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Predicting Virulence Factors in Filamentous Fungi

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Predicting Virulence Factors in Filamentous Fungi TECHNISCHE UNIVERSITAT¨ MUNCHEN¨ Lehrstuhl f¨urGenomorientierte Bioinformatik Predicting virulence factors in filamentous fungi: Regulation and evolution of secondary metabolism gene clusters Christian Martin Konrad Sieber Vollst¨andigerAbdruck der von der Fakult¨atWissenschaftszentrum Weihenstephan f¨urErn¨ahrung,Landnutzung und Umwelt der Technischen Universit¨atM¨unchen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. W. Liebl Pr¨uferder Dissertation: 1. Univ.-Prof. Dr. H.-W. Mewes 2. Univ.-Prof. Dr. K. Jung, (Ludwig-Maximilians-Universit¨atM¨unchen) Die Dissertation wurde am 13.11.2014 bei der Technischen Universit¨atM¨unchen eingereicht und durch die Fakult¨at Wissenschaftszentrum Weihenstephan f¨ur Ern¨ahrung,Landnutzung und Umwelt am 20.01.2015 angenommen. i Abstract Pathogenic filamentous fungi constitute a health risk to humans and animals all over the world. Most of these fungi provide a diverse repertoire of bioactive small molecules like antibiotics and mycotoxins which play a key role in diseases but are also utilized as drugs and growth factors of plants. Especially Fusarium species are known to be involved in many plant diseases that lead to large agricultural and economic damage. Genes that encode enzymes of a secondary metabolism pathway usually are locally clustered on the chromosome. The rapidly increasing number of available fungal genomes enables comparative genomics studies for the identification of host specific virulence factors. Furthermore, large-scale genomic mining for gene clus- ters of novel bioactive compounds has become feasible. In this work the genome sequence of the rice pathogen Fusarium fujikuroi alongside with an extensive comparative analysis to Fusarium species with di- verse host specificities is presented. To better understand the regulation of sec- ondary metabolism gene clusters and virulence associated genes, transcriptomic, proteomic and epigenetic measurements taken under virulence inducing conditions are integrated. A significant genome wide correlation between gene expression and protein abundance could be determined that is even higher when focusing on secondary metabolism genes alone. Epigenetic regulation of gene clusters by dif- ferential chromatin acetylation at different physiological conditions is discovered. Furthermore, influence of the histone deacetylase Hda1 and the global regulator Sge1 on secondary metabolism could be shown by analyzing experimental data of deletion mutants. Genome shaping and plasticity is heavily influenced by transposable elements. In silico prediction of repetitive interspersed sequences revealed two highly abun- dant repeat families that occur exclusively in two distinct phylogenetic branches inside the Fusarium phylum. Extensive evidence for the repeat induced point mutation (RIP) mechanism that inactivates transposable elements in F. fujikuroi is presented. Expression data analysis revealed an inverse correlation between the amount of RIP-specific point mutations and expression intensities of transposable elements. ii Recent genome analysis studies on filamentous fungi revealed a diversity of pu- tative secondary metabolism genes. At the same time only a small fraction of syn- thesized compounds is known. To determine the amount of secondary metabolism genes that is involved in biosynthetic pathways, putative secondary metabolism gene clusters based on statistical overrepresentation of secondary metabolism re- lated functions are predicted. Additionally, evidence in terms of co-expression by available experimental data and computed conserved promoter motifs is presented. In collaboration with experimental biologists it was possible to identify previously unknown compounds and verify the regulatory function of overrepresented pro- moter motifs. The presence of gene clusters between even closely related fungi differs consid- erably in most of the cases and the evolutionary origin of many biosynthetic genes is yet unknown. In an extensive ortholog analysis of predicted clusters of 22 Fusar- ium species different evolutionary processes are observed, such as individual gene loss and horizontal gene transfer, that are possibly responsible for the non-uniform phylogenetic distribution of gene clusters. In addition shuffling of pathway genes between gene clusters, alternative tailoring of a common signature enzyme as well as duplication and divergence of clusters is observed. The results of this work not only identify species-specific virulence related fea- tures and give insight into the regulation of secondary metabolism, but also reveal evolutionary processes that shed light on the origin of secondary metabolism gene clusters. iii Zusammenfassung Pathogene filament¨osePilze stellen ein Gesundheitsrisiko f¨urMensch und Tier auf der ganzen Welt dar. Dazu tr¨agtdas vielf¨altigeRepertoire an kleinen bioaktiven Molek¨ulenbei, das von vielen dieser Pilze synthetisiert wird. Die sogenannten Sekund¨armetabolite bieten ein Selektionsvorteil f¨urdie Organismen, sind aber nicht essentiell f¨urderen Entwicklung. Beispiele sind Antibiotika, Phytohormone und Pilzgifte, die eine wichtige Rolle bei Krankheiten spielen oder als Medikamente in der Medizin sowie als Wachstumsfaktoren f¨urPflanzen Verwendung finden. Vor allem Pilze der Gat- tung Fusarium sind in vielen Pflanzenkrankheiten involviert, die zu großem land- wirtschaftlichen und somit auch wirtschaftlichen Schaden f¨uhren. Gene, die Enzyme eines Sekunde¨armetabolismus Pathways kodieren, sind in der Regel in direkter chromosomaler Nachbarschaft zueinander in Form von Genclustern angeordnet. Die schnell wachsende Anzahl an verf¨ugbaren Pilz-Genomsequenzen erm¨oglicht neue, vergleichende genomische Studien zur Identifizierung von wirtsspezifischen Virulenzfaktoren. Dar¨uber hinaus werden genomweite, großformatige Vorhersagen von Gencluster m¨oglich, die noch un- bekannte, bioaktive Stoffe synthetisieren. In dieser Arbeit wird die Genomsequenz des Reispathogens Fusarium fujikuroi zusammen mit einer umfangreichen vergleichenden Analyse mehrerer Fusarium- Arten mit verschiedenen Host-Pr¨aferenzenpr¨asentiert. Um die Regulation von Sekund¨armetabolismus Genclustern und Virulenz-Genen besser zu verstehen, wer- den Daten aus Transkriptomik-, Proteomik- und Epigenetik- Experimenten zu Virulenz induzierenden Bedingungen integriert. Eine genomweite Korrelation zwischen Genexpressionsintensit¨at und quantifizierten Proteinen konnte bestimmt werden, die besonders stark bei Sekunde¨armetabolismusgenen zu beobachten ist. Des weiteren wird eine epigenetische Regulation der Gencluster gezeigt, die auf unterschiedliche Chromatin-Acetylierung unter verschiedenen physiologischen Bedingungen zur¨uckgef¨uhrt werden konnte. Dar¨uber hinaus konnte ein regulatorischer Einfluss der Histon-Deacetylase Hda1 und des globalen Regulators Sge1 auf den Sekund¨armetabolismus durch experimentelle Daten von Deletionsmutan- iv ten gezeigt werden. Transposons und Repeats beeinflussen die Struktur und Plastizit¨at eines Genoms. Die in silico Vorhersage von repetitiven Sequenzen resultierte in zwei sehr pr¨asenten Repeat-Familien, die ausschließlich in zwei monophyletischen Grup- pen innerhalb der Fusarium Phylogenie vorkommen. Hinweise f¨urden aktiven Abwehrmechanismus RIP (repeat-induced point mutation), welcher Transposons inaktiviert, konnten in F. fujikuroi gefunden werden. Eine Expressionsdaten- Analyse zeigte eine inverse Korrelation zwischen der Anzahl an RIP-spezifischen Punktmutationen und der Transposon Expressions Intensit¨at. In aktuellen Analysen von vollst¨andigsequenzierten Pilzgenomen wurde eine Vielzahl von vermeintlichen Sekund¨armetabolismusgenen vorhergesagt. Gleich- zeitig ist aber nur ein kleiner Teil an synthetisierten Metaboliten bekannt. Um putative Sekunde¨armetabolismusgene zu bestimmen, die Teil eines Biosynthese- Pathways darstellen, wurde eine Gencluster Vorhersage durchgef¨uhrt,welche auf statistischer Uberrerpesentation¨ von Sekund¨armetabolismus Funktionen beruht. Zus¨atzlich wurden Evidenzen zur Coexpression, in Form von experimentellen Daten und konservierten Promotor-Motiven, mit einbezogen. In Kooperation mit experimentellen Biologen konnten bisher unbekannte Metabolite identifiziert und die regulatorische Funktion ¨uberrepr¨asentierter Promotor Motive best¨atigt werden. Selbst zwischen nah verwandten Species unterscheidet sich die Pr¨asenz von Genclustern im Genomvergleich teilweise erheblich, wobei die Herkunft vieler Biosynthesegene unbekannt ist. In einer umfangreichen Analyse der vorherge- sagten Orthologen Cluster von 22 Fusarium-Arten wurden verschiedene putative evolution¨areAbl¨aufeidentifiziert, die m¨oglicherweise verantwortlich f¨urdie un- gleichm¨aßigephylogenetische Verteilung der Gencluster sind. Diese umfassen den individuellen Verlust von Genclustern, aber auch horizontalen Gentrans- fer. Zus¨atzlich konnte die Fusion von Pathwaygenen zweier Gencluster, unter- schiedliche Tailoring Enzyme um ein gemeinsames Signature-Enzym und eine Du- plikation mit Divergenz eines Clusters beobachtet werden. Die Ergebnisse dieser Arbeit identifizieren nicht nur artspezifische Virulen- zfaktoren und geben Einblick in die Sekunde¨armetabolismus-Regulation, son- dern zeigen auch evolution¨are Prozesse, die Licht auf die Entstehung der Sekund¨armetabolismus Gencluster werfen. v Acknowledgements A lot of people accompanied and supported me during the last years. First, I would like to thank my supervisor Prof. Dr. Hans-Werner Mewes for his support, advice
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