Yellow dwarf disease resistance related gene MS1 and application thereof

Abstract

The invention discloses a yellow dwarf disease resistance related gene MS1 and application thereof, and belongs to the technical field of biology. The invention discloses for the first time that the MS gene is a core positive regulation factor of plant broad-spectrum antiviral property through systematic research. Experiments prove that replication and diffusion of various pathogens such as BYDV, BSMV, TRV, PVX and BSCTV can be remarkably inhibited by up-regulating MS expression of plants such as wheat, barley and tobacco, and resistance does not depend on virus types. Mechanism research shows that MS silencing is achieved by enhancing PTGS-mediated RNA silencing and other basic resistance. The discovery breaks through the traditional mindset of'enhancing resistance genes', and a brand new strategy of'removing resistance inhibition factors' is created. The invention proves that MS inhibition can realize multiple virus resistance and does not damage growth and development.

Description

Technical Field

[0001] This invention relates to the field of gene breeding technology, and in particular to the MS1 gene, which is related to resistance to yellow dwarf disease, and its application. Background Technology

[0002] Global agricultural production suffers approximately $220 billion in economic losses annually due to plant diseases (Savary et al., 2019), with plant viral diseases accounting for as much as $30 billion annually due to their diverse types, rapid mutation, and lack of broad-spectrum control methods (Nicaise, 2014). Climate change further exacerbates the spread and severity of viruses, posing a serious challenge to food security (Pequeno et al., 2024). Current virus control mainly relies on chemical pesticides (such as insecticides to block aphids and other vectors) and the breeding of traditional resistant varieties. However, the former causes environmental pollution and ecological imbalance, while the latter, because resistance genes are mostly targeted at single virus strains, are easily rendered ineffective due to rapid viral evolution (Wu et al., 2024). This contradiction is particularly pronounced in cereal crops: taking barley yellow dwarf virus (BYDVs) as an example, it is persistently transmitted by aphids, infecting crops such as wheat and barley and causing typical symptoms such as leaf yellowing, stunted growth, and decreased photosynthetic efficiency (Miller & Lozier, 2022). This results in a yield reduction of 13-45 kg / ha for every 1% incidence rate in wheat (McKirdy et al., 2002), and seedling infection can lead to root degeneration and delayed heading (Choudhury, 2017). In major producing areas such as the North China Plain in China, BYDV occurs in combination with multiple viruses such as yellow mosaic virus and stripe mosaic virus, and overlaps with rust, Fusarium head blight, and insect pests to form a "biological stress network" (Li et al., 2024; Wang et al., 2024), rendering single control strategies completely ineffective. Therefore, discovering new broad-spectrum, persistent, and environmentally compatible antiviral targets has become an urgent need in international agricultural science and technology.

[0003] While plants have developed multi-layered defense mechanisms (such as transcriptional silencing TGS / PTGS) through long-term evolution, many identified antiviral genes are strain-specific and struggle to cope with viral population variations (Li et al., 2023; Jones, 2021). Recent studies have shown that basal metabolic pathways may confer broad-spectrum resistance to hosts by regulating immune signaling networks (Wu et al., 2024), with the methionine cycle attracting significant attention due to its central role. In this cycle, methionine synthase (MS) catalyzes the synthesis of methionine (MET) from homocysteine ​​(HCY) and 5-methyltetrahydrofolate (5-MTHF), which is then converted to S-adenosylmethionine (SAM) by S-adenosylmethionine synthase (SAMS)—the sole methyl donor for DNA / histone methylation and a direct precursor for ethylene synthesis (Hesse & Hoefgen, 2003; Rahikainen et al., 2018). Notably, a fierce battle ensues between the virus and the host over SAM metabolism: silencing repressors (VSRs) encoded by geminiviruses, such as CaLCuV-AL2 and BSCTV-C2 / C3, inhibit TGS-mediated viral genome silencing by interfering with SAM synthesis or methyltransferase degradation (Raja et al., 2008; Chen et al., 2020); the C4 protein of Multan cotton leaf curl virus CLCuMuV directly inhibits SAMS activity, promoting viral infection (Ismayil et al., 2018); while overexpression of rice OsSAMS1 increases ethylene levels, conversely enhancing susceptibility to rice dwarf virus (RDV) (Zhao et al., 2017). These findings reveal that key enzymes in the methionine cycle possess a double-edged sword effect in viral defense, and their precise regulatory mechanisms remain to be elucidated.

[0004] In higher plants, metastatic enzymes (MS) are all cobalamin-independent (MetE) and are the rate-limiting enzymes of the methionine cycle (Eckermannet et al., 2000). Interestingly, MS exhibit opposite functions in the defense against different pathogens: in fungal diseases, rice OsMETS1 stabilizes ethylene synthesis through the deubiquitinating enzyme PICI1, positively regulating resistance to rice blast fungus (Zhai et al., 2022); however, in bacterial diseases, overexpression of Arabidopsis thaliana AtMetE1 leads to increased genome-wide DNA methylation, which in turn increases susceptibility to Pseudomonas syringae (Gonzalez & Vera, 2019). More importantly, the mechanism of MS in viral interactions is almost unknown—although there are reports of transcriptional fluctuations in MetE after infection with citrus leprosy virus and soybean mosaic virus (Babu et al., 2008; Freitas-Astúa et al., 2007), whether it directly regulates antiviral activity, its direction of action, and its molecular pathways remain scientifically unknown. This gap has severely hampered the progress of designing broad-spectrum antiviral crops based on metabolic engineering. Summary of the Invention

[0005] The purpose of this invention is to provide the MS1 gene, which is related to resistance to barley yellow dwarf virus, and its applications, in order to solve the problems existing in the prior art. The protein encoded by the MS1 gene enhances the resistance of wheat and tobacco to viruses such as barley yellow dwarf virus (BYDV), barley stripe virus (BSMV), and potato virus X (PVX), and expands its applications in plant breeding.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of the MS1 gene or its encoded protein in improving plant disease resistance.

[0007] The second technical solution of the present invention is the application of the MS1 gene or recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the MS1 gene in the cultivation of plant varieties with high disease resistance.

[0008] The third technical solution of the present invention is a method for improving the disease resistance of plants by overexpressing the MS1 gene or increasing the level of the protein it encodes, thereby improving the disease resistance of plants.

[0009] The fourth technical solution of the present invention is a method for cultivating plant varieties with high disease resistance, which involves overexpressing the MS1 gene using a recombinant vector containing the MS1 gene, an expression cassette, a transgenic cell line or recombinant bacteria.

[0010] Based on the above technical solution, the present invention has the following technical effects: This invention, through systematic research, reveals for the first time that the MS gene is a core positive regulator of broad-spectrum antiviral resistance in plants. Experiments have demonstrated that upregulating MS expression in wheat, barley, and tobacco significantly inhibits the replication and spread of multiple pathogens, including BYDV, BSMV, TRV, PVX, and BSCTV, with resistance independent of virus type. Mechanistic studies show that MS silencing enhances basal resistance, such as PTGS-mediated RNA silencing. This discovery breaks through the traditional mindset of "enhancing resistance genes" and pioneers a novel strategy of "removing resistance inhibitors." Compared to targeting downstream enzymes such as SAMS, directly regulating MS has significantly broader spectrum and stability—for example, in rice, OsSAMS1 overexpression increases viral susceptibility (Zhao et al., 2017), while this invention confirms that MS inhibition can achieve multiple viral resistances without impairing growth and development. Attached Figure Description

[0011] Figure 1 To determine AtMS1 as the interacting protein of BYDV 17K VSR, (a) shows six specific peptides marked in red identified in previous IP-MS experiments (Wang et al., 2024), which were mapped to AtMS1. This protein consists of 765 amino acid residues, with a predicted molecular weight of 84.36 kDa, and is encoded by the Arabidopsis locus AT5G17920. (bd) The interaction between BYDV 17K and AtMS1 was verified using yeast two-hybrid (Y2H, b), luciferase complementation (SLC, c), and co-immunoprecipitation (Co-IP, d) experiments. Negative controls were empty AD and BD vectors used in the Y2H experiment in yeast (Saccharomyces cerevisiae) (b), and nLUC-GUS and GFP-cLUC used in the SLC experiment in tobacco (N. benthamiana) (c). In (d), the immunoprecipitation (IP) products were prepared from total protein extracted from transgenic strains tA_GFP (expressing free GFP) or tA_17K-GFP (producing 17K-GFP fusion protein) using the polyclonal anti-MS antibody developed in this paper (see below). BYDV 17K was detectable in the IP products from tA_17K-GFP but not in the products from tA_GFP.

[0012] Figure 2MS protein identity and similarity analysis for different species. The accession number and predicted molecular mass are in parentheses.The chromosomal location and predicted molecular mass of each MS protein (provided in parentheses), where 1HvMS1 (HORVU4Hr1G002270, 84.43 kDa), 2HvMS2 (HORVU5Hr1G006780, 84.56 kDa), 3TaMS1A (TraesCS4A02G298700, 84.44 kDa), 4TaMS1B (TraesCS4B02G014700, 84.39 kDa), 5TaMS1D (TraesCS4D02G012900, 84.31 kDa), 6TaMS2A (TraesCS5A02G024900, 84.55 kDa), 7TaMS2B (TraesCS5B02G022800, 84.62 kDa), 8 TaMS2D (TraesCS5D02G031300, 84.55 kDa), 9 OsMS1 (Os12g0623900, 84.58 kDa), 10 OsMS2 (Os12g0624000, 84.67 kDa), 11 ZmMS1 (Zm00001d033480, 84.47 kDa), 12 ZmMS2 (Zm00001d013644, 84.62 kDa), 13 ZmMS3 (Zm00001d031128, 84.49 kDa), 14 AtMS1 (AT5G17920, 84.36 kDa), 15 AtMS2 (AT3G03780, 84.58 kDa), 16 AtMS3 (AT5G20980, 85.43 kDa), 17 NbMSa (Niben101Scf03634g04009, 84.58 kDa), 18 NbMSb (Niben101Scf07438g04014, 84.50 kDa), 19 NbMSc (Niben101Scf09725g00022, 84.52kDa), 20 NbMSd (Niben101Scf01812g02025, 84.61 kDa), 21 NbMSe (Niben101Scf00054g01013, 83.33 kDa), 22 NbMSf (Niben101Scf12280g00002, 77.52kDa), 23 NbMSg (Niben101Scf09813g00005, 83.04 kDa), 24 NbMSh (Niben101Scf05629g01024, 73.04 kDa).

[0013] Figure 3 To weaken defense against BYDV through BSMV-mediated HvMS silencing. (a, b) Changes in HvMS transcription levels (a) and HvMS protein abundance (b) in barley after different BYDV infection days (DPI) were detected by RT-qPCR and Western blotting, respectively. (c) BSMV-mediated gene silencing significantly reduced HvMS gene expression. BSMV-EV represents the empty vector control, while BSMV-HvMS represents the recombinant virus used to silence HvMS1 and HvMS2. (d) Overall disease severity and plant height were compared among four groups of barley plants (unsilenced or silenced HvMS, with or without BYDV infection). Barley plants were first infected with BSMV-EV or BSMV-HvMS, and then inoculated with aphids carrying or not carrying BYDV. (e, f) Transcriptional levels of the BYDV capsid protein gene (CP) were determined by RT-qPCR (e), and the abundance of BYDV 17K protein (f) was detected by Western blotting in the four plant groups shown in (d). Primers used in (a) and (c) recognized HvMS1 and HvMS2. The anti-MS polyclonal antibody in (b) detected MS protein in monocotyledonous and dicotyledonous plants (Figure S1). Actin was used as a loading control (b and f). Values ​​shown (a, c, and e) are mean ± standard deviation (SD), with each dataset containing three or more biological replicates. Statistical analysis was performed using the Studentt test (c and e) or the LSD paired multiple comparison test (a), with different letters indicating significant differences (P ≤ 0.05). The intensity of protein bands (b and f) was quantified using ImageJ (https: / / imagej.net / software / imagej / ). Scale bar, 5 cm. The results shown are typical results from three independent experiments.

[0014] Figure 4 This study analyzed TaMS expression induced by BYDV and the interaction between TaMS and BYDV 17K in wheat. (a) SLC analysis in *N. benthamiana* showed that all six common wheat TaMS members (TaMS1A, TaMS1B, TaMS1D, TaMS2A, TaMS2B, and TaMS2D) interacted with BYDV 17K, with nLUC-GUS and GFP-cLUC as negative controls. (b, c) Changes in TaMS transcription levels (b) and TaMS protein abundance (c) at six different time points after BYDV inoculation were determined by RT-qPCR and Western blot analysis, respectively. In (b), RT-qPCR analysis showed that after BYDV inoculation... TaMSExpression level trends. In (c), Western blot analysis shows the trend of TaMS expression levels after BYDV inoculation. Protein band intensity was quantified using ImageJ (https: / / imagej.net / software / imagej / ). The results shown are typical of three independent experiments.

[0015] Figure 5 The interaction between BYDV 17K VSR and barley and wheat MS proteins was investigated. In (ad), the interaction between BYDV 17K and barley HvMS1 / HvMS2 was revealed using yeast two-hybrid (Y2H, a), luciferase complementation (SLC, b), bimolecular fluorescence complementation (BiFC, c), and co-immunoprecipitation (Co-IP, d). Negative controls included empty AD and BD vectors (a), nLUC-GUS and GFP-cLUC (b), and GUS-nYFP and GUS-cYFP (c). In (c), the H2B-mCherry fusion protein was co-expressed as a nuclear marker. In (d), total protein extracted from simulated controls or BYDV-infected barley plants (variety GoldenPromise) was immunoprecipitated (IP) using the anti-MS antibody developed in this study. BYDV 17K was found in the IP product derived from virus-infected plants (BYDV), but not in the product of the simulated control. (e) Co-immunoprecipitation assays showed the interaction between BYDV 17K and wheat TaMS1 / TaMS2. The experimental method was similar to (d), but the plant used was common wheat (variety Fielder). Scale bar, 40 μm. The results shown represent typical results from three independent experiments.

[0016] Figure 6The interaction of barley HvMS1 protein with five different RNA and DNA viral vectors (VSRs) was investigated. (a) SLC experiments showed the interaction of five VSRs (BSMV γb, PVX 25K, TRV 16K, BSCTV C2, and TBSV P19) with HvMS1. Negative controls in these experiments were nLUC-GUS and GFP-cLUC. (b) The interaction between HvMS1 and the five VSRs was verified by Co-IP experiments. Agrobacterium strains carrying constructs expressing FLAG-labeled HvMS1 or HA-labeled VSRs (γb, 25K, 16K, C2, or P19) co-infiltrated tobacco (N. benthamiana) leaf cells. After 48 hours, total protein was extracted from the infiltrated tissue and immunoprecipitated (IP) with an anti-FLAG antibody. The presence of VSRs in the IP products was detected by Western blotting with an anti-HA antibody. The results shown represent the consensus of three independent experiments.

[0017] Figure 7 The interaction of HvMS1 with four other RNA and DNA viral VSRs was investigated. (a) The interaction of HvMS1 with the four VSRs (PEBV 12K, WYMV P1, TuMV HC-Pro, and WDV Rep) was demonstrated by SLC experiments (7a). nLUC-GUS and GFP-cLUC were used as negative controls in these experiments. (b) The interaction of HvMS1 with these four VSRs was verified by Co-IP experiments. Agrobacterium strains carrying FLAG-labeled HvMS1 or HA-labeled VSRs (12K, P1, HC-Pro, or Rep) constructs were co-infected into tobacco leaf cells. Total protein was prepared from the infected tissue after 48 hours and immunoprecipitated (IP) with an anti-FLAG antibody. The presence of VSRs in the IP products was detected by Western blotting with an anti-HA antibody. The results shown are representative of three independent experiments.

[0018] Figure 8 The effects of TaMS1 / 2 gene editing mutants on BYDV infection in common wheat. (a) CRISPR / Cas9-mediated editing of three TaMS1 and three TaMS2 homologs using conserved sgRNA target sites is shown; PAM sequences are displayed in red, and Bsu36I restriction endonuclease cleavage sites are underlined. Three mutants were used in this study: MS1... AAbbdd / MS2 AAR49Sdd MS1 aabbdd / MS2 AAR49Sdd and MS1 aabbdd / MS2 aaR49SDD(Table 1, denoted as II-IV in this figure). (b) TaMS protein levels in WT Fielder (I) and the three mutants (II-IV) were compared by immunoblotting analysis using polyclonal anti-MS antibodies. Plants were grown under normal conditions, and samples were collected and analyzed at the three-leaf stage. (c) The relative expression levels of BYDV 17K transcription in WT Fielder and the three mutants were detected by RT-qPCR at 21 days post-infection (DPI), with Fielder's value set as 1 for comparison. (d) Differences in BYDV 17K protein accumulation levels in WT Fielder and the three mutants were detected by immunoblotting at 21 DPI. (e, f) Effects of BYDV infection on root morphology (e) and maximum root length (f) in WT Fielder and the three mutants. Plants were grown hydroponically, and samples were collected and analyzed at 21 DPI. TaMS (b) and 17K (d) band intensities were quantified using ImageJ. The values ​​shown in (c) and (f) are mean ± standard deviation (SD), with each group of data representing three or more biological replicates. Statistical analysis was performed using Student's t-test. The results shown represent typical outcomes from three independent experiments.

[0019] Figure 9 For Ben's tobacco NbMSAnalysis of the defense capabilities of gene-edited mutants against PVX or BSCTV infection. (a) Schematic diagram of the sgRNAs and target sites of the four NbMS members (NbMSa-NbMSd) of *N. benthamiana*, with BclI restriction endonuclease sites underlined and PAM sequences highlighted in red. (b) Nucleotide changes (shown in red) in two nbms double-gene mutants (nbms ac and nbms ad) used for further experiments. (c) Immunoblotting showed that the NbMS protein levels in the nbms ac and nbms ad mutants were lower than the Cas9 / Nb control, detected using a polyclonal anti-MS antibody. These results were obtained from plants grown for three weeks under standard greenhouse conditions. (d, e) Morphological differences (d) and plant height changes (e) of Cas9 / Nb, nbms ac, and nbms ad 14 days after PVX-GFP infection (DPI). Three plant types in (f, g) and (d) were used to detect PVX coat protein gene (CP) transcription level (f) and PVX-mediated GFP expression (g) by RT-qPCR or immunoblotting. (h) Symptoms of Cas9 / Nb, nbmsac, and nbmsad at 20 DPI of BSCTV infection were recorded, with two mutants exhibiting a more severe top-rolling phenotype than Cas9 / Nb. (i) BSCTV infection progression in the two mutants was faster than in Cas9 / Nb, determined by assessing the percentage of infected plants (n = 20 plants per genotype) from 13 to 23 DPI. (j, k) BSCTV DNA accumulation at 20 DPI was compared among the three genotypes by qPCR (j) and DNA hybridization (k). Systematically infected leaves were used in the experiments. In (j), the values ​​for the two mutants were normalized to Cas9 / Nb and set to 1 for comparison. In (k), the locations of the open circular (OC), supercoiled (SC), and single-stranded (SS) DNA of BSCTV are indicated, with ethidium bromide-stained genomic DNA used as a loading control. MS protein bands in (c) and GFP bands in (g) were quantified using ImageJ. Values ​​for each genotype are the mean ± SD (e) of at least 10 different plants, or the mean ± SD of three biological replicates (f and j). Statistical analysis was performed using Student's t-test. Scale bar: 5 cm. Results shown are reproducible in three independent experiments.

[0020] Figure 10 For Ben's tobacco NbMS Analysis of the defense capabilities of gene-edited mutants against TRV infection. (a, b) were compared at 14 days post-TRV-GFP infection (DPI), with the control group (Cas9 / Nb) and two... nbms Double mutant ( nbms ac and nbms ad Comparisons were made regarding morphological differences (a) and plant height variations (b). (c, d) RT-qPCR or Western blot analysis was performed on the TRV capsid protein gene (CP) transcription level (c) and TRV-mediated GFP expression (d) at 14 DPI for the three plant types shown in (a). Values ​​are the mean ± SD of at least 10 plants (b) or three biological replicates (c) for each genotype, with mean values ​​compared using Student's t-test. In (d), Ponceau S staining of the Rubisco large subunit was used as a loading control; GFP band intensity was quantified using ImageJ. The results shown are representative data from three independent experiments.

[0021] Figure 11To enhance antiviral defense in common wheat by overexpressing HvMS1. Specifically, (a) both total MS protein and FLAG-labeled HvMS1 expression were increased in two common wheat overexpression (OE) lines (OE-33 and OE-53). Ponceau S staining of the Rubisco large subunit served as a loading control. (bd) Overall growth morphology (b), plant height (c), and maximum root length (d) of WT Fielder and the two OE lines were assessed 21 days after BYDV infection (DPI). Hydroponic plants of the three genotypes were inoculated with BYDV at the two-leaf stage. (eg) Differences in the accumulation of BYDV subgenomic RNAs (RNA1, RNA2, and RNA3) (e), 17K transcriptional levels (f), and 17K protein abundance (g) were detected in the three plant types shown in (b) by RNA blotting, RT-qPCR, or immunoblotting. (h) BSMV-induced striped mosaic symptoms in leaves of WT Fielder and the two OE lines were recorded at 7 DPI. (i, j) Transcriptional levels (i) and protein abundance (j) of the BSMV capsid protein (CP) gene were analyzed in the three plant types shown in (h) by RT-qPCR or immunoblotting. In (b), 25S rRNA hybridization served as a loading control. In (g) and (j), immunoassay of wheat actin served as a loading control. In (a), (g), and (j), the intensity of the protein bands was quantified using ImageJ. The values ​​shown in (c), (d), (f), and (i) are mean ± SD, and each data point was from four or more biological replicates, and statistical analysis was performed using the Student's t-test. Scale bar, 3 cm. The results presented are representative of three independent experiments.

[0022] Figure 12 Tobacco overexpression of HvMS1 enhances antiviral defense. Among them, (a) two... N. benthamianaIncreased abundance of total MS protein and expression of the HA marker HvMS1 were observed in the overexpression lines (OE-19 and OE-26). Ponceau S staining of the Rubisco large subunit was used as a loading control. (b, c) Morphological differences (b) and plant height changes (c) among different plants 14 days after PVX-GFP infection: N. benthamiana (Nb, as wild-type control), OE-19, and OE-26. (d, e) Differences in PVX capsid protein gene (CP) transcription levels (d) and PVX-mediated GFP expression (e) were detected in the three plants shown in (b). (f) Compared with Nb, the BSCTV infection process in the two OE lines was slower, determined by calculating the percentage of infected plants from day 13 to day 23 of infection for each genotype (n = 20 plants for each genotype). (g) BSCTV infection symptoms of Nb, OE-19, and OE-26 at 20 days post-infection, with significantly reduced leaf tip curling phenotype in the two OE lines. (h, i) Comparison of BSCTV DNA accumulation in the three genotypes at 20 days post-infection by qPCR (h) and DNA blotting (i). Systemically infected leaves were used in the experiments. In (h), values ​​for the two OE lines were normalized to Nb (set to 1 for comparison). In (i), open circular (OC), supercoiled (SC), and single-stranded (SS) DNA sites of BSCTV were marked, with EB-stained genomic DNA used as a loading control. MS protein bands in (a) and GFP bands in (e) were quantified using ImageJ. Values ​​are the mean ± SD of at least 10 different plants (c) or three biological replicates (d and h) for each genotype. Statistical analysis was performed using Student's t-test. Scale bar, 5 cm. Three independent experiments showed high consistency.

[0023] Figure 13 To enhance host defense against TRV in tobacco through overexpression of HvMS1. (a, b) Morphological differences (a) and plant height variations (b) of HA-tagged HvMS1 protein expression in wild-type control (Nb) and two different OE lines (OE-19 and OE-26) were analyzed at 21 days after inoculation (DPI). (c, d) Transcriptional levels of the TRV capsid protein gene (CP) and TRV-mediated GFP expression (d) were determined by RT-qPCR or Western blot analysis in the three plant types shown in (a) at 21 DPI. Data for each genotype are presented as mean ± standard deviation from at least 10 plants (b) or three biological replicates (c), with means statistically compared using the Student t-test. In (d), Ponceau S staining of the Rubisco large subunit served as a loading control; GFP band intensity was quantified using ImageJ. The results shown were reproducible in three independent experiments.

[0024] Figure 14 HvMS1 was used to suppress the function of six different viral silencing repressors (VSRs). (a) lists 10 VSRs and their corresponding viruses, belonging to five viral families and possessing positive-sense single-stranded RNA (ssRNA) or single-stranded DNA (ssDNA) genomes. (b) HvMS1 suppressed the function of six viral VSRs (BYDV 17K, BSMV γb, TRV 16K, PVX 25K, BSCTV C2, and TBSV P19). (c, d) The effects of infiltration with 35S::GUS or 35S::HvMS1 on GFP transcription levels (c) and GFP protein abundance (d) in the presence of different VSRs were detected by RT-qPCR or Western blotting three days after the second infiltration. In (c), relative values ​​are taken as the mean ± standard deviation (SD) of three biological replicates, with 35S::GUS (set to 1) as the standard. In (d), GFP band intensity was quantified using ImageJ. The molecular weight standards marked with * and ** are 30 kDa and 55 kDa, respectively. Statistical analysis was performed using Student's t-test (****, P<0.001). The results shown are reproducible in three independent experiments.

[0025] Figure 15 HvMS1 was used to inhibit the function of four different viral silencing repressors (VSRs). (a) The inhibition of gene silencing function of four VSRs (PEBV 12K, WYMV P1, TuMV HC-Pro, and WDV Rep) by HvMS1. (b, c) The effects of inoculation with 35S::GUS or 35S::HvMS1 constructs on GFP transcription levels (b) and GFP protein abundance (c) in the presence of different VSRs. RT-qPCR or Western blot analysis was performed three days after secondary inoculation. In (b), relative values ​​normalized to 35S::GUS (set to 1) are the mean ± standard deviation of three biological replicates. In (c), GFP band intensity was quantified by ImageJ. Molecular weight standards marked * and ** are 30 and 55 kDa, respectively. Statistical analysis was performed using Student's t-test (****, P < 0.001). The results shown represent typical results from three independent experiments. Detailed Implementation

[0026] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0027] This invention provides the application of the MS1 gene or its encoded protein in improving plant disease resistance.

[0028] In some specific embodiments, the nucleotide sequence of the HvMS1 gene is shown in SEQ NO 1, and the protein sequence it encodes is shown in SEQ NO 2.

[0029] In some specific implementations, overexpression of the MS1 gene or increasing the level of its encoded protein can enhance the plant's disease resistance.

[0030] In some specific implementations, the disease resistance includes plant diseases caused by barley yellow dwarf virus, barley stripe virus, potato virus X, tobacco brittleness virus, pea early brown virus, wheat yellow mosaic virus, turnip mosaic virus, wheat dwarf virus, and beet severe crooked top virus.

[0031] In some specific implementations, the disease resistance includes resistance to plant diseases caused by barley yellow dwarf virus, potato virus X, tobacco brittle virus, and beet severe crescent virus.

[0032] The present invention also provides the application of the MS1 gene or recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in the cultivation of plant lines with high disease resistance.

[0033] This invention also provides a method for improving plant disease resistance by overexpressing the MS1 gene or increasing the level of its encoded protein.

[0034] This invention also provides a method for cultivating plant strains with high disease resistance, which involves overexpressing the MS1 gene using a recombinant vector containing the MS1 gene, an expression cassette, a transgenic cell line, or a recombinant bacterium.

[0035] Virus-resistant crops bred using MS gene editing (such as CRISPR / Cas9-mediated silencing or weak allelic mutations) can fundamentally solve the problems of chemical pesticide pollution and resistance loss. Taking wheat-growing areas in China as an example, MS technology can simultaneously improve crop tolerance to multiple biological stresses (e.g., reducing ethylene sensitivity can alleviate Fusarium head blight damage) against major viruses such as BYDV and yellow mosaic virus, as well as multiple stresses. It is estimated that if the incidence of BYDV in wheat is reduced by 50%, the annual yield increase in the North China Plain could reach 420 million kg (calculated based on a median yield reduction of 19 kg / ha / 1% incidence rate). Against the backdrop of global climate change exacerbating virus transmission (Pequeno et al., 2024), this technology provides an innovative and sustainable solution for ensuring food security.

[0036] Example 1 MS genes are highly conserved in plants. Previous research indicated that the protein P4 (17K) encoded by BYDV ORF4 is a multifunctional protein and a viral silencing repressor (Jin et al 2020, 2022). Based on this, our laboratory prepared Arabidopsis overexpression transgenic lines 17K-GFP and GFP driven by inducible promoters. IP-MS / MS analysis of these overexpression materials identified several candidate proteins interacting with 17K. One of these candidate proteins is the Arabidopsis methionine synthase AtMS1 (the identified peptide sequence is (SEQ ID NO.3): YLFAGVVDGR). Figure 1 (a) This protein is the terminal enzyme in the methionine cycle for the synthesis of methionine. The gene encoding this protein is AT5G17920, which is 2298 bp in length, and the predicted molecular weight of the protein is approximately 84.4 kDa.

[0037] To further verify whether there is an interaction between 17K and AtMS1, the yeast two-hybrid (Y2H) assay, luciferase protein complementation assay (LCA), and co-immunoprecipitation (Co-IP) were used. The results showed that BYDV-GAV 17K interacts with AtMS1.

[0038] In the Y2H experiment, AtMS1 was linked to AD (pGADT7), and BYDV-GAV 17K was linked to BD (pGBKT7). Negative and positive controls were also included to verify the reliability of the results. The yeast two-hybrid experiment results showed that BYDV-GAV 17K and AtMS1 can interact (…). Figure 1 (b)

[0039] In the LCA experiment, AtMS1 was linked to CLuc (pCambia1300-CLuc) in AtMS1-CLuc, BYDV-GAV17K was linked to NLuc (pCambia1300-NLuc) in 17K-NLuc, and three control groups—CLuc-GFP with NLuc-Gus, 17K-NLuc with CLuc-GFP, and AtMS1-CLuc with NLuc-Gus—were used to verify the reliability of the results. Luciferase protein complementation experiments showed that BYDV-GAV 17K and AtMS1 can interact (…). Figure 1 (c)

[0040] In the Co-IP experiment, seedlings were first cultured in 1 / 2 MS medium. After one week, the seedlings were transferred to 1 / 2 MS medium containing estradiol (β-ES, 5 μM) and cultured for 48 h. Residual medium was removed, and samples were collected. The samples were thoroughly ground, lysis buffer was added, and the mixture was incubated on ice for 30 min, then at 12000 rpm for 20 min. The supernatant (protein lysis buffer) was collected, and this process was repeated once. 100 μL of the supernatant was retained for input. GFP-Beads were prepared and added to the supernatant protein lysis buffer. The mixture was incubated at 4°C for 4 h by rotation. The GFP-Beads were washed three times, and 40 μL of 4× Loading buffer was added. The mixture was boiled at 95°C for 5 min, and the protein was separated using 12% SDS-PAGE. Protein immunoassays were performed using anti-GFP, anti-HvMS1, and anti-BYDV 17K antibodies. The results showed that in Arabidopsis thaliana 17K-GFP overexpression materials, 17K and AtMS1 could interact (…). Figure 1 (d).

[0041] Since Arabidopsis is not a natural host of BYDV, we searched the genome databases of Arabidopsis, wheat, barley, maize, rice and Nicotiana benthamiana. Three homologous genes were found in the Arabidopsis database: AtMS1 (AT5G17920), AtMS2 (AT3G03780) and AtMS3 (AT5G20980). Two barley homologs were retrieved from the Poaceae database: HvMS1 (HORVU4Hr1G002270) and HvMS2 (HORVU5Hr1G006780); two wheat homologs: TaMS1 and TaMS2. Since wheat is a hexaploid crop, each has three subgenomic homologs: TaMS1-A (TraesCS4A02G298700), TaMS1-B (TraesCS4B02G014700), TaMS1-D (TraesCS4D02G012900), TaMS2-A (TraesCS5A02G024900), TaMS2-B (TraesCS5B02G022800), and TaMS2-D (TraesCS5D02G031300); and three maize homologs: ZmMS1... (Zm00001d033480), ZmMS2 (Zm00001d013644) and ZmMS3 (Zm00001d031128); two rice homologs are OsMS1 (Os12g0623900) and OsMS2 (Os12g0624000). In addition, Nicotiana benthamiana also has two homologous genes, namely NbMS1 and NbMS2. Since Nicotiana benthamiana is an allotetraploid crop, NbMS1 has four subgenomes, namely NbMS1a (Niben101Scf03634g04009), NbMS1b (Niben101Scf07438g04014), NbMS1c (Niben101Scf09725g00022) and NbMS1d (Niben101Scf01812g02025).

[0042] Using MatGAT2.01 (Campanella et al., 2003), the identity and similarity of protein sequences from 20 MS proteins of different species were analyzed. The results showed that barley HvMS1 and HvMS2 exhibited high identity and similarity, reaching 92.8% and 95.8%, respectively. Wheat TaMS1 showed 98% and 92% identity and similarity with barley HvMS1, and wheat TaMS2 showed 92% and 92% similarity with barley HvMS2, respectively. Dicotyledonous model plants such as Arabidopsis and tobacco also showed high identity and similarity with gramineous crops, all exceeding 70%, and even reaching 89%. This indicates that MS proteins are highly conserved in higher plants. Figure 2 ).

[0043] Example 2 MS gene expression in cereals is induced by BYDV The HvMS1 gene sequence in this embodiment is SEQ ID NO.1.

[0044]

[0045] Its encoded protein sequence is shown in SEQ ID NO.2: SEQ ID NO.2:

[0046] Barley seedlings 5-7 days after germination were inoculated with aphids carrying BYDV-GAV and those not carrying BYDV-GAV. Samples were taken at 0, 4, 6, 8, 10, and 14 days after inoculation. Changes in HvMS expression levels were detected at the protein level. Within 0-14 days of BYDV infection, HvMS expression gradually increased, reaching a peak at 10 DPI, and then decreased. Figure 3 (a) In the control group (Mock) inoculated with aphids not carrying BYDV, the expression level of HvMS did not change significantly. It is worth noting that the accumulation trend of BYDV 17K protein in susceptible barley plants was highly consistent with that of HvMS protein. RT-qPCR was used to detect... HvMS Similar results were obtained from RNA level detection. HvMS The expression level showed a trend of first increasing and then decreasing. Figure 3 (b) These results indicate that BYDV infection can simultaneously upregulate... HvMS The transcription and protein product levels are synchronized with viral gene expression and genome replication.

[0047] Similarly, using wheat seedlings 5-7 days after germination, after inoculation with BYDV-GAV, the changes in TaMS expression levels at both RNA and protein levels were detected. TaMS expression levels gradually increased with increasing infection days, reaching a peak at 10 DPI, and then decreased. Similar results were obtained for RNA level detection. TaMS The expression level showed a trend of first increasing and then decreasing in uninfected wheat. TaMS There was no obvious trend in the change in expression levels. Figure 4 (bc).

[0048] Given that the expression of barley MS protein is induced by BYDV, various experiments, including Y2H, LCA, bimolecular fluorescence complementation (BiFC), and immunoco-IP, were used to verify the interaction between barley MS protein and BYDV 17K. Figure 5 ad.

[0049] Meanwhile, experiments using LCA and Co-IP confirmed the interaction between wheat MS protein and BYDV 17K. Figure 4 a, Figure 5 (e).

[0050] Example 3 MS gene interacts with VSR proteins of various viruses. As research into different viruses deepens, it has been discovered that viruses possess one or more viral silencing repressors, which are not only pathogenic factors but also effective targets for plant defense against viruses. Therefore, this study used LCA and Co-IP experiments to verify the interaction between viral silencing repressors of seven RNA viruses (BSMV) γb, potato virus X (PVX) P25 (25K), tobacco brittle virus (TRV) 16K, pea early brown virus (PEBV) 12K, wheat yellow mosaic virus (WYMV) P1, turnip mosaic virus (TuMV) HC-Pro, tomato dwarf virus (TBSV) P19, beet severe crooked top virus (BSCTV) C2, wheat dwarf virus (WDV) Rep, and MS proteins, represented by HvMS1. The results showed that there is an interaction between the VSR proteins of these nine viruses and HvMS1. Figure 6-7 ).

[0051] Example 4 Reducing MS gene expression weakens plant resistance to viruses: 1. BSMV-VIGS Silent Barley HvMSs Inoculation with BYDV Trial To further elucidate the biological significance of the interaction between 17K and HvMS, BSMV-VIGS technology (Yuan, 2011) was used to specifically silence HvMS1 and HvMS2 transcript mRNA in barley. After inoculation with BYDV-GAV, the effect of BYDV on silencing the barley HvMS gene was identified. Barley seedlings were rubbed with BSMV-EV (empty vector control) and BSMV-HvMS for 7-10 days. Samples were taken from newly emerged leaves of each individual plant, and total RNA was extracted. After reverse transcription, the silencing effect was detected using universal RT-qPCR primers for detecting HvMS1 and HvMS2 genes. It was found that the expression level of the barley HvMS gene decreased by approximately 50%. Figure 3 (c)

[0052] Plants with a silencing efficiency of 50% were classified into two groups: barley seedlings with HvMS gene silence and barley seedlings without HvMS gene silence. These groups were then inoculated with aphids carrying or not carrying BYDV-GAV, resulting in four groups of barley plants: barley without HvMS gene silence and not infected with BYDV-GAV, barley with HvMS gene silence and not infected with BYDV-GAV, barley with HvMS gene silence and infected with BYDV-GAV, and barley without HvMS gene silence and infected with BYDV-GAV. These plants were then cultured in an incubator for three weeks.

[0053] Twenty-one days after inoculation with BYDV-GAV virus, the BYDV phenotype of barley was observed, and the plant height of the four groups of barley plants was statistically analyzed. The results showed that barley plants with silenced HvMS genes and infected with BYDV-GAV exhibited the most severe disease susceptibility and the shortest plant height compared to the other three groups. Figure 3 (d). RT-qPCR results showed silencing. HvMSs Genes, BYDV viral accumulation increased ( Figure 3 Similar results were obtained from immunoblotting experiments (e). Figure 3 (f)

[0054] To further expand the application scope of MS proteins and explore the defense mechanisms of plants against viruses after gene mutation, mutants were created in wheat and Nicotiana benthamiana, respectively. Conserved gRNAs were designed using CRISPR / Cas9 gene editing technology to simultaneously edit wheat TaMS1 and TaMS2. After identification of the progeny, three mutants were screened: 1 AAbbdd / 2 AABBdd 1 aabbdd / 2 aaBBDD 1 aabbdd / 2 AABBdd To verify gene function in wheat, tobacco brittle virus-mediated gene editing (TRV-VIGE) technology was used to create mutants of tobacco NbMS1. After T1, T2, and T3 screening, two mutants were obtained. nbms1ac and nbms1ad Mutants. A study to verify tobacco resistance to viruses such as PVX and TRV in MS loss-of-function states. Figure 8 ab, Figure 9 (c).

[0055] 2. PVX inoculation test of Benedict's tobacco editing materials PVX-GFP recombinant virus via Agrobacterium infiltration (OD) 600 =0.001) Do not introduce receptor controls WT / Cas9 and double mutants. nbms1 ac and nbms1 ad Viral symptoms and gene expression levels were analyzed in plants of the strains after 14 days. Plant height statistics revealed that the height of nbms1 ac and nbms1 ad plants was significantly lower than that of WT / Cas9 plants. Figure 9 (de). RT-qPCR analysis showed that the RNA accumulation of the PVX CP gene in nbms1 ac and nbms1 ad plants was significantly higher than that in the WT / Cas9 control. Figure 9 Immunoblot analysis revealed that the expression level of PVX-mediated GFP protein was higher in nbms1ac and nbms1ad plants than in the WT / Cas9 control. Figure 9These results indicate that the mutant NbMS gene reduces the defense response of Nicotiana benthamiana to PVX infection, thereby promoting PVX accumulation.

[0056] 3. TRV inoculation test of Benedict's tobacco editing materials TRV-GFP recombinant virus is infiltrated by Agrobacterium tumefaciens (OD). 600 =0.001) were introduced into the receptor control WT / Cas9 and the double mutant, respectively. nbms1 ac and nbms1 ad In the plants of the strains, viral symptoms and gene expression levels were analyzed after 14 days. The mutants nbms1ac and nbms1ad showed significantly stronger symptoms than the WT / Cas9 control, and their plant height was significantly reduced. Figure 10 (ab). The expression level of TRV CP in both mutants was significantly higher than that in the WT / Cas9 control (ab). Figure 10 In the middle (c), TRV-mediated GFP protein expression levels were higher in nbms1 ac and nbms1 ad plants than in the WT / Cas9 control (c). Figure 10 (d). These data indicate that the mutant NbMS gene reduces the defense response of Nicotiana benthamiana to TRV infection, leading to enhanced TRV infectivity.

[0057] 4. BSCTV test on the inoculation of Benedictine tobacco editing materials BSCTV recombinant virus is infiltrated by Agrobacterium tumefaciens (OD). 600 =0.2) Introduced the receptor control WT / Cas9 and double mutant respectively. nbms1 ac and nbms1 ad In plants of the strain, the incidence of BSCTV was statistically analyzed, and the virus accumulation was detected by qPCR and DNA Blot. Mutants were also identified. nbms1 ac and nbms1 ad The onset of symptoms and the incidence rate were significantly earlier than in the WT / Cas9 control group. Figure 9 The expression level of BSCTV in both mutants was significantly higher than that in the WT / Cas9 control. Figure 9 In the middle (j), the accumulated amount of BSCTV virus is in nbms1 ac and nbms1 ad The plant ratio was higher than that of the WT / Cas9 control ( Figure 9 These data indicate that the mutated NbMS gene reduces the defense response of tobacco Benzodiazepines to BSCTV infection, leading to an increase in BSCTV virus accumulation.

[0058] 5. Trial of inoculating wheat editing materials with BYDV Wheat seedlings of uniform growth, both wild-type and mutant, were hydroponically cultured. Three days after transplanting, they were inoculated with wheat aphids carrying BYDV-GAV. Three days later, imidacloprid was sprayed to kill the aphids. Culture continued for 21 days, followed by phenotypic assessment and virus accumulation testing. Twenty-one days after BYDV inoculation, the wild-type seedlings showed significantly better growth vigor than the gene-edited lines. The virus-induced stunting in root length was more severe in the mutant lines than in the control group. The average longest root length of the wheat mutant lines was significantly lower than that of the control group. Figure 8 RT-qPCR analysis revealed elevated BYDV 17K expression in the mutant lines. Figure 8 In the immunoblotting experiment, the protein accumulation level of 17K in the mutant line was significantly higher than that in the wild-type control. Figure 8 (d).

[0059] Silencing HvMSs with BSMV-VIGS followed by inoculation with BYDV, inoculation of wheat gene-edited materials with BYDV, and nbms1 Inoculation with PVX, TRV, and BSCTV using two double mutants showed that MS expression was reduced, virus accumulation in plants was increased, and plant defense against the virus was reduced.

[0060] Example 5 Overexpression of the MS gene increases plant resistance to viruses. To further verify the plant's defense against the virus after overexpressing the MS gene, wheat and Nicotiana benthamiana lines overexpressing HvMS1 were created. After screening at T1, T2, and T3 and immunoblotting, homozygous lines were used for subsequent experiments (Figure 11a, ...). Figure 12 (a)

[0061] 1. Wheat HvMS1 overexpressing lines inoculated with BYDV experiment Seedlings with uniform growth were selected for hydroponics. Three days after transplanting, they were inoculated with the wheat aphid carrying BYDV-GAV. One day after inoculation, the number of aphids on each plant was observed, and additional aphids were added as needed to ensure a consistent number of aphids per plant. Three days later, imidacloprid was sprayed to kill the aphids. Culture continued for 21 days, followed by phenotypic assessment and virus accumulation testing. Twenty-one days after BYDV inoculation, the overexpressing lines showed significantly better growth than the control group, significantly alleviating virus-induced stunting. Plant height and longest root length showed varying degrees of difference; the average plant height and longest root length of the overexpressing lines OE-33 and OE-53 were significantly greater than those of the control group. Figure 11 (middle bd).

[0062] Total RNA was extracted from the overexpression lines and control materials. RNA blot hybridization experiments revealed that the accumulation of BYDV subgenomic RNA1 and RNA3 was significantly reduced in the overexpression lines OE-33 and OE53, and the accumulation level of RNA2 also decreased. Figure 11 (e); RT-qPCR detection revealed that the expression level of BYDV 17K in OE-33 and OE-53 was reduced several times (e). Figure 11 In the immunoblotting assay, the protein accumulation levels of 17K in OE-33 and OE-53 were reduced to 42% and 54% of those in the wild-type control, respectively. Figure 11 (g).

[0063] Overexpression lines OE-33 and OE-53, and the control material Fielder (FLD), were germinated. Seedlings of uniform growth were transplanted into hydroponic containers. When the seedlings reached the stage of one leaf and one bud, BSMV was inoculated into these materials using a friction inoculation method. Mosaic symptoms were observed 5-7 days later. The mosaic symptoms appeared earlier in FLD than in the overexpression lines OE-33 and OE-53. As time progressed, the mosaic symptoms in FLD became more pronounced, while the mosaic symptoms in the overexpression lines OE-33 and OE-53 were milder when infected with BSMV. Figure 11 (h). Taking BSMV CP as a representative, the viral gene expression in three genotypes was compared. The accumulation of BSMV virus in OE-33 and OE-53 plants was analyzed at both RNA and protein levels using RT-qPCR and Western blotting. The results showed that the RNA level and protein product accumulation of BSMV CP were significantly lower than those in the wild-type control plant. Figure 11 (ij), which is consistent with the milder BSMV infection symptoms shown in OE-33 and OE-53 plants.

[0064] 2. PVX inoculation experiment of HvMS1 overexpressing lines in Tobacco Benedictine var. ... PVX-GFP recombinant virus via Agrobacterium infiltration (OD) 600 =0.001) technology was used to introduce wild-type Nicotiana benthamiana (Nb) and two HvMS1-overexpressing tobacco lines (OE19 and OE30). Viral symptoms and gene expression levels were analyzed 14 days later. Phenotypic observation and plant height statistics revealed that HvMS1-overexpressing tobacco showed milder PVX symptoms, more normal growth, and significantly higher plant height than the wild-type control. Figure 12 In tobacco plants overexpressing HvMS1, the expression level of PVX CP was significantly lower than that of the control tobacco plant (bc). Figure 12 In the middle (d), the expression level of GFP protein was also lower than that in the control (d). Figure 12(e). These results indicate that overexpression of HvMS1 in tobacco enhances the defense response of Tobacco Benzoviae to PVX infection, thereby downregulating PVX accumulation.

[0065] 3. TRV inoculation test of HvMS1 overexpressing lines in Tobacco Benedictine bursonii TRV-GFP recombinant virus is infiltrated by Agrobacterium tumefaciens (OD). 600 The TRV-GFP assay was used to introduce wild-type Nicotiana benthamiana and two HvMS1-overexpressing Nicotiana lines. Viral symptoms and gene expression levels were analyzed 20 days later. After TRV-GFP infection, apical bud growth in Nicotiana benthamiana was inhibited, and lateral branch growth was faster than that of the main branch. However, the growth and development of HvMS1-overexpressing Nicotiana benthamiana were less affected, and its plant height was significantly higher than that of the wild-type control. Figure 13 In HvMS1-overexpressing tobacco plants, the expression level of TRV CP was significantly lower than that of the control tobacco (Nicotiana benthamiana). Figure 13 In the middle (c), the expression level of GFP protein was also lower than that in the control (c). Figure 13 (d). These data indicate that overexpression of HvMS1 in tobacco significantly enhances the defense response of Tobacco Benzoviae to TRV infection, leading to a downregulation of TRV accumulation levels.

[0066] 4. BSCTV test of HvMS1 overexpressing lines in Tobacco Benedictine bursonii. BSCTV recombinant virus was introduced into the recipient control WT / Nb and overexpression lines via Agrobacterium infiltration (OD600=0.2). The incidence of BSCTV was statistically analyzed, and viral accumulation was detected by qPCR and DNA Blot. The onset of symptoms and the incidence rate in the overexpression lines were significantly later than those in the WT control. Figure 12 (fg). The accumulation of BSCTV virus in the overexpression lines was lower than that in the WT / Nb control (Figure 12 hj). These data indicate that overexpression of HvMS1 enhances the defense response of Nicotiana benthamiana against BSCTV infection and reduces the accumulation of BSCTV in Nicotiana benthamiana.

[0067] Overexpression of HvMS1 in plants such as wheat and tobacco increases the expression level of HvMS1 and enhances the resistance of these plants to viruses such as BYDV, PVX, TRV, and BSCTV, indicating that the MS gene has certain broad-spectrum disease resistance characteristics.

[0068] Example 6 MS enhances plant resistance to viruses by inhibiting the activity of viral VSR. Based on the results of this invention, it is hypothesized that the host MS protein may interact with the VSR protein of plant viruses to inhibit the activity of their gene silencing repressors, thereby enhancing the host's viral resistance. To verify this hypothesis, this invention used the 16c tobacco line as material and HvMS1 as a representative of plant MS proteins to investigate whether MS expression can broadly inhibit the gene silencing repressor activity of VSR proteins from eight different RNA viruses and two different DNA viruses. The 10 viruses are BYDV-GAV, BSMV, PVX, TRV, PEBV, Wheat yellow mosaic virus (WYMV), Turnip mosaic virus (TuMV), Tomato bushy stunt virus (TBSV), Wheat dwarf virus (WDV), and Beet severe curly topvirus (BSCTV) (Chiu, 2010; Martínez-Priego et al, 2008; Mathioudakis et al, 2018; Mérai, 2006; Okano et al, 2014; Tilsner et al, 2012b; Yelina et al, 2002). Their classification, genomic categories, and VSR protein information are shown in Table 1.

[0069] Table 1. Information on 10 different plant viruses and their VSR proteins To this end, VSR expression sequences from different viral sources were constructed into the T-DNA expression plasmid pEG100, resulting in expression vectors for 10 VSRs: BYDV 17K-pEG100 (35S::17K), TBSV P19-pEG100 (35S::P19), BSMV γb-pEG100 (35S::γb), TRV 16K-pEG100 (35S::16K), PEBV 12K-pEG100 (35S::12K), PVX25K-pEG100 (35S::25K), WYMV P1-pEG100 (35S::P1), TuMV HC-Pro-pEG100 (35S::HC-Pro), BSCTV C2-pEG100 (35S::C2), and WDV Rep-pEG100. (35S::Rep). Different VSR expression vectors were mixed with 35S::GFP and co-expressed in 16c tobacco leaves. It was found that the introduction of all 10 35S::VSR vectors inhibited gene silencing in 16c tobacco, thereby promoting 35S::GFP expression and producing stronger GFP fluorescence than that introduced by 35S::EV (empty vector control). Figure 14-15 Therefore, it was determined that the VSR proteins of these 10 viruses all have significant transcriptional silencing repressor activity in 16c tobacco.

[0070] Based on the above results, this invention further analyzed whether transient expression of HvMS1 could inhibit the gene silencing repressor activity of VSR in 16c tobacco. First, 35S::GUS (expressing β-glucuronidase, used as a negative control) and 35::HvMS1 were injected into 16c tobacco leaves to form a small injection area; 24 h later, 35S::VSR and 35S::GFP were mixed at an OD600 ratio of 0.2 and 0.5, and injected a second time at the same location to form a larger injection area. 2-4 days later, the injection area was irradiated with long-wavelength ultraviolet light, and the GFP green fluorescence signal was detected. Figure 14 (b) In the inner circle treatment that simultaneously expresses HvMS1 and VSR, the GFP green fluorescence signal decreased, while in the inner circle treatment that simultaneously expresses GUS and VSR, the GFP green fluorescence signal did not change significantly. Therefore, it is speculated that HvMS1 expression can downregulate the gene silencing repressor activity of VSR.

[0071] Subsequently, the transcriptional level of GFP under different treatments was examined. The results showed that expression of 35S::17K, 35S::γb, 35S::16K, 35S::25K, 35S::C2, or 35S::P19 reduced GFP mRNA expression by approximately 30%, while expression of 35S::γb, 35S::12K, 35S::P1, or 35S::C2 led to a 10%–15% reduction in mRNA expression. Figure 14 (c)

[0072] Further examination of GFP protein level changes in different experimental combinations showed that the presence of 35S::17K, 35S::γb, 35S::16K, 35S::25K, 35S::C2, or 35S::P19 reduced GFP protein levels by approximately 38%–55%. Specifically, 35S::16K or 35S::Rep resulted in a reduction of approximately 38% in GFP protein levels, while 35S::17K and 35S::γb reduced protein levels by approximately 55% and 45%, respectively. Figure 14 (d). Therefore, it was determined that the HvMS1 protein can effectively inhibit the silencing repressor activity of the VSRs of these 10 viruses in 16c tobacco.

[0073] Similarly, the transcriptional level of GFP under different treatments was examined, and the results showed that GFP mRNA expression levels decreased by approximately 29%–55% under the expression of 35S::12K, 35S::P1, 35S::HC-Pro, and 35S::Rep. Figure 15 (ab). Further examination of GFP protein level changes in different experimental combinations showed that the GFP protein level decreased by approximately 29%–55% in the presence of 35S::12K, 35S::P1, 35S::HC-Pro, and 35S::Rep. Among these, 35S::HC-Pro caused a decrease of approximately 55% in GFP protein level, while 35S::12K, 35S::Rep, and 35S::P1 caused decreases of approximately 29%, 32%, and 38%, respectively. Figure 15 (c). Therefore, it was determined that the HvMS1 protein can effectively inhibit the silencing repressor activity of the VSRs of these 10 viruses in 16c tobacco.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of MS1 gene or protein coded by the same in improving disease resistance of plants.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the MS1 gene is shown as SEQ ID NO. 1, and the protein sequence coded by the same is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, Overexpression of the MS1 gene or improvement of the protein level coded by the same can improve the disease resistance of plants.

4. Use according to claim 1, characterized in that, The disease resistance includes plant diseases caused by barley yellow dwarf virus, barley stripe virus, potato virus X, tobacco virus, pea early brown virus, wheat yellow mosaic virus, turnip mosaic virus, wheat dwarf virus and beet severe curly top virus.

5. The use according to claim 1, characterized in that, The disease resistance includes plant diseases caused by barley yellow dwarf virus, potato virus X, tobacco virus and beet severe curly top virus.

6. Use of MS1 gene or recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the same in breeding plant lines with high disease resistance.

7. A method for increasing disease resistance in plants, characterized by, Overexpression of the MS1 gene or improvement of the protein level coded by the same can improve the disease resistance of plants.

8. A method of breeding a plant line with high disease resistance capacity, characterized by, The MS1 gene is overexpressed by using recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the same.

Images