PHOSPHOINOSITIDE BREAKDOWN DURING THE K+/H+ EXCHANGE RESPONSE OF TOBACCO TO PSEUDOMONAS-SYRINGAE PV SYRINGAE

The hypersensitive response (HR) of tobacco to Pseudomonas syringae pv. syringae involves activation of a K+/net H+ exchange response (XR). Dependence of this response on calcium influx suggests that it is triggered by a signal transduction pathway. To test this hypothesis, we studied in vivo phospholipid metabolism during the XR. Suspension-cultured tobacco cells were incubated with H-3- or C-14-labeled fatty acids to radiolabel endogenous lipids. Cells were then inoculated with P. s. pv. syringae or with a hrp mutant that does not induce the HR or the XR in tobacco. In XR-induced cells, radioactivity in phosphatidylinositol decreased 20-30% within 4 hr, relative to cells inoculated with the hrp mutant. In contrast, radioactivity in other phospholipids remained constant or increased over this time period. When tobacco cells were pulse-labeled with Pi-32 during the XR, radioactivity in phosphatidylinositol and phosphatidylinositol 4-phosphate was reduced while radioactivity in other phospholipids was markedly increased. Phosphatidylinositol breakdown, calcium influx, and the XR were inhibited by bromophenacylbromide, a phospholipase inhibitor, and by neomycin, which blocks metabolism of polyphosphoinositides. These results suggest that increased activity of phosphoinositide-specific enzymes, possibly phospholipase C and/or phosphoinositide kinases, play a role in XR activation.