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.
Highlights
Powdery mildew (PM) fungi are obligate biotrophic parasites that are spread worldwide and cause severe diseases in a large variety of horticultural plants, such as cucurbits, tomato, pea, and okra (Martínez-Cruz et al, 2017; Cobos et al, 2018; Fan et al, 2019)
Dense powdery mildew (PM) hyphae were seen on the surface of the D8 leaves, whereas no conidia were detected on the surface of the SSSL508-28 leaves (Figure 1C)
More differentially expressed miRNAs (DEMs) were identified in the inoculated D8 (ID) versus non-inoculated D8 (NID) comparison than in the inoculated SSSL508-28 (IS) versus non-inoculated SSSL508-28 (NIS) comparison (Tables 3, 4), which suggested the miRNA expression levels may vary greatly depending on the lines/varieties
Summary
Powdery mildew (PM) fungi are obligate biotrophic parasites that are spread worldwide and cause severe diseases in a large variety of horticultural plants, such as cucurbits, tomato, pea, and okra (Martínez-Cruz et al, 2017; Cobos et al, 2018; Fan et al, 2019). The disease initiates as thin white spots, first on the surfaces of older leaves and gradually spreading to younger leaves. Grayish white mycelia are visible on severely affected leaves, and such leaves exhibit chlorosis and senescence. The application of fungicide can effectively control the disease at present but is not environmentally friendly and is harmful to consumers’ health because cucumbers are harvested almost daily for fresh market consumption (Fukino et al, 2013; Xu et al, 2016). A deeper understanding of the molecular mechanisms that trigger cucumber plants to recognize and eventually prevent or limit PM infection is required
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