Mechanism Of Negative Regulation Of Tomato Resistance To Fusarium Wilt By Sly-miR482e-3p

Tomato wilt is a soil-borne fungal disease on global scale,elicited by infection of Fusarium oxysporum f.sp.Lycopersici(Fol)and leaving the plant vascular bundles withered with brown plants as well as rotten bulbs and roots,debilitating growth and even death of the whole plant.To date,the molecular mechanisms underlying interplay of Fol and the host remain elusive.Plant small RNAs(sRNAs)are a family of non-coding RNAs with 20-30 nucleotides in length that can regulate the expression of their target genes at the transcript or post-transcription level.Upon pathogenic infection,the expression levels of various endogenous sRNA from plants are perturbed.Our previous studies indicated that the expression level of Sly-miR482e-3p was suppressed as well as its target genes up-regulated in tomato when exposed to Fol.This fundamental finding drives the present investigation with a set of new insights:(1)Sly-miR482e-3p responded to Fol infection at early stages from different resistant varieties;(2)The tissue-specific expression profiles revealed that Sly-miR482e-3p was enriched in the tomato stems and leaves;(3)CRISPR/Cas9 technique was utilized to knock out Sly-miR482e-3p in the susceptible cultivar Moneymaker yielding three positive seedlings including Line2,Line5 and Line6 with 2,9 and 6 nucleotides deletion at the target 1 locus,respectively;(4)The expression level of Sly-miR482e-3p significantly declined and accordingly the mRNA levels of its two target genes(NBS-LRR)were increased;(5)Expressions of other members of the slmR482 family including sly-miR482a,sly-miR482b,sly-miR482c,sly-miR482d-3p,sly-miR482-5p and sly-miR482e-5p were not affected;(6)The resistance of susceptible Moneymaker against Fol was enhanced in Sly-miR482e-3p knockout plants,suggesting Sly-miR482e-3p implicating in the tomato resistance to Fusarium wilt;(7)To unravel the mechanisms of Sly-miR482e-3p engaging in tomato resistance against Fusarium wilt,transcriptome libraries from six groups(MM_H2O,MM_Fol,Line 2 H2O,Line 2 Fol,Line 5 H2O,Line 5_Fol)were established for RNA-seq analysis;(8)The KEGG analysis pointed to the ethylene signaling pathway was modulated in plants devoid of Sly-miR482e-3p and genes related with ethylene synthesis or encoding signaling factors were deferentially expressed;(9)The mRNA levels of ethylene synthase genes(LeACO1 and LeACS3),Ethylene signaling pathway positive regulatory factor genes(LeEIN2 and LeEILl),ethylene transcription response effector genes(LeERFl,LeERF4,LeERF5 and LeERF9)declined relative to wild-type ones;in contrast,ethylene pathway negative regulatory factor genes(LeETR1,LeETR4,LeETR5,LeCTR2 and LeCTR3)were up-regulated as compared to wild type plants;(10)Sly-miR482e-3p may modulate tomato resistance to Fusarium wilt by mediating the ethylene signaling,which evidenced by attenuation the resistance of Motelle and three transgenic lines against Fol with exogenous spraying of ACC(the ethylene precursor).In conclusion,this study not only highlights the negative modulation of Sly-miR482e-3p in mediating ethylene signaling pathway against Fusarium wilt,but also sheds light on molecular underpinnings of Sly-miR482e-3p involved in interactions between Fusarium oxysporum and its host.Our results,theoretically,should lay solid foundation for genetic breeding of novel varieties resistant to Fusarium wilt.