Graduate Theses, Dissertations, and Problem Reports 2009 Ergot alkaloid biosynthesis in Aspergillus fumigatus : Association with sporulation and clustered genes common among ergot fungi Christine M. Coyle West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Coyle, Christine M., "Ergot alkaloid biosynthesis in Aspergillus fumigatus : Association with sporulation and clustered genes common among ergot fungi" (2009). Graduate Theses, Dissertations, and Problem Reports. 4453. https://researchrepository.wvu.edu/etd/4453 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Ergot alkaloid biosynthesis in Aspergillus fumigatus: Association with sporulation and clustered genes common among ergot fungi Christine M. Coyle Dissertation submitted to the Davis College of Agriculture, Forestry, and Consumer Sciences at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics and Developmental Biology Daniel G. Panaccione, Ph.D., Chair Kenneth P. Blemings, Ph.D. Joseph B. Morton, Ph.D. Alan J. Sexstone, Ph.D. Sharon L. Wenger, Ph.D. Division of Plant and Soil Sciences Morgantown, West Virginia 2009 Keywords: Aspergillus fumigatus, ergot alkaloid biosynthesis, sporulation Copyright 2009 Christine M. Coyle ABSTRACT Ergot alkaloid biosynthesis in Aspergillus fumigatus: Association with sporulation and clustered genes common among ergot fungi Christine M. Coyle Ergot alkaloids, indole-derived mycotoxins, interact with multiple monoamine neurotransmitter receptors and cause disease in exposed individuals. They have been well studied in the ergot fungus, Claviceps purpurea, and have been reported in some closely related grass endophytes, as well as the distantly related opportunistic human pathogen Aspergillus fumigatus. A. fumigatus, which sporulates prolifically, produces clavines, specifically festuclavine and fumigaclavines A, B, and C in association with asexual spores and the total mass of alkaloids constitutes over 1% of the spore mass. These alkaloids differ from those of most clavicipitaceous fungi, which consist of different clavines and often more complex lysergic acid derivatives. However, the ergot alkaloid pathways of A. fumigatus and ergot fungi are hypothesized to share early biosynthetic steps, diverging at some point after the formation of the intermediate chanoclavine. A homologue of dmaW, a gene encoding dimethylallyltryptophan synthase in Neotyphodium endophytes, was found in the A. fumigatus genome. By gene knockout analysis, dmaW was shown to be required for ergot alkaloid production in A. fumigatus. Comparison of genes clustered around A. fumigatus dmaW to those clustered with dmaW in the ergot fungi revealed potential homologues that could encode proteins controlling early, shared steps in the pathway. Functional analysis via gene knockout of three additional A. fumigatus genes (easA, easE, and easF) rendered mutants with altered alkaloid profiles, demonstrating their involvement in ergot alkaloid biosynthesis. All mutants lacked normal ergot alkaloid production from the latter part of the pathway; complementation with a functional copy of the respective gene, restored normal ergot alkaloid production in each mutant. Analyses of intermediates positioned the products of easF and easE as the second and third enzymatic steps of the pathway. Knockout of easA caused accumulation of multiple intermediates, including chanoclavine; complementation with the C. purpurea easA gene resulted in accumulation of agroclavine, setoclavine, and its isomer isosetoclavine. These data confirm easA involvement post chanoclavine synthesis and more specifically assign its role to reduction of chanoclavine aldehyde, the branch point of the two lineage-specific pathways. These mutants, due to their differing ergot alkaloid profiles, are valuable for testing the role of specific ergot alkaloids in animal pathogenesis and toxicoses. Elucidation of ergot alkaloid biosynthesis, along with the capacity to control the spectrum of alkaloids produced, may be beneficial to agriculture and medicine. Additional studies demonstrated that production of ergot alkaloids was restricted to conidiating cultures. Disruption of brlA, a regulatory gene involved in conidiation, interfered with conidiophore development, as well as ergot alkaloid production. The association of these toxins with sporulation may offer insight into their ecological significance and utility to the fungus. DEDICATION I would like to dedicate this research to my advisor, Dr. Dan Panaccione. His vision and guidance has made this research possible. He has approached the project with overwhelming insight and enthusiasm, providing encouragement and enlightenment along the way. I appreciate the time and energy he has devoted to teaching and mentoring me as a graduate student. I am extremely grateful for his support, patience, and understanding during my years here at WVU, which made it possible to pursue this degree while balancing life as a mother and wife. It truly has been a privilege to work with Dan. He has helped lay my foundation for success and for this I will be forever thankful. iii ACKNOWLEDGEMENTS It is through the support of others that we can accomplish great things. First, I would like to thank my advisor, Dr. Dan Panaccione, and my committee members, Dr. Sharon Wenger, Dr. Ken Blemings, Dr. Joe Morton, and Dr. Alan Sexstone for their assistance and guidance throughout my Ph.D. pursuit. Dr. Dale Karlson, Dr. Jed Doelling, Dr. Joginder Nath, and Dr. Barton Baker have also provided assistance during my time at WVU, along with the Plant and Soil Science administrative assistants. Many thanks to Sherie Edenborn, Kerry Goetz, Shanthi Mulinti, Dawn Chamberlain, Mike Hamstead, and Brian Lewis for their laboratory assistance. I truly cherish the moments I have spent with my dear colleagues and friends within Environmental Microbiology and Plant Pathology. I am grateful for their support, advice, and camaraderie. I would especially like to recognize Bill Wheeler, Mark Double, Dr. Gary Bissonnette, Sherie Edenborn, Kerry Goetz, Shanthi Mulinti, Dawn Chamberlain, Tiara King, Emily Markle, Zola Msiska, Kelly Mayhew, Brian Lewis, Shawn Kenaley, and Bill Rittenour. In addition, I will never forget the lunch bunch, (This includes you, Dr. Gallegly) for the exhilarating and often enlightening conversations. I have been blessed with the constant love and support of my family and friends. My parents have provided me with endless guidance and encouragement. My siblings have enriched my life through their friendship. I feel fortunate for the encouragement and support of my in-laws over the last several years. Their willingness to care for my daughter, Grace, has made this experience easier. Grace has been a blessing throughout this endeavor. Her sweet and loving nature is a wonderful welcome, especially after a long day. And last, but definitely not least, my husband Andrew. I am so grateful for his support, love, and encouragement, especially this last year. Keeping life together, especially in the last few months, has been a challenge and his support has made it possible. I have had a wonderful experience at WVU and will cherish these memories always! iv Table of Contents ABSTRACT ............................................................................................................................................. ii DEDICATION ......................................................................................................................................... iii ACKNOWLEDGEMENTS ......................................................................................................................... iv LIST OF TABLES ................................................................................................................................... viii LIST OF FIGURES ................................................................................................................................. viii REVIEW OF LITERATURE ........................................................................................................................ 1 OVERVIEW ................................................................................................................................................. 1 ERGOT ALKALOIDS AND THEIR HISTORICAL SIGNIFICANCE ....................................................................... 1 CLASSES OF ERGOT ALKALOIDS .................................................................................................................. 4 ERGOT ALKALOID PRODUCING FUNGI ....................................................................................................... 6 ERGOT ALKALOID SYNTHESIS GENE CLUSTERS .........................................................................................