Identification of differentially expressed mRNA species in Vitis vinifera in reaction to infection by Uncinula necator

[摘要] ENGLISH SUMMARY: Powdery mildew is one of the most important grapevine diseases and is a problem in allthe grapevine producing areas. Losses caused by uncontrolled powdery mildew forceproducers to follow extensive fungicide spray programs to control the disease. Grapevinebreeders has tried to incorporate natural disease resistance into commercial cultivars, buthas been confronted with problems like inbreeding depression, long generation cycle, thepolygenic nature of powdery mildew inheritance and a complex genetic system. As aresult, not much progress has been made to convert existing susceptible cultivars toresistant cultivars.Previous studies attributed V. vinifrera resistance to powdery mildew to several factorssuch as differences in cuticle thickness, increased activity of enzymes involved in ligninbiosynthesis, production of papillae, deposition of silica incrusts, localised necrosis and theactivation of enzymes like chitinases and glucanases (Pratt et al., 1984, Heintz & Blaich,1989, 1990, Eibach, 1994, Clingefeffer & Scott, 1994). We approached this problem byaddressing disease resistance of grapevine cultivars to powdery mildew at the gene-activationlevel. Liang & Pardee (1992) develop a technique, called differential displayPCR (DD-PCR), that enabled us to compare differential gene expression of identical cellsunder altered (infected and not-infected) conditions.The identification of differentially expressed fragments started at the successful cultivationof test plants under identical environmental conditions. The next step was to artificiallyinoculate test plants with powdery mildew and to harvest the infected and control leavesafter a short incubation period. The isolation of RNA from grapevine leaves wasproblematic and had to be optimised during this study. Because DD-PCR was notpreviously used in our lab, the next step was to optimise the technique to suit our labconditions. DD-PCR was then applied to identify differentially expressed genes frominfected grapevine leaves. Differentially expressed fragments were then sequenced andcompared with known gene sequences in sequence databases. Verification of differentialexpression was done using reverse northern blots.Although a relatively high percentage of false positives were obtained with reverse northernblots, 25 differentially expressed mRNA species were isolated and compared with knowngene sequences in sequence databases. These DNA fragments aligned to several knowngene 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 (notsignificant) and L. escu/entum polygalacturonase (not significant).Our study strengthens previous results concerning the grapevine defence reaction inresponse to powdery mildew infection. We observed reactions that are involved in thereinforcement of cell walls (peroxidase, catalase and proline rich proteins), localised celldeath (ferredoxin reductases and catalase) and the production of antifungal compounds(thaumatin-like proteins). It was disappointing that no differentially expressed fragmentshowed significant homology with PR proteins like glucanase and chitinase. It is possiblethat these genes were expressed, but not at Do-PCR detectable levels.Although several general defence-related genes were isolated during this study, it wasdisappointing that no genes, specifically activated by powdery mildew infection, wereisolated. However, several novel differentially expressed fragments were isolated andmight represent novel and important links in the grapevine defence response.

[发布机构] Stellenbosch University