The phytopathogenic interaction between Claviceps purpurea and rye [Elektronische Ressource] : significance of enzymes involved in the response to active oxygen species / vorgelegt von Sabine Ivison
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The phytopathogenic interaction between Claviceps purpurea and rye [Elektronische Ressource] : significance of enzymes involved in the response to active oxygen species / vorgelegt von Sabine Ivison

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203 Pages
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MikrobiologieThe phytopathogenic interaction betweenClaviceps purpurea and rye: significance of enzymesinvolved in the response to active oxygen speciesInaugural-Dissertationzur Erlangung des Doktorgradesder Naturwissenschaften im Fachbereich Biologieder Mathematisch-Naturwissenschaftlichen Fakultätder Westfälischen Wilhelms-Universität Münstervorgelegt vonSabine Ivisonaus Victoria, BC, Kanada-2002-Dekan: Pr. Dr. PaulErster Gutachter: Pr. Dr. TudzynskiZweiter Gutachter: Pr. Dr. MeinhardtTag der mündlichen Prüfung: 17.07.2002Tag der Promotion: 17.07.2002IIndexIndexIndex IAbbreviations VI1 Introduction 11.1 Oxygen toxins: the price of an aerobic lifestyle 11.1.1 The toxicity of oxygen 11.1.2 Sources of active oxygen 31.1.3 Survival as an aerobe 41.1.4 A closer look at two AOS scavenging proteins: Cu,Zn SOD and catalase 61.1.4.1 Cu,Zn SOD 61.1.4.2 Catalase 81.2 Harnessing the evil: AOS in plant pathogenesis 101.2.1 Early response to attack determines compatibility of interaction 101.2.2 Early response AOS production: the oxidative burst 111.2.3 Functions of the oxidative burst 121.2.3.1 Direct antimicrobial activity 121.2.3.2 Oxidative cell wall alterations 131.2.3.3 AOS in gene activation during plant defense 141.2.3.4 Hypersensitive response 151.2.3.5 Other functions of the oxidative burst 161.3 Claviceps purpurea 171.3.1 Pathogenesis on rye 181.3.2 Ergot alkaloids of sclerotia 201.3.

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Published 01 January 2002
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Mikrobiologie
The phytopathogenic interaction between
Claviceps purpurea and rye: significance of enzymes
involved in the response to active oxygen species
Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften im Fachbereich Biologie
der Mathematisch-Naturwissenschaftlichen Fakultät
der Westfälischen Wilhelms-Universität Münster
vorgelegt von
Sabine Ivison
aus Victoria, BC, Kanada
-2002-Dekan: Pr. Dr. Paul
Erster Gutachter: Pr. Dr. Tudzynski
Zweiter Gutachter: Pr. Dr. Meinhardt
Tag der mündlichen Prüfung: 17.07.2002
Tag der Promotion: 17.07.2002IIndex
Index
Index I
Abbreviations VI
1 Introduction 1
1.1 Oxygen toxins: the price of an aerobic lifestyle 1
1.1.1 The toxicity of oxygen 1
1.1.2 Sources of active oxygen 3
1.1.3 Survival as an aerobe 4
1.1.4 A closer look at two AOS scavenging proteins: Cu,Zn SOD and catalase 6
1.1.4.1 Cu,Zn SOD 6
1.1.4.2 Catalase 8
1.2 Harnessing the evil: AOS in plant pathogenesis 10
1.2.1 Early response to attack determines compatibility of interaction 10
1.2.2 Early response AOS production: the oxidative burst 11
1.2.3 Functions of the oxidative burst 12
1.2.3.1 Direct antimicrobial activity 12
1.2.3.2 Oxidative cell wall alterations 13
1.2.3.3 AOS in gene activation during plant defense 14
1.2.3.4 Hypersensitive response 15
1.2.3.5 Other functions of the oxidative burst 16
1.3 Claviceps purpurea 17
1.3.1 Pathogenesis on rye 18
1.3.2 Ergot alkaloids of sclerotia 20
1.3.3 The molecular biology of parasitic growth 22
1.3.3.1 A short summary of past results and present research 22
1.3.3.2 Implications for the involvement of AOS in pathogenesis 24
1.3.3.3 Focal points of research 25
2 Experimental Procedures 26
2.1 Strains 26
2.1.1 E. coli 26
2.1.2 Claviceps 26
2.1.3 Rye 27
2.2 Culture Conditions 27
2.2.1 E.coli 27
2.2.2 Claviceps 28
2.2.2.1 Cultivation of sporulating mycelia from C. purpurea (20-1) 28
2.2.2.2 Cultivation for protein, RNA and DNA analysis of C. purpurea 28
2.2.2.3 Cultivation for induction of alkaloid in axenic culture 28
2.2.2.4 Cultivation of C. fusiformis 28
2.2.3 Rye 29IIIndex
2.3 Vectors, libraries and primers 29
2.3.1 Vectors 29
2.3.2 Libraries 30
2.3.3 Primers 31
2.3.3.1 PCR and RT-PCR Primers 31
2.3.3.2 Sequencing primers 32
2.4 General procedures in molecular genetics 32
2.4.1 Isolation of DNA and RNA 32
2.4.1.1 Isolation of lambda-DNA 32
2.4.1.2 Isolation of genomic DNA from Claviceps 33
2.4.1.3 Isolation of total RNA from Claviceps 33
2.4.1.4 Isolation of mRNA from Claviceps 33
2.4.1.5 Isolation of plasmid DNA 33
2.4.1.6 Determination of RNA and DNA concentrations 34
2.4.2 Restriction, precipitation and washing of DNA 34
2.4.3 Gel electrophoresis of DNA and RNA 34
2.4.4 Isolation of DNA from agarose gels 35
322.4.5 Hybridisation of DNA and RNA with α- P-dCTP-labelled probes 35
2.4.5.1 Radio-labelling of DNA fragments 35
2.4.5.2 Synthesis of radio-labelled cDNA from mRNA template 35
2.4.5.3 DNA-DNA hybridisation 36
2.4.5.4 RNA-DNA hybridisation 36
2.4.6 Analysis of DNA and RNA on membranes 36
2.4.6.1 Downward blotting of DNA and RNA 36
2.4.6.2 Dot Blots 36
2.4.6.3 Preparation and screening of bacterial colony filters 36
2.4.6.4 First- and second-round preparation of phage plaque filters for genomic and cDNA library
screening 37
2.4.7 Excision of phagemid vectors from lambda-ZAP vectors 38
2.4.8 PCR and RT-PCR 38
2.4.9 Cloning of DNA fragments 39
2.4.9.1 Ligation of DNA fragments into cloning vectors 39
2.4.9.2 Transformation of ligated vectors into E. coli and selection of recombinantclones 39
2.4.10 DNA sequencing 40
2.5 Creation and differential screening of a copper-induced cDNA-library 41
2.5.1 Creation of a copper-induced cDNA-library 41
2.5.1.1 Cultivation of mycelia 41
2.5.1.2 Synthesis of the cDNA library 41
2.5.2 Differential cDNA screening 42
2.5.2.1 Non-stringent differential cDNA screening 43
2.5.2.2 Stringent differential screening 43
2.6 Induction of mycelia for northern and protein analysis 44
2.6.1 Induction with copper and iron 44
2.6.2 Induction with AOS 45
2.6.2.1 Induction with hydrogen peroxide 45
2.6.2.2 Induction with sources of superoxide 45
2.6.3 Induction with polyols 46IIIIndex
2.7 Protein-biochemical methods 46
2.7.1 Extraction of proteins from C. purpurea 46
2.7.2 Determination of protein concentration in extracts 47
2.7.3 Isoelectric focussing (IEF) gel electrophoresis 47
2.7.4 Silver staining of protein gels 47
2.7.5 Zymogram techniques 48
2.7.5.1 Staining for SOD activity 48
2.7.5.1.1 Inhibition of Cu,Zn SOD activity with DDC 48
2.7.5.2 Staining for catalase activity 48
2.7.6 Western Blotting 49
2.7.6.1 SDS-PAGE 49
2.7.6.2 Blotting of gel proteins and Ponceau S staining 49
2.7.6.3 Immuno-staining of blot proteins 49
2.7.7 Determination of mycelial contamination of cell wall extracts with G6PDH 50
2.8 Generation and analysis of mutant strains 51
2.8.1 Transformation of C. purpurea strain 20-1 and derivatives 51
2.8.2 Cultivation and analysis of C. purpurea transformants 52
2.8.3 Pathogenicity assays 52
2.8.4 Testing paraquat sensitivity of wild type and mutant strains 53
2.9 Computer-based analysis of DNA and protein sequences 53
3R esults 55
3.1 Cloning and analysis of a cell-wall associated Cu,Zn SOD in Claviceps
purpurea 55
3.1.1 Identification of a CfSOD1-homologue in Claviceps purpurea 55
3.1.2 Analysis and localisation of SOD activity in axenic culture 56
3.1.2.1 SOD activity in culture filtrate, cell wall and mycelia of C. purpurea 56
3.1.2.2 Analysis of cytoplasmatic contamination in the cell wall fraction by measurement of
G6PDH activity 57
3.1.2.3 Direct comparison of SODA activity in cell wall and mycelial protein fractions 58
3.1.2.4 Determination of SOD-type using a Cu,Zn SOD inhibitor 58
3.1.3 Isolation and sequencing of a Cu,Zn SOD from C. purpurea 59
3.1.3.1 Heterologous PCR using cDNA template 59
3.1.3.2 Isolation and sequencing of the cpsod1 gene 60
3.1.3.2.1 Genomic Southern with the cpsod1 probe 60
3.1.3.2.2 Isolation and sequencing of genomic cpsod1 60
3.1.3.2.3 Promotor analysis 62
3.1.4 Expression analysis of cpsod1 63
3.1.4.1 Expression of cpsod1 in axenic culture 63
3.1.4.2 In planta expression analysis of cpsod1 using RT-PCR 65
3.1.5 Creation of mutants lacking cpsod1 for functional analysis 66
3.1.5.1 Construction of the cpsod1 disruption vector pDV1 66
3.1.5.2 Transformation of C. purpurea and analysis of pDV1-4 transformants with PRC 67
3.1.5.3 Analysis of T1-73 69
3.1.5.3.1 Southern analysis of T1-73 69
3.1.5.3.2 Protein analysis of T1-73 69IVIndex
3.1.5.4 Complementation of ∆cpsod1 70
3.1.5.4.1 Construction of the complementation vectors pComp1 and pComp1. 70a c
3.1.5.4.2 Screening pComp1 transformants for integration of functional cpsod1 71
3.1.5.4.3 Southern and western analysis of the selected complementation mutants c∆sod1
and c∆sod2 72
3.1.6 Analysis of ∆cpsod1 phenotypes in axenic and parasitic culture 73
3.1.6.1 Axenic growth of ∆cpsod1 with and without oxidative stress 74
3.1.6.2 Testing the virulence of ∆cpsod1 on rye 75
3.1.6.2.1 Pathogenicity assays with ∆cpsod1 75
3.1.6.2.2 In planta analysis of SOD activities in the ∆cpsod1 and WT strains 76
3.2 Characterisation and functional analysis of a putative catalase gene 78
3.2.1 Sequencing of cpcat2 78
3.2.2 Analysis of the -derived protein and promotor region 79
3.2.3 Southern analysis of cpcat2 81
3.2.4 Expression analysis of cpcat2 82
3.2.4.1 Northern analysis in the pathogenic strain 20-1 and the alkaloid production strain P1 82
3.2.4.2 Catalase activity in strain P1 82
3.2.4.3 In planta expression of cpcat2 83
3.2.5 Targeted inactivation of cpcat2 83
3.2.5.1 Construction of replacement vector pRV-1 84
3.2.5.2 Transformation and analysis of RF-cpcat2 transformants with PCR 84
3.2.5.3 Protein analysis of ∆cpcat2 in axenic culture 87
3.2.6 Pathogenicity assays with ∆cpcat2 88
3.3 Searching for new genes involved in AOS detoxification: Differential cDNA
screening with and without copper 90
3.3.1 Creation of a copper-induced cDNA library 90
3.3.2 Differential screening of the copper-induced cDNA library 91
3.3.2.1 Results of non-stringent differential screening 91
3.3.2.2 Results of stringent differential screening 93
3.3.2.2.1 Descriptions of the sequenced clones and their homologies 95
3.3.2.2.2 Summary: stringent differential screening 99
3.3.2.3 Northern analysis of isolated cDNAs 100
3.3.2.3.1 Clones which are not induced by copper 102
3.3.2.3.2 Clones induced by copper: could induction be Fenton-mediated? 102
3.3.3 Further characterisation of some of the clones which emerged in the differential
screening 103
3.3.3.1 Isolation and sequencing of genomic regions corresponding to the cDNA clones 104
3.3.3.1.1 X12a, the putative hydrophobin (cph1) 104
3.3.3.1.2 X46, the putative Zn-binding dehydrogenase (cpdh1) 105
3.3.3.1.3 X8b, the Cu,Zn SOD-like protein (cpsod-ish) 109
3.3.3.2 Promotor analysis of the sequenced cDNA clones 110
3.3.3.2.1 Promotor analysis of cph1 111
3.3.3.2.2 Promotor analysis of cpdh1 (app. A4) 112
3.3.3.2.3 Promotor analysis of cpsod-ish 112
3.3.3.3 In planta expression analysis of the sequenced cDNA clones 112
3.3.3.3.1 cph1 113
3.3.3.3.2 cpdh1 113
3.3.3.3.3 cpsod-ish 114VIndex
4 Discussion 115
4.1 The Cu,Zn SOD of C. purpurea 115
4.1.1 Localisation of CpSOD1 116
4.1.2 Regulation of cpsod1 119
4.1.3 The importance of cpsod1 in axenic culture 121
4.1.4 Role of cpsod1 during pathogenesis 122
4.2 Yet more catalases: role in parasitic growth and biosynthesis of ergot alkaloids 124
4.2.1 The putative protein of cpcat2 125
4.2.2 Significance of for axenic and parasitic growth 127 cpcat2
4.2.2.1 Catalase: phase-specific expression and redundancy 127
4.2.2.2 Cpcat2 is not required for pathogenicity 128
4.2.2.3 A role for catalase in morphogenetic differentiation? 129
4.2.3 and the biosynthesis of ergot alkaloids 131Cpcat2
4.2.3.1 Putative trans-acting factors 132
4.2.4 Conclusions: 135 cpcat2
4.3 From metals to the oxidative stress response 136
4.3.1 Implications of the transcriptional response to copper, iron and H O 1372 2
4.3.2 Preliminary analysis of selected genes isolated in the course of differential cDNA
screening 139
4.3.2.1 cph1: a metal-responsive hydrophobin? 139
4.3.2.1.1 Hydrophobins, oxidative stress and metals 140
4.3.2.1.2 Aspects of cph1 regulation 141
4.3.2.2 cpdh1: a putative polyol dehydrogenase 142
4.3.2.3 cpsod-ish: encodes a protein similar to Cu,Zn SODs 144
4.3.3 Could the response to metals and metal-mediated oxidative stress be relevant for
pathogenicity? 144
4.4 Conclusions: AOS and beyond 145
5 Summary 148
6 References 150
Appendix A: Genomic Sequences 170
Appendix B: cDNA Sequences 177
Appendix C: Chemicals 186
Appendix D: Media 189
Acknowledgements 190
Curriculum Vitae 191
VIAbbreviations
Abbreviations
BCIP 5-bromo-4-chloro-3-indolylb beta
phosphateD difference
bp base pairsl lambda
BSA bovine serum albumine? registered trademark
C carbonS sum
C cytosine?C degrees Celsius
C. purpurea Claviceps purpureamg microgram
C. fulvum Cladiosporum fulvumml microlitre
C. fusiformis Claviceps fusiformismM micromolar (micromol/l)
C. neoformans Cryptococcus neoformansmmol micromol Ca calcium
ms microsecond ca. circa
(v/v) volume/volume cAMP cyclic adenosine
(w/v) weight/volume monophosphate
A absorption CAT catalase
A adenine Cd cadmium
A. nidulans Aspergillus nidulans cDNA complementary DNA
A. niger Aspergillus niger CDPK calcium-dependent protein
A absorption at 340 nm340 kinase
aa amino acids Ce cerium
ABA abscisic acid Cf (or CF) in genes, refers to
ABARE abscisic acid response claviceps fusiformis
element Cl chlorine
ABC ATP-binding cassette cm centimetre
Ac acetate CMS cytoplasmic male sterile
Acc.# accession number CODE-HOP consensus-degenerate
ACE activation of copper enzyme hybrid oligonucleotide
acyl-coA acetyl-coenzyme A primers
Ag silver cp in genes, refers to
AIDS acquired immune deficiency claviceps purpurea
syndrome C-terminus carboxy-terminus
AnCF A. nidulans CCAAT factor CTT cytosolic catalase
AOS active oxygen species Cu copper
app. appendix Cu(I) copper (oxidation state I)
approx. approximately CuRE copper response element
APS ammonium persulfate CW cell wall
ATCC American type culture CWDE cell wall degrading enzymes
collection CYB cytochrome b2
ATP adenosine triphosphate d days
AT-rich adenine and thymine rich DAB diaminobenzidine
av avirulence dATP 2'-deoxyadenosine
BCA bathocuproine disolfonic 5'-triphosphate
acid
VIIAbbreviations
dCTP 2'-deoxycystidine Fe SOD iron-containing SOD
5'-triphosphate Fe iron
ddATP 2',3'-dideoxyadenosine-5'- Fe(III) iron (oxidation state III)
triphosphate FEBS federation of European
DDC diethyldithiocarbamate biochemical societies
ddCTP 2',3'-dideoxycystidine FEMS
5'-triphosphate microbiological societies
ddGTP 2',3'-dideoxyguanosine FeRE iron response element
5' Fig. (or fig.) figure
ddH O double destilled H O FMN flavin mononucleotide2 2
ddTTP 2',3'-dideoxythymidine g gram
5'-triphosphate G guanine
DEPC diethyl pyrocarbonate G-6-P glucose-6-phosphate
dGTP 2'-deoxyguanosine 5'- G6PDH
triphosphate dehydrogenase
DMF dimethyl GFP green fluorescent protein
DMSO dimethylsulfoxide GPx glutathione peroxidase
DNA desoxyribonucleic acid GR glutathione reductase
dNTP desoxynucleotide GSH glutathione (reduced)
triphosphate GSSG glutathione (oxidised)
dpi days post inoculation GST glutathione-S-transferase
DS differential screening h hour
DTT 1,4-dithiothreitol H hydrogen
dTTP 2'-deoxythymidine HAP heme activating protein
5'-triphosphate HI homologous integration
e base of natural logarithms HR hypersensitive response
-e electron HRE heat response element
E enzyme HSF heat shock factor
E. amylovora Erwinia amylovora HSP heat shock protein
E. coli Escherichia coli i.e. id est (that is to say)
e.g. exempli gratia (for example) IEF isoelectric focusing
EC-SOD extracellular SOD IMD isopropyl malate
Ed. (or Eds.) editor (or editors) dehydrogenase
EDTA ethylenediamine tetra acetic IRE iron response element
acid ITPG isopropylthio-b-D-
EMBL European molecular biology galactoside
laboratory JA jasmonic acid
EMBO K in DNA, guanine or thymine
organisation K potassium
ER endoplasmatic reticulum kB kilobase
EST expressed sequence tag kDa kilodalton
et al. et alii kg kilogram
EtBr ethidium bromide K Michealis-Menton constantm
etc. et cetera (and so on) l litre
eV electronvolt LB Luria Bertani medium
F. neoformans Filobasidiella neoformans LBM LB with magnesium sulfate
FASEB federation of American LBMM LBM with maltose
societies for experimental Li lithium
biology LSD lysergic acid diethylamide