The protein PcF from P. cactorum: further properties of possible functional significance

PcF protein from the plant-pathogenic oomycete Phytophthora cactorum is a novel protein which is strongly toxic to host plants and elicits Phenylalanine Ammonia Lyase and PR-proteins expression in tomato plant (1,2). PcF comprises 52 residues and shows no sequence homology with proteins of known function (1,2). Despite PcF remarkable bioactivity, both its role in the pathogen physiology and the biochemical mechanism of its toxicity are still unknown. Immunocytochemical studies on PcF protein translocation, throughout the pathogen lifecycle, may help elucidate its role in pathogen-plant interaction (3). In addition, since calcium ion fluxes are involved in plant response to pathogens (4), we also tested whether PcF might bind calcium ions. MATERIALS AND METHODS Specific anti-PcF polyclonal antibodies were raised in rabbit, and used for immunocytochemical investigations on different pathogen specimens corresponding to different lifecycle stages, processed for fluorescence microscopy and TEM (5). Calcium ion-binding was tested with a radioactive-overlay assay (6): pure PcF protein was immobilized onto a PVDF membrane, incubated with radioactive CaCl2 and exposed to X-ray film. For quantitation, the exposed film was analysed by densitometry. RESULTS Both immunofluorescence and immunogold labeling showed that the PcF protein is located mainly on the external and internal layers of the cell wall of the pathogen hyphae. Preliminary ultrastructural observations on zoospores suggest that PcF might be secreted to the cell wall at the onset of encystment, consistent with the formation of new cell wall. The radioactive-overlay assay revealed that the protein is able to bind calcium with an apparent 1:1 stoichiometry. This property may affect calcium signalling taking place at the host plant cell upon pathogen perception, possibly resulting in plant toxicity.