UNIVERSITE MONTPELLIER II SCIENCES ET TECHINIQUES DU LANGUEDOC THESE Présentée pour l‘obtention du titre de Docteur en Sciences de l’Université Montpellier II Formation Doctorale : Biologie Intégrative des Plantes Ecole Doctorale : SIBAGHE – Systèmes Intégrés en Biologie, Agronomie, Géosciences, Hydro sciences et Environnement par Alessandra Ferreira RIBAS Characterization of a resistance locus to coffee leaf rust (Hemileia vastratrix) in coffee trees: genomic organization, diversity and development of tools for functional gene validation Soutenue publiquement devant le jury composé de, Mme Françoise ADAN-BLONDON (INRA), Rapporteur Mme Valérie GEFFROY (INRA), Examinatrice M. Emmanuel DOUZERY (UM2), Examinateur M. Gilles PILATE (INRA), Rapporteur M. Hervé ETIENNE (CIRAD), Co-Directeur de thèse M. Philippe LASHERMES (IRD), Co-Directeur de thèse ABREVIATIONS BAC Bacterial Artificial Chromosome 6-BA: 6-benzylaminopurine CaMV: cauliflower mosaic virus CC Coiled-Coil CGA candidate gene approach CG candidate gene CLR Coffee Leaf Rust CP medium: callus proliferation medium DNA Deoxyribonucleic acid 2,4-D: 2,4-dichlorophenoxyacetic acid ECP medium: embryogenic callus production medium ETI Effector-Triggered Immunity ETS Effector-Triggered Susceptibility GFP Green Fluorescent Protein HR Hypersensitive Response HPTII hygromycin phosphotransferase II i.e. id est Ka non-synonymous substitutions Ks synonymous substitutions LRR Leucine-Rich Repeats LTR Long Terminal Repeat M medium: maturation medium MAMP Microbe-Associated Molecular Pattern MAS market assisted selection NBS Nucleotide-Binding site NPTII Neomycin Phosphotransferase III OD: optical density PAMP Pathogen-Associated Molecular Pattern PCR Polymerase Chain Reaction PEMs: proembryogenic masses PTI PAMP-Triggered Immunity QTL Quantitative-Trait Locus R: regeneration medium RLK Receptor-Like Kinase RLP Receptor-Like Protein SD: standard deviation SNP single nucleotide polymorphism TAIR: the Arabidopsis information resources TIR Toll and Interleukin-1 Receptor SUMMARY LIST OF FIGURES .................................................................................................................... 7 LIST OF TABLES .................................................................................................................... 11 General Introduction ................................................................................................................ 13 CHAPTER I ............................................................................................................................... 14 LITERATURE REVIEW ......................................................................................................... 14 PART I ....................................................................................................................................... 15 1. Generalities about Coffee ................................................................................................. 15 1.1 Coffee consumption, cultivation and economical trade .................................................... 15 1.2 Coffee taxonomy ............................................................................................................... 16 1.3 Characteristics of cultivated coffee species ....................................................................... 19 1.4 Cytological aspects of Coffea ............................................................................................ 22 1.5 Speciation and genome features of C. arabica .................................................................. 22 1.6 Coffee arabica genetic diversity and breeding .................................................................. 23 1.7 Interspecific hybridization and coffee introgression ......................................................... 26 2. Coffee leaf rust: the main coffee disease ............................................................................. 28 2.1 The genus Hemileia ........................................................................................................... 28 2.2 Coffee leaf rust (CLR) symptoms ..................................................................................... 29 2.3 CLR epidemic ................................................................................................................... 31 2.4 Disease Management......................................................................................................... 32 2.4.1 Chemical control ........................................................................................................ 33 2.4.2 Biological control ....................................................................................................... 33 2.5.1 CLR resistance genes ................................................................................................. 35 2.5.2 Physiological races of Hemileia vastatrix .................................................................. 36 3. Identification of molecular markers associated to CLR .................................................... 39 3.1 Partial resistance to CLR ................................................................................................... 41 PART II ...................................................................................................................................... 42 1. Mechanisms of disease resistance in plants ..................................................................... 42 1.1 Non host resistance............................................................................................................ 42 1.1.1 PAMP/MAMP-triggered immunity (PTI/MTI) ......................................................... 44 1.2 Host resistance (Effector-Triggered Immunity (ETI)) ...................................................... 45 2. Modes of plant-pathogen interaction ............................................................................... 49 2.1 Direct Interaction .............................................................................................................. 49 2.2 The Guard Model (indirect R-Avr) interactions ................................................................ 50 2.3 Decoy model ..................................................................................................................... 51 3. Avirulence genes (effectors) .............................................................................................. 53 4. Resistance Genes (R genes) ............................................................................................... 54 4.1 Classes of Resistance Gene ............................................................................................... 58 4.1.1 RLP and RLK class of R genes .................................................................................. 58 4.2.1 The function of main domains in NB-LRR proteins .................................................. 61 5. How NBS-LRR proteins recognize pathogens and activate defense responses? ......... 66 6. Expression of R proteins ................................................................................................... 67 7. Plant-pathogen co evolution ............................................................................................. 68 7.1 Evolution of R genes at plant population level ................................................................. 68 7.2 Evolutionary patterns R genes clusters ............................................................................. 71 8. Wide genome analysis of NBS-LRR encoding genes ...................................................... 72 8.1 Organization of NBS-LRR genes into plant genomes ...................................................... 75 9. Mechanisms of R-genes diversification ........................................................................... 77 9.1 Duplication ........................................................................................................................ 79 9.1.1 Duplication by polyploidization ................................................................................. 79 9.2 Sequence recombination ................................................................................................... 81 9.3 Selection ............................................................................................................................ 82 9.4 Other mechanisms that contributed to generate R gene diversification ............................ 84 10. Patterns of R gene function .......................................................................................... 86 11. Comparative analysis of R genes ................................................................................. 88 PART III .................................................................................................................................... 91 1. Identification of candidate genes...................................................................................... 91 1.1 Identification of CG genes based on genomic synteny ..................................................... 91 1.2 Identification of CG using positional cloning strategy ..................................................... 93 2. Functional validation of CG ............................................................................................. 95 2.1 RNA interference .............................................................................................................. 95 2.2