The transformation of grapevine with yeast glucanase and chitinase genes
Vivier, M A
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology
Marais, E M
The Institute for Wine Biotechnology (IWBT) chose the subject of manipulated disease resistance as main theme for the grapevine improvement programme. In this broad subject we are currently focusing on resistance to fungal infections as well as resistance to viral infections in collaboration with Dr Burger from the Department of Genetics, University Stellenbosch. Several approaches have been implemented to potentially enhance disease resistance against the major fungal pathogens that cause grey mould, downy mildew and powdery mildew. One of these approaches involves the use of polygalacturonase-inhibiting proteins (PGIPs) (Winetech funded project, IWBT 04-03). In this application, as well as another project (see project IWBT 04-07), different approaches are suggested to obtain the same goal, namely that of enhanced fungal resistance in grapevine.
Plants have evolved a number of mechanisms to curb attacks by pathogenic fungi, bacteria, viruses and insects. These include structural barriers, such as waxy cuticles or strategically positioned hydrolyzing enzymes and/or antimicrobial compounds that function to prevent colonisation of the tissue. Plants also use induced or active cellular defence mechanisms to prevent further colonisation of the tissue when the structural barriers of the host have been breached. Active defence responses are induced by all classes of plant pathogens and typically follow a cascade effect. The primary response is usually elicited in the cells directly in contact with the pathogen, subsequently leading to a secondary response in surrounding cells due to diffusible elicitor molecules originating from the primary responses. The final stage of the active defence response is associated with systemic acquired resistance hormonally induced throughout the plant.
Most of the strategies employed to manipulate disease resistance in plants involve up-regulation of one or more of the aforementioned defence strategies. Proteins present at low levels in healthy plants but more abundantly produced during pathogen attack have been termed pathogenesis-related (PR) proteins. This diverse group of proteins includes hydrolytic enzymes such as chitinases and ß-1,3-glucanases. Several classes of plant glucanases and chitinases have been identified and their suspected antifungal activities have been confirmed by in vitro tests. These proteins are able to break down the structural polysaccharides in fungal cell walls and their encoding genes have therefore been targeted as antifungal genes in various genetic manipulation procedures.
Similarly, in grapevine, a direct correlation between the activity of ß-1,3-glucanases and chitinases and the resistance rating of different grape genotypes to powdery mildew (Indium tuckerii) has been found. Glucanase and chitinase enzymes purified from leaves of a resistant cultivar showed inhibition to powdery mildew in a bioassay, confirming their antifungal properties. Genes encoding chitinase and/or ß-1,3-glucanase proteins from various sources have been overexpressed in several plant spp., resulting in increased resistance to fungal pathogens in the transgenic plants.
In this project the aim was to utilise glucanase and chitinase encoding genes from Saccharomyces cerevisiae as possible PR-proteins in grapevine.
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