PURIFICATION AND CHARACTERIZATION OF CACTOREIN, A PHYTOTOXIN SECRETEDBY PHYTOPHTHORA-CACTORUM

An extracellular, hydrophilic, thermostable and non-dialysable phytoto xin was purified to homogeneity from culture fluids of Phytophthora ca ctorum with a yield of 2.7 mg l(-1). The toxin is an acidic protein wi th a M(r) of 10 000 and a pI of 3.65. The amino acid composition was d etermined and resembles that of characterized members of the elicitin family of Phytophthora phytotoxins. Toxicity was evaluated with a cell viability assay using tobacco cell suspensions, and a LD(50) value an d a lethal dose of 38 and > 100 nM respectively, were determined for t he purified toxin. When applied to tobacco plantlets, concentrations a s low as 100 nM resulted in wilting and desiccation. Mature leaf tissu e reacted hypersensitively to the toxin, with localized necrosis; and produced pathogenesis-related proteins in response to the toxin. The t oxin induced rapid extracellular alkalinization in cultured tobacco ce lls and elicited active defence responses with concentration dependent induction of sesquiterpenoid phytoalexins. Rishitin, phytuberol and c apsidiol accumulated in the medium to concentrations of 25, 14, and 63 nmol g(-1) cells, 24 hr after exposure to a toxin concentration of 40 nM. Higher concentrations of toxin resulted in a loss of cell viabili ty and eventual cell death.