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<p>1. To dissect the role of alternative sigma factors in regulating virulence factors in Erwinia amylovora. In addition to housekeeping sigma factor RpoD (?70), E. amylovora genome contains all major sigma factors, including RpoE, RpoH, RpoN, and RpoS. The alternative sigma factors RpoN (?N or ?54) and stationary sigma factor RpoS (?S or ?38) and their associated modulation proteins will be the focus of this study. We will determine the role of alternative sigma factors in virulence of E. amylovora. </p>
<p>2. To determine the role of rsmAB in regulating virulence factors in Erwinia amylovora. It has been reported that in Pectobacterium, rsmB, which encodes a regulatory small RNA, binds to and neutralizes the degradation effect of RsmA on hrpL mRNA. However, our preliminary data indicates otherwise in E. amylovora. We will determine what role of rsmAB in virulence of E. amylovora. </p>
<p>3. To demonstrate the role of protein lysine acetylation in Erwinia amylovora virulence. Several lysine acetylated proteins associated with E. amylovora virulence were identified in our lab as lysine acetylation (LysAc) proteins, which included three proteins involved in amylovoran production (AmsJ, ThiI, and GalE) and two T3SS-related proteins (HsvB and YopH). However, their roles in virulence and metabolism are unclear. We will determine the role of protein acetylation in virulence of E. amylovora.</p>
<p>NON-TECHNICAL SUMMARY: <br/>Fire blight, caused by the bacterial pathogen E. amylovora, is a destructive disease of apple and pear. In the United States, regional losses to fire blight and cost of control average over $100 million annually. The use of streptomycin, an antibiotic that targets the blossom blight phase, has been recommended until recent years when the occurrence of streptomycin resistance has rendered this antibiotic ineffective. Moreover, this also raises concerns over the potential impact of agricultural use of antibiotics on human health. Without streptomycin, there are no other reliable fire blight disease control measures available. Thus, new strategies for controlling fire blight are critical for preventing severe losses in susceptible orchards in the near-term along with pursuing strategies for long-term management of fire blight. Sigma factors, small
RNA, and protein post-translational modification are part of the global regulatory networks in bacteria. Sigma factors, including alternative sigma factors, are essential transcription initiation factors that direct RNA polymerase to bind specific promoter regions. It is also known that many sRNAs are members of regulatory circuits involved in stress response, virulence and metabolism. Furthermore, it is well established that acetylation of proteins can influence their binding to DNA, thus impacting gene expression. However, for plant pathogenic bacteria, studies involving sigma factors, small RNA and protein lysine acetylation have been very limited. Therefore, there is a critical need to conduct research in these new and emerging research areas by determining the role of small RNA, sigma factors and protein lysine acetylation in regulating virulence factors in E. amylovora. The
knowledge gained will further improve our fundamental understanding of how E. amylovora infects hosts and cause disease, and allow us to develop strategies for disease control and reduce the economic losses. The knowledge could also be extended to other enterobacterial systems such as E. coli and Salmonella, which are great threats to human health and food safety.
<p>APPROACH: <br/>We will employ bacterial genetics, bioinformatics, genomics, and sequencing approaches to elucidate the roles of alternative sigma factors, small RNA and protein lysine acetylation in regulating virulence factors in E. amylovora. Many procedures used previously in my lab will also apply to this project, including immature pear and apple shoots as plant materials to test virulence of bacterial wild type and mutants, generation of mutants using PCR-based red-cloning, GFP-reporter system to monitor the promoter activities of target genes, quantitative real-time PCR-based approach to determine gene expression, CPC method to measure amylovoran production, microarray-based global transcriptomic profiling, as well as other routine microbiology methodology. In addition, site-directed mutagenesis will be used to manipulate the lysine residues of transcriptional
regulators or metabolic enzymes that are found to be acetylated to study its function in situ; and electrophoretic mobility shift assays (EMSA) and phosphorylation assays will be used for DNA binding assays in vitro. Finally, to reconstruct the regulatory network, functional genomic approaches including microarray will be used to determine the regulatory node of different regulators in the complicated regulatory networks.
<p>PROGRESS: 2012/10 TO 2013/09
<p>Target Audience:<br/>Scientists in the fire blight research community and related enterobacterial areas. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Presentations at professional meetings. What do you plan to do during the next reporting period to accomplish the goals? We will continue working on the role of small RNA and RNA-binding protein in the virulence of E. amylovora, inlcuding generating mutants.