Application of OsVAL2 gene in improving broad-spectrum disease resistance of rice

Abstract

The application belongs to the field of molecular biology, and particularly relates to application of OsVAL2 gene in improving broad-spectrum disease resistance of rice. The application provides application of the OsVAL2 gene, and the application includes application in preventing and treating plant diseases and / or enhancing resistance of plants to pathogenic bacteria. An amino acid sequence coded by the OsVAL2 gene is shown as SEQ ID NO. 1, and a CDS sequence of the OsVAL2 gene is shown as SEQ ID NO. 2. The application proves that the OsVAL2 is involved in regulating an immune response pathway of rice to a fungal disease rice blast, a fungal disease sheath blight and a bacterial disease bacterial leaf blight. The application provides important gene resources and a molecular theoretical basis for developing new rice varieties with broad-spectrum disease resistance.

Description

Technical Field

[0001] This application belongs to the field of molecular biology, specifically relating to the application of the OsVAL2 gene in improving broad-spectrum disease resistance in rice. Background Technology

[0002] Rice (Oryza sativa L.) is a major global food crop, and the stability and sustainability of its yield are directly related to food security. During its growth and development, rice is frequently infected by various pathogens, including Magnaphalthe oryzae, Xanthomonas oryzae pv. oryzae, and Rhizoctonia solani. These pathogens cause diseases that disrupt plant physiological metabolism, reduce photosynthesis, and result in significant yield losses. Currently, there is a lack of broad-spectrum resistance rice varieties to control these multiple pathogen infections. Existing resistance genes are mostly pathogen-specific, making it difficult to address the complex situation of multiple diseases occurring simultaneously in the field.

[0003] From the perspective of plant immune mechanisms, rice resistance to pathogens mainly relies on two layers of the immune system: pathogen-associated molecular pattern-triggered (PTI) immune response and effector-triggered (ETI) immune response. These two layers of the immune system are interconnected through a series of signal transduction elements, jointly regulating the disease resistance response. Known immune signaling events include calcium ion flow, reactive oxygen species bursts, activation of the mitogen-activated protein kinase (MAPK) cascade, and transcription factor-mediated reprogramming of defense gene expression. In addition, the cross-talk of plant hormone signaling pathways such as salicylic acid and jasmonic acid plays a key role in the establishment and maintenance of systemically acquired resistance. However, the key genetic loci that simultaneously regulate resistance to multiple pathogens in rice still lack systematic identification, the synergistic mechanism of their encoded proteins in integrating the PTI and ETI signaling networks remains unclear, and the interaction network between downstream immune signaling pathways and hormone pathways needs further elucidation.

[0004] The B3 transcription factor family in plants is a large and diverse family of gene regulators. Its members share a signature B3 DNA-binding domain, enabling them to specifically recognize and bind to G / A enriched sequences in cis-elements. This family plays a crucial "molecular switch" role in several key processes of the plant life cycle. Based on their structural characteristics and functional specificity, the B3 family can be mainly divided into several subfamilies, the most well-known of which are ARF (auxin-responsive factor), ABI3 / VP1 (abscisic acid-insensitive 3 / viviparous 1), and LAV (including RAV1 / RAV2 / VAL). The ARF subfamily is a core regulator of auxin signal transduction in plants. By binding to auxin-responsive elements in the promoter regions of target genes, it directly activates or inhibits the expression of downstream genes, thereby precisely regulating cell elongation, division, and organogenesis, such as lateral root formation and leaf development. The ABI3 / VP1 subfamily plays a central role in seed development and maturation, integrating multiple hormonal signals such as abscisic acid and gibberellin to regulate the accumulation of seed storage proteins, the acquisition of dehydration tolerance, and the induction of dormancy, ensuring seed germination under suitable conditions. Members of the LAV subfamily frequently participate in responses to biotic and abiotic stresses, as well as regulating flowering time. Furthermore, the B3 family is widely involved in photomorphogenesis, vernalization pathways, and the establishment of embryonic development patterns; however, there are no reports of VAL2 subfamily transcription factors from the B3 family participating in resistance to multiple diseases, i.e., VAL2 transcription factor-mediated broad-spectrum disease resistance.

[0005] In the process of co-evolution between plants and pathogens, different pathogens have evolved highly specific pathogenic strategies to evade or suppress the basic immune system in order to successfully colonize rice. Therefore, it is crucial to discover and utilize genes with broad-spectrum disease resistance. Identifying such genes and analyzing their regulatory networks is of core guiding significance for breeding new rice varieties with durable and broad-spectrum disease resistance to cope with the complex and ever-changing pathogen populations in the field. Summary of the Invention

[0006] To address the aforementioned problems, this application constructed OsVAL2 knockout mutants and overexpression transgenic lines in susceptible rice. Resistance phenotypic analysis through inoculation with multiple pathogens, including *Oryza sativa*, *Rhizoctonia solani*, and *Bacterium oxysporum*, revealed that OsVAL2 significantly affects rice resistance to these three major diseases. This application provides important genetic resources and a molecular theoretical basis for developing broad-spectrum disease-resistant rice varieties.

[0007] On the one hand, this application provides the application of the OsVAL2 gene in the prevention and control of plant diseases and / or the enhancement of plant resistance to pathogens, wherein the amino acid sequence encoded by the OsVAL2 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the CDS sequence of the OsVAL2 gene is shown in SEQ ID NO.2.

[0009] In this application, the rice OsVAL2 gene has NCBI accession number LOC4344293, a full-length transcribed sequence of 6599 bp, of which the CDS coding region is 2850 bp, and the CDS sequence is shown in SEQ ID NO.2. This gene encodes 949 amino acids, and the OsVAL2 protein sequence has NCBI number XP_015646778.1, and its amino acid sequence is shown in SEQ ID NO.1.

[0010] Furthermore, the application includes increasing the expression level of the OsVAL2 gene, thereby improving the plant's ability to control diseases and / or enhancing the plant's resistance to pathogens.

[0011] Furthermore, the plant pathogens include rice blast fungus, Xanthomonas auricula-judae, and Rhizoctonia solani.

[0012] Furthermore, the plant diseases shown include rice blast, bacterial blight, and sheath blight.

[0013] Furthermore, the plant in question is rice.

[0014] In this application, rice blast resistance specifically refers to rice's ability to defend against infection by the pathogenic fungus *Magnaporthe oryzae*. This disease is infectious throughout the entire growth cycle, damaging organs and tissues such as leaves, stems, and panicles. Typical symptoms include: spindle-shaped lesions with a grayish-white center and brown edges on leaves; panicle infection leads to impaired grain development, and in severe cases, total crop failure. This disease typically causes 10%-30% yield loss, but in extreme cases, it can reach 40%-50%. Some rice varieties carry susceptible genes such as ROD1, which can suppress the immune response; while resistant varieties are less susceptible to infection due to the lack of corresponding susceptible genes.

[0015] In this application, resistance to rice sheath blight refers to the rice's ability to resist the pathogenicity of Rhizoctonia solani. This disease begins to appear during the tillering stage of rice, with typical symptoms including: initially, small, dark green, water-soaked spots appear on the leaf sheaths near the water surface, which expand into elliptical, mottled spots as the disease progresses, characterized by brown edges and a light brown to grayish-white center. Severe infections result in large, irregular lesions that can extend to the leaves and stems, leading to premature leaf senescence, lodging, and ultimately yield losses such as reduced seed setting rate and decreased grain plumpness.

[0016] In this application, bacterial blight resistance specifically refers to the defense mechanism of rice against the pathogenicity of Xanthomonas oryzae pv. oryzae (Xoo). Bacterial blight of rice caused by this Gram-negative bacterium is a highly destructive bacterial disease. Its resistance genetic characteristics are controlled by a single gene or a few major genes; for example, the rice Xa23 gene is a typical major gene for bacterial blight resistance. Bacterial blight can occur at any stage of rice growth, mainly on the leaves and leaf sheaths. Initially, small, translucent yellow spots appear on the edges of the rice leaves. Later, these spots expand along one or both sides of the leaf margin, or along the midrib, becoming wavy yellow-green or gray-green lesions. The boundary between the diseased and healthy parts becomes increasingly clear. After several days, the lesions turn grayish-white and curl inwards, spreading rapidly through the air via wind, rain, insects, or other means, eventually infecting the entire rice field. From a distance, it appears as a withered, sallow landscape, hence the name bacterial blight.

[0017] On the other hand, this application also provides a method for improving the ability of plants to control diseases and / or enhancing the resistance of plants to pathogens, the method comprising increasing the expression level of the OsVAL2 gene in the plant, thereby improving the ability of plants to control diseases and / or enhancing the resistance of plants to pathogens.

[0018] Furthermore, the plant pathogens include rice blast fungus, Xanthomonas auricula-judae, and Rhizoctonia solani.

[0019] Furthermore, the plant diseases mentioned include rice blast, bacterial blight, and sheath blight.

[0020] Furthermore, the plant in question is a grass, preferably rice.

[0021] Compared to existing technologies, this application constructed OsVAL2 knockout mutants and overexpression transgenic lines in susceptible rice. Resistance phenotypic analysis was conducted by inoculating the rice with multiple pathogens, including *Oryza sativa*, *Sheath blight*, and *Bacterium oxysporum*. The results showed that OsVAL2 significantly affects rice resistance to these three major diseases. Specifically, the length of rice blast lesions on leaves of the overexpression line Ubi::OsVAL2-FLAG#1-#2 was significantly reduced compared to the wild-type TP309; the length of sheath blight lesions on leaf sheaths of the overexpression line Ubi::OsVAL2-FLAG#1-#2 was significantly reduced compared to the wild-type TP309; and the length of bacterial blight lesions on leaves of the overexpression line Ubi::OsVAL2-FLAG#1-#2 was significantly reduced compared to the wild-type TP309. Therefore, this demonstrates that OsVAL2 positively regulates rice resistance to *Oryza sativa*, *Sheath blight*, and *Bacterium oxysporum*. This provides a solid theoretical basis for constructing broad-spectrum disease resistance in rice at the molecular level and applying it to design breeding, while also having significant scientific implications for deepening the understanding of the immune regulatory network of monocotyledonous plants. Attached Figure Description

[0022] Figure 1 The images show the structural diagrams of the recombinant plasmids that overexpress the OsVAL2 gene; where A is the diagram of plasmid pUN1301-OsVAL2-Flag and B is the diagram of plasmid pUN1301-OsVAL2-GFP.

[0023] Figure 2 Genotyping results of OsVAL2 knockout materials and protein level identification results of OsVAL2 overexpression materials.

[0024] Figure 3 Fluorescence images showing OsVAL2 localization in the nucleus, cytoplasm, and cell membrane of rice cells; A shows transiently transformed tobacco cells, revealing that OsVAL2 has localization in the nucleus, cytoplasm, and cell membrane, with the reported nuclear marker gene OsNLS connected to the red fluorescent tag Mcherry as an indicator, scale bar 50 μm; B shows root cells of the stable transgenic material pUN1301-OsVAL2-GFP, revealing that OsVAL2 has localization in the nucleus, cytoplasm, and cell membrane, scale bar 5 μm.

[0025] Figure 4 This figure shows the results of OsVAL2 positive regulation of rice resistance to rice blast fungus. In Figure A, OsVAL2 positively regulates rice resistance to rice blast fungus. This was achieved by inoculating OsVAL2 knockout lines, overexpressing lines, and wild-type TP309 with rice blast fungus in vitro, and statistically analyzing disease incidence 7-10 days after inoculation. Figure B shows the disease incidence as reflected by measuring the length of lesion expansion. All measured data are presented using scatter plots. Data analysis employed a two-tailed t-test, with asterisks indicating significant differences (**P<0.01, *P<0.05).

[0026] Figure 5 This figure shows the results of OsVAL2 positive regulation of rice resistance to sheath blight. In Figure A, OsVAL2 positively regulates rice resistance to sheath blight. Sheath blight was inoculated into OsVAL2 knockout lines, OsVAL2 overexpression lines, and wild-type TP309. Disease incidence was assessed seven days after inoculation. Figure B shows the disease incidence as reflected by measuring the length of lesion expansion. All measured data are represented and presented using scatter plots. Data analysis employed a two-tailed t-test, with asterisks indicating significant differences (**P<0.01, *P<0.05).

[0027] Figure 6Figure A shows the results of OsVAL2 positive regulation of rice resistance to bacterial blight. A represents the positive regulation of rice resistance to bacterial blight by OsVAL2. This was achieved by inoculating OsVAL2 knockout lines, overexpression lines, and wild-type TP309 with bacterial blight race PXO99A. Fourteen days later, disease incidence was statistically analyzed, and representative photos of diseased leaves with lengths near the average were selected. Figure B shows the statistical analysis of bacterial blight incidence. Disease incidence was characterized by measuring the length of lesions extending downwards from the incision site. All measurements were represented by points. Data analysis used a two-tailed t-test, with asterisks indicating significant differences (**P<0.01, *P<0.05).

[0028] Figure 7 Agronomic traits such as plant height and plant type of OsVAL2 rice. Morphological images of wild-type TP309, OsVAL2 knockout plants, and OsVAL2 overexpressing plants growing in the field. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: unless otherwise specified in the following embodiments, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0030] Experimental materials The wild type of the Japonica rice variety TP309 (TP309) is identified by the following pathogens: rice blast fungus (M. oryzae): TH12; Rhizoctonia solani Kühn: highly pathogenic strain RH-9; and Xanthomonas oryzae pv. Oryzae, Xoo: PXO99A.

[0031] Example 1: Construction of OsVAL2 gene knockout / overexpression plasmid 1. OsVAL2 gene and protein structure The rice OsVAL2 gene has NCBI accession number LOC4344293, with a full-length transcribed sequence of 6599 bp, including a 2850 bp CDS coding region, as shown in SEQ ID NO.2. This gene encodes 949 amino acids. The OsVAL2 protein sequence has NCBI number XP_015646778.1, and its amino acid sequence is shown in SEQ ID NO.1.

[0032] 2. Construction of rice OsVAL2 gene knockout plasmid Knockout target sites for OsVAL2 were designed based on the genomic DNA sequence of OsVAL2, and the conserved region agtgcatgaacgccgcgtg (SEQ ID NO.3) was selected as the target site. The double-stranded DNA sequence was chemically synthesized using primers OsVAL2-KO-1T-F: 5'-GGCAggtgcatgaacgccgcgtg-3' (SEQ ID NO.4) and OsVAL2-KO-1T-R: 5'-AAACcacgcggcgttcatgcacc-3' (SEQ ID NO.5). The sequence was then assembled into the U3 promoter by enzyme digestion, ligation, and PCR. Subsequently, it was assembled into the binary vector pYLCRISPR / Cas9 using the Golden Gate and Gibson Assembly cloning methods to obtain the rice OsVAL2 gene knockout recombinant plasmid pYLCRISPR / Cas9-OsVAL2, which was used to transform TP309 plants to obtain OsVAL2 knockout material.

[0033] 3. Construction of rice OsVAL2 gene expression vector 3.1 Amplification of the target sequence Using rice TP309 cDNA as a template, the target fragment OsVAL2 was amplified using high-fidelity DNA polymerase KOD FX (TOYOBO, Cat#KFX-101). The PCR system is shown in Table 1.

[0034] Table 1. PCR system for amplifying the target fragment OsVAL2

[0035] PCR reaction program: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, 68 ℃ extension at 1 kb / min, approximately 35 cycles; 68 ℃ extension for 10 min; 16 ℃ for 1 min. Primers used for the PCR reaction are shown in Table 2. The PCR products were purified by agarose gel electrophoresis and used for subsequent expression vector preparation.

[0036] Table 2 Primer sequences used for amplifying the target fragment

[0037] 3.2 Enzyme digestion and ligation The enzyme digestion systems of pUN1301-GFP and pUN1301-Flag plasmids are shown in Table 3.

[0038] Table 3 Plasmid digestion system

[0039] After gently mixing and briefly centrifuging, incubate in a 37°C water bath for 30-60 minutes.

[0040] Next, the enzyme digestion products were purified by 1% agarose gel electrophoresis, recovered, and their concentration was determined. Recombinant reactions were then performed (20 μL) according to the instructions of the ClonExpress® II One Step Cloning Kit (Vazyme, Cat#C112-02). The system is shown in Table 4, and the PCR instrument was kept at 37℃ for 30 min.

[0041] Table 4 Plasmid recombination reaction system

[0042] The map of the recombinant plasmid pUN1301-OsVAL2-Flag is as follows: Figure 1 As shown in Figure A, the spectrum of the recombinant plasmid pUN1301-OsVAL2-GFP is as follows. Figure 1 As shown in B.

[0043] Example 2: Obtaining Gene-Edited / Overexpression Rice Lines 1. Induction of callus tissue in mature rice embryos 1) Use a threshing machine to remove the husks from the rice seeds, and manually screen and discard any moldy or deformed seeds.

[0044] 2) Add a small amount of 75% ethanol, shake by hand for about 30 seconds, and rinse once with sterile water.

[0045] 3) Add 25-30% (v / v) antifomin solution and shake on a shaker at 200 rpm for 30 min.

[0046] 4) Rinse with sterile water 5-6 times, 5-10 minutes each time.

[0047] 5) Place the seeds on sterile filter paper to absorb the moisture on the seed surface, and use sterile tweezers to sow the seeds on NBD medium to induce callus.

[0048] NBD rice screening medium (1L): NB Basal Medium (PhytoTech) 4.1 g, sucrose 30 g, glutamine 0.5 g, proline 0.5 g, hydrolyzed casein 0.5 g, 1 mL 2,4-D solution (1 mg / mL), pH 5.8, and solids require the addition of 4.5 g / L plant gel.

[0049] 6) After culturing in the dark for about 14 days, remove the endosperm, plumule, and radicle. The resulting callus can be used for transgenic or subculture. Subculture is performed every two weeks, and the number of subcultures depends on the state of the callus.

[0050] 2. Preparation of Agrobacterium-mediated transformation broth

[0051] 1) The constructed plasmids (recombinant plasmid pYLCRISPR / Cas9-OsVAL2, recombinant plasmid pUN1301-OsVAL2-Flag, and recombinant plasmid pUN1301-OsVAL2-GFP) were chemically transformed into EHA105 competent cells and cultured at 28°C for two days.

[0052] 2) Select a single clone and place it in 5 mL of LB liquid medium containing the corresponding antibiotic for culture. Incubate at 28°C with shaking for 48 h.

[0053] 3) Take 1 mL of the overnight culture and transfer it to 15 mL of AB (20 mg / L Rif + 50 mg / L Kan + 100 mg / L AS) liquid medium. Incubate at 28°C until OD600 = 0.5 (about 4 h). AB liquid medium (1 L): KH2PO4 3 g, NaH2PO4 1 g, NH4Cl 1 g, MgSO4·7H2O 300 mg, KCl 150 mg, CaCl2 10 mg, FeSO4·7H2O 2.5 mg, glucose 5 g.

[0054] 3. Co-culture of rice callus with Agrobacterium tumefaciens culture

[0055] 1) Centrifuge the bacterial culture at 4000 rpm for 10 min and discard the supernatant.

[0056] 2) Resuspend the bacterial cells in AAM containing 100 mg / L AS until the bacterial OD600 reaches 0.4-0.6.

[0057] 3) Co-culture the bacterial solution with rice callus for 20 min, shaking occasionally.

[0058] 4. Filtering Blot the callus tissue dry with sterile filter paper and transfer it to a selection medium containing hygromycin and carbenicillin to screen for resistant callus tissue. Change the medium every two weeks.

[0059] Screening media: S1: 100 mg / L carbenicillin + 30 mg / L hygromycin, S2: 100 mg / L carbenicillin + 40 mg / L hygromycin, S3: 100 mg / L carbenicillin + 50 mg / L hygromycin.

[0060] 5. Differentiation Selected rice callus tissues were transferred to rice differentiation medium and cultured under light. The medium was changed every two weeks until the callus differentiated into seedlings.

[0061] MS rice differentiation medium (1L): M&S BASAL MEDIUM w / VITAMINS (PhytoTech) 4.43 g, sucrose 30 g, 6-BA 3 mg / L, NAA 0.5 mg / L, pH 6.3, solids require the addition of 4.5 g / L plant gel.

[0062] 6. Rooting Transfer the seedlings from the differentiation medium to the rooting medium. After about 2 weeks of growth, remove the seedlings, wash off the agar medium, and culture them in water for 7 days before transplanting them into the soil.

[0063] 1 / 2MS rice rooting medium (1L): M&S BASAL MEDIUM w / VITAMINS (PhytoTech) 2.165 g, sucrose 20 g, pH 6.3, solids require the addition of 4.5 g / L plant gel.

[0064] 1. PCR identification Genomic DNA was extracted from the leaves of the regenerated plants, and PCR amplification was performed on the regenerated plants using the primers shown in Table 5. The amplified products were then sequenced.

[0065] Table 5 Primers for identifying regenerated rice plants

[0066] Based on the above identification, two mutant plants were screened from the regenerated plants and named CR-osval2#1 and CR-osval2#2, respectively. Sequencing analysis showed that, compared with the wild-type TP309 genomic DNA, [the following characteristics were observed]. Figure 2 As shown in Figure A, plant CR-OsVAL2#1 has a 46bp deletion in the gene encoding the OsVAL2 protein, while plant CR-OsVAL2#2 has a two-base deletion in the gene encoding the OsVAL2 protein. Simultaneously, overexpression materials of OsVAL2 driven by the strong promoter Ubiquitin, Ubi::OsVAL2-FLAG#1-#4, were obtained. Figure 2 As shown in Figure B, Western blotting identified high expression of Ubi::OsVAL2-FLAG#1-#4.

[0067] Example 3: OsVAL2 is located in the nucleus of rice cells 1) The CDS sequence of the OsVAL2 protein was amplified and constructed into the 1301-GFP vector. After successful sequencing, the plasmid was transformed into Agrobacterium GV3101, shaken, and preserved.

[0068] 2) After centrifugation to collect the bacteria, resuspend the bacteria in a conversion solution containing 10 mmol / L MgCl2, 10 mmol / L MES (pH 5.6) and 150 μmol / L acetylsylgenone, and let it stand in the dark for 3 hours.

[0069] 3) Select tobacco materials with a growth cycle of 3-4 weeks, choose healthy young leaves, and use a syringe with the needle removed to inject the mixed Agrobacterium transformation solution into the leaf tissue from the back of the tobacco leaf. After growing in the dark for several hours, culture under normal light conditions.

[0070] 4) After culturing for 32 hours, take leaves and observe the fluorescence using Confocal.

[0071] like Figure 3 As shown, OsVAL2 is located within the rice cell nucleus. In Figure A, transient transformation of tobacco reveals that OsVAL2 has nuclear localization. A GFP tag was fused to the gene terminal, and tobacco leaves were transiently transformed. Fluorescence distribution was observed 32 hours later. The previously reported nuclear marker gene NLS was tagged with the red fluorescent tag Mcherry as an indicator. (Scale bar 50 μm). Figure B shows observations of the root tips of stable transgenic rice materials, revealing that OsVAL2 has localization in the rice cell nucleus, cytoplasm, and cell membrane. (Scale bar 5 μm).

[0072] Example 4: OsVAL2 positively regulates rice resistance to rice blast fungus. 1) Field-harvested seeds are dried in a 42℃ oven for 4-7 days and then exposed to sunlight for a week. They are then soaked in water at room temperature for 24 hours, after which the soaking water is discarded. The seeds are rinsed three times with clean water, and then spread on damp paper towels to retain moisture. The seeds will germinate after 2 days at room temperature. When the seed buds reach approximately 0.5 cm in size, they can be planted in greenhouse soil or hydroponic containers. The rice cultivation environment is 28℃ with 12 hours of light and 12 hours of darkness.

[0073] 2) Activate rice blast fungus race TH12 using CM medium. Place a filter paper disc containing the rice blast fungus in the center of the CM solid medium. Incubate at 28℃ for 7-10 days to produce conidia (12 days of light / 12 days of darkness). Once the surface of the CM medium is covered with mycelium, wash off the spores with purified water containing 0.02% Tween 20, filter through double-layer gauze, and prepare a spore suspension. The spore concentration used for inoculation is 5 × 10⁻⁶. 5 The spores per ml averaged 30-50 per field of view under a 10×10x microscope.

[0074] 3) In vitro inoculation of OsVAL2 knockout lines, overexpression lines, and wild-type TP309 with rice blast fungus was carried out. The disease incidence was investigated 7-10 days after inoculation. The disease incidence and size of the lesions were recorded by statistically analyzing the length of the lesions and calculating the amount of fungal growth on the leaves.

[0075] The results are as follows Figure 4 As shown, the length of rice blast lesions on leaves of the knockout lines CR-osval2#1 and CR-osval2#2 was significantly increased compared to the wild-type TP309; while the length of rice blast lesions on leaves of the overexpressing line Ubi::OsVAL2-FLAG#1-#2 was significantly decreased compared to the wild-type TP309. Therefore, this demonstrates that OsVAL2 positively regulates rice resistance to the fungal disease rice blast fungus.

[0076] Example 5: OsVAL2 positively regulates rice resistance to sheath blight pathogen. 1) Take the sclerotia of the blight pathogen preserved in the laboratory and grow them on PDA solid medium at 28°C. PDA medium (1L): 200 g of potato (cut into small pieces and boiled in water until the potato pieces are soft and mushy, filtered through four layers of gauze), 20 g of glucose, and 15 g / L agar powder added to the solid medium.

[0077] 2) Once the sclerotia have grown new hyphae, cut off the well-grown and uncontaminated hyphae with a sterile blade and place them on a new PDA medium for further cultivation.

[0078] 3) Repeat step 2 until there are no other microbial contaminations on the culture medium, and continue culturing for 2-3 days until sclerotia are produced.

[0079] 4) Cut the toothpicks into small pieces of about 2 cm and sterilize them.

[0080] 5) Remove the sclerotia and place them on PDA medium. After culturing for 1-2 days, spread them on sterilized toothpicks and continue to grow for 2-3 days. When the mycelium has covered the toothpicks, they can be used to inoculate the rhizoctonia solani.

[0081] 6) Rice can be inoculated with sheath blight pathogen from about 2 months into its growth until before the booting stage. When inoculating, use tweezers to remove a toothpick and insert it into the second and third leaf sheaths from the bottom of the rice plant.

[0082] 7) The incidence of sheath blight can be observed 7 days after inoculation.

[0083] The results are as follows Figure 5 As shown, the length of leaf sheath lesions in the knockout lines CR-osval2#1 and CR-osval2#2 was significantly increased compared to the wild-type TP309, while the length of leaf sheath lesions in the overexpression line Ubi::OsVAL2-FLAG#1-#2 was significantly decreased compared to the wild-type TP309, demonstrating that OsVAL2 positively regulates the resistance of rice to the fungal disease sheath blight.

[0084] Example 6: OsVAL2 positively regulates rice resistance to bacterial blight. 1) Take the Xoo strain preserved in the laboratory, activate it 2-3 times on a PSA plate, and incubate it at 28℃ for 48-72 hours.

[0085] 2) After a single colony has grown, pick a single colony and transfer it to PSA liquid medium and shake gently for 1-2 days.

[0086] 3) Pipette 100 μL of bacterial culture onto a PSA plate, spread it evenly with a clean spreader, invert the plate and incubate at 28°C for 48-72 hours. The bacterial blight pathogen that grows can be used for rice inoculation.

[0087] 4) Scrape the white leaf blight that has grown on the PSA medium with sterile water and dilute it to an OD value of 1.0.

[0088] 5) Dip scissors in the bacterial solution and cut the tip of the rice leaf at an angle 1-2 cm downwards.

[0089] 6) Measure the length of the leaf lesions 12-14 days after inoculation.

[0090] The results are as follows Figure 6 As shown, the leaf lesion length of the knockout lines CR-osval2#1 and CR-osval2#2 was significantly increased compared to the wild-type TP309, while the leaf lesion length of the overexpression line Ubi::OsVAL2-FLAG#1-#2 was significantly decreased compared to the wild-type TP309. Therefore, it is demonstrated that OsVAL2 positively regulates the resistance of rice to the bacterial disease Bacillus thuringiensis.

[0091] Example 7: OsVAL2 does not affect rice agronomic traits. Observations on the effects of OsVAL2 on agronomic traits of rice revealed that, for example Figure 7 As shown, there was no significant difference in plant height between the OsVAL2 overexpression lines Ubi::OsVAL2-FLAG#1-#2 and the knockout lines CR-osval2#1 and CR-osval2#2 and the wild type, indicating that OsVAL2 does not affect the normal growth and development of rice in stimulating plant immune responses.

[0092] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. The application of the OsVAL2 gene in the control of rice diseases and / or the enhancement of rice resistance to pathogens, characterized in that, The amino acid sequence encoded by the OsVAL2 gene is shown in SEQ ID NO.

1. The application includes increasing the expression level of the OsVAL2 gene, thereby improving the ability of rice to control diseases and / or enhancing the resistance of rice to pathogens, including rice blast fungus, Xanthomonas auricula-judae, and Rhizoctonia solani, and the diseases including rice blast, bacterial blight, and sheath blight.

2. The application according to claim 1, characterized in that, The CDS sequence of the OsVAL2 gene is shown in SEQ ID NO.

2.

3. A method for improving the ability of rice to control diseases and / or enhancing the resistance of rice to pathogens, characterized in that, The method includes increasing the expression level of the OsVAL2 gene in rice, thereby improving the rice's ability to control diseases and / or enhancing its resistance to pathogens. The amino acid sequence encoded by the OsVAL2 gene is shown in SEQ ID NO.

1. The pathogens include rice blast fungus, Xanthomonas auricula-judae, and Rhizoctonia solani. The diseases include rice blast, bacterial blight, and sheath blight.

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