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A Genomic and Proteomic Investigation of the Plant Pathogen Armillaria Mellea: Buried Treasure Or Hidden Threat?

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A Genomic and Proteomic Investigation of the Plant Pathogen Armillaria Mellea: Buried Treasure Or Hidden Threat? A Genomic and Proteomic Investigation of the Plant Pathogen Armillaria mellea: Buried Treasure or Hidden Threat? Cassandra Collins B.Sc. In fulfilment of the degree of Doctor of Philosophy Thesis submitted to the National University of Ireland Department of Biology August 2013 Head of Department: Supervisors: Professor Paul Moynagh Professor Sean Doyle Dr. David Fitzpatrick Table of Contents Table of Contents ...................................................................................................... ii Declaration of Authorship .............................................................................................. xiv Acknowledgements ......................................................................................................... xv Publications and Presentations ....................................................................................... xvi Abbreviations ...............................................................................................................xviii Summary ........................................................................................................................ xix Chapter 1 Introduction ................................................................................................. 1 1.1 General introduction ....................................................................................... 1 1.1.1 Ascomycota ............................................................................................. 4 1.1.1.1 Ascomycete genomes and proteomes ................................................. 5 1.1.2 Basidiomycota ......................................................................................... 8 1.1.2.1 Basidiomycete genome sequencing and proteomics. ....................... 14 1.1.2.2 Investigation of basidiomycete pathogenicity and virulence strategies .......................................................................................................... 15 1.1.2.3 Enzymes with biotechnological application ..................................... 20 1.1.2.4 Wood degradation in basidiomycetes ............................................... 21 1.1.2.5 Brown-rot basidiomycetes ................................................................ 22 1.1.2.6 White-rot basidiomycetes ................................................................. 25 1.1.2.7 Other cell wall component degradation by basidiomycetes species. 30 1.2 Armillaria spp. .............................................................................................. 32 1.2.1 Taxonomy .............................................................................................. 33 1.2.2 Lifecycle of Armillaria spp. .................................................................. 35 1.2.3 Armillaria mellea pathogenicity and virulence. .................................... 38 1.2.3.1 Signs and symptoms of infection by A mellea. ................................ 39 1.2.3.2 Damage caused by A. mellea ............................................................ 41 1.2.4 Bioluminescence, secondary metabolites, and known protein activities .. ............................................................................................................... 43 1.2.4.1 Bioluminescence in A. mellea .......................................................... 44 ii 1.2.4.2 Diketopiperazines ............................................................................. 44 1.2.4.3 Anti-inflammatory activity ............................................................... 44 1.2.4.4 Antimicrobial activity ....................................................................... 45 1.2.4.5 Antioxidant activity .......................................................................... 45 1.2.4.6 A. mellea immunostimulating activity .............................................. 46 1.2.4.7 A. mellea indole compounds............................................................. 46 1.2.4.8 A. mellea known proteins and their activities ................................... 47 1.2.4.9 Sesquiterpene aryl-esters .................................................................. 47 1.2.5 Recent molecular studies on Armillaria mellea. ................................... 48 1.2.5.1 Processing sites involved in intron splicing of Armillaria natural product genes .................................................................................................. 48 1.2.5.2 Structure and Cytotoxicity of Arnamial and Related Fungal Sesquiterpene Aryl Esters ............................................................................... 49 1.2.5.3 Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea. .......................................................................................................... 50 1.2.5.4 A rapid infection assay for Armillaria and real-time PCR quantitation of the fungal biomass in planta ................................................... 50 1.2.5.5 Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria. ................................. 51 1.2.5.6 Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases ............................................ 51 1.2.6 Knowledge deficiency of A. mellea biology ......................................... 52 1.3 Thesis objectives ........................................................................................... 55 Chapter 2 Materials and Methods .............................................................................. 56 2.1 Materials ....................................................................................................... 56 2.1.1 DNA isolation ........................................................................................ 56 2.1.2 3 M Sodium Acetate .............................................................................. 56 2.1.3 Media and Agar ..................................................................................... 56 2.1.3.1 Potato dextrose broth (PDB) ............................................................ 56 iii 2.1.3.2 Potato dextrose agar (PDA) .............................................................. 57 2.1.3.3 Malt extract agar (MEA) .................................................................. 57 2.1.3.4 Trace elements .................................................................................. 57 2.1.3.5 50 X Salt solution ............................................................................. 57 2.1.3.6 100 X Ammonium Tartrate .............................................................. 57 2.1.3.7 Sterilising Solution ........................................................................... 57 2.1.3.8 300 mM L-glutamine ........................................................................ 57 2.1.3.9 Carbon Sources ................................................................................. 58 2.1.3.10 Sterilised Vitis vinifera (grapevine) ................................................ 58 2.1.3.11 Culture of A. mellea on MEA with autoclaved C. albicans ........... 58 2.1.3.12 Minimal Media (MMA) agar.......................................................... 58 2.1.3.13 Minimal Media (MML) liquid........................................................ 58 2.1.3.14 Phosphate Buffered Saline (PBS) ................................................... 59 2.1.3.15 Phosphate Buffer Saline 0.05 % Tween (PBST) ............................ 59 2.1.3.16 Bradford Solution ........................................................................... 59 2.1.4 Solutions for pH Adjustment ................................................................. 59 2.1.4.1 5 M Hydrochloric Acid (HCl) .......................................................... 59 2.1.4.2 5 M Sodium Hydroxide (NaOH) ...................................................... 59 2.1.5 A. mellea protein extraction ................................................................... 59 2.1.5.1 Lysis buffer 1 – A. mellea 2-DE protein extraction.......................... 59 2.1.5.2 Lysis Buffer 2 – A. mellea whole cell lysate (WCL) protein extraction ......................................................................................................... 59 2.1.5.3 50 mM Tris Buffer pH 7.5 ................................................................ 59 2.1.5.4 100 mM ammonium bicarbonate (NH4HCO3) ................................. 60 2.1.5.5 10 mM Ammonium bicarbonate (NH4HCO3) .................................. 60 2.1.5.6 50 mM Potassium phosphate (KH2PO4 pH 7.5) ............................... 60 2.1.5.7 1 M DL-Dithiothreitol (DTT) stock ................................................. 60 2.1.5.8 1 M Iodoacetamide stock.................................................................. 60 2.1.5.9 Trypsin Diluent ................................................................................. 60 iv 2.1.5.10 Trypsin solution WCL (400 ng/µl) ................................................. 60 2.1.5.11 Trypsin solution WCL agar (65 ng/µl) ........................................... 60 2.1.5.12 Trypsin solution in-gel digestion (13 ng/µl)................................... 60 2.1.5.13 80 % (v/v) Glycerol solution .......................................................... 60 2.1.5.14 Supernatant organic extraction buffer ............................................ 61 2.1.5.15 100 % (w/v) Trichloroacetic acid
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