Das Projekt "Funktionale Analyse von Efflux-Transportern phytopatogener Pilze" wird vom Umweltbundesamt gefördert und von Universität Halle-Wittenberg, Institut für Pharmazie durchgeführt. Höhere Pflanzen wehren phytopathogene Pilze häufig mit Hilfe niedermolekularer, für Pilze toxischer Sekundärstoffe, sog. Phytoalexine oder Phytoanticipine, ab. Um dem zu begegnen, können Pilze Efflux-Transporter synthetisieren und so die intrazelluläre Konzentration der an-tifungalen Sekundärmetabolite unter der Toxizitätsgrenze halten. Das Projekt hat das Ziel, über Komplementation von Transporter-defizienten Hefemutanten Transporter-Gene des Bohnenpathogens Colletotrichum lindemuthianum und des Tomaten- und Kartoffelpathogens C. coccodes zu klonieren und deren Funktion während der Pathogenese zu untersuchen. Ferner sollen pflanzliche Wirkstoffe identifiziert werden, die Efflux-Transporter inhibieren und so die Phytoalexin- oder Phytoanticipin-basierte pflanzliche Abwehr verstärken. Ein Fernziel der geplanten Arbeiten ist die Herstellung transgener Tomaten, die Flavone mit inhibitorischer Aktivität gegen pilzliche Efflux-Transporter synthetisieren. Mit solchen transgenen Pflanzen oder durch direkten Einsatz pflanzeneigener Flavonoide, die das Abwehrpotenzial der Pflanzen stärken, könnte die Intensität des klassischen chemischen Pflanzenschutzes reduziert werden. Weitere Ziele sind die Aufklärung der Proteinstruktur und der Protein-Substrat-Interaktionen pilzlicher Transporter, die in Kooperationen mit der Biophysik und der Biochemie angestrebt werden.
Das Projekt "Regulation of AtPGP1-mediated auxin transport by phosphorylation" wird vom Umweltbundesamt gefördert und von Universität Zürich, Institut für Pflanzenbiologie, Abteilung Physiologie und Mikrobiologie durchgeführt. Auxin - principally indole-3-acetic acid (IAA) - has proven as unique signaling molecule virtually controlling all plant developmental processes. Recent research has concentrated on the fascinating feature auxin being transported in a directed or polar fashion. Polar auxin transport (PAT) is regulated at the cellular level and is apparently both a product and determinant of cellular polarity. Auxin unloading is thought to be mediated by protein complexes that are characterized by members of the p-plycoprotein (PGP) and pin-shaped (PIN) protein families. The establishment of auxin gradients is controlled by reversible protein phosphorylation, however, the individual targets of protein kinases and phosphatases are unknown. Several lines of evidence point to components of auxin efflux complexes and/or NPA-binding proteins as targets of phosphorylation. While PIN proteins are apparently unlikely candidates two findings favor PGP as targets: PGP1 has been shown recently to catalyze the primary active export of auxin and to be modulated by NPA binding. Moreover, in a recent phosphoproteomic approach, PGP1 has been demonstrated to be phosphorylated in conserved phosphorylation sites in a so-called regulatory linker domain. This domain is known to modulate the activity mammalian PGPs by phosphorylation via PKC. In this project we envisage to demonstrate that PGP1-mediated auxin transport is modulated by phosphorylation in its regulatory linker domain. Phosphoproteomic data, a yeast-based mutant screen, and site directed mutagenesis will be used to determine the impact of phosphorylation on transport activity. The outcome of the yeast work will allow us to engineer relevant phosphorylation sites in the linker domain of PGP1 that alter protein activity and/or location. Additionally, TILLING technology will be used to identify relevant point mutations in the linker domain. Finally, in order to identify plant-borne kinases/phosphatases responsible for (de)phosphorylation of PGP1, a classical yeast two-hybrid screen using the linker domain as bait will be carried out. In an inverse approach, the phosphorylation status of PGP1 will be upon will be determined biochemically and by mass spectrometry applying physiological, chemical and genetic tools. The outcome should provide a deep insight into the regulation of auxin transport via PGPs and the establishment of local auxin gradients controlling virtually all steps of plant development. Transfer of this knowledge might later on open new strategies for the directed genetic or chemical manipulation of plant development.