Identification and characterization of cucumber microRNAs in response to Pseudoperonospora cubensis infection

• Forty-two miRNAs showed differentially expressions in P. cubensis-infected leaves.
• Four miRNAs were validated for responding to P. cubensis infection.
• miRNA-mediated target gene cleavage was confirmed experimentally.
• The target expression levels negatively correlated with these four miRNAs.

MicroRNAs (miRNAs) regulate the expression of genes related to several stress responses, including fungal infection, in plants. However, the miRNA-mediated gene regulatory networks in cucumbers that respond to Pseudoperonospora cubensis stress remain unexplored. In this study, the miRNA expression patterns in response to P. cubensis stress in cucumbers were investigated through high-throughput sequencing. A total of 123 known miRNAs and 4 novel miRNAs were identified, and their corresponding expressions were detected in mock- and P. cubensis-inoculated leaves. Three novel and 39 known miRNAs were found to be differentially expressed in P. cubensis-infected leaves. The results of 5′-RLM-RACE confirmed that miR164b, miR156h, miR171e, miR160b, and miR159f targeted No Apical Meristem domain protein, squamosa promoter binding protein-like class transcription factor, GRAS family transcription factor, Auxin response factor ARF16, and a conserved gene of unknown function, respectively. The expression patterns of these miRNAs were also determined through quantitative reverse transcription polymerase chain reaction (qRT-PCR). All of these miRNAs, except for miR156h, can respond to P. cubensis infection in cucumber leaves. In addition, the results of qRT-PCR revealed that the targets negatively correlated with their corresponding miRNAs (miR164b, miR171e, miR160b, and miR159f).