Viral disease resistance gene and application of excellent natural variation thereof

Abstract

The invention discloses a virus disease resistance gene and application of excellent natural variation of the virus disease resistance gene, and belongs to the technical field of biological breeding and vegetable production. The anti-ToCV function of the SlAVE3 gene is identified for the first time, the antiviral mechanism of the SlAVE3 gene for targeted degradation of virus p59 protein through a UPS system and regulation and control of transcription positive feedback circulation is clarified, and the blank of tomato ToCV resistance gene identification is filled. The invention provides a mutation form of the SlAVE3, clarifies the influence of key site mutation on the function of the SlAVE3, and provides a target for optimizing the antiviral activity of the SlAVE3 through a gene editing technology. The excellent haplotype SpAVE3 derived from wild tomatoes is explored, the ToCV resistance of the excellent haplotype SpAVE3 is obviously superior to that of SlAVE3 of cultivated tomatoes, and a precious gene resource is provided for disease-resistant breeding of tomatoes.

Description

Technical Field

[0001] This invention relates to the fields of biological breeding and vegetable production technology, specifically to the application of a viral disease resistance gene and its superior natural variations. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes are one of the world's highest-yielding vegetable crops, with an annual total production exceeding 180 million tons, playing a vital role in vegetable production and consumption. However, viral diseases are one of the major pests affecting tomato production, causing agricultural losses of up to $30 billion globally each year. Tomatoes infected with viruses can suffer yield losses of 70%-95%, seriously threatening the security of tomato supply.

[0003] Tomato chlorosis virus (ToCV) belongs to the family Longiviridae ( Closteroviridae ) genus Trichoviridae ( Crinivirus Tomato virus (ToCV) is a significant RNA virus that harms tomato production. Transmitted semi-persistently by whiteflies, infection causes tomato leaves to turn yellow and chlorotic, reduces photosynthetic efficiency, and impairs fruit development, severely impacting tomato yield and quality. Currently, no tomato germplasm is completely immune to ToCV, and traditional breeding methods struggle to develop highly ToCV-resistant tomato varieties. Therefore, identifying tomato's own antiviral genes and their superior variants, and using molecular breeding techniques to directionally improve ToCV resistance, is a crucial approach to addressing the threat of ToCV.

[0004] The ubiquitin-proteasome system (UPS) is an important protein degradation pathway in plants, playing a crucial role in antiviral defense. It specifically recognizes and degrades viral proteins through a three-tiered enzymatic reaction involving E1 (ubiquitin activator), E2 (ubiquitin conjugator), and E3 (ubiquitin ligase), thereby inhibiting viral infection. The E3 ubiquitin ligase determines the substrate specificity of the UPS and is a core component of antiviral defense. The tomato genome contains 1500-2100 E3 ubiquitin ligase genes, but the antiviral functions of most of these genes have not yet been identified.

[0005] Wild tomatoes, through long-term natural selection, have accumulated a wealth of superior genes for disease resistance, serving as an important germplasm bank for the genetic improvement of cultivated tomatoes. During tomato domestication, some disease-resistance-related genes may have undergone sequence variations under selective pressure, leading to a decrease in the disease resistance of cultivated tomatoes. Therefore, identifying superior haplotypes of virus-resistant genes from wild tomatoes and elucidating their functional variation mechanisms is of great significance for tomato disease-resistant breeding. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of a viral disease resistance gene and its superior natural mutations.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of tomato AVE3 protein in either (1) or (2) below: (1) Improve plant resistance to tomato chlorosis virus; (2) Preparation of drugs for the prevention and control of tomato chlorosis virus disease; The tomato AVE3 protein is any one of the following proteins (A1)-(A3): (A1) The SlAVE3 protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The SpAVE3 protein with the amino acid sequence shown in SEQ ID NO.4; (A3) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

[0008] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0009] A second aspect of the present invention provides the use of the gene encoding the tomato AVE3 protein in either (1) or (2) below: (1) Improve plant resistance to tomato chlorosis virus; (2) Cultivate plant varieties resistant to tomato chlorosis virus; The gene encoding the tomato AVE3 protein is any of the nucleic acid molecules shown in (i)-(iv) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1; (ii) Nucleic acid molecules other than (i) that encode the SlAVE3 protein shown in SEQ ID NO.2; (iii) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.3; (iv) Nucleic acid molecules other than (i) that encode the SpAVE3 protein shown in SEQ ID NO.4.

[0010] In the above applications, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA or mRNA.

[0011] In the above applications, the plant is preferably a plant of the genus *Tomato*.

[0012] This invention is the first to discover the E3 ubiquitin ligase gene in cultivated tomatoes. SlAVE3 ( Solanum lycopersicum Antiviral E3 ubiquitin ligase 3) has anti-ToCV function. SlAVE3 The gene-encoded SlAVE3 protein specifically recognizes the ToCV p59 protein (a viral movement protein and effector), mediating p59 degradation through ubiquitination modification, thereby inhibiting viral intercellular movement and pathogenicity. Therefore, overexpression of SlAVE3... SlAVE3 Genes can increase the resistance of tomatoes to ToCV.

[0013] Furthermore, based on SlAVE3 Genes, this invention further from wild tomatoes Solanum pimpinellifolium The superior haplotype SpAVE3 of SlAVE3 was identified, and its coding region sequence is shown in SEQ ID NO.3. The amino acid sequence of the encoded SpAVE3 protein is shown in SEQ ID NO.4. Compared with cultivated tomato SlAVE3, it has two non-synonymous mutations: the 43rd amino acid in the coding region is mutated from serine (Ser) to threonine (Thr) (corresponding to the A→T mutation in the nucleic acid sequence); the 199th amino acid in the coding region is mutated from glutamine (Gln) to arginine (Arg) (corresponding to the T→C mutation in the nucleic acid sequence).

[0014] SpAVE3 haplotype has the following functional advantages: (1) Binding to the p59 protein of ToCV can mediate the degradation of p59 more efficiently; (2) After overexpressing the sequence of cultivated tomato, its resistance to ToCV is significantly better than that of tomato lines expressing SlAVE3, which is manifested in a reduced virus transmission rate, milder disease symptoms and reduced yield loss.

[0015] A third aspect of the present invention provides the use of a substance that regulates the expression of the gene encoding the tomato AVE3 protein or a substance that regulates the activity and / or content of the tomato AVE3 protein in at least one of the following (1)-(3): (1) Improve plant resistance to tomato chlorosis virus; (2) Preparation of drugs for the prevention and control of tomato chlorosis virus disease; (3) Cultivate plant varieties resistant to tomato chlorosis virus.

[0016] In this invention, the regulation is to increase, enhance, or improve.

[0017] In this invention, the enhancement, increase or upregulation of the expression level of the coding gene of the protein described above in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the protein described above, is achieved by introducing the coding gene of the protein described above into the recipient plant.

[0018] In some preferred embodiments, the substance regulating the expression of the gene encoding tomato AVE3 protein, or the substance regulating the activity and / or content of tomato AVE3 protein, can be any of the following: C1) An expression cassette containing the gene encoding the tomato AVE3 protein; C2) A recombinant vector containing the gene encoding the tomato AVE3 protein, or a recombinant vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing the gene encoding the tomato AVE3 protein, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) A transgenic plant cell line containing the gene encoding the tomato AVE3 protein, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing the gene encoding the tomato AVE3 protein, or transgenic plant tissue containing the expression cassette described in C1).

[0019] Recombinant expression vectors containing the coding gene for tomato AVE3 protein can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0020] When constructing a recombinant plant expression vector using the tomato AVE3 protein coding gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0021] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0022] A fourth aspect of the present invention provides a method for improving plant resistance to tomato chlorosis virus, comprising the step of overexpressing the gene encoding the tomato AVE3 protein in the plant.

[0023] In the above method, the encoding gene of tomato AVE3 protein in plants can be overexpressed by exogenously introducing the gene encoding tomato AVE3 protein.

[0024] A fifth aspect of the present invention provides a method for cultivating a tomato variety resistant to tomato chlorosis virus, comprising the following steps: The gene encoding the tomato AVE3 protein was transferred into wild-type tomato to obtain transgenic tomatoes that overexpressed the gene encoding the tomato AVE3 protein; the transgenic tomatoes showed higher resistance to tomato chlorosis virus than tomato chlorosis virus. To cultivate tomato varieties resistant to tomato chlorosis virus by self-pollinating genetically modified tomatoes or by crossing them with other tomatoes resistant to tomato chlorosis virus.

[0025] The beneficial effects of this invention are: (1) This invention is the first to identify SlAVE3The gene's anti-ToCV function was identified, clarifying its antiviral mechanism of targeting and degrading viral p59 protein through the UPS system and regulating the positive feedback loop of transcription, filling the gap in the identification of ToCV resistance genes in tomatoes.

[0026] (2) This invention provides a mutant form of SlAVE3, elucidates the impact of key site mutations on its function, and provides a target for optimizing the antiviral activity of SlAVE3 through gene editing technology.

[0027] (3) This invention has discovered an excellent haplotype SpAVE3 derived from wild tomatoes, whose resistance to ToCV is significantly better than that of cultivated tomatoes SlAVE3, providing valuable genetic resources for tomato disease resistance breeding.

[0028] (4) This invention establishes transgenic breeding, gene editing breeding and molecular marker-assisted breeding methods based on the SlAVE3 gene and its superior variants. These methods can rapidly cultivate new ToCV-resistant tomato germplasm, improve breeding efficiency, reduce the harm of ToCV to tomato production, and have important economic and application value. Attached Figure Description

[0029] Figure 1 Results of yeast two-hybrid experiments verifying the interaction between SlAVE3 and p59.

[0030] Figure 2 Experimental results of bimolecular fluorescence complementation verifying the interaction between SlAVE3 and p59.

[0031] Figure 3 The ubiquitination and degradation effect of SlAVE3 on p59; in the figure, a is the result of in vitro ubiquitination experiment and b is the result of plant ubiquitination experiment.

[0032] Figure 4 ToCV infection of WT, slave3 and SlAVE3 - Disease symptom assessment of OE plants.

[0033] Figure 5 Comparison of the degradation ability of SlAVE3 haplotype SpAVE3 and SlAVE3 on p59.

[0034] Figure 6 Comparison of ToCV resistance phenotypes between SlAVE3 overexpression lines, SpAVE3 transformed lines, and wild-type tomatoes. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0037] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The ToCV used in this invention, with its Accession Numbers of Tomato chlorosis virus (ToCV) infectious clones (RNA1, NC_007340.1; RNA2, NC_007341.1), is described in the literature: Zhao D, Xia T, Zhou T, Zhao L, Zhu X, Gong B, 2025. Inoculation method and disease evaluation of tomato chlorotic virus (ToCV) in Solanum lycopersicum. Vegetable Research 5: e006.

[0038] Example 1: SlAVE3-p59 interaction and ubiquitination verification 1. Test method: The SlAVE3 protein (shown in SEQ ID NO.2) was truncated at position 245 to generate SlAVE3Δ1 (1-244 amino acids, containing a low-complexity region, amino acid sequence as shown in SEQ ID NO.6, corresponding coding sequence as shown in SEQ ID NO.5) and SlAVE3Δ2 (245-334 amino acids, containing coiled-coil and RING domains). Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments were performed, respectively; where: The experimental method for yeast two-hybrid (Y2H) was as follows: Y2H strain was cultured in the dark on YPDA medium for 3 days to prepare Y2H competent cells. The successfully constructed AD / BD recombinant plasmid containing the complete or partial coding regions of p59 (GeneID: 3607997) and SlAVE3, carrier DNA, and 1 × TE / LiAc / PEG4000 were transferred into the Y2H competent cells. The cells were gently mixed and incubated at 30°C for 30 min, inverting every 10 min to mix. DMSO was then added, and the cells were incubated at 42°C for 15 min, mixing every 5 min. The cells were centrifuged at 2284 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of YPDA. The cells were cultured on a shaker at 30°C for 1 h, and then centrifuged at 12000 rpm for 30 s. The supernatant was discarded, and the cells were resuspended in 200 μL of sterile water and cultured on two-deficient and four-deficient media. After culturing at 30℃ in the dark for 3-5 days, observe the interaction.

[0039] The bimolecular fluorescence complementation (BiFC) assay was performed as follows: Recombinant plasmids of nYFP and cYFP successfully ligated to p59 and SlAVE3, along with empty nYFP and cYFP vectors, were transformed into Agrobacterium. The Agrobacterium was infiltrated into 4-week-old tobacco leaves. After 48-72 hours, small square pieces approximately 1.5 cm in length and width were cut from the leaves around the inoculation wells using a blade. Imaging was then performed using a Zeiss LSM800 confocal microscope. The excitation wavelength of GFP was 488 nm, and the emission wavelength was 510-550 nm. Images were processed using ZEN blue edition 2.3 software.

[0040] In addition, in vitro and plant ubiquitination experiments were conducted using p59 as a potential substrate, as follows: The reaction mixture contains: ubiquitin (2 μg μL) -1 Wheat E1-His, human E2-His (UBCH5b), or SlAVE2-His, as well as SlAVE3 / SlAVE3H304Y / SpAVE3-Strep, were in a reaction buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 5 mM ATP, 2 mM DTT). After incubation at 30°C for 2 hours, ubiquitination was detected by Western blotting using anti-Strep and anti-Ub antibodies. For substrate ubiquitination, an equimolar amount of GST-tagged substrate protein was added to the reaction before incubation, and ubiquitination was detected by Western blotting.

[0041] For ubiquitination in plants, the target protein tagged with a flag was expressed in leaves of designated genotypes. Ten hours before total protein extraction, 100 μM MG132 was infiltrated into the leaves. Subsequently, total protein was extracted from the designated genotypes, and ubiquitination was detected as previously described.

[0042] 2. Test Results: The results of the yeast two-hybrid (Y2H) experiment are as follows: Figure 1 As shown; the results of the bimolecular fluorescence complementary assay (BiFC) are as follows. Figure 2 As shown; the results indicate that both full-length SlAVE3 and SlAVE3Δ2 interact with p59, while SlAVE3Δ1 does not; this demonstrates that amino acids 245-334 at the C-terminus of SlAVE3 are the key region for interaction with p59. The absence of this region (SlAVE3Δ2) or mutation at the key site within this region will prevent SlAVE3 from binding to p59 and thus lose its ability to degrade p59.

[0043] Results of in vitro and plant ubiquitination experiments are as follows Figure 3 As shown, the results indicate that p59 is ubiquitinated by SlAVE3.

[0044] Example 2: SlAVE3 Gene function identification 1. Test method: (1) SlAVE3 Construction of gene knockout and overexpression lines: The CDS sequence (SEQ ID NO.1) of the SlAVE3 gene was cloned from ToCV-infected tomato leaves, and a CRISPR-Cas9 vector was constructed. SlAVE3 -OE vector; CRISPR-Cas9 vector and... via Agrobacterium-mediated transformation SlAVE3 - The OE vector was transformed into the tomato cultivar "AilsaCraig, AC" to obtain SlAVE3 knockout lines and SlAVE3 overexpression lines. Details are as follows: Mutant Construction: The slave3 mutant was generated using CRISPR-Cas9 technology. Two sgRNAs targeting the N-terminus of the SlAVE3 coding sequence were designed (using the CRISPOR tool; http: / / crispor.tefor.net / ) and cloned into the pHSE401 vector via homologous recombination using the ClonExpress II one-step cloning kit (catalog number: C112-01, Vazyme, China). The construct was transformed into LBA4404 strain via Agrobacterium-mediated transformation for the transformation of wild-type tomato plants. The mutant was confirmed by PCR and genomic DNA sequencing; homozygotes were used for experiments.

[0045] Construction of overexpression (OE) lines: The SlAVE3 coding sequence was amplified and cloned into the pCAMBIA1300 vector under the CaMV35S promoter, with a GFP tag fused. The construct was transformed into wild-type tomato plants via LBA4404-mediated transformation. The SlAVE3-OE lines were confirmed by PCR and gene expression analysis.

[0046] (2) Pathogen infection: The above-constructed SlAVE3 One-month-old tomato plants from gene knockout and overexpression lines were cultured in a controlled greenhouse (10-13 hours light, 11-14 hours dark, 18-28°C, 60-70% humidity) and used for ToCV infection experiments. GV3101 strains carrying ToCV infection clones were cultured overnight, then precipitated and resuspended in virus inoculation buffer (10 mM MgCl2, 100 mM MES, 200 µM acetosyringone, pH 5.2). These cultures were then placed in the dark for 2 hours. Cultures containing pCa-ToCR1 were mixed with cultures containing pCa-ToCR2 at a 1:1 ratio (OD). 600 (Value: 0.6-0.8). Inoculate onto tomato stems and leaves. After inoculation, the plants continue to grow under the same greenhouse conditions.

[0047] At 28 days post-inoculation (dpi), disease symptoms, disease index, and capsid protein (CP) concentration were assessed to evaluate ToCV resistance. Samples were collected from the second systemic leaf above the inoculation site. For each treatment, 20 plants were randomly selected, and the disease grade of the sample leaves was recorded according to the following levels: Grade N, no obvious symptoms; Grade I, scattered yellow spots (<20% of leaf area); Grade II: obvious yellow spots (20%–50% of leaf area); Grade III: numerous yellow spots (50%–80% of leaf area); Grade IV: almost complete loss of green in the mesophyll tissue (80%–100% of leaf area). Disease incidence was calculated as the percentage of plants in each disease grade. The disease index was determined using the following formula: Disease index = ∑(d×n) / (N×D), where d is the representative value of each disease level, n is the number of plants in that level, N is the total number of plants assessed, and D is the highest disease level.

[0048] Total RNA and protein were extracted from the pool of mixed sample leaves, and CP concentration was analyzed by immunoblotting.

[0049] 2. Test Results: The results are as follows Figure 4 As shown, slave3 strain ( slave3# 1. slave3# 2) The leaf chlorosis and yellowing symptoms and disease index were significantly more severe than those of WT; SlAVE3 -OE strain ( SlAVE3 -OE # 1. SlAVE3 -OE # 2) The leaf chlorosis, yellowing symptoms, and disease index of the WT were significantly milder than those of the WT. Figure 4 a, b).

[0050] Virus content test results show: slave3 strain ( slave3# 1. slave3# 2) The viral load was significantly higher than that of WT; SlAVE3 -OE strain ( SlAVE3 -OE # 1. SlAVE3 -OE # 2) The viral load was significantly lower than that of WT.

[0051] Example 3: Functional Verification of Superior Haplotype SpAVE3 1. Test method: (1) Virus-mediated gene expression From wild tomatoes Solanum pimpinellifoliumThe SpAVE3 gene (SEQ ID NO.3) was cloned, and PVX-SpAVE3 and PVX-SlAVE3 were constructed respectively for virus-mediated gene expression, as detailed below: Gene expression was performed using a potato virus X (PVX) vector. The coding sequence of the target gene was amplified and cloned into a PVX vector (PVX-target gene), which was tagged with either GFP or Flag under the guidance of the CaMV35S promoter. An empty PVX vector (PVX-EV) served as a control. Recombinant vectors constructed in the LBA4404 strain were used to infect germinated tomato seeds of a specific genotype.

[0052] WT / PVX-EV: WT is inoculated with an unloaded PVX. slave3 / PVX-EV: The slave3 knockout line constructed in Example 2 was inoculated with an empty PVX vector. slave3 / PVX-SlAVE3: The slave3 knockout line constructed in Example 2 was inoculated with PVX-SlAVE3. slave3 / PVX-SpAVE3: The slave3 knockout line constructed in Example 2 was inoculated with PVX-SpAVE3.

[0053] Before any experiments can be conducted, the seedlings need to undergo gene expression testing.

[0054] (2) Examination of the degradation ability of p59: Cell-free degradation assay. Total protein was extracted from tomato leaves of a specified genotype. Equal volumes of the protein extract were incubated with purified substrate protein (recombinant GST-tagged target protein) and 10 mM ATP for a specified time period. MG132 (50 μM) was added to inhibit the 26S proteasome. Proteins were isolated by SDS-PAGE and immunoblotting was performed using a specific tag antibody. Actin was used as a control.

[0055] (3) ToCV inoculation experiment: One-month-old tomato plants (WT / PVX-EV, slave3 / PVX-EV, slave3 / PVX-SlAVE3, and slave3 / PVX-SpAVE3) were cultured in a controlled greenhouse (10-13 hours of light, 11-14 hours of darkness, 18-28 degrees Celsius, and 60-70% humidity) and used for ToCV infection experiments.

[0056] At 28 days post-inoculation (dpi), disease symptoms, disease index, and capsid protein (CP) concentration were assessed to evaluate ToCV resistance. The method was the same as in Example 2.

[0057] 2. Test Results: The comparison results of the degradation ability of the superior haplotype SpAVE3 and SlAVE3 on p59 are as follows: Figure 5 As shown, the results indicate that SpAVE3 mediates the degradation of p59 more efficiently than SlAVE3.

[0058] ToCV inoculation experiment results are as follows Figure 6 As shown, the results indicate that the PVX-SpAVE3 transformed lines exhibit milder disease symptoms and superior resistance to ToCV compared to the PVX-SlAVE3 lines.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of tomato AVE3 protein in the following (1) or (2): (1) Improve plant resistance to tomato chlorosis virus; (2) Preparation of drugs for the prevention and control of tomato chlorosis virus disease; The tomato AVE3 protein is any one of the following proteins (A1)-(A3): (A1) The SlAVE3 protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The SpAVE3 protein with the amino acid sequence shown in SEQ ID NO.4; (A3) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

2. The application according to claim 1, characterized in that, The plant in question is a member of the genus Tomato.

3. The application of the gene encoding the tomato AVE3 protein in the following (1) or (2): (1) Improve plant resistance to tomato chlorosis virus; (2) Cultivate plant varieties resistant to tomato chlorosis virus; The gene encoding the tomato AVE3 protein is any of the nucleic acid molecules shown in (i)-(iv) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1; (ii) Nucleic acid molecules other than (i) that encode the SlAVE3 protein shown in SEQ ID NO.2; (iii) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.3; (iv) Nucleic acid molecules other than (i) that encode the SpAVE3 protein shown in SEQ ID NO.

4.

4. The application according to claim 3, characterized in that, The plant in question is a member of the genus Tomato.

5. The application of substances that regulate the expression of the gene encoding tomato AVE3 protein or substances that regulate the activity and / or content of tomato AVE3 protein in at least one of the following (1)-(3): (1) Improve plant resistance to tomato chlorosis virus; (2) Preparation of drugs for the prevention and control of tomato chlorosis virus disease; (3) Cultivate plant varieties resistant to tomato chlorosis virus.

6. The application according to claim 5, characterized in that, The substance that regulates the expression of the gene encoding tomato AVE3 protein, or the substance that regulates the activity and / or content of tomato AVE3 protein, is any one of the following: C1) An expression cassette containing the gene encoding the tomato AVE3 protein; C2) A recombinant vector containing the gene encoding the tomato AVE3 protein, or a recombinant vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing the gene encoding the tomato AVE3 protein, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) A transgenic plant cell line containing the gene encoding the tomato AVE3 protein, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing the gene encoding the tomato AVE3 protein, or transgenic plant tissue containing the expression cassette described in C1).

7. The application according to claim 5 or 6, characterized in that, The regulation refers to increasing, strengthening, or raising.

8. A method for improving plant resistance to tomato chlorosis virus, characterized in that, include: Steps to overexpress the gene encoding the tomato AVE3 protein in plants.

9. The method according to claim 8, characterized in that, The gene encoding the tomato AVE3 protein was overexpressed in plants by exogenously introducing the gene encoding the tomato AVE3 protein.

10. A method for cultivating a tomato variety resistant to tomato chlorosis virus, characterized in that, Includes the following steps: The gene encoding the tomato AVE3 protein was transferred into wild-type tomato to obtain transgenic tomatoes that overexpressed the gene encoding the tomato AVE3 protein; the transgenic tomatoes showed higher resistance to tomato chlorosis virus than tomato chlorosis virus. To cultivate tomato varieties resistant to tomato chlorosis virus by self-pollinating genetically modified tomatoes or by crossing them with other tomatoes resistant to tomato chlorosis virus.

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