Identification Of The Mirna Induced Or Suppressed By Xanthomonas Campestris PV. Campestris In Arabiodopsis Thaliana

miRNA(miRNA)is the endogenous non-coding RN A with the length of-22nt(nucleotide)in eukaryotic cells.miRNA is involved in various cellular processes including cell growth and development,tissue differentiation,tumorigenesis and cell apoptosis.In addition,miRNA has been shown playing important role in the defense response against the infection of plant pathogens.Arabidopsis thaliana is one of the model plant for studying the molecular basis of the interaction between host plant and plant pathogens.It has been known that,several Arabidopsis miRNAs are involved in the defense response against the infection of Pseudonmonas syringae pv.tomato(Pst).Xanthomonas campestris pv.campestris(Xcc)is the causal agent of black rot disease of cruciferous plants,and it is able to infect Arabidopsis thaliana.However,no miRNA involved in the defense response against Xcc has been reported so far.It has been shown that expression of the miRNA involved in defense response usually induced or suppressed by the pathogen.The aim of this study is to identify the Arabidopsis thaliana miRNA whose expression is induced or suppressed by Xcc,and thus provided the first step for studying the role of miRNA in the defense response against Xcc.For this,we firstly determine the expression profiles of miRNA at different stages of Xcc infection by using the high-throughput RNA-seq technology.And then,identify the candidate miRNAs that is induced or suppressed by Xcc by using differential expression analysis.Subsequently,we use the quantitative Northern blot analysis to validate the candidate miRNAs.Before conducting high-throughput RNA-seq,we established an inoculation method,vacuum filtration,which is suitable for determination of the transcriptome changes in both the host Arabidopsis thaliana and the pathogen Xcc during their interaction.Then,we determined the expression profiles of miRNA in Arabidopsis thaliana Colombia 0 eco-type(Col-0)at the time points of 2,6,12 and 24 hours after infected by Xcc wild type strain and the hrcV gene mutants(△hrcV).The results of differential expression analysis show that the expression of 10 miRNAs is induced by Xcc and thus consider as candidate Xcc-induced miRNAs.Results of quantitative Northern blot analysis show that the expression of miR166 and miR393 is increased significantly after 2 hours post-inoculation ofXcc,while the expression of miR160 and miR156 is increased significantly after 24 hours post-inoculation,indicating that these miRNAs are induced by Xcc.In addition,3 novel Arabidopsis miRNAs are confirmed by Northern blot analysis.Taken together,in this work,we systemically identified the miRNAs that are induced or suppressed by Xcc infection in Arabidopsis thaliana buy using the RNA-Seq technique,and found that miR156,miR160,miR166 and miR393 were induced by Xcc.This is the first report on identifying Xcc-induced or-suppressed miRNAs in Arabidopsis thaliana.Our results provided the first step for studying the role of miRNA in the defense response against Xcc.