Molecular and metabolic investigation into the fungal-fungal interaction between the soilborne plant pathogen Rhizoctonia solani and the mycoparasite Stachybotrys elegans Rony Chamoun Plant Science Department McGill University, Montreal April 2013 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Ph.D. © Rony Chamoun 2013 TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................ II LIST OF TABLES .............................................................................................. VIII LIST OF FIGURES .............................................................................................. IX LIST OF ABBREVIATIONS ............................................................................... XI ABSTRACT ........................................................................................................ XIII RÉSUMÉ ............................................................................................................. XV ACKNOWLEDGMENTS ................................................................................ XVII PREFACE AND CONTRIBUTIONS OF AUTHORS ..................................... XIX Contribution to Science...................................................................................... XIX CHAPTER 1 ............................................................................................................1 1. INTRODUCTION ...............................................................................................1 1.1. HYPOTHESES .................................................................................................2 1.2. OBJECTIVES ...................................................................................................3 CHAPTER 2 ............................................................................................................4 2.1. LITERATURE REVIEW .................................................................................4 2.1.2. BIOLOGICAL CONTROL ........................................................................4 2.1.3. IMPROVEMENT OF BIOCONTROL MECHANISMS ...........................5 2.1.4. ANTAGONISTIC MECHANISMS ...........................................................6 2.1.4.1. Mycoparasitism ................................................................................6 2.1.4.1.1. Cell Wall Degrading Enzymes (CWDEs) and their encoding genes ............................................................................................................7 2.1.4.1.2. Genes induced during mycoparasitism .......................................9 2.1.4.1.3. Host reaction in response to mycoparasitism ...........................11 2.1.5. RHIZOCTONIA SOLANI ..........................................................................12 2.1.5.1. Biocontrol of R. solani ...................................................................12 2.1.6. STACHYBOTRYS ELEGANS ....................................................................13 2.1.7. MYCOPARASITISM OF SCLEROTIA ..................................................14 2.1.8. SIGNAL TRANSDUCTION GENES ......................................................16 II 2.1.8.1. G-PROTEIN ...................................................................................16 2.1.8.2. Cyclic Adenosine Monophosphate (cAMP) ...................................17 2.1.8.3. MAP kinase ....................................................................................18 2.1.9. METABOLISM ........................................................................................20 2.1.9.1. Classes of secondary metabolites ...................................................21 2.1.9.1.1. Terpenes ...................................................................................21 2.1.9.1.2. Polyketides ...............................................................................21 2.1.9.1.3. Non-ribosomal peptides (NRP) ................................................22 2.1.9.1.3.1. Gliotoxin and gliovirin .......................................................22 2.1.9.1.3.2. Peptaibols ...........................................................................22 2.1.9.2. Metabolic profiling of pathosystems ..............................................23 2.1.10. CONCLUSION .......................................................................................25 CONNECTING STATEMENT BETWEEN CHAPTERS 2 AND 3....................26 CHAPTER 3 Expression of genes of Rhizoctonia solani and the biocontrol Stachybotrys elegans during mycoparasitism of hyphae and sclerotia ..................27 3.1. ABSTRACT ....................................................................................................28 3.2. INTRODUCTION ..........................................................................................29 3.3. MATERIALS AND METHODS ....................................................................30 3.3.1. Organisms and cultures conditions ...........................................................30 3.3.2. Confrontation assays .................................................................................31 3.3.3. Dual culture of S. elegans with hyphae of R. solani .................................31 3.3.4. Dual culture of S. elegans with the sclerotia of R. solani .........................32 3.3.5. Light microscope observation of the mycoparasitic process ....................32 3.3.6. RNA extraction and retrotranscription (RT) .............................................33 3.3.7. Primer design ............................................................................................33 3.3.8. Transcripts abundance by real time quantitative RT-PCR .......................34 3.3.9. Data quantification ....................................................................................34 3.4. RESULTS .......................................................................................................35 3.4.1. Light microscopy ......................................................................................35 3.4.2. Quantification of target and reference genes transcripts...........................36 3.4.3. S. elegans transcript abundance during interaction with the host’s hyphae and sclerotia ....................................................................................................36 III 3.4.5. R. solani transcript abundance during interaction with the mycoparasite 37 3.5. DISCUSSION .................................................................................................48 3.5.1. Mycoparasitism-associated genes .............................................................48 3.5.2. Host response genes ..................................................................................50 3.6. ACKNOWLEDGMENTS ..............................................................................52 CONNECTING STATEMENT BETWEEN CHAPTERS 3 AND 4....................53 CHAPTER 4 Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions .......................................................................................54 4.1. ABSTRACT ....................................................................................................55 4.2. INTRODUCTION ..........................................................................................56 4.3. MATERIALS AND METHODS ....................................................................58 4.3.1. Fungal strains, media and culture conditions ............................................58 4.3.2. Experimental set up...................................................................................58 4.3.3. Protein extraction ......................................................................................59 4.3.4. Western blot ..............................................................................................60 4.3.5. Targeted proteomics analyses ...................................................................61 4.3.5.1. Sample preparation for proteomics ................................................61 4.3.5.2. Liquid chromatography-MS/MS ....................................................61 4.3.5.3. Protein identification ......................................................................62 4.3.6. Protein sequence alignment and primers design .......................................62 4.3.7. Nucleic acid extraction and manipulation.................................................63 4.3.8. Southern blot analysis ...............................................................................64 4.3.9. Phylogenetic analysis ................................................................................64 4.3.10. Isolation of RNA and reverse transcription ............................................64 4.3.11. Quantitative RT-PCR conditions ............................................................65 4.4. RESULTS .......................................................................................................66 4.4.1. Activation of ERK1/2 during interaction with R. solani and in response to starvation condition ........................................................................................66 4.4.2. A phosphoproteomic approach identified one phosphopeptide with a TXY motif, which is activated during mycoparasitism and in response to nutrient starvation ........................................................................................................67