Development Of A New Biological Agent Against Tomato Wilt Disease Based On RNA Interference Technology

Tomato is one of the most important vegetable in the world.Tomato wilt disease is a major fungal disease which is caused by Fusarium oxysporum f.sp.Lycopersici(FOL)leading to serious economic losses in the global agricultural industry.MiRNA is a non-coding single-stranded RNA molecule about 20-24 nucleotides encoded by an endogenous gene and is involved in the regulation of post-transcriptional gene expression.In this study,we e1ployed two near-isogentic cultivar,susceptive cultivar Moneymaker and resisitant cultivar Motelle,to identify trans-kingdom miRNA from pathogen by infected with FOL.(1)Four libraries with tomato roots were constructed after 24 h treating with FOL/H2O including_MM H2O,MM_FOL,Mot_H2O and Mot FOL.(2)FOL-specific miRNA:folmiR1 was screened in the infected tomato by bioinformatics.It was confirmed by Northern blot and qRT-RCR that folmiR1 was enriched in the FOL-treated tomato.To exclude the interference of FOL miRNAs,we isolated protoplasts from infected tomato roots to detected folmiR1.Our results showed that folmiR1 presented in tomato root protoplasts.It indicates that folmiR1 was delivered into host cells during the pathogen infection.(3)Four target genes of folmiR1 were predicted in tomato genome.Most of these predicted genes were nagetive regulated by FOL.(4)By transient co-expression folmiR1 and target genes in Nicotiana tabacum,Solyc06g007430 and Solyc07g065090 were confirmed regulate by folmiR1 at transcription level.(5)To explore the function of Solyc06g007430 and Solyc07g065090,we silenced partially these two genes in resistant cultivar Motelle by virus-induced gene silencing(VIGS).VIGS plants infected by FOL results indicated that Solyc06g007430 and Solyc07g065090 participated in the resistance of tomato.(6)Based on the results above,we overexpressed Solyc06g007430 and Solyc07g065090 in susceptible cultivar Moneymaker,and knock-outed by CRISPR/cas 9 in resistant cultivar Motelle for further study the resistant function to tomato wilt disease.AGO,DCL and RDR family genes modulated the process of RNA inteferance to regulate the expression of target gene in plant.These homologous genes in tomato genome were knocked down by VIGS in the susceptile cultivar Moneymaker,and then infected by FOL.The VIGS plants was partially silenced with Ago1、Ago4a and Ago6 showed a relative resistance to FOL.These results indicated that folmiR1 may interact with Ago1、Ago4a or Ago6 to mediate the resistance to FOL in tomato.Small RNA can be loaded to specific protein to form RNA-induccd silencing complex(RISC)to trigger RNAi and regulate the target genes.RNA-dcpcndcnt RNA polymerase(RDR)is a critical component RNAi by genrating dsRNA.We constructed RDR1 mutant by homologous recombination in FOL.Our results indicated that RDR1 was involved in pathogenicity.Therefore,RDR1 can be used as a target for RNAi to prevent the tomato wilt disease.In brief,our study identified the components in the folmiR1-mediated signaling pathway during tomato-F.oxysporum interaction,and reveal the related mechanism.Our studies should shed light on the breeding program of the disease resistance tomato.The discovery of the critical pathogenic gene RDR1 is a potential strategy to develop a new fungicide to tomato wilt disease.