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Laney Graduate School

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THE BROAD-HOST-RANGE PATHOGENESIS OF AGROBACTERIUM TUMEFACIENS: SUCCESSFUL COUPLING THE MOTIONS AND DOMAIN INTERACTIONS WITHIN VIRA TO INTEGRATE SIGNAL SENSING

Lin, Yi-Han (2011)
Dissertation (203 pages)
Committee Chair / Thesis Adviser: Lynn, David
Committee Members: Gallivan, Justin ; Lutz, Stefan
Research Fields: Chemistry, Biochemistry
Keywords: Agrobacterium tumefaciens; VirA/VirG; two-component system; phenol; signal integration; GAF; ratchet motion
Program: Laney Graduate School, Chemistry
Permanent url: http://pid.emory.edu/ark:/25593/b4kn0

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

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