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

Van der Merwe, Andreas Stephanus (1999-12)

Thesis (M.Sc.Agric.) -- University of Stellenbosch, 1999.

Thesis

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.

AFRIKAANSE OPSOMMING: Witroes is een van die mees belangrike wingerdsiektes en is 'n probleem in al die wingerdverbouingsgebiede. Verliesse wat veroorsaak word deur onbeheerde witroesinfeksies verplig produsente om 'n omvattende fungisied spuitprogram te volg om die siekte te beheer. In die verlede het druif-telers gepoog om natuurlike siekteweerstand in kommersiele kultivars te inkorporeer, maar was gekonfronteer met probleme soos inteling depressie, lang generasie siklus, die poligeniese aard van witroesweerstand en 'n komplekse genetiese sisteem. Gevolglik is weinig vordering gemaak om bestaande vatbare kultivars om te skakel na witroesbestande kultivars. Vorige studies het bewys dat V. vinifera weerstand teen witroes veroorsaak word deur verskeie faktore soos verskille in die kutikula, 'n verhoogde aktiwitiet van ensieme betrokke in lignien biosintese, produksie van papilla, neerlegging van silika invoegsels, gelokaliseerde nekrose, en die aktivering van ensieme soos kutinase en glukanse ( Pratt et al., 1984, Heintz & Blaich, 1989, 1990, Eibach, 1994, Clingeleffer & Scott, 1994). In hierdie studie is bogenoemde probleem benader deur te kyk na siekteweerstand van druifkultivars op geen-aktiveringsvlak. 'n Tegniek genaamd "Differrensiele Vertonings Polimerase Kettingreaksie" (DVP) is vir hierdie doel aangewend. Bogenoemde tegniek is in 1992 deur Liang & Pardee ontwikkel en word gebruik om differensiele geen-uitdrukking van geneties identiese selle in verskillende omgewingstoestande (geinfekteer en niegeinfekteer) met mekaar te vergelyk. Die identifikasie van differensieel uitgedrukte fragmente begin by die suksesvolle verbouing van toetsplante onder identiese omgewingstoestande. Die volgende stap was om toetsplante kunsmatig te infekteer met witroes en dan die geinfekteerde- en ongeinfekteerde blare na 'n kort inkubasie periode te oes. Die isolasie van RNS vanaf druifblare was problematies en moes ge-optimiseer word gedurende die studie. Aangesien DVP nie voorheen in ons laboratoruim gebruik is nie, was die volgende doelwit die optimisering van die tegniek in ons laboratoruim toestande. DVP was toe aangewend om differensieel uitgedrukte gene wat in geinfekteerde wingerblare uitgedruk is, te identifiseer. Die nukleotied volgorde van bogenoemde fragmente is toe bepaal en vergelyk met bekende nukleotied volgordes in databassise. Die bevestiging van die differensiele uitdrukking van hierdie fragmente is gedoen deur Omgekeerde Noordelike Blot (ONB) analises. Alhoewel 'n redelike hoe persentasie vals positiewe verkry is met ONB, is 25 differensieel uitgedrukte fragmente geisoleer en vergelyk met bekende geen-volgordes in databassise. Hierdie DNS fragmente het homologie getoon met verskeie bekende geenvolgordes soos V.vinifera, Z. mays ferredoksin III, C. sativus and B. napus katalase, A. thaliana peroxidase (nie- betekenisvol) en L. esculentum poli-galakturonase (niebetekenisvol). Ons studie bevestig en versterk vorige resultate rakende die siekteweerstandsreaksie van wingerdplante in reaksie op infeksie deur witroes. Tydens die studie is reaksies waargeneem wat betrokke is by die versterking van selwande (peroksidases, katalase en prolien-ryke proteine), gelokaliseerde sel dood (ferredoksien reduktases en katalase) en die produksie van anti-fungiese bestandele (thaumatin-agtige proteine). Dit was teleurstellend dat geen differensieel-uitgedrukte fragmente, betekenisvolle homologie getoon het met patogeen-verwante proteine soos glukanase en kutinase nie. Dit is egter moontlik dat laasgenoemde gene wel uitgedruk was, maar nie teen OVP waarnemingsvlakke nie. Alhoewel verskeie algemene siekteweerstandsverwante gene gedurende die studie geisoleer is, was dit teleurstellend dat geen gene wat spesifiek deur witroes infeksie geaktiveer word, geisoleer is nie. Verskeie onbekende geen-fragmente is egter tydens die studie geisoleer en kan moontlik nuwe en onbekende skakels wees in die siekteweerstandsrespons van wingerdplante.

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