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Interplay of Verticillium Signaling Genes Favoring Beneficial Or Detrimental Outcomes

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Interplay of Verticillium Signaling Genes Favoring Beneficial Or Detrimental Outcomes Interplay of Verticillium signaling genes favoring beneficial or detrimental outcomes in interactions with plant hosts Dissertation For the award of the degree “Doctor rerum naturalium” of the University of Goettingen within the doctoral program “Plant Responses To Eliminate Critical Threats“ of the Georg-August University School of Science (GAUSS) submitted by Jessica Starke from Celle May 2019 Thesis Committee and members of the Examination Board Referee: Prof. Dr. Gerhard H. Braus Department of Molecular Microbiology and Genetics University of Goettingen 2nd referee: apl. Prof. Dr. Kai Heimel Department of Molecular Microbiology and Genetics University of Goettingen 3rd referee: Prof. Ph.D. James W. Kronstad Michael Smith Laboratories University of British Columbia Vancouver Prof. Dr. Andrea Polle Department of Forest Botany and Tree Physiology University of Goettingen Prof. Dr. Petr Karlovsky Department of Molecular Phytopathology and Mycotoxin Research University of Goettingen Prof. Dr. Volker Lipka Department of Plant Cell Biology University of Goettingen Date of oral examination: July 22nd 2019 I Declaration of independence Herewith I declare that the dissertation entitled “Interplay of Verticillium signaling genes favoring beneficial or detrimental outcomes in interactions with plant hosts” was written on my own and independently without any other aids and sources than indicated. Jessica Starke Goettingen, 2019 II This work was accomplished in the group of Prof. Dr. Gerhard H. Braus, at the Department of Molecular Microbiology and Genetics at the Institute of Microbiology and Genetics, University of Goettingen. Parts of this work will be published in: Harting R*, Starke J*, Kusch H, Pöggeler S, Maurus I, Schlüter R, Landesfeind M, Bulla I, Nowrousian M, de Jonge R, Stahlhut G, Hoff K, Aßhauer KP, Thürmer A, Stanke M, Daniel R, Morgenstern B, Thomma BPHJ, Kronstad JW, Braus-Stromeyer SA, Braus GH (2019) A 20 kb Lineage Specific genomic region tames virulence in pathogenic allodiploid Verticillium longisporum. Manuscript in preparation. (* contributed equally) Starke J, Harting R, Maurus I, Bremenkamp R, Kronstad JW, Braus GH (2020) Unfolded protein response and scaffold independent pheromone MAP kinase signalling control Verticillium dahliae growth, development and plant pathogenesis. BioRxiv 941450. doi:10.1101/2020.02.10.941450. III IV Table of Contents Table of Contents Summary ..............................................................................................................................1 Zusammenfassung ..............................................................................................................2 1 INTRODUCTION ........................................................................................... 5 1.1 Verticillium spp. – colonizers of the plant´s vascular system ................................5 1.1.1 The ascomycete genus Verticillium ..................................................................5 1.1.2 Life cycle of Verticillium.....................................................................................6 1.2 Niche adaptation ....................................................................................................8 1.2.1 Verticillium dahliae Lineage Specific regions ...................................................9 1.2.2 Interspecific hybridization of Verticillium longisporum ....................................10 1.3 Mitogen-activated protein kinase signaling .........................................................12 1.3.1 Fus3 and Kss1 MAPK signaling in Saccharomyces cerevisiae .....................13 1.3.2 Scaffolds in Fus3/Kss1 MAPK signaling in filamentous ascomycetes ..........15 1.3.3 Fus3/Kss1 MAPK signaling in plant pathogenic fungi ....................................15 1.4 The unfolded protein response ............................................................................17 1.4.1 The unfolded protein response pathway in Saccharomyces cerevisiae ........17 1.4.2 The unfolded protein response pathway in pathogenic fungi ........................19 1.5 Oxylipins...............................................................................................................21 1.5.1 Oxylipins in fungal development .....................................................................21 1.5.2 Oxylipins in plant-fungus interactions .............................................................23 1.6 Aim of the study ...................................................................................................26 2 MATERIALS AND METHODS ....................................................................... 29 2.1 Organisms and their cultivation ...........................................................................31 2.1.1 Cultivation of bacteria .....................................................................................31 2.2.2 Cultivation of Verticillium strains .....................................................................31 2.2 Strains, plasmids and primers .............................................................................32 2.2.1 Bacterial strains ...............................................................................................32 2.2.2 Verticillium strains ...........................................................................................32 2.2.3 Plants ..............................................................................................................34 2.2.4 Primers, Plasmid, and strain constructions ....................................................34 2.2.4.1 Primers ............................................................................................................34 2.2.4.2 Plasmids ..........................................................................................................38 2.2.4.3 Plasmid and strain construction of V. longisporum strains ............................40 2.2.4.4 Plasmid and strain construction of V. dahliae strains ....................................41 2.3 Microbiological methods ......................................................................................46 2.3.1 Transformation of Escherichia coli .................................................................46 2.3.2 Transformation of Agrobacterium tumefaciens ..............................................46 2.3.3 Transformation of Verticillia ............................................................................46 2.3.4 Phenotypic analysis and stress tests..............................................................47 2.3.5 Growth quantification ......................................................................................47 2.3.6 Microsclerotia observation and quantification of melanization .......................47 2.3.7 Conidiospore quantification ............................................................................48 2.3.8 Localization assay ...........................................................................................48 2.3.9 Plant infection assays .....................................................................................48 V Table of Contents 2.3.9.1 Brassica napus infection assay with V. longisporum ..................................... 48 2.3.9.2 Solanum lycopersicum infection assay with V. dahliae .................................. 49 2.3.9.3 Disease rating criteria ..................................................................................... 49 2.3.9.4 Stem assay ..................................................................................................... 50 2.3.9.5 Arabidopsis thaliana root colonization assay ................................................. 50 2.4 Nucleic acid methods .......................................................................................... 51 2.4.1 Nucleic acid purification .................................................................................. 51 2.4.2 Polymerase chain reaction ............................................................................. 52 2.4.3 Agarose gel electrophoresis ........................................................................... 53 2.4.4 Abscence confirmation of the Vl43LS20kb region in V. longisporum Vl32 .... 53 2.4.5 Primer walking................................................................................................. 53 2.4.6 Complementary DNA amplification................................................................. 54 2.4.7 Quantitative reverse transcriptase PCR ......................................................... 54 2.4.8 Verification of intron-exon structures .............................................................. 55 2.4.9 Southern hybridization .................................................................................... 55 2.5 Protein methods ................................................................................................... 56 2.5.1 Protein extraction ............................................................................................ 56 2.5.2 Determination of protein concentration by Bradford assay ............................ 56 2.5.3 SDS-PAGE ...................................................................................................... 57 2.5.4 Immunoblot hybridization ................................................................................ 57 2.6 Sequence analyses ............................................................................................. 58 3 RESULTS ................................................................................................
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