Impact of exogenous caffeine on regulatory networks of microRNAs in response to Colletotrichum gloeosporioides in tea plant.

Anthracnose, caused by Colletotrichum gloeosporioides is one of the most serious diseases of tea plant [Camellia sinensis (L.) O. Kuntze]. MicroRNAs are key modulators of gene expression in defense responses and plant immunity; although, foliar application of exogenous caffeine in anthracnose disease control management has proven to be effective, miRNA-mediated regulatory mechanisms underlying caffeine-induced plant defense response to C. gloeosporioides remain unexplored in tea plant. Using high-throughput-sequencing, 24 miRNA sequencing data sets and 8 degradome data sets were generated from the susceptible cultivar Longjing43 (LJ43) and the resistant cultivar Zhongcha108 (ZC108) leaves treated with CK (Water), C. gloeosporioides-inoculation (CgI), exogenous caffeine (CN) and CgI + CN. Using sRNA sequencing, 424 conserved miRNAs and 417 novel miRNAs were identified; of these, 146 and 130 miRNAs were differentially expressed under CgI + CN treatment in the LJ43 and ZC108, respectively. Degradome sequencing identified 599 targets predicted to be cleaved by 210 conserved and 70 novel miRNAs. Majority of the annotated targets were found to involve in regulation of transcription factors, oxidation-reduction and metabolic process for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. The expression pattern of eight miRNAs and their targets were validated by qRT-PCR, and correlation analysis of csn-miR164a_R+1_1ss21AG/NAC-17 and csn-miR396b-5p/GRF-1 showed highly significant negative R-value at 7th dpi under CgI + CN in the LJ43. This study provides important insights into the novel approach of exogenous caffeine-induced miRNAs dynamically exerts its fungicidal activity through regulating JA/ET signaling pathway, thereby accurately switch on LJ43 susceptibility nature to resistance activity against C. gloeosporioides infection. In order to identify the microRNAs and their target genes in the tea plant, a total of 24 sRNA libraries and 8 degradome libraries were constructed from the susceptible cultivar Longjing (LJ43) and anthracnose-resistant cultivar Zhongcha (ZC108) leaves collected after 7 days of the mock-inoculated control (LJCK and ZCCK), the C. gloeosporioides-inoculated treatment (LJCg and ZCCg), the exogenous caffeine treatment (LJCN and ZCCN) and the C. gloeosporioides inoculation with exogenous caffeine treatment (LJCgCN and ZCCgCN); three independent replicates of each treatment were sequenced for sRNA sequencing using the Illumina Hiseq2500 platform.