Johannes Gutenberg-Universität Mainz Institut für Biotechnologie und Wirkstoff-Forschung IDENTIFICATION AND CHARACTERIZATION OF NOVEL FACTORS INVOLVED IN DIMORPHISM AND PATHOGENICITY OF FUNGAL WHEAT PATHOGEN ZYMOSEPTORIA TRITICI Dissertation to attain the academic degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology, Johannes Gutenberg University Mainz submitted by Alexander Yemelin born on October 19th, 1986 in Nikolaev (Ukraine) Mainz, April 2017 Johannes Gutenberg-Universität Mainz Institut für Biotechnologie und Wirkstoff-Forschung IDENTIFIZIERUNG UND CHARAKTERISIERUNG VON NEUEN DIMORPHISMUS- UND PATHOGENITÄTS-RELEVANTEN FAKTOREN IN ZYMOSEPTORIA TRITICI Dissertation zur Erlangung des akademischen Grades „Doktor der Naturwissenschaften“ (doctor rerum naturalium, Dr.rer.nat.) am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz vorgelegt von Alexander Yemelin geb. am 19.10.1986 in Nikolaev (Ukraine) Mainz, April 2017 | 2 Hiermit erkläre ich, Alexander Yemelin, die vorliegende Arbeit ohne fremde Hilfe und nur unter Verwendung der angegebenen Hilfsmittel und Quellen angefertigt zu haben. Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient. Kaiserslautern, April 2017 X | 3 Gewidmet meiner Familie INDEX | 4 Table of contents Summary .............................................................................................................................................. 7 Zusammenfassung ............................................................................................................................ 9 List of Abbreviations .................................................................................................................... 11 List of tables ..................................................................................................................................... 13 List of figures ................................................................................................................................... 13 1. Introduction ........................................................................................................................ 16 1.1 General Introduction ...................................................................................................................... 16 1.2 Septoria leaf blotch disease ......................................................................................................... 17 1.3 Infection cycle of Zymoseptoria tritici and some cytological aspects regarding infection-related morphogenesis ....................................................................................................... 18 1.4 Zymoseptoria tritici as a model organism .............................................................................. 22 1.5 Known virulence factors in Zymoseptoria tritici and current studies ......................... 24 1.6 Dimorphism as pathogenicity-relevant process ................................................................. 28 1.7 Future challenges and actuality of the current study ........................................................ 32 1.8 Scope of the thesis ........................................................................................................................... 33 2. Material and methods ...................................................................................................... 35 2.1 Material ............................................................................................................................................... 35 2.1.1 Chemicals and media components .................................................................................. 35 2.1.2 Antibodies and enzymes .................................................................................................. 36 2.1.3 Reaction kits .................................................................................................................... 36 2.1.4 Solutions and buffers ....................................................................................................... 36 2.1.5 Culture media .................................................................................................................. 38 2.1.6 Organisms used in this study ........................................................................................... 40 2.1.7 Vectors used in this study ................................................................................................ 42 2.1.8 Oligonucleotides used in this study ................................................................................. 44 2.2 Molecular biological methods ...................................................................................................... 45 2.2.1 Isolation and preparation of nucleic acids ...................................................................... 45 2.2.2 Polymerase chain reaction (PCR) ..................................................................................... 47 2.2.3 Restriction of DNA and gel electrophoresis .................................................................... 50 2.2.4 Ligation and DNA cloning methods ................................................................................. 52 2.2.5 Transformation ................................................................................................................ 55 2.2.6 Southern Blot ................................................................................................................... 58 I N D E X | 5 2.2.7 Transcriptome analysis using RNA-Seq ........................................................................... 60 2.2.8 qRT-PCR analysis .............................................................................................................. 60 2.3 Methods for phenotypic characterization of Zymoseptoria tritici strains .................. 61 2.3.1 Conidiogenesis ................................................................................................................. 61 2.3.2 Biomass production ......................................................................................................... 61 2.3.3 Examination of vegetative growth behaviour on different media .................................. 61 2.3.4 Spore germination test .................................................................................................... 62 2.3.5 Pathogenicity assays ........................................................................................................ 62 2.3.6 Micro-/Macroscopic analysis and photographic documentation of results ................... 62 2.3.7 Spore Lysis Test................................................................................................................ 63 2.4 Bioinformatic analysis and data mining .................................................................................. 63 2.4.1 RNA-Seq data analysis considering expression of known pathogenicity related genes . 63 2.4.2 Multiple sequence alignment and phylogenetic analysis of Zymoseptoria tritici protein sequences ................................................................................................................................. 63 2.4.3 Gene ontology and gene enrichment analysis ................................................................ 64 2.4.4 Homology modelling and structure comparison ............................................................. 64 2.4.5 RNA-Seq data processing ................................................................................................. 65 2.4.6 Retrieval of target sequences and basic analysis ............................................................ 66 3. Results ................................................................................................................................... 67 3.1 Generation of a transformant library by Agrobacterium tumefaciens mediated transformation.......................................................................................................................... 67 3.2 Analysis of the frequency and mode of the T-DNA integration in the genome of Zymoseptoria tritici ................................................................................................................... 68 3.3 Identification and characterization of "dimorphic switch" - deficient transformants ....... 70 3.4 Molecular identification of T-DNA tagged gene loci .......................................................... 71 3.5 Transcriptome analysis of selected strains during the dimorphic transition on artificial medium ..................................................................................................................................... 87 3.6 Extended phenotypic analysis of the generated mutants ................................................ 101 3.7 Localization studies using fluorescence microscopy ........................................................ 118 3.8 In planta gene expression analysis of the candidate genes ............................................. 123 3.9 In planta expression of chloroperoxidase-encoding genes derived from RNA-Seq ......... 124 3.10 Identification of novel gene ZtPTF1 encoding an essential transcription factor ............ 126 3.11 In silico analysis of predictive binding sites of ZtPtf1p and deduction of potential function..................................................................................................................................