Identification And Functional Study Of Non-conventional Peptide NCP38 Against Maize Dwarf Mosaic Disease

Maize is the most important grain,forage,and economic crop in China,occupying a pivotal position in both agricultural production and the national economy.However,in recent years,there has been a growing trend in the occurrence of maize diseases,posing a severe threat to both the yield and quality of maize.Among these diseases,maize virus disease,which often exhibit migratory,intermittent,and explosive patterns of epidemics,represents one of the primary obstacles to maize production.Specifically,maize dwarf mosaic disease(MDMD)caused by the sugarcane mosaic virus(SCMV)has become the most severe and extensively prevalent virus disease affecting both domestic and foreign maize production areas.Consequently,it is critical to discover novel,highly effective disease-resistance molecules for green prevention and control of MDMD to safeguard maize production.Peptides are a class of small molecules composed of 2-100 amino acids.They can be classified based on their sources into conventional peptides(CPs)derived from conventional open reading frames(ORFs)and non-conventional peptides(NCPs)from non-conventional ORFs,such as intergenic regions,UTR regions,and intron regions.Previous studies have mainly focused on the functions of CPs in various plant biological processes,including growth,development,and defense.Recent research in humans and animals has also demonstrated that NCPs play important roles in various biological processes.However,there have been no reports on the study of NCPs on plant resistance to virus diseases.Therefore,exploring NCPs with crucial functions in MDMD resistance and analyzing their role in regulating resistance mechanisms can provide novel insights for the scientific prevention and control of the disease.The laboratory established the plant peptidogenomic methods firstly in the early stage and identified numerous NCPs in maize.In this study,we established one identification and screening system to discover the new NCP candidates resistance to MDMD using maize NCPs as the research object.Finally,we identified two non-conventional peptides,NCP38 and NCP1988,that can enhance maize resistance to the MDMD.Additionally,we conducted transcriptomics and molecular biology experiments to explore the resistance mechanism mediated by NCP38.The main research findings are as follows:1.We established a screening system to identify NCPs that confer resistance to MDMD.Firstly,maize NCPs were synthesized and detected,and a 10 μM NCPs solution was prepared.B73 maize seeds were soaked in this solution(with sterile water as a control)for 24 hours before being transferred to nutrient substrate soil.The maize seedlings were grown until the third leaf stage,after which the second fully expanded leaf was immersed in the 10 μM NCPs solution for 24 hours.Next,the second leaf of the maize seedling was rubbed with a crude extract of SCMV.On the tenth day after inoculation,the chlorophyll content and phenotype of the maize leaves were examined and observed,and the second leaf infected with SCMV was collected for the determination of SCMV accumulation.By comparing the differences in SCMV RNA and coat protein(CP)content in maize leaves inoculated with SCMV under NCP treatment and control conditions,we would identify candidate NCPs related to MDMD resistance.2.Using the above established screening system,we screened the NCP function on the resistance to MDMD from 54 maize NCPs.Through symptom observation of the second systematically infected leaf,chlorophyll content measurement,and comparative analysis of SCMV RNA,we found that treatment with NCP38 and NCP1988 significantly reduced maize leaf symptoms compared to the control group,and significantly increased the relative chlorophyll content.Fluorescence quantitative PCR was used to measure virus RNA levels in the second systematically infected leaf of maize,infected with SCMV after NCP treatment,which showed that NCP38 and NCP1988 treatment reduced SCMV RNA levels by approximately 50% and 25%,respectively,compared to the control group.We selected the most effective NCP against SCMV,NCP38,for further research.Western blot analysis showed that NCP38 treatment reduced SCMV CP levels by approximately 75% in the second systematically infected leaf after SCMV inoculation.These findings suggest that NCP38 can significantly reduce virus levels in maize and enhance resistance to MDMD.3.By transcriptome sequencing,1259 differentially expressed genes were identified in SCMV_CK control group and SCMV_NCP38 treatment group,of which 896 were upregulated and 363 were down-regulated under NCP38 treatment conditions.We verified the nine differentially expressed genes in the transcriptome data using the fluorescence quantitative PCR method,and the results showed that the differentially expressed genes in the transcriptome data matched with the fluorescence quantitative PCR results,indicating that the transcriptome data were accurate and reliable.Further GO functional enrichment analysis of differentially expressed genes revealed that differentially expressed genes were significantly enriched in defense response,lipid response,hydrogen peroxide metabolism,abscisic acid signaling pathway and algal sugar synthesis process.The enrichment analysis of differentially expressed genes using KEGG revealed that they were mainly involved in plant pathogen interaction pathway,benzyl propane biosynthesis pathway and plant MAPK signaling pathway.Through in-depth analysis of the differentially expressed genes in these pathways,we found that WRKY transcription factor,MAPK kinase,ethylene synthesis/response and peroxidase were all up-regulated in the SCMV_NCP38-treated group compared to the control group.These results suggest that NCP38-mediated maize dwarf Mosaic resistance may be closely related to the up-regulated expression of disease-resistance defense genes.4.To deeply explore the mechanism of NCP38-induced resistance to MDMV,We verified the genes related to the antiviral genes of RNAi pathway in maize and found that NCP38 did not affect the gene expression level of the RNAi pathway,implying that NCP38-mediated resistance to maize dwarf mosaic disease is not mediated through the RNAi pathway.And then we verified the expression level of genes related to disease resistance defense in NCP38-treated maize leaves using real-time fluorescence quantitative PCR and found that NCP38 treatment significantly induced the expression of genes related to the signaling pathway of plant immune process,including WRKY transcription factors(WRKY12,WRKY38,WRKY105),ethylene insensitive 3-like 5 protein(EIL8),ethylene-responsive transcription factor 1B(EREB58)associated with ethylene synthesis,salicylate/benzoate carboxyl methyltransferase(AOMT1),and peroxidase-related genes(Peroxidase3,Peroxidase12,Peroxidase45,Peroxidase52).This result suggests that NCP38-mediated resistance to maize dwarf mosaic disease may be associated with the genes of these immune defense response pathways.5.In order to verify whether NCP38 can activate immune defense response,we measured the ROS content in maize leaves treated with NCP38 alone for 24 hours,and it was found that H2O2 accumulation and peroxidase activity increased significantly after NCP38 treatment.Finally,by NBT and DAB staining and H2O2 content determination,it was found that the accumulation of H2O2 decreased significantly in maize leaves inoculated with SCMV after NCP38 treatment.These results suggested that NCP38 mediated maize resistance to maize dwarf mosaic disease by regulating the accumulation of H2O2 and thereby regulating the resistance response.These results indicate that the NCP38-mediated pathway of disease resistance is related to H2O2.In summary,we established a screening system for non-traditional peptides related to dwarf leaf disease resistance in maize and identified two non-traditional peptides(NCP38 and NCP1988)that significantly enhanced resistance to dwarf mosaic disease in maize,reducing SCMV RNA levels by 50% and 25%,respectively,and reducing SCMV CP levels by 75%for NCP38.Furthermore,we selected the most effective NCP38 treated samples for transcriptomic study,and combined with molecular biological experiments,we demonstrated that NCP38 could enhanced maize resistance to dwarf mosaic disease by regulating the H2O2-mediated resistance pathway.This study fills a gap in the research of non-conventional peptides on the maize virus disease resistance and provides a new idea for green control of maize dwarf mosaic disease.