AN ABSTRACT OF THE DISSERTATION OF Edward W. Davis II for the degree of Doctor of Philosophy in Molecular and Cellular Biology presented on June 7, 2017. Title: Phylogeny and Evolution of Gall-Associated Plant Pathogenic Bacteria. Abstract approved: ______________________________________________________ Jeff H. Chang Gall-associated phytopathogens have unique evolutionary histories that have shaped both their modes of infection and genomic structures. Pathogenicity of the gall- associated plant pathogens of the Rhodococcus, Agrobacterium, and Rathayibacter genera is mediated by horizontally acquired virulence loci. The relative ease of gain and loss of the virulence loci has confounded accurate characterization of these bacteria, especially those characterizations made prior to the use of molecular markers for genotyping. Work presented in this thesis uses whole genome guided approaches to discern the intra- and inter- species genetic diversity in the three genera described. Rhodococcus fascians is the causal agent of leafy gall disease. We tested the hypothesis that R. fascians is comprised of a single species level group and show that two distinct clades with up to six species level groups are able to cause symptoms consistent with leafy gall disease. These data reveal previously unknown chromosomal diversity within this group of phytopathogenic bacteria. Four lines of evidence that make use of whole genome sequences indicate that the species are acted on by distinct selective pressures and have unique evolutionary histories. Further, a set of horizontally acquired virulence loci is correlated with the pathogenic phenotype, regardless of chromosomal lineage. These data suggest that delimitation of phytopathogenic Rhodococcus isolates requires careful consideration with respect to both the chromosomal genotype as well as the presence of virulence loci. Rathayibacter spp. are vector mediated plant pathogens that infect nematode galls in the seed heads of several grass species. Rathayibacter toxicus, a Select Agent in the U.S., has been shown to produce corynetoxins, nucleoside antibiotics that also cause neurotoxic effects in mammals, when infected by a bacteriophage. As R. toxicus encodes a clustered regularly interspersed short palindromic repeats (CRISPR) system that acts as an adaptive immune system against foreign DNA in bacteria, we studied the CRISPR array evolution for insights into the ecology of bacteria-phage interactions. We predicted that over half of the spacers encoded in R. toxicus are functional in targeting the phage, while the bacteria still show susceptibility to the phage. These data suggest a complex homeostatic interaction whereby neither bacteria nor phage populations are eliminated. Further, our results challenge past conclusions on the efficacy of CRISPR-mediated immunity. Lastly, we generated a set of tools to facilitate comparative genomics research and accurate species and strain identification. Data are integrated into a website (http://gall-id.cgrb.oregonstate.edu/) to facilitate their use by all researchers, regardless of computational proclivity. ©Copyright by Edward W. Davis II June 7, 2017 All Rights Reserved Phylogeny and Evolution of Gall-Associated Plant Pathogenic Bacteria. by Edward W. Davis II A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented June 7, 2017 Commencement June 2017 Doctor of Philosophy dissertation of Edward W. Davis II presented on June 7, 2017 APPROVED: _____________________________________________________________________ Major Professor, representing Molecular and Cellular Biology _____________________________________________________________________ Director of the Molecular and Cellular Biology Program _____________________________________________________________________ Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. _____________________________________________________________________ Edward W. Davis II, Author ACKNOWLEDGEMENTS I owe so much to so many people; I was certainly very fortunate to receive all of the support that I did throughout my time as a graduate student. I would like to thank my advisor, Jeff Chang, for his guidance, support, and the opportunity to make mistakes. I have gained more insight from Jeff over the past five years than I could have ever imagined. I would also like to thank my committee members Dee Denver, Taifo Mahmud, Tom Sharpton, and Gary DeLander. Thank you to the BPP department, the MCB program and the CGRB. Thank you to Joyce Loper and Tom Wolpert for giving me excellent advice throughout my graduate student career. I need to extend a special thank you to Melodie Putnam and members of the OSU Plant Clinic; I would not have worked on Rhodococcus, Rathayibacter, or Agrobacterium without you. Thank you to Marilyn Miller and Walt Ream; my time working with you on Agrobacterium was an pleasure. Thanks to my lab mates from the Chang lab, past and present: Allie Creason, Elizabeth Savory, Alex Buchanan, Jason Cumbie, Meesha Pena, Alex Weisberg, Chih-Feng Wu, Skylar Fuller, Michael Belcher, Dani Stevens, and others. It was certainly a team effort, and I’m glad to have had your help and friendship. Last, and most importantly, I would like to thank my family. My children, Lexie and Landon, are what keep me going every day. And without my wife, Marie, I would not be here. She is my rock and this is all possible because of her support. CONTRIBUTION OF AUTHORS Chapter 1. EWD wrote the chapter. Chapter 2. EWD and ALC contributed equally. EWD, ALC, MLP, OMV and JHC contributed to the conception and design of the work, analysis of data, and wrote and approved the manuscript. ALC, EWD, MLP, and OMV also contributed to the acquisition of isolates and/or genome sequences. Chapter 3. EWD and AJW contributed equally. EWD, AJW, JFT, NJG and JHC contributed to the conception and design of the work, analysis of the data, and wrote and approved the manuscript. EWD wrote software to collect sequences, do analyses, and generate the sequence database. AJW and JFT designed the website interface. EWD and AJW contributed to acquisition of isolates and/or genome sequences, and wrote software for WGS pipeline. AJW added virulence gene search, and generated databases for the website. Chapter 4. EWD, MLP, LDL, MSB, AJS, BKS, TDM, DGL, WLS, EER, and JHC contributed to the conception and design of the work and wrote and approved the manuscript. EWD, LDL, MSB, and JHC contributed to analysis of data. EWD, MLP, MSB, AJS, BKS, TDM, DGL, WLS, EER also contributed to the acquisition of isolates and/or genome sequences. Chapter 5. EWD wrote the chapter TABLE OF CONTENTS Page Chapter 1 – Introduction: Reconciliation of Species Concepts, Horizontal Gene Transfer, and Genome Evolution .................................................................................. 1 SPECIES CONCEPTS AND APPLICATION TO BACTERIA .............................. 2 BACTERIAL SPECIES DELIMITATION .............................................................. 5 OPERATIONAL METHODS FOR CLASSIFYING BACTERIA ........................ 10 CORE, FLEXIBLE, AND PAN-GENOME ............................................................ 11 HORIZONTAL GENE TRANSFER AND SYMBIOSIS ...................................... 13 TAXONOMIC CLASSIFICATION AND NOMENCLATURE............................ 16 BIBLIOGRAPHY ................................................................................................... 17 Chapter 2 – Use of whole genome sequences to develop a molecular phylogenetic framework for Rhodococcus fascians and the Rhodococcus genus ............................ 22 ABSTRACT ............................................................................................................ 23 INTRODUCTION ................................................................................................... 23 MATERIAL AND METHODS ............................................................................... 27 Isolation of phytopathogenic Rhodococcus.......................................................... 27 Nucleic acid preparations ..................................................................................... 27 Next-generation sequencing, assembly, and annotation ...................................... 27 Phylogenetic analyses .......................................................................................... 28 Bioinformatic analyses ......................................................................................... 30 RESULTS ................................................................................................................ 31 Whole genome-based phylogeny supports multiple lineages of plant pathogenic Rhodococcus......................................................................................................... 31 Alternative whole-genome based analyses support the multiple lineages of Rhodococcus......................................................................................................... 33 A molecular phylogeny based on whole genome sequences provides a framework for resolving the Rhodococcus genus ................................................................... 35 ANI provides a framework for resolving the Rhodococcus genus ....................... 37 DISCUSSION .........................................................................................................