University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2020 More than the sum of their parts: Building a framework for understanding host-microbe interactions in Medicago sativa Katherine Mackenzie Moccia University of Tennessee, Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Environmental Microbiology and Microbial Ecology Commons Recommended Citation Moccia, Katherine Mackenzie, "More than the sum of their parts: Building a framework for understanding host-microbe interactions in Medicago sativa. " PhD diss., University of Tennessee, 2020. https://trace.tennessee.edu/utk_graddiss/6154 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Katherine Mackenzie Moccia entitled "More than the sum of their parts: Building a framework for understanding host-microbe interactions in Medicago sativa." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Sarah L. Lebeis, Major Professor We have read this dissertation and recommend its acceptance: Heidi Goodrich-Blair, Alison Buchan, James Fordyce Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) More than the sum of their parts: Building a framework for understanding host-microbe interactions in Medicago sativa A Dissertation Presented for the Doctorate of Philosophy Degree The University of Tennessee, Knoxville Katherine Mackenzie Moccia December 2020 Copyright © 2020 Katherine Mackenzie Moccia. “More than the sum of their parts: Building a framework for understanding host-microbe interactions in Medicago sativa” All rights reserved. ii DEDICATION This dissertation is dedicated to my parents, Kevin Moccia and Regina Gallagher, my brother James Moccia, my uncle Jim Gallagher, and my grandmother Mary Gallagher. The five of you have always encouraged me be who I am, and not what other people wanted me to be. What a gift you have given me. My strength and my endurance come from you all. Thank you. iii ACKNOWLEDGEMENTS First and foremost, I want to express my profound gratitude for my advisor Sarah Lebeis. Sarah has taught me much of what I know about microbiology, from the best controls to how to write method sections that people can actually follow. But what I learned from Sarah goes far beyond the field of microbiology. Sarah has a perspective in science that allows for abounding optimism. Although it is easy to brush it off as a positive perspective, it comes from a much deeper place. When an experiment fails, Sarah is unphased. She does not despair, but instead comes with the question “What did we learn?”. This small question provides the opportunity to look beyond the present experiment and expand out to the whole. I recently heard someone describe a PhD as one who creates new knowledge. Sarah has taught me that failure is not an unfortunate misstep on the way to new knowledge, it is new knowledge itself. I learned to embrace this viewpoint from Sarah, and it continues to teach me to be a better scientist every day. On the topic of becoming a better scientist, Michelle Larsen has played such an integral role in forming the scientist I am today, that I am not sure I can express my gratitude with words. As a high school student, I was nervous, quiet, and wholly unsure of what to do in a laboratory. Michelle, along with JoAnn Tufariello, Oren Mayer, and many other members of the William Jacobs Lab, have taught me the principals of microbiology. Many of the lessons I learned from all of you, I have taught to my own students. Michelle, I hope that one day I give a high school student the opportunities that you have given me. For now, my deepest appreciation will have to suffice. iv I thank Alison Buchan, Gary LeClair and Steve Wilhelm and anyone else who was involved with providing me a spot in the REU. The REU program not only demonstrated how exciting science can be, but also taught me that a scientific career can be a part of a well-balanced life. My experience within that program gave me the confidence to apply to graduate school. While in graduate school, Heidi Goodrich-Blair and James Fordyce have both been incredibly helpful members of my committee and I thank them both for their patience while I found my project and their insightful experimental suggestions once I solidified my aims. My secret committee member, Veronica Brown, has been exceptionally helpful in troubleshooting problems and providing words of encouragement. Next Generation Sequencing has been a huge part of my dissertation, and Veronica has much of what I know on this subject. My lovely lab mates, David Grant, Bridget O’Banion and Alexandra Gates all deserve acknowledgement. David is an excellent scientist, and an even better lab mate. He is always available to help troubleshoot, and I have benefitted from his knowledge of molecular biology many times. The talented Bridget O’Banion who, among other things, has an incredibly critical mind for experimental design. She has made my first paper better because of her suggested controls, and for that I am profoundly grateful. I have thoroughly enjoyed discussing scientific papers and ideas Alexandra Gates, as she is able to quickly get to the crux of a paper while understanding pitfalls. This is a highly sought- after skill that I have benefitted from. Her analysis of scientific papers provided me with many a citation within this dissertation. Thank you all! v This dissertation would not have been possible without the wonderful undergraduates and post-baccalaureate students who have helped me. Andrew Willems, my first undergraduate, has been both a friend and an amazing scientist. His enthusiasm for science is rare, and it has been a joy to work with him. Alex Demetros, another friend and scientist, was a great help to my third research chapter, and my final years in graduate school. I always looked forward to teaching him and hearing his thoughts about the world. I thank my REU students, Alicia Flores, Alexi Girod, Kayla Bonilla, as well as the other students I have had the pleasure of working with, Makaila Gray and Erin Yi, for all of their hard work that moved many a project forward! Lizzie Larsen and Maddy Hwee have shared in my scientific journey and both inspired and comforted me along the way with their curiosity and determination. Emily Stern and Bethany Zulick have patiently listened to me complain about graduate school and wisely reminded me that it is not interminable! I thank my all friends, both new ones formed in graduate school and ones from seemingly the beginning of time, for their support, kindness, and love. Finally, I want to thank my husband, Spiro Papoulis. I could write a whole other dissertation on just how much you have helped me. For now, I will just say you have been my biggest advocate, and my sweetest solace. vi ABSTRACT This dissertation seeks to understand plant-microbe interactions in the agriculturally relevant plant Medicago sativa from three distinct vantage points within microbiology. Within the plant microbiome, we examine how primer usage and the application of peptide nucleic acids impacts 16S and 18S rRNA gene sequencing. In doing so, we design a novel peptide nucleic acid, PNA, and test its impact using multiple primers and sequencing protocols. Once microbial sequencing methodology is established, we generate a synthetic consortium of bacterial isolates from M. sativa leaves and modulate nitrogen levels to better understand microbial structure. Drop out communities, where we remove one member at a time, elucidate what community members colonize to high levels, and how they change the microbial community when present. Using this approach, we uncover how, and which microbes can consistently colonize plants across nutrient conditions. Further, we examine multiple genetic approaches to investigate potential genetic mechanisms behind plant colonization, such as high throughput sequencing techniques such as randomly barcoded transposon sequencing (RB-TnSeq) and traditional transposon mutagenesis. By using a variety of approaches within biology, we elucidate plant-microbe interactions in alfalfa. vii TABLE OF CONTENTS Chapter 1: Understanding plant microbe interactions in Medicago Sativa ........................ 1 Chapter Contributions: .................................................................................................... 2 Introduction: .................................................................................................................... 2 Alfalfa and its role in the United States ...................................................................... 2 Challenges and limitations within alfalfa research ..................................................... 4 One approach towards improving alfalfa research ..................................................... 7 Chapter 2- Microbiome technologies and their impact on plant microbe research