Announcement of Final Thesis Defense Plant Pathology M.S. Degree : Elise W. Straub
August 10, 2026 1:00PM - 2:00PM
Computational prediction and validation of small RNA binding sites in Erwinia amylovora
August 10, 2026
Time: 1:00 pm in FST 162
Members of the Examining Committee and their Department:
- Dr. George W. Sundin – Plant, Soil, and Microbial Sciences
- Dr. Michelle Hulin – Plant, Soil, and Microbial Sciences
- Dr. Sarah Lebeis – Plant, Soil, and Microbial Science
ABSTRACT
The bacterium Erwinia amylovora, the causal agent of fire blight disease of Rosaceous plants, results in major yield loss in apple and pear growing systems worldwide. During the fire blight disease cycle of fire blight, E. amylovora employs major phenotypic switches between motile and sessile lifestyles to successfully colonize and infect the host plants. The shifting phenotypes employed by the pathogen require careful control manifested by many regulatory factors, one of which is small, non-coding RNA (sRNAs). The conserved, Hfq-dependent sRNA ArcZ occurs the order Enterobacterales and plays a core role in the regulation of E. amylovora virulence factors such as Type III secretion, biofilm formation, motility, and oxidative stress. In this study we used multiple types of RNA-RNA interaction (RRI) prediction software to generate an expansive list of putative target genes for ArcZ within E. amylovora. RRIs were evaluated for their binding site location, hybridization energy, and secondary structure to produce a subset of likely RRIs in both virulence and housekeeping genes to be used as a framework for future exploration of the ArcZ regulatory network. Additionally, we found high numbers of putative targets in virulence genes, supporting the keystone role of ArcZ in virulence regulation. We also found that benchmarks such as statistical outputs and hybridization energies generated by computational algorithms do not always accurately predict the biological relevance of sRNA-target interactions. From our computational predictions, we isolated the hofC gene, a putative pilus gene active in the irreversible attachment stage of biofilm formation. We found the hofC gene and its associated operon present across the Enterobacterales order, likely having been transmitted vertically from a common ancestor. We generated mutants of the sRNA chaperone protein Hfq, arcZ, hofC, and the ArcZ-hofC binding site in E. amylovora. Using both static and continuous flow cell attachment assays, we found that ArcZ regulates hofC in a direct RRI and that disruption of the hofC gene under flow conditions attenuates the biofilm formation of E. amylovora in a similar manner to that of a biofilm negative control. Our results indicate that hofC is necessary to produce a stable and mature biofilm, that ArcZ is active in the regulation of this process, and that there is a direct interaction between the sRNA and target mRNA. Our work demonstrates the usefulness of computational predictions of RNA interactions and how they can be a beginning to in vitro experimental design. Additionally, validated RRIs, such as between ArcZ and hofC can be used as a benchmark to evaluate future prediction algorithms and data sets. As both computational and experimental methods expand, the many factors regulating key bacterial processes, such as sRNA that module virulence factors, will become more defined and our understanding of the complexity of these networks will continue to develop.
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