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.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a rapid and sensitive method for analyzing microRNA (miRNA) expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA) were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis) using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA), respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants.

Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms.