THE BROAD-HOST-RANGE PATHOGENESIS OF AGROBACTERIUM TUMEFACIENS: SUCCESSFUL COUPLING THE MOTIONS AND DOMAIN INTERACTIONS WITHIN VIRA TO INTEGRATE SIGNAL SENSING Pubblico
Lin, Yi-Han (2011)
Abstract
ABSTRACT
THE BROAD-HOST-RANGE PATHOGENESIS OF AGROBACTERIUM
TUMEFACIENS: SUCCESSFUL COUPLING THE MOTIONS AND
DOMAIN
INTERACTIONS WITHIN VIRA TO INTEGRATE SIGNAL SENSING
By
Yi-Han Lin
Agrobacterium tumefaciens has provided a nice example of a
wide-host-range
pathogen mediating the only known inter-kingdom gene transfer
strategy for transferring
higher plants. The system employs a simple two-component system
(TCS) scheme to
perceive and integrate multiple plant-derived signals and regulates
virulence. The
VirA/VirG two-component protein adopts a conserved phosphorylation
catalytic core, as
other TCS proteins, but incorporates signal regulation by
coordinating with three
regulatory domains. In this dissertation, I have attempted to
dissect the motions and
molecular mechanisms of these regulatory domains. I started by
analyzing the interface
of the critical phenol sensing linker domain and the kinase core,
and the data supports a
rotational motion propagating through what appears to be a rigid
helical bundle, spanning
from the linker domain to the kinase. The phenol binding within the
linker domain and
signal specificity were probed, both computationally and
genetically, and the data
indicates a possible long-range motion connecting the helical
interface to the proposed
phenol binding site. I further analyzed the mechanisms that
coordinate the other two
regulatory domains: the periplasmic domain for sugar sensing and
the C-terminal receiver
domain for regulation of the phenol response. A critical
convergence point regulating
both phenol perception and sugar perception was identified at a
single tyrosine residue,
aa293. The role of this point in mediating the wide-host-range
properties of the pathogen
is discussed.
The findings in this dissertation shed light on protein evolution,
grounded in the
mechanism of inner-domain motion regulation and the long-range
inter-domain
coordination, to allow for this truly remarkable pathogen to
emerge. New experimental
approaches have emerged from this dissertation that may well define
the plant signaling
landscape and the intricate system-wide chemistry that occurs at
the host-pathogen
interface. This unique insight into plant-bacterial interaction
continues to define the
intricate cellular commitments important for pathogen resistance
and eukaryotic cell
development.
Table of Contents
TABLE OF CONTENTS
CHAPTERS
PAGE
1. THE PRINCIPLE OF AGROBACTERIUM TUMEFACIENS
1
PATHOGENESIS AND THE VIRA/VIRG TWO-COMPONENT
SYSTEM
Agrobacterium pathogenesis and the plant-derived
signals
The VirA/VirG TCS
Phenol perception by the linker domain and the linker structure
model
2. MEDIATING THE ROTATIONAL MOTION BETWEEN THE
25
PHENOL SENSING LINKER DOMAIN AND THE KINASE IN VIRA
2
Introduction
25
The HAMP domain
Materials and Methods
32
Results
45
GCN4 fusions
Library screen of constitutive on mutants in aa426-437 (linker
4)
Kinase coiled-coil insertion
Discussion
73
3. GENETIC,
COMPUTATIONAL,
AND
BIOPHYSICAL
80
CHARACTERIZATION OF PHENOL PERCEPTION OF THE LINKER
DOMAIN
Introduction
80
The GAF domain
Phenol specificity of different Agrobacterium strains and
Y293T
Materials and Methods
94
Results
103
Computation modeling phenol binding in GAF
Genetic analysis of phenol perception
About this Dissertation
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