THE EVOLUTION OF SECONDARY METABOLISM REGULATION AND PATHWAYS IN THE ASPERGILLUS GENUS By Abigail Lind Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Informatics August 11, 2017 Nashville, Tennessee Approved: Antonis Rokas, Ph.D. Tony Capra, Ph.D. Patrick Abbot, Ph.D. Louise Rollins-Smith, Ph.D. Qi Liu, Ph.D. ACKNOWLEDGEMENTS Many people helped and encouraged me during my years working towards this dissertation. First, I want to thank my advisor, Antonis Rokas, for his support for the past five years. His consistent optimism encouraged me to overcome obstacles, and his scientific insight helped me place my work in a broader scientific context. My committee members, Patrick Abbot, Tony Capra, Louise Rollins-Smith, and Qi Liu have also provided support and encouragement. I have been lucky to work with great people in the Rokas lab who helped me develop ideas, suggested new approaches to problems, and provided constant support. In particular, I want to thank Jen Wisecaver for her mentorship, brilliant suggestions on how to visualize and present my work, and for always being available to talk about science. I also want to thank Xiaofan Zhou for always providing a new perspective on solving a problem. Much of my research at Vanderbilt was only possible with the help of great collaborators. I have had the privilege of working with many great labs, and I want to thank Ana Calvo, Nancy Keller, Gustavo Goldman, Fernando Rodrigues, and members of all of their labs for making the research in my dissertation possible. I have been supported by a National Library of Medicine training grant T15LM007450 during my time at Vanderbilt, which has also given me the opportunity to travel to conferences and present my work. I am grateful to both the Department of Biomedical Informatics and the National Library of Medicine for their support. I am grateful to my family and friends for their support, love, and encouragement. My husband Nathan has helped me in more ways than I can possibly name during the last five years, but especially by encouraging me during times of self-doubt. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................................ ii LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii LIST OF ABBREVIATIONS ........................................................................................................ ix Chapter I. INTRODUCTION ........................................................................................................................1 Ecological roles of fungal secondary metabolites .......................................................................1 Secondary metabolic pathways in fungal genomes .....................................................................5 Regulation of fungal secondary metabolism................................................................................7 Chapter previews .........................................................................................................................9 II. EXAMINING THE EVOLUTION OF THE REGULATORY CIRCUIT CONTROLLING SECONDARY METABOLISM AND DEVELOPMENT IN THE FUNGAL GENUS ASPERGILLUS ..........................................................................................................................12 Authors .......................................................................................................................................12 Introduction ................................................................................................................................12 Methods......................................................................................................................................16 Genome sequences and orthogroup definitions for A. nidulans, A. fumigatus, A. oryzae, and A. niger .............................................................................................................................16 Gene category definitions .......................................................................................................17 Strains and culture conditions .................................................................................................17 RNA extraction .......................................................................................................................18 RNA sequencing .....................................................................................................................18 RNA-seq read alignment and differential gene expression ....................................................19 Statistical analyses ..................................................................................................................19 Results ........................................................................................................................................20 The majority of SM gene clusters in Aspergillus are species-specific ...................................20 Aspergillus SM genes are significantly less conserved than genes for primary metabolism .24 VeA regulates the same biological processes as well as the same fraction of the genome in both A. nidulans and A. fumigatus ....................................................................................26 MtfA’s regulatory role is smaller in scope in A. fumigatus compared to A. nidulans ............29 Regulation of similar processes regardless of gene conservation ...........................................30 Discussion ..................................................................................................................................32 Aspergillus secondary metabolic genes and gene clusters are largely species-specific ...33 The evolution of the circuit regulating secondary metabolism and development ............35 iii III. REGULATION OF SECONDARY METABOLISM BY THE VELVET COMPLEX IS TEMPERATURE-RESPONSIVE IN ASPERGILLUS ...........................................................38 Authors .......................................................................................................................................38 Introduction ................................................................................................................................38 Methods......................................................................................................................................41 Strains and culture conditions .................................................................................................41 RNA isolation .........................................................................................................................41 RNA sequencing .....................................................................................................................42 Gene expression analysis ........................................................................................................42 Functional enrichment analysis...............................................................................................43 Gene cluster expression ..........................................................................................................43 Data availability ......................................................................................................................44 Results ........................................................................................................................................44 Temperature shift changes the expression of 10% of all genes and of more than half of the genes in SM gene clusters ................................................................................................44 VeA regulates a much large number of genes at 37ºC than at 30ºC .......................................46 LaeA regulates similar numbers and types of genes at 30ºC and 37ºC ..................................48 VeA and LaeA have greater regulatory overlap at 37ºC than at 30ºC ....................................50 Many SM gene clusters are regulated by both VeA and LaeA at 37ºC, but only by LaeA at 30ºC ..................................................................................................................................53 Discussion ..................................................................................................................................55 IV. TRANSCRIPTIONAL REGULATORS OF ASEXUAL DEVELOPMENT CONTROL BOTH SPORE AND HYPHAL SECONDARY METABOLIC PATHWAYS IN ASPERGILLUS FUMIGATUS ..................................................................................................59 Authors .......................................................................................................................................59 Introduction ................................................................................................................................59 Methods......................................................................................................................................61 Growth conditions and RNA isolation .....................................................................................61