Hydrogen Peroxide Yields during the Incompatible Interaction of Tobacco Suspension Cells Inoculated with Phytophthora nicotianae

Rates of H2O2 production by tobacco suspension cells inoculated with zoospores from compatible or incompatible races of the pathogen Phytophthora nicotianae were followed by direct measurement of oxygen evolution from culture supernatants following catalase addition. Rates of $\text{HO}_{2}{}^{\cdot}/\text{O}_{2}{}^{-}$ production were compared by following the formation of the formazan of sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate. In the incompatible interaction only, both reactive oxygen species (ROS) were produced by the cultured host cells in a minor burst between 0 and 2 h and then in a major burst between 8 and 12 h after inoculation. Absolute levels of H2O2 could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H2O2 being formed via dismutation of $\text{HO}_{2}{}^{\cdot}/\text{O}_{2}{}^{-}$. The effects of inhibitors of endogenous Cu/Zn superoxide dismutase (diethyldithiocarbamate) and catalase (3-amino-1,2,4-triazole and salicylic acid) were also examined. Yields of ROS in the presence of the inhibitors diphenylene iodonium, allopurinol, and salicylhydroxamic acid suggest that ROS were generated in incompatible host responses by more than one mechanism.