1Department of Biological and Physical Sciences, University of Southern Queensland, Toowoomba, Queensland 4350, Australia; 2Department of Botany, University of Melbourne, Parkville, Victoria 3052, Australia

Background and objectives
A variety of responses may occur when a plant cell is challenged by an incompatible pathogen, including the production of reactive oxygen species (ROS) and the hypersensitive reaction (HR) [1, 2]. We are currently investigating the role of ROS in disease resistance using suspension-cultured cells of Nicotiana tabacum cultivars that are either resistant or susceptible to infection by particular isolates of the fungus Phytophthora parasitica var. nicotianae. We have recently established that this is the first demonstration of the expression of cultivar-race specificity in tobacco cell cultures challenged by Oomycete zoospores.

Superoxide production is measured by means of a new tetrazolium assay [3] and compared with hydrogen peroxide production (which was measured by both fluorescence techniques and the evolution of oxygen after the addition of catalase). ROS was produced specifically in the incompatible interaction immediately prior to the HR.

The involvement of calcium in ROS production and the HR was investigated. In addition, inhibitors of various host enzymes have been applied to the system to determine which host proteins are involved in ROS production.

Materials and methods
The assay system involves dispensing 1-ml aliquots of 4-day-old tobacco cells (cvs Hicks and NC2326) suspended in 10-mM phosphate buffer (+1% sucrose) into 24-well culture plates, 2 h prior to inoculation with zoospores from Phytophthora parasitica nicotianae (Ppn) isolates 4974 (race 0) and 9201 (race 1). Resistance is elicited in NC2326 cells by inoculation with zoospores of Ppn 4974, while a susceptible response is observed in NC2326 cells inoculated with Ppn 9201 and Hicks cells inoculated with either isolate. The specific response of this system is currently being compared with non-specific responses observed when glucan elicitors isolated from Ppn are added to cells from both cultivars.

To indicate production of superoxide, the tetrazolium dye XTT is added and its reduction to XTT formazan monitored [3, 4] in the presence and absence of the superoxide scavengers superoxide dismutase (SOD) and Mn(III)desferal. Production of hydrogen peroxide was monitored using the fluorescent dye pyranine or an oxygen electrode.

Results and discussion
During the resistant interaction, ROS are produced in two bursts, 0-2 h and 8-12 h after zoospore addition. ROS production is negligible during susceptible interactions or in control treatments. Superoxide scavengers inhibit dye reduction and the HR significantly, suggesting a role for superoxide in potentiating the HR. The second burst of superoxide appears essential to the HR.The hydrogen peroxide scavenger catalase does improve viability, but not to the same extent, suggesting that hydrogen peroxide does not play as great a role in initiating the HR.

Exogenous calcium (CaCl2) had no significant effect on ROS production, but modulators of endogenous calcium did have a significant effect. EGTA (a chelator of Ca2+) and LaCl3 (a specific calcium-channel blocker) lower both ROS production and the extent of the HR in the avirulent response and during a non-specific elicited response, while the Ca2+ ionophore A23187 enhances ROS production. The effects of these compounds suggest a role for endogenous Ca2+ in ROS production and the HR.

DPI, a suicide inhibitor of mammalian NAD(P)H oxidase, completely inhibits the first superoxide burst but only partially inhibits the second burst and the HR in an avirulent interaction. Allopurinol, an inhibitor of xanthine oxidase, partially inhibits both bursts and the HR, but to a lesser extent, while SHAM, an inhibitor of cell-wall peroxidases, significantly inhibits both superoxide bursts and partially inhibits the HR. The possibility exists, therefore, that there is more than one source of superoxide during the respiratory burst. More than one source of superoxide also appears to exist during the respiratory burst of cells elicited non-specifically with glucans, while preliminary results of the hydrogen peroxide assays further support this possibility.

1. Low PS, Merida JR, 1996. Physiologia Plantarum 96, 533-542.
2. Sutherland MW, 1991. Physiological and Molecular Plant Pathology 39, 79-93.
3. Able AJ, Guest DI, Sutherland MW, 1998. Plant Physiology (in press).
4. Sutherland MW, Learmonth BA, 1997. Free Radical Research 27, 283-289.