Characterisation of the water expulsion vacuole in Phytophthora nicotianaezoospores

The water expulsion vacuole (WEV) in zoospores of Phytophthora nicotianae a nd other members of the Oomycetes is believed to function in cell osmoregul ation. We have used videomicroscopy to analyse the behaviour of the WEV dur ing zoospore development, motility and encystment in P. nicotianae. After c leavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 +/- 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The comm encement of WEV operation in sporangia coincides with the reduction of zoos pore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV in P. nicoti anae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towa rds vacuolar H+-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma m embrane. An antibody directed towards plasma membrane H+-ATPase shows the p resence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in mi crosomal fractions from P. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these: cells and has shown th at they are similar to those in true fungi. Inhibition of the vacuolar H+-A TPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H+-ATPase plays an important role in water ac cumulation by the spongiome in oomycete zoospores, as it does in other prot ists.