Two types of calcium signalling in legume-rhizobia symbiosis

6 ACKNOLEDGEMENTS 7 LIST OF TABLES 9 LIST OF FIGURES 10 LIST OF ABBREVIATIONS 12 CHAPTER 1. GENERAL INTRODUCTION 15 NITROGEN AVAILABILITY: A GLOBAL PROBLEM 15 SYMBIOTIC NITROGEN FIXATION 15 BRIEF OVERVIEW OF LEGUME-RHIZOBIUM SYMBIOSIS 17 EARLY STEPS OF LEGUME-RHIZOBIUM SYMBIOSIS: A MOLECULAR COMMUNICATION 17 FLAVONOIDS 17 NOD FACTOR 18 NOD FACTOR RECOGNITION IN THE PLANT 22 RECEPTOR-LIKE KINASES 23 LECTIN NUCLEOTIDE PHOSPHOHYDROLASE 24 EARLY PLANT RESPONSES TO NOD FACTORS 25 MEMBRANE DEPOLARIZATION AND PH CHANGE 25 CALCIUM RESPONSES IN NOD FACTOR SIGNALLING 26 CALCIUM SIGNALLING IN PLANT CELLS 26 ION FLUXES 27 CALCIUM SPIKING 29 GENES INVOLVED IN THE ACTIVATION OF CALCIUM SPIKING 33 LRR RECEPTOR-LIKE KINASE 33 ION CHANNELS REQUIRED FOR CALCIUM SPIKING 34 THE ROLE OF THREE NUCLEOPORINS PROTEINS REQUIRED FOR CALCIUM SPIKING 36 PHOSPHOLIPID SIGNALLING 36 GENES INVOLVED IN THE PERCEPTION OF CALCIUM SPIKING 37 A CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE 38 TRANSCRIPTION FACTORS TRANSDUCE THE SIGNAL DOWNSTREAM OF CCAMK 38 ROOT HAIR DEFORMATION 39 BACTERIAL INFECTION 42 NODULE FORMATION 45 PLANT HORMONES 45 MYCORRHIZAL SYMBIOSIS 47 RESEARCH OBJECTIVE OF THIS THESIS 50 CHAPTER 2. MATERIALS AND METHODS 51 PLANT MATERIAL 51 BACTERIAL STRAINS 51 MEDIA 51 ANTIBIOTICS 52 CHEMICALS 52 PLANT GROWTH CONDITIONS 53 M. TRUNCATULA SEED STERILISATION AND PLANT GROWTH 53 L. JAPONICUS SEED STERILISATION AND PLANT GROWTH 53 PREPARATION OF NOD FACTOR 54 NOD FACTOR EXTRACTION FROM LIQUID MEDIUM 54 HPLC ANALYSIS AND MASS SPECTROMETRY QUANTIFICATION OF NOD FACTOR 54 BIOLOGICAL ESTIMATION OF NOD FACTOR ACTIVITY 56 PHYSIOLOGICAL TECHNIQUES 57 ROOT HAIR DEFORMATION 57 ENOD11-GUS INDUCTION ASSAY 57 CALCIUM IMAGING WITH OREGON GREEN 58 PLANT PREPARATION 58 DYE PREPARATION 58 NEEDLE PREPARATION 58 MICROINJECTION 59 IMAGING BY EPIFLUORESCENT MICROSCOPE 59 CALCIUM IMAGING WITH THE CAMELEON SYSTEM YC 2.1 59 CALCIUM IMAGING WITH THE NUCLEAR-TARGETED CAMELEON NUPYC2.1 60 AGROBACTERIUM RHIZOGENES-MEDIATED ROOT TRANSFORMATION 60 CALCIUM IMAGING 61 MOLECULAR BIOLOGICAL TECHNIQUE 61 RNA EXTRACTION AND DNASEI TREATMENT 61 QUANTITATIVE RT-PCR 62 CHAPTER 3. ANALYSIS OF THE EFFECT OF NOD FACTOR STRUCTURE ON CALCIUM FLUX 63 INTRODUCTION 63 RESULTS 70 ANALYSIS OF NOD FACTOR-INDUCED CALCIUM FLUX IN ROOT HAIR CELLS USING YC 2.1 70 TITRATION OF CALCIUM INFLUX AND CALCIUM SPIKING USING YC 2.1 73 ANALYSIS OF CALCIUM FLUX INDUCTION BY NODFL NOD FACTOR 75 ANALYSIS OF CALCIUM FLUX INDUCTION BY NODL NOD FACTOR 79 DISCUSSION 80 CHAPTER 4. ANALYSIS OF CALCIUM RESPONSES IN INFECTION MUTANTS 83 INTRODUCTION 83 RESULTS 86 ANALYSIS OF CALCIUM FLUX USING MICROINJECTION OF OREGON GREEN 86 TITRATION OF CALCIUM FLUX USING MICROINJECTION OF OREGON GREEN 88 ANALYSIS OF CALCIUM FLUX IN M. TRUNCATULA MUTANTS LACKING INFECTION THREADS 90 ANALYSIS OF CALCIUM RESPONSES IN MT RIT1 AND LJ NAP1-1, AND PIR1-1 MUTANTS 95 DISCUSSION 100 CHAPTER 5. L. JAPONICUS SYMRK-14 FORMS NODULES BUT IS IMPAIRED FOR CALCIUM SPIKING 107 INTRODUCTION 107 RESULTS 112 LJSYMRK-14 IS IMPAIRED FOR NOD FACTOR-INDUCED CALCIUM SPIKING 112 DISCUSSION 119 CHAPTER 6. LNP IS A NEW COMPONENT REQUIRED FOR NOD-FACTOR SIGNALLING IN L. JAPONICUS 127 INTRODUCTION 127 RESULTS 134 ANTISENSE SUPPRESSION OF LNP EXPRESSION RESULTS IN A NOD AND MYC PHENOTYPE 134 EXPRESSION OF NIN IS IMPAIRED IN LNP ANTISENSE LINE 138 LNP IS REQUIRED FOR ACTIVATION OF NOD FACTOR-INDUCED CALCIUM SPIKING 140 THE 5’D LNP ANTISENSE LINE IS DEFECTIVE FOR CALCIUM FLUX 142 ROOT HAIR DEFORMATION IS NORMAL IN LNP ANTISENSE LINE 144 DISCUSSION 146 CHAPTER 7. GENERAL DISCUSSION AND CONCLUSIONS 155 THE NOD FACTOR SIGNALLING PATHWAY IN NODULATION 155 A LINK BETWEEN CALCIUM SPIKING AND BACTERIAL INFECTION 158 A ROLE FOR CALCIUM FLUX IN INFECTION THREAD INITIATION 160 SYMRK FUNCTION: A BRANCH POINT 162 ACTIVATION OF CORTICAL RESPONSES 163 CONCLUSIONS 165