Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)