University of Kentucky UKnowledge Theses and Dissertations--Plant Pathology Plant Pathology 2013 CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON-LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE John H. Starnes 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 Starnes, John H., "CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON-LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE" (2013). Theses and Dissertations--Plant Pathology. 6. https://uknowledge.uky.edu/plantpath_etds/6 This Doctoral Dissertation is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant Pathology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. John H. Starnes, Student Dr. Mark L. Farman, Major Professor Dr. Lisa J. Vaillancourt, Director of Graduate Studies CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON‐LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE ___________________________________________________________________ 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 John Howard Starnes Lexington, KY Director: Dr. Mark L. Farman, Professor of Plant Pathology 2013 Copyright © John Howard Starnes 2013 ABSTRACT OF DISSERTATION CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON‐LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE The filamentous ascomycete fungus Magnaporthe oryzae is a pathogen of over 50 genera of grasses. Two important diseases it can cause are gray leaf spot in Lolium perenne (perennial ryegrass) and blast in Oryza sativa (rice). The telomeres of M. oryzae isolates causing gray leaf spot are highly variable, and can spontaneously change during fungal culture. In this dissertation, it is shown that a rice‐infecting isolate is much more stable at the telomeres than an isolate from gray leaf spot. To determine the molecular basis of telomere instability several gray leaf spot isolates telomeres were cloned, which revealed two non‐LTR retrotransposons inserted into the telomere repeats. The elements have been termed Magnaporthe oryzae Telomeric Retrotransposons (MoTeRs). These elements do not have poly‐A tails common to many other non‐LTR retrotransposons, but instead have telomere like sequences at their 5’ end that allow them to insert into telomeres. Intact copies of MoTeRs were restricted to the telomeres of isolates causing gray leaf spot. Surveys for the presence of these elements in M. oryzae showed they were present in several host‐specialized forms including gray leaf spot isolates, but were largely absent in the rice blast isolates. The absence of MoTeRs in rice blast isolates, which are relatively stable by comparison, suggested that the telomere instability in gray leaf spot isolates could be due to MoTeRs. Analyzing spontaneous alterations in telomere restriction fragment profiles of asexual progeny revealed that MoTeRs were involved. Expansion and contraction of MoTeR arrays were observed and account for some telomere restriction profile changes. New telomere formation in asexual progeny followed by MoTeR addition was also observed. Based on this evidence, MoTeRs are largely responsible for the high variability of telomere restriction profiles observed in GLS isolates. KEYWORDS: Non‐LTR, retrotransposon, telomere, gray leaf spot, Magnaporthe oryzae ____John Howard Starnes_______________ Student’s Signature ____February 06, 2013__________________ Date CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON‐LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE By John Howard Starnes ___Dr. Mark L. Farman_____________ Director of Dissertation ___Dr. Lisa J. Vaillancourt__________ Director of Graduate Studies ___February 6th, 2013______________ ACKNOWLEDGEMENTS The production of this dissertation would not have been possible without the support and guidance of many wonderful individuals. I want to thank my family for their unfailing support in my endeavors. The assistance and training by current and past members of the Farman laboratory was invaluable. My advisor, Dr. Mark Farman, has been instrumental in facilitating my professional development. He has provided me with greater insight into molecular genetics, plant pathology, and scientific thinking. Dr. David Thornbury was a boundless resource in the laboratory. He provided technical assistance and advice whenever needed. Melanie Heist provided an extra pair of ears, eyes, and hands in any complications that I experienced while completing my work. The members of my committee (Dr. Christopher Schardl, Dr. Paul Vincelli, and Dr. Randal Voss) have provided needed support and direction during my pursuits, and were very patient in my progress. I was able to pursue my research effectively with funding from the Department of Plant Pathology and the Research Challenge Trust Fund Fellowship. Funding for the research described in this dissertation was provided by the National Science Foundation. iii TABLE OF CONTENTS Acknowledgement......................................................................................................................... iii List of Tables.................................................................................................................................... iv List of Figures................................................................................................................................... viii CHAPTER ONE: Introduction and Literature Review..................................................... 1 1.1 Introduction................................................................................................................ 1 1.1 Host‐specialization of M. oryzae........................................................................ 4 1.2 Biology and population dynamics of M. oryzae.......................................... 7 1.3 Transposable elements......................................................................................... 11 1.3.1 Distribution of Class I transposable elements in M. oryzae... 15 1.3.2 Distribution of Class II transposable elements in M. oryzae. 19 1.4 Implications of TEs in the variability of M. oryzae.................................... 20 1.5 Telomere variability in M. oryzae..................................................................... 22 1.6 Variability of GLS pathogens and the presence of MoTeRs................... 27 1.7 Overview of dissertation research................................................................... 29 CHAPTER TWO: Materials and Methods.............................................................................. 34 2.1 Fungal cultures......................................................................................................... 34 2.2 Plant inoculation and conidia collection....................................................... 34 2.3 Small scale DNA extraction................................................................................. 35 2.4 Large scale DNA preparation.............................................................................. 36 2.5 End‐enriched clone library preparation........................................................ 36 2.6 Cloning of MoTeR Bands unlinked to telomeres........................................ 38 2.7 Primer design............................................................................................................ 39 2.8 Hybridization probes............................................................................................. 39 2.9 Southern hybridization.......................................................................................