Browsing by Subject "Effector"
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Item Analysis of secreted proteins of Magnaporthe grisea and the search for protein effectors(Texas A&M University, 2007-09-17) Shang, YueMagnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.Item Directing cell migration by dynamic control of laminar streams(2010-12) Moorjani, Samira Gian; Shear, Jason B.; Morrisett, Richard; Zhang, John X.; Aldrich, Richard W.; Zaman, MuhammadInteractions of cells with their chemical microenvironments are critical to many polarized processes, including differentiation, migration, and pathfinding. To investigate such cellular events, tools are required that can rapidly reshape the microscopic chemical landscapes presented to cultured cells. Existing chemical dosing technologies rely on use of pre-fabricated chemical gradients, thus offering static cell-reagent interactions. Such interactions are particularly limiting for studying migration and chemotaxis, during which cells undergo rapid changes in position, morphology, and intracellular signaling. This dissertation describes the use of laminar streams, containing cellular effector molecules, for precise delivery of effectors to selected subcellular regions. In this approach, cells are grown on an ultra-thin polymer membrane that serves as a barrier to an underlying reagent reservoir. By using a tightly-focused pulsed laser beam, micron-diameter pores can be ablated in the membrane upstream of desired subcellular dosing sites. Emerging through these pores are well-defined reagent streams, which dose the targeted regions. Multiple pores can be ablated to allow parallel delivery of effector molecules to an arbitrary number of targets. Importantly, both the directionality and the composition of the reagent streams can be changed on-the-fly under a second to present dynamically changing chemical signals to cells undergoing migration. These methods are applied to study the chemotactic responses of neutrophil precursor cells. The subcellular localization of the chemical signals emerging through pores is found to influence the morphological evolution of these motile cells as they polarize and migrate in response to rapidly altered effector gradients.Item Identification of C. burnetii Type IV Secretion Substrates Required for Intracellular Replication and Coxiella-Containing Vacuole Formation(2014-05-05) Weber, MaryCoxiella burnetii is a Gram-negative intracellular pathogen that encodes a specialized type IVb secretion system (T4SS) which is essential for intracellular replication, Coxiella-containing vacuole (CCV) formation, modulation of apoptosis, and effector translocation. To identify T4SS candidate substrates, we used an enhanced bioinformatics guided approach. Expression of 234 T4SS candidate substrates as TEM1 ?-lactamase fusions identified 53 substrates that were translocated in a Dot/Icm-dependent manner. Large scale screens aimed at identifying localization and function revealed that several of these substrates traffic to distinct subcellular compartments and interfere with crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Of these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells, but displayed normal growth in axenic culture. Given their confirmed role in intracellular replication and CCV formation, we named 5 of these substrates CirA-E (Coxiella effector for intracellular replication). To identify the pathways targeted by these crucial substrates, S. cerevisiae strains were co-transformed with pYEp13 yeast genomic library and the T4SS substrates. Using this approach we identified multiple members of the Rho family of GTPases as suppressors of Cbu0041 (CirA) toxicity. Overexpression in mammalian cells resulted in cell rounding, detachment, and reduced stress fibers. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation.