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Genomic analysis of secondary metabolism in Ramularia collo-cygni, causative agent of Ramularia leaf spot disease of barley François Mathieu Didier Dussart Thesis submitted to the University of Edinburgh for the degree of Doctor of Philosophy March 2017 Declaration I hereby declare that this thesis has been composed by me and that the work is my own, except as acknowledged by means of references. This thesis has not been submitted for any other degree or professional qualification except as specified. François Mathieu Didier DUSSART Table of contents List of Figures ............................................................................................................. v List of Tables ............................................................................................................. ix List of abbreviations ................................................................................................. xi Acknowledgment ..................................................................................................... xiv Abstract ..................................................................................................................... xv Lay summary .......................................................................................................... xvii Chapter 1: General introduction .............................................................................. 1 1.1. Crop fungal diseases in history .................................................................... 2 1.2. Barley: a resilient crop ................................................................................. 3 1.2.1. History of barley ................................................................................................ 3 1.2.2. Barley production ............................................................................................... 4 1.2.3. Main uses of barley ............................................................................................ 5 1.2.4. Threats to barley production .............................................................................. 6 1.2.4.1. Abiotic stresses .......................................................................................... 6 1.2.4.2. Biotic stresses ............................................................................................ 7 1.3. Ramularia leaf spot: an emerging leaf disease of barley .............................. 9 1.3.1. Taxonomy of Ramularia leaf spot ...................................................................... 9 1.3.2. Distribution of Ramularia collo-cygni ............................................................. 10 1.3.3. Ramularia leaf spot symptoms ......................................................................... 11 1.3.4. Host range of Ramularia collo-cygni ............................................................... 13 1.3.5. Consequences of Ramularia leaf spot .............................................................. 14 1.3.6. Life cycle of Ramularia collo-cygni ................................................................ 14 1.3.7. Control methods for Ramularia leaf spot ......................................................... 16 1.3.7.1. Seed treatment ......................................................................................... 16 1.3.7.2. Varietal resistance ................................................................................... 16 1.3.7.3. Fungicide treatments ............................................................................... 17 1.4. Fungal secondary metabolites .................................................................... 18 1.4.1. Secondary metabolites...................................................................................... 18 1.4.2. History of fungal secondary metabolites .......................................................... 19 1.4.3. Importance of fungal secondary metabolites .................................................... 20 1.4.3.1. Pharmaceutical products .......................................................................... 20 1.4.3.2. Harmful secondary metabolites ............................................................... 21 1.4.4. Role of secondary metabolites in fungal biology ............................................. 22 1.4.5. Rubellins: secondary metabolites produced by Ramularia collo-cygni ........... 23 1.5. Aims and objectives ................................................................................... 27 i Chapter 2: Identification and characterisation of secondary metabolite gene clusters in Ramularia collo-cygni ............................................................................ 28 2.1. Biosynthesis of fungal secondary metabolites ........................................... 29 2.1.1. The polyketide pathway ................................................................................... 32 2.1.1.1. Mechanism of polyketide biosynthesis .................................................... 32 2.1.1.2. Classification of fungal polyketide synthases ......................................... 34 2.1.1.3. Specific domains from non-reducing polyketide synthetases.................. 35 2.1.1.4. Release of the polyketide from the core enzyme ..................................... 35 2.1.2. The non-ribosomal peptide pathway ................................................................ 36 2.1.2.1. Non-ribosomal peptide elongation mechanism ....................................... 36 2.1.2.2. Classification of NRPS ............................................................................ 37 2.1.2.3. Peptide modification ................................................................................ 38 2.1.3. The hybrid PKS/NRPS pathway ...................................................................... 39 2.1.4. The role of secondary metabolism in plant pathogenic fungi .......................... 40 2.1.5. Objectives ......................................................................................................... 41 2.2. Materials and Methods ............................................................................... 42 2.2.1. Ramularia collo-cygni genome data ................................................................ 42 2.2.2. Identification of core genes .............................................................................. 42 2.2.2.1. Identification of putative core genes in Ramularia collo-cygni .............. 42 2.2.2.2. Secondary metabolites core gene domain analysis .................................. 44 2.2.2.3. Localisation of secondary metabolites core genes in the genome ........... 44 2.2.3. Identification of putative secondary metabolism gene clusters ........................ 44 2.2.4. Detailed investigation of selected secondary metabolite-related gene clusters 45 2.2.5. Phylogenetic study of secondary metabolites core genes................................. 45 2.3. Results ........................................................................................................ 47 2.3.1. Identification of secondary metabolism core genes in Ramularia collo-cygni 47 2.3.1.1. Identification of putative polyketide synthases and non-ribosomal peptide synthetases .................................................................................................................... 47 2.3.1.2. Identification of putative functional core genes ...................................... 51 2.3.2. Identification of putative secondary metabolism gene clusters ........................ 57 2.3.3. Characterisation of putative secondary metabolites clusters ............................ 65 2.3.3.1. Phylogenetic study ................................................................................... 65 2.3.3.2. Detailed study of selected clusters ........................................................... 74 2.4. Discussion ................................................................................................ 115 Chapter 3: Expression of Ramularia collo-cygni secondary metabolism-related core genes during the development of Ramularia leaf spot on barley seedling ..... .................................................................................................................................. 123 3.1. Secondary metabolites and disease development .................................... 124 3.1.1. Phytotoxins in disease development .............................................................. 124 3.1.1.1. Role of host specific toxins ................................................................... 124 3.1.1.2. Role of non-host specific toxins ...........................................................