Identification of differentially expressed biomarkers in the host-parasite interaction in the Brassica napus - Verticillium longisporum system
Final Report Abstract
The devastating soil-borne fungal pathogen Verticillium longisporum (VL) is host-specific to Brassicaceae including oilseed rape (Brassica napus). VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem. VL reacts to the presence of B. napus xylem sap with the production of six distinct up-regulated and eight down-regulated proteins visualized by 2-dimensional gel electrophoresis. Identification of ten proteins by mass spectrometry revealed that all up-regulated proteins are involved in oxidative stress response. The catalase peroxidase VlCPEA was the most up-regulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical corroborating the diploid or „amphihaploid‟ status of the fungus. Knock-downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease. We show that VL is a hybrid between V. dahliae and near relative of V. albo-atrum. VL isolates carry only one type of rDNA, A1xD3 isolates carry the rDNA of V. dahliae, whereas the A1xD1 isolates contain an rDNA which has high homology to the rDNA of V. albo-atrum.
Publications
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(2009) Study of genes of the phytopathogenic fungus Verticillium longisporum involved in the colonization of xylem vessels of its host Brassica napus. PhD thesis
Seema Singh
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(2010) Pilzwachstum in der lebenden Rapspflanze auf dem Prüfstand – Systematische Analyse einer Pilz- Pflanzeninteraktion: Genom, Proteom und Metabolom. GenomXPress 3.10, 4-6
Braus-Stromeyer S. A. & G. H. Braus
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(2010) Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem. Applied Microbiology and Biotechnology 85: 1961-1976
Singh, S., S. A. Braus-Stromeyer, C. Timpner, V. T. Tran, G. Lohaus, M. Reusche, J. Knüfer, T. Teichmann, A. v.Tiedemann & G. H. Braus
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(2011) Adhesion of the rapeseed pathogen Verticillium longisporum to its host Brassica napus. PhD thesis
Van Tuan Tran
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(2012) Communication between the plant pathogen V. longisporum and its host Brassica napus. PhD theis
Christian Timpner
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(2012) Transcriptional activation and production of tryptophan-derived secondary metabolites in Arabidopsis roots contributes to the defense against the fungal vascular pathogen Verticillium longisporum. Mol Plant
Iven T, Konig S, Singh S, Braus-Stromeyer SA, Bischoff M, Tietze LF, Braus GH, Lipka V, Feussner F, Droge-Laser W
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The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease. Molecular Plant Microbe Interaction 25, 569-581
Singh S, Braus-Stromeyer SA, Timpner C, Valerius O, von Tiedemann A, Karlovsky P, Druebert C, Polle A, Braus GH
