University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2018 Identification and functional characterization of effectors from an anther smut fungus, Microbotryum lychnidis-dioicae. Venkata Swathi Kuppireddy University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Molecular Genetics Commons Recommended Citation Kuppireddy, Venkata Swathi, "Identification and functional characterization of effectors from an anther smut fungus, Microbotryum lychnidis-dioicae." (2018). Electronic Theses and Dissertations. Paper 3078. https://doi.org/10.18297/etd/3078 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. 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IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF EFFECTORS FROM AN ANTHER SMUT FUNGUS, MICROBOTRYUM LYCHNIDIS-DIOICAE By Venkata Swathi Kuppireddy M.Sc., Biology, University of Louisville, 2016 A Dissertation Submitted to the Faculty of the College of Arts and Sciences of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Department of Biology, Division of Molecular, Cellular and Developmental Biology, University of Louisville Louisville, Kentucky December 2018 Copyright 2018 by Venkata Swathi Kuppireddy © All rights reserved IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF EFFECTORS FROM AN ANTHER SMUT FUNGUS, MICROBOTRYUM LYCHNIDIS-DIOICAE By Venkata Swathi Kuppireddy M.Sc., Biology, University of Louisville, 2016 A Dissertation Approved on November 14, 2018 By the following Dissertation Committee: _________________________________________ Dr. Michael H. Perlin, Principal Advisor _________________________________________ Dr. David Schultz _________________________________________ Dr. Brian Wattenberg _________________________________________ Dr. Carolyn Klinge _________________________________________ Dr. Paul Himes ii DEDICATION This work is dedicated to my beloved family My parents, brother & my loving husband Thank you for helping to give me the life that I love today iii ACKNOWLEDGMENTS I am highly indebted to my advisor, Dr. Michael Perlin for accepting me into his lab and allowing me to learn all that I learned today. He is extremely patient in answering all the questions and being very supportive in this incredible journey. I thank my committee members Dr. David Schultz, Dr. Brian Wattenberg, Dr. Carolyn Klinge and Dr. Paul Himes for their valuable comments and inputs in directing my research and encouraging me towards my goal. I also thank Dr. Michelle Barati for her patience and for all the hours that she sat with me for confocal microscopy assistance and western blots and Dr. Liang Bao for technical support with the SEM. I am grateful to the suggestions of Dr. Ronald Atlas for helping me to develop my teaching abilities, Dr. Mark Running for his valuable inputs and suggestions in my research, and Dr. Gary Cobbs for his help with statistical analysis. I want to extend my heartful gratitude to all the undergraduates Brittany Carman, Catarina Cahill, Laura Irons, Matthew Carruthers, Joseph Paul Ham, Ankur Patel, Patrick Soucy and Medha Desai for their incredible support. I also want to thank my close comrades Parul Singh, Richa Singhal, Sanaya Bamji, Ming Chang Tsai, Joseph Paul Ham, Sunitha Khanal, Lalu Vijaya Krishna Pillai for all the smiles that made this journey less stressful. I would like to offer special thanks to my dear friend Susan Toh, who, although no longer with us, continues to remain as an inspiration, forever. Finally, I like iv to thank fellow Goat lab members William Beckerson, Margaret Wallen and Hector Mendoza, Roxanne Leiter for their companionship throughout these years. I like to appreciate and thank the support of the Biology department Ms. Doris Meadows, Judi Felli, Terri Norris, and Charice Johnson and the Professors Dr. Ronald Fell and Dr. Perri Eason in supporting my graduate career. Finally, I would like to take this opportunity to express my gratitude to my parents, brother, my husband and my daughter for their unconditional love and support in this arduous journey. v ABSTRACT IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF EFFECTORS FROM AN ANTHER SMUT FUNGUS, MICROBOTRYUM LYCHNIDIS-DIOICAE Venkata Swathi Kuppireddy November 14, 2018 Microbotryum lychnidis-dioicae causes anther smut fungus in its host plant, Silene latifolia . The goal of this work is to identify and characterize the virulence determinants of this pathogen to better understand the molecular basis behind this host-pathogen interaction. This work studied for the first time the key effectors in the mechanism of infection by this fungal species. Using, yeast two-hybrid screens, I have identified the host plant interaction partners for the effector, MVLG_01732. A second effector MVLG_05720, interacts with other fungal proteins that appear to facilitate the fungal establishment and colonization during the infection. Our findings indicate that a third effector, MVLG_04106, could serve as a transcriptional regulator to promote infection. To further characterize the role of the effector, MVLG_01732, I have conducted heterologous expression studies in A. thaliana followed by infection assays with the pathogen Pseudomonas syringae . Our results indicate that this effector has a role in the early bolting of flowers in A. thaliana, this finding provides an important clue about the vi role of MVLG_01732 in advancing host plant floral development, which is known to occur in infected S. latifolia flowers. Here, we have identified a key molecular link between a fungal effector and the developmental change it triggers in the host plant. Infection assays reveal that this effector might play a role in promoting pathogen growth. I have also examined the response to this effector by the host by expressing the His- tagged effector in S. latifolia in an experiment designed to model the mechanism of infection in the native habitat. This is the first time that this approach of delivering the candidate effector protein has been carried out in planta and aimed to provide information about the previously unidentified interacting partners from the host. Overall, this dissertation body aimed to increase the number of available genetic tools to study M. lychnidis-dioicae and will serve as a valuable resource for future investigators along with furthering our understanding of the infection mechanism. vii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iv ABSTRACT ....................................................................................................................... vi LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi CHAPTER I AN OVERVIEW OF THE EFFECTORS .................................................... 1 CHAPTER II IDENTIFICATION AND INITIAL CHARACTERIZATION OF THE EFFECTORS OF AN ANTHER SMUT FUNGUS AND POTENTIAL HOST TARGET PROTEINS ....................................................................................................................... 42 Overview ....................................................................................................................... 42 Introduction ................................................................................................................... 43 Results ........................................................................................................................... 45 Discussion ..................................................................................................................... 57 Materials and Methods .................................................................................................. 66 Conclusions ................................................................................................................... 76 CHAPTER III FUNCTIONAL CHARACTERIZATION OF THE MICROBOTRYUM LYCHNIDIS-DIOICAE EFFECTORS IN A HETEROLOGOUS HOST MODEL ......... 77 Overview ....................................................................................................................... 77 Introduction ................................................................................................................... 78 Materials and Methods .................................................................................................. 80 Results ........................................................................................................................... 91 Discussion ................................................................................................................... 103 CHAPTER IV EXPRESSION OF MVLG_01732 HIS TAGGED EFFECTOR IN SILENE LATIFOLIA TO IDENTIFY NATIVE HOST INTERACTORS ..................... 107 Introduction ................................................................................................................