Successful bacterial pathogens manipulate crucial intracellular host processes
as a virulence strategy. One particular potent mechanism utilized by bacterial
phytopathogens is to inject virulence factors (effectors) directly into the host cell. While
many effectors have been identified and shown to suppress plant immune responses,
very few have well-characterized enzymatic activities or host targets. To overcome the
challenges of functional analysis of effectors, I designed two heterologous screens to
characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae.
Specifically, my objective was to identify those P. syringae effectors that target
evolutionarily conserved host proteins or processes and to subsequently elucidate the
molecular mechanisms of these effectors. The first heterologous screen that I
performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human
cells to identify potential interacting host proteins. The second heterologous screen
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utilized a high-throughput genomics approach in yeast, known as the pathogenic
genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous
approach, I have identified HopZ1a as the first bacterial phytopathogen effector that
binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the
eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin.
In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the
secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase
activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and
a conserved lysine residue (K289). Using the second heterologous screen in yeast, I
have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK)
signaling cascades. Together, my work has identified novel eukaryotic targets and
elucidated the virulence functions of HopZ1a.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/35068 |
| Date | 28 February 2013 |
| Creators | Lee, Amy Huei-Yi |
| Contributors | Guttman, David S., Desveaux, Darrell |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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