Alaryan, Maryam Mahdi, authorCharkowski, Amy O., advisorStewart, Jane, committee memberRoberts, Robyn, committee memberBorlee, Brad, committee member2025-09-012025-09-012025https://hdl.handle.net/10217/241865https://doi.org/10.25675/3.02185This dissertation investigates the role of proteases in the virulence of Pectobacterium carotovorum, a significant bacterial pathogen responsible for economically devastating soft rot diseases in numerous plant hosts. Proteases are crucial enzymes that enable bacterial pathogens to degrade host tissues, thus facilitating infection and colonization. Despite extensive study, significant gaps persist regarding protease regulation, precise roles in virulence, and effective diagnostic and management strategies. Initially, we thoroughly reviewed the current knowledge on bacterial proteases, particularly emphasizing their classification, regulatory pathways, and mechanisms involved in host interactions. Subsequently, we systematically optimized protease activity detection assays, leading to improved protocols for reliably identifying protease activity in P. carotovorum. These optimized assays provided foundational tools for accurately characterizing protease-related phenotypes. To identify genetic factors controlling virulence, a mutant library of P. carotovorum WPP14 was constructed using mini-Tn5 transposon mutagenesis. Phenotypic screening identified critical mutations affecting protease production, motility, and pectate lyase activity, notably disruptions in gacS, purM, metA, and prtW genes. Transcriptomic analyses further confirmed extensive regulatory roles for these genes, highlighting their impact on secretion systems, metabolic pathways, and stress responses. Complementation experiments validated these genes' direct roles in regulating virulence-associated traits. Functional evaluations of cloned proteases from P. carotovorum WPP14 were conducted to assess interactions with plant-derived inhibitors from Solanum chacoense. Experimental assays revealed limited inhibitory effects, with measurable inhibition observed only for Lon protease under specific conditions. Complementary computational analyses using AlphaFold3 supported these findings, predicting weak interactions between most cloned proteases and the tested inhibitors. These results highlight the need for further refinement and optimization of protease inhibitors as a potential strategy for managing soft rot diseases. Additionally, a comprehensive review of related soft rot pathogens Dickeya and Musicola outlined current diagnostic methods, biochemical differentiation techniques, and molecular identification tools, identifying limitations in existing methods. These findings underscore the need for enhanced diagnostic tools and methodologies to better detect and manage these pathogens effectively. Collectively, this research advances the understanding of protease-mediated virulence mechanisms in P. carotovorum, identifies key genetic targets for potential disease management strategies, and provides improved methodologies for pathogen detection and virulence assessment. These insights lay critical groundwork for developing effective control measures against economically significant soft rot diseases.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Text