microRNAs and their targets engage in apple-Valsa mali interaction

miRNAs were important regulators involving in plant-pathogen interactions. However, their roles in apple response to Valsa canker pathogen (Valsa mali, Vm) infection were poorly understood. In this study, we constructed two miRNA libraries using the bark tissues of apple twig (Malus domestica Borkh “Fuji”) inoculated with Vm (IVm) and PDA medium (control, BMd). Among the all miRNAs, 23 miRNAs were specifically isolated in BMd and 39 miRNAs were specifically isolated in IVm. Compared with BMd, the expression of 294 miRNAs decreased, and 172 miRNAs increased in IVm, respectively. We also identified the target genes of these miRNAs using degradome sequencing technology. In total, 353 differentially expressed miRNAs during the pathogen infection were detected to target 1 077 unigenes with 2 251 cleavage sites. Based on GO and KEGG analysis, the genes were found to be mainly related to transcription regulation and signal transduction. We further selected 17 miRNAs and 22 corresponding target genes to detect the expression profiles during pathogen infection. The results indicate that most of them are involved in apple twig-Vm interaction. What’s more, miRNAs and their corresponding target genes seem to regulate the apple twig-Vm interaction by forming many complicated regulation networks. It is worth that a conserved miRNAs mdm-miR482b, which was down regulated in IVm compared with BMd, has 14 potential target genes, and most of them were disease resistance related genes. More important, the feedback regulation of sRNA pathway in apple twig was much more complex and critical in the interaction between apple bark tissue and V. mali. The results provide insights into the crucial functions of miRNAs in the woody plant, apple tree-Vm interaction. miRNAs were isolated using high-throughput sequence analysis and their corresponding target genes were identified using degradome sequencing technology.

微RNA（microRNA，miRNA）是参与植物-病原物互作的重要调控因子。然而，其在苹果应对苹果树腐烂病病原（*Valsa mali*，Vm）侵染过程中的功能却鲜有研究报道。本研究以接种Vm的苹果枝条树皮组织（栽培品种‘富士’，*Malus domestica* Borkh ‘Fuji’）为材料，分别构建了接种组（IVm）与对照组（BMd，接种马铃薯葡萄糖琼脂PDA培养基）的两个miRNA文库。在所获得的全部miRNA中，23个仅在对照组BMd中特异性检出，39个仅在接种组IVm中特异性检出。与对照组BMd相比，接种组IVm中有294个miRNA表达量下调，172个miRNA表达量上调。本研究同时利用降解组测序技术，对上述miRNA的靶基因进行了鉴定。总计共有353个在病原侵染过程中差异表达的miRNA，靶向调控1077个单基因簇（unigene），共计2251个剪切位点。通过基因本体（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析发现，这些靶基因主要参与转录调控与信号转导过程。本研究进一步选取了17个miRNA及其对应的22个靶基因，对其在病原侵染过程中的表达谱进行了验证分析。实验结果表明，其中多数参与苹果枝条与Vm的互作过程。此外，miRNA与其靶基因通过构建复杂的调控网络，参与调控苹果枝条与Vm的互作过程。值得注意的是，保守miRNA mdm-miR482b在接种组IVm中表达量较对照组BMd下调，其拥有14个潜在靶基因，且多数靶基因与抗病相关。更为重要的是，在苹果树皮组织与Vm的互作过程中，小RNA（small RNA，sRNA）通路的反馈调控机制更为复杂且关键。本研究结果为解析木本植物苹果与Vm的互作过程中miRNA的核心功能提供了新的视角。本研究通过高通量测序技术分离得到miRNA，并利用降解组测序技术鉴定了其对应的靶基因。