Identification of MicroRNAs and Their Targets That Respond to Powdery Mildew Infection in Cucumber by Small RNA and Degradome Sequencing

Abstract

Powdery mildew (PM) is a prevalent disease known to limit cucumber production worldwide. MicroRNAs (miRNAs) are single-stranded molecules that regulate host defense responses through posttranscriptional gene regulation. However, which specific miRNAs are involved and how they regulate cucumber PM resistance remain elusive. A PM-resistant single-segment substitution line, SSSL508-28, was developed previously using marker-assisted backcrossing of the PM-susceptible cucumber inbred D8 line. In this study, we applied small RNA and degradome sequencing to identify PM-responsive miRNAs and their target genes in the D8 and SSSL508-28 lines. The deep sequencing resulted in the identification of 156 known and 147 novel miRNAs. Among them, 32 and six differentially expressed miRNAs (DEMs) were detected in D8 and SSSL508-28, respectively. The positive correlation between DEMs measured by small RNA sequencing and stem-loop quantitative real-time reverse transcription–polymerase chain reaction confirmed the accuracy of the observed miRNA abundances. The 32 DEMs identified in the PM-susceptible D8 were all upregulated, whereas four of the six DEMs identified in the PM-resistant SSSL508-28 were downregulated. Using in silico and degradome sequencing approaches, 517 and 20 target genes were predicted for the D8 and SSSL508-28 DEMs, respectively. Comparison of the DEM expression profiles with the corresponding mRNA expression profiles obtained in a previous study with the same experimental design identified 60 and three target genes in D8 and SSSL508-28, respectively, which exhibited inverse expression patterns with their respective miRNAs. In particular, five DEMs were located in the substituted segment that contained two upregulated DEMs, Csa-miR172c-3p and Csa-miR395a-3p, in D8 and two downregulated DEMs, Csa-miR395d-3p and Csa-miR398b-3p, in SSSL508-28. One gene encoding L-aspartate oxidase, which was targeted by Csa-miR162a, was also located on the same segment and was specifically downregulated in PM-inoculated D8 leaves. Our results will facilitate the future use of miRNAs in breeding cucumber varieties with enhanced resistance to PM.