Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity

Generation of superoxide anion (O2−) by potato tuber protoplasts following treatment with hyphal wall components (HWC) of Phytophthora infestans was demonstrated by the superoxide dismutase (SOD)-sensitive reduction of extracellular cytochrome c or nitroblue tetrazolium (NBT). In the presence of NADPH ability of protoplasts to reduce cytochrome c was enhanced within 2 min after adding HWC and then proceeded as a sigmoidal curve. Addition of SOD during the course of the reaction prevented further reduction of cytochrome c. In the reaction mixture without NADPH or HWC, little reducing activity was detected. A similar reduction of NBT was also found in the protoplasts treated with HWC. Water-soluble glucans from compatible races of P. infestans, which are known as suppressors of hypersensitivity, inhibited the activation of reducing activity when they were applied before elicitation of the reaction with HWC. The glucans from incompatible races caused little inhibition. Of the tested inhibitors of hypersensitive cell death, SH-binding reagent and NADP+inhibited the reducing activity whereas respiratory inhibitors, NaN3, antimycine A, and 2,4-dinitrophenol were ineffective. These results indicate that O2− generation and NADPH oxidation occur on the plasma-membrane upon incompatible recognition of host cells leading to hypersensitive cell death. Suppression of the NADPH-dependent OZ- generation may be involved in compatible relationships in the potato—P. infestans system.