Screening the genome of grapevine leafroll associated virus-2 for proteins with RNA silencing suppressor activity and the construction of a tandem silencing vector to induce simultaneous silencing of two genes

Engelbrecht, M. (2006-04)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: Grapevine is one of the oldest food plants and was first exploited from in the wild and later cultivated by man. Grapevine viruses are among the most important pathogens of grapevine and one of these viruses termed Grapevine .!:eafroll-§ssociated Y'.irus (GLRaV) is regarded as one of the most harmful grapevine viruses. The virus is responsible for the disease called leafroll disease which affects the South African wine industry causing losses of millions of Rands annually. Existing control measures focus on prevention by utilising virus-free propagation material and integrated control of the insect vectors. Virus resistant grapevine by means of genetic modification seems to be a realistic approach in solving grapevine diseases, especially leafroll disease. A natural occurring plant mechanism called post transcriptional gene silencing (PTGS) can be exploited to help in the process of obtaining transgenic virus resistant grapevine. PTGS is a sequence-specific defence system of the plant that targets alien RNA (transgenes, endogenous genes and cytoplasmically replicating viruses) for degradation (Dunoyer et al., 2002; Vanitharani et al., 2003; Waterhouse et al., 2001a). However viruses have evolved a counter defence mechanism against PTGS by encoding suppressor proteins able to suppress RNA silencing (Thomas et al., 2003). Very few suppressor proteins have been identified in grapevine viruses. In this study the GLRaV-2 genome was screened for a suppressor protein able to reverse or to prevent the onset of PTGS. A constitutively expressed green fluorescent protein (GFP) gene was silenced in transgenic Nicotiana benthamiana plants (line 16c) by agro-infiltration, using a second GFP-construct. The GFP-silenced plants were inoculated with a strain of GLRaV-2 to screen for a suppressor able to reverse PTGS. Individual GLRaV-2 genes were isolated and cloned into an intermediate PCR cloning vector, followed by subsequent cloning into a plant expression vector. These constructs were transformed into Agrobacterium tumefaciens strains GV3101 and C58C1 and were agro-infiltrated into silenced transgenic or co-infiltrated into transgenic N. benthamiana plants (16c) in two different expression assays. It was found in both the silencing reversal assay and the transient assay that the p24 protein of GLRaV-2 possessed suppressor activity. An attempt was made to corroborate the fluorescence assays by screening infiltrated plants for the presence of GFP siRNAs, which would be a telltale sign that silencing has occurred. Unfortunately (and probably due to technical problems) these experiments failed to yield signals in the Northern blot analysis. The second part of this study was to construct a tandem silencing vector to serve as "proof of concept to show that two genes can be silenced simultaneously in a plant. The primary construct, pHanViralGFP-SAS was constructed by performing a rapid direct reverse transcription reaction (RDOT-RT-PCR) with primers containing 5' -extension restriction sites to facilitate subsequent cloning and to amplify a gene fragment from the GLRaV-2 genome. A portion of the GFP was obtained by a polymerase chain reaction (PCR) from the plasmid vector pBIN mGFPS-ER, also using primers containing restriction sites. The fragments obtained in the individual reactions were ligated into an intermediate PCR cloning vector, followed by the subsequent cloning into corresponding sites in the pHannibal vector, in sense and anti-sense orientations. The silencing cassette was removed from the pHannibal vector and ligated into pART27. The final construct, pSilencer-SAS, was transformed into A. tumefaciens strains GV3101 and C58C1 and transgenic (16c) and non-transgenic N. benthamiana plantlets, of which some were infected with GLRaV-2, were agro-infiltrated with these Agrobacterium strains. Results obtained showed that the tandem silencing vector was successful in silencing two genes simultaneously, justifying the construction of a tandem vector. The effectivity of the vector can now be tested by inserting genes from two different viruses.

AFRIKAANSE OPSOMMING: Wingerd is een van die oudste plante wat al benut word vandat dit wild gegroei het tot en met vandag wat dit verbou word. Wingerdvirusse is van die mees belangrike patogene van wingerd en een van die virusse, bekend as Wingerd rolblaar geassosieerde virus (GLRaV) word beskou as een van die mees vernietigende virusse van wingerd. Die virus is verantwoordelik vir die siekte bekend as rolblaar en veroorsaak jaarliks verliese van miljoene Rande vir die SuidAfrikaanse wynbedryf. Bestaande beheermeganismes maak gebruik van virusvrye voortplantingsmaterial en van die chemiese beheer van insekvektore verantwoordelik vir die oordrag van siektes. Genetiese manipulasie van wingerd lyk op die oomblik na een van die mees realistiese benaderings om virusbestande wingerd te verkry en so wingerdsiektes, soos rolblaar, te bekamp. 'n Meganisme wat natuurlik in plante voorkom en wat post transkripsionele geen uitowing (PTGS) genoem word kan gebruik word in die proses om virusbestande transgeniese wingerd te maak. PTGS is 'n volgordespesifieke beskermingssisteem in die plant wat vreemde RNA (transgene, endogene gene en sitoplasmiese repliseerende virusse) teiken vir degradasie (Dunoyer et al., 2002; Vanitharani et al., 2003; Waterhouse et al., 2001a). Virusse het deur middel van evolusie onderdrukkerprote'iene geproduseer as n teenvoeter teen PTGS (Thomas et al., 2003). Baie min uitdowings gene is al geidentifiseer in wingerd virusse. In hierdie studie is die genoom van die GLRaV-2 virus deursoek vir 'n onderdrukkersproteine wat PTGS kan omkeer of dit heeltemal kan verhoed. 'n Konstitatiewe uitgedrukte groen fluoreseerende prote'ien (GFP) is uitgedoof in transgeniese Nicotiana benthamiana plante (lyn 16c) deur middel van agro-infiltrasie met 'n tweede GFP konstruk. Die GFP uitgedoofde plante is ge'inokuleer met die GLRaV-2 virusras om te toets vir die teenwoordigheid van 'n onderdrukkersprote'ien wat PTGS kan omkeer. lndividuele GLRaV-2 gene is ge'isoleer uit die virus uit en gekloneer in 'n intermediere vektor gevolg deur die klonering in 'n plant uitdrukkingsvektor. Hierdie konstrukte is getransformeer in die Agrobacterium tumefaciens rasse, GV3101 en C58C1 en is in uitgedoofde transgeniese plante deur middel van agroinfiltrasie ingesit of is in transgeniese N. benthamiana plante (16c) getransformeer afhangende van die uitdrukkings toets sisteem gebruik. Dit is gevind in beide die "reverse" en "transient" uitdowings toets sisteem dat die p24 prote'ien van die GLRaV-2 onderdrukkings aktiwiteit besit. Daar was probeer om die fluoreseerende toets resultate in die geinfiltreerde plante met die teenwoordigheid van siRNAs te ondersteun. Ongelukkig kon geen seine verkry word in die Noordelike kladtegniek nie en was dit hoogs waarskynlik as gevolg van tegniese probleme. In die tweede deel van die studie is 'n tandem uitdowingsvector gemaak wat as 'n bewys van beginsel sal dien. Die vektor wys dat twee gene gelyktydig uitgedoof kan word in 'n plant. Die primere konstruk, pHannibalGFP-SAS, bestaan uit 'n GLRaV-2 fragment tesame met 'n GFP fragment in 'n sin en anti-sin orientasie. Die GLRaV-2 fragment is geamplifiseer deur 'n vinnige direkte tru-transkriptase reaksie uit tevoer met inleiers wat 'n spesifieke restriksie ensiem snyplek op die 5' kant dra om klonering later te vergemaklik. Die GFP fragment is verkry vanaf die pBIN mGFP5-ER vektor deur 'n polimerase ketting reaksie (PCR) te doen met inleiers wat weereens spesifieke restriksie-ensiem snyplekke dra. Die individuele fragmente verkry in die reaksies is geligeer in 'n intermediere PCR klonerings vektor, wat gevolg is deur die klonering van die fragmente in ooreenstemmende plekke in die pHannibal vektor in sin en anti-sin orientasies. Die uitdowingskasset is verwyder uit die pHannibal vektor en is geligeer in die pART27 plant transformasievektor. Die finale konstruk, pSilencer-SAS, is getransformeer in die A. tumefaciens rasse GV3101 en C58C1. Transgeniese (16c) en nie- transgeniese N. benthamiana plante, waarvan sommige met GLRaV-2 geinfekteer was, is met die finale Agrobacterium rasse geinfekteer. Resultate het gewys dat die tandem uitdowingsvektor wel gewerk het om twee gene gelyktydig uit te doof. Die effektiwiteit van die vektor kan nou getoets word met gene van twee verskillende virusse.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/50640
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