Date of Award
Bachelor of Science
Dr. B. Daniel Pierce
The soil bacterium Agrobacterium tumefaciens causes tumors in plants through interkingdom gene transfer. This transfer is initiated upon a wounding event that results in the release of plant signaling factors such as phenols and sugars that are recognized by protein machinery in the periplasm and inner membrane of A. tumefaciens. The histidine kinase protein VirA, in combination with the periplasmic protein ChvE, recognizes these signals and initiates induction of virulence genes via a signaling pathway, culminating with the insertion of tumor-inducing T-DNA into the wounded plant cells. While the interaction between the Periplasmic domain of VirA and sugar-bound ChvE is well-characterized, less is understood about the VirA Linker domain, which is critical for phenol reception. Indeed, experiments using VirA truncations have found that the Linker domain is essential in the recognition process of phenols through a putative direct binding. We have begun to investigate novel VirA mutations and the effect they have on signal recognition through -galactosidase assays. Through the production and isolation of the VirA Linker domain, we aim to further characterize this region through identifying its secondary structure using circular dichroism. In addition, models have predicted that the Linker region is homologous to GAF domains, which are known to bind small ligands. Here, we currently have developed a model that predicts where phenols may bind in this region but solving the structure of the Linker domain will be necessary to fully test these predictions. In addition, we aim to directly test the interactions between the Linker domain and phenols by using fluorescence-based thermal shift assays. These assays will monitor protein folding and stability in the presence and absence of phenols at varying temperatures. Together, these data contribute to a further understanding of how phenol interaction with VirA results in autophosphorylation and signal propagation.
Garofalo, Jessica, "Investigation of the VirA Linker Domain to Characterize its Phenol Interactions" (2023). Honors Theses. 1726.