Appressorienbildung von Ustilago maydis auf hydrophoben Oberflächen: Regulation durch Membranproteine

Ustilago maydis ist der Erreger des Maisbeulenbrandes. Die pathogene Entwicklung wird durch Fusion kompatibler Zellen und der Bildung eines dikaryotischen Filaments initiiert. Auf der Pflanzenoberfläche bildet U. maydis Appressorien aus, die das Eindringen des Pilzes in die Pflanze ermöglichen. Die Appressorienbildung wird durch die Hydrophobizität der Blattoberfläche und Cutin Monomere stimuliert. Die pathogene Entwicklung von U. maydis wird durch eine konservierte, zum FG (filamentous growth)-Signalweg in Saccharomyces cerevisiae homologe, MAP-Kinase Kaskade gesteuert. In Hefe agieren zwei Plasmamembranproteine, Sho1p und Msb2p, an der Spitze des MAP-Kinase Signalwegs. In dieser Arbeit wurden Sho1- und Msb2-verwandte Proteine in U. maydis untersucht. Es konnte gezeigt werden, dass Sho1 und Msb2 essentiell für die Virulenz von U. maydis sind. Genetische Analysen ergaben, dass Sho1 und Msb2 oberhalb der pathogenitätsrelevanten MAP-Kinase Kaskade agieren. Für Sho1 wurde zudem gezeigt, dass es die MAP-Kinase Kpp6 destabilisiert, indem es direkt mit der N-terminalen Domäne von Kpp6 interagiert. Dies dient wahrscheinlich der Feinregulation der Kpp6 Aktivität. Es konnte nachgewiesen werden, dass Sho1 und Msb2 spezifisch die Appressorienbildung regulieren, aber für die Zellfusion sowie filamentöses Wachstum nicht benötigt werden. Ferner waren sho1 und msb2 Mutanten während der morphologischen Differenzierung auf hydrophoben Oberflächen eingeschränkt, während die Reaktion auf Cutin Monomere nicht beeinträchtigt war. Dies deutet darauf hin, dass Sho1 und Msb2, die beide in der Plasmamembran lokalisieren, bei der Perzeption von Oberflächen beteiligt sind. Msb2, welches zur Familie der Transmembranmucine gehört, wird in ein zelluläres und ein extrazelluläres Fragment prozessiert. Beide Proteindomänen werden für die Funktion von Msb2 benötigt. Da die hoch glycosylierte extrazelluläre Domäne an die Umgebung abgegeben wird, werden zusätzliche Funktionen von Msb2 in der extrazellulären Matrix vermutet, wie z.B. die Vermittlung der Adhäsion zwischen Filamenten und Oberflächen. Transkriptomanalysen unter Appressorien-induzierenden in vitro Bedingungen zeigten, dass Sho1 und Msb2 notwendig für die Expression von potentiell sekretierten Zellwand-degradierenden Enzymen sind. Ferner wurden sekretierte Effektoren, die essentiell für die biotrophe Interaktion von U. maydis mit seiner Wirtspflanze sind, in Abhängigkeit von Sho1 und Msb2 exprimiert. Dies zeigt, dass U. maydis durch Sho1 und Msb2 auf die biotrophe Entwicklung vorbereitet wird, noch während die Hyphen auf der Pflanzenoberfläche wachsen. Da Sho1 und Msb2 in phytopathogenen Pilzen konservierte Proteine sind, könnten sie generelle Virulenzfaktoren darstellen

The British community in occupied Cairo, 1882-1922.

Though officially ruled by the Ottoman Entire, Egypt was under British occupation between 1882 and 1922. Most studies about the British in Egypt during this time focus on the political and administrative activities of British officials based on government documents or their memoirs and biographies. This thesis focuses on various aspects of the British community in Cairo based on sources that have been previously overlooked such as census records, certain private papers, and business, newspaper, military and missionary archives. At the outset, this discussion introduces demographic data on the British community to establish its size, residential location and context among other foreign communities and the wider Egyptian society. Then it deliberates on the occasional ambiguous boundaries that identified members of the community from non-members as well as the symbols and institutions that united the community. Ensuing chapters on the community's socio-occupational diversity and criminal activities suggest that the British community in Cairo was not homogeneous. The community consisted of not only law-abiding upper middle class officials but of an assortment of businessmen, missionaries, and working-class maids and labourers; some of whom were involved in crimes and misdemeanours. The analysis concludes by investigating the diversity of reactions of Cairo's Britons to the challenge of World War I and the subsequent revolutionary period of 1919-1922. Due to time and space constraints, the discussion concentrates on the British community in Cairo, since for the most part, more Britons resided in Cairo than Alexandria. However, where appropriate to the thesis' key themes, data on the British in Alexandria will be included

Septation of Infectious Hyphae Is Critical for Appressoria Formation and Virulence in the Smut Fungus Ustilago Maydis

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis

Multiple Plant Surface Signals are Sensed by Different Mechanisms in the Rice Blast Fungus for Appressorium Formation

Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance