Previously we reported an antifungal protein specific to Pythium porphyrae, a causative agent of red rot disease afflicting seaweed Porphyra spp. This study was carried out to identify the antifungal mechanism of the antifungal protein to P. porphyrae. When we first examined the effect of an anti-Pythium protein (SAP) on the P. porphyrae cell walls, SAP did not decompose the six structural polysaccharides in Pythium cell walls. However, hyphal growth was significantly inhibited in Pythium cells treated with 50 μg/ml of SAP by MTT assay. Protoplasmic leakage was observed in P. porphyrae hyphae treated with SAP for 1 h, followed by hyphal swelling and disintegration, using SYTOX Green, and SAP permeabilized the membrane of P. porphyrae in a dose-dependent manner. Treating P. porphyrae cells with SAP in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a membrane-depolarizing agent, significantly reduced the membrane permeability to SYTOX Green. Moreover, a similar effect was observed when the P. porphyrae cells were treated with SAP in the presence of MgCl2. In contrast, identical treatment in the presence of KCl significantly increased the membrane permeability to SYTOX Green. These results suggested that anti-Pythium mechanism of SAP was related to alteration of the membrane permeability in P. porphyrae.
