Publikationen - Stress- und Entwicklungsbiologie

An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50 value of 4.7 nM, obtained from ligand binding assays. No crosslinking of 125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of 125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of 125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response.

cDNA sequences encoding the 42 kDa glycoprotein elicitor from the oomycete, Phytophthora megasperma, that induces the defense response in parsley have been cloned and sequenced. The 5′ end of the mRNA matches a consensus derived from sequences surrounding the transcription initiation sites of seven other oomycete genes. The major transcript of 1802 nucleotides contains a 529-codon open reading frame, which was predicted to encode a 57 kDa precursor protein. On the basis of peptide sequencing, the N-terminus of the mature protein is at position 163, suggesting that proteolytic processing events, in addition to signal peptide cleavage, generate the protein purified from the fungal culture filtrate. Expression studies in Escherichia coli with the cDNA as well as smaller subfragments demonstrated that a region of 47 amino acids located in the C-terminal third of the protein was sufficient to confer elicitor activity. The gene encoding the elicitor was found to be a member of a multigene family in P. megasperma. Homologous families of differing sizes were found in all eight other Phytophthora species tested, but not in other filamentous fungi including other Oomycetes. No significant similarity of the elicitor preprotein to sequences present in the databases has yet been detected.