Method for preventing and treating maize chlorotic mottle virus by using thiamine and application of thiamine

Abstract

The invention belongs to the field of plant protection, and particularly relates to application of thiamine in prevention and treatment of maize chlorotic mottle viruses. The molecular mechanism of thiamine for enhancing the host antiviral effect is determined through transcriptome sequencing, and it is found that maize chlorotic mottle virus (MCMV) infection can affect transcriptional changes of genes related to a thiamine metabolic pathway, and consequently biosynthesis of plant endogenous thiamine is inhibited; after MCMV infection, P31 and ZmTHIC interact to change positioning of ZmTHIC, so that synthesis of thiamine is inhibited; the resistance of corn to MCMV is enhanced by exogenous spraying of thiamine, the thiamine enhances plant defense reaction by inducing an MAPK pathway, and induces a phenylpropane biosynthesis pathway to promote synthesis of plant cell wall lignin so as to inhibit infection of MCMV; therefore, external application of thiamine can promote corn growth, enhance plant defense reaction and increase lignin synthesis to increase virus disease resistance.

Description

Technical Field

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

[0002] Maize chlorotic mottle virus (MCMV) belongs to the maize chlorotic mottle virus genus (machlomovirus) of the tomato bushy stunt virus family (tombusviridae), and its virus particles are regular icosahedrons with a diameter of about 30nm. MCMV mainly infects corn, sugarcane, and millet, and is mainly transmitted by vectors such as thrips, and can also be transmitted by mechanical friction and seeds. MCMV infection alone will cause mild chlorotic mottle symptoms at the base of corn leaves, and then the symptoms will gradually develop upward to the tip of the leaf. In severe cases, the chlorotic parts will connect into pieces to form leaf spots. When MCMV is co-infected with a virus of the potyviridae family, it will cause a serious corn virus disease, maize lethal necrosis disease (MLND), resulting in serious losses in corn yield. 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 read-through protein P111 of P50, of which sgRNA1 can encode P7a, P7b, P31, and cp, and sgRNA2 does not encode protein. P31 is the main pathogenic factor of MCMV to help the virus move systemically. P31 protein can hijack ZmCAT1 to enhance the accumulation of P31 and weaken SA-mediated defense; P31 protein can also directly target ZmPAO1 and enhance enzyme activity to offset Zma-miR167-mediated defense to a certain 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. 2022Jun 1; 189(2): 1065-1082.).

[0003] Thiamine, also known as vitamin B1 (vtamin B1, VB1), is an essential micronutrient for all organisms. Its two synthetic precursors: 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and 4-methyl-5-(2-hydroxyethyl)-thiazole phosphate are catalyzed by pyrimidine synthase (THIC) and thiazole synthase (THI1), respectively. Thiamine has three forms: thiamine pyrophosphate (TMP), thiamine diphosphate (TPP) and thiamine triphosphate (TTP). Thiamine pyrophosphate is the activated form of thiamine. As a coenzyme factor of important enzyme reactions such as pyruvate dehydrogenase complex (PDHC), α-ketoglutarate dehydrogenase complex (KGDHC) and transketolase (TK), it participates in many metabolic activities and plays a key role. Thiamine is essential for all organisms. Plant seedlings lacking thiamine will lose their green color, and in severe cases, they will die. It plays an important role in plant growth and development, biotic and abiotic stresses (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, pathogenicity-related proteins will accumulate rapidly (Boubakri H, Gargouri M, Mliki A, et al. Vitamins for enhancing plantresistance.Planta.2016Sep;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 and promote plant virus infection. This indicates that there is a connection between thiamine biosynthesis and antiviral ability in plants.However, this discovery does not prove that thiamine can directly resist viruses, but suggests a potential role for thiamine in plant defense against viruses. The molecular mechanism of its broad-spectrum disease resistance remains to be revealed (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.) As a naturally occurring nutrient, thiamine can be used to improve plant resistance to viral diseases, which has higher environmental compatibility and is conducive 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] In order to solve the above problems, the present invention provides a method and application of thiamine for preventing and controlling maize chlorotic mottle virus.

[0005] The technical solution of the present invention is achieved in this way:

[0006] This application found that spraying thiamine enhances corn's resistance to MCMV. To further clarify the molecular mechanism of thiamine enhancing host antiviral effects, we performed transcriptome sequencing on four groups of treatments: healthy plants, MCMV-infected plants, thiamine-sprayed plants, and plants inoculated with MCMV after thiamine spraying. The results showed that MCMV infection would affect the transcriptional changes of genes related to the thiamine metabolic pathway, resulting in the inhibition of the biosynthesis of endogenous thiamine in plants. A comparative analysis of the treatments of spraying thiamine and inoculating MCMV with the treatment of inoculating MCMV alone found that thiamine can enhance plant defense responses by inducing the MAPK pathway, and induce the phenylpropanoid biosynthesis pathway to promote the synthesis of plant cell wall lignin, thereby inhibiting MCMV infection.

[0007] The present application also found that the key pathogenic factor of MCMV, P31 protein, interacts with the key gene ZmTHIC for thiamine synthesis. The endogenous ZmTHIC of the plant is silenced using a CMV-mediated gene silencing system. After silencing ZmTHIC, the accumulation of MCMV is higher than that of the control plants. LC-MS determination found that both ZmTHIC silencing and MCMV infection can lead to a decrease in the endogenous thiamine content of the plant. After MCMV infection, P31 interacts with ZmTHIC and changes the localization of ZmTHIC, thereby inhibiting the synthesis of thiamine.

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

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

[0010] Preferably, the thiamine solution improves the resistance of plants to maize chlorotic mottle virus by regulating the MAPK signaling pathway and the lignin synthesis pathway.

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

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

[0013] Preferably, the plant is corn.

[0014] In a second aspect, the above method is used in the prevention and control of maize chlorotic mottle virus.

[0015] A third aspect is the application of the above method in promoting plant growth.

[0016] Fourthly, the above method is used to improve the broad-spectrum antiviral ability of plants.

[0017] A fifth aspect is the application of the above method in 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 is corn.

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

[0021] This application found that spraying thiamine can enhance the resistance of corn to MCMV. Transcriptome sequencing was performed to clarify the molecular mechanism by which thiamine enhances the host's antiviral effect. The results showed that MCMV infection affects the transcriptional changes of genes related to the thiamine metabolic pathway, resulting in the inhibition of the biosynthesis of endogenous thiamine in plants. At the same time, we found that the key pathogenic factor of MCMV P31 protein interacted with the key gene ZmTHIC for thiamine synthesis. Silencing the endogenous ZmTHIC gene in plants resulted in a higher accumulation of MCMV than in control plants. LC-MS determination showed that both ZmTHIC silencing and MCMV infection would lead to a decrease in the endogenous thiamine content in plants. After MCMV infection, the localization of ZmTHIC was changed through the interaction between P31 and ZmTHIC, thereby inhibiting the synthesis of thiamine. Analysis of the treatments of inoculating MCMV after spraying thiamine and inoculating MCMV alone showed that thiamine can enhance plant defense responses by inducing the MAPK pathway and promote the synthesis of plant cell wall lignin by inducing the phenylpropanoid biosynthesis pathway, thereby inhibiting MCMV infection. In summary, exogenous thiamine increases resistance to viral diseases by promoting corn growth, enhancing plant defense responses and increasing lignin synthesis. The molecular mechanism of action shows that thiamine produces a broad-spectrum disease resistance by inducing plant immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The effect of exogenous spraying of thiamine on MCMV infection; A is the disease symptoms of leaves under different treatments, B is the relative expression level of MCMV, C is the MCMV accumulation amount detected by Western-blotting, and D is the physiological index of each treatment.

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

[0025] Figure 3Verification of the interaction between ZmTHIC and MCMV P31; A is the yeast two-hybrid assay of ZmTHIC, B is the yeast two-hybrid assay of ZmTHI1, C is the immunoprecipitation detection of P31-GFP and ZmTHIC-Myc, D is the bimolecular fluorescence complementation experiment of MCMV-P31 and ZmTHIC, E is the subcellular localization map of ZmTHIC in corn protoplasts, F is the subcellular localization map of ZmTHIC and MCMVP31 in Nicotiana benthamiana, and G is the subcellular colocalization map of ZmTHIC and MCMV P31 in Nicotiana benthamiana.

[0026] Figure 4 The figure shows the effect of ZmTHIC silencing on MCMV infection and thiamine; A is the growth status of corn inoculated with MCMV after ZmTHIC silencing, B is the relative expression level of ZmTHIC, C is the relative expression level of MCMV, D is the accumulation level of MCMV detected by Western-blotting, and E is the thiamine content.

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

[0028] Figure 6 Schematic diagram of the molecular mechanism of action of thiamine. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Example

[0032] 1. Materials and methods

[0033] (1) Test plants

[0034] N. benthamiana was grown under a photoperiod of 16 h light / 8 h dark and a temperature of 24 °C in the greenhouse of the College of Plant Protection, Henan Agricultural University.

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

[0036] (2) Plasmid construction:

[0037] The full-length coding sequence (CDS) of ZmTHIC was amplified from B73 maize. For Y2H assay, the full-length ZmTHIC and ZmTHI1 CDS were subcloned into pGADT7 (Clontech, Beijing, China), and the full-length CDS of MCMV-P31 was cloned into pGBKT7; for BiFC analysis, 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 analysis, 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 the GFP and RFP vectors; for virus-induced gene silencing (VIGS) assay, the DNA fragment of ZmTHIC was cloned into the CMV vector to obtain the VIGS vector CMV-ZmTHIC. All vectors were confirmed by DNA sequencing.

[0038] (3) Transcriptome sequencing analysis:

[0039] Four biological replicates of healthy plants, MCMV-infected plants, thiamine-sprayed plants, and thiamine-sprayed and MCMV-inoculated plants were sampled for total RNA extraction and transcriptome sequencing. The transcriptome was sequenced on the Illumina Hiseq 2500 platform of BioMarker Technologies Co, Ltd. Further reads containing >10% and >50% of the entire 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 are estimated as the number of fragments per kb transcript per 1 million fragments mapped. DESeq2 software was used to analyze |log2FC|>1 and FDR<0.05 as described above. The FDR was obtained by correcting the p-value for the significance of the difference. Gene ontology enrichment analysis was used to rank the functions of DEGs by the top GO package in R.

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

[0041] The paired plasmids were transformed into yeast strain Y2H using the Matchmaker Gold Y2H system (Clontech, Beijing, China) according to the manufacturer's instructions. Transformants were cultured on selective medium lacking leucine (L) and tryptophan (W) (SD-LW) for 3 days. The co-transformants were inoculated into a 20 mg mL -1 X-α-gal and 500 μg mL -1 Aba supplements lacking L, W, histidine (H) and adenine (A) (SD-LWHA) were grown on culture medium to test possible interactions for 4 days.

[0042] (5) Bimolecular fluorescence complementation (BiFC) assay:

[0043] The corresponding plasmids were transformed into Agrobacterium strain GV3101. 600 The concentration of 0.6 was co-infiltrated into leaves of 4-week-old Nicotiana benthamiana in different combinations. The samples were observed at 48 hpi using a Zeiss LSM710 laser scanning microscope. YFP was excited at 514 nm and captured at 565-585 nm. At least 3 independent experiments were performed.

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

[0045] 48 h after agroinfiltration, fluorescence signals were observed with a Zeiss LSM710, and total proteins were extracted with lysis buffer. For Co-IP assays, 15 μL GFP-Trap agarose beads (GenScript, Nanjing, China) were incubated with 2 mL crude protein at 4 °C for 10 h and collected as described previously. The dilution of Myc and GFP antibodies was 1:5000. Total cellular proteins were 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 phenylmethylsulfonyl fluoride). The beads were collected and washed five times with wash buffer and analyzed by Western blotting. The blots were washed thoroughly and visualized using a chemiluminescent detection system (YESEN, Shanghai, China).

[0046] (7) VIGS assay:

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

[0048] (8) Chemical treatment:

[0049] To treat corn plants with exogenous thiamine, two-leaf, one-heart stage corn plants were sprayed with 10 mM and 50 mM thiamine diluted in ddH2O. The control treatment was sprayed with ddH2O. One day after treatment, corn plants were inoculated with MCMV as described above. Changes in morphology, plant height, root length, and virus accumulation of corn plants were examined. The experiment was repeated 3 times.

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

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

[0052] Total RNA was extracted from leaves of Nicotiana benthamiana and corn leaves using Trizol Plus (TaKaRa, Dalian, China). First-strand cDNA was synthesized from 500 ng of total RNA in a total volume of 10 μL using HiScript IIIQ RT SuperMix kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. Then, 1.0 μL of the diluted cDNA was used for RT-qPCR analysis in a total volume of 10 μL 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 previously described, 2 -ΔΔCT Methods Relative gene expression was calculated. The experiment was repeated 3 times.

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

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

[0059] (12) Western blot analysis:

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

[0061] (13) Statistical analysis

[0062] Data are the mean of three independent experiments. Data were analyzed by Student's t-test. Values ​​with P < 0.05 are indicated by asterisks, and values ​​with P < 0.01 are indicated by double asterisks.

[0063] 2. Results and Analysis

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

[0065] First, we investigated whether exogenous thiamine would affect maize resistance to MCMV. We sprayed the leaves of maize plants with 10mM thiamine, 50mM thiamine, and ddH2O, respectively. On the second day after treatment, MCMV was inoculated by rubbing the second new leaf of maize, and symptoms were observed one week after virus inoculation. The results showed that maize plants treated with 10mM thiamine and 50mM thiamine could alleviate the symptoms of MCMV compared with the control ( Figure 1 A). Western-blotting and RT-qPCR analysis results showed that the accumulation of MCMV CP in maize leaves was reduced after thiamine treatment ( Figure 1 B, C), considering that the effect of reducing the dosage of thiamine was similar after treatment with 10mM and 50mM thiamine, we chose 10mM thiamine for subsequent experiments. In addition, we also tested the plant height, root length, fresh weight and dry weight of corn, and found that there was no significant difference in plant height, aboveground fresh weight and aboveground dry weight between corn plants treated with thiamine and healthy plants treated with ddH2O. After thiamine treatment, there was no significant difference in root length, but the fresh weight and dry weight of the roots increased ( Figure 1 D). In summary, thiamine can effectively inhibit the infection of MCMV. Spraying thiamine will not affect the growth of the aboveground part of corn, but will promote the growth of roots.

[0066] (2) MCMV infection inhibits thiamine synthesis

[0067] In order to study the molecular mechanism of thiamine in preventing and controlling MCMV infection, we set up four different treatment groups for transcriptome analysis, namely, 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 down-regulated genes and 51.2% (4380) were up-regulated genes ( Figure 2 B). KEEG analysis showed that MCMV regulated multiple signaling pathways and also affected the transcriptional expression of genes related to the thiamine metabolic pathway ( Figure 2C). We analyzed the differentially expressed genes in the thiamine metabolic pathway. After MCMV infection, thiamine phosphate synthase (TH1), TMP phosphatase (TH2), and thiamine pyrophosphokinase (TPK) were up-regulated, while pyrimidine synthase (THIC), thiazole synthase (HEP-T synthase, THI1), 1-deoxy-D-xylulose-5-phosphate pyruvate lyase (DXS), and adenylate kinase (ADK) were down-regulated ( Figure 2 D and 2E). We detected the relative transcription levels of 9 DEGs involved in the thiamine metabolic pathway by RT-qPCR and obtained the same results as the transcriptome data ( Figure 2 F). We also tested the endogenous thiamine content of plants after MCMV infection. The results showed that MCMV infection caused a decrease in endogenous thiamine in corn plants ( Figure 2 G). In conclusion, these results indicate that MCMV infection affects the thiamine metabolic pathway in maize and reduces the thiamine content in the plant.

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

[0069] MCMV infection inhibits the synthesis of thiamine. We speculate that MCMV inhibits the synthesis of thiamine by hijacking key proteins in thiamine synthesis. In order to screen whether maize thiamine synthesis-related proteins interact with MCMV viral proteins, we used two key synthesis precursor proteins of thiamine, ZmTHIC and ZmTHI1, and viral genes for Y2H screening. The viral protein P7b, P31, P32, P50, and CP coding sequences were cloned into the pGBKT7 vector, respectively, and the ZmTHIC and ZmTHI1 protein coding sequences were cloned into the pGADT7 vector, respectively. After the constructed vectors were co-transformed into yeast, we screened for interaction between ZmTHIC and P31 proteins, but ZmTHI1 did not interact with P31 ( Figure 3 A and 3B). To further verify the interaction between ZmTHIC and P31, we performed co-immunoprecipitation assays, constructed P31-GFP and ZmTHIC-Myc vectors, and performed Agrobacterium-mediated transient expression, using GFP and ZmTHIC-Myc as negative controls. The results showed that P31-GFP interacted with ZmTHIC-Myc 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 N. benthamiana leaves. We observed YFP fluorescence in N. benthamiana leaves 2 days post-infiltration (dpi) ( Figure 3 D). The results showed that ZmTHIC was not localized in chloroplasts after interacting with P31, but in intracellular aggregates. In order to observe the localization of ZmTHIC, the leaves of Nicotiana benthamiana and corn protoplasts were observed. ZmTHIC was localized in chloroplasts ( Figure 3 E and 3F). P31 is localized to the cytoplasmic membrane in Nicotiana benthamiana leaves ( Figure 3 F). When ZmTHIC-RFP and P31-GFP were co-expressed, ZmTHIC-RFP was not completely localized in chloroplasts ( Figure 3 G). The results showed that MCMV P31 interacted with ZmTHIC and altered the localization of ZmTHIC.

[0070] (4) ZmTHIC silencing promotes MCMV infection

[0071] To further study the role of ZmTHIC in MCMV infection, we used CMV-mediated gene silencing to silence endogenous ZmTHIC in plants and inoculated MCMV ( Figure 4 A). We used RT-qPCR to detect the silencing efficiency of ZmTHIC at 10 dpi. 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 by friction inoculation. After 8 days, it was found that CMV-ZmTHIC corn plants showed more severe mosaic symptoms than CMV-GFP corn plants ( Figure 4 A). Next, we analyzed the viral accumulation of MCMV CP by RT-qPCR and Western blotting, and the results showed that the MCMV viral accumulation was significantly increased in maize plants with silenced ZmTHIC expression ( Figure 4 C and 4D). At the same time, we detected the thiamine content in ZmTHIC-silenced plants after inoculation with MCMV. The results showed that both ZmTHIC-silence and MCMV inoculation would lead to a decrease in the thiamine content in the plants ( Figure 4 E) In summary, MCMV utilizes P31 to hijack ZmTHIC and suppress endogenous thiamine levels 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 MCMV after thiamine spraying. The results showed that a total of 423 DEGs were obtained when thiamine was used to prevent MCMV infection compared with the control, of which 58.9% (249) were down-regulated genes and 41.1% (174) were up-regulated genes ( Figure 5 A). KEGG analysis was performed on the above differentially expressed genes ( Figure 5 D), the results showed that the differentially expressed genes were mainly involved in phenylpropanpid biosynthesis and plant MAPK signaling pathway. We analyzed the differentially expressed genes in the MAPK pathway, and the results showed that 9 differentially expressed genes in the MAPK pathway changed significantly after thiamine treatment to prevent MCMV infection ( Figure 5 B). We detected the transcriptional levels of two MAPKKK17 in the MAPK pathway by RT-qPCR, and the results were the same as the transcriptome data, and both were upregulated ( Figure 5 C). In the treatment of thiamine against MCMV infection, the differentially expressed genes in the phenylpropanoid biosynthesis pathway were significantly enriched and all showed an up-regulated trend. The phenylpropanoid pathway is the main biosynthesis pathway of lignin ( 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 pathway catalyzed by PAL is essential for the initial step of lignin synthesis and provides the basis for all subsequent branches and resulting metabolites; CAD is a key enzyme in lignin biosynthesis, which catalyzes the last step of lignin monool synthesis; activated lignin monomers are oxidatively polymerized under the catalysis of POX to form lignin polymers. These genes lead to the production of four metabolites: p-hydroxy-phenylLignin, guaiacyl lignin, 5-hydroxyguaiacyl lignin, and syringyl lignin ( Figure 5 F). In conclusion, thiamine can improve the resistance of maize to MCMV by regulating the MAPK signaling pathway and the phenylpropanoid lignin biosynthesis pathway.

[0074] The molecular mechanism of action of thiamine of the present invention is shown in Figure 6 , exogenous spraying of thiamine can trigger plant immune response and enhance the resistance of corn to MCMV. Thiamine induces plant immune response to produce broad-spectrum disease resistance; ZmTHIC interacts specifically with MCMV P31 protein, changing the subcellular localization of ZmTHIC from chloroplasts to intracellular aggregates. We speculate that MCMV infection hijacks ZmTHIC through P31 and changes the localization of ZmTHIC, thereby inhibiting thiamine synthesis and increasing the host's susceptibility to MCMV. This study found that inoculation of MCMV after application of thiamine can induce the expression of MAPK pathway-related genes such as MAPKKK17.1 and MAPKKK17.2 to activate the plant's defense response. It is worth mentioning that thiamine can also upregulate the expression of genes related to the phenylpropanoid synthesis pathway such as PAL, β-GLUE, CAD and POX, which are involved in regulating the synthesis and accumulation of lignin in secondary cell walls. Lignin plays a major role in plant development and resistance to pathogens, but the role of lignin in viral infection is still unclear. The present invention provides a reference for the process of lignin defense against viral diseases. In summary, MCMV inoculation after exogenous thiamine application mainly regulates plant antiviral defense response by inducing the MAPK signaling pathway and the synthesis of lignin.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for controlling maize chlorotic mottle virus with thiamine, characterized in that: The steps are: spray thiamine solution on the leaves of plants.

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

3. The method according to claim 2, characterized in that: The thiamine solution improves the resistance of plants to maize chlorotic mottle virus by regulating the MAPK signaling pathway and the 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 the genes MAPKKK17.2 and MAPKKK17.

3.

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

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

7. Use of the method according to any one of claims 1 to 6 in preventing and controlling maize chlorotic mottle virus.

8. Use of the method according to any one of claims 1 to 6 in improving the broad-spectrum antiviral ability of plants.

9. Use of the method according to any one of claims 1 to 6 in increasing the lignin content of plants.

10. The use according to claim 9, characterized in that: The lignin includes one or more of p-hydroxyphenyl lignin, guaiacyl lignin, 5-hydroxyguaiacyl lignin and syringyl lignin.

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