Evaluation of two pathogen-derived resistance strategies for Grapevine leafroll-associated virus 3

Suidgeest, Faira (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is one of the most economic important viral diseases affecting grapevine. Grapevine leafroll associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of the leafroll associated virus family. To prevent the spread of GLD, management strategies such as rogueing and insect vector control are required to limit crop losses. Alternative control strategies based on genetic modification of the grapevine genome, such as pathogen-derived resistance (PDR), is proven to be effective in conferring resistance to several viruses. Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs (amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host and the development of an amiRNA-mediated silencing validation system. In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and #17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus titres of all grafted plants were quantified relative to two reference genes using RT-qPCR. Results were evaluated by comparing the relative virus titre of each transgenic plant line to that of the non-modified control plant line. Results showed that resistance levels of plant line #3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more susceptible to the virus. The second part of the study was the construction and validation of amiRNAs targeting GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were quantified to determine the silencing efficiency of the amiRNAs. Results showed that the amiRNAs were successful in silencing the GFP target construct, however, they were not specific in silencing exclusively their corresponding target. These amiRNA constructs are ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD infected grapevines.

AFRIKAANSE OPSOMMING: Wingerd rolblaar siekte (GLD), wat veroorsaak word deur die lede van die familie Closteroviridae, is een van die ekonomies mees belangrike virus siektes van wingerd. Grapevine leafroll-associated virus 3 (GLRaV-3), van die genus Ampelovirus, is die mees wydverspreide lid van die rolblaar geassosieerde virus familie. Om die verspreiding van GLD te voorkom, is bestuur strategieë, soos die verwydering van geïnfekteerde plante en insekvektor beheer, ’n vereiste om oes verliese te beperk. Alternatiewe beheer strategieë gebaseer op genetiese modifikasie van die wingerdgenoom, soos patogeen-afgeleide weerstand (PDR), is bewys om effektief te wees in die verlening van weerstand teen verskeie virusse. Daarom was die fokus van hierdie studie om patogeen-afgeleide weerstand strategieë vir GLRaV-3 te evalueer met behulp van die volgende twee benaderings; 1) die evaluering van transgeniese plante wat 'n disfunksionele GLRaV-3 hitte-skok proteïen 70 homoloog (HSP70h) uitdruk, ten einde weerstand te verleen teen GLRaV-3, en 2) die konstruksie van kunsmatige mikroRNAs (amiRNAs) om te gebruik as 'n instrument vir die ondrukking van spesifieke genoomvolgordes van GLRaV-3 in die wingerd gasheer en die ontwikkeling van ’n stelsel om amiRNA-bemiddelde onderdrukking te bevestig. In die eerste deel van hierdie studie, is ses transgeniese plant lyne (plant lyne # 1, # 3, # 9, # 14, # 15 en # 17) sowel as 'n nie-gemodifiseerde gesonde plant lyn, geïnokuleer met GLRaV- 3 deur enting van ogies van elk op GLRaV-3 besmette plantmateriaal. Na ongeveer vyf maande, is GLRaV-3 virus konsentrasies van alle ingeënte plante gekwantifiseer relatief tot twee verwysing gene deur gebruik te maak van tru-transkripsie kwantitatiewe PCR (RTqPCR). Resultate is geëvalueer deur die relatiewe virus konsentrasie van elke transgeniese plant lyn te vergelyk met dié van die nie-gemodifiseerde kontrole lyn. Resultate het getoon dat weerstand vlakke van plant lyn # 3 beduidend verbeter is (> 99%) en merkwaardig is plant lyn # 14 bewys om meer vatbaar vir die virus te wees. Die tweede deel van die studie was die konstruksie en bevestiging van kunsmatige mikroRNAs (amiRNAs) wat GLRaV-3 genoomvolgordes teiken. Twee 21 nt streke van GLRaV-3 is suksesvol geïnkorporeer in die ruggraat van wingerd mikroRNA vvi167b. Verder is teiken konstrukte ontwikkel deur die inkorporering van ooreenstemmende GLRaV-3 teiken genoomvolgordes in die 3'UTR (3’ ongetransleerde area) van 'n groen fluoressensie proteïen (GFP) geen. Daarna is die teiken konstrukte gesamentlik geïnfiltreer met die gekonstrueerde amiRNA in Nicotiana benthamiana en GFP uitdrukkingsvlakke is gekwantifiseer deur die onderdrukkingsdoeltreffendheid van die amiRNAs te bepaal. Resultate het getoon dat die amiRNAs suksesvol was in die onderdrukking van die GFP teiken konstruk, maar hulle was egter nie-spesifiek in die eksklusiewe onderdrukking van die ooreenstemmende teiken. Hierdie amiRNA konstrukte is ideaal vir verdere virus studies om die doeltreffendheid van GLRaV-3 onderdrukking in GLD besmette wingerdstokke te bepaal.

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