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Prof. Dr. Barbara Reinhold-Hurek Tag des öffentlichen Kolloquiums: 24.05.2013 Table of contents Page 1 Table of contents Table of contents.............................................................................1 I Zusammenfassung.................................................................6 II Summary.................................................................................9 1 Introduction ............................................................................. 11 1.1 Bicoloured deceiver (Laccaria bicolor) as model organism ............................... 11 1.1.1 Lifecycle of Basidiomycota ................................................................................................ 13 1.1.1.1 The tetrapolar mating system of Laccaria ......................................................... 15 1.1.2 Laccaria bicolor - an ectomycorrhiza forming model fungus ............................................. 17 1.1.3 Carbohydrate acquisition in ectomycorrhizal fungi ............................................................ 18 1.2 Carbohydrates in fungi ...................................................................................... 19 1.2.1 Sugar phosphorylation....................................................................................................... 19 1.2.2 Glycogen and trehalose in fungi ........................................................................................ 20 1.2.2.1 Glycogen metabolism ........................................................................................ 21 1.2.2.2 Trehalose metabolism ....................................................................................... 22 1.2.2.2.1 Trehalose biosynthesis by the TPS-complex ............................................... 22 1.2.2.2.2 Trehalose metabolism by trehalose phosphorylase ..................................... 24 1.2.2.2.3 Degradation of trehalose by trehalases ........................................................ 24 1.2.3 Carbohydrates as fungal storage compounds .................................................................. 25 1.2.3.1 Trehalose as carbon store in ectomycorrhizal symbiosis ................................. 26 1.2.4 Function of trehalose in fungi in response to abiotic stress .............................................. 26 1.2.5 Regulation of fungal carbohydrate metabolism ................................................................. 27 1.3 RNA interference as a tool to manipulate gene expression in Laccaria ............ 29 1.4 Aim of the thesis ................................................................................................ 30 2 Materials and Methods ........................................................... 32 2.1 Bioinformatics .................................................................................................... 32 2.1.1 Programs and databases .................................................................................................. 32 2.1.2 Online sources ................................................................................................................... 32 2.1.3 Identification of genes involved in trehalose metabolism .................................................. 33 Table of contents Page 2 2.2 Statistical analysis ............................................................................................. 34 2.3 Organisms and plasmids ................................................................................... 34 2.3.1 Organisms ......................................................................................................................... 34 2.3.2 Plasmids .......................................................................................................................... 34 2.3.2.1 E. coli cloning vectors ....................................................................................... 34 2.3.2.2 The fungal transformation vector pBGgHg ....................................................... 35 2.4 Culture of organisms ......................................................................................... 36 2.4.1 Bacteria .......................................................................................................................... 36 2.4.2 Laccaria bicolor ................................................................................................................. 37 2.5 Molecular biological methods ............................................................................ 37 2.5.1 Isolation of nucleic acids.................................................................................................... 37 2.5.1.1 Isolation of total RNA ........................................................................................ 37 2.5.1.2 Isolation of genomic DNA .................................................................................. 38 2.5.2 Amplification of DNA fragments by PCR ........................................................................... 39 2.5.3 Gel-electrophoresis of nucleic acids .................................................................................. 39 2.5.3.1 RNA ................................................................................................................... 39 2.5.3.2 DNA ................................................................................................................... 40 2.5.3.3 Visualisation of DNA/RNA ................................................................................. 40 2.5.4 Cloning of DNA fragments ................................................................................................. 40 2.5.4.1 DNA digestion ................................................................................................... 40 2.5.4.2 Isolation of DNA fragments from agarose gels ................................................. 40 2.5.4.3 Ligation of DNA fragments into plasmids .......................................................... 41 2.5.4.4 Plasmid preparation from Escherichia coli ........................................................ 41 2.5.4.5 Sequencing of DNA ........................................................................................... 42 2.5.4.6 PCR amplification of genes of interest .............................................................. 42 2.5.4.7 PCR amplification of genomic DNA fragments and introduction of novel restriction enzyme digestion sites ............................................................................................... 43 2.5.5 Quantification of gene expression ..................................................................................... 44 2.5.5.1 Isolation of total RNA ........................................................................................ 44 2.5.5.2 cDNA synthesis ................................................................................................. 45 2.5.5.3 Quantitative RT-PCR (qPCR) ........................................................................... 45 2.5.5.3.1 Primer selection for qPCR ............................................................................ 45 2.5.5.3.2 Quantification of gene expression using 18S rRNA as external standard.... 46 2.5.6 Transformation of Escherichia coli .................................................................................... 47 2.5.6.1 Generation of transformation competent E. coli ................................................ 47 2.5.6.2 Transformation of competent E. coli ................................................................. 47 2.5.7 Transformation of Agrobacterium tumefaciens ................................................................. 48 Table of contents Page 3 2.5.7.1 Generation of transformation competent A. tumefaciens ................................. 48 2.5.7.2 Transformation of competent A. tumefaciens ................................................... 48 2.5.8 Transformation of Laccaria bicolor .................................................................................... 48 2.5.9 Fungal growth assays ........................................................................................................ 51 2.5.10 Formation of dikaryotic mycelia from monokaryotic Laccaria strains ............... 52 2.5.10.1 Microscopic analysis of fused fungal strains ..................................................... 53 3 Results ..................................................................................... 54 3.1 Selection of genes for RNAi suppression .......................................................... 54 3.2 Identification of potential MSN-like factors ........................................................ 58 3.3 Growth behaviour of Laccaria bicolor wild type mycelia .................................... 60 3.4 Construction of fungal transformation vectors ................................................... 65 3.4.1 Isolation of genomic DNA from Laccaria bicolor ............................................................... 65 3.4.1.1 Genomic DNA fragments as template for construction of inverted repeats ...... 65 3.4.2 Integration of inverted