Download Agrobacterium tumefaciens and Plant Cell - igem

Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Agrobacterium tumefaciens
and Plant Cell Interactions
and Activities Required for
Interkingdom
Macromolecular Transfer
Colleen A. McCullen1,2 and Andrew N. Binns1
1
Department of Biology and Plant Sciences Institute, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6018; email: [email protected]
2
Current address: National Institutes of Health, Bethesda, Maryland 20892
Annu. Rev. Cell Dev. Biol. 2006. 22:101–27
Key Words
First published online as a Review in
Advance on May 18, 2006
virA-virG two-component system, signal integration, type IV
secretion, VirB complex
The Annual Review of
Cell and Developmental Biology is online at
http://cellbio.annualreviews.org
This article’s doi:
10.1146/annurev.cellbio.22.011105.102022
c 2006 by Annual Reviews.
Copyright !
All rights reserved
1081-0706/06/1110-0101$20.00
Abstract
Host recognition and macromolecular transfer of virulencemediating effectors represent critical steps in the successful transformation of plant cells by Agrobacterium tumefaciens. This review
focuses on bacterial and plant-encoded components that interact to
mediate these two processes. First, we examine the means by which
Agrobacterium recognizes the host, via both diffusible plant-derived
chemicals and cell-cell contact, with emphasis on the mechanisms
by which multiple host signals are recognized and activate the virulence process. Second, we characterize the recognition and transfer
of protein and protein-DNA complexes through the bacterial and
plant cell membrane and wall barriers, emphasizing the central role
of a type IV secretion system—the VirB complex—in this process.
101
ANRV288-CB22-05
ARI
28 September 2006
21:44
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
Contents
INTRODUCTION . . . . . . . . . . . . . . . . .
HOST RECOGNITION . . . . . . . . . . .
Virulence Gene Expression . . . . . . .
Attachment of Agrobacteria
to Host Cells . . . . . . . . . . . . . . . . . .
MACROMOLECULAR
TRANSPORT . . . . . . . . . . . . . . . . . . .
Targeting of Substrates . . . . . . . . . . .
Mechanism of Export . . . . . . . . . . . . .
Substrate Entry into Host Cells . . .
102
103
104
110
111
111
113
116
INTRODUCTION
Tumor-inducing
(Ti) plasmid: large
plasmid found in
virulent strains of A.
tumefaciens that
encodes several of
the proteins required
for tumorigenesis
Transferred DNA
(T-DNA): region of
the Ti plasmid of A.
tumefaciens between
23 bp direct repeats
processed into
single-stranded form
and transported into
host plant cells
during infection
Opine: sugar–amino
acid conjugates
synthesized by A.
tumefaciens–
transformed plant
cells that can be
metabolized by A.
tumefaciens cells
carrying a Ti plasmid
102
Agrobacterium tumefaciens is a broad-hostrange pathogen, initiating tumors on plants of
most dicotyledonous genera and some monocots as well (DeCleene & DeLay 1976). These
tumors no longer require the continuous presence of the inciting bacterium for proliferation (White & Braun 1942) demonstrating
that the plant cells have been transformed.
The molecular basis of this transformation
revolves around the activities of a large
(∼200 kb) tumor-inducing (Ti) plasmid resident in virulent strains (for reviews of earlier literature, see Binns & Thomashow 1988,
Braun 1982, Hooykaas & Beijersbergen 1994,
Zambryski 1988). Specifically, a portion of the
Ti plasmid, the transferred DNA (T-DNA),
delineated by 23 base pair (bp) border repeats,
is transferred into the plant cells, integrated
into the nuclear DNA, and expressed. This
results in the production of enzymes that catalyze (a) plant hormone synthesis, responsible for tumor growth, and (b) the formation
of novel amino acid–sugar conjugates, termed
opines. The synthesis of opines by the transformed cells provides the selective advantage that drives the evolution of this system:
Opines can serve as carbon and nitrogen
sources for the inducing strain of bacterium
but not for (or for few) other strains (Tempé
& Petit 1982). The capacity for gene transfer led to the development of A. tumefaciens
McCullen
·
Binns
as a gene vector: Virtually any DNA cloned
into the T-DNA can be transferred into
plant cells. Moreover, the elimination of the
T-DNA genes responsible for tumorous
growth ensures that the transformed cells can
be regenerated into fertile plants that transmit
the engineered DNA to progeny (Hooykaas &
Schilperoort 1992, Newell 2000). Using such
modified strains and appropriate selectable
markers, the host range over which DNA can
be transferred now includes species ranging
from other bacteria, fungi, virtually all classes
of plants, and even to some mammalian cells
(Lacroix et al. 2006).
The intense mechanistic analysis of
Agrobacterium-mediated transformation has
established Agrobacterium as an important
model system, at the forefront in understanding how pathogens recognize hosts and
deliver macromolecules into target cells, resulting in disease. From these studies a basic model for the cellular transformation of
plants by A. tumefaciens has been developed
(Figure 1). The cellular intricacies of this
transformation are remarkable. We arbitrarily
break them into the following steps: (a) chemical recognition of host and activation of virulence gene expression, (b) physical recognition and interaction between bacterium and
host, (c) production of transferred substrates
and transfer machinery, (d ) transfer of substrates out of the bacterium and into the
host cell, (e) movement of substrates into nucleus, ( f ) integration of T-DNA into host
genome, and (g) expression of T-DNA. This
oversimplified list leaves out several potentially crucial steps. For example, what steps
does the host take to fend off the pathogen?
Is the bacterium suppressing host defenses,
and if so, how? Once T-DNA transfer has occurred, what are the metabolic consequences
for the bacterium? Detailed consideration of
all these steps is not possible given space limitations; however, numerous recent reviews
have examined many of them (Brencic &
Winans 2005, Christie et al. 2005, Gelvin
2003, Palmer et al. 2004, Tzfira & Citovsky
2002). Here we focus on the following
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Figure 1
General model of Agrobacterium-mediated transformation of a plant cell. See text for details.
question: How do the bacterium and plant
get together so as to achieve macromolecular transfer? Specifically, we seek to explore
first, the chemical and physical interactions
between bacterium and host that set the stage
for transfer and second, the mechanisms by
which cellular barriers are breached so as to
facilitate macromolecular transfer.
HOST RECOGNITION
Agrobacterium strains are widely distributed
in nature. Quite capable of thriving in the
soil independent of hosts, most isolates do
not contain a Ti plasmid (e.g., Krimi et al.
2002). Nevertheless, the capacity for a strain
to induce tumors yields a clear selective
advantage in that the tumor produces a
dedicated food source (the opines) for the
inciting bacterium. Yet equally clearly, the activities of the bacterium necessary to transform the plant are complex and energetically
taxing. Thus, the commitment by the bacterium to the virulence-inducing processes
should be carefully regulated and should occur
only when a competent host is recognized and
available. The first steps in understanding this
control were made in studies that identified
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
103
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
vir genes: genetic
loci on the Ti
plasmid of A.
tumefaciens that
encode proteins
necessary to
recognize plant
signals as well as to
process and transfer
the T-DNA into
host cells
Sensor kinase:
inner membrane
component of a
two-component
regulatory system
that, in response to
signals, controls
phosphorylation
status of a response
regulator
Response
regulator:
transcription factor
component of a
two-component
regulatory system
that binds to DNA
and activates
transcription of
target genes upon
phosphorylation by a
sensor kinase
104
21:44
several Ti plasmid–borne operons outside the
T-DNA that were required for virulence, the
vir genes (Figure 1) (Klee et al. 1983, Stachel
& Nester 1986). Analysis of vir gene expression revealed that, with the exception of
virA and virG, the vir genes are essentially
silent unless they are cocultured with plant
cells (Stachel & Nester 1986). Interestingly,
this induction does not require the attachment of the bacteria to the plant cells but
is rather dependent on molecules exuded by
them. Conversely, several studies reveal that
attachment to the host is necessary for transformation and is mediated by chromosomally
encoded Agrobacterium genes (Douglas et al.
1982, Lippincott & Lippincott 1969). Thus,
host recognition by Agrobacterium resulting in
transformation is composed of two independent processes: virulence gene activation and
attachment to the host cell.
Virulence Gene Expression
vir gene activation during coculture with
plant cells requires two genes, virA and virG
(Stachel & Nester 1986), and although virA
and virG are constitutively expressed at low
levels, they are also highly induced, in an
autoregulatory fashion (Winans et al. 1988).
virA and virG have significant homology to
genes encoding two-component regulatory
systems in which a sensor kinase responds
to signal input and mediates activation of a
response regulator by controlling the latter’s
phosphorylation status (Wolanin et al. 2002).
Several of the fundamental biochemical steps
carried out by two-component systems are
seen in the VirA/VirG system (Figure 2). In
this case, the membrane-bound sensor kinase,
VirA, autophosphorylates at a conserved histidine residue and transfers this phosphate to
a conserved aspartate residue on the cytoplasmic response regulator, VirG. VirG-PO4
binds at specific 12 bp DNA sequences of the
vir promoters (vir boxes) and activates transcription (Brencic & Winans 2005). In contrast to most two-component systems controlling virulence systems of pathogens, signals
McCullen
·
Binns
from the host responsible for the activation
of the VirA/VirG system have been defined.
These are phenols, aldose monosaccharides,
low pH, and low PO4 (Brencic & Winans
2005, Palmer et al. 2004). The phenols are
absolutely required to induce vir gene expression, whereas the other signals sensitize the
bacteria to the phenols. This raises two important questions: How does the system recognize, integrate, and transduce multiple signals, and what is the biological significance of
the multiple signals necessary for optimal vir
gene activation?
Activating signals. Of the inducing signals,
low PO4 and low pH can activate virG
expression—independent of VirA—through
the activities of the complex virG promoter
(Winans 1990). Recent evidence suggests the
pH regulation of this promoter may be mediated through a separate pH-sensing twocomponent system, ChvG/ChvI. This system is chromosomally encoded and required
for both vir gene expression and virulence
(Li et al. 2002), although its mechanism has
not been elaborated.
The identification of phenols as inducers of vir gene expression was first achieved
Figure 2
The ChvE/VirA/VirG signal transducing system
(note that stoichiometry of ChvE:VirA is not
known). See text for details.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Phenols
CH3
O
O
O
CH3
O
O
H3C
O
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
OCH3
O
HO
OH
OCH3
OCH3
(+)-DDF (inactive)
(–)-DDF (active)
H
Sugars
CH2OH
O
H
OH
OH
OH
acid
H
OH
HO
O
H
OH
H
HO
H
H
HO
H
O
OH
H
D-glucoronic
H
H
HO
H
H
HO
COOH
H
OCH3
H
HO
AS (active)
H
O
OCH3
H
H3CO
O
H
O
H3C
CH3
O
H
OH
H
OH
H
D-glucose
H
OH
L-arabinose
H
H
HO
HO
OCH3
R, R-MMCP (inactive)
OCH3
S, S-MMCP (active)
Figure 3
Representative phenols and sugars (pyranose forms) capable of serving as signals to induce vir gene
expression. Active versus inactive enantiomers of DDF and MMCP are shown. AS, acetosyringone;
DDF, dehydrodiferulate dimethylester; MMCP, 1-hydroxymethyl-2-(4-hydroxy-3methoxyphenyl)-cyclopropane.
via the examination of exudates from cultured roots and leaf protoplasts (Stachel et al.
1985). 3,5-Dimethoxyacetophenone, acetosyringone (AS), was present at high levels in
these exudates, and synthetic AS had high inducing activity in assays carried out in the absence of plant cells (Figure 3). A diverse set
of synthetic and naturally occurring phenols,
derived from the phenylpropanoid pathway
in plants, is active (reviewed in Palmer et al.
2004). Several important structural features
of the active molecules have been identified.
First, the aromatic hydroxyl is essential. Second, monomethoxy derivatives are more active than those lacking methoxy substitutions
on the phenol ring, but dimethoxy derivatives invariably are the most active of these
three classes. Third, the potential capacity of
the group para to the aromatic hydroxyl to
hydrogen bond (e.g., with a polar or acidic
group of the receptor) generally is associated with higher activities, and chirality at this
carbon center is critical for inducing activity
(Figure 3). These structural features led to
the proposal of a proton transfer model of
phenol perception (Hess et al. 1991). This
model envisions that the transfer of a proton from the aromatic hydroxyl to a base on
the receptor is a key step in the activation
cascade. Although numerous studies utilizing
structural variations of the inducer, as well as
specific inhibitors of vir gene expression, are
consistent with the model ( Joubert et al. 2002,
Lee et al. 1992, Zhang et al. 2000), final resolution awaits the identification of the phenolbinding site in the receptor (see below).
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
AS: acetosyringone
105
ANRV288-CB22-05
ARI
28 September 2006
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
chv genes:
chromosomal loci of
A. tumefaciens
required for
virulence
21:44
The role of sugars in the process of vir
gene induction was recognized through both
physiological and genetic studies. The response to inducing sugars is twofold: First,
the VirA/VirG system induces at significantly
lower doses of the phenol (e.g., 0.5 µM AS
versus 10–20 µM AS), and second, the maximal level of vir induction at saturating concentrations of phenol increases by five- to tenfold (Cangelosi et al. 1990, Shimoda et al.
1990). Mutations at a chromosomal virulence locus—chvE—and in virA can drastically affect this response (Banta et al. 1994,
Cangelosi et al. 1990, Lee et al. 1992, Shimoda
et al. 1993). Structure/function studies indicate several aldoses are inducers, the capacity
to form a pyranose ring form is required, and
the acidic sugars exhibit the highest activity
on a molar basis (Ankenbauer & Nester 1990,
Shimoda et al. 1990) (Figure 3).
A commonality, therefore, of both the phenol and sugar signaling is the diversity of plant
compounds recognized. This likely plays a
role in achieving the broad host range exhibited by Agrobacterium. The phenols, which
are absolutely required for vir gene activation,
may be especially important in this regard. Although ubiquitous in higher plants, phenols
with varying substitution patterns can be specific for particular genera or species (Dixon
et al. 2002). Thus, the capacity of the virinducing system to recognize diverse phenols
allows the expression of the virulence machinery across a broad range of host plants
and, conceivably, different cell types within
the plant.
VirA and signal perception. VirA is a
dimeric membrane-bound protein and is
found in this state even in the absence of signals (Pan et al. 1993). Several domains of the
protein have been identified (Figure 4) in
both structural and genetic studies (reviewed
in Brencic & Winans 2005). Each monomer
contains a small N-terminal cytoplasmic domain, two transmembrane (TM) domains surrounding a 180-amino-acid periplasmic (P)
domain, and a large cytoplasmic domain. Se-
106
McCullen
·
Binns
quence and functional analyses (see below)
have shown the latter to be composed of three
domains—a linker (L) domain involved in
phenol perception; the kinase (K) domain that
includes the conserved histidine that is phosphorylated as well as an ATP-binding site; and
a receiver (R) domain that has some sequence
homology, including the conserved aspartate,
to the N-terminal region of VirG.
The means by which the various inducing signals are perceived have been defined
with varying degrees of success. In the case of
the inducing sugars, the role of a chromosomally encoded, periplasmic protein, ChvE,
seems clear. This protein has significant homology to a broad range of periplasmic sugarbinding proteins, and strains lacking it can
no longer respond to the vir-inducing effects
of the sugars (Cangelosi et al. 1990). Several
studies indicate ChvE works in concert with
VirA’s periplasmic domain to sensitize VirA
to phenolic inducers when a sugar is present
(Banta et al. 1994, Chang & Winans 1996,
Shimoda et al. 1993). Currently, the best evidence for the ChvE/VirA interaction comes
through the isolation of a suppressor mutation in ChvE that restores sugar sensitivity to
a strain carrying a point mutation in the VirA
periplasmic domain (Shimoda et al. 1993).
Physical evidence of the VirA/ChvE interaction has not been reported, nor is there biochemical evidence of the sugar-binding activity of the ChvE protein. Intriguingly, ChvE
also participates in sugar uptake and chemotaxis, indicating it must interact with other
proteins as well (Ankenbauer & Nester 1990).
The physical basis of phenol perception is
less clear. Genetic analysis suggests the linker
domain is critical in this process (Chang &
Winans 1992, Turk et al. 1994). The best evidence for this is that a version of VirA containing only the L and K domains (VirALK )
is a phenol-responsive form of VirA, whereas
VirAK , although it has a low level of constitutive activity, is not (Chang & Winans
1992). Whereas the linker domain is implicated in phenol perception, no physical evidence of phenol binding to it, or any other
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Figure 4
Model of signal integration and activity by the ChvE/VirA signal transducing proteins. For simplicity
only one VirA monomer is presented. See text for details. AS, acetosyringone.
domain of VirA, has been reported. Studies utilizing either phenol derivatives that are
specific, irreversible inhibitors of vir gene expression or phenol affinity chromatography
protocols identified several phenol-binding
proteins, but these did not include VirA (Dyé
& Delmotte 1997, Lee et al. 1992). No evidence for the involvement of the phenolbinding proteins in vir gene activation has
been obtained. Phenol-hypersensitive mutant
strains of Agrobacterium have been isolated,
and although the gene(s) responsible for the
mutant phenotype has not been identified, it is
not Ti plasmid linked (Campbell et al. 2000).
Genetic evidence supporting the model of the
phenol directly interacting with VirA comes
in two forms. First, some strains of Agrobacterium detect a different range of phenols, and
this capacity tracks with the virA genes of
those strains (Lee et al. 1995, 1996). Second,
a phenol-responsive VirA/VirG system has
been reconstituted in Escherichia coli (Lohrke
et al. 2001). Unless there are phenol-binding
proteins that also exist in E. coli and can productively bind to, and activate, VirA, then
VirA must sense the phenol directly. Final
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
107
ANRV288-CB22-05
ARI
28 September 2006
LZ: leucine zipper
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
LKR: linker, kinase,
and receiver domains
(of VirA)
21:44
resolution of this question awaits the demonstration and characterization of the phenol:VirA interaction.
The third signal input mediated by VirA is
low pH (e.g., pH 5.5). Melchers et al. (1989)
showed that a large periplasmic deletion resulted in a VirA molecule that could induce vir
gene expression (in the presence of phenols)
at pH 7, a normally noninducing condition
(Melchers et al. 1989). More recent studies
demonstrate that both an intact periplasmic
domain (Chang & Winans 1996) and ChvE
(and sugars) (Gao & Lynn 2005) must be
present to have VirA-mediated pH control.
Although the ChvE/sugar interaction appears
necessary for pH regulation, the sugar and pH
signal can be uncoupled by a small deletion in
the periplasmic domain of VirA (Banta et al.
1994, Gao & Lynn 2005). The physical mechanism of pH perception is not known. Because
ChvE supports arabinose utilization at neutral
pH (A. Wise, G. Nair, and A. Binns, unpublished data), sugar binding must not be pH dependent. Thus, the interaction of ChvE with
VirA or the response of VirA to this interaction may be affected by pH.
VirA and signal integration and transduction. The modular nature of VirA has greatly
assisted studies examining the integration of
the signals and their transduction. Extensive
genetic analysis has yielded a model for domain effects on kinase activity (Figure 4). Partially purified kinase domain has the capacity
to phosphorylate VirG in vitro, and the expression of this domain (VirAK ) in Agrobacterium activates vir gene expression, though at
a low level and without signal control (Chang
& Winans 1992, Jin et al. 1990). The addition
of the linker domain results in a form, VirALK ,
that exhibits constitutive inducing activity in
vivo but that can also be stimulated by phenols, indicating a positive effect by these signals (Chang & Winans 1992). However, because wild-type VirA is inactive (i.e., no vir
gene expression) in the absence of signals,
other domains must have a repressive effect on
activity. One example of this is the periplasmic
108
McCullen
·
Binns
domain. Although insensitive to sugar, VirA
with a large periplasmic deletion (VirA!P ) results in the gain of ability to induce at neutral
pH and to induce high levels of vir gene expression in response to phenols in the absence
of sugars (Banta et al. 1994, Lee et al. 1992,
Melchers et al. 1989). This suggests that the
periplasmic domain exerts a repressive effect
on the cytoplasmic portion of VirA. Sugar and
pH modulate ChvE interaction with VirA so
as to relieve this repression, resulting in maximal sensitivity and responsiveness to the phenols. The receiver domain also has a repressive
function in relation to the kinase activity of
VirA. VirA!R no longer requires phenols for
activation, although it is still dependent on pH
and sugar signaling (Chang & Winans 1992,
1996). Interestingly, the expression of the receiver domain in trans to VirA!R restored
the wild-type phenotype (Chang & Winans
1996).
The dimeric state of the VirA protein
is typical for most membrane-bound sensor histidine kinases and important for function. Genetic evidence strongly suggests that
ATP bound to one VirA monomer phosphorylates the other (Brencic et al. 2004b).
The importance of the dimeric state in signal
transduction has also been demonstrated. The
expression of an inactive version of VirA containing a point mutation in the linker domain
and an inactive version of VirA containing
a mutation (H474Q) at the conserved histidine resulted in an active, phenol-responsive
dimer (Toyoda-Yamamoto et al. 2000). Similarly, coexpression of the sugar-nonresponsive
VirAE255L along with the null VirAH474Q
yields a strain with a sugar-responsive phenotype (Wise et al. 2005). As noted above, versions of VirA lacking the transmembrane and
periplasmic domains have low activity even at
high AS levels. This may be a result of relatively poor dimerization of such constructs.
Fusion of the leucine zipper (LZ) of the
GCN4 transcription factor to the N terminus of the linker domain (at amino acid 285 of
VirApTiA6 ) resulted in the protein VirALZ-LKR ,
which shows significantly higher activity than
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
VirALKR (Wang et al. 2002). This increased
activity was accompanied by an increased capacity of VirALZ−LKR to form cross-linkable
dimers in comparison with VirALKR . Interestingly, the exact position of the LZ fusion
is critical: When fused at position 293—just
into a predicted helical region of the linker
domain—the resultant VirALZ(0)LKR is locked
into an active but phenol-nonresponsive state.
This, and the analysis of LZ fusions at the
same site but with an additional three or
four amino acids that could force interaction at alternate faces of the proposed dimer
helices, led to a model that suggests the
rotation of the helices of the interacting
monomers relative to one another is crucial in
the phenol-mediated accumulation of VirGPO4 , leading to the activation of vir gene
expression (Palmer et al. 2004, Wang et al.
2002).
What steps in the VirA/VirG phosphotransfer process are signal mediated? Partially
purified VirAK phosphorylates VirG in vitro
but not in a signal-regulated fashion ( Jin et al.
1990). Recent studies have utilized in vivo
phosphorylation protocols in which strains
carrying epitope-tagged versions of VirALKR
or VirG were exposed to 32 P-labeled phosphate in the presence or absence of inducing signals followed by affinity purification
and analysis (Mukhopadhadyay et al. 2004).
These studies demonstrate that VirA is phosphorylated at both the kinase and receiver domains in the absence of inducing signals, and
this phosphorylation is dependent on the conserved histidine of the kinase domain. Importantly, labeled VirG-PO4 was observed only
when phenol inducers were present, and this
was dependent on the conserved aspartate of
VirG. This strongly suggests phenol signals
recognized by VirA-PO4 result in the stimulation of phosphotransfer to VirG, although the
effects on dephosphorylation via a VirA phosphatase activity must be considered (Brencic
et al. 2004b). Because these studies were carried out on VirALKR , the role of the periplasmic domain, ChvE, and the sugar and pH
signals on the phenol-independent phospho-
rylation of VirA remains an important unanswered problem.
Wounds, infection, and virulence gene expression. Wounds on host plants are common sites of transformation by A. tumefaciens
(Braun 1952). Although the wound may simply be a portal of entry, other specific processes may occur at the wound site and facilitate transformation. The identification of
the vir-inducing signals described above has
provided one likely explanation for the importance of the wound site: High activity of the
phenylpropanoid pathway, low pH, and sugars associated with cell wall synthesis are routinely associated with wound repair (Baron &
Zambryski 1995). Braun (1952) proposed that
cell divisions at the wound site are an essential component of the wound response necessary for transformation, and numerous subsequent studies indicate that dividing plant cells
are more efficiently transformed than quiescent cells (e.g., Sangwan et al. 1992). Thus the
multiple signal input regulating VirA function
may be optimizing vir induction at the most
vulnerable site for transformation: the wound.
Two recent papers have established, however, that transformation can occur in unwounded tobacco seedlings (Brencic et al.
2005, Escudero & Hohn 1997), and in one
case, Brencic et al. (2005) observed vir gene
expression in the bacteria in the absence of
wounding. This raises important questions
concerning the infection process and the role
of wounding. First, what is the relative timing
and frequency of vir induction and transformation at wounded versus unwounded sites?
This is a nontrivial question because delivering the bacteria without wounding may result
in different cell types being exposed to them
(Escudero & Hohn 1997). Second, although
a great deal is known about the chemistry of
the inducing signals, their distribution in the
plant over both time and space—which we
term the signal landscape—is less clear. Both
chemical and biological methods are needed
to establish this and to determine if it is altered by developmental processes, including
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
109
ANRV288-CB22-05
ARI
28 September 2006
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
T-pilus: surface
appendage of A.
tumefaciens composed
of proteins encoded
in the virB region of
the Ti plasmid and
proposed to be
involved in physical
interaction with
plant cells
rat mutants:
mutants in A.
thaliana plants that
confer a phenotype
of resistance to
transformation by A.
tumefaciens
21:44
the wound response and possibly the inciting bacteria themselves (e.g., Brencic et al.
2004a). Third, quantitative estimation of cell
cycle activity in target tissues—wounded or
unwounded, infected or uninfected—needs to
be examined and compared with transformation frequencies in those areas.
Changes in gene expression in plants as a
result of Agrobacterium infection suggest that
plant antimicrobial defense processes may be
suppressed (Ditt et al. 2005, Veena et al. 2003),
and genes involved in cell growth and division
(e.g., histones) may be activated (Veena et al.
2003). A recent report (Dunoyer et al. 2006)
indicates that RNA silencing pathways are
active during Agrobacterium infections. Early
after the infiltration of leaf tissues with
Agrobacterium, small interfering RNAs (siRNAs) directed against incoming T-DNA
genes are produced by the plant tissues, but
tumors ultimately arising from the infection
are virtually devoid of them. Moreover, the
expression of other siRNAs and certain microRNAs targeting regulators of the plant auxin
response is suppressed in tumors and, importantly, in uninfected leaf tissue stimulated to
divide in culture. These results suggest that
tumor development requires the transformed
cells to overcome host defenses initiated to
combat the incoming foreign DNA. They
also raise the intriguing possibility that wound
sites—which are sites of active cell division—
represent an opportunity for Agrobacterium to
transfer DNA into cells that are less likely to
mount a T-DNA silencing defense.
Attachment of Agrobacteria
to Host Cells
That DNA and proteins are transferred from
A. tumefaciens to plant cells strongly suggests
an intimate association between pathogen and
host cells. Although obviously critical, this is
one of the least-characterized sets of cellular processes in the entire interaction (for review of earlier literature, see Gelvin 2000).
Microscopic examination of bacteria interacting with the plant cells indicates a signifi110
McCullen
·
Binns
cant propensity to attach in a polar fashion
(Smith & Hindley 1978). Quantitative estimation of binding by Agrobacterium to plant
cells has revealed two types of interactions:
a nonspecific, nonsaturable, aggregation-like
interaction readily removed via washes with a
buffered salt solution and a specific, saturable
interaction (200–1000 bacteria per plant cell)
impervious to such washing (Gurlitz et al.
1987, Neff & Binns 1985). The specific attachment of A. tumefaciens to plant cells is not
dependent on the Ti plasmid (Douglas et al.
1982, Neff & Binns 1985). This rules out the
T-pilus (see below) as the causal agent in the
stable attachment process. Three chromosomally encoded bacterial genes [chvA, chvB, and
pscA (exoC )] involved in the synthesis and/or
localization of periplasmic β1-2 glucan are required for attachment, although their role in
this process is not understood (Cangelosi et al.
1987, 1989; Douglas et al. 1985; Thomashow
et al. 1987; Zorreguieta et al. 1988). Recently,
a series of genes were identified as attachment
deficient (att genes), and all mapped to a
29 kb region of the genome (Matthysse 1987,
Matthysse et al. 2000). The role of these
genes in the attachment process has, however,
been questioned because they are located on a
542 kb plasmid, pAtC58 (Goodner et al. 2001,
Wood et al. 2001), which is not required for
virulence (Hynes et al. 1985, Nair et al. 2003).
Early studies revealed that the exposure
of A. tumefaciens cells to soluble pectic plant
cell wall fractions renders them less capable of
binding specifically to plant cells and less capable of inducing tumors (reviewed in Gelvin
2000). This suggests that they may contain a
receptor-like component, but molecular characterization of such an entity has not occurred.
Recent genomic studies, however, are beginning to provide new insight into possible plant
molecules involved in the attachment process.
Many Arabidopsis thaliana mutants have been
isolated that are recalcitrant to Agrobacterium
transformation (rat mutants) (Zhu et al. 2003).
Some of these mutants are plants to which
Agrobacterium can no longer bind efficiently.
The best characterized of these is a mutant
ANRV288-CB22-05
ARI
28 September 2006
21:44
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
with an insertion in the promoter region of an
arabinogalactan protein—likely to be found in
the cell wall—and bacteria bind poorly to root
cells of this plant (Nam et al. 1999, Zhu et al.
2003). Following up on these leads will be
important in characterizing the recognition
processes and physical interaction of Agrobacterium and host cells.
MACROMOLECULAR
TRANSPORT
Following the activation of the virulence
genes and attachment to the plant cells,
A. tumefaciens transports several different
molecules into the host during infection, including DNA and protein substrates. These
substrates need to cross the inner membrane, periplasm/peptidoglycan wall, and
outer membrane, as well as the plant host cell
wall and membrane. A specialized transporter
complex of the VirB proteins and VirD4 is
employed to get the substrates across the bacterial cell envelope. The VirB complex is a
prototypical type IV secretion system (T4SS),
a class of transporters found across a broad
range of gram-negative bacteria and involved
in the conjugative transfer of plasmids between bacteria as well as the translocation
of virulence factors from pathogens to host
cells during infection (for recent reviews, see
Cascales & Christie 2003, Nagai & Roy 2003,
Schroder & Lanka 2005).
The VirB complex is composed of at least
12 proteins: VirB1–11 and VirD4 (Figure 5).
These proteins are required for virulence,
associate with the cell envelope, and form
a multisubunit envelope-spanning structure
(Christie et al. 2005). Substrates transported
into host cells by the VirB complex include
the VirD2-T-strand, VirE2, VirE3, VirF, and
VirD5 (Vergunst et al. 2005). VirD2 nicks
the T-DNA at the border repeats, is covalently bound to the 5# end, and is likely
transported with the T-strand into the plant
cell, where it is involved in nuclear import
and integration of the T-DNA into the host
genome (Gelvin 2003). VirE2 is a single-
stranded DNA-binding protein that can coat
the length of the T-strand in vitro (Christie
et al. 1988, Citovsky et al. 1988). It interacts with the T-DNA in the plant cell cytoplasm and also has roles in nuclear import
and integration (Gelvin 2003). VirF function
is not well characterized, but it may be involved in the degradation of host cell factors
during infection (Schrammeijer et al. 2001,
Tzfira et al. 2004). Intriguingly, the VirB/D4
complex can also transport the broad-hostrange, mobilizable plasmid RSF1010 to either
plants or other agrobacteria, demonstrating
that the conjugative intermediate (MobA-Rstrand) must also be a substrate (Beijersbergen
et al. 1992, Buchanan-Wolloston et al. 1987).
Type IV secretion
system (T4SS):
multisubunit
transporter that
transports protein
and DNA across a
bacterial cell
envelope into a
recipient cell
VirD2-T-strand:
transport-competent
form of the T-DNA
that is single
stranded and
covalently bound to
VirD2 at the 5# end
Targeting of Substrates
The A. tumefaciens virB-encoded T4SS transports the substrates described above across
the bacterial cell envelope. Recent work has
Figure 5
Generalized scheme representing the VirB/D4 complex, depicting
localization and some of the known interactions between members of the
complex. Asterisks indicate proteins shown to interact with the transported
DNA substrate, and arrows indicate the order of DNA transfer through the
complex. See text for details.
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
111
ANRV288-CB22-05
ARI
28 September 2006
Coupling protein:
inner membrane
component of a
T4SS that typically
contains an
ATP-binding motif
and recruits
substrates to the
T4SS
21:44
allowed us to begin to answer some critical
questions, including the following: How are
these substrates targeted for export? How do
they interact with the transport complex? Specific regions of targeted substrates that are important for these processes have been identified. These export signals are found near the
C termini of the substrates and are necessary
in mediating the interaction of substrates with
the T4SS.
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
Export signals. Genetic studies have revealed export signals in the VirE2 and VirF
transported substrates. The insertion of a
FLAG tag at the C terminus of VirE2, or the
truncation of the C-terminal 18 amino acids
of VirE2, renders the protein nonfunctional
in A. tumefaciens, although it can still bind
single-stranded DNA (Simone et al. 2001).
However, transgenic plants producing these
C-terminal mutant forms of VirE2 can complement the virulence of a virE2− strain of A.
tumefaciens. The finding that these forms of
VirE2 are functional in the plant but not in
the bacterium led to the prediction that the
mutations disrupted a region of amino acids
required for translocation, such as a secretion
signal.
Vergunst et al. (2000) used fusion of VirE2
and VirF to the Cre recombinase to examine directly the transport of these substrates
in the absence of T-DNA. In this assay, the
transport of Cre-VirE2 or Cre-VirF fusions
into host plant cells resulted in a recombination event that conferred kanamycin resistance to host tissues, and this transport was
shown to be dependent on the VirB/VirD4complex. In a subsequent study, Cre::VirD2
transfer into plant cells, in the absence of TDNA, was observed, although at a very low
level (Vergunst et al. 2005). The success of
this approach has resulted in the discovery of
two other secreted factors: Both VirE3 and
the last 50 amino acids of VirD5 directed Cre
secretion into plant cells (Schrammeijer et al.
2003, Vergunst et al. 2005). VirE3 may function in the plant to aid nuclear localization
of VirE2 (Lacroix et al. 2005), and sequence
112
McCullen
·
Binns
analysis suggests that VirD5 has homology to
host transcription factors (Schrammejer et al.
2000).
C-terminal fusions of VirE2 blocked its
translocation to host cells, whereas fusion of
Cre to the N terminus of VirE2 was permissive for transport (Vergunst et al. 2000),
consistent with the presence of a C-terminal
secretion signal in VirE2. Additionally, the
C-terminal 37 amino acids of VirF and the
C-terminal 50 amino acids of VirE2 and
VirE3 are sufficient to mediate transport of
these fusion proteins to plants (Vergunst et al.
2000, 2003). The minimal component of VirF
required to direct Cre translocation to plants
is the C-terminal 10 amino acids, and several arginines within this region are required
for transport (Vergunst et al. 2005). A possible consensus sequence was identified in the
C termini of substrates secreted by the VirB
complex: R-X(7)-R-X-R-X-R (Vergunst et al.
2005). Importantly, subsequent studies of substrates in other systems that use T4SSs for
transport of DNA and/or virulence factors
have shown that they, too, utilize C-terminal
sequences as export signals (Hohlfeld et al.
2006, Luo & Isberg 2004, Nagai et al. 2005,
Schulein et al. 2005).
Substrate interactions with the coupling
protein. Substrates destined for export by
a T4SS must access the complex and interact with it in a fashion that facilitates the
transport process (Gomis-Ruth et al. 2004).
All T4SSs that mobilize DNA-protein complexes, and many that mobilize only proteins,
have a putative NTPase that is proposed to
serve as a coupling protein that recruits substrates to the transport complex. Genetic and
biochemical evidence have demonstrated that
the specificity of transfer by a T4SS is a result of the specific interaction of a transported substrate with the cognate coupling
protein (Hamilton et al. 2000, Llosa et al.
2003, Schroder et al. 2002). Additionally, the
evidence strongly suggests that the coupling
proteins also interact with other members
of the T4SS complex (Cascales & Christie
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
2004a, Llosa et al. 2003), thereby recruiting
the transported substrates to the transport
complex (see below).
Because of its homology to coupling
proteins of plasmid conjugation systems,
VirD4 is the proposed coupling protein of
the A. tumefaciens T4SS (Hamilton et al.
2000). VirD4 is required for both T-strand
and VirE2 transfer to host cells, associates
with the inner membrane, and contains
ATP-binding motifs necessary for virulence
(Kumar & Das 2002, Okamoto et al. 1991).
Although there is no in vitro evidence
for specific interactions between VirD4 and
VirD2, Cascales & Christie (2004b) obtained in vivo evidence for the interaction
of VirD4 with the single-stranded T-strand
via modification of chromatin immunoprecipitation methodology, called transferred-DNA
immunoprecipitation (TrIP). They treated
cells of A. tumefaciens, induced to express the
vir genes and hence produced both VirD2T-strand and the VirB/D4 complex, with
formaldehyde. Amplifiable DNA from the Tstrand, but not other parts of the Ti plasmid, was coimmunoprecipitated from lysates
of such cells by anti-VirD4 antibodies. Mutants expressing VirD4 but not VirD2—and
therefore not producing VirD2-T-strands—
did not yield amplifiable DNA upon immunoprecipitation.
The C-terminal secretion signal identified on substrates of the A. tumefaciens T4SS
is likely important for their interaction with
the translocation apparatus. Atmakuri et al.
(2003) demonstrated binding of VirE2 to
the coupling protein, VirD4, in vivo. Importantly, they found that the truncation of the
C-terminal 100 amino acids of VirE2 disrupts interaction with VirD4, suggesting a
role for C-terminal secretion signals in this interaction. Beyond VirD4’s involvement in the
translocation of DNA substrates by the T4SS,
the requirement of VirD4 for VirE2 translocation specifically demonstrates that the coupling protein plays an important role in the
export of non-DNA substrates. Additionally,
molecules such as the conjugative intermedi-
ate of RSF1010 and the Osa protein of plasmid pSa have recently been shown to inhibit Agrobacterium virulence by specifically
blocking T-DNA and VirE2 interactions with
VirD4 (Cascales et al. 2005, Lee et al. 1999,
Stahl et al. 1998). All these data support a
model in which VirD4 mediates the transport
of VirD2-T-strand and VirE2 by providing
both access to the complex and, most likely,
energy for transport (Cascales et al. 2005,
Llosa et al. 2002) (see below).
TrIP: transferred
DNA
immunoprecipitation
Mechanism of Export
The VirB complex is a membrane-bound
macromolecular structure that mediates substrate translocation out of the cell. Known
structural elements and protein-protein interactions within this complex are outlined
below, along with exciting new work that describes substrate contacts with particular proteins of the VirB complex.
VirB and VirD4 proteins form a channel
across the cell envelope. All the VirB proteins and VirD4 associate with the membrane
system of Agrobacterium and with each other
to form the VirB transport complex. Similar
to VirD4, VirB4 and VirB11 associate with
the inner membrane and exhibit homology
to NTP-binding proteins. Mutations in the
NTP-binding sites of these three proteins
render them unable to complement for virulence (Berger & Christie 1993, Kumar & Das
2002, Stephens et al. 1995). VirD4, VirB4,
and VirB11 have been coimmunoprecipitated
and do not require their ATP-binding domains to interact (Atmakuri et al. 2004). However, ATP hydrolysis by one or more of these
proteins may provide energy for transporter
biogenesis or substrate translocation (see
below).
Analysis of the VirB complex inner
membrane–localized proteins, or homologs
thereof, is beginning to provide a structural
context for their activities. Purified homologs
of VirD4 form hexameric rings with crystal structures similar to that of F1-ATPase,
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
113
ARI
28 September 2006
21:44
VirB11 homologs also form hexameric ring
structures, and VirB4 is predicted to form hexamers on the basis of computational analysis (Krause et al. 2000, Machon et al. 2002,
Middleton et al. 2005, Yeo et al. 2000). All
these rings have been suggested to form
pores in the inner membrane that could mediate the transport of substrates across this
barrier, although evidence for this is not
available. VirB6 is also an inner membrane
protein and has been shown to have multiple membrane-spanning and periplasmic domains whose functions are under intense
scrutiny ( Jakubowski et al. 2004; Judd et al.
2005a,c) (see below).
These four inner membrane proteins have
been connected physically to other VirB proteins located in the periplasm and/or outer
membranes, thereby participating in a cell
envelope–spanning structure. VirB10, a protein that fractionates with both the inner and
outer membranes, interacts with the inner
membrane–associated VirD4 as well as with
VirB9 and VirB7, which fractionate with the
outer membrane (Cascales & Christie 2004a,
Thorstenson et al. 1993, Ward et al. 1990),
forming a transenvelope complex. VirB10 interaction with VirD4 does not require VirD4’s
ATP-binding site, nor does it require VirB4
or VirB11, suggesting that ATP hydrolysis is
not necessary for this step in biogenesis of
the secretion apparatus (Cascales & Christie
2004a). However, VirB10 undergoes an apparent conformational change (as detected by
protease susceptibility) that requires VirD4
and VirB11 Walker A sites. ATP hydrolysis by VirD4 and VirB11 may induce this
conformational change in VirB10, resulting
in VirB10 association with VirB7 and VirB9
(Cascales & Christie 2004a). Similarly, VirB4
and VirB6 also participate in contacts with
more distal portions of the secretion apparatus. A VirB4 homolog from Brucella suis
was shown to interact with the B. suis VirB8,
a periplasmic protein with an inner membrane anchor, and immunoprecipitation studies characterizing solubilized membrane proteins of A. tumefaciens demonstrate that VirB6
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
114
McCullen
·
Binns
interacts with VirB7 and VirB9 ( Jakubowski
et al. 2004, Yuan et al. 2005).
VirB7, VirB8, VirB9, and VirB10 are
widely considered the core components of the
transporter apparatus (Christie 2001, Kado
2000, Zupan et al. 2000) and have been shown
to interact with themselves or each other via
disulfide bonds or other means as indicated by
chemical cross-linking and yeast two-hybrid
analysis (reviewed in Christie et al. 2005).
Consistent with the hypothesis that these proteins form a membrane-bound core is that
VirB7–10, along with VirB4 and VirB6, have
been shown to comigrate as a +600 kDa complex in both blue native gel electrophoresis
and gel filtration of solubilized membrane
proteins from AS-induced strains (Krall et al.
2002, Yuan et al. 2005). Thus, the core complex, connected with the inner membrane
components (as described above), forms a cell
envelope–spanning structure and may function as the conduit through which substrates
transit the envelope system.
The formation of a readily isolatable Ti
plasmid–encoded pilus on the surface of A.
tumefaciens cells seems to be an important
component of plant infection. This T-pilus is
composed primarily of the processed VirB2
pilin protein but also contains VirB5 and
VirB7 (Lai & Kado 1998, Lai et al. 2002,
Sagulenko et al. 2001b, Schmidt-Eisenlohr
et al. 1999). A 270 kDa complex containing
these proteins has been observed in blue
native polyacrylamide gel electrophoresis and
gel filtration analyses of detergent-solubilized
membrane protein complexes isolated from
A. tumefaciens (Krall et al. 2002). A recent
study demonstrated (a) the association of
VirB3, VirB6, and VirB8 with this complex
and (b) that the complex’s formation is
dependent on VirB4 (Yuan et al. 2005). This
is distinct from the +600 kDa core complex
observed in the same experiments and raises
the intriguing possibility that these groups of
proteins may be associating as independent
subcomplexes.
The localization of VirB complex components to specific sites on the cell surface
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
is under intense investigation. In wild-type
strains, all the VirB/D4 complex proteins, as
well as the T-pilus, localize to the cell poles
( Jakubowski et al. 2004; Judd et al. 2005a,b;
Kumar & Das 2002; Lai et al. 2000). These
results are consistent with observations of the
polar attachment of bacteria to plant cells.
The VirD4, VirB3, VirB4, VirB8, and VirB11
proteins, when expressed individually, localize to the cell poles. Of these, VirB8 plays a
critical role in nucleating assembly and the polar localization of the core complex described
above, as well as the other VirB complex proteins ( Judd et al. 2005b, Kumar et al. 2000).
Loading of the VirB complex. Significant recent findings by Cascales & Christie
(2004b) using the TrIP assay described above
have clarified our understanding of the DNA
substrate translocation from VirD4 into the
VirB complex. This study demonstrated binding of the T-strand to specific subunits of
the VirB complex (VirB2, -6, -8, -9, and -11)
and a proposed sequence of such interactions:
First, the T-strand interacts with VirD4, followed by binding to VirB11, then to VirB6 and
VirB8, and finally interacts with VirB2 and
VirB9. The interaction of the T-strand with
VirD4 does not require any VirB proteins,
whereas binding to VirB11 requires VirD4
and VirB7 and binding to VirB6 and VirB8
requires VirB4. The final step—the interaction of the T-strand with VirB9 and VirB2—
requires all the VirB proteins.
These data are consistent with the spatial distribution of the VirB complex components (Figure 5): The inner membrane–
associated components interact with the
T-strand first, followed by periplasmic- and
then outer membrane–associated components. Additionally, the results are consistent
with many of the physical interactions between VirB/D4 complex proteins previously
identified. These include, for example, the
ability of VirD4 and VirB11 to be coimmunoprecipitated and the requirement of VirD4
for VirB11 to become associated with the
T-strand (Atmakuri et al. 2004, Cascales &
Christie 2004a). Similarly, VirB4 is known to
interact with VirB11 and VirB8 and is required for the T-strand to associate with both
these proteins (Atmakuri et al. 2004, Cascales
& Christie 2004b, Ward et al. 2002, Yuan et al.
2005). A periplasmic loop of VirB6 is required
for binding to the T-DNA, whereas other regions of VirB6 are required for T-DNA binding to VirB8, VirB2, and VirB9 ( Jakubowski
et al. 2004). These data suggest that VirB6
may not function in the translocation of the
T-DNA across the inner membrane but rather
that it may interact with the substrate in the
periplasm and mediate its interactions with
the distal portion of the translocon.
The energy requirements for some of the
substrate interactions with the VirB complex described above have also been defined
(Atmakuri et al. 2004). VirD4, VirB4, and
VirB11 Walker A sites are dispensable for Tstrand interaction with VirD4 and VirB11.
However, they are required for the interaction of substrates with the proteins further
along in the periplasmic and outer membrane locations. ATP hydrolysis by one or all
the NTPases may be required for substrate
translocation across the inner membrane or
movement to and through the periplasm.
For example, VirB10 and VirB9 interact in
a manner dependent on the NTPase activities of VirD4 and VirB11, and all of these
are required for translocation of the T-strand
to VirB9 (Atmakuri et al. 2004, Cascales &
Christie 2004a). Detailed models of possible energy expenditure during transport have
been presented (Christie et al. 2005, Llosa
et al. 2002).
As mentioned above, A. tumefaciens transports DNA and protein substrates to host cells
via the T4SS. Are DNA and protein transported together? Although VirE2 is a singlestranded DNA-binding protein that interacts
with the T-strand in vitro and in the plant,
increasing evidence supports the model that
VirE2 and the T-DNA are exported independently and associate in the plant cell (Binns
et al. 1995, Sundberg et al. 1996). Certainly,
VirE2 can be exported in the absence of the
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
115
ARI
28 September 2006
21:44
VirD2-T-strand and vice versa (Citovsky et al.
1992, Otten et al. 1984, Vergunst et al. 2000).
Furthermore, the C-terminal mutant forms
of VirE2 that are, themselves, not secreted
but are capable of binding single-stranded
DNA do not interfere with T-DNA or wildtype VirE2 export (Simone et al. 2001).
These experiments suggest that VirE2 and the
T-strand are exported independently. Supporting this is TrIP analysis (Cascales &
Christie 2004b) that indicates VirE2 and the
T-strand do not interact in the cytoplasm of
A. tumefaciens and thus cannot be transported
as a complex.
The TrIP analyses have provided important information about the interaction of the
T-strand with the VirB complex. A critical
issue that remains to be addressed is if and
how substrate interactions with the complex,
as well as protein:protein interactions within
it, might be altered when the bacterial cells
are in contact with the host. To date, the TrIP
analysis has been carried out on cells cultured
in liquid medium under vir-inducing conditions. Yet to the best of our knowledge, no secretion of substrates by the VirB complex into
the extracellular environment has been observed, strongly supporting the concept that
the interaction of the bacterium with the host
cell alters the activity of the complex.
Additionally, although the studies described above help us understand the interaction of the T-strand with the VirB/D4
complex, they do not address the question of
protein transport. For example, are protein
substrates folded or unfolded for targeting to
the VirB complex, and are they transported
in a folded or unfolded manner? Are protein
substrates interacting with the same complex
components as the T-strand? One of the substrates, VirD2, is likely folded before translocation because it nicks at the border repeats
and catalyzes the formation of a covalent bond
with the 5# end of the T-strand. VirE2 transport requires the activities of a chaperone,
VirE1, which is necessary for VirE2 translation and stability and may keep VirE2 from
prematurely folding or binding the single-
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
116
McCullen
·
Binns
stranded DNA of the T-strand (Deng et al.
1999, Sundberg & Ream 1999, Sundberg et al.
1996, Zhao et al. 2001). However, VirE1 is
not specifically required for the recognition of
the C-terminal secretion signal by the T4SS
(Vergunst et al. 2003). The issue of the interaction of protein substrates with the VirB
complex components besides VirD4 has not
been addressed and represents a major future
challenge.
Substrate Entry into Host Cells
The means by which the substrates traverse
the host cell wall and membrane barriers is
not at all clear. In the case of T4SS-mediated
plasmid transfer, the pilus mediates the interaction between donor and recipient, followed by the fusion of outer membranes in
a mating junction that, interestingly, appears
not to contain pilus proteins (Schroder &
Lanka 2005). The mechanism by which the
transferred conjugal intermediate traverses
the bacterial wall and inner membrane is not
known.
Even less is known about VirB-mediated
transfer across host cell barriers. As noted
above, the rat mutants of A. thaliana represent an important collection of plants deficient in various activities that appear critical to
Agrobacterium-mediated transformation. The
classes of mutants include two phenotypic
groups—those that exhibit transient expression of the T-DNA but not stable integration and tumorigenesis and those that do not
show any sign of (or show vastly reduced)
transformation (Zhu et al. 2003). The latter group includes any plant that would be
deficient in activities necessary for Agrobacterium to interact with the host wall and membrane systems so as to facilitate transfer of
substrates. For example, activities that could
be disrupted are the T-pilus interaction with
the plant wall (or membrane) systems, potential undefined host-pathogen interactions that
may signal the VirB complex to initiate transfer, and the interaction of host gene products
with transferred substrates in order to move
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
them through the host wall and membrane.
Further analysis of these plants should be most
informative.
The range of hosts that can be transformed by Agrobacterium is extensive and
growing constantly; protocols generated for
Agrobacterium-mediated transformation of organisms range from bacteria and fungi
through virtually all classes of plants and even
to certain mammalian cells (Lacroix et al.
2006). This suggests that the specificity of
physical interactions between pathogen and
host required to breach the host wall and
membrane barriers may be less important
than expected. With regard to specificity, it is
important, however, to consider the issue of
efficiency. For example, frequencies of VirBmediated RSF1010 plasmid transfer from
Agrobacterium to Agrobacterium range from
10−8 to 10−4 (transconjugants/recipient),
whereas the frequency of transfer and the stable integration of T-DNA from Agrobacterium
to plant cells can be as high as 10−1 to 100 per
plant cell under ideal conditions (Binns 1991,
Bohne et al. 1998). The converse of this is also
true: Interagrobacterial RSF1010 transfer by
an IncP plasmid T4SS is several orders of
magnitude higher in efficiency than the VirBmediated transfer (Stahl et al. 1998). Thus,
T4SSs likely evolve to exploit specific natural
host cell surface components while retaining
the capacity to interact productively but less
efficiently with a wide variety of host surfaces.
Studies on the VirB-mediated conjugative transfer of the broad-host-range plasmid
RSF1010 between agrobacteria provide clues
regarding the recipient barriers to transfer
that may be crucial. Remarkably, the expression of a subset of the VirB complex proteins
(VirB1–4 and VirB7–10) in recipient agrobacteria can increase the virB-mediated RSF1010
transfer frequency by three to four log orders (Bohne et al. 1998, Liu & Binns 2003).
The general nature of this activity has been
shown by the expression of T4SS components from other bacteria in A. tumefaciens,
which yield the same result (Carle et al. 2006).
Although the mechanism responsible for the
VirB-mediated increase in recipient activity
is not known, these results provide evidence
that recipient barriers can be limiting to DNA
transfer, particularly in cases where the T4SS
in question has evolved to provide optimal
interaction/translocation with an alternative
recipient.
An obvious and important structure likely
to play a (specific?) role in the interaction with
the plant cell is the T-pilus, yet little is known
about its activities. Most current models of
the pilus in T4SSs directing plasmid conjugation suggest that it is involved in recipient
recognition and the development of so-called
mating pairs, that is, pulling the cells together
to form a mating junction that is characterized in gram-negative bacteria by the fusion of
outer membranes (Schroder & Lanka 2005).
Interestingly, there is no evidence of the
pilus at such junctions. That certain VirB6,
VirB9, and VirB11 mutants do not produce
a T-pilus but can transport some substrates
( Jakubowski et al. 2003, 2004; Sagulenko et al.
2001a) suggests that a fully developed pilus
is not an absolute requirement for substrate
transfer. How might the T-pilus (or other
bacterial proteins) interact with particular recipient wall or membrane components to facilitate transfer? Many of the Arabidopsis rat
mutants described above may be deficient in
such surface molecules (Zhu et al. 2003). A
yeast two-hybrid screen using the major pilus
subunit as a bait has identified potential candidate receptors (Hwang & Gelvin 2004), and
further analysis of these will be interesting.
Finally, in vitro formation of VirE2 pores
in artificial membrane systems has led to the
intriguing proposal that this protein may insert into the host cell membrane and help mediate transfer of itself and/or other substrates
(Duckely & Hohn 2003). Although this is not
consistent with the observation of VirD2-Tstrand transfer into host cells in the absence of
VirE2 (Yusibov et al. 1994), it raises intriguing possibilities for the movement of macromolecules across the plant membrane barrier
that should be the focus of further biochemical and biophysical analysis.
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
117
ANRV288-CB22-05
ARI
28 September 2006
21:44
SUMMARY POINTS
1. A. tumefaciens is a model system for understanding how pathogens recognize hostderived signals to activate virulence machinery and how T4SSs recognize and transport DNA and proteins to host cells.
2. Diverse families of chemical signals are recognized by the ChvE/VirA/VirG system,
thereby broadening the natural host range of Agrobacterium.
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
3. The VirA protein varies its response to phenols as a function of other signals—sugar
and pH. This capacity for multiple signal recognition and integration may optimize
the system for the activation of virulence machinery at wound sites, which appear to
be the most efficient sites of transformation.
4. Virulence-promoting proteins and protein-DNA complexes are recruited to the
VirB/D4 complex via VirD4 recognition of C-terminal targeting signals.
5. The VirB/D4 complex spans the bacterial envelope, and the path of transported DNA
into this complex has been defined, as has a variety of protein-protein interactions
necessary for this movement.
FUTURE ISSUES
1. Physical analysis of signal interaction with ChvE and VirA and how this affects VirA
conformation and activity represent the next steps in understanding signal integration
and the activation of virulence gene expression.
2. Physical recognition steps leading to attachment of the bacterium to plant cells are
poorly defined, although new tools—in the form of Arabidopsis mutants that do not
bind the pathogen—are being developed.
3. Interactions of protein and DNA substrates with components of the VirB/D4 complex
after the bacterium has attached to the host cell need to be characterized.
4. The means by which transported substrates are moved across the host wall and membrane barriers are unknown, and both genetic and biochemical analyses of this step
lie ahead.
ACKNOWLEDGMENTS
We thank Zhenying Liu, Arlene Wise, and Gauri Nair for critically reading earlier versions of
this manuscript and David Lynn and his lab members for stimulating discussions about host
recognition systems, and we acknowledge NSF and NIH for support.
LITERATURE CITED
Ankenbauer RG, Nester EW. 1990. Sugar-mediated induction of Agrobacterium tumefaciens
virulence genes: structural specificity and activities of monosaccharides. J. Bacteriol.
172:6442–46
118
McCullen
·
Binns
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Atmakuri K, Cascales E, Christie PJ. 2004. Energetic components VirD4, VirB11 and
VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion.
Mol. Microbiol. 54:1199–211
Atmakuri K, Ding Z, Christie PJ. 2003. VirE2, a type IV secretion substrate, interacts with the
VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol. Microbiol. 49:1699–
713
Banta LM, Joerger RD, Howitz VR, Campbell AM, Binns AN. 1994. Glu-255 outside the
predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone
perception by Agrobacterium tumefaciens. J. Bacteriol. 176:3242–49
Baron C, Zambryski PC. 1995. The plant response in pathogenesis, symbiosis and wounding:
variations on a common theme? Annu. Rev. Genet. 29:107–29
Beijersbergen A, Dulk-Ras AD, Schilperoort RA, Hooykaas PJJ. 1992. Conjugative transfer
by the virulence system of Agrobacterium tumefaciens. Science 256:1324–27
Berger BR, Christie PJ. 1993. The Agrobacterium tumefaciens virB4 gene product is an essential
virulence protein requiring an intact nucleoside triphosphate-binding domain. J. Bacteriol.
175:1723–34
Binns AN. 1991. Transformation of wall deficient cultured tobacco protoplasts by Agrobacterium
tumefaciens. Plant Physiol. 96:498–506
Binns AN, Beaupré CF, Dale EM. 1995. Inhibition of VirB-mediated transfer of diverse
substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J. Bacteriol.
177:4890–99
Binns AN, Thomashow MF. 1988. Cell biology of Agrobacterium infection and transformation
of plants. Annu. Rev. Microbiol. 42:575–606
Bohne J, Yim A, Binns AN. 1998. The Ti plasmid increases the efficiency of Agrobacterium
tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid. Proc.
Natl. Acad. Sci. USA 95:7057–62
Braun AC. 1952. Conditioning of the host cell as a factor in the transformation process in
crown gall. Growth 16:65–74
Braun AC. 1982. A history of the crown gall problem. In Molecular Biology of Plant Tumors, ed.
G Kahl, J Schell, pp. 155–210. New York: Academic
Brencic A, Angert ER, Winans SC. 2005. Unwounded plants elicit Agrobacterium vir gene
induction and T-DNA transfer: Transformed plant cells produce opines yet are tumor
free. Mol. Microbiol. 57:1522–31
Brencic A, Eberhard A, Winans SC. 2004a. Signal quenching, detoxification and mineralization
of vir gene-inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens. Mol.
Microbiol. 51:1103–15
Brencic A, Winans S. 2005. Detection and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiol. Mol. Biol. Rev. 69:155–94
Brencic A, Xia Q, Winans SC. 2004b. VirA of Agrobacterium tumefaciens is an intradimer
transphosphorylase and can actively block vir gene expression in the absence of phenolic
signals. Mol. Microbiol. 52:1349–62
Buchanan-Wolloston V, Passiatore JE, Cannon F. 1987. The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature (London) 328:172–75
Campbell AM, Tok J, Zhang J, Wang Y, Stein M, et al. 2000. Xenognosin sensing in virulence:
Is there a phenol receptor in Agrobacterium tumefaciens? Chem. Biol. 7:65–76
Cangelosi GA, Ankenbauer RG, Nester EW. 1990. Sugars induce the Agrobacterium virulence
genes through a periplasmic binding protein and a transmembrane signal protein. Proc.
Natl. Acad. Sci. USA 87:6708–12
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
119
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
First
demonstration of
the interaction of a
DNA substrate
with the VirB
T4SS, via the
development and
use of the TrIP
assay.
Provides first clear
evidence for four
functional domains
of the VirA protein.
120
21:44
Cangelosi GA, Hung L, Puvanesarajah V, Stacey G, Ozga AD, et al. 1987. Common loci for
Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their role
in plant interactions. J. Bacteriol. 169:2086–91
Cangelosi GA, Martinetti G, Leigh JA, Lee CC, Theines C, Nester EW. 1989. Role of Agrobacterium tumefaciens ChvA protein in export of β-1,2-glucan. J. Bacteriol. 171:1609–15
Carle A, Höppner C, Ahmed Aly K, Yuan Q, den Dulk-Ras A, et al. 2006. The Brucella suis type
IV secretion system assembles in the cell envelope of the heterologous host Agrobacterium
tumefaciens and increases plasmid pLS1 recipient competence. Infect. Immun. 74:108–17
Cascales E, Atmakuri K, Liu Z, Binns AN, Christie PJ. 2005. Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein. Mol. Microbiol. 58:565–79
Cascales E, Christie PJ. 2003. The versatile bacterial Type IV secretion systems. Nature Rev.
Microbiol. 1:137–49
Cascales E, Christie PJ. 2004a. Agrobacterium VirB10, an ATP energy sensor required for type
IV secretion. Proc. Natl. Acad. Sci. USA 101:17228–33
Cascales E, Christie PJ. 2004b. Definition of a bacterial type IV secretion pathway for
a DNA substrate. Science 304:1170–73
Chang CH, Winans SC. 1992. Functional roles assigned to the periplasmic, linker,
and receiver domains of the Agrobacterium tumefaciens VirA protein. J. Bacteriol.
174:7033–39
Chang CH, Winans SW. 1996. Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene. J. Bacteriol. 178:4717–20
Christie PJ. 2001. Type IV secretion: intercellular transfer of macromolecules by systems
ancestrally related to conjugation machines. Mol. Microbiol. 40:294–305
Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E. 2005. Biogenesis,
architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol.
59:451–85
Christie PJ, Ward JE, Winans SC, Nester EW. 1988. The Agrobacterium tumefaciens virE2
gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J.
Bacteriol. 170:2659–67
Citovsky V, DeVos G, Zambryski P. 1988. A novel single stranded DNA binding protein,
encoded by the virE locus, is produced following activation of the A. tumefaciens T-DNA
transfer process. Science 240:501–4
Citovsky V, Zupan J, Warnick D, Zambryski P. 1992. Nuclear localization of VirE2 protein
in plant cells. Science 256:1802–5
DeCleene M, DeLay J. 1976. The host range of crown gall. Bot. Rev. 42:389–466
Deng W, Chen L, Peng WT, Liang X, Sekiguchi S, et al. 1999. VirE1 is a specific molecular
chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.
Mol. Microbiol. 31:1795–807
Ditt RF, Nester EW, Comai L. 2005. The plant cell defense and Agrobacterium tumefaciens.
FEMS Microbiol. Lett. 247:207–13
Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSR, Wang L. 2002. The phenylpropanoid
pathway and plant defense—a genomics perspective. Mol. Plant Pathol. 3:371–90
Douglas CJ, Halperin W, Nester EW. 1982. Agrobacterium tumefaciens mutants affected in
attachment to plant cells. J. Bacteriol. 152:1265–75
Douglas CJ, Staneloni RJ, Rubin RA, Nester EW. 1985. Identification and genetic analysis of
an Agrobacterium tumefaciens chromosomal virulence region. J. Bacteriol. 161:850–60
Duckely M, Hohn B. 2003. The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang
of T-DNA transfer. FEMS Microbiol. Lett. 223:1–6
McCullen
·
Binns
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Dunoyer P, Himber C, Voinnet O. 2006. Induction, suppression and requirement of
RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nature
Genet. 38:258–63
Dyé F, Delmotte FM. 1997. Purification of a protein from Agrobacterium tumefaciens strain
A348 that binds phenolic compounds. Biochem. J. 321:319–24
Escudero J, Hohn B. 1997. Transfer and integration of T-DNA without cell injury in the host
plant. Plant Cell 9:2135–42
Gao R, Lynn DG. 2005. Environmental pH sensing: resolving the VirA/VirG two component
inputs for Agrobacterium pathogenesis. J. Bacteriol. 187:2182–89
Gelvin SB. 2000. Agrobacterium and plant genes involved in T-DNA transfer and integration.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:223–56
Gelvin SB. 2003. Agrobacterium-mediated plant transformation: the biology behind the “genejockeying” tool. Microbiol. Mol. Biol. Rev. 67:16–37
Gomis-Rüth FX, Sola M, de la Cruz F, Coll M. 2004. Coupling factors in macromolecular
type-IV secretion machineries. Curr. Pharm. Des. 10:1551–65
Goodner B, Hinkle G, Gattung S, Miller N, Blanchard M, et al. 2001. Genome sequence
of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science
294:2323–28
Gurlitz RHG, Lamb PW, Matthysse AG. 1987. Involvement of carrot surface proteins in
attachment of Agrobacterium tumefaciens. Plant Physiol. 83:564–68
Hamilton CM, Lee H, Li PL, Cook DM, Piper KR, et al. 2000. TraG from RP4 and TraG
and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system
of pTiC58. J. Bacteriol. 182:1541–48
Hess KM, Dudley MW, Lynn DG, Joerger RD, Binns AN. 1991. Mechanism of phenolic
activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial
sensor/response systems. Proc. Natl. Acad. Sci. USA 88:7854–58
Hohlfeld S, Pattis I, Puls J, Plano GV, Haas R, Fischer W. 2006. A C-terminal translocation
signal is necessary, but not sufficient, for type IV secretion of the Helicobacter pylori CagA
protein. Mol. Microbiol. 59:1624–37
Hooykaas PJJ, Beijersbergen AGM. 1994. The virulence system of Agrobacterium tumefaciens.
Annu. Rev. Phytopathol. 32:157–79
Hooykaas PJJ, Schilperoort RA. 1992. Agrobacterium and plant genetic engineering. Plant Mol.
Biol. 19:15–38
Hwang HH, Gelvin SB. 2004. Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell 16:3148–67
Hynes MF, Simon R, Puhler A. 1985. The development of plasmid-free strains of Agrobacterium tumefaciens Ti plasmid by using incompatability with a Rhizobium meliloti plasmid
to eliminate pAtC58. Plasmid 13:99–105
Jakubowski S, Krishnamoorthy V, Cascales E, Christie PJ. 2004. Agrobacterium tumefaciens
VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion
system. J Mol. Biol. 341:961–77
Jakubowski S, Krishnamoorthy V, Christie PJ. 2003. Agrobacterium tumefaciens VirB6 protein
participates in formation of VirB7 and VirB9 complexes required for Type IV secretion.
J. Bacteriol. 185:2867–78
Jin S, Roitsch T, Ankenbauer RG, Gordon MP, Nester EW. 1990. The VirA protein of
Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation. J.
Bacteriol. 172:525–30
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
Demonstrates
siRNAs produced
in response to
initial infection by
virulent
Agrobacterium
strains are
suppressed in
tumors and
dividing cells.
121
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
First report of the
isolation of VirB
subcomplexes,
besides the
extracellular
T-pilus, and
co-migration of
transported
substrates with
these complexes.
Demonstrates that
VirB/D4 complex
proteins are
localized at the
poles of A.
tumefaciens cells.
122
21:44
Joubert P, Beaupére D, Leliévre P, Wadouachi A, Sangwan RS, Sangwan-Norreel BS. 2002.
Effects of phenolic compounds on Agrobacterium vir genes and gene transfer induction—a
plausible molecular mechanism of phenol binding protein activation. Plant Sci. 162:733–43
Judd P, Mahli D, Das A. 2005a. Molecular characterization of the Agrobacterium tumefaciens
DNA transfer protein VirB6. Microbiology 151:3483–92
Judd PK, Kumar RB, Das A. 2005b. Spatial location and requirements for the assembly of
the Agrobacterium tumefaciens type IV secretion apparatus. Proc. Natl. Acad. Sci. USA
102:11498–503
Judd PK, Kumar RB, Das A. 2005c. The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole. Mol. Microbiol. 55:115–24
Kado CI. 2000. The role of the T-pilus in horizontal gene transfer and tumorigenesis. Curr.
Opin. Microbiol. 3:643–48
Klee HJ, White FF, Iyer VN, Gordon MP, Nester EW. 1983. Mutational analysis of the
virulence region of an Agrobacterium tumefaciens Ti plasmid. J. Bacteriol. 153:878–83
Krall L, Wiedemann U, Unsin G, Weiss S, Domke N, Baron C. 2002. Detergent extraction identifies different VirB protein subassemblies of the type IV secretion
machinery in the membranes of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci.
USA 99:11405–10
Krause S, Barcena M, Pansegrau W, Lurz R, Carazo JM, Lanka E. 2000. Sequence-related
protein export NTPases encoded by the conjugative transfer region of RP4 and by the
cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc.
Natl. Acad. Sci. USA 97:3067–72
Krimi Z, Petit A, Mougel C, Dessaux Y, Nesme X. 2002. Seasonal fluctuations and longterm persistence of pathogenic populations of Agrobacterium spp. in soils. Appl. Environ.
Microbiol. 68:3358–65
Kumar RB, Das A. 2002. Polar location and functional domains of the Agrobacterium
tumefaciens DNA transfer protein VirD4. J. Bacteriol. 43:1523–32
Kumar RB, Xie YH, Das A. 2000. Subcellular localization of the Agrobacterium tumefaciens
T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore.
Mol. Microbiol. 36:608–17
Lacroix B, Tzfira T, Vainstein A, Citovsky V. 2006. A case of promiscuity: Agrobacterium’s
endless hunt for new partners. Trends Genet. 22:29–37
Lacroix B, Viadya M, Tzfira T, Citovsky V. 2005. The VirE3 protein of Agrobacterium mimics
a host cell function required for plant genetic transformation. EMBO J. 24:428–37
Lai EM, Chesnokova O, Banta LM, Kado CI. 2000. Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium
tumefaciens. J. Bacteriol. 182:3705–16
Lai EM, Eisenbrandt R, Kalkum M, Lanka E, Kado CI. 2002. Biogenesis of T pili in Agrobacterium tumefaciens requires precise VirB2 propilin cleavage and cyclization. J. Bacteriol.
184:327–30
Lai EM, Kado CI. 1998. Processed VirB2 is the major subunit of the promiscuous pilus of
Agrobacterium tumefaciens. J. Bacteriol. 180:2711–17
Lee K, Dudley MW, Hess KM, Lynn DG, Joerger RD, Binns AN. 1992. Mechanism of
activation of Agrobacterium virulence genes: identification of phenol-binding proteins.
Proc. Natl. Acad. Sci. USA 89:8666–70
Lee LY, Gelvin SB, Kado CI. 1999. pSa causes oncogenic suppression of Agrobacterium by
inhibiting VirE2 protein export. J. Bacteriol. 181:186–96
McCullen
·
Binns
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Lee YW, Jin S, Sim WS, Nester EW. 1995. Genetic evidence for direct sensing of phenolic
compounds by the VirA protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA
92:12245–49
Lee YW, Jin S, Sim WS, Nester EW. 1996. The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein.
Gene 179:83–88
Li L, Jia Y, Hou Q, Charles TC, Nester EW, Pan SQ. 2002. A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and
Ti plasmid. Proc. Natl. Acad. Sci. USA 99:12369–74
Lippincott BB, Lippincott JA. 1969. Bacterial attachment to a specific wound site as an essential
stage in tumor initiation by Agrobacterium tumefaciens. J. Bacteriol. 97:620–28
Liu Z, Binns AN. 2003. Functional subsets of the VirB Type IV transport complex proteins
involved in the capacity of Agrobacterium tumefaciens to serve as a recipient in virB-mediated
conjugal transfer of plasmid RSF1010. J. Bacteriol. 185:3259–69
Llosa M, Gomis-Rüth FX, Coll M, de la Cruz F. 2002. Bacterial conjugation: a two step
mechanism for DNA transport. Mol. Microbiol. 45:1–8
Llosa M, Zunzunegui S, de la Cruz F. 2003. Conjugative coupling proteins interact with
cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate
relaxosomes. Proc. Natl. Acad. Sci. USA 100:10465–70
Lohrke SM, Yang H, Jin S. 2001. Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia
coli. J. Bacteriol. 183:3704–11
Luo ZQ, Isberg RR. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system
identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101:841–46
Machon C, Rivas S, Albert A, Goni FM, de la Cruz F. 2002. TrwD, the hexameric traffic
ATPase encoded by plasmid R388, induces membrane destabilization and hemifusion of
lipid vesicles. J. Bacteriol. 184:1661–68
Matthysse AG. 1987. Characterization of nonattaching mutants of Agrobacterium tumefaciens.
J. Bacteriol. 169:313–23
Matthysse AG, Yarnall H, Boles SB, McMahan S. 2000. A region of the Agrobacterium tumefaciens chromosome containing genes required for virulence and attachment to host cells.
Biochim. Biophys. Acta 1490:208–12
Melchers LS, Regensburg-Tuı̈nk TJG, Bourret RB, Sedee NJA, Schilperoort RA, Hooykaas
PJJ. 1989. Membrane topology and functional analysis of the sensory protein VirA of
Agrobacterium tumefaciens. EMBO J. 8:1919–25
Middleton R, Sjolander K, Krishnamurthy N, Foley J, Zambryski P. 2005. Predicted hexameric
structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site
during type IV secretion. Proc. Natl. Acad. Sci. USA 102:1685–90
Mukhopadhadyay A, Gao R, Luynn DG. 2004. Integrating signal input from multiple signals:
the VirA/VirG two-component system of Agrobacterium tumefaciens. Chembiochem 5:1535–
42
Nagai H, Cambronne ED, Kagan JC, Amor JC, Kahn RA, Roy CR. 2005. A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to
host cells. Proc. Natl. Acad. Sci. USA 102:826–31
Nagai H, Roy CR. 2003. Show me the substrates: modulation of host cell function by type IV
secretion systems. Cell Microbiol. 5:373–83
Nair G, Liu Z, Binns AN. 2003. Re-examining the role of the accessory plasmid pAtC58 in
the virulence of Agrobacterium tumefaciens strain C58. Plant Physiol. 133:989–99
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
Demonstrates
expression of VirA,
VirG and RpoA of
A.tumefaciens in E.
coli results in
phenol-activatable
vir gene expression
and strongly
supports the model
that VirA
recognizes the
inducing phenols
directly.
123
ARI
28 September 2006
21:44
Nam J, Myosore KS, Zheng C, Knue MK, Matthysee AG, Gelvin SB. 1999. Identification of
T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium.
Mol. Gen. Genet. 261:429–38
Neff NT, Binns AN. 1985. Agrobacterium tumefaciens interaction with suspension-cultured
tomato cells. Plant Physiol. 77:35–42
Newell CA. 2000. Plant transformation technology—developments and applications. Mol.
Biotechnol. 16:53–65
Okamoto S, Toyoda-Yamamoto A, Ito K, Takebe I, Machida Y. 1991. Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol. Gen.
Genet. 228:24–32
Otten L, De Greve H, Leemans J, Hain R, Hooykaas P, Schell J. 1984. Restoration of virulence
of vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal
and mutant Agrobacterium strains. Mol. Gen. Genet. 195:159–63
Palmer AG, Gao R, Maresh J, Erbiol WK, Lynn DG. 2004. Chemical biology of multihost/pathogen interactions: chemical perception and metabolic complementation. Annu.
Rev. Phytopathol. 42:439–64
Pan SQ, Charles T, Jin S, Wu Z, Nester EW. 1993. Preformed dimeric state of the sensor
protein VirA is involved in plant-Agrobacterium signal transduction. Proc. Natl. Acad. Sci.
USA 90:9939–43
Sagulenko E, Sagulenko V, Chen J, Christie PJ. 2001a. Role of the Agrobacterium VirB11
ATPase in T-pilus assembly and substrate selection. J. Bacteriol. 183:5813–25
Sagulenko V, Sagulenko E, Jakubowski S, Spudich E, Christie PJ. 2001b. VirB7 lipoprotein is
exocellular and associates with the Agrobacterium tumefaciens T pilus. J. Bacteriol. 183:3642–
51
Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel B. 1992. Characterization
of competent cells and early events of Agrobacterium-mediated genetic transformation of
Arabidopsis thaliana. Planta 188:439–56
Schmidt-Eisenlohr H, Domke N, Angerer C, Wanner G, Zambryski PC, Baron C. 1999.
Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium
tumefaciens. J. Bacteriol. 181:7485–92
Schrammeijer B, den Dulk-Ras A, Vergunst AC, Jurado Jacome E, Hooykaas PJ. 2003. Analysis
of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae
as a model: evidence for transport of a novel effector protein VirE3. Nucleic Acids Res.
31:860–68
Schrammeijer B, Risseeuw E, Pansegrau W, Regensburg-Tuink TJ, Crosby WL, Hooykaas
PJ. 2001. Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant
homologs of the yeast Skp1 protein. Curr. Biol. 11:258–62
Schroder G, Krause S, Zechner EL, Traxler B, Yeo HJ, et al. 2002. TraG-like proteins of DNA
transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane
gate for exported substrates? J. Bacteriol. 184:2767–79
Schroder G, Lanka E. 2005. The mating pair formation system of conjugative plasmids: a
versatile secretion machinery for transfer of proteins and DNA. Plasmid 54:1–25
Schulein R, Guye P, Rhomberg TA, Schmid MC, Schroder G, et al. 2005. A bipartite signal
mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.
Proc. Natl. Acad. Sci. USA 102:856–61
Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Katayama M, et al. 1990. Control of
expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules
and monosaccharides. Proc. Natl. Acad. Sci. USA 87:6684–88
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
124
McCullen
·
Binns
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
21:44
Shimoda N, Toyoda-Yamamoto A, Shinsuke S, Machica Y. 1993. Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. J. Biol. Chem. 268:26552–58
Simone M, McCullen CA, Stahl LE, Binns AN. 2001. The carboxy-terminus of VirE2 from
Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded
type IV transport system. Mol. Microbiol. 41:1283–93
Smith VA, Hindley J. 1978. Effect of agrocin 84 on attachment of Agrobacterium tumefaciens to
cultured tobacco cells. Nature 276:498–500
Stachel SE, Messens E, Van Montagu M, Zambryski P. 1985. Identification of the signal
molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium
tumefaciens. Nature 318:624–29
Stachel SE, Nester EW. 1986. The genetic and transcriptional organization of the vir region
of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J. 5:1445–54
Stahl LE, Jacobs A, Binns AN. 1998. The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirB-dependent export of VirE2. J. Bacteriol.
180:3933–39
Stephens KM, Rousch C, Nester E. 1995. Agrobacterium tumefaciens VirB11 protein requires a
consensus nucleotide-binding site for function in virulence. J. Bacteriol. 177:27–36
Sundberg C, Meek L, Carroll K, Das A, Ream W. 1996. VirE1 protein mediates export of the
single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant
cells. J. Bacteriol. 178:1207–12
Sundberg CD, Ream W. 1999. The Agrobacterium tumefaciens chaperone-like protein, VirE1,
interacts with VirE2 at domains required for single-stranded DNA binding and cooperative interaction. J. Bacteriol. 181:6850–55
Tempé J, Petit A. 1982. Opine utilization by Agrobacterium. In Molecular Biology of Plant Tumors,
ed. G Kahl, J Schell, pp. 451–59. New York: Academic
Thomashow MF, Karlinsey JE, Marks JR, Hurlbert RE. 1987. Identification of a new virulence
locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell
attachment. J. Bacteriol. 169:3209–16
Thorstenson YR, Kuldau G, Zambryski PC. 1993. Subcellular localization of seven VirB proteins of Agrobacterium tumefaciencs: implications for the formation of a T-DNA transport
structure. J. Bacteriol. 175:5233–41
Toyoda-Yamamoto A, Shimoda N, Machida Y. 2000. Genetic analysis of the signal sensing
region of the histidine protein kinase VirA of Agrobacterium tumefaciens. Mol. Gen. Genet.
263:939–47
Turk SCHJ, van Lange RP, Regensburg-Tuink TJG, Hooykaas PJJ. 1994. Localization of the
VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium
tumefaciens. Plant Mol. Biol. 25:899–907
Tzfira T, Citovsky V. 2002. Partners-in-infection: host proteins involved in the transformation
of plant cells by Agrobacterium. Trends Cell Biol. 12:121–29
Tzfira T, Vaidya M, Citovsky V. 2004. Involvement of targeted proteolysis in plant genetic
transformation by Agrobacterium. Nature 431:87–92
Veena, Jiang H, Doerge RW, Gelvin SB. 2003. Transfer of T-DNA and Vir proteins to plant
cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating
transformation and suppresses host defense gene expression. Plant J. 35:219–36
Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT, Regensburg-Tuı̈nk
TJG, Hooykaas PJJ. 2000. VirB/D4 dependent protein translocation from Agrobacterium into plant cells. Science 290:979–82
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
First direct
demonstration of
protein transport
by the VirB/D4
complex into plant
cells.
125
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
ANRV288-CB22-05
ARI
28 September 2006
Proposes a model
and provides data
suggesting the
rotation of helices
in the linker region
of VirA is critical to
vir gene activation
by phenols.
Detailed analysis of
the resistance to
Agrobacterium
transformation
(rat) mutants of
Arabidopsis
thaliana.
126
21:44
Vergunst AC, van Lier MC, den Dulk-Ras A, Hooykaas PJJ. 2003. Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not
require VirE1. Plant Physiol. 133:978–88
Vergunst AC, van Lier MC, den Dulk-Ras A, Stuve TA, Ouwehand A, Hooykaas PJJ.
2005. Positive charge is an important feature of the C-terminal transport signal of the
VirB/D4-translocated proteins of Agrobacterium. Proc. Natl. Acad. Sci. USA 102:832–
37
Wang Y, Gao R, Lynn DG. 2002. Ratcheting up vir gene expression in Agrobacterium
tumefaciens: coiled-coil in histidine kinase signal transduction. Chembiochem 3:311–
17
Ward DV, Draper O, Zupan JR, Zambryski PC. 2002. Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies.
Proc. Natl. Acad. Sci. USA 99:11493–500
Ward JE, Dale EM, Nester EW, Binns AN. 1990. Identification of a VirB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J. Bacteriol. 172:5200–
10
White PR, Braun AC. 1942. A cancerous neoplasm of plants: autonomous bacteria-free crown
gall tissue. Cancer Res. 2:597–17
Winans SC. 1990. Transcriptional induction of an Agrobacterium regulatory gene at tandem
promoters by plant-released phenolic compounds, phosphate starvation, and acidic growth
media. J. Bacteriol. 172:2433–38
Winans SC, Kerstetter RA, Nester EW. 1988. Transcriptional regulation of the virA and virG
genes of Agrobacterium tumefaciens. J. Bacteriol. 170:4047–54
Wise AA, Voinov L, Binns AN. 2005. Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium
tumefaciens. J. Bacteriol. 187:213–23
Wolanin PM, Thomason PA, Stock JB. 2002. Histidine kinases: key signal transducers outside
the animal kingdom. Genome Biol. 3:1–8
Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, et al. 2001. The genome of the
natural genetic engineer Agrobacterium tumefaciens. Science 294:2317–23
Yeo HJ, Savvides SN, Herr AB, Lanka E, Waksman G. 2000. Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol. Cell 6:1461–
72
Yuan Q, Carle A, Gao C, Sivanesan D, Aly KA, et al. 2005. Identification of the VirB4VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems. J. Biol. Chem.
280:26349–59
Yusibov VM, Steck TR, Gupta V, Gelvin SB. 1994. Association of single-stranded transferred
DNA from Agrobacterium tumefaciens with tobacco cells. Proc. Natl. Acad. Sci. USA 91:2994–
98
Zambryski P. 1988. Basic processes underlying Agrobacterium-mediated DNA transfer to plant
cells. Annu. Rev. Genet. 22:1–30
Zhang J, Boone L, Kocz R, Zhang C, Binns AN, Lynn DG. 2000. At the maize/Agrobacterium
interface: natural factors limiting host transformation. Chem. Biol. 7:611–
21
Zhao Z, Sagulenko E, Ding Z, Christie PJ. 2001. Activities of virE1 and the VirE1 secretion
chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV
secretion pathway. J. Bacteriol. 183:3855–65
Zhu Y, Nam J, Humara JM, Mysore KS, Lee Y, et al. 2003. Identification of Arabidopsis
rat mutants. Plant Physiol. 132:494–505
McCullen
·
Binns
ANRV288-CB22-05
ARI
28 September 2006
21:44
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
Zorreguieta A, Geremia RA, Cavaignac S, Cangelosi GA, Nester EW, Ugalde RA. 1988.
Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene.
Mol. Plant Microbe Interact. 1:121–27
Zupan J, Muth TR, Draper O, Zambryski P. 2000. The transfer of DNA from Agrobacterium
tumefaciens into plants: a feast of fundamental insights. Plant J. 23:11–28
www.annualreviews.org • Agrobacterium/Plant Cell Interactions
127
Contents
ARI
12 September 2006
8:3
Contents
Annual Review of
Cell and
Developmental
Biology
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
Volume 22, 2006
From Nuclear Transfer to Nuclear Reprogramming: The Reversal of
Cell Differentiation
J.B. Gurdon ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1
How Does Voltage Open an Ion Channel?
Francesco Tombola, Medha M. Pathak, and Ehud Y. Isacoff ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 23
Cellulose Synthesis in Higher Plants
Chris Somerville ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 53
Mitochondrial Fusion and Fission in Mammals
David C. Chan ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 79
Agrobacterium tumefaciens and Plant Cell Interactions and Activities
Required for Interkingdom Macromolecular Transfer
Colleen A. McCullen and Andrew N. Binns ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 101
Cholesterol Sensing, Trafficking, and Esterification
Ta-Yuan Chang, Catherine C.Y. Chang, Nobutaka Ohgami,
and Yoshio Yamauchi ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 129
Modification of Proteins by Ubiquitin and Ubiquitin-Like Proteins
Oliver Kerscher, Rachael Felberbaum, and Mark Hochstrasser ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 159
Endocytosis, Endosome Trafficking, and the Regulation of Drosophila
Development
Janice A. Fischer, Suk Ho Eun, and Benjamin T. Doolan ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 181
Tight Junctions and Cell Polarity
Kunyoo Shin, Vanessa C. Fogg, and Ben Margolis ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 207
In Vivo Migration: A Germ Cell Perspective
Prabhat S. Kunwar, Daria E. Siekhaus, and Ruth Lehmann ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 237
Neural Crest Stem and Progenitor Cells
Jennifer F. Crane and Paul A. Trainor ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 267
vii
Contents
ARI
12 September 2006
8:3
Of Extracellular Matrix, Scaffolds, and Signaling: Tissue Architecture
Regulates Development, Homeostasis, and Cancer
Celeste M. Nelson and Mina J. Bissell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 287
Intrinsic Regulators of Pancreatic β-Cell Proliferation
Jeremy J. Heit, Satyajit K. Karnik, and Seung K. Kim ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 311
Epidermal Stem Cells of the Skin
Cédric Blanpain and Elaine Fuchs ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 339
Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF FLORIDA on 02/13/07. For personal use only.
The Molecular Diversity of Glycosaminoglycans Shapes Animal
Development
Hannes E. Bülow and Oliver Hobert ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 375
Recognition and Signaling by Toll-Like Receptors
A. Phillip West, Anna Alicia Koblansky, and Sankar Ghosh ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 409
The Formation of TGN-to-Plasma-Membrane Transport Carriers
Frédéric Bard and Vivek Malhotra ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 439
Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and
Mechanisms
Roland Lill and Ulrich Mühlenhoff ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 457
Intracellular Signaling by the Unfolded Protein Response
Sebastián Bernales, Feroz R. Papa, and Peter Walter ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 487
The Cellular Basis of Kidney Development
Gregory R. Dressler ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 509
Telomeres: Cancer to Human Aging
Sheila A. Stewart and Robert A. Weinberg ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 531
The Interferon-Inducible GTPases
Sascha Martens and Jonathan Howard ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 559
What Mouse Mutants Teach Us About Extracellular Matrix Function
A. Aszódi, Kyle R. Legate, I. Nakchbandi, and R. Fässler ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 591
Caspase-Dependent Cell Death in Drosophila
Bruce A. Hay and Ming Guo ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 623
Regulation of Commissural Axon Pathfinding by Slit and its Robo
Receptors
Barry J. Dickson and Giorgio F. Gilestro ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 651
Blood Cells and Blood Cell Development in the Animal Kingdom
Volker Hartenstein ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 677
Axonal Wiring in the Mouse Olfactory System
Peter Mombaerts ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 713
viii
Contents