1School of Botany, University of Melbourne, Parkville 3052, Australia; 2Department of Biology, University of Southern Queensland, Toowoomba 4350, Australia

Background and objectives
Plant and animal cells challenged by pathogens undergo an oxidative burst, rapidly generating and releasing reactive oxygen species at the cell surface. Reactive oxygen species, including the superoxide anion and hydrogen peroxide, are involved in a range of environmental responses in plants, although their precise role in disease resistance remains unclear [1, 2]. We examined the relationship between superoxide release and plant disease resistance responses, including hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin, capsidiol, in infected tobacco seedlings and cell suspension cultures. Preliminary studies have shown that race-cultivar specificity in whole plants is retained in suspension cultures [3].

Materials and methods
Seedlings of the near-isogenic N. tabacum cvs Hicks (susceptible) and NC2326 (resistant), or suspension cell cultures of these cultivars maintained on MS liquid medium at 24C, were inoculated with zoospores of the black shank pathogen, Phytophthora parasitica var. nicotianae, or the non-host pathogen, Phytophthora palmivora. Inoculated seedlings or suspension cells were examined at regular intervals from inoculation until pathogen sporulation in compatible interactions (ca 30 h). Superoxide release was monitored by staining with nitroblue tetrazolium (NBT) for 15 min before each observation. Inhibition of staining by superoxide dismutase (SOD) or its catalytic analogue, Mn(IV)-desferal, indicates stain specificity for the superoxide anion. In some experiments catalase was included to inhibit hydrogen peroxide release. Phytoalexin accumulation was monitored using gas chromatography, and cell viability was monitored using hypertonic neutral red staining.

Results and conclusions
Seedlings or cell suspension cultures of N. tabacum respond to inoculation with incompatible strains of P. parasitica var. nicotianae or P. palmivora by rapidly releasing the superoxide anion in a zone surrounding the infection court. Superoxide release, indicated by SOD-inhibited NBT reduction, is more intense in incompatible or non-host interactions than in compatible interactions. Later defence responses in cell suspension cultures, including cytoplasmic aggregation, nuclear migration, hypersensitive cell death, deposition of lignin and callose and phytoalexin accumulation, are also more rapid and intense in incompatible interactions. There were temporal differences in the response of seedlings compared to cell suspensions, but the overall pattern of responses was similar. This correlation supports the hypothesis that superoxide release plays a role in the activation of defence responses, possibly through its ability to cause host cell membrane damage, leading to hypersensitive cell death and other responses.

We tested this hypothesis by blocking superoxide release using the naturally occurring enzyme SOD or its analogue, Mn(IV)-desferal. When present during attempted penetration and host colonization, both suppress capsidiol accumulation. SOD is less active than Mn(IV)-desferal, possibly because its size excludes intimate contact with the plant cell membrane, the putative site of superoxide release. Catalase addition did not affect capsidiol accumulation alone, but did enhance the suppression due to SOD. These results indicate that the superoxide anion is the physiologically active form of active oxygen in this interaction, while hydrogen peroxide, formed by the degradation of superoxide, may have a supplementary effect. Superoxide release appears to be a key link in the signalling cascade leading to the elicitation of phytoalexins and other defence responses in this interaction. Further studies will examine the mechanism by which superoxide release elicits host defence responses.

1. Low PS, Merida JR 1996. Physiologia Plantarum 96, 533-542.
2. Sutherland MW 1991. Physiological and Molecular Plant Pathology 39, 79-93.