter paper. Incubate in the dark at 28°C for 3 days to obtain the co-cultivated callus.

[0052] 4) Screening and differentiation of resistant callus

[0053] The co-cultivated callus was washed with an appropriate amount of sterile water to remove residual Agrobacterium on the surface, placed on a screening medium, cultured in the dark at 26°C for screening, and transferred to a new screening medium after two weeks to continue screening for two weeks. Select callus in good condition after two rounds of screening, transfer it to a differentiation medium flat plate, culture in the dark for 3 days, and then transfer it to a light incubator (15hr / day) for light culture. The seedlings that were differentiated were visible after one month. When the differentiated seedlings grew to about 2cm, they were transferred to a rooting medium in a conical flask and continued to be cultured for about two weeks. Select seedlings with good growth and well-developed root systems, transplant them into the soil after washing the culture medium from the roots with tap water, collect seeds, and obtain T1 generation transgenic rice seeds. Sowing gave T1 generation transgenic rice.

[0054] T1 rice seeds were initially screened with G418 (the pCambia2300 vector carries the G418 resistance selection gene). Germinated seeds indicated the vector had been transferred into the rice plants. The germinated seeds were planted in soil and allowed to grow for two weeks. 0.1 g of leaves were then collected and ground into a powder using liquid nitrogen.

[0055] Add 200 μl of protein extraction buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) to the leaf powder of the transgenic rice line overexpressing OsAGO12, incubate on ice for 10 min, boil at 100°C for 10 min, centrifuge at 4°C and 12,000 rpm for 10 min, take the supernatant, perform SDS-PAGE, transfer to the membrane, and detect by Western blotting. SDS-PAGE and Western Blot were performed according to the known methods and product instructions. The antibody used was anti-FLAG-HRP (sigma), and the antibody anti-Actin was used to detect the endogenous Actin protein of rice as an internal reference. Figure 1 The ones with a band at 120 kDa were positive, indicating that the target gene was transferred and the protein was expressed. Two overexpression lines #4 and #19 were selected for subsequent disease resistance analysis experiments (Zhonghua 11 in the figure is wild-type rice, used as a negative control).

[0056] Example 3: Obtaining OsAGO12 loss-of-function mutant rice

[0057] 1. Construction of expression vector

[0058] 1) According to the website http: / / skl.scau.edu.cn / and the PAM site at the end of the sequence is NGG, a 20 bp specific target sequence targeting the N-terminus of the OsAGO12 coding region was selected as follows: 5'-GCGGCGCGTCGACCCGTAGG-3';

[0059] 2) Design primers according to the target DNA sequence and add BsaI restriction sites at both ends of the primers. The primer sequence is: UF: 5'-CTCCGTTTTACCTGTGGAATCG-3',

[0060] AGO12-gRT1: 5'-GCGGCGCGTCGACCCGTAGGgttttagagctagaaat-3',

[0061] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3',

[0062] AGO12-OsU3T1: 5'-CCTACGGGTCGACGCGCCGCgccacggatcatctgc-3';

[0063] B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3',

[0064] BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3';

[0065] 3) Referring to the CRISPR / Cas9 gene editing vector construction protocol in the reference (A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant. 2015), three rounds of PCR amplification were performed using the intermediate vector as a template with three primer pairs: UF / AGO12-OsU3T1, AGO12-gRT1 / gRNA-R, and B1' / BL, respectively, to obtain an expression cassette with an adapter. The intermediate vector contained a sgRNA (5'-GCGGCGCGTCGACCCGTAGG-3') that specifically targeted the OsAGO12 exon, which guides Cas9 to cleave the target gene OsAGO12, causing mutations and loss of function. After amplification, the final product was purified and recovered by gel excision. The final vector pYLCRISPR / Cas9, restriction enzymes, and T4 ligase were added, and cleavage and ligation were performed according to a specific system.

[0066] 4) Transform Escherichia coli strain DH5α and coat with kanamycin-resistant medium to obtain transformants. The plasmids of the transformants were extracted and sent for sequencing. The positive transformants were the final recombinant vector, named pYLCRISPR / Cas9-AGO12:sgRNA.

[0067] 2. Obtaining OsAGO12 CRISPR / Cas9 Rice

[0068] 1) Callus culture and transformation

[0069] Basically the same as Example 2, except that the transfer vector is pYLCRISPR / Cas9-AGO12:sgRNA.

[0070] 2) Identification of OsAGO12 loss-of-function mutants in rice

[0071] Leaf powder from OsAGO12 loss-of-function mutant rice lines was used to extract genomic DNA using the High-Efficiency Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No. DP350). PCR was then performed using 0.5 g of genomic DNA as a template and primers AGO12-CF and AGO12-CR. The PCR products were directly sequenced and then aligned. Figure 2 As shown, DNA sequencing results showed that two positive loss-of-function mutant strains were named ago12#1 and ago12#2, among which ago12#1 inserted an "A" between bases 332 and 333 in the OsAGO12 coding region, and ago12#2 deleted 5bp between bases 327 and 333 in the OsAGO12 coding region, both of which caused the premature termination of the OsAGO12 amino acid sequence, resulting in the loss of OsAGO12 protein.

[0072] AGO12-CF: 5'-TCGTCGGCGAGCGGCAAG-3',

[0073] AGO12-CR: 5'-TTGTCCCGCGACTTGCACCAG-3'.

[0074] Example 4: Overexpression of OsAGO12 can improve rice resistance to RGDV, while functional loss of OsAGO12 can reduce rice resistance to RGDV

[0075] 1) RGDV infection was identified by quantitative RT-PCR (qRT-PCR) using the expression level of RGDVPns11. Leaves of T1 generation OsAGO12-overexpressing rice, OsAGO12 loss-of-function mutant rice, and wild-type rice Zhonghua 11 were inoculated with RGDV-carrying electric leafhoppers (the pathogen is Rice gall dwarf virus). Thirty plants of each type of rice were inoculated and cultured at 30°C during the day, 22°C at night, and 60% humidity. Three electric leafhoppers were inoculated on each plant. After three days of feeding, the leafhoppers were caught and the fed rice plants were cultured in a sunny greenhouse (natural light, temperature. Two transgenic rice lines were used in the experiment to verify each other).

[0076] Four weeks after infection, infected rice leaf powder was extracted and Trizol (Invitrogen) was added to each sample. RNA was extracted according to the manufacturer's instructions. Genomic DNA was then digested with RQ1 DNase (Promega, Catalog No. M610A) according to Table 1 below. Two micrograms of digested RNA was then used for reverse transcription qRT-PCR. For specific methods, see Invitrogen M-MLV Reverse Transcriptase (Catalog No. 28025-021). OsEF1α was used as an internal reference with primers EF1α-F and EF1α-R. The expression of RGDV Pns11 was detected using primers Pns11-F and Pns11-R.

[0077] Table 1 Digestion system

[0078]

[0079] EF1α-F: 5'-GCACGCTCTTTCTTGCTTTCACTCT-3'

[0080] EF1α-R: 5'-AAAGGTCACCACCATACCAGGCTT-3',

[0081] Pns11-F: 5'-GGTGGTATTGATTGACGCATGT-3',

[0082] Pns11-R: 5'-TCGGTCTCATCGTTCCCATCTT-3'.

[0083] The results are as follows Figure 3As shown in the figure, the accumulation of RGDV Pns11 in the T1 generation of OsAGO12-overexpressing rice (AGO12 OE) was lower than that in the wild-type rice Zhonghua11, while the accumulation of RGDV Pns11 in the OsAGO12 loss-of-function mutant was higher than that in the wild-type rice Zhonghua11 (asterisks indicate significant differences). Therefore, overexpression of OsAGO12 can enhance rice resistance to RGDV, while loss of OsAGO12 function can increase rice susceptibility to RGDV.

[0084] 2) Determine the incidence of RGDV after inoculation by phenotype

[0085] Four weeks after infection, symptoms were observed in 30 T1 generation OsAGO12 overexpressing rice plants (AGO12OE#4 and AGO12OE#19), 30 OsAGO12 loss-of-function mutant rice plants (ago12#1 and ago12#2), and 30 wild-type rice plants Zhonghua 11 (RGDV-infected plants exhibited dwarfism, increased tillering, curled leaf tips, and notched leaf margins). The number of symptomatic plants was counted, and the infection rate was calculated as (number of phenotypical plants / total number of plants) × %. The results are summarized in Table 2:

[0086] Table 2 shows the statistical results of virus infection rate of transgenic rice after virus infection

[0087]

[0088] As can be seen from Table 2, OsAGO12 overexpression rice has a lower susceptibility rate, while OsAGO12 loss-of-function mutant rice has a relatively high susceptibility rate. In addition, we took photos of different strains of susceptible rice, such as Figure 4 Figure 2 shows disease symptoms in rice plants overexpressing OsAGO12 and in OsAGO12 loss-of-function mutants four weeks after infection. As can be seen, OsAGO12-overexpressing rice exhibits milder disease symptoms, with less dwarfing compared to wild-type rice; whereas OsAGO12 loss-of-function mutant rice exhibits more severe disease symptoms, with a greater degree of dwarfing (AGO12OE represents OsAGO12-overexpressing rice, and ago12 represents OsAGO12 loss-of-function mutant rice). Compared to wild-type rice, OsAGO12-overexpressing rice is more resistant to disease, while OsAGO12 loss-of-function mutant rice is more susceptible to disease.

[0089] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Use of an OsAGO12 protein or a gene encoding an OsAGO12 protein in regulating plant resistance to rice gall dwarf virus, characterized in that: The amino acid sequence of the OsAGO12 protein is shown in SEQ ID NO.2, the nucleotide sequence of the gene encoding the OsAGO12 protein is shown in SEQ ID NO.1, and the plant is rice.

2. The use according to claim 1, characterized in that: By overexpressing OsAGO12 protein or increasing the expression level of the gene encoding OsAGO12 protein, the resistance of plants to rice gall stunt virus can be improved.

3. The use according to claim 2, characterized in that: By making the OsAGO12 protein function ineffective or down-regulating the expression of the OsAGO12 protein encoding gene, the plant's resistance to rice gall stunt virus is reduced.

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