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Title:Posttranscriptional and posttranslational regulation of virulence in erwinia amylovora
Author(s):Lee, Jae Hoon
Director of Research:Zhao, Youfu
Doctoral Committee Chair(s):Zhao, Youfu
Doctoral Committee Member(s):Huber, Steven; Schroeder, Nathan; Chen, Li-Qing
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Erwinia amylovora
virulence
Lon
ClpXP
CsrA
two-component system
gene regulatory network
Abstract:Erwinia amylovora is the causal agent of fire blight, the most destructive bacterial disease of the Rosaceae family plants. Two virulence factors, the type III secretion system (T3SS) and the exopolysaccharide (EPS) amylovoran, are strictly required for its pathogenicity. Our previous studies have determined the role of several transcription factors in the regulation of E. amylovora virulence; however, molecular mechanisms of virulence regulation at the posttranscriptional and posttranslational levels have still remained elusive. In this dissertation, our goal was to understand new regulatory mechanisms in E. amylovora virulence. First, we characterized the molecular mechanism of Lon protease-mediated virulence regulation. Mutation of the lon gene caused the amylovoran overproduction, the increased T3SS expression and the non-motile phenotype. In the absence of Lon, abundance and stability of the HrpS/HrpA and RcsA proteins were significantly increased, and the resulting accumulation of the RcsA/RcsB proteins influenced the expression of flhD, hrpS and csrB. In addition, lon expression is under the control of the RNA-binding protein CsrA, possibly at both the transcriptional and post-transcriptional levels, suggesting a possible interplay between Lon and the Csr system. Second, we examined the role of ClpXP protease in virulence regulation and its potential interaction with Lon. Mutation in clpXP diminished the T3SS expression, amylovoran production and motility, resulting in delayed disease progress. Highly accumulated RpoS proteins were detected in the clpXP mutant, and mutation of rpoS in the clpXP mutant background restored virulence to the wild-type level. These suggest that ClpXP-dependent RpoS degradation positively affects virulence traits. In addition, lack of both ClpXP and Lon resulted in significantly reduced virulence independently of RpoS level, suggesting that ClpXP and Lon are indispensable for full virulence. Third, transcriptional regulation mechanism of the hrpS gene, encoding the essential T3SS activator, was examined. We found that the hrpS gene contains two promoters driven by HrpX/HrpY and the Rcs system, respectively. IHF also positively regulates hrpS expression through directly binding to the hrpX promoter and positively regulating hrpX/hrpY expression. Moreover, hrpX expression was down-regulated in the ppGpp-deficient mutant and the dksA mutant, but up-regulated when the wild-type strain was treated with serine hydroxamate, suggesting that ppGpp might induce hrpX/hrpY and hrpS expression. Furthermore, CsrA positively regulates hrpS expression mainly through the Rcs system. These results suggest that E. amylovora recruits multiple stimuli-sensing systems to regulate hrpS and T3SS gene expression. Fourth, we examined the global effect of CsrA and determined potential molecular mechanisms of CsrA-dependent virulence regulation in E. amylovora. Using REMSA, direct interaction between CsrA protein and csrB sRNA was confirmed, while CsrA did not bind to the transcripts of T3SS activators, hrpL and hrpS. Transcriptomic analyses under the T3SS-inducing condition revealed that mutation in csrA led to differential expression in more than 20% genes in the genome. Of these, T3SS genes and those required for cell growth and viability were significantly down-regulated, explaining the pleiotropic defects in the csrA mutant. On the other hand, the csrB mutant exhibited significant up-regulation of the major virulence genes, further suggesting antagonistic effects of csrB on CsrA. Through REMSA combined with site-directed mutagenesis and LacZ reporter gene assay, three CsrA targets (flhD, rcsB and relA) were identified that positively regulate E. amylovora virulence. Overall, this dissertation demonstrates that E. amylovora employs multiple layers of gene regulatory networks to effectively control the expression of virulence factors.
Issue Date:2018-04-09
Type:Thesis
URI:http://hdl.handle.net/2142/101271
Rights Information:Copyright 2018 Jae Hoon Lee
Date Available in IDEALS:2018-09-04
Date Deposited:2018-05


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