Suppression and triggering of Arabidopsis immunity by Albugo species TORSTEN SCHULTZ-LARSEN Thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy September 2012 © This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom, may be published without the author’s prior written consent. Table&of&Content& Abstract ................................................................................................................................. 5 Acknowledgements ............................................................................................................... 6 Publications arising from this work ...................................................................................... 7 List of abbreviations .............................................................................................................. 8 1. INTRODUCTION ........................................................................................................... 11 1.1 Interactions between plants and microbes ................................................................. 11 1.2 PTI signaling ............................................................................................................. 13 1.2.1 Pathogen associated molecular paterns .............................................................. 13 1.2.2 PAMP recognition receptors .............................................................................. 13 1.2.3 Downstream signaling events and attenuation of signaling ............................... 15 1.3 Mechanisms of effector delivery ............................................................................... 16 1.3.1 General strategies to suppress host defense ....................................................... 16 1.3.2 The type III secretion system ............................................................................. 16 1.3.3 Effector secretion from haustoria forming pathogens ........................................ 17 1.3.4 Effector delivery in P. falciparum ..................................................................... 19 1.3.5 Uptake of effectors into plant cells .................................................................... 21 1.4 Identification of effectors in oomycetes .................................................................... 23 1.4.1 Introduction to oomycete genomics ................................................................... 23 1.4.2 Oomycete effectors are modular proteins .......................................................... 24 1.4.2.1 RXLR effectors ........................................................................................... 24 1.4.2.2 Crinkler effectors ........................................................................................ 26 1.4.3 Extracellular effectors ........................................................................................ 28 1.5 Biochemical functions of effectors inside the plant cell ........................................... 31 1.6 Plant surveillance of host processes .......................................................................... 34 1.6.1 Mechanisms of R-protein function ..................................................................... 34 1.6.2 The guard hypothesis ......................................................................................... 36 1.6.3 Decoy model ...................................................................................................... 38 1.6.4 The zig-zag-zig model ........................................................................................ 40 1.6.5 Events downstream of R-protein recognition .................................................... 41 1.7 Origin of the oomycetes ............................................................................................ 42 1.7.1 Albugo sp. .......................................................................................................... 43 1.7.2 The life cycle of Albugo sp. ............................................................................... 43 1.7.3 Arabidopsis resistance to A. candida and A. laibachii ....................................... 44 1.8 Aims of this research ................................................................................................. 46 2. General Materials and Methods ...................................................................................... 47 2.1 Plants ......................................................................................................................... 47 2.1.1 Plant growth ....................................................................................................... 47 2.1.2 Seed sterilization ................................................................................................ 48 2.1.3 Arabidopsis ecotypes and mutants used in this study ........................................ 48 2.1.4 Arabidopsis transformation ................................................................................ 48 2.1.5 Agrobacterium tumefaciens transient expression ............................................... 49 2.1.6 Reactive oxygen measurements ......................................................................... 50 2.2 Plant pathogens ......................................................................................................... 50 2.2.1 List of bacterial and oomycete strains ................................................................ 50 2.2.1.1 Oomycetes isolates ...................................................................................... 50 2.2.1.2 Bacterial strains ........................................................................................... 51 2.2.2 Bacterial strains and growth conditions ............................................................. 51 2.3 Antibiotics ................................................................................................................. 52 2.4 Microbial methods .................................................................................................... 52 ! 1! 2.4.1 Hpa Waco9 infection ......................................................................................... 52 2.4.2 A. laibachii and A. candida infection ................................................................. 52 2.4.3 Pseudomonas syringae infection ........................................................................ 53 2.4.4 Quantification of bacterial populations within leaves ........................................ 53 2.4.5 Trypan blue staining of Hpa structures .............................................................. 54 2.4.6 Quantification of Hpa conidiophores on adult trypan blue stained leaves ........ 54 2.4.7 Fluorescent detection of aerial Hpa, powdery mildew and P. infestans ............ 54 2.4.7.1 Staining of Hpa, powdery mildew and P. infestans structures .................... 54 2.4.7.2 Imaging Hpa, Powdery mildew and P. infestans structures with Uvitex 2B ................................................................................................................................. 55 2.4.7.3 Quantification of Hpa conidiophores on cotyledons using uvitex 2B ........ 55 2.4.8 Infection of N. benthamiana leaves with Phytophthora sp. ............................... 56 2.4.8.1 Rye A plates ................................................................................................ 56 2.4.8.2 Phytophthora growth and infection ............................................................. 56 2.5 Molecular biology methods ....................................................................................... 56 2.5.1 DNA ................................................................................................................... 56 2.5.1.1 Rapid DNA isolation for amplicons less than 1000 bp ............................... 56 2.5.1.2 High molecular weight DNA isolation from Albugo sp. and Arabidopsis . 57 2.5.1.3 Ethanol precipitation of DNA ..................................................................... 57 2.5.1.4 Plasmid isolation ......................................................................................... 57 2.5.1.5 DNA Digestion ............................................................................................ 57 2.5.1.6 Polymerase chain reaction (PCR) ............................................................... 58 2.5.1.7 Agarose gel electrophoresis and gel extraction ........................................... 60 2.5.1.8 Cloning ........................................................................................................ 60 2.5.1.9 DNA sequencing ......................................................................................... 61 2.5.1.10 Site directed mutagenesis .......................................................................... 61 2.5.2 RNA ................................................................................................................... 61 2.5.2.1 Isolation of total RNA ................................................................................. 61 2.5.2.2 3’ and 5’ RACE PCR .................................................................................. 62 2.5.3 Bacteria .............................................................................................................. 62 2.5.3.1