Phytotoxic Nep1-like proteins from the necrotrophic fungus Botrytis cinerea associate with membranes and the nucleus of plant cells

Nep1-like proteins (NLPs), produced by an array of unrelated microorganisms, are phytotoxic for dicotyledonous plant cells but their mode of action has not yet been established. Two paralogous NLPs from the necrotrophic plant pathogenic fungus Botrytis cinerea were characterized, designated BcNEP1 and BcNEP2. Both proteins were produced in the heterologous host Pichia pastoris and purified to homogeneity. The localization of fluorescently labelled proteins was studied and mechanisms of cell death were investigated in protoplasts and suspension cells. Purified BcNEP1 and BcNEP2 caused necrosis in all dicotyledonous plant species tested, but not in monocotyledons. A synthetic heptapeptide comprising a sequence (GHRHDWE) that is conserved in all NLPs did not cause symptoms and was unable to interfere with necrosis induction by BcNEP1 and BcNEP2 proteins. Fluorescently labelled BcNEP1 and BcNEP2 proteins were associated with plasma membranes and the nuclear envelope, as well as in the nucleolus of responding plant cells. A strong hydrogen peroxide (H(2)O(2)) accumulation was observed in chloroplasts. The death process was characterized by TUNEL assays as apoptosis, necrosis or intermediate forms of both. BcNEP1- and BcNEP2-induced cell death execution could not be abolished by specific inhibitors. These results provide further information on mechanisms of NLP-inflicted cell death.