High-Throughput Sequencing Identifies Novel and Conserved Cucumber (Cucumis sativus L.) microRNAs in Response to Cucumber Green Mottle Mosaic Virus Infection

Abstract

Seedlings of Cucumis sativus L. (cv. 'Zhongnong 16') were artificially inoculated with Cucumber green mottle mosaic virus (CGMMV) at the three-true-leaf stage. Leaf and flower samples were collected at different time points post-inoculation (10, 30 and 50 d), and processed by high throughput sequencing analysis to identify candidate miRNA sequences. Bioinformatic analysis using screening criteria, and secondary structure prediction, indicated that 8 novel and 23 known miRNAs (including 15 miRNAs described for the first time in vivo) were produced by cucumber plants in response to CGMMV infection. Moreover, gene expression profiles (p-value <0.01) validated the expression of 3 of the novel miRNAs and 3 of the putative candidate miRNAs and identified a further 82 conserved miRNAs in CGMMV-infected cucumbers. Gene ontology (GO) analysis revealed that the predicted target genes of these 88 miRNAs, which were screened using the psRNATarget and miRanda algorithms, were involved in three functional categories: 2265 in molecular function, 1362 as cellular components and 276 in biological process. The subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the predicted target genes were frequently involved in metabolic processes (166 pathways) and genetic information processes (40 pathways) and to a lesser degree the biosynthesis of secondary metabolites (12 pathways). These results could provide useful clues to help elucidate host-pathogen interactions in CGMMV and cucumber, as well as for the screening of resistance genes.

Fig 1. Confirmation of CGMMV-infection in cucumber samples (cv. Zhongnong 16).

A, CGMMV particles in infected cucumber leaves observed by SEM. B. Gel electrophoresis of RT-PCR using CGMMV specific primers. M, DNA ladder; 1, Sample from uninfected plants; 2, Blank control; 3, Sample from infected plant; 4, Infected leaves obtained from CAIQ used as a positive control.

Fig 2. Length distribution of mapped sequence reads from cucumber plants infected with CGMMV.

Fig 3. Length distribution of cucumber pre-miRNAs identified in the current study.

Fig 4. Secondary structure of novel cucumber miRNAs predicted by bioinformatic analysis using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).

Fig 5. Overview of the microarray analysis.

Chips prepared with miRNAs from cucumber and conserved miRNAs from species closely related to cucumber were probed with sRNA extracted from the leaves and flowers of cucumbers at different time points post inoculation with CGMMV. A, B and C, The proportions of genes annotated by GO analysis in three categories: molecular function, biological process and cellular component, respectively. D. Results from the KEGG analysis, which classified the target genes into different functional pathways [37, 38].