Publikationsserver der Universitätsbibliothek Marburg

Titel: The role of Stp1, a secreted effector, in the biotrophic interaction of Ustilago maydis and its host plant maize
Autor: Liang, Liang
Weitere Beteiligte: Kahmann, Regine (Prof. Dr.)
Veröffentlicht: 2012
URI: https://archiv.ub.uni-marburg.de/diss/z2013/0078
DOI: https://doi.org/10.17192/z2013.0078
URN: urn:nbn:de:hebis:04-z2013-00780
DDC: Biowissenschaften, Biologie
Titel(trans.): Die Rolle des sekretierten Effektors Stp1 in der biotrophen Interaktion von Ustilago maydis und seiner Wirtspflanze Mais
Publikationsdatum: 2013-02-20
Lizenz: https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Ustilago zeae, Ustilago maydis secreted effector

Summary:
Secreted effectors play crucial roles during the establishment of the biotrophic interaction between Ustilago maydis and maize. In a previous study (Schipper, 2009) it had been demonstrated that a deletion of the stp1 effector gene resulted in a complete loss of virulence symptoms in maize infection and that such mutants elicited a hypersensitive response. This distinguishes stp1 from most other secreted effectors that are either dispensable for pathogenicity or have only a minor effect on virulence. This study focuses on the functional analysis of Stp1. A mutational analysis showed that the conserved N- and C-terminal domains of Stp1 can be separately expressed but are both required for Stp1 protein function. The long central variable domain was demonstrated to be dispensable yet may promote fungal growth in vascular bundles. stp1 homologs from closely related smut fungi of U. maydis could replace stp1 in U. maydis, indicating a conserved function. Stp1Δ136-432 lacking the central domain could be purified to homogeneity and was stable, while the isolated C-terminal domain, Stp1433-515, was unstable after purification. This could suggest that N- and Cterminal domains of Stp1 stabilize each other. Stp1-HA expressed by U. maydis was detected in the nucleus of plant cells by immunolocalization suggesting that Stp1 may suppress plant defense responses by affecting the transcription of respective genes. Both cytoplasmic and apoplastic maize proteins were identified as interaction partners of Stp1 by yeast two-hybrid assays using Stp1Δ136-432 as bait, suggesting that Stp1 may be an effector with both apoplastic and cytoplasmic functions The C-terminus of Stp1 as well as Stp1Δ136-432 could inhibit the activity of a maize extracellular cysteine protease, Sip3, which was identified as one of the apoplastic interaction partners. The interactions between Stp1 and the cytoplasmic interactors Sip9, a cell number regulator 8, Sip16, a CCR4-NOT transcription complex subunit, Sip19, a serine/threonine-protein kinase and Sip21, a VIP2 protein were verified with full-length cDNA but await to be confirmed by other techniques. RNA-Seq analysis demonstrated that several early defense response genes are not induced by stp1 mutants expressing the N-terminus of Stp1 while stp1 mutants expressing the Cterminus of Stp1 triggered even stronger plant defense responses than stp1 mutants during colonization. This suggests that N- and C-terminal domains of Stp1 have distinct functions.

Zusammenfassung:
Bei der Etablierung der biotrophen Interaktion zwischen Ustilago maydis und Mais spielen sekretierte Effektoren eine entscheidende Rolle. In einer vorausgegangenen Studie(Schipper, 2009) konnte gezeigt werden, dass die Deletion des stp1 Effektor-Gens zu einem vollständigen Verlust der Virulenz führt und dass entsprechende Mutanten eine hypersensitive Reaktion auslösen. Diesbezüglich unterscheidet sich stp1 von den meisten anderen untersuchten Effektoren, die entweder keinen oder nur einen geringen Beitrag zur Virulenz des Pilzes leisten. Diese vorliegende Arbeit befasst sich mit der funktionellen Analyse von Stp1. Mutationsanalysen zeigten, dass die konservierten N- und C-terminalen Domänen von Stp1 zwar als separate Polypeptide exprimiert werden können, aber die Gegenwart beider Domänen nötig ist, um die Stp1 Funktion zu komplementieren. Es wurde nachgewiesen, dass die zentrale variable Domäne von Stp1 keine essentielle Funktion übernimmt, allerdings könnte diese Domäne für die Proliferation des Pilzes entlang der Leitbündel eine Rolle spielen. stp1 Homologe aus mit U. maydis nahe verwandten Brandpilzen waren in der Lage, die Funktion von stp1 in U. maydis zu komplementieren, was für eine konservierte Funktion spricht. Eine Stp1 Version, bei der die zentrale Domäne deletiert wurde (Stp1Δ136-432), konnte bis zur Homogenität aufgereinigt werden und erwiess sich als stabil, während die isolierte C-terminale Domäne, Stp1433-515, nach der Aufreinigung instabil war. Diese Beobachtung könnte darauf hinweisen, dass sich die N- und C-terminalen Domänen von Stp1 gegenseitig stabilisieren. Durch Immunlokalisierung konnte ein Stp1-HA Fusionsprotein im Zellkern von Pflanzenzellen detektiert werden, die mit dem Stamm SG200stp1-HA infiziert waren. Demzufolge könnte Stp1 auf die Transkription von Genen Einfluss nehmen und dadurch die pflanzliche Abwehrreaktion unterdrücken. In einem Hefe-zwei-Hybrid-System, bei dem Stp1 als Köder benutzt wurde, konnten sowohl apoplastische als auch cytoplasmatische Mais Proteine als Stp1-Interaktoren identifiziert werden. Dies könnte bedeuten, dass Stp1 ein Effektorprotein mit dualer Funktion zum einen im Apoplasten und zum anderen im pflanzlichen Zytoplasma ist. Sowohl die gereinigte C-terminale als auch die N-terminale Domäne von Stp1war in der Lage, die Aktivität der extrazellulären Mais Cystein-Protease Sip3 zu inhibieren, die zuvorals ein apoplastischer Interaktionspartner von Stp1 identifiziert werden konnte. Die Interaktion zwischen Stp1 und den cytoplasmatischen Interaktoren Sip9 (cell number regulator 8), Sip16(CCR4-NOT Transkriptionskomplex Untereinheit), Sip19(Serine/Threonin- Kinase) und demVIP2 Protein Sip21 konnten nach Expression der jeweiligen Gene in voller Länge verifiziert werden, müssen aber zukünftig noch durch weitere Methoden bestätigt werden. RNA-Seq Analysen des Transkriptoms infizierter Mais Blätter zeigten, dass einige der frühen pflanzlichen Abwehrgene, die nach Infektion mit einem stp1-Deletions Stamm induziert werden, Hingegen führte die Infektion mit einem stp1-Deletions Stamm komplementiert durch den Stp1 C-Terminus sogar zu einer stärkeren Abwehrreaktion als sie nach Infektion mit dem stp1- Deletionsstamm beobachtet wurde. Dies weist darauf hin, dass die N- und C-terminale Domänen von Stp1 unterschiedliche Funktionen erfüllen könnten.

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