Profiling of miRNA responses to viral coinfections in grapevine (Vitis vinifera L.)

Abstract

Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops worldwide and is susceptible to numerous pathogens, including viruses. To date, more than 100 viruses have been identified in grapevine, several of which cause severe disease symptoms that negatively affect yield and fruit quality (1). Viral infections can also alter the expression of microRNAs (miRNAs), a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level (2, 3). In this study, we investigated how viral infections influence miRNAexpression in the grapevine cultivars Refošk and Zeleni Sauvignon. In addition, degradome sequencing was performed to identify and analyze potential miRNAtarget transcripts.

Small RNAs were extracted from virus-free and virus-infected plants of two cultivars, Zeleni Sauvignon and Refošk. Sequencing was performed on the Ion Proton™ platform (Ion Torrent™, Life Technologies). In sRNA-seq datasets, viruses were detected using the VirusDetect pipeline. miRNAexpression was analyzed using three miRNAprediction tools: miRador, ShortStack and miRDeep2, and the integrated outputs from all three tools were used for differential expression analysis. Expression of selected miRNAs was further assessed by stem-loop RT-qPCR, and degradome sequencing was performed to confirm cleavage of target transcripts.

Small RNAsequencing generated between 6.3 and 25 million reads per library. No viruses were detected in virus-free samples, whereas infected samples contained viruses GPGV, GRSPaV, and GRVFV. Differential expression analysis identified 6 differentially expressed miRNAs in Refošk and 16 in Zeleni Sauvignon. Stem-loop RT-qPCR confirmed differential expression for 10 of 12 tested miRNAs in Refošk and 5 of 15 in Zeleni Sauvignon. Degradome sequencing identified miRNA-mediated cleavage of numerous transcripts, including transcripts involved in hormone signaling and plant–pathogen interaction pathways.

References

(1) Fuchs M. Grapevine viruses: Did you say more than a hundred? J. Plant Pathol. 2024 Dec 16; 107:217–227.

(2) Pantaleo V, Vitali M, Boccacci P, Miozzi L, Cuozzo D, Chitarra W, et al. Novel functional microRNAs from virus-free and infected Vitis vinifera plants under water stress. Sci Rep. 2016 Feb 2; 6:20167.

(3) Wang J, Mei J, Ren G. Plant microRNAs: Biogenesis, homeostasis, and degradation. Front. Plant Sci. 2019 Mar 27; 10:360.