Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of phytophthora parasitica var nicotianae

Abstract

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L. ) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2-) production based on reduction of the tetrazolium dye sodium,3'-(1-[phenylamino-carbonyl]-3, 4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2./O2-) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2./O2- production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2./O2- was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 x 10(-15) mol min-1 cell-1 were observed. The HO2./O2- scavengers O2- dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2- production is a necessary precursor to the HR.

Figure 1

The viability of tobacco suspension-cultured cells inoculated with compatible and incompatible zoospores from Ppn. Data are means ± se of 20 replicates from six independent experiments.

Figure 2

Cumulative reduction of XTT by tobacco suspension-cultured cells inoculated with Ppn zoospores. cv NC2326 cells were inoculated with Ppn 4974 and Ppn 9201 zoospores, representing the incompatible and compatible interactions, respectively. cv Hicks cells were inoculated with Ppn 4974, also representing the compatible interaction. XTT (5 × 10⁻⁴ m) was added at 0 h and its formazan was allowed to accumulate. Data are means ± se of 20 replicates from six independent experiments.

Figure 3

The effect of O₂⁻ scavengers on the cumulative reduction of XTT by tobacco suspension cells inoculated with Ppn. Assays were performed as described in Figure 2 except that 100 units of SOD or 100 equivalent SOD units of Mn(III)desferal was added when required at 0 h. Data are means ± se of 10 replicates from three independent experiments.

Figure 4

Effect of radical scavengers on viability of inoculated tobacco suspension cells at 18 h postinoculation. The assay was as described in Figure 1. Data are means ± se of 12 replicates from three independent experiments.

Figure 5

Comparison of the scavenging abilities of SOD and Mn(III)desferal during the HR. The y axis is the percentage inhibition of XTT reduction relative to the absence of the scavenger 18 h postinoculation of cv NC2326 with Ppn 4974. Relative enzyme units were determined by the xanthine/xanthine oxidase assay of Faulkner et al. (1994). Data are means ± se of eight replicates from two independent experiments.

Figure 6

The reduction of Cyt c and the effect of SOD and Mn(III)desferal on the reduction of Cyt c by tobacco suspension cells inoculated with zoospores from Ppn. Cyt c (20 μm) was added at 0 h and its reduced product was allowed to accumulate. When required, 100 units of SOD or 100 equivalent SOD units of Mn(III)desferal was added at 0 h. Data are means ± se of 12 replicates from four independent experiments.

Figure 7

Effect of cell aggregation on viability of inoculated cells (A) and on reduction of XTT (B) at 18 h postinoculation. Data are means ± se of nine replicates from three independent experiments. White bars, Unfiltered; black bars, filtered.