Biological and chemical induction of systemic resistance in the barley powdery mildew pathosystem [Elektronische Ressource] / vorgelegt von Kate Morrissey

Prl20

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2006
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Biological and chemical induction of
systemic resistance in the barley powdery
mildew pathosystem











Inaugural-Dissertation

zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf







vorgelegt von

Kate Morrissey

aus Cirencester

November 2006 Aus dem Institut für Chemie und Dynamik der Geosphäre (ICG-III)
des Forschungszentrums Jülich







































Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf
Referent: Jun. Prof. Dr. I. Janzik
Koreferent: Prof. Dr. U. Schurr
Tag der mündlichen Prüfung: 25 Januar 2007

2 Contents
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Contents

Contents...................................................................................................................................... 1
Abstract 3
Zusammenfassung...................................................................................................................... 5
Abbreviations ............................................................................................................................. 7
1 Introduction ........................................................................................................................... 11
1.1 Induced resistance ..........................................................................................................11
1.2 Signalling mechanisms................................................................................................... 15
1.3 Chemical activators ........................................................................................................ 17
1.4 Biological inducers.18
1.5 Barley-powdery mildew interaction............................................................................... 19
1.6 Aims ............................................................................................................................... 20
2 Materials and Methods.. 21
2.1 Biological material.21
2.1.1 Plant......................................................................................................................... 21
2.1.2 Fungus ..................................................................................................................... 21
2.1.3 Bacteria.................................................................................................................... 21
2.1.4 Plasmids................................................................................................................... 22
2.2 Chemicals ....................................................................................................................... 23
2.2.1 Primer information .................................................................................................. 23
2.3 Equipment.... 24
2.4 Cultivation of biological material................................................................................... 24
2.4.1 Plant material........................................................................................................... 24
2.4.2 Fungal material........................................................................................................ 25
2.4.3 Bacterial material .................................................................................................... 25
2.5 Chemical pre-treatment 25
2.6 Bacterial pre-infiltration................................................................................................. 26
2.7 Fungal inoculation..........................................................................................................26
2.8 Harvesting of plant material........................................................................................... 27
2.9 Data analysis................................................................................................................... 27
2.10 Molecular biological methods...................................................................................... 27
2.10.1 RNA isolation 27
2.10.2 cDNA synthesis..................................................................................................... 28
2.10.3 PCR amplification of cDNA probes...................................................................... 28
2.10.4 Gel extraction ........................................................................................................ 29
2.10.5 Cloning and sequencing of PCR Products ............................................................ 29
2.10.6 DIG labelling of probes......................................................................................... 31
2.10.7 Northern analysis and hybridisation 32
2.11 Microarray analysis ...................................................................................................... 33
2.11.1 DNA array ............................................................................................................. 33
2.11.2 Microarray preparation.......................................................................................... 34
2.11.3 Fluorescent probes................................................................................................. 34
2.11.4 Hybridisation and scanning................................................................................... 35
2.12 In situ detection of reactive oxygen species (ROS) ..................................................... 36
2.13 HPLC analysis of soluble phenolics............................................................................. 37
2.14 Intercellular wash fluid (ICWF) 37
2.15 Protein determination ................................................................................................... 37
2.16 G6PDH activity assay .................................................................................................. 38
2.17 Protein precipitation ..................................................................................................... 38
1 Contents
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2.18 SDS-PAGE...................................................................................................................39
2.19 Coomassie blue staining............................................................................................... 39
2.20 2D electrophoresis........................................................................................................ 39
2.20.1 First dimension...................................................................................................... 40
2.20.2 Second dimension.................................................................................................. 40
2.20.3 Visualisation.......................................................................................................... 41
2.20.4 Evaluation and picking of spots ............................................................................ 41
2.20.5 In-gel digest........................................................................................................... 41
2.20.6 Mass spectrometry analysis of the trypsin fragments ........................................... 42
3 Results ................................................................................................................................... 43
3.1 Testing the system for systemic resistance induction .................................................... 43
3.1.1 Chemical inducers ................................................................................................... 46
3.1.2 Biolog.................................................................................................. 48
3.1.3 Comparison of biological and chemical resistance inducers................................... 56
3.2 Investigations of the responsible mechanisms and signalling cascades......................... 60
3.2.1 Oxidative burst ........................................................................................................ 60
3.2.2 Phenolic accumulation ............................................................................................ 63
3.2.3 Signal transduction 69
3.2.4 Chemical induced resistance ................................................................................... 73
3.2.5 Pathogenesis-related proteins.................................................................................. 74
3.2.6 Microarray...................................................................