REGULATION OF THE OXIDATIVE BURST AND THE HYPERSENSITIVE RESPONSE IN TOBACCO CELLS CHALLENGED WITH ZOOSPORES FROM PHYTOPHTHORA PARASITICA VAR. NICOTIANAE
AJ ABLE1, MW SUTHERLAND1 and DI GUEST2
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
Superoxide production is measured by means of a new tetrazolium assay  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
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
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.