Differential protein expression in response to the phytoalexin brassinin allows the identification of molecular targets in the phytopathogenic fungus Alternaria brassicicola

The effects of the cruciferous phytoalexin brassinin on the protein expression patterns of the phytopathogenic fungus Alternaria brassicicola were investigated. Cell-free protein extracts of mycelia of A. brassicicola induced with brassinin at 0.50 and 0.10 mm were fractionated, and the proteins in soluble fractions were separated by two-dimensional electrophoresis. Spots corresponding to differentially expressed proteins were digested and analysed by liquid chromatography-electrospray ionization-mass spectrometry. The number of differentially expressed proteins was significantly higher in mycelia treated with brassinin at 0.50 mm (96 protein spots) than in mycelia treated with brassinin at 0.10 mm (18 protein spots). The majority of differentially expressed proteins included proteins involved in metabolism, processing, synthesis and several heat shock proteins (HSPs). Brassinin concentrations below 0.30 mm induced HSP90, a protein involved in the regulation of morphogenetic signalling in fungi, suggesting that 0.30 mm is a minimal concentration of brassinin necessary for the protection of brassicas against A. brassicicola. These results reveal that HSP90 is a potential target for inhibition in stressed A. brassicicola and confirm that brassinin has strong detrimental effects on A. brassicicola, suggesting that its detoxification by the fungus suppresses an important defence layer of the plant.