A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/90321 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Molecular genetics of interactions between Xanthomonas campestris pv. raphani and Arabidopsis thaliana Vânia Isabel Horta de Passo Thesis submitted for the degree of Doctor of Philosophy in Plant and Environmental Sciences School of Life Sciences, University of Warwick August 2016 TABLE OF CONTENTS LIST OF FIGURES ................................................................................................. vi LIST OF TABLES ................................................................................................... ix LIST OF ABBREVIATIONS ................................................................................ xii ACKNOWLEDGEMENTS .................................................................................. xiv DECLARATION ................................................................................................... xvi SUMMARY ........................................................................................................... xvii CHAPTER 1. General introduction ........................................................................ 1 1.1 Xanthomonas, a genus of diverse bacterial plant pathogens ......................... 3 1.2 Pathogenic variants of Xanthomonas campestris ............................................ 6 1.2.1 Xanthomonas campestris pv. raphani ........................................................ 10 1.2.2 Xanthomonas campestris pv. campestris .................................................... 12 1.3 Pathogenicity factors of Xanthomonas .......................................................... 13 1.4 Plant immunity ................................................................................................ 14 1.5 Predicted gene-for-gene models to explain interactions between Xanthomonas campestris pathovars raphani and campestris, and Brassicaceae hosts .......................................................................................................................... 16 1.6 The plant model Arabidopsis thaliana ............................................................ 19 1.7 Molecular basis of resistance in Arabidopsis thaliana to Xanthomonas campestris ................................................................................................................. 20 1.8 Aims and objectives ......................................................................................... 22 CHAPTER 2. Phenotypic variation within Arabidopsis thaliana for resistance to Xanthomonas campestris pv. raphani .................................................................... 23 2.1 INTRODUCTION ........................................................................................... 24 2.2 MATERIALS AND METHODS .................................................................... 26 2.2.1 Arabidopsis thaliana accessions and growth conditions ............................ 26 2.2.2 Bacterial strains .......................................................................................... 30 2.2.3 Inoculum preparation .................................................................................. 32 2.2.4 Plant inoculations ........................................................................................ 32 2.2.5 Assessment of interaction phenotypes ........................................................ 33 2.2.6 Phenotyping experiments ............................................................................ 33 2.3 RESULTS ......................................................................................................... 35 2.3.1 Symptom development of Xanthomonas campestris strains in Arabidopsis thaliana .................................................................................................................. 35 i 2.3.2 Evaluation of the interaction phenotypes of Arabidopsis thaliana and Xanthomonas campestris ....................................................................................... 37 2.3.3 Interactions between twenty-two Arabidopsis thaliana accessions and Xanthomonas campestris strains ............................................................................ 39 2.3.4 Differential responses of Arabidopsis thaliana accessions to races of Xanthomonas campestris pv. raphani .................................................................... 42 2.3.5 Interactions between eighty-eight Arabidopsis thaliana accessions and the Xanthomonas campestris pv. raphani strain HRI8305 .......................................... 43 2.4 DISCUSSION .................................................................................................. 45 CHAPTER 3. Genetic characterization of resistance in Arabidopsis thaliana to Xanthomonas campestris pv. raphani .................................................................... 49 3.1 INTRODUCTION ........................................................................................... 50 3.2 MATERIALS AND METHODS .................................................................... 52 3.2.1 Bacterial strains .......................................................................................... 52 3.2.2 Plant materials ............................................................................................ 52 3.2.3 Phenotyping ................................................................................................ 53 3.2.4 Cross-pollination and self-pollination of Arabidopsis thaliana plants ....... 54 3.2.5 DNA extraction ........................................................................................... 55 3.2.6 DNA amplification ..................................................................................... 55 3.2.7 DNA sequencing and sequence analysis .................................................... 56 3.2.8 Molecular markers and genotyping of Arabidopsis thaliana plants derived from crosses between Columbia and Niederzenz accessions ................................ 56 3.2.9 Genotyping of Arabidopsis thaliana T-DNA insertion mutants ................ 58 3.2.10 Inheritance and segregation analysis .......................................................... 59 3.2.11 Statistical analysis for interval mapping ..................................................... 60 3.2.12 Clones for plant transformation .................................................................. 61 3.2.13 Arabidopsis thaliana transformation .......................................................... 63 3.2.14 Measurement of bacterial growth in planta ................................................ 64 3.2.15 Sequence analysis of the Arabidopsis thaliana gene At3g57710 ............... 65 3.3 RESULTS ......................................................................................................... 66 3.3.1 Mapping of resistance to Xanthomonas campestris pv. raphani race 2 (strain HRI8305) in MAGIC lines of Arabidopsis thaliana ............................................. 66 3.3.2 Inheritance and coarse-scale mapping of RXCR1 and RXCR2 ................... 69 3.3.3 Fine-mapping of RXCR1 ............................................................................. 73 3.3.4 Identification of RXCR1 gene by loss- and gain-of-function experiments . 77 3.3.5 In planta growth of Xanthomonas campestris pv. raphani strain HRI8305 ................................................................................................................. 82 3.3.6 RXCR1 alleles of resistant and susceptible Arabidopsis thaliana accessions ................................................................................................................ 84 ii 3.4 DISCUSSION .................................................................................................. 86 CHAPTER 4. Characterization of Xanthomonas campestris strains associated with outbreaks of a leaf spot and blight disease in vegetable brassicas in Mauritius ................................................................................................................. 90 4.1 INTRODUCTION ........................................................................................... 91 4.2 MATERIALS AND METHODS .................................................................... 94 4.2.1 Isolation of bacteria from leaf samples ....................................................... 94 4.2.2 Bacterial strains .......................................................................................... 94 4.2.3 DNA amplification and sequencing ............................................................ 96 4.2.4 Sequence similarity searches ...................................................................... 98 4.2.5 Phylogenetic analysis .................................................................................. 98 4.2.6 Plant material and growth conditions ......................................................... 98 4.2.7 Pathogenicity and race typing assays ......................................................... 99 4.1 RESULTS ......................................................................................................