Funktionelle Charakterisierung von apoplastischen Proteinen in Brassica napus und Arabidopsis thaliana nach Infektion mit Verticillium longisporum

Abstract

Verticillium longisporum is a soil-borne pathogenic fungus which is restricted to the family of Brassicaceae. V. longisporum is a vascular pathogen which colonizes the xylem of its host plants. Therefore, main defence reactions are expected in the extracellular matrix. Previously, the extracellular proteome of infected and mock inoculated Brassica napus and Arabidopsis thaliana plants was analysed and compared. In B. napus a glucanase, a peroxidase, a chitinase and a PR-4 protein were induced following infection. In A. thaliana three different peroxidases, a serine-carboxypeptidase, an α-galactosidase and a germin-like protein (GLP3) were more abundant after infection. In contrast, a lectin-like protein was downregulated. The aim of this study was to functionally characterize those differentially expressed extracellular proteins in B. napus and A. thaliana in relation to physiological and anatomical alterations. To test the hypothesis that defence related proteins in the xylem sap of B. napus could influence V. longisporum fungal proliferation was investigated in xylem sap of infected and mock inoculated plants. The growth of the fungus was restricted in xylem sap of infected plants compared to controls. This growth behaviour cannot be attributed to nutrient deficiencies in xylem sap of infected plants. Furthermore, the effect of salicylic acid on the growth of the fungus was studied. Salicylic acid was reported to increase after V. longisporum infection in the xylem sap of B. napus. But there was no effect on the proliferation of V. longisporum. In additional experiments the influence of xylem sap proteins on fungal growth was tested. Proliferation of V. longisporum was strongly enhanced in protein-free xylem sap regardless whether the sap was from infected or non-infected oil seed rape plants. These results show that the constitutive presence of defence proteins in xylem sap suppresses uncontrolled proliferation of V. longisporum. Since some of the differentially expressed candidate proteins in A. thaliana are suggested to play a role in cell wall remodelling and modifications infected and mock inoculated plants were investigated anatomically. Infection with V. longisporum led to the production of hyperplastic xylem in the hypocotyl as well as in leaves. Moreover, transdifferentiation of mesophyll cells into xylem vessels was observed in the leaves. The biological role of this observations remains unclear and needs to be investigated in future studies. To investigate which proteins are responsible for the immune response against V. longisporum in A. thaliana, functional analysis were performed. For this purpose, homozygous plants of T-DNA insertion lines were isolated and challenged with V. longisporum. Most of them displayed a similar pathophenotype like the wildtype. However, one line with a reduced expression of the germin-like protein 3 (GLP3) showed enhanced susceptibility to the fungus. Further experiments with amiRNA lines supported these results. Moreover, glp3-overexpressing plants with a constitutively higher glp3 transcript level were more resistant to V. longisporum. Also the amount of fungal DNA within the plants was significantly reduced in infected overexpressing plants compared to wildtype plants. The next step then was to characterize the function of GLP3 for the plant-pathogen interaction. Growth experiments with heterologous expressed GLP3 showed that the protein was able to attenuate proliferation of V.longisporum in vitro. The results indicate that GLP3 mediates resistance in A. thaliana by restricting the growth of the fungus directly. The molecular mechanisms of glp3 action is unclear and should be investigated in further experiments.