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

Thesis (PhD)--Stellenbosch University, 2016.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, though Grapevine leafroll-associated virus 3 is considered the leading causative agent due to its consistent association with GLD. To better understand the disease and develop effective control strategies, it is necessary 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 normal development and defence responses to biotic and abiotic stresses in plants. Therefore, the aim of this study was to characterise the sRNA species in healthy and infected grapevine to contribute to the growing database of sRNAs present in Vitis vinifera. Microarray analysis and next-generation sequencing was used to identify sRNA species in Chardonnay, Chenin blanc, Cabernet Sauvignon and own-rooted Cabernet Sauvignon plants. Differential expression of sRNAs was evaluated to identify sRNAs associated with GLRaV-3 infection. The modulation of the differentially expressed microRNAs (miRNAs) was validated with stemloop RT-qPCR assays. Transcriptome NGS was also performed to validate the differential expression of the predicted miRNA targets, and to identify metabolic pathways modulated in response to GLRaV-3 independently from sRNA regulation. The transcriptome NGS transcripts that were differentially expressed in all cultivar groups, and transcripts that anti-correlated with miRNA expression, were validated with RT-qPCR assays. These highthroughput approaches identified several differentially expressed sRNAs and (target) genes in infected plants. The anti-correlation of miRNA expression and putative target expression were shown for two miRNAs. Cultivar specificity was identified in the sRNA and gene expression analyses, and both approaches identified Chenin blanc-specific responses. This comparison of symptomatic and asymptomatic GLRaV-3-infected plants provides the first insight into the disease symptom inhibition observed in certain cultivars. The differentially expressed genes identified in all cultivar groups, using the NGS transcriptome data, provides a collection of genes displaying a potentially universal molecular response against GLRaV-3. These genes showed strong associations with cell wall biosynthesis and signalling during pathogen recognition. This study has contributed significantly to the knowledge of sRNAs produced in grapevine and significantly extended the existing sRNA reference database for grapevine. The knowledge generated in this study can be utilised as potential targets for grapevine functional studies, and be translated into potential management strategies to control the disease. A better understanding of both the host defence and viral counter-defence strategies can lead to the prevention of virus replication or the impaired ability of the virus to induce pathogenesis in plants.AFRIKAANS OPSOMMING: Een van die belangrikste virussiektes van wingerd wêreldwyd is wingerd-rolblaarsiekte (GLD). 'n Aantal virusse van die familie Closteroviridae hou verband met hierdie siekte, maar Grapevine leafroll-associated virus 3 (GLRaV-3) word beskou as die hoof-bydraende faktor van GLD as gevolg van die korrelasie met GLD. Om die siekte beter te verstaan en effektiewe beheer toe te pas, is dit nodig om die molekulêre interaksie tussen die virus en die plant te ondersoek. Klein RNA (sRNA) molekules het getoon dat hulle 'n belangrike rol speel in die geenregulering van normale plant ontwikkeling, asook tydens die plant se verdediging teen biotiese en abiotiese stresfaktore. Die doel van hierdie studie was dus om die sRNA spesies in gesonde en geïnfekteerde wingerdstokke te karakteriseer en sodoende by te dra tot die snelgroeiende databasis van Vitis vinifera sRNAs. ‘n Mikro-DNA-volgorde-raamwerk analise, asook nuwe-generasie volgordebepaling (NGS) is gebruik om sRNAs spesies te identifiseer in die kultivars Chardonnay, Chenin blanc, Cabernet Sauvignon en eie-gewortelde Cabernet Sauvignon plante. Daarna is die differensiële uitdrukking van sRNAs geëvalueer om sodoende sRNAs te identifiseer wat verbandhou met GLRaV-3 infeksie. Die regulering van die differensiële uitgedrukte mikroRNA’s (miRNAs) is bevestig met stam-lus tru-transkripsie kwantitatiewe polimerase ketting reaksie (stemloop-RT-qPCR) toetse. Transkriptoom-NGS is ook uitgevoer om die differensiële uitdrukking van die miRNAs se voorspelde teikens te valideer en om gemoduleerde metaboliese paaie in reaksie op GLRaV-3, onafhanklik van sRNA regulasie, te identifiseer. Die transkriptoom NGS gene wat differensieël uitgedruk was in al die kultivar groepe, asook die gene wat ’n anti-korrelasie getoon het met miRNA uitdrukking, is bevestig met RT-qPCR toetse. Hierdie hoë-deurset benaderings het verskeie sRNAs en gene geïdentifiseer wat differensieël uitgedruk was in geïnfekteerde plante. Die anti-korrelasie van miRNA uitdrukking en voorspelde teiken uitdrukking is geïdentifiseer vir twee miRNAs. Kultivar-spesifisiteit was geïdentifiseer in beide die sRNA en geenuitdrukking analises en beide benaderings het ook Chenin blanc-spesifieke reaksies geïdentifiseer. Hierdie vergelyking van simptomatiese en asimptomatiese GLRaV-3-geïnfekteerde plante bied die eerste insig in die simtoom-inhibisie wat waargeneem word in sekere kultivars. Die differensieël-uitgedrukte gene wat geïdentifiseer was in alle kultivar groepe, met behulp van die NGS transkriptoom data, bied 'n versameling van gene wat ‘n potensiële universele molekulêre reaksie toon teen GLRaV-3. Hierdie gene het sterk assosiasies met selwand-biosintese en patogeen herkenningseine. Hierdie studie het aansienlik bygedra tot die kennis van wingerd-geassosieerde sRNAs en dra beduidend by tot die uitgebreide sRNA databasis. Die kennis wat gegenereer is in hierdie studie kan gebruik word om teikens te identifiseer vir wingerd funksionele studies en om verwerk te word na potensiële strategieë om die siekte te beheer. 'n Beter begrip van beide die gasheer verdedigings- en die virale teen-verdedigingstrategieë, kan lei tot die voorkoming van virus replikasie of ‘n verlaging in die vermoë van die virus om die siekte in plante te veroorsaak