The Taxonomic and Functional Nature of Plant-Associated Microbiomes

THE TAXONOMIC AND FUNCTIONAL NATURE OF PLANT-ASSOCIATED MICROBIOMES Scott MacKay Yourstone A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Bioinformatics and Computational Biology. Chapel Hill 2017 Approved by: Jeffery Dangl Corbin Jones Piotr Mieczkowski Fernando Pardo-Manuel de Villena Jan Prins ©2017 Scott MacKay Yourstone ALL RIGHTS RESERVED ii ABSTRACT Scott MacKay Yourstone: The Taxonomic and Functional Nature of Plant-Associated Microbiomes (Under the direction of Jeffery Dangl and Corbin Jones) Microbes live in close association with eukaryotes and have substantial impacts on fitness and well-being of their hosts. In plants, microbes can colonize soil adjacent to plant roots and can even survive inside of plant tissues. They can have either positive or negative effects on plant fitness and therefore show potential for use as an agricultural tool. However, our current understanding of how these microbial communities are formed and how they function is limited. The work described in this dissertation reports novel insights regarding plant-associated microbiomes along with new and improved methods for observing their associations with plants. Chapter 1 gives a brief introduction to plant associated microbiomes and the common methods used to study them. Chapter 2 outlines colonization patterns of Arabidopsis thaliana associated microbiomes. The taxa that colonize Arabidopsis roots endophytically are distinct from those colonizing the soil surrounding the roots (rhizosphere) and unplanted bulk soil. This suggests that plants modulate these communities and possibly select for microbes that provide specific fitness advantages. Endophytic communities of plants grown in different soils exemplify how soil type is the primary factor in determining the taxa found in these communities. However, there are core taxa that are consistently found in the endophyte compartment regardless of soil type and other factors. These core microbes are potential candidates that are actively selected by iii plants. Communities associated with different Arabidopsis ecotypes and ages have only minor differences. Chapter 3 presents improved methods for profiling taxa in plant-associated microbiomes by utilizing two techniques. First, PCR amplification clamps designed from peptide nucleic acids are used to reduce plant chloroplast contamination. Removing unwanted chloroplast contamination reduces the cost of sequencing by increasing the yield of usable, bacterial 16S reads. Second, 16S amplicons are tagged with a unique DNA oligo (i.e. molecule tag) prior to PCR amplification. After PCR amplification and sequencing, reads having the same molecule tag likely originated from the same DNA template. Therefore, discrepancies between these reads are presumably sequencing errors and can be corrected bioinformatically. Identifying and correcting these sequencing errors can be performed using the MTToolbox software described in Chapter 4. Correcting sequencing errors using molecule tagged reads and MTToolbox substantially reduces the number of spurious singleton OTUs. Chapter 5 compares the functional profiles of Arabidopsis rhizospheres against those in the bulk soil and also describes novel methods for comparing across metagenomes. These methods can report functional differences between microbiomes at a global level or for specific taxa. For example, transcription-related functions were identified as frequently enriched in the rhizosphere across a broad diversity of taxa. Alternatively, synthesis of cyclic beta-1,2-glucans were identified as rhizosphere enriched in multiple taxa among a small group of betaproteobacteria despite other betaproteobacteria having different enrichment patterns. Additionally, rare functions in these metagenomes were more likely to be classified as rhizosphere enriched suggesting that microbes are constantly evolving new mechanisms for iv rhizosphere colonization. Therefore, identifying taxa specific enrichments patterns is important for understanding mechanisms associated with rhizosphere colonization. Collectively, the methods and insights described in this document expand our understanding of plant microbiomes and generate important hypotheses for future examination. v To my parents. vi ACKNOWLEDGEMENTS This work represents collaborative efforts from a large number of people. First I would like to acknowledge and thank my major professors—Jeff Dangl and Corbin Jones. Working with them has been a pleasure. They have provided countless hours of help and tutelage that has contributed to my growth as a scientist. Without their efforts this would have been impossible. One of the best things about working in the Dangl lab is the great people that I get to associate with on a daily basis. The Dangl lab members are all accomplished scientists. Their advice and assistance has been invaluable. I have thoroughly enjoined the numerous scientific discussions I have had with each of them. Many current and former lab members have contributed useful suggestions, advice, and efforts to this work. I respect and admire their work ethic and commitment to exceptional science. Beyond having a fantastic working relationship with members of the Dangl lab, I consider them some of my best friends. I am also grateful for my committee members—Jeff Dangl, Corbin Jones, Jan Prins, Piotr Mieczkowski, and Fernando Pardo-Manuel de Villena. I have had the privilege of working with each of them individually and greatly appreciate the time and energy they have committed on my behalf. I also thank the institutions that provided financial support for this work. These include the University of North Carolina at Chapel Hill, and its Graduate School and Department of Biology; the DOE Joint Genome Institute; the National Science Foundation; the Howard Hughes Medical Institute; and the Gordon and Betty Moore Foundation. vii Lastly, I would like to thank my family for their support and encouragement over the last seven years—especially my parents and my wife, Erin. They have provided unwavering support in all aspects of my life, and have helped me become the best version of myself. Due to the efforts of all these wonderful people I am a better scientist and better person than when I began this work seven years ago. I can’t thank them enough. viii TABLE OF CONTENTS LIST OF ABBREVIATIONS ...................................................................................................... xiii LIST OF FIGURES ..................................................................................................................... xiv CHAPTER 1: INTRODUCTION ....................................................................................................1 2.1 Impact and Significance of microorganisms ......................................................................... 1 2.2 Observing the microbiome using the 16S ribosomal gene .................................................... 2 2.3 Observing the microbiome using shotgun sequencing .......................................................... 6 CHAPTER 2: DEFINING THE CORE ARABIDOPSIS THALIANA ROOT MICROBIOME ..................................................................................................................12 2.1 Overview ............................................................................................................................. 12 2.2 Introduction ......................................................................................................................... 13 2.3 Results ................................................................................................................................. 14 2.4 Methods ............................................................................................................................... 27 2.4.1 General strategy ....................................................................................................................... 27 2.4.2 Soil collection and analysis ........................................................................................................ 28 2.4.3 Seed sterilization and germination ........................................................................................... 28 2.4.4 Seedling growth ........................................................................................................................ 29 2.4.5 Harvesting ................................................................................................................................. 30 2.4.6 DNA extraction .......................................................................................................................... 32 2.4.7 PCR ............................................................................................................................................ 33 2.4.8 454 pyrotag sequencing ............................................................................................................ 33 2.4.9 Primer test and technical reproducibility ................................................................................. 34 2.4.10 Primer specificity sequence .................................................................................................... 35 2.4.11 Sequence processing pipeline and assignment of OTUs ......................................................... 35 2.4.12 Detection of differentially enriched OTUs by the GLMM ......................................................

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