1.10.9
CHARACTERIZATION OF THE WATER EXPULSION VACUOLE OF PHYTOPHTHORA ZOOSPORES

HJ MITCHELL and AR HARDHAM

Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia

Background and objectives
Species of Phytophthora cause many serious plant diseases, including the highly destructive late blight of potato. In Australia, Phytophthora infection is estimated to cause an annual loss in excess of $200 million [1]. As well as infecting economically important crop plants, species of Phytophthora also damage native Australian plants, causing extensive vegetation changes in large areas of forest and heathland.

Disease dissemination is achieved by the production of large numbers of motile zoospores which are able to swim chemotactically towards a host plant. The zoospores are wall-less cells and their outer surface is the plasma membrane. They are therefore unable to build up turgor pressure, and so water that enters the cell down the osmotic gradient is expelled through the action of a water expulsion vacuole. The water expulsion vacuole complex forms during cleavage of multinucleate sporangia to form the uninucleate zoospores, a process that takes about 30 min. The main central vacuole contracts approximately once every 6 s throughout the life of the zoospore. On contact with a host plant, the zoospores encyst, secreting adhesive and forming a cellulosic cell wall. Wall formation allows turgor pressure to build up and the water expulsion vacuole disappears. Expulsion of water from the central vacuole is thought to occur by an actin-based contraction; however, the molecular basis for water accumulation in the vacuole is completely unknown. As part of a study of the properties of Phytophthora zoospore biology, we have analysed H+-ATPase activity in zoospore membranes. The work aims to provide information which will aid development of effective control strategies for Phytophthora diseases.

Results and conclusions
Biochemical studies of H+-ATPases in membrane fractions from Phytophthora nicotianae zoospores have revealed the presence of vacuolar type H+-ATPase activity in addition to plasma membrane type H+-ATPase activity. An antiserum raised against a 60-kDa subunit of the vacuolar H+-ATPase from oat roots [2] cross-reacts with a polypeptide of similar size in extracts from zoospores of P. nicotianae. Immunofluorescent labelling of zoospores shows a reaction with components in an annular ring in the anterior of the cell at the site occupied by the water expulsion vacuole. This site is also labelled in cleaving sporangia. Immunogold localization confirmed that the antiserum reacts with the reticulate tubular network that surrounds the large central vacuole of the water expulsion vacuole complex. Inhibition of the vacuolar type H+-ATPase activity in zoospores with concanamycin A leads to premature encystment of the zoospore, suggesting that the vacuolar type H+-ATPase may play a role in the osmoregulation of the zoospore.

Further studies are focusing on the location of the vacuolar type H+-ATPase in other stages of the Phytophthora life cycle.

References
1. Irwin JAG, Cahill DM, Drenth A, 1995. Australian Journal of Agricultural Research 46, 1311-1337.
2. Ward JA, Reinders A, Hse H-T, Sze H, 1992. Plant Physiology 99, 161-169.