Publikationsserver der Universitätsbibliothek Marburg

Titel:Functional characterization of the Ustilago maydis virulence gene scp2
Autor:Krombach, Sina
Weitere Beteiligte: Kahmann, Regine (Prof. Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0051
DOI: https://doi.org/10.17192/z2017.0051
URN: urn:nbn:de:hebis:04-z2017-00515
DDC:580 Pflanzen (Botanik)
Titel(trans.):Funktionelle Charakterisierung des Virulenzgen Scp2 (sterol carrier protein 2) von U. maydis
Publikationsdatum:2016-12-20
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
peroxisome, Peroxisom, Corn smut, Maisbeulenbrand

Summary:
The causative agent of the corn smut disease Ustilago maydis infects its host plant Zea mays by specialized infection structures, so-called appressoria, which are formed upon perception of chemical and physical stimuli on the leave surface. During the colonization process U. maydis secretes effector proteins that help to establish a biotrophic interaction. These effector proteins harbor an N-terminal hydrophobic secretion signal that targets them to the classical secretory pathway. In recent years, however, the existence of unconventionally secreted proteins has been uncovered which reach the extracellular space independently of the classical ER-Golgi system. In the present study the non-specific lipid transfer protein Scp2 (sterol carrier protein 2) of U. maydis was analyzed, which was identified as a putative candidate for unconventional protein secretion. Scp2 lacks a classical N-terminal signal peptide but exhibits a peroxisomal targeting signal (PTS1). A quantitative real-time PCR approach revealed that scp2 is up-regulated during early stages of plant colonization. Microscopic analyses demonstrated that the ability of scp2 deletion strains to form appressoria on artificial surfaces was significantly decreased. Furthermore, deletion of scp2 caused a virulence defect that appeared to result from a reduced efficiency of plant cuticle penetration. These defects are unlikely to result from deficiency in peroxisomal β- oxidation. In contrast to scp2 deletion strains, the infection of maize plants with a strain overexpressing scp2 under the cmu1 promoter triggered strong plant defense reactions. Two Scp2 paralogs were shown to localize in peroxisomes but deletion of the respective genes revealed no effect on U. maydis virulence. With the help of colony secretion assays it was demonstrated that small amounts of Scp2 are unconventionally secreted. The export of Scp2 via the classical ER-Golgi route, however, could not complement the virulence phenotype of the scp2 mutant strain, suggesting that the virulence defect is unconnected to the extracellular population of the protein. Surprisingly, peroxisomes and lipid droplets in the scp2 deletion strains displayed an altered distribution during filamentation on parafilm and on the plant surface. Based on these results, it is proposed that Scp2 affects appressorium development by influencing the distribution of peroxisomes and lipid droplets and thus constitutes a novel player in plant surface penetration.

Zusammenfassung:
Der Maisbeulenbranderreger Ustilago maydis infiziert seine Wirtspflanze Zea mays mithilfe spezialisierter Infektionsstrukturen, sogenannter Appressorien. Die Ausbildung von Appressorien wird durch die Wahrnehmung chemischer und physikalischer Stimuli auf der Blattoberfläche induziert. Im Verlauf der Kolonisierung sekretiert U. maydis Effektor-Proteine die zur Etablierung der biotrophen Interaktion beitragen. Diese Effektoren besitzen ein N-terminales hydrophobes Sekretionssignal, welches den Transport über den klassischen Sekretionsweg vermittelt. In den vergangenen Jahren wurde jedoch eine Vielzahl unkonventionell sekretierter Proteine identifiziert, die den Extrazellularraum unabhängig vom ER-Golgi-System erreichen. In der vorliegenden Studie wurde das unspezifische Lipid-Transfer Protein Scp2 (sterol carrier protein 2) von U. maydis analysiert, welches als potentieller Kandidat für unkonventionelle Proteinsekretion identifiziert wurde. Scp2 besitzt kein N-terminales Signalpeptid, verfügt jedoch über eine peroxisomale Zielsteuerungssequenz (PTS1). Quantitative Echtzeit-PCR zeigte eine Induktion der scp2-Expression in frühen Stadien der Pflanzen-Kolonisierung. Mikroskopische Untersuchungen ergaben, dass scp2 Deletionsstämme eine stark eingeschränkte Fähigkeit zur Appressorienbildung auf künstlichen Oberflächen aufweisen. Des Weiteren führte die Deletion von scp2 zu einem Virulenzdefekt, der auf eine reduzierte Penetration der pflanzlichen Cuticula zurückgeführt werden konnte. Die beobachteten Defekte wurden dabei vermutlich nicht durch eine gestörte peroxisomale β- Oxidation verursacht. Im Gegensatz zu scp2-Deletionsstämmen führte die Infektion von Maispflanzen mit Stämmen, die scp2 unter dem cmu1 Promoter überexprimierten, zu starken Pflanzenabwehrreaktionen. Zwei weitere Scp2-ähnliche Proteine konnten in Peroxisomen nachgewiesen werden, doch die Deletion der beiden Gene hatte keinen Einfluss auf die Virulenz von U. maydis. Mithilfe von Koloniesekretions-Analysen konnte bestätigt werden, dass Scp2 in geringen Mengen unkonventionell sekretiert wird. Der Export von Scp2 über die klassische ER-Golgi-Route führte jedoch nicht zu einer Komplementation des Virulenz-Phänotyps eines scp2-Deletionsstammes, was vermuten lässt, dass der Virulenzdefekt nicht in Verbindung mit der extrazellulären Population des Proteins steht. Erstaunlicherweise wiesen scp2- Deletionsstämme eine veränderte Verteilung von Peroxisomen und Lipidtropfen in Filamenten auf Parafilm und auf der Pflanzenoberfläche auf. Daher wird vermutet, dass Scp2 an der Appressorienbildung beteiligt ist, indem es die Verteilung von Peroxisomen und Lipidtropfen beeinflusst. Scp2 könnte somit einen neuen Faktor für die Penetration der Pflanzenoberfläche darstellen.

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