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An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana
Thesis (MScAgric)--Stellenbosch University, 2025.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613751151165440 |
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| access_status_str | Open Access |
| author | Kruger, Kian |
| author2 | Burger, Johan |
| author_browse | Burger, Johan Kruger, Kian |
| author_facet | Burger, Johan Kruger, Kian |
| author_sort | Kruger, Kian |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/133117 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:41:06.301Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/133117 An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana Kruger, Kian Burger, Johan Campa, Manuela Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics & Institute of Plant Biotechnology. Grapes -- Diseases and pests -- South Africa Grapevine leafroll virus -- Economic aspects Plant viruses -- Control RNA interference Nicotiana benthamiana -- Growth Plant antiviral proteins UCTD Thesis (MScAgric)--Stellenbosch University, 2025. Kruger, K. 2025. An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/0e7629f8-92d7-40c0-937c-d3b17f044140 ENGLISH ABSTRACT: Grapevine is one of the most significant perennial fruit crops worldwide. However, many economically significant diseases jeopardise the global wine industry annually. In South Africa grapevine leafroll disease (GLD) is considered to be the most economically significant virus disease impacting the local industry. Losses related to the disease range from reduction of fruit yield and quality to a decrease in plant vigour. Although producers employ measures like rogueing, insect vector control and the use of virus-free propagation material, these efforts merely attenuate disease spread without providing a sustainable resolution. The Ampelovirus, grapevine leafroll-associated virus 3 (GLRaV-3) is considered the main etiological agent of GLD. Other viruses, such as grapevine virus A (GVA) and grapevine virus B (GVB), share insect vectors with GLRaV-3 and are frequently transmitted together, with it being hypothesised that GLRaV-3 may assist GVA and GVB transmission. A promising strategy employed by researchers to confer virus resistance involves the application of exogenous dsRNA to activate the endogenous RNAi mechanism. This eukaryotic RNAi mechanism can be exploited to target virus-genes to induce temporary virus resistance in plants treated by the application of double stranded RNA (dsRNA). To test this concept, the experimental model plant Nicotiana benthamiana and GVA were chosen as proxy for the grapevine/GLRaV-3 pathosystem. GVA (unlike GLRaV-3) can infect N. benthamiana, which has a short life cycle; and a GVA infectious clone was available. The study aimed to produce, extract, and apply dsRNA targeting coat protein structural gene of GVA to induce resistance in treated plants. First, two in vivo dsRNA-producing constructs targeting the coat protein gene of GVA were assembled - one produced hairpin-RNA and the other transcribed two complementary ssRNA strands that were subsequently annealed to form dsRNA. Both dsRNA products were purified from induced E. coli cultures using a phenol-based extraction and purification method. Subsequently, both dsRNA products were successfully applied to N. benthamiana plants using either high-pressure spraying equipment or mechanical inoculation. Exogenously applied dsRNA treatments demonstrated robust virus resistance in treated plants, indeed a significant decrease in GVA_CP gene expression was observed. Additionally, treated plants remained asymptomatic for at least 30 days post-treatment. Findings also suggested that dsRNA stability was maintained for less than three days post-treatment. The findings of this study demonstrated the potential of this methodology to induce virus resistance in N. benthamiana and the potential it has as a field deployable biopesticide to provide virus resistance. AFRIKAANSE OPSOMMING: Wingerd is een van die belangrikste meerjarige vrugtegewasse wêreldwyd. Tog bedreig baie ekonomies noemswaardige siektes jaarliks die globale wynbedryf. In Suid-Afrika word rolbladsiekte (GLD) beskou as die mees ekonomies relevante virussiekte wat die plaaslike industrie beïnvloed. Verliese wat met die siekte verband hou, wissel van 'n afname in vrug-opbrengs en -kwaliteit tot 'n vermindering in die groeikrag van plante. Alhoewel produsente maatreëls soos die verwydering van besmette plante, die beheer van insekvektore en die gebruik van virusvrye voortplantingsmateriaal toepas, verminder hierdie pogings slegs die verspreiding van die siekte sonder om 'n volhoubare oplossing te bied. Die Ampelovirus, grapevine leafroll associated virus 3 (GLRaV-3), word as die hoof-etiologiese agent van GLD beskou. Ander virusse, soos grapevine virus A (GVA) en grapevine virus B (GVB), deel insekvektore met GLRaV-3 en word dikwels saam oorgedra. Daar word ook vermoed dat GLRaV-3 moontlik die oordrag van GVA en GVB kan fasiliteer. ’n Belowende strategie wat deur navorsers gebruik word om virusweerstand te verleen, behels die toediening van eksogene dsRNA om die endogene RNAi-meganisme te aktiveer. Hierdie eukariotiese RNAi-meganisme kan benut word om virus-gene te teiken en tydelike virusweerstand in plante te veroorsaak deur die toediening van dubbelstring RNA (dsRNA). Om hierdie konsep te toets, is die eksperimentele modelplant Nicotiana benthamiana en GVA gekies as ’n plaasvervanger vir die wingerd/GLRaV-3-patostelsel. GVA kan, in teenstelling met GLRaV-3, N. benthamiana infekteer, dit het ’n kort lewensiklus, en ’n infektiewe GVA-kloon was beskikbaar. Die studie het ten doel gehad om dsRNA te produseer, te ekstraeer en toe te dien om die kapsiedproteïen strukturele geen van GVA te teiken en weerstand in behandelde plante te induseer. Eerstens is twee in vivo dsRNA-produserende konstrukte saamgestel wat die kapsiedproteïen-geen van GVA teiken – een het haarnaald-dsRNA geproduseer en die ander het twee komplementêre ssRNA-stringe getranskribeer wat daarna versmelt is om dsRNA te vorm. Beide dsRNA-produkte is uit geïnduseerde E. coli-kulture gesuiwer deur ’n fenol-gebaseerde ekstraksie- en suiweringsmetode. Daarna is albei dsRNA-produkte suksesvol op N. benthamiana-plante toegedien met óf hoëdruk-spuittoerusting óf meganiese inokulasie. Eksogene toegediende dsRNA-behandelings het robuuste virusweerstand in behandelde plante getoon, en’n beduidende afname in GVA_CP-geenuitdrukking is inderdaad waargeneem. Verder het behandelde plante vir minstens 30 dae na behandeling asimptomaties gebly. Bevindings het ook aangedui dat dsRNA-stabiliteit minder as drie dae na behandeling gehandhaaf is. Die resultate van hierdie studie het die potensiaal van hierdie metodologie om virusweerstand in N. benthamiana te induseer uitgewys, asook die moontlikheid om as ’n veldtoepasbare bioplaagmiddel gebruik te word om virusweerstand te bied. Masters 2025-07-18T07:39:08Z 2025-07-18T07:39:08Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/133117 en Stellenbosch University xiii, 70 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Grapes -- Diseases and pests -- South Africa Grapevine leafroll virus -- Economic aspects Plant viruses -- Control RNA interference Nicotiana benthamiana -- Growth Plant antiviral proteins UCTD Kruger, Kian An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title | An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title_full | An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title_fullStr | An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title_full_unstemmed | An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title_short | An RNAi approach to induce GVA virus resistance in Nicotiana benthamiana |
| title_sort | rnai approach to induce gva virus resistance in nicotiana benthamiana |
| topic | Grapes -- Diseases and pests -- South Africa Grapevine leafroll virus -- Economic aspects Plant viruses -- Control RNA interference Nicotiana benthamiana -- Growth Plant antiviral proteins UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/133117 |
| work_keys_str_mv | AT krugerkian anrnaiapproachtoinducegvavirusresistanceinnicotianabenthamiana AT krugerkian rnaiapproachtoinducegvavirusresistanceinnicotianabenthamiana |