| Rice sheath blight is one of the main diseases caused by the necrotrophic pathogen Rhizoctonia solani,which results in a severe declining in rice yield and quality.Nowadays,although some functional genes that confers resistance to R.solani were found,there were not much highly resistant rice cultivars obtained.So,pesticide is still firstly considered to control the rice sheath blight.To gain insight into the molecular mechanisms of the rice defense response to R solani infection,it is conducive to provide data support and molecular basis for molecular breeding,and gain more resistant resources to improve the rice yield and quality.In the present study,we observed the cellular condition of the in,fected leaf sheath by microscope,analyzed the expression profiles of genes and miRNAs by high-throughput sequencing and validated the function of a miRNA by transgene technology,which is helpful to explore the functional genes or miRNAs and the involvement of biological process at the level of transcriptional and post-transcriptional regulation.The results obtained in this study were concluded asfollowed:I.The cytological observation of rice leaf sheath that infected by R.solani was shown that no obvious symptoms were observed at 24 hours post-inoculation(hpi)and a few cells were damaged,while the lesion on the leaf sheath had turned to taupe at 48 hpi and mass of cells were death and destruction,suggesting that the disease may outbreak in a certain stage to result in serious condition rapidly.2.Based on the RNA-seq technology,we have obtained more than 430 million reads by RNA-sequencing and have identified 742 and 2,825 differentially expressed genes(DEGs)by comparing the SB-24 hpi and SB-48 hpi with their controls,respectively.Among these DEGs,a series of jasmonic acid(JA)and ethylene(ET)-related genes,pathogenesis-related(PR)genes,transcription factors(TFs)were found and classified.JA and ET-related genes participated in the biosynthesis of hormones and its derivatives,and delivering signaling to regulate their levels and signal transduction s PR genes found in this study could be classified into 12 families,and most of them were up-regulated with the infection.GO enrichment terms in cellular component have revealed that PR proteins function in cytoplasmic to inhibit the infection process.In addition,three TF families(MYB,ERF and WRKY)took mainly active in regulating the defense response to rice sheath blight,and WRKY genes of which may be act as a positive regulator.By function and pathway analyses,a large number of interrelated secondary metabolism were involved in resistance to R.solani in whole stage and the involvement of TFs may play an important role in the interaction of JA and ET signaling with metabolites and PR proteins.3.Based on the small RNAs sequencing technology,we identified both 33 differential expressed known miRNAs(DEMs)in 24hpi and 48 hpi,respectively,which overall tended to down-regulate in response to R.solani infection.There were 195 and 163 target genes of DEMs separately in 24 hpi and 48 hpi,whose expression changes were examined by transcriptome data,and showed that half of target genes expressed negatively correlated with DEMs.qRT-PCR analysis of part DEMs and target genes expression further confirmed the sequencing results.Function analyses have shown that these target genes were mainly associated with secondary metabolites,stress response and plant hormone signaling.4.Based on the functional analysis of the transgenic technology,a total of 35 positive transgenic rice was obtained in this experiment and the positive ratio was 70%.Over-expressing the osa-miR159b precursor in transgenic rice can significant increased the expression of osa-miR159b and decreased its target gene LOC_Os01g59660.Resistance assay results have shown that the lesion area and disease index of transgenic plant were smaller than that in WT,indicating that increasing the osa-miR159b expression could enhance rice resistance to R.solani.5.Based on the analysis of the unannotated sequences and combined with bioinfomatics tools,we have filtered 127 novel miRNA candidates and 17 of them were further identified by clone sequencing.In addition,approximately 80 miRNAs*or variants were obtained from the precursors of the known miRNAs and the osa-miR529*variant and osa-miR444c.1*variant were validated.There were 141 target genes predicted from the validated novel miRNAs.5' RACE and transient transformation analyses have shown that osa-NmiRl targets to LOC_Os09g34900 that is a chloroplast transport protein,which maybe involve in the biosynthesis of secondary metabolites. |
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