Identification And Functional Analysis Of Important LncRNAs Related To Tomato Late Blight

Tomato is a major horticulture plant widely planted in the world.During its growth and development,it is often harmed by late blight,causeing serious economic losses.Therefore,clarifying the molecular mechanism of tomato resistance to late blight,main resistance molecules and cultivating resistance varieties,are the key to control late blight.As a regulator,IncRNA is involved in various biological processes of plants,including regulating growth and development,responding to abiotic stresses and resisting pathogen infection.However,few studies has been reported that lncRNAs affect in tomato resistance to late blight.In this study,late blight-resistant and susceptible tomatoes were used as materials to identify IncRNA by RNA-Seq,analyze the function by overexpression and silencing methods,and construct regulatory networks with miRNAs and genes.This study provides a theoretical basis for the molecular mechanism of tomato resistance tolate blight pathogen,and also provides candidate molecules for tomato resistance breeding.The main findings and conclusions are as follows:(1)Comparative transcriptome analysis of late blight-resistant and-susceptible tomatoes was used to identify 688 differentially expressed lncRNAs,127 of which may regulate 128 gens.In Chr4,8 lncRNAs locate in Glutaredoxin(GRX)gene cluster,which are antisense lncRNAs.These lncRNAs and GRX genes are divided into 4 modules.LncRNA16397 acted as an antisense transcript of SlGRX22 to regulate its expression,and also induced SlGRX21 expression.In addition,disease symptoms and reactive oxygen species(ROS)accumulation in tomatoes overexpressing lncRNA16397 and SpGRX were fewer and lower than those in wild-type after late blight pathogen infection.This result suggests that lncRNAs involved in the response of tomato to late blight pathogen infection,and lncRNA 16397 induces SlGRXs expression to reduce ROS accumulation and alleviate cell membrane injury,resulting in enhanced resistance to late blight pathogen.(2)Transcriptome analysis of tomato that overexpressed WRKY1 shows that the expression of 22 lncRNAs may be affected by WRKY1 transcription factor.The results of promoter-GUS fusion and yeast one-hybrid assay show that lncRNA33732 are activated by WRKY1 through sequence-specific interactions with the W-Box element in its promoter.The overexpression and silencing analysis of lncRNA33732 in tomato show that lncRNA33732 as a positive regulator enhances tomato resistance to P.infestans by induction of the expression of RESPIRATORY BURST OXlDASE(RBOH)and increase in the accumulation of H2O2.When the expression of RBOH gene is inhibited in tomato plants,H2O2 accumulation decreases and the resistance impaires.These findings suggest that lncRNA33732 regulated by WRKY1 induces RBOH expression to lncrease H2O2 accumulation in early defense reaction of tomato to late blight pathogen infection.(3)Comparative transcriptome analysis of tomato inoculated with and without late blight pathogen allow to find that the expression levels of 1103 genes and 196 lncRNAs are changed.35 genes from 5 transcription factor family resoponse to P.infestans infection,and 88 lncRNAs as endogenous competing RNAs(ceRNAs)can silence 46 miRNAs.Degradome sequence result shows that 11 miRNAs could target 30 genes.These lncRNAs,miRNAs and genes formed 10 regulatory modules.Among them,3 modules affect the expression of transcription factors.Silencing of lncRNA42705 and lncRNA08711 in tomato led to lncreased accumulation of miR159 and decreased accumulation of MYBs,as well as reduced resistance to late blight pathogen Our results demonstrate that TFs are involved in defense reactions and lncRNAs functioned as ceRNAs modulate the effects of miRNAs in tomato and improve plant disease resistance mechanism.In conclusion,this study screens 4 key lncRNAs,identifies transcription factors and miRNAs interacting with them and clarifies their functions,which will provide theoretical basis for revealing the molecular mechanism of tomato late blight resistance,provide candidate molecules for tomato disease resistance breeding.