Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2020 Exploring the genetic basis for host specific virulence and pathogenicity in Erwinia tracheiphila Olakunle Olawole Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Recommended Citation Olawole, Olakunle, "Exploring the genetic basis for host specific virulence and pathogenicity in Erwinia tracheiphila" (2020). Graduate Theses and Dissertations. 18577. https://lib.dr.iastate.edu/etd/18577 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Exploring the genetic basis for host specific virulence and pathogenicity in Erwinia tracheiphila by Olakunle I. Olawole A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Pathology Program of Study Committee: Gwyn A. Beattie, Major Professor Mark L. Gleason Steven A. Whitham Alison E. Robertson Kyaw J. Aung The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2020 Copyright © Olakunle I. Olawole, 2020. All rights reserved. ii DEDICATION To God Almighty – my strength, shield and exceeding great reward; without You by my side, the journey wouldn’t have been this glorious. To my wife, Funmi, who sacrificially stood by me through the thick and thin of graduate school, often offering herself as a ‘cheap labor’ to help with data entry for my numerous plant studies; without you, I wouldn’t have come this far. Thank you so much for your love, patience, understanding, prayers and encouragement during the darkest moments of this journey. To my parent, thank you for your sacrifice to ensure I have access to invaluable educational opportunities. To folks of the New Life Church and the United Nations for Christ, thank you for your spiritual support, and for being such a great family to hang out with. iii TABLES OF CONTENT Page ACKNOWLEDGEMENTS v ABSTRACT vi CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation organization 2 Literature review 3 Research rationale and significance 14 References 15 CHAPTER 2. SUBSPECIES DELINEATION OF ERWINIA TRACHEIPHILA 21 STRAINS ON THE BASIS OF WHOLE-GENOME SEQUENCE AND PHYSIOLOGICAL DATA Abstract 21 Introduction 22 Materials and methods 24 Results 34 Discussion 48 References 54 CHAPTER 3. THE EFFECTORS EOP1 AND DSPE ARE DRIVERS OF HOST- 58 SPECIFICITY AND PATHOGENICITY AMONG ERWINIA TRACHEIPHILA STRAINS Abstract 58 Introduction 59 Materials and methods 63 Results 70 Discussion 83 References 88 CHAPTER 4. ERWINIA TRACHEIPHILA SUBSPECIES DIFFERENCES IN HOST 94 SPECIFICITY ARE ASSOCIATED WITH HOST-SPECIFIC DIFFERENCES IN HRPA EXPRESSION Abstract 94 Combined Introduction, Results and Discussion 95 References 102 iv CHAPTER 5. IN PLANTA EXPRESSION PROFILING AND FUNCTIONAL 104 ANALYSIS OF ERWINIA TRACHEIPHILA EFFECTORS Abstract 104 Introduction 105 Materials and methods 108 Results 117 Discussion 129 134 References CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS 141 References 146 v ACKNOWLEDGEMENTS A big thank you to my advisor, Dr. Gwyn A. Beattie, for her patience and support as a mentor for research, teaching, and professional development. I have learned a lot from your wealth of knowledge and experience in this field. Thank you so much! To my committee members Dr. Mark L. Gleason, Dr. Steven A. Whitham, Dr. Alison E. Robertson and Dr. Kyaw J. Aung, thank you for your feedback and guidance. I am grateful to all of the past and present colleagues in the Beattie and Halverson laboratories. Your suggestions and comments during joint laboratory meetings are greatly appreciated. Specifically, I would like to thank Dr. Chiliang Chen for his guidance on optimizing bacterial mutagenesis protocols which set a very good background for me to work on this project. I also appreciate the assistance of Dr. Benzhong Fu, Sharon Raquel Badilla Arias and Kephas Mphande during my gazillion plant studies. Finally, I would like to thank my funding sources – College of Agriculture and Life Sciences, Iowa State University and the Iowa State University Presidential Fellowship. vi ABSTRACT Erwinia tracheiphila is among the few plant pathogenic species of Erwinia retained in this genus after several other species have been re-assigned to new genera. E. tracheiphila strains fall into clades that exhibit differences in host-specific virulence based on their ability to rapidly wilt muskmelon but not wilt squash (Et-melo clade) or to rapidly wilt squash and less rapidly wilt muskmelon (Et-C1 and Et-C2 clades). We characterized whole genome sequences and physiological traits of 24 E. tracheiphila strains in two of these clades, leading us to propose that the Et-melo and Et-C1 clades be delineated as E. tracheiphila subsp. tracheiphila and E. tracheiphila subsp. lentilata, respectively. Here, we explored how the genetic basis of E. tracheiphila sub-speciation is associated with host-specific virulence. We identified the distribution of the type III secreted effector (T3SE) repertoire of two host-specific strains, SCR3 (Et-melo) and BHKY (Et-C1), and characterized the expression profile of selected effectors in planta using RT-qPCR. We explored the role of one highly expressed effector, Eop1, as a host- specificity candidate, and found that loss of eop1 did not alter the virulence of two Et-melo strains or an Et-C1 strain on their respective hosts. However, over-expression of eop1 from an Et-melo strain in an Et-C1 strain increased its virulence on muskmelon, but not on squash, indicating that Eop1 functions as a host-specific virulence factor. The expression profiling in planta also highlighted poor expression of a key T3SS pilus gene, hrpA, in Et-melo in squash. We demonstrated experimentally that over-expression of hrpA in an Et-melo strain enabled this vii strain to infect squash, demonstrating that host-specific hrpA expression is another factor contributing to host-specific virulence in E. tracheiphila. We also examined the role of the effector DspE in pathogenicity, and found that loss of dspE from Et-melo strains significantly reduced, but did not eliminate, virulence on muskmelon, whereas loss of dspE from Et-C1 strains resulted in a complete loss of pathogenicity on squash and muskmelon. Thus, DspE has distinct roles in the two E. tracheiphila clades. This work represents the first characterization of the major molecular drivers of host specificity and pathogenicity among E. tracheiphila strains. Finally, using pyramided mutant analysis, we demonstrated that, consistent with their expression profiles in planta, effectors DspE, OspG, Eop1 and AvrB4 contributed to the virulence of Et- melo on muskmelon, whereas DspE, OspG, Eop1, HopL1 and HopO1 contributed to the virulence of Et-C1 on squash and muskmelon, with highly overlapping functions among many of the effectors. This work thus illustrates the contribution of distinct sets of effectors to the virulence of E. tracheiphila strains on their hosts. This work sets the foundation for a better understanding of the molecular interaction between the E. tracheiphila pathosystem and its cucurbit hosts which may help guide mechanistic approaches that could lead to the development of durable resistant plant cultivars. 1 CHAPTER 1 GENERAL INTRODUCTION The genus Erwinia contains 44 validly published species that are included in the list of prokaryotic names with standing in nomenclature (LPSN), including both pathogenic and non- pathogenic Enterobacteria. Among these, only a few species are known to be plant pathogens, including Erwinia amylovora, E. pyrifoliae, E. psidii, E. papaya, E. aphidicola and E. tracheiphila. These are the causative agents of fire blight of rosaceous plants, bacterial shoot blight of pear in Asia, branch, flower and fruit rot in guava, bacterial crown rot of papaya and fruit spot of pepper and bacterial wilt of cucurbits, respectively. Other species range from commensals to epiphytes. Unlike most plant pathogenic Erwinia species, which cause necrotic diseases on their respective hosts, E. tracheiphila causes a vascular disease. E. tracheiphila is a xylem pathogen that is vectored by cucumber beetles. It replicates in the plant xylem and can induce wilt. Moreover, there is evidence that it can replicate in the digestive tract of its insect vectors. Adaptation to the low-nutrient conditions in the xylem may be one factor driving the observed differences between E. tracheiphila and other plant-associated Erwinia species. Insights from the approximately 5-Mb genome of E. tracheiphila revealed that it is distinct from those of other sequenced Erwinia species in exhibiting pseudogenization of about one-fifth of its coding sequences, influx of mobile genetic elements, and massive acquisition of horizontally transferred genes, including several putative virulence factors. These features suggest that E. tracheiphila recently evolved from a free-living lifestyle outside of the plant into its current lifestyle in which it is