Résumé
Cultured parsley (Petroselinum crispum) cells or protoplasts respond to treatment with a crude cell wall preparation from Phytophthora sojae with the transcriptional activation of the same set of defense-related genes as are activated in parsley leaves upon infection with fungal spores. A 42 kD glycoprotein was purified from the fungal culture filtrate and its elicitor activity was found to reside exclusively in the protein moiety. A core sequence consisting of 13 amino acids (Pep-13) in the glycoprotein was identified, which stimulates the same responses as the crude cell wall elicitor, namely Ca2+ and H+-influxes, effluxes of K+-and CI- ions, an oxidative burst, ethylene production, defense-related gene activation, and formation of phytoalexins. Over-expression in E. coli of the full-length cDNA encoding the protein portion of the 42 kD glycoprotein, as well as of clones in which the 13 amino acids representing Pep-13 were replaced by either 2 or 6 unrelated amino acids, showed that this region is necessary and sufficient for the elicitor activity of the intact glycoprotein. Using (125I)Tyr-Pep-13 as ligand in binding assays a single-class high-affinity binding site in parsley microsomal membranes and protoplasts could be detected. Binding was specific, saturable and reversible. By chemical crosslinking a 91 kD parsley plasma membrane protein was identified to be the receptor of the peptide elicitor. In patch-clamp analyses a calcium- permeable ion channel (LEAC) of large unitary conductance was identified, the activity of which could be modified by elicitor. A series of peptides was tested for their ability to compete for binding of Pep-13 to parsley microsomes, to activate LEAC as well as macroscopic ion fluxes, oxidative burst, and phytoalexin formation. Findings demonstrated a functional link between the various plant responses to elicitor treatment. The use of a series of ion channel inhibitors as well as inhibitors of the oxidative burst revealed that the observed ion fluxes and active oxygen species are an absolute requirement for defense-related gene activation and subsequent phytoalexin formation. Furhtermore, the same ion channel inhibitors were found efficiently inhibit the elicitor-induced oxidative burst. These findings allow us to establish a sequence of events that may constitute part of a signalling cascade triggering pathogen defense plants.