Lipid and Acetate Metabolism Influence Host Colonization by the Fungal Plant Pathogen Ustilago maydis by Scott Cameron Lambie BSc, Trent University, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology & Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2014 © Scott Cameron Lambie, 2014 Abstract Ustilago maydis is an obligate fungal pathogen of maize that causes disease known as the common smut of corn. Haploids with compatible mating loci fuse to form a dikaryotic cell type that is filamentous and pathogenic; invasion of the host by this cell type leads to the formation of tumors which contain diploid teliospores. Colonization of the plant presents numerous challenges for U. maydis because the host environment may be limited in nutrients and plant defense responses lead to the creation of toxic molecules such as reactive oxygen species (ROS). Understanding the mechanisms employed by U. maydis to overcome such obstacles is necessary to develop strategies to fight smut disease and protect crops from disease caused by fungal plant pathogens. This study focused on the characterization of a group of genes encoding phospholipases (PLs), enzymes which have been implicated in virulence, morphogenesis and nutrient acquisition of a number of pathogenic fungi. This work included a characterization of one PL, Lip2, which may function to repair ROS-induced damage of membranes that occurs during host colonization. Mutants defective in lip2 were less virulent in maize seedlings, showed sensitivity to H2O2 and the drugs chloroquine and quinacrine, and were resistant to ionic stress. A transcriptional profile of a Δlip2 mutant suggested that Lip2 contributes to stress responses and carbon metabolism. Furthermore, lipidomic profiles of the Δlip2 mutant revealed changes in lipid composition that may be linked to the mutant phenotypes. Other aspects of central carbon metabolism were also explored including the utilization of acetate as a carbon source by U. maydis and the role of an ATP-citrate lyase, Acl1, in pathogenesis and fungal development. Acetate had a repressive effect on mating and filamentation, and promoted reduced growth and virulence compared to favorable carbon sources ii such as glucose. Furthermore, Acl1 was shown to be essential to cause disease and for growth on glucose. Overall, this study reveals potential mechanisms employed by U. maydis during plant colonization to resist the defense response. It also highlights the utility U. maydis as a model system to understand the metabolic and nutritional aspects of fungal phytopathogenesis. iii Preface The design and execution of the experiments within were performed by myself, Scott Cameron Lambie, under the guidance and supervision of Dr. James W. Kronstad with the following exceptions: primers designed to construct a deletion construct for the lip2 gene were designed by Dr. Jana Klose (Section 2.2.4). Generation and confirmation of acl1 deletion strains was performed by Dr. Emma Griffiths, who also contributed to the characterization of those strains by performing the growth measurements presented in Figure 16 (Sections 3.3.1 and 3.3.2). For the RNA-Seq experiments, library creation and initial bioinformatics processing was performed by Genewiz, Inc. with the assistance of Dr. Janet Chang; details regarding materials and methods for these experiments presented in sections 2.2.17 and 2.2.18 were provided by Genewiz, Inc. GO enrichment analysis was performed by Dr. Daniel Croll, who also provided the details regarding the materials and methods in section 2.2.18. Lipidomics experiments and initial bioinformatics processing were performed by Dr. Jun Han at the Metabolomics Innovation Center, who also provided the details regarding the materials and methods in sections 2.2.19 and 2.2.20. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ..................................................................................................................................x List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................... xvi Dedication ................................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................1 1.1 An Introduction to Ustilago maydis ................................................................................ 1 1.2 Metabolic Adaptations and Lipid Metabolism in U. maydis .......................................... 3 1.3 Phospholipases and Their Role in Fungal Pathogenesis ................................................. 4 1.3.1 An Introduction to Phospholipases ............................................................................. 4 1.3.2 Phospholipases as Virulence Factors in Pathogenic Fungi ......................................... 6 1.3.3 The Platelet Activating Factor Acetylhydrolase ......................................................... 7 1.3.3.1 The PAFAH Family in Fungi ............................................................................. 8 1.3.3.2 Reactive Oxygen Species, Lipid Peroxidation and the Plant Defense Response ………………………………………………………...……………………….10 1.3.3.3 Dealing With the Oxidative Burst as a Fungal Pathogen ................................. 12 1.4 Additional Aspects of Metabolic Adaptations in Fungal Pathogenesis ........................ 16 1.4.1 Alternative Carbon Source Utilization ...................................................................... 16 1.4.2 Acetate Utilization .................................................................................................... 17 v 1.4.3 Acetyl-CoA and its Role as a Central Metabolite ..................................................... 19 1.4.3.1 ATP-Citrase Lyase ............................................................................................ 20 1.4.3.2 The Role of Acl in Fungi .................................................................................. 21 1.5 Thesis Objectives .......................................................................................................... 22 Chapter 2: Lip2 ............................................................................................................................25 2.1 Introduction ................................................................................................................... 25 2.2 Materials and Methods .................................................................................................. 26 2.2.1 Strains and Growth Conditions ................................................................................. 26 2.2.2 DNA Isolation ........................................................................................................... 26 2.2.3 Identification of PLs in the U. maydis Genome ........................................................ 27 2.2.4 Construction of Knockout Cassettes for Targeted Gene Deletion in U. maydis ...... 28 2.2.5 Biolistic Transformation of U. maydis...................................................................... 32 2.2.6 PCR Confirmation of Mutants .................................................................................. 33 2.2.7 Southern Blot Confirmation of Mutants ................................................................... 33 2.2.8 RNA Isolation for qRT-PCR .................................................................................... 35 2.2.9 qRT-PCR Gene Expression Analysis ....................................................................... 37 2.2.10 Growth Measurements .......................................................................................... 37 2.2.11 Mating Assays ....................................................................................................... 37 2.2.12 Virulence Assays .................................................................................................. 38 2.2.13 Spot Assays ........................................................................................................... 39 2.2.14 Sensitivity to Oxidative Stress .............................................................................. 39 2.2.15 Microscopy ........................................................................................................... 40 2.2.16 RNA Isolation for RNA-Seq ................................................................................. 40 vi 2.2.17 Library Preparation for RNA-Seq ......................................................................... 41 2.2.18 RNA-Seq Bioinformatics Analysis ......................................................................