Characterisation of the RAM pathway in Ustilago maydis

Resumen

Abbreviations 9 Introduction 11 1. Topic and model study 13 2. Principal pathways involved in morphology regulation in fungi 2.1. The nutrient-sensing cAMP pathway 2.2. The MAPK cascade 14 14 16 3. The RAM pathway: new signalling network 3.1. Description of the RAM pathway in Saccharomyces cerevisiae 3.2. The RAM pathway is conserved from yeast to human 3.3. Interplay between the RAM pathway and other signalling networks in morphology regulation 17 17 19 21 4. The fungal model Ustilago maydis 4.1. U. maydis life cycle 4.2. The mating type loci 23 23 24 5. Signaling pathways in U. maydis 5.1. The cAMP/PKA pathway 5.2. The pheromone signaling MAPK pathway 5.3. Cell wall integrity MAPK pathway in U. maydis 5.4. Interplay between cAMP and MAPK pathways in U. maydis 26 26 28 29 29 6. The RAM pathway in U. maydis 32 Objectives 35 Materials and Methods 37 1. Strain, media and growth conditions 1.1. Strains and plasmids 1.2. Growth media and conditions 39 39 41 2. Molecular methodology 2.1. Restriction mapping and subcloning 2.2. PCR reaction 2.3. U. maydis transformation 43 43 43 44 4 3. Biochemical techniques 3.1. Genomic DNA extraction 3.2. Biological methods involving RNA 3.3. Biological methods involving Protein 44 44 45 47 4. Microscopy 4.1 Nucleus staining 4.2. Septum staining 48 49 49 5. Mating on water agar assay 50 6. Mating and pheromone stimulation 50 7. Pathogenicity assay 51 8. In silico analysis 51 9. Strain and plasmid constructions 9.1. Constructions realized for RAM pathway components deletions 9.2. Constructions realized for the Ukc1 characterisation 9.3. Constructions realized to show the interaction between Ukc1p and Mob2p 52 52 54 55 Results 57 Section I: The RAM pathway organization in Ustilago maydis 59 1. The central NDR kinase Ukc1 1.1. Sequence analysis 1.2. Ukc1 is involved in morphology, cell separation and virulence regulation 1.3. Ukc1 subcellular localisation 61 61 62 64 2. The Mob2 regulator 2.1. Identification of the Mob2 protein homologue in U. maydis 2.2. Deletion of mob2 presented the same defects as ¿ukc1 cells 2.3. Mob2 forms a complex with Ukc1 66 66 67 68 3. Conservation of other regulatory proteins 3.1. Sequences identification of Tao3, Hym1, Kic1 and Sog2 homologues 3.2. Sog2, Hym1, Tao3 and Nak1 proteins seemed to act in the same protein network as Mob2 and Ukc1 69 70 71 4. Research of an Ace2p RAM target homologue in U. maydis 72 5 Section II: The RAM pathway is involved in morphogenesis regulation 75 1. Cytoskeleton organization in ¿ukc1 cells 1.1. F-actin organisation in ¿ukc1 mutant cells 1.2. Microtubules organisation in cells lacking ukc1 1.3. Septin organisation in ¿ukc1 mutants 77 77 80 81 2. RAM pathway and growth polarity 82 Section III: The RAM pathway is indispensable for Ustilago maydis virulence 85 1. Avirulence of RAM mutant cells 87 2. ¿ukc1 cells were able to induce infective structures 2.1. Conjugative tube formation induced by fuz7DD is not abolished by ukc1 deletion 2.2. ¿ukc1 mutant cells can form an infective tube 88 88 89 3. ¿ukc1 cells are defective in cell-cell fusion 90 4. Ukc1 localises at the extremity of the conjugative tube 91 5. ¿ukc1 cells are defective in pheromone response 92 6. ukc1 deletion affects prf1 transcription 93 7. Constitutive expression of prf1 rescue the pheromone response 94 Section IV: Interplay between the RAM pathway and other protein networks 97 1. Crosstalk between the cAMP and the RAM pathway 1.1. Inhibition of PKA activity induced stronger filamentation of ¿ukc1 cells 1.2. PKA activation reduces filamentation of ¿ukc1 cells 1.3. PKA activation can not rescue the mfa1 transcription defect of ¿ukc1 cells 1.4. crk1 transcription appears deregulated in ¿ukc1 strain 99 99 100 102 103 2. MAPK and RAM pathway in U. maydis 2.1. Pheromone MAPK and RAM pathway 2.2. Crosstalk between the Cell Wall Integrity MAPK and the RAM pathways 104 104 105