Agrobacterium Tumefaciens: and Plant Cell Interactions and Activities Required For Interkingdom Macromolecular Transfer

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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] Current address: National Institutes of Health, Bethesda, Maryland 20892
Annu. Rev. Cell Dev. Biol. 2006. 22:10127 First published online as a Review in Advance on May 18, 2006 The Annual Review of Cell and Developmental Biology is online at http://cellbio.annualreviews.org This articles doi: 10.1146/annurev.cellbio.22.011105.102022 Copyright c 2006 by Annual Reviews. All rights reserved 1081-0706/06/1110-0101$20.00
Key Words
virA-virG two-component system, signal integration, type IV secretion, VirB complex
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 systemthe VirB complexin this process.
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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
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). Specically, 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 acidsugar 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 (Temp e & Petit 1982). The capacity for gene transfer led to the development of A. tumefaciens
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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: sugaramino acid conjugates synthesized by A. tumefaciens transformed plant cells that can be metabolized by A. tumefaciens cells carrying a Ti plasmid
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 modied 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 oversimplied 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, Tzra & Citovsky 2002). Here we focus on the following
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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? Specically, we seek to explore rst, 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 rst steps in understanding this control were made in studies that identied
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www.annualreviews.org Agrobacterium/Plant Cell Interactions
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
several Ti plasmidborne 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.
from the host responsible for the activation of the VirA/VirG system have been dened. 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 signicance of the multiple signals necessary for optimal vir gene activation? Activating signals. Of the inducing signals, low PO4 and low pH can activate virG expressionindependent of VirAthrough 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 identication of phenols as inducers of vir gene expression was rst achieved
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 signicant 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 latters 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 specic 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
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Figure 2 The ChvE/VirA/VirG signal transducing system (note that stoichiometry of ChvE:VirA is not known). See text for details.
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Phenols
O O
CH3
O O
CH3
O
O CH3
O H H H3C O H OCH3 H HO O O OCH3
H3C
H3CO
OCH3 OH
HO OCH3
OCH3
AS (active)
(+)-DDF (inactive)
()-DDF (active)
H HO HO H
Sugars
COOH H H OH HO H
D-glucoronic
CH2OH O H H OH HO H OH OH H H OH H H H O OH HO
H O H OH H H OH OH
HO OCH3
H HO OCH3
acid
D-glucose
L-arabinose
R, R-MMCP (inactive)
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 identied. 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 specic inhibitors of vir gene expression, are consistent with the model ( Joubert et al. 2002, Lee et al. 1992, Zhang et al. 2000), nal resolution awaits the identication of the phenolbinding site in the receptor (see below).
AS: acetosyringone
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chv genes: chromosomal loci of A. tumefaciens required for virulence
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 signicantly lower doses of the phenol (e.g., 0.5 M AS versus 1020 M AS), and second, the maximal level of vir induction at saturating concentrations of phenol increases by ve- to tenfold (Cangelosi et al. 1990, Shimoda et al. 1990). Mutations at a chromosomal virulence locuschvEand 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 specic 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 identied (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-
quence and functional analyses (see below) have shown the latter to be composed of three domainsa 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 dened 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 signicant 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 VirAs 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
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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 specic, irreversible inhibitors of vir gene expression or phenol afnity chromatography protocols identied several phenol-binding proteins, but these did not include VirA (Dy e & 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 identied, 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
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LZ: leucine zipper LKR: linker, kinase, and receiver domains (of VirA)
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 puried 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
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 signicantly higher activity than
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VirALKR (Wang et al. 2002). This increased activity was accompanied by an increased capacity of VirALZLKR to form cross-linkable dimers in comparison with VirALKR . Interestingly, the exact position of the LZ fusion is critical: When fused at position 293just into a predicted helical region of the linker domainthe 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 puried 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 afnity purication 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 specic processes may occur at the wound site and facilitate transformation. The identication 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 efciently 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 spacewhich we term the signal landscapeis less clear. Both chemical and biological methods are needed to establish this and to determine if it is altered by developmental processes, including
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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
the wound response and possibly the inciting bacteria themselves (e.g., Brencic et al. 2004a). Third, quantitative estimation of cell cycle activity in target tissueswounded or unwounded, infected or uninfectedneeds 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 inltration 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 siteswhich 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 signi110 McCullen
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 nonspecic, nonsaturable, aggregation-like interaction readily removed via washes with a buffered salt solution and a specic, saturable interaction (2001000 bacteria per plant cell) impervious to such washing (Gurlitz et al. 1987, Neff & Binns 1985). The specic 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 1-2 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 identied as attachment decient (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 specically 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 efciently. The best characterized of these is a mutant
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with an insertion in the promoter region of an arabinogalactan proteinlikely to be found in the cell walland 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: VirB111 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, Tzra 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.
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Coupling protein: inner membrane component of a T4SS that typically contains an ATP-binding motif and recruits substrates to the T4SS
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? Specic regions of targeted substrates that are important for these processes have been identied. These export signals are found near the C termini of the substrates and are necessary in mediating the interaction of substrates with the T4SS. 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 nding 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
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 sufcient 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 identied 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 specicity of transfer by a T4SS is a result of the specic 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
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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 specic interactions between VirD4 and VirD2, Cascales & Christie (2004b) obtained in vivo evidence for the interaction of VirD4 with the single-stranded T-strand via modication 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. Ampliable 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 VirD2and therefore not producing VirD2-T-strands did not yield ampliable DNA upon immunoprecipitation. The C-terminal secretion signal identied 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 VirD4s involvement in the translocation of DNA substrates by the T4SS, the requirement of VirD4 for VirE2 translocation specically 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 specically 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 membranelocalized proteins, or homologs thereof, is beginning to provide a structural context for their activities. Puried homologs of VirD4 form hexameric rings with crystal structures similar to that of F1-ATPase,
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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 envelopespanning structure. VirB10, a protein that fractionates with both the inner and outer membranes, interacts with the inner membraneassociated 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 VirD4s 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
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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 disulde 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 VirB710, along with VirB4 and VirB6, have been shown to comigrate as a +600 kDa complex in both blue native gel electrophoresis and gel ltration 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 envelopespanning structure and may function as the conduit through which substrates transit the envelope system. The formation of a readily isolatable Ti plasmidencoded 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 ltration 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 complexs 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 specic sites on the cell surface
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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. Signicant recent ndings by Cascales & Christie (2004b) using the TrIP assay described above have claried our understanding of the DNA substrate translocation from VirD4 into the VirB complex. This study demonstrated binding of the T-strand to specic 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 nally 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 nal stepthe 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 rst, followed by periplasmic- and then outer membraneassociated components. Additionally, the results are consistent with many of the physical interactions between VirB/D4 complex proteins previously identied. 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 dened (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
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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 single116 McCullen
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 specically 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 decient in various activities that appear critical to Agrobacterium-mediated transformation. The classes of mutants include two phenotypic groupsthose 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 decient 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 undened 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
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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 specicity 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 specicity, it is important, however, to consider the issue of efciency. For example, frequencies of VirBmediated RSF1010 plasmid transfer from Agrobacterium to Agrobacterium range from 108 to 104 (transconjugants/recipient), whereas the frequency of transfer and the stable integration of T-DNA from Agrobacterium to plant cells can be as high as 101 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 efciency than the VirBmediated transfer (Stahl et al. 1998). Thus, T4SSs likely evolve to exploit specic natural host cell surface components while retaining the capacity to interact productively but less efciently 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 (VirB14 and VirB710) 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 (specic?) 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 decient in such surface molecules (Zhu et al. 2003). A yeast two-hybrid screen using the major pilus subunit as a bait has identied potential candidate receptors (Hwang & Gelvin 2004), and further analysis of these will be interesting. Finally, in vitro formation of VirE2 pores in articial 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.
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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. 3. The VirA protein varies its response to phenols as a function of other signalssugar 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 efcient 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 dened, 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 dened, although new toolsin the form of Arabidopsis mutants that do not bind the pathogenare 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.
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First demonstration of the interaction of a DNA substrate with the VirB T4SS, via the development and use of the TrIP assay. Provides rst clear evidence for four functional domains of the VirA protein.
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First report of the isolation of VirB subcomplexes, besides the extracellular T-pilus, and co-migration of transported substrates with these complexes.
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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.
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Contents
Annual Review of Cell and Developmental Biology Volume 22, 2006
From Nuclear Transfer to Nuclear Reprogramming: The Reversal of Cell Differentiation J.B. Gurdon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 How Does Voltage Open an Ion Channel? Francesco Tombola, Medha M. Pathak, and Ehud Y. Isacoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23 Cellulose Synthesis in Higher Plants Chris Somerville p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 53 Mitochondrial Fusion and Fission in Mammals David C. Chan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 79 Agrobacterium tumefaciens and Plant Cell Interactions and Activities Required for Interkingdom Macromolecular Transfer Colleen A. McCullen and Andrew N. Binns p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 101 Cholesterol Sensing, Trafcking, and Esterication Ta-Yuan Chang, Catherine C.Y. Chang, Nobutaka Ohgami, and Yoshio Yamauchi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 129 Modication of Proteins by Ubiquitin and Ubiquitin-Like Proteins Oliver Kerscher, Rachael Felberbaum, and Mark Hochstrasser p p p p p p p p p p p p p p p p p p p p p p p p p p p 159 Endocytosis, Endosome Trafcking, and the Regulation of Drosophila Development Janice A. Fischer, Suk Ho Eun, and Benjamin T. Doolan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 181 Tight Junctions and Cell Polarity Kunyoo Shin, Vanessa C. Fogg, and Ben Margolis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207 In Vivo Migration: A Germ Cell Perspective Prabhat S. Kunwar, Daria E. Siekhaus, and Ruth Lehmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p 237 Neural Crest Stem and Progenitor Cells Jennifer F. Crane and Paul A. Trainor p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267
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Of Extracellular Matrix, Scaffolds, and Signaling: Tissue Architecture Regulates Development, Homeostasis, and Cancer Celeste M. Nelson and Mina J. Bissell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287 Intrinsic Regulators of Pancreatic -Cell Proliferation Jeremy J. Heit, Satyajit K. Karnik, and Seung K. Kim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 311 Epidermal Stem Cells of the Skin C dric Blanpain and Elaine Fuchs p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 339 e The Molecular Diversity of Glycosaminoglycans Shapes Animal Development Hannes E. Blow and Oliver Hobert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 375 Recognition and Signaling by Toll-Like Receptors A. Phillip West, Anna Alicia Koblansky, and Sankar Ghosh p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 409 The Formation of TGN-to-Plasma-Membrane Transport Carriers Fr d ric Bard and Vivek Malhotra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 439 e e Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and Mechanisms Roland Lill and Ulrich Mhlenhoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457 Intracellular Signaling by the Unfolded Protein Response Sebasti n Bernales, Feroz R. Papa, and Peter Walter p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487 a The Cellular Basis of Kidney Development Gregory R. Dressler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 509 Telomeres: Cancer to Human Aging Sheila A. Stewart and Robert A. Weinberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 531 The Interferon-Inducible GTPases Sascha Martens and Jonathan Howard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 559 What Mouse Mutants Teach Us About Extracellular Matrix Function A. Asz di, Kyle R. Legate, I. Nakchbandi, and R. F ssler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 591 o a Caspase-Dependent Cell Death in Drosophila Bruce A. Hay and Ming Guo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 623 Regulation of Commissural Axon Pathnding by Slit and its Robo Receptors Barry J. Dickson and Giorgio F. Gilestro p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 651 Blood Cells and Blood Cell Development in the Animal Kingdom Volker Hartenstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 677 Axonal Wiring in the Mouse Olfactory System Peter Mombaerts p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 713
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Agrobacterium Tumefaciens: and Plant Cell Interactions and Activities Required For Interkingdom Macromolecular Transfer

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Agrobacterium Tumefaciens: and Plant Cell Interactions and Activities Required For Interkingdom Macromolecular Transfer

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Annu. Rev. Cell Dev. Biol. 2006.22:101-127. Downloaded from arjournals.annualreviews.org by INFLIBNET Master on 08/15/09.

For personal use only.

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Virulence Gene Expression

O H H H3C O H OCH3 H HO O O OCH3

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chv genes: chromosomal loci of A. tumefaciens required for virulence

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Attachment of Agrobacteria to Host Cells

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TrIP: transferred DNA immunoprecipitation

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Substrate Entry into Host Cells

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Annual Review of Cell and Developmental Biology Volume 22, 2006

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