DISSECTING THE MULTIMODAL SIGNALING NETWORK MEDIATED BY CYCLIC-DI- GMP IN ERWINIA AMYLOVORA By Roshni Russi Kharadi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pathology―Doctor of Philosophy 2021 DISSECTING THE MULTIMODAL SIGNALING NETWORK MEDIATED BY CYCLIC-DI- GMP IN ERWINIA AMYLOVORA ABSTRACT By Roshni Russi Kharadi Erwinia amylovora is the bacterial phytopathogenic causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. The successful orchestration of infection by E. amylovora within the host entails a coordinated implementation of several different virulence strategies. A key step in the disease cycle of E. amylovora is the transition from a primarily Type III secretion system (T3SS) effectors-dependent phase in the leaf apoplast to a biofilm-dependent phase within xylem vessels. Cyclic-di-GMP (c-di-GMP), a ubiquitous bacterial second messenger mediates this phase transition into a sessile, attached lifestyle within biofilms in E. amylovora. This body of work encompasses several aspects of the complex and multifactorial signaling network dependent on c-di-GMP in E. amylovora. Diguanylate cycles enzymes (encoded by edc genes) dimerize GTP subunits to synthesize c-di-GMP and phosphodiesterase enzymes (encoded by pde genes) hydrolyze c-di-GMP. We found that the deletion of the three active pde genes in E. amylovora, singly and in combinations of two and all three genes, led to a measurable increase in intracellular c-di-GMP levels. In addition, the elevated c-di-GMP levels correlated with increased production of amylovoran, which is the most abundant exopolysaccharide (EPS), and, a pathogenicity factor in E. amylovora. The expression of T3SS, quantified by the transcriptional level of hrpL as well as by virulence measurements in the apple and pear models, was found to be negatively regulated by c- di-GMP. While biofilm formation generally increased with elevated levels of c-di-GMP, the total pde deletion mutant, ΔpdeABC, showed a relative depreciation in the ability to form biofilms, owing to the physical autoaggregative characteristic of this strain when grown in liquid media. Autoaggregation also impaired cell separation post division, leading to the presence of filamentous cells within cellular aggregates. In addition to the EPSs amylovoran and cellulose, EagA, a peptidoglycan hydrolase was found to be a major contributor to the facilitation of autoaggregation in E. amylovora. The eagA/znuABC zinc uptake gene cluster, was found to be transcriptionally regulated by c-di-GMP and the zinc-dependent repressor Zur. Further, we evaluated the impact of a systemic deletion of all genetic components involved in c-di-GMP metabolism, including active and degenerate dgc and pde encoding genes. The resulting mutant, Ea1189Δ12 was found to be impaired in surface sensing and attachment, which are key steps in the initiation of biofilm formation both in-vitro and in-planta. The transcriptomic profile of WT Ea1189 and Ea1189Δ12 at various stages of biofilm development revealed marked differences in critical metabolic and signal transduction pathways. The correlational clustering of phenotypic data gathered from single gene complemented strains generated from Ea1189Δ12, enabled the functional categorization of each of the systemic components. ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. George Sundin for his continued support and guidance throughout this doctoral journey. His motto of pursuing new research avenues stemming from ones own observations has been particularly inspiring, and the basis of the research documented in this dissertation. While pursuing my PhD, I was able to explore exciting new research topics, learn new techniques and most importantly, I was able to do so, while maintaining a great work-life balance. I would also like to acknowledge my fellow Sundin lab group members for their support and teamwork. I would also like to thank my committee members, Dr. Chris Waters, Dr. Monique Sakalidis and Dr. Youfu Zhao for their support, guidance and critical evaluation of my work, which has only enhanced the impact of the research. Finally, the most consistent supporting factor for me throughout this journey has been my family. I would like to thank my parents and my husband for their constant encouragement and their genuine interest in learning about my research. They all know a lot more about c-di- GMP and fire blight now than they ever would have ever conceived possible. iv TABLE OF CONTENTS CHAPTER 1: Dissecting the process of xylem colonization through biofilm formation in Erwinia amylovora ..........................................................................................................1 I. Abstract .........................................................................................................................2 II. Global regulatory impact of c-di-GMP on the E. amylovora disease cycle .................3 CHAPTER 2: Phosphodiesterase genes regulate amylovoran production, biofilm formation, and virulence in Erwinia amylovora ..................................................................7 I. Abstract ..........................................................................................................................8 CHAPTER 3: Physiological and microscopic characterization of cyclic-di-GMP- mediated autoaggregation in Erwinia amylovora ................................................................9 I. Abstract ........................................................................................................................10 CHAPTER 4: Cyclic-di-GMP regulates autoaggregation through the putative peptidoglycan hydrolase, EagA, and, regulates transcription of the znuABC zinc uptake gene cluster in Erwinia amylovora.........................................................................11 I. Abstract ........................................................................................................................12 CHAPTER 5: The cyclic di-GMP network is a global regulator of phase- transition and attachment-dependent host colonization in Erwinia amylovora .................13 I. Abstract .........................................................................................................................14 REFERENCES .................................................................................................................16 v CHAPTER 1 Dissecting the process of xylem colonization through biofilm formation in Erwinia amylovora The abstract presented in subsection I refers to the published article: Kharadi, R.R. and Sundin, G.W., 2020. Dissecting the process of xylem colonization through biofilm formation in Erwinia amylovora. Journal of Plant Pathology, pp.1-9. DOI: 10.1007/s42161-020-00635-x Copyright © 2020, Società Italiana di Patologia Vegetale (S.I.Pa.V.) 1 I. Abstract Erwinia amylovora is the causal agent of fire blight, an economically-important disease affecting apple and pear production worldwide. Initial contact and infection of the host by E. amylovora mainly occurs in flowers, or in young leaves at actively-growing shoot tips. Infection via shoot tips encompasses several distinct steps which include the utilization of a Type III secretion system (T3SS) to establish bacterial populations within the apoplast, infection of the parenchyma, invasion of the xylem, attachment to xylem vessels, biofilm formation, and the eventual colonization of the xylem which manifests outwardly as wilting symptoms in the plant. After E. amylovora gains entry into the xylem, initial attachment to the xylem vessels is mediated by type I fimbriae. Conversely, the small RNA (sRNA) chaperone Hfq and associated sRNA ArcZ negatively regulate attachment and promote biofilm maturation. Attachment and biofilm formation within the xylem are enhanced by the mechanical force emerging from the flow of xylem sap. The second messenger molecule cyclic-di-GMP (c-di-GMP) regulates the transition into the biofilm phase of the infection process of E. amylovora. C-di-GMP also regulates the production of critical exopolysaccharides amylovoran and cellulose, that lend to the structural stability and growth of biofilms within the xylem vessels. In this review, we provide an in-depth evaluation of the process of biofilm formation occurring within the host, as a result of E. amylovora infection. We also provide a model encompassing the different physical and signaling factors involved in biofilm initiation and maturation in E. amylovora, and highlight what needs to be done in the future. 2 II. Global regulatory impact of c-di-GMP on the E. amylovora disease cycle E. amylovora, prevalent in the field, oozing cankers being the primary inoculum source, can infect the host via blossoms and shoot tips (1). The bacterial second messenger, c-di-GMP has been found to affect various regulatory aspects pertaining to fire blight disease progression (2-4). C-di-GMP is chemically characterized by a dimerized form of two GTP subunits, catalyzed by diguanylate cyclase (Dgc) enzymes that contain an active GGDEF domain (5). The hydrolysis of c-di-GMP into the linearized molecule, 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG) is catalyzed by phosphodiesterase (Pde) enzymes that have an active EAL or HD-GYP domain (5). Finally, pGpG can be degraded into GTP subunits by oligoribonucleases (5, 6). The modulation of the activity of these enzymes in response to varied external and internal stimuli can cause changes in the intracellular levels of c-di-GMP (5). Impacts on downstream signaling activity can occur in a c-di-GMP (quantitative) dependent or independent manner. During both blossom infection and during the initial phase of shoot infection occurring within the leaf apoplast, T3SS effectors are critical for effective niche colonization (1, 7). DspE is the most important effector and a pathogenicity factor in E. amylovora (8). C-di-GMP is a negative regulator of the T3SS in E. amylovora (2, 3). HrpL is an alternate sigma factor directly affecting the transcription of the T3SS operonic genes, and is used as an indicator of relative T3SS activity levels (9). In E. amylovora, c-di-GMP negatively regulates hrpL transcription (2). Elevated levels of c-di-GMP have also been corelated with reduced levels of DspE in a tobacco apoplast system (2). Virulence in both apple shoots and immature pear fruit is also negatively regulated by c-di-GMP (2, 3). Specific pathways and/or c-di-GMP dependent effectors involved in the regulation of T3SS, in the context of both blossom infection as well as shoot infection have not been identified. In other bacterial phytopathogenic interactions, c-di-GMP has been 3 found to affect virulence through varied signaling pathways. In Dickeya dadantii, c-di-GMP levels are regulated by the sRNA chaperone Hfq, through the repression of specific Dgc enzymes, GcpA and GcpL (10). This feeds into the global regulatory system of RsmA/RsmB, which can in turn affect the expression of the T3SS through the LysR-type regulator, PecT (10). While Hfq is known to positively regulate virulence in E. amylovora, the mechanism by which it affects T3SS, transcriptionally or post-transcriptionally, is not understood (11). During flower infection, E. amylovora cells are usually deposited on the surface of the stigma and they can proliferate there until they are able to migrate to the nectary at the base of the flower (12-14). While physical forces such as dew and rain droplets can physically move E. amylovora cells from the stigmas to the nectary, flagellar motility is critical for the chemotactic movement towards the organic acid-rich nectary (15-17). In E. amylovora, c-di-GMP has been found to negatively regulate flagellar motility in vitro (2, 3). However, the mechanistic and regulatory specifics of this effect, in the context of tissue-specific host infection, are not fully understood in E. amylovora. In Pseudomonas syringae, high intracellular levels of c-di-GMP have linked to reduced flagellar motility resulting from reduced flagellar synthesis (18). This can in turn suppress FLS2 (Flagellin Sensing 2 pattern recognition receptor)-dependent pattern- triggered immunity (PTI) within the plant, which enables the cells to evade the host’s PTI response (18). In E. amylovora, the PTI response, specifically localized at the flower, is not fully understood. Another critical virulence factor that affects the dynamics of disease progression throughout infection process in E. amylovora is the EPS amylovoran. It is the most abundant EPS, and a pathogenicity factor in E. amylovora (19). Amylovoran biosynthesis is dependent on the 12 gene ams operon in which amsG is the first gene (20). C-di-GMP positively regulates 4 amylovoran production and amsG transcription in E. amylovora (2, 3). All five active Dgc enzymes in E. amylovora, EdcA-E, were found to positively contribute to amylovoran production in vitro (4). The regulatory specifics of this c-di-GMP dependent control of amylovoran production are not well-understood. Elevated levels of c-di-GMP are also associated with increased ooze production in the immature pear fruit (2). Amylovoran, along with cellulose and levan form the bulk of the biofilm matrix within the xylem vessels (4, 19). Cellulose production is positively regulated by c-di-GMP in E. amylovora (4). The Dgc enzymes, EdcB and EdcC are critical in this c-di-GMP dependent regulation of cellulose that occurs through c- di-GMP binding to the cellulose catalytic subunit, BcsA (4). The effect of shifting intracellular levels of c-di-GMP on levan production in E. amylovora has not been studied. E. amylovora cells gain entry into the xylem, either directly, upon being introduced onto the host via externally damaged tissue, or, after channeling through the leaf apoplast, followed by the parenchyma surrounding the xylem and finally into the xylem vessels (21-24). Kharadi and Sundin 2020 covers various aspects of the migration of E. amylovora cells within the host and host factors that influence the development of systemic infection (25). In vitro biofilm studies conducted in a flow-based system have indicated that initial surface sensing and potentially some level of temporary tethering to a surface is dependent upon the flagellar filament in E. amylovora (26). Type IV pili then contribute to a more permanent form of surface attachment, which leads to biofilm development (26). Initial surface sensing, attachment and biofilm development are dependent on c-di-GMP in E. amylovora (26). Other extracellular appendages such as type I fimbriae and curli fimbriae also contribute to biofilm development (27). The Hfq-dependent sRNA, RprA positively regulates dissociation from a biofilm (28). 5 However, the extracellular and intracellular factors that trigger dissociation from a biofilm and systemic spread are not well understood. The current extent of knowledge, specifically pertaining to c-di-GMP based regulation in E. amylovora, has mainly been limited a set of easily measurable phenotypes, including biofilm formation, virulence, amylovoran production and motility (2-4, 29, 30). Most of these phenotypes, barring direct infection measurements in planta, are assessed in vitro. The transcriptional effect of c-di-GMP on critical targets such as hrpL and amsG has also been reported (2, 3). However, the regulatory pathways that are responsible for this transcriptional effect are not fully understood. A primary change in the approach of research focusing on understanding c-di-GMP dependent regulation, should be to develop more in vivo experimental systems that can help understand real-time regulatory changes in the context of the host environment. Additionally, a muti-omics based approach which integrates inputs from the pathogen transcriptome and the proteome during infection, within the host, can help decipher the transcriptional complexity underlying the regulation of critical virulence factors in E. amylovora. 6 CHAPTER 2 Phosphodiesterase genes regulate amylovoran production, biofilm formation, and virulence in Erwinia amylovora This chapter has been published and is accessible at: Kharadi, R.R., Castiblanco, L.F., Waters, C.M. and Sundin, G.W., 2019. Phosphodiesterase genes regulate amylovoran production, biofilm formation, and virulence in Erwinia amylovora. Applied and environmental microbiology, 85(1). DOI: 10.1128/AEM.02233-18 Copyright © 2018, American Society for Microbiology 7 I. Abstract Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule that is an important virulence regulator in the plant pathogen Erwinia amylovora. Intracellular levels of c-di-GMP are modulated by diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP and phosphodiesterase (PDE) enzymes that degrade c-di-GMP. The regulatory role of the PDE enzymes in E. amylovora has not been determined. Using a combination of single, double, and triple deletion mutants, we determined the effect of each of the four putative PDE-encoding genes (pdeA, pdeB, pdeC, and edcA) in E. amylovora on cellular processes related to virulence. Our results indicate that pdeA and pdeC are the two most active phosphodiesterases in virulence regulation in E. amylovora Ea1189. The deletion of pdeC resulted in a measurably significant increase in the intracellular pool of c-di-GMP, and the highest intracellular concentrations of c- di-GMP were observed in the Ea1189∆pdeAC and Ea1189∆pdeABC mutants. The regulation of virulence traits due to the deletion of the pde genes showed two patterns. A stronger regulatory effect was observed on amylovoran production and biofilm formation, where both Ea1189∆pdeA and Ea1189∆pdeC exhibited a significant increase in these two phenotypes in vitro. In contrast, the deletion of two or more pde genes was required to affect motility and virulence phenotypes. Our results indicate a functional redundancy among the pde genes in E. amylovora for certain traits and indicate that the intracellular degradation of c-di-GMP is mainly regulated by pdeA and pdeC, but also suggest a role for pdeB in regulating motility and virulence. 8 CHAPTER 3 Physiological and Microscopic Characterization of Cyclic-di-GMP-Mediated Autoaggregation in Erwinia amylovora This chapter has been published and is accessible at: Kharadi, R.R. and Sundin, G.W., 2019. Physiological and microscopic characterization of cyclic- di-GMP-mediated autoaggregation in Erwinia amylovora. Frontiers in microbiology, 10, p.468. DOI: 10.3389/fmicb.2019.00468 Copyright © 2019 Kharadi and Sundin. 9 I. Abstract The second messenger cyclic-di-GMP (c-di-GMP) is a critical regulator of biofilm formation in the plant pathogen Erwinia amylovora. Phosphodiesterase (PDE) enzymes are responsible for the degradation of intracellular c-di-GMP. Previously, we found that the deletion of one or more of the three PDE enzyme encoding genes (pdeA, pdeB, and pdeC) in E. amylovora Ea1189 led to an increase in biofilm formation. However, in mutants Ea1189ΔpdeAC and Ea1189ΔpdeABC, biofilm formation was reduced compared to the other single and double deletion mutants. Here, we attribute this to an autoaggregation phenotype observed in these two mutants. Examination of Ea1189ΔpdeABC cellular aggregates using scanning electron microscopy indicated that a subset of cells were impaired in cell separation post cell division. Concomitant with this phenotype, Ea1189ΔpdeABC also exhibited increased transcription of the cell-division inhibitor gene sulA and reduced transcription of ftsZ. Ea1189ΔpdeABC showed a significant reduction in biofilm formation, and biofilms formed by Ea1189ΔpdeABC exhibited a distinctive morphology of sparsely scattered aggregates rather than an evenly distributed biofilm as observed in WT Ea1189. Our results suggest that highly elevated levels of c-di-GMP lead to increased cell–cell interactions that contribute to autoaggregation and impair cell-surface interaction, negatively affecting biofilm formation. 10 CHAPTER 4 Cyclic-di-GMP regulates autoaggregation through the putative peptidoglycan hydrolase, EagA, and, regulates transcription of the znuABC zinc uptake gene cluster in Erwinia amylovora This chapter has been published and is accessible at: Kharadi, R.R. and Sundin, G.W., 2020. Cyclic-di-GMP Regulates Autoaggregation Through the Putative Peptidoglycan Hydrolase, EagA, and Regulates Transcription of the znuABC Zinc Uptake Gene Cluster in Erwinia amylovora. Frontiers in microbiology, 11. DOI: 10.3389/fmicb.2020.605265 Copyright © 2020 Kharadi and Sundin. 11 I. Abstract Erwinia amylovora is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. E. amylovora pathogenesis is comprised of distinct type III secretion-dependent and biofilm-dependent stages. Alterations in the intracellular levels of cyclic-di-GMP (c-di-GMP) regulate the transition between the different stages of infection in E. amylovora. We previously reported that hyper-elevation of c-di-GMP levels in E. amylovora Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189ΔpdeABC), resulted in an autoaggregation phenotype. The two major exopolysaccharides, amylovoran and cellulose, were also shown to partially contribute to autoaggregation. In this study, we aimed to identify the c-di-GMP dependent factor(s) that contributes to autoaggregation. We conducted a transposon mutant screen in Ea1189ΔpdeABC and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (Erwinia aggregation factor A), that was found to physiologically contribute to autoaggregation in a c-di-GMP dependent manner. The production of amylovoran was also positively affected by EagA levels. An eagA deletion mutant (Ea1189ΔeagA) was significantly reduced in virulence compared to the wild type E. amylovora Ea1189. eagA is part of the znuABC zinc uptake gene cluster and is located within an operon downstream of znuA. The znuAeagA/znuCB gene cluster was transcriptionally regulated by elevated levels of c-di-GMP as well as by the zinc-dependent transcriptional repressor Zur. We also observed that with an influx of Zn2+ in the environment, the transcription of the znuAeagA/znuBC gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway. 12 CHAPTER 5 The cyclic di-GMP network is a global regulator of phase-transition and attachment- dependent host colonization in Erwinia amylovora This chapter is publicly accessible at : Kharadi, R.R., Selbmann, K. and Sundin, G.W., 2021. The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora. bioRxiv. DOI: 10.1101/2021.02.01.429191 Copyright © owned by the authors. 13 I. Abstract Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. 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