Browsing by Author "Engelbrecht, M."

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    First report of aster yellows phytoplasma in grapevines in South Africa
    (2010) Engelbrecht, M.; Joubert, J.; Burger, J.T.; Engelbrecht, M.; Joubert, J.; Burger, J.T.
    [No abstract available]
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    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
    (Stellenbosch : Stellenbosch University, 2006-04) Engelbrecht, M.; Burger, J. T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics Institute for Plant Biotechnology (IPB).
    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.