Publikationsserver der Universitätsbibliothek Marburg
Titel:Functional characterization of a seven-WD40 repeat protein Rak1 in Ustilago maydis
Autor:Wang, Lei
Weitere Beteiligte: Kahmann, Regine (Prof. Dr. )
Veröffentlicht:2011
URI:https://archiv.ub.uni-marburg.de/diss/z2011/0115
URN: urn:nbn:de:hebis:04-z2011-01159
DOI: https://doi.org/10.17192/z2011.0115
DDC:570 Biowissenschaften, Biologie
Titel (trans.):Funktionale Charakterisierung des sieben-WD40-Domänen Proteins RACK1 in Ustilago maydis
Publikationsdatum:2011-06-28
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:

Summary:
In dem pflanzenpathogenen Brandpilz Ustilago maydis wird die Paarungsreaktion zweier kompatibler Zellen durch ein Pheromon/Rezeptor System koordiniert. Der Pheromon Stimulus wird dabei über ein konserviertes MAP Kinase Modul übermittelt was zur Aktivierung von Prf1 führt, einem essentiellen Regulator für sexuelle und pathogene Entwicklung. Um zusätzliche Komponenten des MAP kinase Signalwegs zu finden, wurde U. maydis Rak1, ein sieben-WD40-Domänen Protein, ein Ortholog von RACK1 in Säugerzellen, untersucht. In U. maydis wird rak1 konstitutiv exprimiert und lokalisiert im Zytoplasma und in der Membran. Rak1 spielt eine Rolle in der Aufrechterhaltung der Zellwandintegrität und in der Regulation des Zellwachstums und konnte den bei erhöhten Temperaturen auftretenden Wachstumsphänotyp von Saccharomyces cerevisiae asc1 Mutanten komplementieren. Die Deletion von rak1 führte zu einer deutlichen Reduktion der Bildung von Konjugationshyphen und resultierte damit in dramatisch reduzierter Paarungseffizienz. Dieser Effekt konnte auf die reduzierte Expression des Pheromon- und des Pheromonrezeptor-Gens zurückgeführt werden. Durch die genetische Aktivierung des MAP kinase Moduls konnte die Bildung von Konjugationshyphen in FB1Drak1 wieder hergestellt werden. Weiterhin konnte die Konjugationshyphenbildung durch konstitutive Expression des Pheromonrezeptor Gens pra1 oder des Pheromon aktivierten Transkriptionsfaktors prf1 und gleichzeitiger Gabe von kompatiblem Pheromon wiederhergestellt werden. In solopathogenen Stämmen führte die Deletion von rak1 zu abgeschwächter Filamentbildung sowie Pathogenität, was durch Expression des kompatiblen bE/bW Heterodimers komplementiert werden konnte. Diese Analyse erlaubte es rak1 genetisch oberhalb von prf1 zu platzieren. Durch Mikroarray-Analyse konnten 201 Gene identifiziert werden, die in dem rak1 Deletionsstamm differenziell reguliert sind. 163 induzierte Gene zeigten eine signifikante Anreicherung in den funktionellen Kategorien Metabolismus, Energie, Virulenz und Stress- bzw. Toxinresistenz. Die 38 reprimierten Gene zeigten eine signifikante Anreicherung im Lipidstoffwechsel, Fermentation und einem MAPK abhängigen Signalweg. Unter den reprimierten Genen in FB1Drak1 wurde rop1 gefunden, ein direkter positiver transkriptioneller Regulator von prf1. Die konstitutive Expression von rop1 in FB1Drak1 führte zur Induktion der Expression von mfa1 und der Pheromon-abhängigen Konjugationshyphenbildung. Zusammenfassend wirkt Rak1 als neuartiger Regulator der rop1 und dadurch prf1 Genexpression während der Kreuzungsreaktion und der pathogenen Entwicklung.

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