Small RNA profiling of grapevine leafroll-associated virus 3 infected grapevine plants

[摘要] ENGLISH ABSTRACT: One of the most important viral diseases of grapevine worldwide is grapevine leafroll disease (GLD).A number of viruses from the family Closteroviridae have been associated with this disease, thoughGrapevine leafroll-associated virus 3 is considered the leading causative agent due to its consistentassociation with GLD. To better understand the disease and develop effective control strategies, it isnecessary to characterise the molecular interactions between the virus and the plant. Small RNA(sRNA) molecules have been shown to play an important role in gene regulation of normaldevelopment and defence responses to biotic and abiotic stresses in plants. Therefore, the aim of thisstudy was to characterise the sRNA species in healthy and infected grapevine to contribute to thegrowing database of sRNAs present in Vitis vinifera. Microarray analysis and next-generationsequencing was used to identify sRNA species in Chardonnay, Chenin blanc, Cabernet Sauvignon andown-rooted Cabernet Sauvignon plants. Differential expression of sRNAs was evaluated to identifysRNAs associated with GLRaV-3 infection. The modulation of the differentially expressedmicroRNAs (miRNAs) was validated with stemloop RT-qPCR assays. Transcriptome NGS was alsoperformed to validate the differential expression of the predicted miRNA targets, and to identifymetabolic pathways modulated in response to GLRaV-3 independently from sRNA regulation. Thetranscriptome NGS transcripts that were differentially expressed in all cultivar groups, and transcriptsthat anti-correlated with miRNA expression, were validated with RT-qPCR assays. These highthroughputapproaches identified several differentially expressed sRNAs and (target) genes in infectedplants. The anti-correlation of miRNA expression and putative target expression were shown for twomiRNAs. Cultivar specificity was identified in the sRNA and gene expression analyses, and bothapproaches identified Chenin blanc-specific responses. This comparison of symptomatic andasymptomatic GLRaV-3-infected plants provides the first insight into the disease symptom inhibitionobserved in certain cultivars. The differentially expressed genes identified in all cultivar groups, usingthe NGS transcriptome data, provides a collection of genes displaying a potentially universal molecular response against GLRaV-3. These genes showed strong associations with cell wallbiosynthesis and signalling during pathogen recognition. This study has contributed significantly tothe knowledge of sRNAs produced in grapevine and significantly extended the existing sRNAreference database for grapevine. The knowledge generated in this study can be utilised as potentialtargets for grapevine functional studies, and be translated into potential management strategies tocontrol the disease. A better understanding of both the host defence and viral counter-defencestrategies can lead to the prevention of virus replication or the impaired ability of the virus to inducepathogenesis in plants.

[发布机构] Stellenbosch University