The introduction of multiple virus resistance in grapevines through virus induced gene silencing


Project Number
GENUS 01-04

Project title
The introduction of multiple virus resistance in grapevines through virus induced gene silencing

Project leader
Burger, J T

Institution
University of Stellenbosch. Department of Genetics

Project description
The aim of this project is the construction of vectors for the introduction of resistance to GLRaV-3 and GRSPaV using gene silencing technology. These vectors will contain: 1) GLRaV-3 CP-, (2) GRSPaV CP-, (3) GRSPaV CP- and GLRaV 3 CP tandem gene fragments. Once these sequences are transformed into the grapevine plant they will be transcribed, and because of their design, will form dsRNA. Upon infection of the plant with the homologous virus(es), the dsRNA will trigger a post transcriptional gene silencing mechanism which will recognise and destroy the viral RNA. This mechanism evolved in plants specifically to combat virus infection, and has proven to be a highly effective system of controlling viruses. A secondary aim is to isolate and express the GRSPaV coat protein gene in E. coli, and to have antibodies made against the purified recombinant protein. Such antibodies are required for reliable and economical screening for this virus.

Towards these goals, the GRSPaV CP gene from a South African strain has been isolated, sequenced and cloned into a protein expression vector, pET14b. The primary construct, pHannibal-SASCon (pHannibal containing both sense and antisense fragments of the GLRaV-3 and GRSPaV CP genes in tandem) has been made and was transferred to the plant transformation vector, pART27. This will be followed by Agrobacterium mediated transformation of Nicotiana benthamiana. Once plants are generated containing the ‘transgene’, effective silencing will be tested by infecting the plant with PVX-GFP-SCon (an infectious potato virus X clone PVX- containing the green fluorescent protein (GFP) gene as well as sense fragments of the GLRaV-3 and GRSPaV CP genes). Silencing of the target genes will be tested using Northern Blots. The two remaining primary vectors are currently being constructed. For the secondary aim of making GRSPaV antibodies, the next step is to express the protein, isolate it and have antibodies made which will be tested with ELISA and possibly Western Blots against infected and uninfected plants.

Poster(s)
Van Eeden, C and Burger, J T. 2002. The introduction of multiple virus resistance in grapevines through virus-induced gene silencing. Poster presented at the Cape Biotechnology Forum. September, Somerset West, South Africa.

Van Eeden, C and Burger, J T. 2002. A study towards the introduction of multiple virus resistance in grapevine through PTGS. Poster presented at the 40th South African Society for Plant Pathology Congress. 20-13 January, Dikhololo, South Africa.

Engelbrecht, M and Burger, J T. 2004. The construction of a tandem silencing vector for grapevine. Poster presented at the 18th Congress of the South African Genetics Society. 4-7 April. Stellenbosch, South Africa.

Van Eeden, C, Freeborough, M-J and Burger, J T. 2004. The introduction of multiple virus resistance in grapevines using PTGS. Poster presented at the 18th Congress of the South African Genetics Society. 4-7 April, Stellenbosch, South Africa.

Presentation(s)
Van Eeden, C and Burger, J T. 2002. The introduction of multiple virus resistance in grapevines through virus-induced gene silencing. Paper presented at the Cape Biotechnology Forum. September, Somerset West, South Africa.

Du Preez, J and Burger, J T. 2002. The construction of an infectious clone of grapevine virus. Paper presented at the 26th National Congress of the South African Society for Enology and Viticulture. 13-15 November, Somerset West, South Africa.

Burger, J T, Van Eeden, C and Engelbrecht, M. 2003. The construction of gene silencing vectors for the introduction of multiple virus resistance in grapevines. Paper presented at the 14th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine. 12-17 September, Locoronto, Italy.

Du Preez, J, Freeborough, M-J and Burger, J T. 2005. The construction of an RNA virus-based expression vector for grapevine. Paper presented at the 3rd International Congress of the South African Society for Enology and Viticulture. 28-30 July, Cape Town, South Africa.

FinalReport.pdf

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