Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Elena Christian - Pennsylvania State University
Co-Author(s): Timothy McNellis, Penn State University, PA; Sara Klee, Penn State University, PA
The gram-negative bacterium Erwinia amylovora is the cause of fire blight, a destructive disease of apples and pears. This project seeks to characterize the ptxR gene in E. amylovora, an uncharacterized gene we believe to be a regulator of virulence. Prior to this work, the McNellis lab of Pennsylvania State University mutated ptxR by the use of transposon mutagenesis and observed a loss of virulence in immature apple halves. The mutant strain was named ptxR::Tn5. PtxR a LysR family transcriptional regulators contains a helix-turn-helix (HTH) DNA-binding domain. Based on its role in human pathogens, we predict that PtxR plays a role in the regulation of virulence factors in E. amylovora. Among E. amylovora’s virulence factors are the siderophore desferrioxamine E and the exopolysacchride (EPS). Siderophore production is often an important factor in pathogenesis as free iron is frequently limited in a host. E. amylovora’s main EPS may protect the pathogen against plant defense reactions, and mutants deficient in amylovoran biosynthesis are nonpathogenic. In this study we observe changes in E. amylovora’s siderophore and amylovoran production as a result of mutating ptxR. Siderophore production is measured by the use of chrome azurol S agar plates. This medium changes color in the presence of strong iron chelators. We observed delayed siderophore production in ptxR::Tn5, suggesting that ptxR regulates siderophore genes. EPS was measured by precipitation with cetylpyridinium chloride. The ptxR::Tn5 mutant displayed reduced EPS production. These reductions in virulence factors may indicate why the ptxR mutant causes reduced disease in apples. Future directions for this research include investigating the region of DNA bound by PtxR’s HTH domain. ChiP-seq is used to identify genome-wide DNA binding sites for transcription factors. Using ChiP-seq, we expect to identify binding sites for PtxR in the E. amylovora genome, including siderophore and EPS genes, and potentially other virulence factors that may be under PtxR regulation. This will allow us to understand the role of PtxR in fire blight disease. PtxR has yet to be described in a plant pathogen, and this allows us to explore the role of this protein in a new pathosystem. Understanding virulence factors can allow scientists to focus their targets when creating methods to control fire blight in agricultural settings.
References: Bellemann, P., Bereswill, S., Berger, S., and Geider, K. (1994). Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis. International Journal of Biological Macromolecules 16, 290-296.Dellagi, A., Brisset, M.-N., Paulin, J.-P., and Expert, D. (1998). Dual role of desferrioxamine in Erwinia amylovora pathogenicity. Molecular Plant-Microbe Interactions 11, 734-742.Perez-Rueda, E., and Collado-Vidas, J. (2000). The repertoire of DNA-binding transcriptional regulators in Escherichia coli K-12. Nucleic Acids Research 28, 1838-1847.
Funder Acknowledgement(s): Funding was provided by USDA NIFA Grant # 2016-67032-25007, titled 'Plant Friends and Foes: Plant Interactions with Other Organisms as Related to Plant Health, Agriculture and Environment', Tim McNellis, Project Director.
Faculty Advisor: Timothy McNellis, email@example.com
Role: My involvement in this research was quite extensive. In the beginning I was responsible for screening mutants to determine if they had a role in Erwinia amylovora’s pathogenicity. However, I was given a clear protocol to follow. After the identification of ptxR, it was my responsibility to find journal articles and design experiments under the guidance of a graduate student. During this process I preformed all molecular techniques required for the characterization of ptxR. These techniques included all gel running, PCR primer design, and ChiP-seq development.