Table_4_Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).xls

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5’RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.

胶孢炭疽菌（Colletotrichum gloeosporioides）引发的炭疽病是茶树（Camellia sinensis）叶片的常见病害。微小RNA（MicroRNAs, miRNAs）作为基因表达的关键调控因子，已被证实可参与植物应对环境胁迫、抗病性、防御反应及植物免疫的调控过程。然而，茶树中miRNA在胶孢炭疽菌胁迫响应中的作用仍未得到阐明。为此，本研究从接种胶孢炭疽菌的茶树叶片及对照叶片中构建了6个miRNA测序数据集与2个降解组数据集。共鉴定得到485个保守miRNA与761个新miRNA，其中239个已知miRNA及369个新miRNA在胶孢炭疽菌胁迫下呈现显著差异表达。通过降解组分析，分别鉴定到389个保守miRNA对应的1134个靶mRNA，以及299个新miRNA对应的596个靶mRNA。降解组分析结果显示，多数预测靶基因与其对应的保守或新miRNA呈负相关关系。通过实时定量PCR（quantitative real-time PCR）验证了12个miRNA及其靶基因的表达水平，其中5个miRNA（PC-5p-80764_22、csn-miR160c、csn-miR828a、csn-miR164a、csn-miR169e）与其各自靶基因（WRKY、ARF、MYB75、NAC及NFY转录因子）的表达谱呈显著负相关。通过5'RLM-RACE进一步验证了5个目标miRNA的预测靶基因。基因本体（Gene Ontology）与代谢通路分析表明，多数靶基因参与茶树抵御胶孢炭疽菌胁迫过程中的生长素通路调控、活性氧清除通路、水杨酸介导通路、受体激酶以及调控植物生长发育与胁迫响应的转录因子相关生物学过程。本研究丰富了茶树中胁迫响应miRNA及其靶基因的资源，为miRNA介导的调控机制提供了新的见解，从而有助于增强茶树对胶孢炭疽菌的感病性。