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

H W Liu,C Q Liang,J Q Li,L X Luo,N Jiang,P F Liu

doi:10.1371/journal.pone.0129002

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.

Highlights

MicroRNAs are evolutionarily conserved endogenous noncoding RNAs [1] that were first discovered in the nematode Caenorhabditis elegans [2]
The putative miRNA sequences were filtered further according to the FE or MEFI criteria listed in Table 1, which yielded a final total of 15,063,156 mappable reads within the 15–30 nt range, of which 95.3% were 23 nt in length (Fig 2)
Only 1.1% of the Small RNA (sRNA) recovered from cucumbers infected with Cucumber green mottle mosaic virus (CGMMV) were 24 nt, which is a huge difference compared with previous studies

Summary

Introduction

MicroRNAs (miRNA) are evolutionarily conserved endogenous noncoding RNAs [1] that were first discovered in the nematode Caenorhabditis elegans [2]. Subsequent studies have revealed that miRNAs can be found in a broad range of species including plants, nematodes, fruit flies, mice and humans. Empirical investigations have recently demonstrated that miRNAs play important regulatory roles in a range of biological and metabolic processes including the development and differentiation of plant organs, as well as in response to external biotic and abiotic stresses [3]. MiRNAs are known to affect processes such as protein degradation, signal transduction, and responses to pathogen invasion as well as the regulation of their own biogenesis [1]. Their mode of action appears to involve post-transcriptional gene regulation by the repression of translation or cleavage of targeted mRNAs

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