Browsing by Author "Van der Merwe, Andreas Stephanus"

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    Identification of differentially expressed mRNA species in Vitis vinifera in reaction to infection by Uncinula necator
    (Stellenbosch : Stellenbosch University, 1999-12) Van der Merwe, Andreas Stephanus; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH SUMMARY: Powdery mildew is one of the most important grapevine diseases and is a problem in all the grapevine producing areas. Losses caused by uncontrolled powdery mildew force producers to follow extensive fungicide spray programs to control the disease. Grapevine breeders has tried to incorporate natural disease resistance into commercial cultivars, but has been confronted with problems like inbreeding depression, long generation cycle, the polygenic nature of powdery mildew inheritance and a complex genetic system. As a result, not much progress has been made to convert existing susceptible cultivars to resistant cultivars. Previous studies attributed V. vinifrera resistance to powdery mildew to several factors such as differences in cuticle thickness, increased activity of enzymes involved in lignin biosynthesis, production of papillae, deposition of silica incrusts, localised necrosis and the activation of enzymes like chitinases and glucanases (Pratt et al., 1984, Heintz & Blaich, 1989, 1990, Eibach, 1994, Clingefeffer & Scott, 1994). We approached this problem by addressing disease resistance of grapevine cultivars to powdery mildew at the gene-activation level. Liang & Pardee (1992) develop a technique, called differential display PCR (DD-PCR), that enabled us to compare differential gene expression of identical cells under altered (infected and not-infected) conditions. The identification of differentially expressed fragments started at the successful cultivation of test plants under identical environmental conditions. The next step was to artificially inoculate test plants with powdery mildew and to harvest the infected and control leaves after a short incubation period. The isolation of RNA from grapevine leaves was problematic and had to be optimised during this study. Because DD-PCR was not previously used in our lab, the next step was to optimise the technique to suit our lab conditions. DD-PCR was then applied to identify differentially expressed genes from infected grapevine leaves. Differentially expressed fragments were then sequenced and compared with known gene sequences in sequence databases. Verification of differential expression was done using reverse northern blots. Although a relatively high percentage of false positives were obtained with reverse northern blots, 25 differentially expressed mRNA species were isolated and compared with known gene sequences in sequence databases. These DNA fragments aligned to several known gene sequences like, V. vinifera proline rich proteins 1 & 2, thaumatin-like proteins, Z. mays ferredoxin III, C. sativus and B. napus catalase, A. thaliana peroxidase (not significant) and L. escu/entum polygalacturonase (not significant). Our study strengthens previous results concerning the grapevine defence reaction in response to powdery mildew infection. We observed reactions that are involved in the reinforcement of cell walls (peroxidase, catalase and proline rich proteins), localised cell death (ferredoxin reductases and catalase) and the production of antifungal compounds (thaumatin-like proteins). It was disappointing that no differentially expressed fragment showed significant homology with PR proteins like glucanase and chitinase. It is possible that these genes were expressed, but not at Do-PCR detectable levels. Although several general defence-related genes were isolated during this study, it was disappointing that no genes, specifically activated by powdery mildew infection, were isolated. However, several novel differentially expressed fragments were isolated and might represent novel and important links in the grapevine defence response.