MicroRNAs-targets In Apple Involved In Response To Infection Of Pathogen Valsa Mali

Apple Valsa canker caused by Valsa mali is widespread in China.It is a destructive disease and brings huge economic losses,hindering the development of the apple industry.However,it has always been in a state of passive control because of the poorly understanding of the pathogenic mechanism and host resistance.The interaction mechanism between apple trees and V.mali is very important for disease control and disease resistance breeding.MicroRNAs are small non-coding RNAs that involved in the plant disease resistance by regulating genes expression at the post-transcriptional level.In this study,sRNA libraries were constructed by small RNA deep-sequencing technology using total RNA isolated from “Fuji” apple tree twig bark tissues inoculated with V.mali and PDA medium.The conserved miRNAs were identified based on the known miRNAs in miRBase21.0 database,the new miRNAs were predicted by characteristics of miRNAs/pre-miRNAs,combining the genome information of apple.And their expression were analyzed.Degradome libraries were constructed by degradome high-throughput sequencing technology using total RNA same to the RNA used for small RNA sequencing library construction.The targets of the differentially expressed miRNAs were analyzed to understand the roles of apple miRNAs in apple-V.mali interactions.The main results are as follows:1.Two sRNA libraries(BMd and IVm)were constructed.In total,187 conserved miRNAs from 138 precursors and 1189 new candidate miRNAs from 1178 precursors were identified.In BMd,had 173 conserved miRNAs from 137 precursors and 1164 new candidate miRNAs from 1157 precursors;In IVm,had 174 conserved miRNAs from 131 precursors and 1179 new candidate miRNAs from 1175 precursors.2.The expression of apple miRNAs were different between BMd and IVm.23 and 39 miRNAs were specifically expressed in the BMd and IVm,respectively.113 and 40 candidate miRNAs showed up-and down-regulation in Vm-infected barks compared with the mock-inoculated control(p ? 0.01,|log2(fold change)| ? 1).3.Two degradome libraries(TBMd and TBVm)were constructed.1077 genes were identified,to be the target of 353 miRNAs(including 158 conserved mi RNAs),and 106 of them were regulated by the differentially expressed miRNAs(p?0.01).Most are transcription factors,some resistance genes and a small number of unknown functions sequences.4.The enrichment results of all target-genes showed that the mainly biological process were "regulation of transcription","apoptosis","plant-type hypersensitive response","defense response" and "auxin-mediated signaling pathway",etc.The targets of the differentially expressed miRNAs were mainly enriched in "transcription","auxin-activated signaling pathway","nucleus","SCF ubiquitin ligase complex" and classified 124 KEGGs pathway,the most abundantly enriched pathways include "cysteine and methionine metabolism","ribosome","regulation of autophagy",and "protein export",etc.5.The regulatory network of miRNAs and their targets was very complex.Based on the detected targets,we found that the one-to-one regulation model was not prevalent and feedback regulation of the sRNA pathway.The conserved miRNA mdm-miR482 b in apple could target multiple resistant genes,and it showed down-regulation in Vm-infected barks.9 miRNAs could target ATL2,and ATL2 showed up-regulation in Vm-infected barks.These miRNAs/targets on the cross point may be essential in the disease response signal network.