Date of Graduation
8-2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Degree Level
Graduate
Department
Cell & Molecular Biology
Advisor/Mentor
Egan, Martin J.
Committee Member
Lewis, Jeffrey A.
Second Committee Member
Fan, Chenguang
Third Committee Member
Wang, Yong
Keywords
Appressoria; M. oryzae; Rice blast; Septin ring
Abstract
The fungus Magnaporthe oryzae causes a devastating disease of rice and wheat, called blast, which poses a major threat to food security across the planet. To enter plant tissue, M. oryzae produces a remarkable pressure-generating infection cell called an appressorium, which mechanically ruptures the tough cuticle of host leaves through the application of enormous physical force directed onto a narrow penetration structure emerging from its base. Importantly, appressorium form and functionality depends on the regulated assembly of a toroidal ring-like structure, composed of septins, which forms within its base. Septins are a family of membrane-associated GTP-binding proteins that polymerize into linear non-polar filaments, that can be further organized into an array of higher-order structures at the cell cortex. Recent advancements in microscopy techniques and biochemical studies have contributed to the growing understanding of septin structure, remodeling, and function in diverse cell types. However, the precise molecular events and regulatory factors of septin polymerization and the formation of distinct higher-order architectures are not fully characterized. In a previous study, we identified 63 putative septin proximal interacting proteins, one of which was a Rho GDP-dissociation inhibitor, MoRdi1. In this dissertation, the role of MoRdi1 in appressorium morphogenesis was investigated. Gene deletion experiments revealed that MoRdi1 plays a role in vegetative growth, sporulation, and spore morphology. Additionally, MoRdi1 was found to be essential for the organization of F-actin and the geometry of higher-order septin rings, ultimately impacting the pathogenicity of M. oryzae. Furthermore, the involvement of Rho3 GTPase in M. oryzae in development and pathogenicity was examined. It was determined that Rho3 GTPase is not required for appressoria formation but plays a role in the localization of the F-actin network and is essential for full pathogenicity. In contrast, Cdc42 GTPase was found to be dispensable for septin and actin dynamics during appressorium development. Additionally, a fluorescence microscopy-bases screen was designed to identify novel proteins that regulate the organization and remodeling of septins in M. oryzae. To facilitate this screening, a novel entry strain was engineered, enabling the visualization of septin higher-order structures, nuclei, and the microtubule cytoskeleton in live cells. Random mutagenesis protocols were optimized, using both chemical and UV-based approaches, to mutate the entry strain, followed by phenotypic screening using fluorescence microscopy, and whole genome sequencing to identify causal mutations. Here, it is described how the future implementation of this genome-wide screening approach may provide improved understanding of molecular mechanisms governing septin dynamics during infection related development in M. oryzae.
Citation
Proko, R. (2023). Investigating and Characterizing Septin Cellular Regulators During Infection Related Development in M. oryzae. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4851
