Inhalt

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   Index
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   Abbreviations
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  1 Introduction
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  1.1 Oxygen toxins: the price of an aerobic lifestyle
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   1.1.1 The toxicity of oxygen
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   1.1.2 Sources of active oxygen
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   1.1.3 Survival as an aerobe
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   1.1.4 A closer look at two AOS scavenging proteins: Cu,ZN SOD and catalse
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  1.2 Harnessing the evil: AOS in plant pathogenesis
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   1.2.1 Early response to attack determines compatibility of interaction
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   1.2.2 Early response AOS production: the oxidative burst
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   1.2.3 Functions of the oxidative burst
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   1.3 Claviceps purpurea
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   1.3.1 Pathogenesis on rye
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   1.3.2 Ergot alkaloids of sclerotia
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   1.3.3 The molecular biology of parasitic growth
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  2 Experimental Procedures
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  2.1 Strains
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   2.1.1 E. coli
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   2.1.2 Claviceps
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   2.1.3 Rye
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  2.2 Culture conditions
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   2.2.1 E. coli
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   2.2.2 Claviceps
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   2.2.3 Rye
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  2.3 Vectors, libraries and primers
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   2.3.1 Vectors
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   2.3.2 Libraries
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   2.3.3 Primers
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  2.4 General procedures in molecular genetics
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   2.4.1 Isolation of DNA and RNA
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   2.4.2 Restriction, precipitation and washing of DNA
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   2.4.3 Gel electrophoresis of DNA and RNA
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   2.4.4 Isolation of DNA from agarose gels
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   2.4.5 Hybridisation of DNA and RNA with α-32P-dCTP-labelled probes
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   2.4.6 Analysis of DNA and RNA on membranes
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   2.4.7 Excision of phagemid vectors from lambda-ZAP vectors
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   2.4.8 PCR and RT-PCR
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   2.4.9 Cloning of DNA fragments
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   2.4.10 DNA sequencing
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  2.5 Creation and differential screening of a copper-induced cDNA-library
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   2.5.1 Creation of a copper-induced cDNA library
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   2.5.2 Differential cDNA screening
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  2.6 Induction of mycelia for northern and protein analysis
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   2.6.1 Induction with copper and iron
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   2.6.2 Induction with AOS
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   2.6.3 Induction with polyols
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  2.7 Protein-biochemical methods
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   2.7.1 Extraction of proteins from C. purpurea
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   2.7.2 Determination of protein concentration in extracts
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   2.7.3 Isoelectric focussing (IEF) gel electrophoresis
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   2.7.4 Silver staining of protein gels
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   2.7.5 Zymogram techniques
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   2.7.6 Western blotting
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   2.7.7 Determination of mycelial contamination of cell wall extracts with G6PDH
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  2.8 Generation and analysis of mutant strains
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   2.8.1 Transformation of C. purpurea strain 20-1 and derivatives
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   2.8.2 Cultivation and anlysis of C. purpurea transformants
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   2.8.3 Pathogenicity assays
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   2.8.4 Testing paraquat sensitivity of wild type and mutant strains
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   2.9 Computer-based analysis of DNA and protein sequences
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  3 Results
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  3.1 Cloning and analysis of a cell-wall associated Cu,Zn SOD in Claviceps purpurea
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   3.1.1 Identification of a CfSOD1-homologue in Claviceps purpurea
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   3.1.2 Analysis and localisation of SOD activity in axenic culture
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   3.1.3 Isolation and sequencing of a Cu, Zn SOD from C. purpurea
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   3.1.4 Expression analysis of cpsod1
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   3.1.5 Creation of mutants lacking cpsod1 for functional analysis
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   3.1.6 Analysis of Δcpsod1 phenotypes in axenic and parasitic culture
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  3.2 Characterisation and functional analysis of a putative catalase gene
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   3.2.1 Sequencing of cpcat2
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   3.2.2 Analysis of the cpcat2-derived protein and promotor region
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   3.2.3 Southern analysis of cpcat2
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   3.2.4 Expression analysis of cpcat2
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   3.2.5 Targeted inactivation of cpcat2
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   3.2.6 Pathogenicity assays with Δcpcat2
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  3.3 Searching for new genes involved in AOS detoxification: Differential cDNA screening with and without copper
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   3.3.1 Creation of a copper-induced cDNA library
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   3.3.2 Differential screening of the copper-induced cDNA library
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   3.3.3 Further characterisation of some of the clones which emerged in the differential screening
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  4 Discussion
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   4.1 The Cu,Zn SOD of C. purpurea
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   4.1.1 Localistaion of CpSOD1
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   4.1.2 Regulation of cpsod1
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   4.1.3 The importance of cpsod1 in axenic culture
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   4.1.4 Role of cpsod1 during pathogenesis
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   4.2 Yet more catalases: role in parasitic growth and biosynthesis of ergot alkaloids
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   4.2.1 The putative protein of cpcat2
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   4.2.2 Significance of cpcat2 for axenic and parasitic growth
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   4.2.3 Cpcat2 and the biosynthesis of ergot alkaloids
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   4.2.4 Conclusions: cpcat2
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   4.3 From metals to the oxidative stress response
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   4.3.1 Implications of the transciptional response to copper, iron and H2O2
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   4.3.2 Preliminary analysis of selected genes isolated in the course of differential cDNA screening
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   4.3.3 Could the response to metals and metal-mediated oxidative stress be relevant for pathogenicity?
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   4.4 Conclusions: AOS and beyond
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   5 Summary
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   6 References
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   App. A: Genomic Sequences
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   App. B: cDNA Sequences
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   App. C: Chemicals
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   App. D: Media
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   Acknowledgements
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   Curriculum Vitae