Proteomics of Aspergillus nidulans sexually differentiated cells Dissertation for the award of the degree “Doctor rerum naturalium” Division of Mathematics and Natural Sciences of the Georg-August University Göttingen submitted by Benedict Dirnberger from Vienna (Austria) Göttingen 2018 Es leuchtet! Seht! – Nun lässt sich wirklich hoffen, dass, wenn wir aus viel hundert Stoffen durch Mischung – denn auf Mischung kommt es an – den Menschenstoff gemächlich komponieren, in einen Kolben verlutieren und ihn gehörig kohobieren, so ist das Werk im Stillen abgetan. Faust II (Johann Wolfgang von Goethe) Thesis Committee Members: Member of the Thesis Committee: Professor Dr. Gerhard H. Braus (Reviewer I) Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August University Goettingen Member of the Thesis Committee: Professor Dr. Stefanie Pöggeler (Reviewer II) Department of Genetics of Eukaryotic Microorganisms, Institute of Microbiology and Genetics, Georg-August University Goettingen Member of the Thesis Committee: Dr. Oliver Valerius Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August University Goettingen Examination Board Members: Member of the Examination Board: Professor Dr. Heike Krebber Department of Molecular Genetics, Institute of Microbiology and Genetics, Georg- August University Goettingen Member of the Examination Board: Professor Dr. Kai Heimel Department of Microbial Cell Biology, Institute of Microbiology and Genetics, Georg- August University Goettingen Member of the Examination Board: Privatdozent Dr. Michael Hoppert Department of General Microbiology, Institute of Microbiology and Genetics, Georg- August University Goettingen Date of oral examination: 04.07.2018 I This doctoral thesis was performed in the group of Professor Dr. Gerhard H. Braus. Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August University Goettingen. Affirmation I declare that my doctoral thesis was written on my own having used only the listed resources and tools. Erklärung Die vorgelegte Arbeit wurde von mir selbständig angefertigt und nur die angegebenen Hilfsmittel wurden benutzt. Alle Stellen, die dem Wortlaut oder dem Sinne nach anderen Werken entnommen wurden, sind durch Angabe der Quelle kenntlich gemacht worden Göttingen am 14.05.2018 Benedict Dirnberger II Table of contents Summary ................................................................................................................... 1 Zusammenfassung ................................................................................................... 2 1. Introduction .......................................................................................................... 4 1.1. The genus Aspergillus ............................................................................................. 4 1.1.1. Aspergillus section Nidulantes ........................................................................................ 4 1.1.2. Aspergillus nidulans: A model organism for fungal development and secondary metabolism .................................................................................................................................... 5 1.1.3. Economic impact of Aspergilli ......................................................................................... 6 1.2. Morphological aspects ............................................................................................. 7 1.2.1. Hyphae ................................................................................................................................ 7 1.2.2. Hülle cells ........................................................................................................................... 7 1.2.3. Cleistothecia ....................................................................................................................... 9 1.2.4. Conidiophores .................................................................................................................. 10 1.3. Factors influencing fungal development .............................................................. 11 1.3.1. Environmental factors ..................................................................................................... 11 1.3.1.1. Light favors asexual differentiation ........................................................................ 13 1.3.1.2. Sexual differentiation is promoted by darkness .................................................. 13 1.3.1.3. Nutrients .................................................................................................................... 14 1.3.2. Endogenous factors ........................................................................................................ 14 1.3.2.1. Primary metabolism ................................................................................................. 15 1.3.2.2. Secondary metabolism............................................................................................ 16 1.4. Specialized metabolism in fungal development ................................................... 17 1.4.1. The monodictyphenone (mdp) / xanthone (xpt) secondary metabolite gene clusters ......................................................................................................................................... 17 1.4.1.1. Monodictyphenone is a precursor for the synthesis of xanthones ................... 18 1.4.2. LaeA as a factor that coordinates fungal development and secondary metabolism ...................................................................................................................................................... 21 1.4.2.1. LaeA methyltransferase .......................................................................................... 21 1.4.2.2. LaeA methyltransferase promotes Hülle cell formation ..................................... 22 1.4.2.3. LaeA regulates the monodictyphenone (mdp) and other secondary metabolite gene clusters ........................................................................................................................... 24 1.4.3. Relationship between fungal development and secondary metabolism ................. 25 III 1.4.3.1. Secondary metabolites that activate sporulation ................................................ 25 1.4.3.2. Pigments ................................................................................................................... 25 1.4.3.3. Secondary metabolites to protect the fungus against fungivory and other environmental threats ............................................................................................................ 26 1.5. Aim of this work ...................................................................................................... 27 2. Materials and methods ...................................................................................... 28 2.1. Materials .................................................................................................................. 28 2.1.1. Strains ............................................................................................................................... 28 2.1.2. Plasmids ........................................................................................................................... 29 2.1.3. Primers .............................................................................................................................. 29 2.1.4. Chemicals and equipment ............................................................................................. 31 2.1.5. Solutions and growth media .......................................................................................... 35 2.1.5.1. Solutions .................................................................................................................... 35 2.1.5.2. Growth media ........................................................................................................... 36 2.2. Methods ................................................................................................................... 37 2.2.1. Cultivation of Aspergillus nidulans ................................................................................ 37 2.2.1.1. Hülle cells from solid agar plate: cleistothecia-rolling technique ...................... 37 2.2.1.2. Hülle cells from submerged cultures ..................................................................... 39 2.2.2. DNA methods ................................................................................................................... 40 2.2.2.1. Genetic transformation procedure ......................................................................... 40 2.2.2.2. Plasmid DNA isolation from Escherichia coli ....................................................... 40 2.2.2.3. Aspergillus nidulans DNA extraction ..................................................................... 41 2.2.2.4. Ligation of DNA fragments ..................................................................................... 41 2.2.2.5. PCR (Polymerase chain reactions) ....................................................................... 42 2.2.2.6. Gelelectrophoresis of DNA ..................................................................................... 42 2.2.2.7. Purification of amplified DNA ................................................................................. 43 2.2.2.8.