Identification And Regulatory Mechanism Of MiRNAs In The Interaction Between Maize And Fusarium Verticillioides

Fusarium verticillioides is the main pathogen of maize ear rot.It not only causes massive yield losses and affects maize quality,but also produces fumonisin which is toxic to humans and livestock.At present,the prevention of ear rot involves the use of fungicides and the cultivation of resistant varieties.However,excessive using fungicides can increase the drug resistance of pathogens.It not only reduces the control effect but also causes concern about the harm of chemicals to the environment.At the same time,it is a long-term process to breed crop resistant varieties.And the maize resistant to Fusarium is scarce in production,which can not meet the breeding needs.Therefore,it is important to clarify the pathogenic mechanism of F.verticillioides and maize resistance in plant pathogen interaction.It is urgent to explore new strategies and new ways to control ear rot.miRNA is a class of endogenous non-coding small RNA,which regulates the expression of endogenous genes at transcriptional or post-transcriptional.It can also trans-kingdom regulate and participate in the plant pathogen interaction.In recent years,with the identification of fungal milRNAs and the analysis of the biological function of plant miRNAs,it is urgent to figure out the pathogenic milRNA of F.verticillioides and study the mechanism of maize miRNAs.This study aimed to identify miRNAs in F.verticillioides and maize then clarify their target genes by small RNA sequencing（sRNA-seq）,degradome sequencing and transcriptome sequencing（RNA-seq）.The regulatory relationship between them was clarified by qPCR,Nicotiana benthamiana co-transformation and 5’ RLM-RACE.Maize miR528b-5p,which could not only target the genes in F.verticillioides but also regulate the expression of endogenous gene.Meanwhile milRNAs in F.verticillioides were identified.Molecular biology,genetic,biochemistry assays and phytopathology were used to clarify the mechanism of miRNAs participating in F.verticillioides pathogenesis and maize disease resistance.It will provide a theoretical basis for new strategies and new ways to control maize ear rot.The main results are as follows:1.Identification miRNAs involved in the interaction between maize and F.verticillioides.A total of 51 milRNAs were identified which could be mapped to the genome of F.verticillioides.284 known miRNA and 6571 novel miRNA were identified in maize.28 maize miRNAs were significantly differentially expressed.2.The related genes of F.verticillioides infection and resistance of maize were identified and the interaction process between them was clarified.RNA-seq and bioinformatics analysis were performed on maize after inoculated with F.verticillioides at 0,4.12 and 72 hours.It was found that the growth of F.verticillioides was mainly confined to intercellular of maize at the early stage of infection,which triggered Ca2+signaling pathway and reactive oxygen species（ROS）burst in maize.F.verticillioides continue to colonize and expand in maize at the later stage of infection.While,maize participate in the interaction through MAPK signaling pathway,plant-pathogen interaction and hormone signal transduction pathways.3.Maize miRNAs could trans-kingdom and negatively regulate their target genes in F.verticillioides.13 F.verticillioides genes were identified as target genes for 8 maize known differentially expressed miRNAs（DEMIs）and 458 genes were predicted to be 17 differentially expressed novel miRNA in maize.Lesion area was slightly inhibited and the expression of target genes decreased by application of exogenous ds-miR528-5p and ds-miR171h-3p.The results indicated that miRNA participated in the process of disease resistance.The expression of maize miRNA and their target genes in F.verticillioides were analyzed by quantitative real time PCR（qPCR）,and the results showed that the trends of miRNA was opposite to that of their target genes.The regulatory relationship between miR528b-5p and FVEG₀2035,miR408b-3p and FVEG₀2164,miR171h-5p and FVEG₀9695 was verified by N.benthamiana co-transformation.The cleavage site of miR528b-5p in FVEG₀2035 was identified by degradome sequencing and 5’RLM-RACE.The results indicate that miR528b-5p negatively regulate the expression of FVEG 02035.4.Further studies showed that miR528b-5p inhibited the infection of F.verticillioides by targeting FVEG₀2035 which encoding twice transmembrane protein.It was found that FVEG₀2035（named FvTTP）contained two transmembrane domains and located on cell membrane through domain analysis and subcellular localization.To explorethe role of FvTTP in the pathogenity of F.verticillioides,gene knockout mutants（ΔFvTTP）and complementation transformants（ΔFvTTP-C）were constructed by protoplast transformation.The pathogenicity showed that FvTTP deletion affected hyphae separation length,sporulation,FB production and pathogenicity,but did not affect hyphae growth.cell integrity and the activity of cell wall degrading enzymes.The content of SA and the expression of PR1 in maize inoculated with ΔFvTTP strain were higher than inoculated with WT.5.miR528b-5p inhibites the expression of new transcript of ZmRING-PHD.A total of 108 maize mRNA were predicted to be the targets of 10 maize DEMIs by degradome sequencing.It was found that the target genes were mainly involved in ABC transport,amino acid biosynthesis,tyrosine metabolism,peroxisome and autophagy.The regulatory relationship between miR528b-5p and ZmRING-PHD（Zm00001d047676）,miR408b-3p and Zm00001d041236 was verified by N.benthamiana co-transformation.The cleavage site of miR528b-5p in PEX（new transcript）of ZmRING-PHD was further identified by degradome sequencing and 5’ RLM-RACE.The expression of ZmRING-PHD PEX decreased and the expression of T1,T2 and T3 transcripts increased after inoculated with F.verticillioides.Meanwhile,the expression of ZmRING-PHD_PEX decreased and other transcripts increased by application of exogenous ds-miR528-5p.All transcripts in ZmRING-PDH transposon insertion mutant were down-regulated,but the influence on T2 and T3 was the largest.It showed that the mutant was more sensitive and susceptible to F.verticillioides.The expression of PRs is higher in B73 than ring-phd after inoculated with F.verticillioides.6.It was the first time to identify F.verticillioides milRNAs.It was found that the production of milRNAs depended on FvDicer2 and participated in fungal pathogenesis through negatively regulate their target genes.333 genes in maize were identified as target genes of F.verticillioides milRNAs which were enriched in plant hormone signal transduction,plant pathogen interaction,MAPK signaling pathway,mRNA surveillance pathway,glycolytic/gluconeogenesis pathway,spliceosome,butanoate metabolism and other pathways.The expression level of milRNAs and their target genes were detected by stem-loop qPCR and the expression trend of them were opposite.FvDicer2 gene deletion and complementation transformants were constructed by protoplast transformation.ΔFvDicer2 exhibited a reduction in pathogenicity on maize kernels and affected the production of milRNAs.These results indicated that the synthesis of milRNAs depends on FvDicer2 and negatively regulate its target genes in maize.