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Title:Characterization of major virulence regulators of Erwinia amylovora
Author(s):Li, Wenting
Advisor(s):Zhao, Youfu
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):Erwinia amylovora
amylovoran
type III secretion system (T3SS)
Two-component signal transduction systems (TCSTs)
EvnZ/OmpR
GrrS/GrrS
sigma factors
RpoN
Abstract:Erwinia amylovora is the causal agent of the devastating fire blight disease which is a major concern to the apple and pear industry. Fire blight costs millions of dollars of economic losses all over the world. Exopolysaccharide (EPS) amylovoran and type III secretion system (T3SS) are two major virulence factors in E. amylovora. However, how these virulence factors are regulated is not completely understood. In bacteria, gene expression is mainly regulated at the transcription initiation level and its core RNA polymerase (RNAP) requires sigma factors for promoter recognition and initiation. In this study, we investigated the role of several sigma factors in regulating virulence gene expression in E.amylovora. Early studies have shown that hrp-type III secretion (T3SS) in E. amylovora is regulated by HrpS, a member of the σ54 enhancer binding proteins, and the master regulator HrpL, which belongs to the ECF subfamily of σ factors. Other sigma factors characterized included RpoN, a nitrogen limitation σ54 factor, and its modulation protein YhbH. Our results showed that mutations in hrpS, hrpL, rpoN and yhbH resulted in nonpathogenic phenotype in host plant and no hypersensitive response in non-host tobacco. Consistently, expression of T3SS genes including hrpL, dspE, hrpN and hrpA was barely detected in hrpS, hrpL, rpoN and yhbH mutants. Amylovoran (EPS) production was higher in these mutants than that of wild type (WT) strain, indicating sigma factors may also play roles in regulating exopolysaccharide production. These results suggest that sigma factors in E. amylovora are important virulence regulators and sigma factor cascade exists in its regulatory networks. Two-component signal transduction systems (TCSTs) in E. amylovora play a major role in virulence and in regulating amylovoran production, including EnvZ/OmpR and GrrS/GrrA, two widely distributed systems in gamma-proteobacteria. While both systems negatively control amylovoran biosynthesis, deletion mutants of envZ/ompR and grrA/grrS have opposite swarming motility phenotypes. In order to determine how the two systems interact, two triple mutants, envZ/ompR/grrA (ERA) and envZ/ompR/grrS (ERS) were generated. Our results showed that both triple mutants had slightly increased virulence on apple shoots as compared to that of wild type (WT) as well as mutants deleting a single system. In an in vitro amylovoran assay, amylovoran production was significantly increased in the two triple mutants, indicating the two systems synergistically regulate amylovoran production. In consistent with amylovoran production, amsG gene expression was expressed significantly higher in the triple mutants in vitro than those in WT as well as mutants deleting a single system. In contrast, exopolysaccharide levan was significantly reduced in the triple mutants compared with that of WT and deletion of a single system. In addition, the triple mutants showed reduced swarming motility on swarming plates compared to that of grrA/grrS mutants and WT strain, but moved slightly faster than that of envZ/ompR mutants, indicating that the two systems antagonistically regulate swarming motility in E. amylovora. Furthermore, type III secretion (T3SS) genes were significantly upregulated in the triple mutants as well as deletion of a single system than that of the WT strain. These results indicate that EnvZ/OmpR and GrrS/GrrA systems play major roles in virulence and in regulating virulence gene expression.
Issue Date:2013-02-03
URI:http://hdl.handle.net/2142/42460
Rights Information:Copyright 2012 Wenting Li
Date Available in IDEALS:2013-02-03
2015-02-03
Date Deposited:2012-12


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