University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2004 STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI Caroline Machado University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Machado, Caroline, "STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI" (2004). University of Kentucky Doctoral Dissertations. 433. https://uknowledge.uky.edu/gradschool_diss/433 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Caroline Machado The Graduate School University of Kentucky 2004 STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Caroline Machado Lexington, Kentucky Director: Dr. Christopher L. Schardl, Professor of Plant Pathology Lexington, Kentucky 2004 Copyright by Caroline Machado 2004 ABSTRACT OF DISSERTATION STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway. KEYWORDS: Neotyphodium endophytes, Ergot alkaloid biosynthesis, Cre/lox, dmaW, marker exchange Caroline Machado Date: 5/06/04 STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI By Caroline Machado Christopher L. Schardl _____________________________ Director of Dissertation Lisa J. Vaillancourt ______________________________ Director of Graduate Studies 5/06/04 ______________________________ RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctors degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. DISSERTATION Caroline Machado The Graduate School University of Kentucky 2004 STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS FUNGI DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Caroline Machado Lexington, Kentucky Director: Dr. Christopher L Schardl, Professor of Plant Pathology Lexington, Kentucky 2004 Copyright by Caroline Machado 2004 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor Dr. Christopher L. Schardl for his constant encouragement, motivation and patient guidance throughout the dissertation period. I would like to thank my committee members Dr. Lisa Vaillancourt, Dr. Tom Pirone, Dr. Lowell Bush, Dr. Deane Falcone and Dr. Tim Phillips for their great help and valuable suggestions. I also would like to thank Dr. Daniel Panaccione from West Virginia University for guiding me in ergot alkaloid analysis at his facility. I am thankful to Dr. Pete Mirabito from the Department of Biology, University of Kentucky for providing me the plasmid containing cre gene. I would like to thank Dr. Jinghong Wang who helped me to get started with the project, Dr. Christina Moon for tips on GPS system and Jimmy Blankenship for teaching me the computer skills and for the friendship and moral support throughout the graduate student career. I appreciate all the help provided by all the Schardl lab members; for creating a very friendly working atmosphere in the laboratory, you are the best. I also would like to thank Walter Hollin and Al Byrd for their great help in the laboratory. Thanks to Jenny Wiseman for helping me with sequencing work. I am indebted to my husband Arun, whose constant support and motivation helped me to reach the goal, thanks Arun for taking over the kitchen, feeding me and for the inspiration during the most difficult time of writing dissertation. I would like to express my deep gratitude to my mummy and papa for all the sacrifices, hard work and love, which made me reach this stage. I also would like to thank my brother and sister for all the support and encouragement. I like to thank my mother-in-law for her support and blessings. I would like to thank all the faculty and staff in the Department of Plant Pathology for the support and encouragement during the course of study. iii TABLE OF CONTENTS Acknowledgements ...........................................................................................................iii List of Tables ....................................................................................................................vi List of Figures ...................................................................................................................vii List of Files .......................................................................................................................viii Chapter One: Introduction Ergot alkoloids and their biosynthesis....................................................................1 Ergotism......................................................................................................1 Ergot alkoloid producing species .................................................................1 Ergot alkaloid biosynthesis ..........................................................................1 Regulation of the pathway ...........................................................................3 Genetics of ergot alkaloid production...........................................................3 Grass endophyte symbiosis ....................................................................................4 Endophyte biology.......................................................................................4 Alkaloid produced by the endophytes ..........................................................5 Benefits of grass endophyte symbiosis.........................................................6 Livestock toxicosis ......................................................................................6 Prevention of livestock toxicosis..................................................................7 Chapter Two: Gene disruption by marker exchange of dmaW in Neotyphodium sp. Lp1 Introduction ...........................................................................................................12 Materials and Methods...........................................................................................13 Results...................................................................................................................17 Discussion .............................................................................................................19 Chapter Three: Knockout of dmaW in Neotyphodium coenophialum using a Cre/lox system Introduction ...........................................................................................................25