Identifizierung und Charakterisierung extrazellulärer Proteine unter dem Einfluss von Verticillium longisporum in Arabidopsis thaliana und Raps (Brassica napus)

Identification and characterization of extracellular proteins in Arabidopsis thaliana and Brassica napus

by Saskia Floerl
Doctoral thesis
Date of Examination:2007-11-01
Date of issue:2008-03-04
Advisor:Prof. Dr. Andrea Polle
Referee:Prof. Dr. Christiane Gatz
Referee:Dr. Susanne Behrens
Referee:Prof. Dr. Wolfgang Liebl
Persistent Address: http://dx.doi.org/10.53846/goediss-3408

 

 

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Abstract

English

Verticillium species are soil-borne, xylem-colonizing fungi with worldwide distribution in temperate and subtropical regions. They cause vascular disease in many agriculturally important crops. Verticillium longisporum is specialized on Brassicaceae e. g. cauliflower, turnip and rapeseed. In europe V. longisporum became an increasing problem with the growing area of oilseed rape production. This thesis aims to extend the knowledge about the interaction of V. longisporum and its host plants. The major goal was the characterization of the extracellular proteome of Arabidopsis thaliana and Brassica napus influenced by V.longisporum and in this context the description of infection symptoms. B. napus was chosen as example for an economically important crop, while the model plant A. thaliana provided the possibility for molecular analysis. The characterization of infection symptoms showed a reduced A. thaliana rosette area and a reduced shoot height with B. napus. In leaves of both plant species the chlorophyll content and activity of photosystem II were reduced by V. longisporum infection. Quantification of Verticillium-DNA by real time PCR resulted in no direct interrelation between symptome development and amount of fungus in overground plant parts, a hint that not the direct contact with the fungus but direct or indirect signals lead to the symptome development. Electrolyte leakage measurements showed that cell membranes were not demaged by the fungus at the timepoint of proteome analysis. Soluble apoplastic and xylem sap proteins from V. longisporum inoculated A. thaliana and B. napus leaves were separated by one- and two-dimensional electrophoresis. With both methods we observed differences in the protein pattern of Verticillium-inoculated and control plants. Proteins were identified by mass spectrometry and NCBI database search. In the leaf apoplast of V. longisporum infected A. thaliana a serine carboxypeptidase, an a-galactosidase, a germin-like protein and three peroxidases with increased expression and a lectin-like protein with decreased expression were found. The results for A. thaliana proteome analysis were verified by quantitative real time PCR. The B. napus apoplast under V. longisporum influence showed increased expression of a glucanase, a chitinase, a peroxidase and a PR-4 proteine. The glucanase and the PR-4 proteine also occurred in xylem sap with increased expression. Because increased expression of chitinases were often described in connection with Verticillium infection, here also in B. napus, the absence of increasingly expressed chitinases in the A. thaliana apoplast was amazing. Genexpression analysis for an acidic endochitinase (At5g24090) showed an obviously increased expression post V. longisporum infection. Though the overexpression of this gene didn
 
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