The construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines

[摘要] ENGLISH ABSTRACT:Viruses are some of the most important pathogens of grapevines. There are no effectivechemical treatments, and no grapevine- or other natural resistance genes have been discoveredagainst grapevine infecting viruses. The primary method of grapevine virus control isprevention by biological indexing and molecular- and serological screening of rootstocks andscions before propagation. Due to the spread of grapevine viruses through insect vectors, andin the case of GRSPaV the absence of serological screening, these methods of virus controlare not always effective. In the past several methods, from cross-protection to pathogenderived resistance (PDR), have been applied to induce plant virus resistance, but withinconsistent results. In recent years the application of post-transcriptional gene silencing(PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistancehas achieved great success. The Waterhouse research group has designed planttransformation vectors that facilitate specific virus resistance through PTGS. The primaryfocus of this study was the production of virus specific transformation vectors for theintroduction of grapevine virus resistance. The Waterhouse system has been successfullyutilised for the construction of three transformation vectors with the pHannibal vector asbackbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned ina complementary conformation upstream and downstream of an intron sequence. Theprimary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaVand GLRaV-3 and was designed for the introduction of multiple-virus resistance. For theconstruction of the primary vector the GRSPaV CP gene was isolated from RSP infectedgrapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann-LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector(pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassettecontaining the complementary CP gene segments of both GRSPaV and GLRaV-3 was clonedinto pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana(SRI), through A. tumefaciens mediated transformation. Unfortunately potentialtransformants failed to regenerate on rooting media; hence no molecular tests were performedto confirm transformation. Once successful transformants are generated, infection with arecombinant virus vector (consisting of PYX, the GFP gene as screenable marker and thecomplementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for theefficacy of the vectors to induce resistance. A secondary aim was added to this project whena need was identified within the South African viticulture industry for GRSPaV specificantibodies to be used in serological screening. To facilitate future serological detection ofGRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b)within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used togenerate GRSPaV CP specific antibodies.

[发布机构] Stellenbosch University