Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. u. engl. Sprache |
| Beschreibung: | 137 Bl. Ill., graph. Darst. |
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| adam_text | SANJA MATERN (NEE CURCIN)
GLUTATHIONE-BASED REDOX SWITCH(ES) MODULATE
TOBACCO DEFENSE RESPONSES AGAINST
PSEUDOMONAS SYRINGAE
DISSERTATION SUBMITTED TO
THE COMBINED FACULTIES OF THE NATURAL SCIENCES AND OF MATHEMATICS OF
THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY
FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES
HEIDELBERG, 2014
HTTP://D-NB.INFO/1063292735
TABLE OF CONTENT
SUMMARY IZUSAMMENFASSUNG 9
SUMMARY 9
ZUSAMMENFASSUNG 10
1. INTRODUCTION 11
1.1 THE PLANT IMMUNE SYSTEM 12
1.1.1 DEAD OR ALIVE - PLANT PATHOGENS HAVE DIFFERENT LIFESTYLES 14
1.1.2 SIGNAL PERCEPTION - PATTERN-TRIGGERED AND EFFECTOR-TRIGGERED
IMMUNITY.... 16
1.1.3 DOWNSTREAM SIGNALING MEDIATED BY MITOGEN-ACTIVATED PROTEIN
KINASES... 18
1.1.4 DEFENSE RESPONSE EVENTS 22
1.1.5 SMALL-MOLECULE HORMONES NETWORK PLANT IMMUNITY 23
1.2 SENSING STRESS RESPONSE BY REDOX SIGNALING 27
1.2.1 ROS PRODUCTION AND SCAVENGING MACHINERY 27
1.2.2 ROS IN BIOTIC INTERACTIONS 29
1.2.3 GLUTATHIONE AS A MULTIFUNCTIONAL MOLECULE - BIOSYNTHESIS AND
COMPARTMENTATION 30
1.2.2 ROLE OF GLUTATHIONE IN ABIOTIC STRESS 32
1.2.3 ROLE OF GLUTATHIONE IN BIOTIC STRESS 33
2. AIMS OF THE STUDY 34
3. RESULTS 36
3.1 GLUTATHIONE ACCUMULATION, DISTRIBUTION AND REDOX
HOMEOSTASIS 36
3.1.1 CYTOSOLIC AND PLASTIDIC EXPRESSION OF STGCL-GS IN TOBACCO BOOSTS
GLUTATHIONE CONTENT 36
3.1.2 GLUTATHIONE REDUCTASE ACTIVITY INCREASES WITH EXPANSION OF
GLUTATHIONE
POOL 39
3.1.3 HGLS SHOW DISTINCT OXIDATIVE SIGNATURE WITHOUT ACCUMULATION OF
H2O2....4O
3.1.4 IN SITU GLUTATHIONE REDOX POTENTIAL IN CYTOPLASM AND RATIOMETRIC
MEASUREMENT OF ROGFP2 42
3.1.4.1 VISUALISATION OF ROGFP2 FLUORESCENCE CHANGE BY CLSM AND PROBE
CALIBRATION 42
3.1.4.2 SPATIAL FLUORESCENCE RATIO REPRESENTATION IN THE CYTOSOL AND
GLUTATHIONE
REDOX POTENTIAL 45
3.1.5 INCREASED GLUTATHIONE POOL MAY REACH THE LIMIT OF ANTIOXIDANT
CAPACITY ...47
3
TABLE OF CONTENT
3.2 MODULATION OF PLANT IMMUNITY BY GLUTATHIONE 48
3.2.1 IMMUNITY SIGNALING MODULATION IN THE ABSENCE OF BIOTIC STRESS 48
3.2.1.1 LINES WITH HIGH GLUTATHIONE LEVEL HAVE INCREASED EXPRESSION OF
DEFENSE-
RESPONSIVE GENES 49
3.2.1.2 HGLS DO NOT ELEVATE FREE SALICYLIC ACID 51
3.2.2 IMPOSED GLUTATHIONE PRIMES DEFENSE RESPONSE AGAINST BACTERIUM
PSEUDOMONAS SYRINGAE 52
3.2.2.1. HIGH GLUTATHIONE-INDUCED IMMUNITY DIFFERENTIALLY AFFECTS
DEFENSE AGAINST
ADAPTED PATHOVARS OF PSEUDOMONAS SYRINGAE 52
3.2.2.2 INCOMPATIBLE BACTERIAL INFECTION WITH NON-ADAPTED PATHOGENS
LEADS TO
STRONGER HR LESIONS IN TRANSGENIC LINES 54
3.2.2.3 HIGH GLUTATHIONE LINES ARE PRIMED FOR CALLOSE DEPOSITION 56
3.2.2.4 IN HGLS, HYPERSENSITIVE RESPONSE (HR) OCCURS FASTER THAN IN WILD
TYPE
PLANTS 57
3.2.2.5 OXIDATIVE STATUS CHANGES UPON INFECTION IN WILD TYPE AND HGLS 62
3.2.2.6 REDOX STATE CHANGES DIFFERENTIALLY FOLLOWING COURSE OF THE
INFECTION IN
HGLS AND WILD TYPE 65
3.2.3 NOVEL TARGET OF GLUTATHIONE REDOX IN IMMUNITY SIGNALING 67
3.2.3.1 MAP KINASES, SIPK AND WIPK ARE CONSTITUTIVELY ACTIVATED IN HGLS
67
3.2.3.2 IS GLUTATHIONE/REDOX CHANGE INDUCING ACTIVITY OF TOBACCO MAPKS?
68
3.2.3.3 DIFFERENTIAL REGULATION OF SIPK AND WIPK ACTIVITY UPON INFECTION
IN THE
WILD TYPE AND HGLS 70
3.2.3.4 VIRUS-INDUCED GENE SILENCING OF SIPK AND WIPK 72
3.2.3.5 A. THALIANA MUTANTS PAD2 AND CAT2 DIFFERENTIALLY ACTIVATE MAPKS
UPON
FLG22 TREATMENT 74
3.3 HEAVY METAL ANALYSIS IN TOBACCO PLANTS 76
4. DISCUSSION 78
4.1 HIGH GLUTATHIONE STGCL-GS EXPRESSING LINES: ADVANTAGES AND
LIMITS 78
4.1.1 ADVANTAGES OF EXPLORING TOBACCO IMMUNITY OVER ARABIDOPSIS 78
4.1.2 HIGH GLUTATHIONE INCREASES BOTH OXIDANT AND ANTIOXIDANT CAPACITY
79
4.2 UNCOUPLED GLUTATHIONE ACCUMULATION IMPACTS ON CELLULAR REDOX
HOMEOSTASIS..81
4.2.1 EVIDENCE FOR AN OXIDATIVE SHIFT IN THE CYTOSOL OF HGLS 81
4.2.1 OXIDATION OF THE CYTOSOL FOLLOWS BACTERIAL INFECTION 82
4.3 HIGH GLUTATHIONE MODULATES PLANT DEFENSE RESPONSES 83
4.3.1 HIGH GLUTATHIONE AND REDOX CHANGE INDUCE DEFENSE-RESPONSIVE GENES
WITHOUT INCREASE IN FREE SA 83
4.3.2 HIGH GLUTATHIONE IN TOBACCO BOOSTS IMMUNE RESPONSE IN HOST AND
NON-HOST
INFECTIONS 85
TABLE OF CONTENT
4.3.3 CALLOSE DEPOSITION RESPONSE IS PRIMED IN HGLS 86
4.3.4 HGLS DO NOT INCREASE DEFENSE METABOLITES SCOPOLIN AND SCOPOLETIN
87
4.4 HIGH GLUTATHIONE CONTENT CAUSES MAP KINASES ACTIVATION 87
4.4.1 DO ROS ACTIVATE MAPK SIGNALING MODULE? 87
4.4.2 GLUTATHIONE REDOX CHANGE INDUCE ACTIVATION OF TOBACCO AND
ARABIDOPSIS
MAPKS 88
4.4.3 POTENTIAL TARGETS FOR REDOX MODULATION OF MAPK PATHWAY 89
5. CONCLUSION & OUTLOOK 91
6. MATERIALS AND METHODS 93
6.1 ORGANISMS AND CULTURE CONDITIONS 93
6.1.1 PLANT MATERIAL AND CULTIVATION 93
6.1.2 BACTERIAL STRAINS 94
6.1.3 MEDIA 94
6.1.4 ANTIBIOTICS AND SELECTION 95
6.2.0 NUCLEIC ACID TECHNIQUES 96
6.2.1 EXTRACTION OF PLASMID DNAFROM BACTERIAL CULTURE 96
6.2.2 PLANT RNA EXTRACTION AND CDNA SYNTHESIS 96
6.2.3 ANALYSIS OF DNA AND RNA CONCENTRATION 96
6.2.4 GEL ELECTROPHORESIS FOR DNA AND RNA ANALYSIS 96
6.2.5 POLYMERASE CHAIN REACTION (PCR) 96
6.2.5.1 STANDARD PCR CONDITIONS 96
6.2.5.2 QUANTITIVE PCR ANALYSIS 97
6.2.5.3 OLIGONUCLEOTIDES FOR PCR AND QPCR 97
6.2.6 RESTRICTION DIGEST 99
6.2.7 PURIFICATION OF DNA FRAGMENTS AFTER PCR OR RESTRICTION DIGEST 99
6.2.8 RECOMBINATION-BASED CLONING OF DNA FRAGMENTS 99
6.2.9 DNA SEQUENCING 99
6.2.10 CLONING PROCEDURE FOR GRX:ROGFP IN THE CYTOSOL AND PLASTIDS 99
6.3 BACTERIAL AND PLANT TRANSFORMATION 100
6.3.1 PREPARATION OF ELECTROCOMPETENT E. COLI AND A. TUMEFACIENS CELLS
100
6.3.2 TRANSFORMATION OF E. COLI AND A. TUMEFACIENS CELLS 100
6.3.3 TRANSIENT TRANSFORMATION OF N. BENTHAMIANA VIA AGROBACTERIUM LEAF
INFILTRATION 100
6.3.4 STABLE TRANSFORMATION OF N. TABACUM VIA LEAF DISC TRANSFORMATION
101
6.3.5 VIRUS INDUCED GENE SILENCING (VIGS) 101
TABLE OF CONTENT
6.4 PROTEIN TECHNIQUES 102
6.4.1 PROTEIN ISOLATION AND QUANTIFICATION 102
6.4.2 ENZYME ACTIVITY ASSAYS 102
6.4.2.1 GLUTATHIONE REDUCTASE ASSAY 102
6.4.2.2 CATALASE ACTIVITY 102
6.4.3 IMMUNOBLOT ANALYSIS OF STGCL-GS PROTEIN EXPRESSION 103
6.4.4 MAPK ACTIVATION ASSAY AND IMUNOBLOTTING 104
6.5 PATHOGEN TESTS AND INFECTION ANALYSIS 104
6.5.1 PLANT INFECTION 104
6.5.2 FREE SALICYLIC ACID AND TOTAL SALICYLIC ACID MEASUREMENT 104
6.5.3 ELECTROLYTE LEAKAGE ASSAY 105
6.5.4 DETECTION OF CALLOSE DEPOSITION 105
6.5.5 H2O2 DETECTION AND DAB STAINING 105
6:5.6. SCOPOLIN AND SCOPOLETIN MEASUREMENTS 105
6.5.7 DETERMINATION OF SUCROSE, GLUCOSE AND FRUCTOSE 106
6.6 GLUTATHIONE QUANTIFICATION AND REDOX IMAGING 107
6.6.1 THIOL EXTRACTION AND HPLC QUANTIFICATION 107
6.6.2 CONFOCAL LASER SCANING MICROSCOPY IMAGING AND RATIOMETRIC ANALYSIS
... 108
6.7 METAL ANALYSIS 110
6.8 STATISTICAL ANALYSIS 110
7. SUPPORTING INFORMATION 111
8. ABBREVIATIONS 116
9. LITERATURE 119
10. ACKNOWLEDGEMENT 136
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| spelling | Matern, Sanja Verfasser (DE-588)1063393353 aut Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae [presented by Sanja Matern née Curcin] 2014 137 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. u. engl. Sprache Heidelberg, Univ., Diss., 2014 Pseudomonas syringae (DE-588)4279840-1 gnd rswk-swf Stressresistenz (DE-588)4326741-5 gnd rswk-swf Tabak (DE-588)4136670-0 gnd rswk-swf Glutathion (DE-588)4157715-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Stressresistenz (DE-588)4326741-5 s Glutathion (DE-588)4157715-2 s Tabak (DE-588)4136670-0 s Pseudomonas syringae (DE-588)4279840-1 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027679545&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Matern, Sanja Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae Pseudomonas syringae (DE-588)4279840-1 gnd Stressresistenz (DE-588)4326741-5 gnd Tabak (DE-588)4136670-0 gnd Glutathion (DE-588)4157715-2 gnd |
| subject_GND | (DE-588)4279840-1 (DE-588)4326741-5 (DE-588)4136670-0 (DE-588)4157715-2 (DE-588)4113937-9 |
| title | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae |
| title_auth | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae |
| title_exact_search | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae |
| title_full | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae [presented by Sanja Matern née Curcin] |
| title_fullStr | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae [presented by Sanja Matern née Curcin] |
| title_full_unstemmed | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae [presented by Sanja Matern née Curcin] |
| title_short | Glutathione based redox switch(es) modulate tobacco defense responses against Pseudomonas syringae |
| title_sort | glutathione based redox switch es modulate tobacco defense responses against pseudomonas syringae |
| topic | Pseudomonas syringae (DE-588)4279840-1 gnd Stressresistenz (DE-588)4326741-5 gnd Tabak (DE-588)4136670-0 gnd Glutathion (DE-588)4157715-2 gnd |
| topic_facet | Pseudomonas syringae Stressresistenz Tabak Glutathion Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027679545&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT maternsanja glutathionebasedredoxswitchesmodulatetobaccodefenseresponsesagainstpseudomonassyringae |