Browsing by Author "Joubert, Dirk Albert, 1973-"

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    Development of an Agrobacterium vitis transformation system for grapevine
    (Stellenbosch : Stellenbosch University, 2000-03) Joubert, Dirk Albert, 1973-; Pretorius, I. S.; Vivier, Melane A.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis.
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    Regulation of the Vitis vinifera PGIP1 gene encoding a polygalacturonase-inhibiting protein
    (Stellenbosch : Stellenbosch University, 2004-03) Joubert, Dirk Albert, 1973-; Vivier, Melane A.; Pretorius, I. S.; De Lorenzo, G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation.