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Intermediate Steps of Loline Alkaloid Biosynthesis

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Intermediate Steps of Loline Alkaloid Biosynthesis University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS Jerome Ralph Faulkner 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 Faulkner, Jerome Ralph, "INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS" (2011). University of Kentucky Doctoral Dissertations. 209. https://uknowledge.uky.edu/gradschool_diss/209 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 JEROME RALPH FAULKNER THE GRADUATE SCHOOL UNIVERSITY OF KENTUCKY 2011 INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS DISSERTATION A dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Jerome Ralph Faulkner Lexington, Kentucky Director: Dr. Christopher L. Schardl, Professor Plant Pathology Lexington, Kentucky 2011 Copyright © Jerome Ralph Faulkner 2011 ABSTRACT OF DISSERTATION INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1- aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually methylated and formylated or acetylated on the 1-amine. In previously published studies lolines were shown to be derived from the amino acids L-proline and L-homoserine. In addition the gene cluster involved in loline-alkaloid biosynthesis has also been characterized. In this dissertation a survey of plant-endophyte symbioses revealed a phenotype with only N-acetylnorloline. This phenotype provided insights into loline alkaloid production. This dissertation focuses on determining the steps to loline biosynthesis after the amino acid precursors. The study involves feeding isotopically labeled potential precursors to loline-alkaloid-producing cultures of Neotyphodium uncinatum, as well as RNA interference (RNAi) of N. uncinatum genes for steps in the pathway. Synthesized deuterated compounds were fed to loline-alkaloid-producing cultures of N. uncinatum to test their possible roles as precursors or intermediates in the loline-alkaloid pathway. N-Formylloline was extracted from the cultures and assayed by GCMS for incorporation of the deuterium label. The results indicated that N-(3-amino, 3-carboxy)propylproline and exo-1-aminopyrrolizidine are intermediates in the loline- alkaloid biosynthetic pathway. Plasmids were also designed for expression of double- stranded RNA homologous to loline-alkaloid biosynthesis genes, and introduced by transformation into N. uncinatum. This RNAi strategy resulted in fungal transformants altered in loline-alkaloid profiles. The RNAi results indicated that N-acetyl-1- aminopyrrolizidine is the intermediate before oxygen bridge formation. Based on the results of this study and the likely roles of the loline-alkaloid biosynthesis genes inferred from signature sequences of their predicted protein products, I propose a pathway of bond formation steps in loline-alkaloid biosynthesis. KEYWORDS: Loline, endophyte, Epichloë, biosynthesis, pyrrolizidine Jerome Ralph Faulkner ___________________________________ Student’s Signature August 16, 2011 ___________________________________ Date INTERMEDIATE STEPS OF LOLINE ALKALOID BIOSYNTHESIS By Jerome Ralph Faulkner Christopher L. Schardl _______________________________ Director of Dissertation Lisa Vaillancourt _______________________________ Director of Graduate Studies August 16, 2011 _______________________________ Date RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be 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 the 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. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date ABSTRACT OF DISSERTATION JEROME RALPH FAULKNER THE GRADUATE SCHOOL UNIVERSITY OF KENTUCKY 2011 ACKNOWLEDGEMENTS I would first like to thank my Lord and Savior Jesus Christ because without His grace I would not be the person that was able to do this work. I would like to sincerely thank my major professor and supervisor Christopher Schardl. Thank you for your guidance before, during and after this work. I am positive that your helpful suggestions, assistance and knowledge are the sole reasons that I was able to accomplish this feat. Your kind leadership, encouraging words, and keen research mind has always impressed me and I hope that I take some of those traits along with me in my personal and professional life. I would like to thank the members of my graduate committee Lisa Vaillancourt, Pradeep Kachroo and Robert Grossman for their assistance in this process and for helping to keep me on the right track to reach this final goal. I would also like to thank George Wagner for being my outside examiner and remaining keeping this commitment. I would like to express my gratitude and thanks to my family. Thank you Carol, Cameron and Jordan for being there for me and giving me the support I needed. I would like to thank my mom and sister for helping me throughout my college years. I am not good at expressing my feelings but to all of my family thank you for believing and supporting me. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................... iii LIST OF TABLES ................................................................................................. vi LIST OF FIGURES .............................................................................................. vii LIST OF FILES ..................................................................................................... ix CHAPTER 1: INTRODUCTION Endophytes of Grasses .................................................................................1 Alkaloids of grass-endophyte symbiosis .....................................................2 Loline alkaloid history .................................................................................3 Loline alkaloid effects..................................................................................6 Loline production in culture.........................................................................7 Genetic analysis of LOL cluster ...................................................................9 CHAPTER 2: LOLINE COMPOSITION IN SELECTED ENDOPHYTES INTRODUCTION .................................................................................................15 MATERIALS AND METHODS ...........................................................................17 Plant Material .............................................................................................17 Tissue print immunoblot assay ..................................................................18 Loline alkaloid analysis .............................................................................18 Gas chromatography mass spectrometry ...................................................19 Isolation of endophytes ..............................................................................20 Fungal DNA extraction ..............................................................................20 PCR primers and conditions ......................................................................21 PCR product sequencing ............................................................................21 Sequence analysis ......................................................................................22 RESULTS ..............................................................................................................23 Tissue print immunoblot (TPIB) results ....................................................23 Loline alkaloids in plant material ..............................................................23 LOL cluster genes PCR results ..................................................................24 Phylogenetic analysis of lolC.....................................................................25 DISCUSSION ........................................................................................................26 CHAPTER 3: INCORPORATION OF POTENTIAL BIOSYNTHETIC INTERMEDIATES IN LOLINE ALKALOID-PRODUCING CULTURES INTRODUCTION .................................................................................................43 MATERIALS AND METHODS ...........................................................................47 Deuterated compounds...............................................................................47 Fungal culture conditions and application of labeled compounds .............47
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