Molecular Mechanism Of Distinct Pathogenicity Among Different Variants Of Hop Stunt Viroid

Viroids are small,circular,non-coding infectious RNAs that have the ability to replicate autonomously when infecting higher plants.Their genomic RNAs range in size from 246 to 434 nucleotides(nt),and some viroids can infect economically important crops and cause devastating diseases.Although the interaction of viroid and host had been studied for years,the mechanism is not clear yet.In this study,hop stunt viroid(HSVd)/cucumber(Cucumis sativus cv.'Suyo')system was used to study the mechanism of viroid-host interaction.1.Analysis of pathogenicity differences of different HSVd mutants.HSVd-g was isolated from grapevine.It became a new variant(HSVd-h)after inoculated to hop for 15 years.The nucleotides at positions of 25,26,54,193 and 281 were different between HSVd-h and HSVd-g,and the pathogenicity of the two variants were mild and severe in their indicator plant(cucumber,Cucumis sativus cv.'Suyo'),respectively.Two series of mutants were constructed based on HSVd-h and HSVd-g.The virulence of HSVd-h series of mutants were:HSVd-g>HSVd-h>HSVd-h54>HSVd-h193>HSVd-h281>HSVd-h25>HSVd-h26,and the virulence of HSVd-g series of mutants were:HSVd-g54>HSVd-g193>HSVd-g>HSVd-g281>HSVd-g(25-26)>HSVd-g25>HSVd-g26>HSVd-h.This indicated that the pathogenicity of mutants with nucleotides at positions of 25 and 26 changed was significantly different from their wild type variants while the pathogenicity of mutants with nucleotides at positions of 54,193 and 281 changed was slightly different from their wild type variants.Therefore,the pathogenicity of HSVd is determined by multiple nucleotides,and each nucleotide has a different degree of action.2.Transcriptomic analysis of cucumber plants inoculated with different variants of HSVd.The most severe mutant HS Vd-g54 in HSVd-g series of mutants and the mild variant HSVd-h were inoculated to cucumber,and the global transcriptomic changes were analyzed through RNA sequencing(RNA-seq).The results showed that HSVd-g54 infection induced earlier and severe symptom than HSVd-h.HSVd infection triggered basal defense responses,depressed photosynthesis,disrupted phytohormone homeostasis,might induce the accumulation and signal transduction of salicylic acid(SA),and induced the expression of CsRDR1 genes,especially CsRDRlcl and CsRDRlc2,in cucumber plants.3.Effects of different HSVd variants on miRNAs expression of cucumber plants.Cucumber plants infected with HSVd-h,HSVd-g54 and phosphate buffer were sequenced.A total of 65 known miRNAs and 32 novel miRNAs were indentified.Comparing with mock-inoculated cucumber plants,the expression level of 31 known miRNAs and 21 novel miRNAs changed significantly in HSVd-infeced cucumbers.Target genes were predicted to these differentially expressed miRNAs,and 147 target genes were acquired corresponding to 29 known miRNAs.The function of these miRNAs and their target genes were analyzed.The results showed that the target genes of miR408 encoded basic blue proteins.And basic blue proteins can bind to copper to transfer electron in photosynthetic system.The up-regulation of expression of miR408 might reduce the accumulation of basic blue proteins in HSVd-infected cucumbers.Therefore,photosynthesis of cucumber may be repressed by HSVd through the activity of miR408.In addition,the splice sites of cme-miR408 in the target gene LOC101220519 was determined by 5' RLM-RACE,and the results showed that the main splice sites were located in the bases 10-11 and 11-12 of cme-miR408.