Cerato-platanin, a phytotoxic protein from Ceratocystis fimbriata: expression in Pichia pastoris, purification and characterization.

Cerato-platanin (CP) is a phytotoxic protein secreted by the Ascomycete Ceratocystis fimbriata f.sp. platani. This Ascomycete causes canker stain which is a severe disease with a high incidence in the European Platanus acerifolia. CP probably plays a role in the disease, eliciting defence-related responses in the host plants. CP is a 120 amino acid protein, containing 40% hydrophobic residues and two S-S bridges. In the EMBL data bank CP is the first member of a new fungal protein family known as the Cerato-Platanin Family. The N-terminal region of CP shows a high similarity with that of cerato-ulmin, a phytotoxic protein produced by the Ophiostoma species and that belongs to the hydrophobin family. Hydrophobins are hydrophobic proteins secreted by many saprophytic or pathogenic fungi and have a remarkable ability to self-assemble into a rodlet structure takes part in physiological and/or pathological processes. The methyltrophic yeast Pichia pastoris was used to obtain a high-level expression of recombinant CP (rCP) and the pPIC9 vector was chosen to bring about extra-cellular secretion of the protein. The preliminary structural and functional characterization presented here reveals no significant differences between the native and the recombinant protein. We also show that CP self-assembles in solution. The availability of rCP will allow its three-dimensional structure to be determined, facilitating an understanding of the role of CP in the pathogenesis of canker stain. It is also an excellent model for investigating the mechanism of action of the other proteins related to CP.