Molecular characterization of a fungal aldehyde dehydrogenase in the Tricholoma vaccinumspruce ectomycorrhiza [Elektronische Ressource] / von Theodore Asiimwe

Molecular characterization of a fungal aldehyde
dehydrogenase in the Tricholoma vaccinum-
spruce ectomycorrhiza










Dissertation
Zur Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)



Vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
Der Friedrich-Schiller-Universitä Jena











Von Theodore Asiimwe (MSc)
Geboren am 23.12.1973 in Uganda









































Gutachter (Reviewers):
1. Prof. Dr. Erika Kothe (Friedrich Schiller University, Jena, Germany)
2. Prof. Dr. Wilhelm Boland (Max Planck-Chemical Ecology, Jena, Germany)
3. Prof. Dr. Uwe Nehls (Bremen University, Bremen, Germany)

Tag der öffentlichen Verteidigung (Day of public defense): 03-05-2010CONTENTS 1
TABLE OF CONTENT
TABLE OF CONTENT........................................................................................................... 1
ABBREVIATIONS .................................................................................................................. 3
SUMMARY............................................................................................................................... 4
ZUSAMMENFASSUNG ......................................................................................................... 6
1. INTRODUCTION................................................................................................................ 8
1.1. Mycorrhiza (general overview) .................................................................................... 8
1.2. Ectomycorrhiza ............................................................................................................. 9
1.2.1. Structure and function .............................................................................................. 9
1.2.2. Ectomycorrhizal fungi............................................................................................ 10
1.2.3. Molecular plant-fungal interaction......................................................................... 11
1.3. Aldehyde dehydrogenases as metabolically versatile enzymes ............................... 11
1.3.1. Metabolic roles of aldehyde dehydrogenase .......................................................... 11
1.3.2. Regulation of aldehyde dehydrogenase.................................................................. 14
1.3.3. Possible roles of aldehyde dehydrogenase in ectomycorrhiza 15
1.4. Indole-3-acetic acid in metabolism ............................................................................ 16
1.4.1. Metabolic roles, biosynthesis, and regulation of indole-3-acetic acid ................... 16
1.4.2. Role of indole-3-acetic acid in ectomycorrhiza development................................ 18
1.5. Fungal transformation................................................................................................ 20
1.5.1. Transformation (general overview)........................................................................ 20
1.5.2. Transformation methods ........................................................................................ 20
1.6. Aims of the study......................................................................................................... 21
2. MATERIALS AND METHODS....................................................................................... 23
2.1. Materials ...................................................................................................................... 23
2.1.1. Organisms and plasmids......................................................................................... 23
2.1.2. Chemicals, enzymes and antibiotics....................................................................... 24
2.1.3. Solutions, reagents and buffers .............................................................................. 24
2.1.4. Media...................................................................................................................... 25
2.1.5. Oligonucleotides..................................................................................................... 26
2.2. Methods........................................................................................................................ 27
2.2.1. Culture conditions for different organisms ............................................................ 27
2.2.2. Analysis of IAA production by T. vaccinum.......................................................... 29
2.2.3. Quantification of IAA effects on m and mycorrhiza ............................. 30
2.2.4. Molecular biology methods.................................................................................... 31
2.2.4.1 Nucleic acids preparations ............................................................................... 31
2.2.4.2 DNA-DNA hybridization by Southern blotting............................................... 32
2.2.4.3 Polymerase chain reaction (PCR) .................................................................... 34
2.2.4.4 DNA modifications .......................................................................................... 36
2.2.4.5 DNA sequencing .............................................................................................. 36
2.2.4.6 Sequence analysis............................................................................................. 37
2.2.4.7 Reconstructing phylogeny................................................................................ 37
2.2.4.8 Transformation of different organisms ............................................................ 38
2.2.5. Protein methods...................................................................................................... 39
2.2.5.1 Protein preparation ........................................................................................... 39
2.2.5.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) .................................. 40
2.2.5.3 Western blot analysis ....................................................................................... 40
2.2.5.4 Enzyme activity assays..................................................................................... 40
2.2.6. Microscopy............................................................................................................. 41
2.2.6.1 Specimen preparation 41 CONTENTS 2
2.2.6.2 Object viewing ................................................................................................. 42
2.2.7. Statistical analysis .................................................................................................. 42
3. RESULTS............................................................................................................................ 43
3.1. Isolation and characterization of ald1 from T. vaccinum ........................................ 43
3.1.1. Isolation of ald1 ..................................................................................................... 43
3.1.2. Sequence analysis of ald1 ...................................................................................... 44
3.1.3. Phylogenetic relationships between fungal aldehyde dehydrogenases .................. 46
3.1.4. Verification of ald1 cDNA by heterologous expression in E. coli ........................ 50
3.1.5. Induction of ald1 expression by alcohol and aldehyde-related stress.................... 51
3.2. Analysis of ald1 gene function by overexpression in T. vaccinum .......................... 54
3.2.1. Construction of ald1 gene overexpression vectors ................................................ 54
3.2.2. Agrobacterium tumefaciens-mediated transformation of T. vaccinum .................. 56
3.2.3. In vitro mitigation of stress by ald1 ....................................................................... 59
3.3. Role of indole-3-acetic acid in biology of T. vaccinum ............................................. 60
3.3.1. Effect of IAA on T. vaccinum hyphal morphology 61
3.3.2. Effect of IAA on fungal growth......................................................... 64
3.4. Biosynthesis of indole-3-acetic acid in T. vaccinum.................................................. 66
3.5. Role of indole-3-acetic acid in ectomycorrhiza formation....................................... 69
3.5.1. Influence of IAA on T. vaccinum-spruce ectomycorrhizal development .............. 69
3.5.2. Effect of fungal and IAA inoculation on plant growth and development 71
4. DISCUSSION ..................................................................................................................... 73
4.1. Characterization of an ectomycorrhizal fungal aldehyde dehydrogenase............. 73
4.1.1. The structure and phylogeny of ald1...................................................................... 73
4.1.2. Regulation of ald1 gene expression ....................................................................... 75
4.1.3. The function of ald1 in T. vaccinum 77
4.2. Indole-3-acetic acid in ectomycorrhiza .