Dokument

Zusammenfassung:
Infektionen von Wirtspflanzen durch pathogene Pilze erfordert polares Spitzenwachstum, ein Prozess, der die kontinuierliche polare Anlieferung von Zellwandkomponenten und Enzymen, wie Chitinsynthasen (CHS), entlang des Cytoskeletts benötigt. Klasse V CHS sind als Virulenzfaktoren für die pathogene Entwicklung essentiell. Diese potentiellen molekularen Motoren bestehen aus einer Myosin-Motordomäne (MMD), die mit einer CHS-Domäne fusioniert sind. Letztere ist am Aufbau der pilzlichen Zellwand beteiligt, die Rolle der MMD bleibt bisher ungeklärt. Eine nahe liegende Rolle könnte die MMD-vermittelte Anlieferung sekretorischer Vesikel (Chitosomen) zur Wachstumszone sein. In der vorliegenden Arbeit wurde der Einfluss beider Domänen in Mcs1, der Klasse V CHS in U. maydis, untersucht. Durch quantitative Analysen von Krankheitssymptomen, Besiedlung der Pflanze und Auswertungen morphometrischer Parameter wurde gezeigt, dass beide Domänen essentielle, jedoch ungleiche Rollen, für die pathogene Entwicklung von U. maydis spielen. Während der Phänotyp der G3Mcs1Chsdead-Mutante dem der Deletionsmutante ähnelt, konnte die Besiedelung des Pflanzengewebes durch Stämme, die Defekte in der Motordomäne aufwiesen, noch teilweise erfolgen. mcs1-Deletionsmutanten und CHS-aktivitätsdefekte Stämme werden schnell durch das pflanzliche Abwehrsystem erkannt und getötet. Mutanten bei denen die MMD deletiert wurden verursachen nur eine abgeschwächte Abwehrreaktion. Der Verlust der Klasse V CHS Aktivität führt vermutlich zu gravierenden Mängeln in der Zellwandzusammensetzung, wodurch polares in planta Wachstum gestört und der Infektionsprozess verlangsamt wird. Eine Folge dessen ist eine starke pflanzliche Abwehrreaktion, die durch die Bildung von H2O2 und lokalem Zelltod charakterisiert ist. Mikroskopische Untersuchungen zeigten, dass die apikale Mcs1-Lokalisierung von der eigenen MMD abhängt. Jedoch wurde auch beobachtet, dass noch wenige Chitosomen die Plasmamembran erreichen. Dadurch kann intrazelluläres Hyphenwachstum in begrenztem Maße erfolgen und U. maydis kann die Pflanzenzelle besiedeln. Eine Strukturanalyse verdeutlichte, dass trotz geringer Sequenzidentitäten die Mcs1 MMD der Struktur veröffentlichter Myosinmotoren ähnelt. Des Weiteren konnte belegt werden, dass die Mcs1 MMD an Aktin bindet und in der Lage ist Dimere auszubilden. In in vivo Motilitätsversuche wurde nachgewiesen, dass sich Mcs1-gebundende Chitosomen schnell und bi-direktional über lange Strecken bewegen und kurz bevor sie sekretiert werden in subapikalen Bereichen der Plasmamembran verharren. Die Insertion in die Membran erfolgt dabei selten und zufällig und es wurde gezeigt, dass die Verweildauer am Apex bei MMD-defizienten Mutanten signifikant verkürzt war. Wohingegen die apikale Mcs1-Akkumulation von F-Aktin und der eigenen MMD abhängt, spielen bei der Motilität von Chitosomen sowohl Aktin als auch Mikrotubuli eine Rolle und ist nicht abhängig von der MMD. Diese Ergebnisse zeigen deutlich, dass die eigene MMD nicht für die apikale Anlieferung verantwortlich ist, sondern eher lokale Funktionen in der Exozytose von Chitosomen übernimmt.

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