The interaction of harpin(Pss), with plant cell walls

Erwinia amylovora and Pseudomonas syringae pv. syringae produce elicitors of the hypersensitive reaction (HR), harpin(Ea) and harpin(Pss), respectively. Harpin(Ea) causes K+ efflux and extracellular alkalinization in suspension-cultured cells of tobacco. These responses are associated with disease resistance. We treated living, fixed, and permeabilized cells and protoplasts from tobacco suspension cultures with harpin(Pss), antiharpin(Pss) antibody, and fluorochrome-tagged antibody and examined them with confocal laser microscopy. The fluorescent signal was localized in the outer part of the cell and was not observed in protoplasts. EGTA, a chelating agent that extracts Ca++ and pectins from cell walls, blocked harpin(Pss) binding, as evidenced by the absence of fluorescent signal. The pH of the external medium of suspension cultures alkalinized in response to harpin(Pss) and P. s. syringae. Bacteria and harpin(Pss)-induced alkalinization of the extracellular medium were also completely blocked upon EGTA treatment. Protoplasts alkalinized the medium at much reduced levels in response to P. syringae pv. syringae and did not alkalinize the medium in response to harpin(Pss). These results suggest that the cell wall is crucial for RR induction in the suspension culture cell.