Hydrogen peroxide yields during the incompatible interaction of tobacco suspension cells inoculated with Phytophthora nicotianae

Abstract

Rates of H(2)O(2) 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 HO(2)(*)/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 H(2)O(2) could not be accurately measured due to its metabolism by host cells, but results are consistent with the majority of H(2)O(2) being formed via dismutation of HO(2)(*)/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.

Figure 1

Percentage relative pyranine fluorescence supernatant of inoculated cells as a measure of H₂O₂ production. Pyranine (10 μg mL⁻¹) was added to the supernatant of tobacco cells at the time of sampling the supernatant. Relative fluorescence of the supernatants of cv ‘North Carolina’ (NC) cells inoculated with Pn 9201 (compatible) or Pn 4974 (incompatible) zoospores was determined by dividing by the fluorescence observed in unchallenged control treatments at the same time point. Data represent means ± se of n = 6 from three experiments.

Figure 2

ROS production during the incompatible interaction. HO₂^./O₂⁻ was detected using Mn(III)desferal-inhibitable XTT reduction and H₂O₂ estimated using an oxygen electrode. Data represent means ± se of n = 18 from six experiments for HO₂^./O₂⁻ and n = 8 from three experiments for H₂O₂ data.

Figure 3

The effect of CAT on viability of cells. Viability of cv NC2326 control cells and cv NC2326 cells inoculated with zoospores from Pn 4974 (incompatible) or Pn 9201 (compatible) was determined after 12 h. White bars represent the addition of 400 units of CAT. Black bars indicate the absence of CAT. Data represent means ± se of n = 8 from three experiments.

Figure 4

The effect of inhibitors of endogenous CAT and SOD on ROS production during the incompatible interaction. The effect of 1 mm SA on HO₂^./O₂⁻ (A) and H₂O₂ (B) production and of 1 mm ATZ and 1 mm DDC on H₂O₂ production (B) was monitored. Data represent means ± se of n = 9 from two experiments for SA data and n = 6 from two experiments for ATZ and DDC results.

Figure 5

The effect of Cu/Zn SOD inhibition on the viability of challenged cells. DDC (1 mm) and/or MnSOD (100 units) was added at time of zoospore (or water) addition, and the viability of cells assayed at 18-h post inoculation. Data represent means ± se of n = 6 from two experiments.

Figure 6

ROS production during an incompatible interaction in the presence of possible source enzyme inhibitors. Twenty micromolar DPI, 500 μm allopurinol, or 2 mm SHAM was added at 0 h and HO₂^./O₂⁻ production (A) and H₂O₂ production (B) monitored over 12 and 10 h, respectively. Data represent means ± se of n = 9 from three experiments for A and of n = 8 from three experiments for B.

Figure 7

The effect of possible source enzyme inhibitors on cell viability. Twenty micromolar DPI, 500 μm allopurinol, or 2 mm SHAM was added at 0 h and cell viability measured at 12 h post inoculation. Data represent means ± se of n = 8 from three experiments.