Publikationsserver der Universitätsbibliothek Marburg
Titel:Charakterisierung der Tryptophan-abhängigen Pigmentsynthese am Modellorganismus Ustilago maydis und Identifizierung von Wirtsspezifitätsfaktoren in den phytopathogenen Brandpilzen Ustilago maydis und Sporisorium reilianum
Autor:Zuther, Katja
Weitere Beteiligte: Bölker, Michael (Prof. Dr.)
Veröffentlicht:2009
URI:https://archiv.ub.uni-marburg.de/diss/z2010/0484
DOI: https://doi.org/10.17192/z2010.0484
URN: urn:nbn:de:hebis:04-z2010-04844
DDC: Biowissenschaften, Biologie
Titel (trans.):Characterisation of the Tryptophan dependent pigment synthesis in the model organism Ustilago maydis and Identification of host specificity factors in the phytopathogenic smut fungi Ustilago maydis and Sporisorium reilianum
Publikationsdatum:2010-09-22
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Sporisorium reilianum, Ustilago zeae, Mais, Tryptophan, Pigments, Hirse, Tryptophanderivate, Wirtsspezifität, Phytoalexine, Pigmente, Phytoalexin, Host specificity, Sprorisorium reilianum, Pityriasis versicolor, Tryptophanstoffwechsel, Malassezia furfur, Head smut disease

Zusammenfassung:
Im ersten Teil der vorliegenden Arbeit wurde am Modellorganismus Ustilago maydis der Tryptophan-abhängige Biosyntheseweg untersucht, der zur Generierung von Indolpigmenten mit biologischen Effekten führt, die erstmals für den Pityriasis versicolor-assoziierten Hautpilz Malassezia furfur nachgewiesen worden waren. Mittels eines revers-genetischen Ansatzes konnte gezeigt werden, dass die Tryptophan-Aminotransferase Tam1 das einzige für die Pigmentbiosynthese verantwortliche Enzym repräsentiert. Die aufgereinigte heterolog exprimierte Tam1 aus U. maydis katalysierte die Umsetzung von Tryptophan und Phenylpyruvat zu Indolpyruvat und Phenylalanin. Ein im Vorfeld der Arbeit durchgeführter vorwärts-genetischer Ansatz hatte zur Identifikation von Mutanten geführt, die unfähig waren, in Anwesenheit von Tryptophan Pigmente zu bilden. Diese Mutanten waren in dem Sulfitreduktase-Gen sir1 betroffen. In vitro-Experimente mit aufgereinigter Tam1 zeigten, dass auch 2-Oxo-4-Methylthiobutanoat, die 2-Oxosäure von Methionin, als Substrat für Tam1 dienen kann, was die Tryptophan-Deaminierung an den Schwefelmetabolismus koppelt. Die Experimente zeigten, dass Indolpyruvat spontan, entweder alleine oder in Kombination mit Tryptophan, in wässriger Umgebung zu einer Vielzahl von farbigen Substanzen reagierte, unter denen sich auch mindestens vier der für M. furfur beschriebenen Substanzen mit biologischen Effekten befanden. Experimente an Keratinozyten-Zellkultur zeigten, dass Indolpyruvatabhängige biologische Effekte wie vermindertes Proliferationsvermögen über den humanen Arylhydrocarbonrezeptor vermittelt werden könnten. Im zweiten Teil der Arbeit wurden mittels Genomvergleich Gene aus Sporisorium reilianum identifiziert, die an der Ausprägung der Wirtsspezifität beteiligt sein könnten. S. reilianum existiert in zwei Varietäten (SRM und SRH), wovon SRM bevorzugt auf Mais und SRH ausschließlich auf Hirse zur Sporenbildung kommt. Um Faktoren aus S. reilianum zu identifizieren, die die Virulenz auf Mais unterstützen und gleichzeitig die Virulenz auf anderen Wirtspflanzen schwächen, wurden die in den Genomen der Maispathogene SRM und U. maydis vorkommenden Gene nach Abwesenheit in den Genomen verwandter Pathogenen anderer Wirtspflanzen untersucht. Dabei wurde ein Gen aus S. reilianum identifiziert, sr13864, das vermutlich für ein sekretiertes Protein kodiert. Mittels RT-PCR wurde gezeigt, dass sr13864 während der biotrophen Phase exprimiert wird. U. maydis-Stämme, in denen das homologe Gen von sr13864, um00823, deletiert wurde, zeigten reduzierte Virulenz auf Mais. Während Expression von um00823 in der um00823-Deletionsmutante den Virulenzdefekt vollständig komplementieren konnte, konnte heterologe Expression von sr13864 in der um00823-Deletionsmutante die reduzierte Virulenz nur teilweise aufheben. Expression von sr13864 in SRH führte zu einer erhöhten Abwehrreaktion auf Hirse in Form von roten Läsionen entlang der Leitbündel, die wahrscheinlich auf die Bildung von 3-Desoyanthocyanidin-Phytoalexin zurückzuführen sind. Damit scheint sr13864 ein Wirtsspezifitätsfaktor mit gleichzeitiger Virulenzfunktion für die Infektion von Mais und Avirulenzfunktion für die Infektion von Hirse zu sein.

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