Methods and applications of thiamine in the prevention and control of maize chlorotic mottle virus

Abstract

This invention belongs to the field of plant protection, specifically relating to the application of thiamine in the control of maize chlorotic mottle virus (MCMV). This invention elucidates the molecular mechanism by which thiamine enhances the host's antiviral activity through transcriptome sequencing. It was found that MCMV infection affects the transcriptional changes of genes related to the thiamine metabolic pathway, leading to the inhibition of endogenous thiamine biosynthesis in plants. After MCMV infection, the interaction between p31 and ZmTHIC alters the localization of ZmTHIC, thereby inhibiting thiamine synthesis. Exogenous application of thiamine enhances maize's resistance to MCMV. Thiamine inhibits MCMV infection by inducing the MAPK pathway to enhance plant defense responses and by inducing the phenylpropanone biosynthesis pathway to promote lignin synthesis in plant cell walls. Therefore, exogenous application of thiamine can promote maize growth, enhance plant defense responses, and increase lignin synthesis, thereby increasing resistance to viral diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant protection, and specifically relates to a method for preventing and controlling maize chlorotic mottle virus. Background Technology

[0002] Maize chlorotic mottle virus (MCMV) belongs to the genus *machlomovirus* within the family Tomatobushviridae. Its virus particles are approximately icosahedral in diameter, around 30 nm. MCMV primarily infects maize, sugarcane, and millet, mainly spreading through vectors such as thrips, but can also be transmitted through mechanical friction and seeds. MCMV infection alone causes mild chlorotic mottle symptoms at the base of maize leaves, which gradually progress upwards to the leaf tips. In severe cases, the chlorotic areas merge to form leaf spots. When MCMV co-infects with viruses from the family Potyviridae, it causes a severe maize viral disease, maize lethal necrosis disease (MLND), resulting in significant yield losses. The MCMV genome is a single-stranded, positive-sense RNA virus containing two subgenomes: sgRNA1 and sgRNA2. The MCMV genome directly encodes P32, P50, and the P50 readthrough protein P111. Among them, sgRNA1 can encode P7a, P7b, P31, and cp, while sgRNA2 does not encode a protein. P31 is the main pathogenic factor of MCMV, which helps the virus carry out systemic movement. The P31 protein can hijack ZmCAT1 to enhance the accumulation of P31 and weaken SA-mediated defense. The P31 protein can also directly target ZmPAO1 and enhance enzyme activity to counteract Zma-miR167-mediated defense to some extent (Liu X, Liu S, Chen X, et al. Maize miR167-ARF3 / 30-polyamine oxidase 1module-regulated H2O2 production confers resistance to maize chlorotic mottlevirus. Plant Physiol. 2022 Jun 1; 189(2):1065-1082.).

[0003] Thiamine, also known as vitamin B1 (VB1), is an essential micronutrient for all organisms. Its two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and 4-methyl-5-(2-hydroxyethyl)-thiazolium phosphate, are catalyzed by pyrimidine synthase (THIC) and thiazolium synthase (THI1), respectively. Thiamine exists in three forms: thiamine monophosphate (TMP), thiamine diphosphate (TPP), and thiamine triphosphate (TTP). Thiamine pyrophosphate is the activated form of thiamine. As a coenzyme factor in important enzymatic reactions such as pyruvate dehydrogenase complex (PDHC), α-ketoglutarate dehydrogenase complex (KGDHC), and transketolase (TK), it participates in many metabolic activities and plays a crucial role. Thiamine is essential for all organisms; thiamine deficiency in plant seedlings will cause chlorosis, and in severe cases, death. It plays an important role in plant growth and development, as well as in biotic and abiotic stress (Dong W, Stockwell VO, Goyer A. Enhancement of Thiamin Content in Arabidopsis thaliana by Metabolic Engineering. Plant Cell Physiol. 2015 Dec; 56(12):2285-96.). Thiamine can trigger the plant's defense system. When thiamine is applied exogenously to prevent pathogen infection, pathogen-related proteins accumulate rapidly (Boubakri H, Gargouri M, Mliki A, et al. Vitamins for enhancing...). Plant resistance. Planta. 2016 Sep; 244(3):529-43. doi:10.1007 / s00425-016-2552-0. Epub2016Jun17. PMID:27315123.); Studies have shown that the 17K protein of barley yellow dwarf virus-GAV (BYDV-GAV) can disrupt thiamine biosynthesis, thereby promoting plant virus infection. This indicates that there is a link between thiamine biosynthesis and antiviral ability in plants.However, this discovery does not prove that thiamine can directly fight viruses, but rather suggests a potential role of thiamine in plant defense against viruses. The molecular mechanism by which it has a broad spectrum of disease resistance remains to be elucidated (Han X, Yang X, Chen S, et al. Barley yellow dwarf virus-GAV 17K protein disrupts thiamine biosynthesis to facilitate viral infection in plants. Plant J. 2024 Jul; 119(1):432-444.). Thiamine, as a naturally occurring nutrient, has higher environmental compatibility when used to improve plant resistance to viral diseases, which is beneficial to sustainable agricultural development. However, its mechanism of action in plants is not yet fully understood, so further research is needed to lay the foundation for its application. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a method and application of thiamine for the prevention and control of maize chlorotic mottle virus.

[0005] The technical solution of this invention is implemented as follows:

[0006] This application found that thiamine spraying enhances maize's resistance to MCMV. To further clarify the molecular mechanism by which thiamine enhances the host's antiviral effect, we performed transcriptome sequencing on four treatment groups: healthy plants, MCMV-infected plants, thiamine-sprayed plants, and plants inoculated with MCMV after thiamine spraying. The results showed that MCMV infection affects the transcriptional changes of genes related to the thiamine metabolic pathway, leading to the inhibition of endogenous thiamine biosynthesis in plants. Comparative analysis of thiamine-sprayed plants inoculated with MCMV and plants inoculated with MCMV alone revealed that thiamine can enhance plant defense responses by inducing the MAPK pathway and promote the synthesis of lignin in plant cell walls by inducing the phenylpropanone biosynthesis pathway, thereby inhibiting MCMV infection.

[0007] This application also discovered an interaction between the key MCMV pathogenic factor P31 protein and the key thiamine synthesis gene ZmTHIC. Using a CMV-mediated gene silencing system, endogenous ZmTHIC in plants was silenced, resulting in higher MCMV accumulation compared to control plants. LC-MS analysis revealed that both ZmTHIC silencing and MCMV infection led to a decrease in endogenous thiamine levels in plants. Following MCMV infection, the interaction between P31 and ZmTHIC alters the localization of ZmTHIC, thereby inhibiting thiamine synthesis.

[0008] Based on this, the present invention proposes a method for controlling maize chlorotic mottle virus with thiamine, the steps of which are: spraying thiamine solution on plant leaves.

[0009] Preferably, the concentration of the thiamine solution used is 10-50 mM.

[0010] Preferably, the above-mentioned thiamine solution enhances the plant's resistance to maize chlorotic mottle virus by regulating the MAPK signaling pathway and lignin synthesis pathway.

[0011] Preferably, the above-mentioned regulation of the MAPK signaling pathway is achieved by upregulating the expression levels of genes MAPKKK17.2 and MAPKKK17.3.

[0012] Preferably, the above-mentioned regulation of lignin synthesis pathway is achieved by upregulating the levels of phenylalanine ammonia-lyase, β-glucosidase, cinnamyl alcohol dehydrogenase, and peroxidase.

[0013] Preferably, the plant mentioned above is corn.

[0014] Secondly, the above methods are applied in the prevention and control of maize chlorotic mottle virus.

[0015] Thirdly, the above methods are applied to promoting plant growth.

[0016] Fourthly, the above methods are applied to improving the broad-spectrum antiviral capabilities of plants.

[0017] Fifthly, the above methods are applied to increasing the lignin content of plants.

[0018] Preferably, the lignin comprises one or more of p-hydroxyphenyl lignin, guaiacyl lignin, 5-hydroxyguaiacyl lignin, and syringyl lignin.

[0019] Preferably, the plant mentioned above is corn.

[0020] The present invention has the following beneficial effects:

[0021] This application found that spraying thiamine can enhance maize's resistance to MCMV. To clarify the molecular mechanism by which thiamine enhances the host's antiviral effect, transcriptome sequencing was performed. The results showed that MCMV infection affects the transcriptional changes of genes related to the thiamine metabolic pathway, leading to the inhibition of endogenous thiamine biosynthesis in plants. Meanwhile, we found that the key pathogenic factor P31 protein of MCMV interacts with the key gene ZmTHIC in thiamine synthesis. Silencing the endogenous ZmTHIC gene in plants led to a higher accumulation of MCMV than in control plants. LC-MS analysis showed that both ZmTHIC silencing and MCMV infection resulted in a decrease in endogenous thiamine content in plants. After MCMV infection, the interaction between P31 and ZmTHIC altered the localization of ZmTHIC, thereby inhibiting thiamine synthesis. Analysis of treatments involving MCMV inoculation after thiamine spraying versus MCMV inoculation alone revealed that thiamine can inhibit MCMV infection by inducing the MAPK pathway to enhance plant defense responses and by inducing the phenylpropanone biosynthesis pathway to promote lignin synthesis in plant cell walls. In conclusion, exogenous thiamine increases resistance to viral diseases by promoting maize growth, enhancing plant defense responses, and increasing lignin synthesis. The molecular mechanism indicates that thiamine exerts a broad-spectrum disease resistance effect by inducing plant immune responses. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The effect of exogenous thiamine spray on MCMV infection is shown in Figure A, where A represents leaf symptoms under different treatments, B represents the relative expression level of MCMV, C represents the MCMV accumulation detected by Western blotting, and D represents the physiological indicators of each treatment.

[0024] Figure 2 Transcriptome analysis of MCMV and CK treatment groups; where A is PCA analysis diagram, B is differential gene volcano diagram, C is KEGG enrichment analysis diagram, D is thiamine metabolic pathway diagram, E is cluster analysis heatmap, F is the relative transcriptional level of 9 DEGs, and G is the relative content of endogenous thiamine after MCMV infection.

[0025] Figure 3To verify the interaction between ZmTHIC and MCMV P31; where A is the yeast two-hybrid assay of ZmTHIC, B is the yeast two-hybrid assay of ZmTHI1, C is the immunoprecipitation assay of P31-GFP and ZmTHIC-Myc, D is the bimolecular fluorescence complementation assay of MCMV-P31 and ZmTHIC, E is the subcellular localization map of ZmTHIC in maize protoplasts, F is the subcellular localization map of ZmTHIC and MCMV P31 in Nigeriensis, and G is the subcellular co-localization map of ZmTHIC and MCMV P31 in Nigeriensis.

[0026] Figure 4 The effects of ZmTHIC silencing on MCMV infection and thiamine are shown in Figure A, where A represents maize growth after ZmTHIC silencing and MCMV inoculation; B represents the relative expression level of ZmTHIC; C represents the relative expression level of MCMV; D represents the MCMV accumulation detected by Western blotting; and E represents the thiamine content.

[0027] Figure 5 Transcriptome analysis of MCMV and MCMV+thiamine treatment groups; where A is the differential gene volcano plot, B is the cluster analysis heatmap, C is the relative expression level of MAPKKK17, D is the KEGG enrichment analysis plot, E is the phenylpropanoid metabolism and lignin synthesis pathway, and F is the relative expression level of genes related to the phenylpropanoid pathway and lignin synthesis pathway.

[0028] Figure 6 This is a schematic diagram illustrating the molecular mechanism of thiamine's action. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] Example

[0032] 1. Materials and Methods

[0033] (1) Test plants

[0034] Nicotiana benthamiana was grown in a greenhouse at the College of Plant Protection, Henan Agricultural University, under conditions of a photoperiod of 16 hours of light / 8 hours of darkness and a temperature of 24°C.

[0035] Maize (Zea mays L.) inbred line B73 was grown in a greenhouse at 22°C. The plant photoperiod was 16 h light / 8 h dark and the relative humidity was 60%. The maize leaves were inoculated with a virus by rubbing them.

[0036] (2) Plasmid construction:

[0037] The full-length coding sequence (CDS) of ZmTHIC was amplified from B73 maize. For Y2H assays, the full-length CDS of ZmTHIC and ZmTHI1 were subcloned into pGADT7 (Clontech, Beijing, China), and the full-length CDS of MCMV-P31 was cloned into pGBKT7. For BiFC assays, the full-length CDS of ZmTHIC was cloned into the cYFP vector, and the full-length CDS of P31 was cloned into the nYFP vector. For Co-IP assays, the full-length CDS of ZmTHIC was cloned into the Myc tag vector, and the full-length CDS of P31 was cloned into the GFP vector. To observe the subcellular localization of ZmTHIC, the full-length CDS of ZmTHIC was cloned into GFP and RFP vectors. For virus-induced gene silencing (VIGS) assays, the ZmTHIC DNA fragment was cloned into the CMV vector, resulting in the VIGS vector CMV-ZmTHIC. All vectors were confirmed by DNA sequencing.

[0038] (3) Transcriptome sequencing analysis:

[0039] Four biological replicates were performed on four groups of plants: healthy plants, MCMV-infected plants, thiamine-sprayed plants, and plants inoculated with MCMV after thiamine spraying, for the extraction of total RNA and transcriptome sequencing. Transcriptome sequencing was performed on the Illumina Hiseq 2500 platform of BioMarker Technologies Co., Ltd. Further reads containing >10% and >50% of the total reads and with a quality value of Q ≤ 10 were removed. High-quality clean data following the above series of quality controls are provided in FASTQ format. Gene expression levels were estimated as the number of fragments per kb transcript per million fragments plotted. DESeq2 software was used for analysis of |log2FC| > 1 and FDR < 0.05, as previously described. FDR was obtained by adjusting for p-values ​​to determine significance. Gene ontology enrichment analysis was performed to rank the functions of DEGs by top GO packages in R.

[0040] (4) Yeast two-hybrid (Y2H) assay:

[0041] Use the Matchmaker Gold Y2H system (Clontech, Beijing, China) according to the manufacturer's instructions. Transform paired plasmids into yeast strain Y2H. Culture the transformants for 3 days on a selective medium lacking leucine (L) and tryptophan (W) (SD-LW). Inoculate the co-transformants into a medium containing 20 mg / mL of [unspecified medium]. -1 X-α-gal and 500 μg mL -1 Aba was tested on supplement-deficient medium lacking L, W, histidine (H) and adenine (A) (SD-LWHA) for 4 days to test possible interactions.

[0042] (5) Measurement using bimolecular fluorescence complementary technique (BiFC):

[0043] The corresponding plasmids were transformed into Agrobacterium strain GV3101. 600 YFP was co-infiltrated into leaves of 4-week-old Nicotiana benthamiana at a concentration of 0.6 in different combinations. Samples were observed at 48 hpi using a Zeiss LSM710 laser scanning microscope. YFP was excited at 514 nm and captured in the 565–585 nm range. At least three independent experiments were performed.

[0044] (6) Co-immunoprecipitation (CO-IP) assay:

[0045] Forty-eight hours after Agrobacterium infiltration, fluorescence signals were observed using a Zeiss LSM710, and total protein was extracted using lysis buffer. For Co-IP assays, 15 μL of GFP-Trap agarose beads (GenScript, Nanjing, China) were incubated with 2 mL of crude protein at 4°C for 10 hours, and collected as previously described. Myc and GFP antibodies were diluted 1:5000. Total cellular protein was extracted from fresh leaves in lysis buffer (1 M Tris, pH 7.5, 5 M NaCl, 20% [v / v] Triton X-100, 50% glycerol, 1 mM benzyl sulfonyl fluoride). Beads were collected and washed five times with washing buffer, and analyzed by Western blotting. The blot membrane was thoroughly washed and visualized using a chemiluminescence detection system (YESEN, Shanghai, China).

[0046] (7) VIGS determination:

[0047] For CMV-based VIGS assays, we used CMV vectors. Derivatives of plasmids pCMV101, pCMV301, and pCMV201-2bN81 were introduced into Agrobacterium strain GV3101, respectively. CMV-based VIGS was performed as previously described. ZmTHIC silencing efficiency was assessed at 10 dpi. Then, the plants were inoculated with the virus. One week after MCMV inoculation, changes in virus accumulation in maize plants were examined.

[0048] (8) Chemical treatment:

[0049] To treat maize plants with exogenous thiamine, 10 mM and 50 mM thiamine diluted with ddH2O were sprayed onto maize plants at the two-leaf-one-heart stage. The control treatment was spraying with ddH2O. One day after treatment, maize plants were inoculated with MCMV as described above. Changes in maize plant morphology, plant height, root length, and virus accumulation were examined. The experiment was repeated three times.

[0050] (9) Thiamine determination: Thiamine was extracted from the leaves of control, virus-inoculated maize plants, GFP-silenced maize plants, GFP-silenced and virus-inoculated maize plants, THIC-silenced maize plants, and THIC-silenced and virus-inoculated maize plants, and measured by ultra-high performance LC-triple quadrupole mass spectrometry at Suzhou Grans Biotechnology Co., Ltd. Four samples were analyzed for each of the three biological replicates. The experiment was independently repeated three times.

[0051] (10) RNA extraction and RT-qPCR:

[0052] Total RNA was extracted from leaves of *Nicotiana benthamiana* and leaves of maize using Trizol Plus (TaKaRa, Dalian, China). First-strand cDNA was synthesized from 500 ng of total RNA using the HiScript IIIQ RT SuperMix kit (Vazyme, Nanjing, China) according to the manufacturer's instructions, with a total volume of 10 μL. Then, 1.0 μL of the diluted cDNA was used for RT-qPCR analysis in a 10 μL volume using ChamQ SYBR qPCR premix (Vazyme, Nanjing, China) on a PCR machine (Thermo Fisher, USA).

[0053] Table 1 Primer sequences used for RT-qPCR

[0054]

[0055]

[0056] The primers used in RT-qPCR were designed using an online tool (https: / / www.genscript.com.cn / tools / real-time-pcr-taqman-primer-design-tool) and are listed in Table 1. As mentioned earlier, 2 primers were used. -ΔΔCT The method was used to calculate relative gene expression. The experiment was repeated three times.

[0057] (11) Isolation and transfection of maize protoplasts:

[0058] The isolation and transfection of maize mesophyll cell protoplasts were reported as described previously. Maize B73 cells were cultured in the dark at 25°C for 14 days. The upper-middle part of the first true leaf was removed and cut into 4-6 mm fine filaments to isolate protoplasts. The plasmid to be transformed was added to the protoplasts. The cells were then cultured in the dark for 18-72 hours.

[0059] (12) Western blot analysis:

[0060] Soluble proteins from *Nicotiana benthamiana* were extracted using lysis buffer (1.0 M Tris-HCl pH 6.8, 10% SDS, ddH2O, β-mercaptoethanol). Maize leaf tissues were collected and homogenized using protein extraction buffer (250 M Tris-HCl pH 6.8, 10% SDS, 0.5% BPB, 50% glycerol, 5% β-mercaptoethanol). The crude extract was centrifuged at 12000 g for 10 min. Proteins in the extract were separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. Antibody or antiserum was diluted 1:5000. Hybridization signals were detected using an enhanced chemiluminescence system (eECL Western Blot Kit) (YEASEN, Shanghai, China).

[0061] (13) Statistical Analysis

[0062] The data are the average of three independent experiments. Data were analyzed using Student's t-test. Values ​​p < 0.05 are indicated by an asterisk (*), and values ​​p < 0.01 are indicated by a double asterisk (*).

[0063] 2. Results and Analysis

[0064] (1) Exogenous application of thiamine effectively inhibits MCMV infection.

[0065] First, we investigated whether external application of thiamine affected maize resistance to MCMV. We sprayed maize plant leaves with 10 mM thiamine, 50 mM thiamine, and ddH2O, respectively. The day after treatment, MCMV was inoculated by rubbing the second new leaf of the maize plant. Symptoms were observed one week after inoculation. The results showed that, compared with the control, maize plants treated with 10 mM thiamine and 50 mM thiamine showed reduced MCMV symptoms. Figure 1 A). Western blotting and RT-qPCR analyses showed that thiamine treatment reduced the accumulation of MCMV CP in maize leaves (A). Figure 1 (B, C) Considering the need to reduce the dosage of thiamine and the similar effects of 10mM and 50mM thiamine treatments, we chose 10mM thiamine for subsequent experiments. Furthermore, we measured the plant height, root length, fresh weight, and dry weight of maize. We found no significant differences in plant height, aboveground fresh weight, and aboveground dry weight between thiamine-treated maize plants and ddH2O-treated healthy plants. After thiamine treatment, there was no significant difference in root length, but the fresh and dry weight of the roots increased. Figure 1 D). In conclusion, thiamine can effectively inhibit MCMV infection. Spraying thiamine will not affect the aboveground growth of corn, but it will promote root growth.

[0066] (2) MCMV infection inhibits thiamine synthesis

[0067] To investigate the molecular mechanism of thiamine in controlling MCMV infection, we set up four different treatment groups for transcriptome analysis: MCMV infection alone, thiamine spraying, thiamine spraying followed by MCMV inoculation, and control B73 maize plants. Figure 2 A). We analyzed the differentially expressed genes (DEGs) in maize plants infected with MCMV alone and in control plants. The results showed that a total of 8558 DEGs were obtained after MCMV infection, of which 48.8% (4178) were downregulated and 51.2% (4380) were upregulated. Figure 2 B). KEEG analysis showed that MCMV regulates multiple signaling pathways and also affects the transcriptional expression of genes related to the thiamine metabolism pathway. Figure 2C). We analyzed differentially expressed genes in the thiamine metabolic pathway. After MCMV infection, thiamine phosphate synthase (TH1), thiamine phosphate phosphatase (TH2), and thiamine pyrophosphokinase (TPK) were upregulated, while pyrimidine synthase (THIC), thiazole synthase (HEP-T synthase (THI1), 1-deoxy-D-xylulose-5-phosphate lyase (DXS), and adenylate kinase (ADK) were downregulated. Figure 2 D and 2E). We detected the relative transcriptional levels of nine DEGs involved in the thiamine metabolic pathway using RT-qPCR, obtaining results consistent with those obtained from the transcriptome data (D and 2E). Figure 2 F). We also examined the endogenous thiamine content in plants after MCMV infection, and the results showed that MCMV infection led to a decrease in endogenous thiamine in maize plants (F). Figure 2 In summary, these results indicate that MCMV infection affects the thiamine metabolic pathway in maize and reduces thiamine content in plants.

[0068] (3) Verification of the interaction between ZmTHIC and MCMV P31

[0069] MCMV infection inhibits thiamine synthesis, and we hypothesize that MCMV inhibits thiamine synthesis by hijacking key proteins involved in thiamine synthesis. To screen whether maize thiamine synthesis-related proteins interact with MCMV viral proteins, we performed Y2H screening using two key thiamine precursor proteins, ZmTHIC and ZmTHI1, with viral genes. The coding sequences for viral proteins P7b, P31, P32, P50, and CP were cloned into the pGBKT7 vector, and the coding sequences for ZmTHIC and ZmTHI1 were cloned into the pGADT7 vector. After co-transforming the constructed vectors in yeast, we screened for interactions between ZmTHIC and P31 proteins, but no interaction between ZmTHI1 and P31. Figure 3 (A and 3B). To further verify the interaction between ZmTHIC and P31, we performed immunoprecipitation assays, constructed P31-GFP and ZmTHIC-Myc vectors respectively, and performed Agrobacterium-mediated transient expression, using GFP and ZmTHIC-Myc as negative controls. The results showed that P31-GFP and ZmTHIC-Myc interact in plants ( Figure 3C). Finally, we performed a bimolecular fluorescence complementation experiment. We constructed MCMV-P31 and ZmTHIC into 35S-nYFP and 35S-cYFP, respectively, to generate P31-nYFP and cYFP-ZmTHIC. P31-nYFP and cYFP-ZmTHIC were transformed with Agrobacterium and co-infiltrated into *Tobacco Benedict* leaves. We observed YFP fluorescence in *Tobacco Benedict* leaves 2 days (dpi) after infiltration. Figure 3 D). The results showed that ZmTHIC, after interacting with P31, did not localize in chloroplasts but rather as intracellular aggregates. To observe the localization of ZmTHIC, observations were made on tobacco leaves and maize protoplasts, where ZmTHIC was localized in chloroplasts ( Figure 3 E and 3F). P31 is located in the cell membrane in tobacco leaves (E and 3F). Figure 3 F). When ZmTHIC-RFP and P31-GFP are co-expressed, ZmTHIC-RFP is not completely localized in chloroplasts (F). Figure 3 G). The results show that MCMV P31 interacts with ZmTHIC and alters the localization of ZmTHIC.

[0070] (4) ZmTHIC silencing promotes MCMV infection.

[0071] To further investigate the role of ZmTHIC in MCMV infection, we used a CMV-mediated gene silencing system to silence endogenous ZmTHIC in plants and then inoculated them with MCMV. Figure 4 A). We used RT-qPCR to detect the silencing efficiency of ZmTHIC at 10 dpi, and the results showed that ZmTHIC was indeed silenced in CMV-ZmTHIC plants, with a silencing efficiency of about 90%. Figure 4 B). CMV-ZmTHIC and control plants were inoculated with MCMV via friction grafting. Eight days later, observation revealed that CMV-ZmTHIC maize plants exhibited more severe mosaic symptoms compared to CMV-GFP maize plants. Figure 4 A). Next, we analyzed the viral accumulation of MCMV CP using RT-qPCR and Western blotting. The results showed that the viral accumulation of MCMV was significantly increased in maize plants with ZmTHIC expression silence (A). Figure 4 C and 4D). Simultaneously, we examined the thiamine content in ZmTHIC-silenced plants after MCMV inoculation, and the results showed that both ZmTHIC silencing and MCMV inoculation led to a decrease in thiamine content in the plants. Figure 4 E). In summary, MCMV uses P31 to hijack ZmTHIC and inhibit the content of endogenous thiamine in plants.

[0072] (5) Thiamine inhibits MCMV infection by regulating the MAPK pathway and lignin synthesis.

[0073] To further clarify how thiamine inhibits MCMV infection, we analyzed the transcriptome data of plants infected with MCMV alone and those infected after thiamine spraying. The results showed that thiamine control of MCMV infection yielded a total of 423 DEGs compared to the control, of which 58.9% (249) were downregulated and 41.1% (174) were upregulated. Figure 5 A). KEGG analysis was performed on the differentially expressed genes mentioned above. Figure 5 D), the results showed that the differentially expressed genes were mainly involved in phenylpropanoid biosynthesis and the plant MAPK signaling pathway. We analyzed the differentially expressed genes in the MAPK pathway, and the results showed that nine differentially expressed genes in the MAPK pathway changed significantly after thiamine treatment to control MCMV infection. Figure 5 B). We used RT-qPCR to detect the transcriptional levels of two MAPKKK17 molecules in the MAPK pathway, and the results were consistent with the transcriptome data, showing that both were upregulated. Figure 5 C). Thiamine treatment for MCMV infection showed significant enrichment of differentially expressed genes in the phenylpropane synthesis pathway, all of which showed an upregulated trend. The phenylpropane pathway is the main pathway for lignin synthesis. Figure 5 E). The expression of genes related to phenylalanine ammonia-lyase (PAL), β-glucosidase (β-GLUE), cinnamyl-alcoholdehydrogenase (CAD), and peroxidase (POX) in this pathway was significantly upregulated. The PAL-catalyzed pathway is essential for the initial step of lignin synthesis and provides the foundation for all subsequent branches and resulting metabolites. CAD is a key enzyme in lignin biosynthesis, catalyzing the final step in the synthesis of lignin monools. Activated lignin monomers undergo oxidative polymerization under POX catalysis to form lignin polymers. These genes lead to the production of four metabolites: p-hydroxy-phenyl lignin, guaiacyl lignin, 5-hydroxyguaiacyl lignin, and syringyl lignin. Figure 5 F). In summary, thiamine can enhance maize's resistance to MCMV by regulating the MAPK signaling pathway and the phenylpropane-lignin synthesis pathway.

[0074] A schematic diagram of the molecular mechanism of action of thiamine in this invention is shown below. Figure 6 Exogenous application of thiamine can trigger a plant immune response, enhancing maize's resistance to MCMV. Thiamine exerts a broad-spectrum disease resistance effect by inducing a plant immune response. ZmTHIC specifically interacts with the MCMV P31 protein, altering its subcellular localization from chloroplasts to intracellular aggregates. We hypothesize that MCMV infection inhibits thiamine synthesis and increases host susceptibility to MCMV by hijacking ZmTHIC with P31 and altering its localization. This study found that MCMV inoculation after thiamine application can induce the expression of MAPK pathway-related genes such as MAPKKK17.1 and MAPKKK17.2, thereby activating the plant's defense response. Notably, thiamine can also upregulate the expression of genes related to the phenylpropane synthesis pathway, such as PAL, β-GLUE, CAD, and POX, which are involved in regulating the synthesis and accumulation of lignin in the secondary cell wall. Lignin plays a major role in plant development and pathogen resistance, but its role in viral infection remains unclear. This invention provides a reference for understanding the process by which lignin defends against viral diseases. In summary, MCMV inoculation following thiamine application primarily regulates plant antiviral responses by inducing the MAPK signaling pathway and lignin synthesis.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for controlling maize chlorotic mottle virus using thiamine, characterized in that, The steps are as follows: spray thiamine solution onto the plant leaves.

2. The method according to claim 1, characterized in that: The concentration of the thiamine solution used is 10-50 mM.

3. The method according to claim 2, characterized in that: The thiamine solution enhances plant resistance to maize chlorotic mottle virus by regulating the MAPK signaling pathway and lignin synthesis pathway.

4. The method according to claim 3, characterized in that: The regulation of the MAPK signaling pathway is achieved by upregulating the expression levels of genes MAPKKK17.2 and MAPKKK17.

3.

5. The method according to claim 4, characterized in that: The regulation of lignin synthesis pathway is achieved by upregulating the levels of phenylalanine ammonia-lyase, β-glucosidase, cinnamyl alcohol dehydrogenase, and peroxidase.

6. The method according to claim 5, characterized in that: The plant in question is corn.

7. The application of the method according to any one of claims 1-6 in the prevention and control of maize chlorotic mottle virus.

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