Characterisation of microRNA expression profiles of Vitis vinifera in response to grapevine leafroll-associated virus 3 infection

Aldrich, Dirk Jacobus (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is endemic to all grape-growing regions of the world and is considered the most significant grapevine viral disease. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the primary cause of GLD and in South African vineyards five genetic variant groups (I, II, III, VI and VII) have been confirmed. Small RNAs (sRNAs) have been shown to play a significant role in a plant’s response to biotic and abiotic stress. This has led to a growing interest in evaluating sRNAs, such as microRNAs (miRNAs), for their role in mediating gene regulation in response to virus infections. In this study, stem-loop RT-qPCR probe-based assays were utilised for miRNA quantitation in GLRaV-3 positive and negative grapevines. A set of own-rooted Cabernet Sauvignon plants representing GLRaV-3 variant groups I, II, III and VI has been established from cuttings of highly symptomatic GLRaV-3 infections found in commercial vineyards. These plants were sampled and screened to yield the first data set. Additionally, young Cabernet Sauvignon plants were established and graft-inoculated with single infections of the five known variants of GLRaV-3 found in South African vineyards. All these plants were maintained in a climate-controlled greenhouse and sampled twice, six months apart, to yield two data sets. A fourth data set comprised of GLRaV-3 positive and negative Cabernet Sauvignon plants sampled from various vineyards in Stellenbosch. Eleven miRNAs were quantified in both infected and healthy grapevine samples. Putative miRNA targets were predicted and annotated using in silico analyses. These targets were subsequently quantified in both greenhouse and field samples using a SYBR Green RT-qPCR assay. This study validated statistically significant differences in virus concentrations, expressed as virus concentration ratios (VCRs), in plants singly infected with different GLRaV-3 variants. Interestingly, no difference in mean VCRs were observed between data sets, despite notable differences in plant age, duration of GLRaV-3 infection, scion/rootstock combination and growing conditions. Several miRNAs showed statistically significant expression modulation between infected and healthy samples. miRNA expression between data sets varied substantially and a greater miRNA/target response was observed in plants with more established GLRaV-3 infections. The lack of significant differences in mean VCRs between data sets, coupled with the consistent modulation of certain miRNAs in plants that have likely been infected for longer is a promising result. This finding could indicate that successful inhibition of further virus replication by plant defence mechanisms occurred and that these miRNAs and their targets are implicated in this response. The predicted targets for these miRNAs are genes involved in disease resistance, apoplastic processes, oxidation-reduction processes and growth and developmental processes. Additionally, possible variant-specific miRNA responses to infection were observed across all data sets, which could aid in elucidating possible biological differences between variants of GLRaV-3.

AFRIKAANSE OPSOMMING: Wingerd-rolblaarsiekte (GLD) is endemies tot alle wingerdstreke ter wêreld en word beskou as een van die mees belangrike virussiektes van wingerd. Grapevine leafroll-associated virus 3 (GLRaV-3) word beskou as die primêre oorsaak van GLD en vyf genetiese variante (I, II, III, VI en VII) van hierdie virus is bevestig in Suid-Afrikaanse wingerde. Verskeie studies het al getoon dat klein RNAs (sRNAs) ‘n belangrike rol speel in plantverdedigingsmeganismes teen biotiese en abiotiese stresfaktore. Die betrokkenheid van sRNAs in hierdie verband het gelei tot ‘n toename in navorsing gerig tot die karakterisering van uitdrukkingspatrone van sRNAs, insluitend mikroRNAs (miRNAs), en die rol wat hierdie molekule speel in die onderliggende geenregulering in plant virussiektes. Hierdie studie het gebruik gemaak van stam-lus tru-transkripsie kwantitatiewe polimerase kettingreaksie (stem-loop RT-qPCR) om miRNA uitrukking in GLRaV-3 geïnfekteerde- en gesonde wingerdstokke te bepaal. ‘n Stel eie-gewortelde Cabernet Sauvignon plante, verteenwoordigend van variantgroepe I, II, III en VI is gevestig vanaf steggies van hoogs-simptomaties plante vanuit kommersiële wingerde. Hierdie plante is gekarakteriseer om die eerste datastel te lewer. Jong Cabernet Sauvignon plante is addisioneel gevestig en geënt met enkel-variant infeksies van die vyf erkende variante van GLRaV-3 in Suid-Afrika. Al hierdie plante is onderhou in ‘n klimaatgekontroleerde glashuis en twee maal gekarakteriseer, ses maande uit mekaar, om twee datastelle te lewer. ‘n Vierde datastel het bestaan uit GLRaV-3-positiewe en –negatiewe Cabernet Sauvignon plante vanuit kommersiële wingerde in die Stellenbosch omgewing. Elf miRNAs is geïdentifiseer in beide geïnfekteerde en ongeïnfekteerde plantmateriaal. Vermeënde miRNA teikengene is voorspel en geannoteer d.m.v in silico analises. Hierdie voorspelde teikengene is daaropvolgend gekwantifiseer in beide glashuis- en veldplante d.m.v ‘n SYBR Green RT-qPCR metode. Hierdie studie het statisties-beduidende verskille in viruskonsentrasie, uitgedruk as virus konsentrasie verhoudings (VCRs) tussen plante geïnfekteer met enkele variantgroepe gevalideer. ‘n Interessante bevinding is die afwesigheid van beduidende verskille in gemiddelede VCRs tussen datastelle, ten spyte van merkbare verskille in plant ouderdom, tydperk van GLRaV-3 besmetting, bostok/onderstok kombinasies en groei-omstandighede. Verskeie miRNAs het statisties-beduidende verskille tussen geïnfekteerde en gesonde plante getoon. Die miRNA-uitdrukking tussen datastelle het ook aansienlik verskil en ‘n meer prominente miRNA/teikengeen respons is gemerk in plante met ‘n meer gevestigde infeksie van GLRaV-3. Die afwesigheid van beduidende verskille in gemiddelde VCRs tussen datastelle tesame met die konsekwente modulasie van sekere miRNAs in plante met meer gevestigde GLRaV-3 infeksie is ‘n bemoedigende resultaat. Hierdie bevinding kan impliseer dat plantverdedigingsmeganismes suksesvol was in die inhibering van verder virusreplikasie oor tyd en dat hierdie miRNAs en hul teikengene betrokke is in hierdie respons. Die voorspelde teikengene van hierdie miRNAs is betrokke in groei- en ontwikkelingsprosesse, apoplastiese prosesse, oksidasie-reduksie en siekteweerstand. Hierdie studie het ook moontlike variant-spesifieke miRNA uitdrukking geïdentifiseer wat kan bydra tot pogings om moontlike biologiese verskille tussen variante van GLRaV-3 te identifiiseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101192
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