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

[摘要] ENGLISH ABSTRACT: Grapevine is one of the oldest food plants and was first exploited from in the wild and latercultivated by man. Grapevine viruses are among the most important pathogens of grapevine andone of these viruses termed Grapevine .!:eafroll-§ssociated Y'.irus (GLRaV) is regarded as one ofthe most harmful grapevine viruses. The virus is responsible for the disease called leafrolldisease which affects the South African wine industry causing losses of millions of Randsannually. Existing control measures focus on prevention by utilising virus-free propagationmaterial and integrated control of the insect vectors. Virus resistant grapevine by means ofgenetic modification seems to be a realistic approach in solving grapevine diseases, especiallyleafroll disease.A natural occurring plant mechanism called post transcriptional gene silencing (PTGS) can beexploited to help in the process of obtaining transgenic virus resistant grapevine. PTGS is asequence-specific defence system of the plant that targets alien RNA (transgenes, endogenousgenes and cytoplasmically replicating viruses) for degradation (Dunoyer et al., 2002; Vanitharaniet al., 2003; Waterhouse et al., 2001a). However viruses have evolved a counter defencemechanism 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 toprevent the onset of PTGS. A constitutively expressed green fluorescent protein (GFP) genewas silenced in transgenic Nicotiana benthamiana plants (line 16c) by agro-infiltration, using asecond GFP-construct. The GFP-silenced plants were inoculated with a strain of GLRaV-2 toscreen for a suppressor able to reverse PTGS. Individual GLRaV-2 genes were isolated andcloned into an intermediate PCR cloning vector, followed by subsequent cloning into a plantexpression vector. These constructs were transformed into Agrobacterium tumefaciens strainsGV3101 and C58C1 and were agro-infiltrated into silenced transgenic or co-infiltrated intotransgenic 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 ofGLRaV-2 possessed suppressor activity. An attempt was made to corroborate the fluorescenceassays by screening infiltrated plants for the presence of GFP siRNAs, which would be a telltalesign 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 ofconcept to show that two genes can be silenced simultaneously in a plant. The primary construct, pHanViralGFP-SAS was constructed by performing a rapid direct reversetranscription reaction (RDOT-RT-PCR) with primers containing 5' -extension restriction sites tofacilitate subsequent cloning and to amplify a gene fragment from the GLRaV-2 genome. Aportion of the GFP was obtained by a polymerase chain reaction (PCR) from the plasmid vectorpBIN mGFPS-ER, also using primers containing restriction sites. The fragments obtained in theindividual reactions were ligated into an intermediate PCR cloning vector, followed by thesubsequent cloning into corresponding sites in the pHannibal vector, in sense and anti-senseorientations. The silencing cassette was removed from the pHannibal vector and ligated intopART27. The final construct, pSilencer-SAS, was transformed into A. tumefaciens strainsGV3101 and C58C1 and transgenic (16c) and non-transgenic N. benthamiana plantlets, ofwhich some were infected with GLRaV-2, were agro-infiltrated with these Agrobacteriumstrains.Results obtained showed that the tandem silencing vector was successful in silencing twogenes simultaneously, justifying the construction of a tandem vector. The effectivity of thevector can now be tested by inserting genes from two different viruses.

[发布机构] Stellenbosch University