Roles of the plant cell wall in powdery mildew disease resistance in Arabidopsis thaliana: PMR5 (POWDERY MILDEW RESISTANT 5) affects the acetylation of cell wall pectin By Candice Cherk Lim A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Shauna Somerville, Chair Professor Patricia Zambryski Associate Professor Mary Wildermuth Professor James Berger Spring 2013 Abstract Roles of the plant cell wall in powdery mildew disease resistance in Arabidopsis thaliana: PMR5 (POWDERY MILDEW RESISTANT 5) affects the acetylation of cell wall pectin by Candice Cherk Lim Doctor of Philosophy in Plant Biology University of California, Berkeley Professor Shauna Somerville, Chair The pmr5 (powdery mildew resistant 5) mutant was found in a screen for genes involved in susceptibility to Golovinomyces cichoracearum, a biotrophic pathogen that infects Arabidopsis. PMR5 is a member of the TBL (TRICHOME BIREFRINGENCE LIKE) family, which is composed of 46 functionally uncharacterized plant-specific proteins. Initial characterization of this mutant showed that pmr5-mediated disease resistance acts independently of the salicylic acid, jasmonic acid, and ethylene signal transduction pathways, and that there are changes in the pmr5 cell wall that may be linked to the gain of resistance in the mutant. Specifically, PMR5 may be affecting cell wall pectin by acetylation. Characterization of the pmr5 cell wall has revealed changes in pectin composition and a decrease in acetylation. This is corroborated by the ability of heterologously expressed PMR5 protein to bind to pectin, with decreased binding affinity to acetylated pectin. The cell wall and disease phenotypes of the pmr5 mutant may be revealing a potential role of PMR5 in the elusive plant cell wall integrity-signaling pathway. This work summarizes the continuing efforts in both determining the biochemical function of PMR5 and investigating the mechanism behind pmr5-mediated disease resistance. 1 Dedication To A.Y.L. and T.Y.L. i Table of Contents Abstract........................................................................................................................................... 1 Dedication........................................................................................................................................ i Table of Contents............................................................................................................................ ii List of Figures................................................................................................................................iii List of Tables .................................................................................................................................. v Acknowledgements........................................................................................................................ vi CHAPTER 1: Defense at the plant cell wall................................................................................... 1 Preface......................................................................................................................................... 2 Part I: Host pathogen warfare at the plant cell wall.................................................................... 2 Part II: An update on trends in plant cell wall defense: the cell wall integrity (CWI) pathway. 9 Figures....................................................................................................................................... 16 CHAPTER 2: Investigating the function of PMR5 (POWDERY MILDEW RESISTANT 5).... 18 Introduction............................................................................................................................... 19 Materials and Methods.............................................................................................................. 20 Results....................................................................................................................................... 28 Discussion................................................................................................................................. 42 Figures....................................................................................................................................... 45 Tables........................................................................................................................................ 82 Appendix................................................................................................................................... 93 CHAPTER 3: Identifying potential pmr5-mediated disease resistance pathway partners ........... 95 Introduction............................................................................................................................... 96 Materials and Methods.............................................................................................................. 96 Results....................................................................................................................................... 97 Discussion................................................................................................................................. 99 Tables...................................................................................................................................... 101 CHAPTER 4: Suppressors of pmr5-mediated disease resistance............................................... 107 Introduction............................................................................................................................. 108 Materials and Methods............................................................................................................ 108 Results..................................................................................................................................... 109 Discussion............................................................................................................................... 112 Figures..................................................................................................................................... 114 Tables...................................................................................................................................... 120 CHAPTER 5: A hypothetical model for pmr5-mediated disease resistance .............................. 131 References Cited ......................................................................................................................... 134 ii List of Figures Figure 1.1. Electron micrographs of different pathogens infecting plant cells .............................16 Figure 1.2. Overview of the plant cell signaling components mediating responses upon pathogen attack .............................................................................................................................................17 Figure 2.1. Modified TBL protein family tree with G. cichoracearum phenotypes .....................45 Figure 2.2. Schematic of pectin structure .....................................................................................46 Figure 2.3. Secondary structure alignments of PMR5, putative alpha-galactosidase, and rhamnogalacturonan acetylesterase ...............................................................................................47 Figure 2.4. PMR5 gene expression generated by the eFP browser................................................48 Figure 2.5. Schematic of PMR5 gene structure .............................................................................49 Figure 2.6. Virus induced gene silencing of PMR5 in Arabidopsis...............................................50 Figure 2.7. Complementation after site-directed mutagenesis of catalytic residues .....................52 Figure 2.8. Principle component analysis of Fourier transform infrared spectroscopy spectra from wild-type and pmr5 cell walls .......................................................................................................53 Figure 2.9. Oligosaccharide mass profiling analysis of xyloglucan composition ........................54 Figure 2.10. Acetic acid assay comparing acetylation of cell walls ..............................................55 Figure 2.11. Saccharification assay comparing cell wall digestability..........................................56 Figure 2.12. Cell wall immunolabeling of stem sections...............................................................57 Figure 2.13. Example of immunolabeling of spotted extracts with LM6 anti-arabinan antibody.58 Figure 2.14. Glycome profiling of rosette leaves .........................................................................59 Figure 2.15. Arabidopsis seed mucilage is rich in rhamnogalacturonan I ....................................60 Figure 2.16. Morphology and phenotypes of transgenic lines used in localization study.............61 Figure 2.17. FTIR spectrum of cell walls from plants expressing 35S:PMR5-GFP ....................62 Figure 2.18. Localization of PMR5 in plants expressing pPMR5:PMR5:GFP.............................63 Figure 2.19. PMR5 is more highly expressed in root cortical cells in plants expressing pPMR5:PMR5:GFP.......................................................................................................................64