Hellweg, Matthias: Molecular biological and biochemical studies of proteolytic enzymes of the cereal pathogen Fusarium graminearum. 2003
Inhalt
- Contents
- Abbreviations
- Introduction
- Introduction to plant disease resistance mechanisms
- The structure of plant cell walls
- Cell wall degrading enzymes
- General classification of proteases
- Fusarium graminearum
- Objectives of the present work
- Materials and Methods
- Materials
- Methods
- Cultivation of Fusarium graminearum in submerged culture
- CM-medium, minimal medium and protease induction medium
- Preculture
- Induction of conidia
- Induction of protease secretion
- Growth rate and kinetics of protease secretion
- Kinetics of initial protease secretion – Mass inoculation
- Variations in protein derived nitrogen levels
- Variations in carbohydrate levels
- Isolated wheat cell wall material as substrate
- Cultivation of Fusarium graminearum on agar plates
- In planta cultivation – Detached leaf petri dish assay
- Protein biochemical methods
- Photometric assay for protease activity
- pH-profile and temperature optimum
- Inhibitor studies
- Cation exchange perfusion chromatography
- SDS polyacrylamide gel elecrophoresis (SDS-PAGE)
- 'Slow' Coomassie staining of acrylamide gels
- Silver staining of acrylamide gels
- Standard zymography
- Blotting zymography
- Molecular biology
- Isolation of fungal DNA
- Standard PCR
- Touch-down PCR
- Inverse PCR
- TAIL PCR
- Standard agarose gel electrophoresis
- Purification of PCR fragments
- Cloning of PCR fragments and colony PCR
- Plasmid purification and sequencing of cloned PCR fragments
- Isolation of fungal RNA
- cDNA production and RT-PCR
- Quantitative expression analysis
- Computer aided methods
- Results
- General characterization of fungal growth
- Optimization of the photometric protease assay
- Development of protease induction medium
- Characterization of growth in protease induction medium
- Growth on PI-agar compared to CM- and minimal-agar
- Submerged culture in PI-medium compared to CM- and minimal medium
- Kinetics of protease secretion
- Overview of protease secretion – daily samples
- Basic characterization of the protease activity induced in PI-medium
- Kinetics of initial protease secretion – Mass inoculation
- Variations in nitrogen and carbohydrate levels – Mass inoculation
- Growth in CM- and PI-medium – Mass inoculation
- pH of the medium during cultivation in PI- and pPI-medium with variations in carbon levels – Mass inoculation
- Protease activity and pH of the medium during growth in minimal medium containing wheat cell wall material – Mass inoculation
- SDS-PAGE of crude media samples
- Partial purification of a protease from PI-medium
- Cloning and sequencing of a protease gene
- Molecular characterization of the obtained gene
- Molecular characterization of the 5'-flanking region of prt1
- Cloning and sequencing of a cDNA fragment of prt1
- Qualitative expression studies
- Discussion
- Selection and evaluation of a suitable proteinacious substrate for protease induction
- Macroscopic and microscopic characterization of growth
- Protease induction - Time course, basic characterization of proteolytic activity and regulation by carbon and nitrogen availability
- Protease induction – Electrophoretic analysis of substrate digestion and identification of induced proteases
- Protease induction during cultivation with cell wall material?
- Prt1 – a gene encoding a subtilisin-like protease
- Comparative overview and outlook
- Abstract
- Zusammenfassung
- References
- Appendix
- Thank you
- Curriculum vitae