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Publications - Stress and Develop Biology

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Publications

Jabs, T.; Tschöpe, M.; Colling, C.; Hahlbrock, K.; Scheel, D.; Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley Proc. Natl. Acad. Sci. U.S.A. 94, 4800-4805, (1997) DOI: 10.1073/pnas.94.9.4800

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2− but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2− rather than H2O2 appears to trigger the subsequent reactions.
Publications

Sacks, W.; Nürnberger, T.; Hahlbrock, K.; Scheel, D.; Molecular characterization of nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora megasperma Molec. Gen. Genet. 246, 45-55, (1995) DOI: 10.1007/BF00290132

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.
Publications

Nürnberger, T.; Nennstiel, D.; Hahlbrock, K.; Scheel, D.; Covalent cross-linking of the Phytophthora megasperma oligopeptide elicitor to its receptor in parsley membranes. Proc. Natl. Acad. Sci. U.S.A. 92, 2338-2342, (1995) DOI: 10.1073/pnas.92.6.2338

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.
Publications

Nürnberger, T.; Nennstiel, D.; Jabs, T.; Sacks, W. R.; Hahlbrock, K.; Scheel, D.; High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses Cell 78, 449-460, (1994) DOI: 10.1016/0092-8674(94)90423-5

An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. megasperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl− effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants.
Publications

Lozoya, E.; Block, A.; Lois, R.; Hahlbrock, K.; Scheel, D.; Transcriptional repression of light-induced flavonoid synthesis by elicitor treatment of cultured parsley cells Plant J. 1, 227-234, (1991) DOI: 10.1111/j.1365-313X.1991.00227.x

Cultured parsley (Petroselinum crispum) cells respond differentially to UV‐containing white light and fungal elicitor. Both stimuli activate the transcription of genes encoding enzymes of partly overlapping phenylpropanoid biosynthetic pathways. Irradiation induces vacuolar accumulation of flavonoids, whereas elicitor treatment stimulates the secretion of furanocoumarins. Simultaneous treatment of parsley cells with UV light and elicitor results in quantitative changes in both responses. Irradiation reduces elicitor‐induced furanocoumarin synthesis, possibly by post‐transcriptional mechanisms, whereas elicitor treatment completely blocks the light‐induced accumulation of flavonoids by repressing the transcription of the chalcone synthase gene. We have identified elicitor‐sensitive regions in the chalcone synthase promoter by transient expression analysis of selected promoter constructs linked to the β‐D‐glucuronidase reporter gene in parsley protoplasts. These regions are identical to those that were found to be sufficient for light inducibility of the chalcone synthase promoter.
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