Doctoral Degrees (Genetics)

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Browsing Doctoral Degrees (Genetics) by Author "Bester, Rachelle"

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    Small RNA profiling of grapevine leafroll-associated virus 3 infected grapevine plants
    (Stellenbosch : Stellenbosch University, 2016-12) Bester, Rachelle; Maree, H. J.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    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.