The EMBO Journal (2010), 1–12 |& 2010 European Molecular Biology Organization | All Rights Reserved 0261-4189/10

THE
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EMBO Member’s Review

New insights into an old story: Agrobacterium-

Andrea Pitzschke1 and Heribert Hirt2,3,* genetically. The factors required for tumour formation are
1
Department of Applied Genetics and Cell Biology, University of Natural
encoded on a large tumour-inducing (Ti) plasmid of virulent
Resources and Applied Life Sciences, Muthgasse 18, Vienna, Austria, Agrobacterium strains. The Ti plasmid also serves as a
2
Department of Plant Molecular Biology, Max F. Perutz Laboratories, source for the transfer DNA (T-DNA), a DNA region that is
University of Vienna, Dr-Bohr-Gasse 9, Vienna, Austria and 3URGV imported into plant cells and integrated into the host
Plant Genomics, INRA-University of Evry, 2 Rue Gaston Crémieux,
chromosomal DNA—resulting in genetic manipulation of
Evry, France
the host. The expression of T-DNA-encoded bacterial genes
Agrobacterium tumefaciens causes tumour formation in in the host cell results in the production of enzymes that
plants. Plant signals induce in the bacteria the expression catalyse the synthesis of plant hormones, which are respon-
of a range of virulence (Vir) proteins and the formation of sible for tumour growth and the formation of novel amino-
a type IV secretion system (T4SS). On attachment to plant acid–sugar conjugates, termed as opines. As opines can serve
cells, a transfer DNA (T-DNA) and Vir proteins are as carbon and sometimes nitrogen sources for Agrobacterium
imported into the host cells through the bacterial T4SS. to the exclusion of most other microorganisms, they provide
Through interaction with a number of host proteins, the a selective advantage for this species (Tempé and Petit, 1982).
Vir proteins suppress the host innate immune system and The capacity for gene transfer into plants has been used to
support the transfer, nuclear targeting, and integration of develop Agrobacterium tumefaciens as a vector for genetic
T-DNA into host cell chromosomes. Owing to extensive manipulation. Engineered DNA segments of interest,
genetic analyses, the bacterial side of the plant– which are first cloned into the T-DNA region of ‘disarmed’
Agrobacterium interaction is well understood. However, plasmids, are then introduced into Agrobacterium and
progress on the plant side has only been achieved recently, subsequently transferred into plants. From these disarmed
revealing a highly complex molecular choreography under plasmids, the genes responsible for tumourous growth have
the direction of the Vir proteins that impinge on multiple been removed, ensuring that the transformed cells can be
processes including transport, transcription, and chromo- regenerated into fertile plants that transmit the engineered
some status of their host cells. DNA to their progeny (Hooykaas and Schilperoort, 1992,
The EMBO Journal advance online publication, 11 February Newell, 2000). By these means, the host range of
2010; doi:10.1038/emboj.2010.8 Agrobacterium has been extended to include other bacterial
Subject Categories: plant biology; microbiology & pathogens species as well as fungi and even some mammalian cells
Keywords: Agrobacterium; plant innate immunity; plant (Lacroix et al, 2006). Under laboratory conditions, normally
tumour formation; T-DNA recalcitrant plants (Ishida et al, 1996; Hiei et al, 1997;
Chen et al, 2006), fungi (Bundock et al, 1995; Abuodeh
et al, 2000), and even human cells (Kunik et al, 2001;
Tzfira et al, 2006) can be transformed by Agrobacterium.
Agrobacterium-mediated transformation serves as an impor-
tant model system for studying host–pathogen recognition
Introduction and delivery of macromolecules into target cells. The
interaction between Agrobacterium and plant cells can be
Agrobacterium species are known as the only organisms divided into several steps: recognition, virulence (Vir) gene
capable of interkingdom gene transfer. This soil-borne expression, attachment to the host cell, targeting of Vir
Gram-negative bacterium is a broad-host range plant patho- factors and T-DNA into the host cell, and chromosomal
gen, which initiates tumour formation on most dicotyledo- T-DNA integration (Figure 1). On chemical recognition of
nous and some monocotyledonous species (DeCleene and plant-derived compounds, Agrobacterium Vir gene expres-
DeLay, 1976). Such tumours do not require the continuous sion is induced, which is followed by the physical interaction
presence of the bacteria for proliferation (White and Braun, between bacterium and plant cells. A bacterial transfer
1942), showing that the plant cells have been transformed machinery is subsequently produced and assembled to
import the de novo produced T-DNA strand along with a
*Corresponding author. URGV Plant Genomics, INRA-University of number of Vir factors into the host cell. Once inside the plant
Evry, 2 Rue Gaston Crémieux, Evry 91057, France. cell, the T-DNA is translocated into the nucleus, in which it
Tel.: þ 33 1 6087 4508; Fax: þ 33 1 6087 4510; integrates into the host chromosome. On expression of
E-mail: [email protected]
T-DNA genes, plant cells are re-programmed for tumour
Received: 11 December 2009; accepted: 19 January 2010 growth and production of opines.

& 2010 European Molecular Biology Organization The EMBO Journal VOL 00 | NO 00 | 2010 1
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

Plant cell

4. Transport of T-DNA
Agrobacterium and vir fators by T4SS
virD2 virE2 virE2
virE2 virD 2 5. Nuclear import
virE2
T-DNA

3. T-DNA synthesis
vir transcription
virB1-11
F
virE2
E 6. T-DNA
vir
D integration
virE2 virE3
C
G
A B virE3 virF
P virF
virG

P
virA

2. VirA/G activation

1. Plant signals Wound

Figure 1 Overview of the Agrobacterium–plant interaction. 1. Plant signals induce 2. VirA/G activation and thereby 3. T-DNA synthesis and vir
gene expression in Agrobacterium. 4. Through a bacterial type IV secretion system (T4SS) T-DNA and Vir proteins are transferred into the plant
cell to assemble a T-DNA/Vir protein complex. 5. The T-DNA complex is imported into the host cell nucleus in which 6. the T-DNA becomes
integrated into the host chromosomes by illegitimate recombination.

Recognition of plant cells as host by system in which VirA is a membrane-bound sensor and VirG
Agrobacterium is the intracellular response regulator (Wolanin et al, 2002).
On signal sensing, the histidine kinase VirA activates VirG
Agrobacterium strains are widely distributed in the soil. through transferring its phosphate to a particular aspartate of
Moreover, most isolates do not contain a Ti plasmid and VirG, thereby activating VirG to function as a transcription
are capable of living independently of a plant host. Yet, as factor. Phosphorylated VirG then binds at specific 12 bp DNA
tumour-produced opines are a specific food source for sequences of the vir gene promoters (vir boxes), thereby
Agrobacterium, the capacity of Agrobacterium strains to in- activating transcription (Brencic and Winans, 2005).
duce such tumours is a clear selective advantage. However, The signals perceived by VirA are phenols, aldose mono-
because plant transformation is a complex process and saccharides, low pH, and low phosphate (Palmer et al, 2004;
energetically demanding, Vir gene expression must be care- Brencic and Winans, 2005). Phenols are indispensable for vir
fully regulated. The identification of vir genes, which are gene induction, whereas the other signals sensitise VirA to
required for virulence, but lie outside the T-DNA (Klee et al, phenol perception, for example sugars allow induction of the
1983; Stachel and Nester, 1986), was a major step towards VirA/VirG system at much lower phenol concentrations and
understanding the transformation process. With the excep- increase the response several-fold (Shimoda et al, 1990). The
tion of virA and virG, the vir genes were found to be identification of phenols, such as acetosyringone, as inducers
essentially silent unless the bacteria are cultured with plant of vir gene expression was achieved through analysis of root
cells (Stachel and Nester, 1986). Although vir gene induction exudates and leaf protoplasts (Stachel et al, 1985).
depends on molecules exuded by the plant, attachment to Acetosyringone is now routinely used for enhancing the
plant cells is necessary for transformation and is mediated by efficiency of Agrobacterium-mediated plant transformation.
chromosomally encoded Agrobacterium genes (Lippincott The capability of the VirA/VirG system to recognise a diver-
and Lippincott, 1969; Douglas et al, 1982). Thus, host recog- sity of phenols and sugars is a likely explanation for the
nition by Agrobacterium resulting in transformation is com- broad-host range exhibited by Agrobacterium.
posed of two independent processes: Vir gene activation and
attachment to the host cell.
Plant entry sites for Agrobacterium
In nature, Agrobacterium attacks mainly wounded tissue
Agrobacterium Vir gene expression (Braun, 1952). A wound site may simply be a portal of
The vir gene activation by plant factors requires two genes, entry, but other specific processes specifically occurring at
virA and virG (Stachel and Nester, 1986), which are consti- these sites are likely to facilitate transformation: wound-
tutively expressed at a basal level, but can become highly secreted compounds such as phenols and sugars induce vir
induced in a feed-forwards manner (Winans et al, 1988). The gene expression. In addition, the latter act as chemotactic
virA and virG genes encode a two-component phospho-relay attractants of Agrobacterium. Thus, wound-specific features

2 The EMBO Journal VOL 00 | NO 00 | 2010 & 2010 European Molecular Biology Organization
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

such as high activity of the phenylpropanoid pathway, low The VirB complex belongs to the class of type IV secretion
pH, and sugars associated with cell wall synthesis/wound systems (T4SS), which are found across a broad range of
repair correlate with enhanced transformation frequency and Gram-negative bacteria and are involved in the conjugative
efficiency (Baron and Zambryski, 1995). Although transfor- transfer of plasmids between bacteria as well as the translo-
mation can also occur in unwounded plants—with cation of Vir factors from pathogens to host cells during
Agrobacterium cultures grown in pre-induction medium infection (Cascales and Christie, 2003).
(Escudero and Hohn, 1997)—it seems that Agrobacterium The VirB complex is composed of at least 12 proteins:
has optimised the VirA/VirG system to respond to signals VirB1–11 and VirD4 and is required for virulence. The
from wound sites. Cell division activity at the wound sites is proteins associate with the cell envelope and form a multi-
thought to be equally important for transformation (Braun, subunit envelope-spanning structure (Christie et al, 2005).
1952). However, cells in the root elongation zone were found The bacterial factors transported into host cells by the VirB
to be the most highly transformable (Yi et al, 2002). Cells of complex include the VirD2-T-DNA, VirE2, VirE3, VirF, and
this non-meristematic zone are not undergoing a normal VirD5 (Vergunst et al, 2005). VirD2 nicks the T-DNA at the 25-
cell cycle, but endoreduplication. nucleotide long repeats that border the T-DNA and VirD2,
then becomes covalently bound to the 50 -end of the T-DNA.
VirD2 seems to be transported with the T-strand into the plant
Host cell attachment by Agrobacterium cell, in which it is involved in nuclear import and integration
As T-DNA and proteins are transferred from A. tumefaciens of the T-DNA into the host genome (Gelvin, 2003). VirE2 is a
into plant cells, an intimate association between pathogen single-stranded DNA-binding protein that can coat the length
and host cell is a prerequisite for transformation. of the T-strand in vitro (Christie et al, 1988; Citovsky et al,
Quantitative-binding assays have revealed a non-specific 1992). It likely interacts with T-DNA in the plant cell cyto-
interaction that is readily removed and a specific interaction plasm and also has functions in nuclear import and integra-
(Neff and Binns, 1985). The specific attachment of A. tume- tion (Gelvin, 2003). Intriguingly, the VirB/D4 complex can
faciens to plant cells is not dependent on the Ti plasmid not only transport Ti-derived T-DNA, but also the broad-host
(Douglas et al, 1982; Neff and Binns, 1985). Instead, it is range plasmid RSF1010 to plants or other Agrobacterium
facilitated by the chromosomally encoded bacterial genes species, showing that conjugative intermediates must also
chvA, chvB, and pscA (exoC), which are involved in the be substrates (Buchanan-Wolloston et al, 1987; Beijersbergen
synthesis and/or localisation of periplasmic b-1,2 glucan et al, 1992).
(reviewed in McCullen and Binns, 2006).
Early studies revealed that the exposure of A. tumefaciens
cells to soluble pectic plant cell wall fractions decreases both
Import of Agrobacterium Vir factors
the specific binding of Agrobacterium to plant cells and into host cells
tumour-induction frequencies (reviewed in Gelvin, 2000), The A. tumefaciens virB-encoded T4SS transports substrates
suggesting the presence of as yet elusive Agrobacterium across the bacterial cell envelope. Certain C-terminal motifs
receptor-like components. Possible candidates are BTI-do- were found to be required for the export of targeted sub-
main proteins that had been isolated from a screen for strates. These export signals mediate the interaction of sub-
VirB2-interacting proteins. Owing to its transient increase strates with the T4SS. The C-termini of VirF, VirE2, and VirE3
immediately after Agrobacterium infection and its preferential are sufficient to mediate transport of fusion proteins to plants
localisation to the periphery of root cells, a direct contact of (Vergunst et al, 2000). The minimal size of VirF required to
BTI1 with the Agrobacterium T-pilus in the initial interaction direct protein translocation to plants is the C-terminal 10
of Agrobacterium with plant cells has been proposed (Hwang amino acids (Vergunst et al, 2005), from which the minimal
and Gelvin, 2004). consensus sequence R-X(7)-R-X-R-X-R required for substrate
Genomic studies are beginning to provide new insight into secretion by the VirB complex could be derived (Vergunst
possible plant molecules involved in the attachment process. et al, 2005).
A number of Arabidopsis mutants have been isolated that are C-terminal fusions of VirE2 blocked its translocation to
recalcitrant to Agrobacterium transformation (rat mutants) host cells (Vergunst et al, 2000). Accordingly, insertion of a
(Zhu et al, 2003) and it was shown that Agrobacterium can no FLAG tag at the C-terminus of VirE2, or truncation of the
longer bind efficiently to some rat mutants. One well-char- C-terminal 18 amino acids of VirE2, renders the protein non-
acterised mutant is affected in the gene encoding a cell wall functional in A. tumefaciens, while not affecting its capability
arabinogalactan protein to which bacteria bind poorly (Nam to bind single-stranded DNA (Simone et al, 2001). However,
et al, 1999; Zhu et al, 2003). Further analysis of these mutants overexpression of such VirE2 C-terminal mutant derivatives
should help to unravel the recognition process and physical in transgenic plants confers susceptibility to transformation
interaction of Agrobacterium and host cells. by an A. tumefaciens virE2-deficient strain, suggesting that
the mutations disrupted a region of amino acids required for
translocation, such as a secretion signal.
Agrobacterium secretion of T-DNA and Vir
In an elegant experimental approach, using fusion proteins
proteins into plant cells of VirE2 or VirF to the Cre recombinase, the transport of
After vir gene activation and attachment of Agrobacterium to these proteins in the absence of T-DNA has been studied
plant cells, a transporter complex formed by VirB proteins (Vergunst et al, 2000). The experiments were designed in
and VirD4 enables Vir proteins and T-DNA to cross the inner such a way that the transport of Cre-VirE2 or Cre-VirF
bacterial membrane, the peptidoglycan layer, and outer fusions into host plant cells results in a recombination
membrane, as well as the plant host cell wall and membrane. event conferring kanamycin resistance to host tissues.

& 2010 European Molecular Biology Organization The EMBO Journal VOL 00 | NO 00 | 2010 3
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

Thus, it could be shown that the transport of bacterial absence of VirD2 NLS. In support of this notion, Gelvin
factors is dependent on the VirB/VirD4-complex. In addition, (1998) observed that an A. tumefaciens virE2 virD2DNLS
VirE3 and the C-terminus of VirD5 were found to mediate double mutant was able to form tumours on VirE2-producing
substrate targeting into host cells (Schrammeijer et al, transgenic tobacco, but not on wild-type tobacco, and sug-
2003; Vergunst et al, 2005). VirE3 may function in the plant gested that the NLS of VirE2 could have a function in
to aid nuclear localisation of VirE2 (Lacroix et al, 2005), and directing T-DNA to the nucleus.
VirD5 may function as transcription factor in the plant cells Several studies suggest additional functions of VirE2 as
(Schrammeijer et al, 2000). transmembrane DNA transporter. VirE2 can insert itself into
artificial membranes and form channels. These channels can
facilitate the efficient transport of ssDNA through membranes
Host cell entry of Agrobacterium factors (Dumas et al, 2001; Duckely et al, 2005). Indeed, as shown by
It is presently unclear how the Vir proteins and the T-DNA biophysical experiments and particle-bombarded tobacco
protein complex traverse the host cell wall and membrane cells transiently expressing VirE2 fusion protein, VirE2
barriers. In T4SS-mediated plasmid transfer, the pilus enables seems to actively pull ssDNA into the host (Grange et al,
the interaction between donor and recipient, followed by the 2008).
fusion of outer membranes in a mating junction (Schroder Despite the prominent function of VirE2 in the transforma-
and Lanka, 2005). The mechanism by which the transferred tion process, some strains of Agrobacterium rhizogenes
conjugal intermediate traverses the bacterial wall and inner lacking this protein can still transfer T-DNA efficiently. This
membrane is not known. Even less is known about VirB- is achieved through GALLS proteins (Hodges et al, 2004,
mediated transfer across host cell barriers. The enormous 2006, 2009). Interestingly, despite their dissimilarity to
host range transformed by Agrobacterium suggests that the VirE2, GALLS protein restored pathogenicity to virE2 mutant
specificity of host–pathogen interaction required to breach A. tumefaciens (Hodges et al, 2004). The GALLS gene en-
the host cell wall and membrane barriers may be less codes for two proteins: full-length GALLS and a C-terminal
important than expected. domain that initiates at an internal in-frame start codon.
Full-length GALLS protein contains domains for ATP binding,
nuclear localisation, and type IV secretion (Hodges et al,
Targeting of Agrobacterium T-DNA into the
2006). In plant cells, interaction of GALLS-FL with VirD2 was
host cell nucleus observed (Hodges et al, 2009). On the basis of these findings,
Once inside the plant cell, the T-DNA must find its way into as well as the nuclear localisation of GALLS-FL and its
the nucleus. Several Agrobacterium Vir proteins, as well as a predicted helicase activity, the authors proposed that
number of plant proteins, seem to be involved in this process GALSS-FL may pull T-strands into the nucleus.
(Figure 2). The proteins VirD2 and VirE2 contain plant-active VirF has been implicated in the degradation of host cell
nuclear localisation signal (NLS) sequences. VirD2, which is factors during infection. In the host nucleus, VirF, in concert
covalently linked to the 50 -end of the T-DNA, contains two with the host proteasome machinery, is believed to mediate
NLS regions, both of which can direct chimeric proteins to the degradation of the T-DNA complex, thus facilitating the
nucleus. Sterical considerations suggest that the bipartite NLS release of the T-DNA and its subsequent choromosomal
in the carboxy-terminus of VirD2 might be biologically im- integration (Schrammeijer et al, 2001; Tzfira et al, 2004a, b).
portant for nuclear targeting of the T-DNA complex (Tinland
et al, 1995).
Functions of Vir proteins in T-DNA
VirE2 protein contains two separate bipartite NLS regions
that can target fusion reporter proteins to plant nuclei
integration
(Citovsky et al, 1992, 1994). Fluorescently labelled single- Relatively little is known about the precise mechanism of
stranded DNA coated with VirE2 and microinjected into plant T-DNA integration into the plant genome or the function
cells localises to the nucleus, whereas naked single-stranded specific proteins have in this process. The major mode
DNA remains in the cytoplasm (Zupan et al, 1996). foreign DNA integrates in plants is by illegitimate recombina-
In agreement with a function of VirD2 and VirE2 in T-DNA tion or non-homologous end-joining; and T-DNA integrates
nuclear guidance, deletion of the VirD2 bipartite NLS resulted into plant chromosomes by a similar mechanism
in almost complete loss of transformation (Rossi et al, 1993), (Paszkowski et al, 1988; Gheysen et al, 1991; Mayerhofer
indicating that VirE2 NLS domains cannot compensate for the et al, 1991). An alternative model proposes that the initial
loss of the VirD2 NLS. Furthermore, VirD2 and VirE2 proteins invasion of plant DNA by the T-DNA is also of importance
were shown to be necessary for nuclear targeting of in vitro for integration (Meza et al, 2002). In addition, analysis of
synthesised T-complexes in permeabilised HeLa cells T-DNA-integration sites suggests the involvement of micro-
(Ziemienowicz et al, 1999). homologies in the integration process (Pelczar et al, 2004).
As shown by Shurvinton et al (1992), the C-terminal NLS Measurements of the relative amounts of transient versus
of VirD2 is essential for virulence, but not for intrabacterial stable expression of reporter genes in Agrobacterium-infected
T-strand production. This NLS does not contribute to targeting plant cells suggests that most T-DNA is not stably integrated
of the T-DNA to the nucleus (Shurvinton et al, 1992; Rossi into chromosmal DNA (Nam et al, 1997). Although T-DNA
et al, 1993; Mysore et al, 1998), and it was suggested that enters the nucleus as a single-stranded molecule, much of the
deletion of the VirD2 NLS may alter the structure of the VirD2 T-DNA likely becomes double stranded, because the conver-
protein such that it can still nick the T-DNA border, but may sion to a transcriptionally competent form requires the synth-
fail to pass through the T4SS or the nuclear pore (Mysore esis of a complementary DNA to the T-strand (Narasimhulu
et al, 1998). VirE2 might provide nuclear targeting in the et al, 1996). It is not yet clear whether the T-DNA integrates

4 The EMBO Journal VOL 00 | NO 00 | 2010 & 2010 European Molecular Biology Organization
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

PAMPs
Receptors

virE3
Importin virE3 MAPK
pBr activation
p
Tumour development genes
V I P1
virF 2. Proteasomal 3. Manipulation of
degradation host gene expression
Importin
VIP1 VIP1
1. Nuclear import virF
VIP2 Defence genes

Histone genes
V I P1 virE2
virE2 virD2
VI
P2 V I P1 virE2 virD2
VI virE2
P2 Importin

4. T-DNA
integration

Figure 2 Molecular details of Agrobacterium and host factors involved in T-DNA transfer and host responses. 1. Nuclear import of the T-DNA
complex along with other Vir proteins. 2. VIP1 becomes activated by PAMP-triggered MAPK activation, but undergoes proteasomal degradation
through VirF. 3. Manipulation of host gene expression by Vir proteins helps 4. T-DNA integration and tumour development as well as
suppression of defence responses.

through single-strand invasion of locally denatured plant Plant factors and defence responses
DNA (Tinland, 1996), or whether the extrachromosomal involved in Agrobacterium tumour
double-stranded T-DNA is the substrate for integration. As
formation
VirD2 is covalently linked to the T-DNA strand, it likely has
some function in the integration process: VirD2 can mediate Although certainly unintentionally, the host plant actively
site-specific cleavage and reversal of it, site-specifically only participates in Agrobacterium transformation. This ‘assis-
(Pansegrau et al, 1993). Ligation of T-DNA to plant DNA is tance’ occurs at several levels: Vir protein/T-DNA import,
rather mediated by host proteins. VirD2 failed to facilitate dissociation of the Vir/T-DNA complex, T-DNA integration,
in vitro ligation-integration reactions in which T-DNA was and re-programming of gene expression for tumour develop-
ligated to a model target sequence, whereas this reaction did ment. A number of host factors that are exploited by
take place in the presence of plant extracts (Ziemienowicz Agrobacterium to achieve transformation have been identi-
et al, 1999). The integration of the 50 end of the T-strand into fied. Major progress has been made through yeast-two-hybrid
plant DNA is generally precise and only a few 50 nucleotides (Y2H) screens for identifying host proteins that interact with
are usually deleted on T-DNA integration into the plant Agrobacterium Vir proteins (Ballas and Citovsky, 1997; Deng
genome (Tinland et al, 1995). This may result from the et al, 1998; Hwang and Gelvin, 2004). Another important
protection from exonucleases that VirD2 offers to the capped progress was achieved through large plant mutant screens
50 T-strand end, as mutations in VirD2 result in imprecise (Zhu et al, 2003; Crane and Gelvin, 2007).
ligation and deletion of the 50 end of the T-DNA to plant
chromosomal DNA (Tinland et al, 1995). Moreover, the o
domain of VirD2, a conserved region outside the NLS, is
Functions of plant proteins in T-DNA
important for tumourigenesis (Shurvinton et al, 1992; Mysore nuclear import
et al, 1998), but has only minor effects on transient T-DNA VirD2 was reported to interact with several members of the
expression (Narasimhulu et al, 1996). Arabidopsis importin a and cyclophilin families in vitro and
The virE2 mutants are extremely attenuated in virulence in the Y2H system (Koncz et al, 1989; Deng et al, 1998; Bako
(Stachel and Nester, 1986). As VirE2 can function as a gated et al, 2003; Bhattacharjee et al, 2008). Importins a are NLS-
pore for the passage of ssDNA (Dumas et al, 2001; Duckely binding proteins of the nuclear import machinery that speci-
et al, 2005; Grange et al, 2008), the severe attenuation of fically interact with the bipartite NLS region of VirD2 and
virE2 mutant Agrobacterium strains might be explained by a may thus facilitate its nuclear import (Ballas and Citovsky,
defect in nuclear host transport. However, the integrated 1997). Deng et al (1998) identified an Arabidopsis cyclophilin
T-DNA molecules transferred from virE2 mutant that interacts in Y2H experiments with a central domain of
Agrobacterium strains also exhibit extensive deletions corre- VirD2. As some cyclophilins have peptidyl-prolyl isomerase
sponding to the 30 ends of the T-strand (Rossi et al, 1996), activity, the authors speculated that this protein might serve
suggesting that nucleolytic protection of the T-strand is also a as a chaperonin to hold VirD2 in a transfer-competent con-
major function of VirE2. formation during T-strand trafficking through the plant cell.

& 2010 European Molecular Biology Organization The EMBO Journal VOL 00 | NO 00 | 2010 5
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

However, this hypothesis is put into question by a recent Nuclear transport of the T-DNA complex
study in which the cyclophilin-binding domain of VirD2 was
VIP1 functions in the shuttling of the T-DNA complex into the
found to be dispensable for virulence (van Kregten et al,
host cell nucleus, but this function can be partially comple-
2009).
mented for by Agrobacterium VirE3, which, similar to VIP1,
Furthermore, VirD2 is subject to post-translational mod-
was found to be capable of binding to VirE2 and IMPa-1
ification. Nuclear targeting of VirD2 is apparently controlled
(Lacroix et al, 2005). In vitro, VIP1 forms ternary complexes
through phosphorylation of a serine residue close to the
with VirE2 and ssDNA (Tzfira et al, 2001). Analysis of an
bipartite NLS (Tao et al, 2004). Alanine substitution of this
Arabidopsis vip1-1 mutant, which produces a truncated VIP1
residue resulted in the predominantly cytoplasmic localisa-
protein, revealed that the C-terminal region of the protein is
tion of a b-glucuronidase (GUS)-VirD2 NLS fusion protein.
required for stable transformation, but dispensable for tran-
Moreover, Tao et al (2004) identified DIG3, a type 2C serine/
sient transformation (Li et al, 2005a). VIP1 is a mobile
threonine protein phosphatase, which negatively affects
protein, which undergoes cytoplasmic-nuclear trafficking in
nuclear import of a GUS-VirD2 NLS fusion protein.
a stress-dependent manner (Djamei et al, 2007). The other
A biochemical approach has led to the identification of a
VirE2-interacting protein, VIP2, shows nuclear localisation
VirD2-interacting kinase, CAK2M (Bako et al, 2003). VirD2
(Tian et al, 2004). The most characteristic feature of VIP2 is
interacts with and is phosphorylated by CAK2M in vivo;
an NOT domain (negative on TATA-less), rendering VIP2 a
CAK2M might correspond to the kinase that regulates
putative transcriptional repressor protein (Anand et al, 2007).
nuclear import of VirD2 (Gelvin, 2000). Another substrate
Virus-induced gene silencing of VIP2 in Nicotiana benthami-
of CAK2M is RNA polymerase II large subunit (RNApolII
ana and characterisation of the Arabidopsis vip2 mutant
CTD), a factor that is responsible for recruiting TATA-box-
revealed that VIP2 is required for stable, but not for transient,
binding proteins (TBP) to actively transcribed regions.
transformation. As shown by Y2H and bimolecular fluores-
Comparative sequence analysis of insert junctions and target
cence complementation (BiFC) studies, VIP2 does not only
sites suggested a preferential integration of the T-DNA into
interact with VirE2, but also with VIP1 (Anand et al, 2007).
promoters of transcribed chromatin domains (Koncz et al,
Microarray analysis revealed a major impairment of the
1989; Mayerhofer et al, 1991; Brunaud et al, 2002; Szabados
transcriptional response of vip2 to Agrobacterium in compar-
et al, 2002). Moreover, experiments in Agrobacterium-trans-
ison with wild-type plants. Moreover, as many as 52 histone/
formed Arabidopsis cells revealed an association of VirD2
histone-associated genes are constitutively repressed in vip2
with TBP, one of the most conserved nuclear proteins in
mutants (Anand et al, 2007). Together, these observations
eukaryotic cells (Nikolov et al, 1992) in transformed
prompted the authors to suggest that the recalcitrancy of vip2
Arabidopsis cells (Bako et al, 2003). A hypothetical scenario
mutants to Agrobacterium infection and the decreased trans-
suggested by the authors is that TBP or CAK2M may target
formation efficiency are due to impaired Agrobacterium-re-
VirD2 to the CTD, thereby controlling T-DNA integration.
ponsive gene induction and constitutive histone gene
However, an alternative explanation for TBP–VirD2 interac-
repression.
tion might have to be found. A genome-wide analysis
Data on some aspects of VirE2 are still somewhat con-
of T-DNA-integration sites in Arabidopsis performed under
troversial. Although Ballas and Citovsky (1997) reported
non-selective conditions does not support the earlier
specific interaction of VirE2 with Arabidopsis importin
concept of preferential T-DNA integration into transcription-
IMPa-1 and nuclear localisation of VirE2, other studies
ally active regions. Instead, Kim et al (2007) found that
showed a predominant cytoplasmic VirE2 localisation
T-DNA integration occurs rather randomly and that the earlier
(Bhattacharjee et al, 2008; Grange et al, 2008) and interaction
reported enrichment of such integration sites in gene-rich or
of VirE2 with several importin isoforms in planta
transcriptionally active regions of chromatin is due to the
(Bhattacharjee et al, 2008). Moreover, impa-4, but not other
selection pressure applied for recovery of T-DNA insertions.
importin mutants, is recalcitrant to transformation. This
However, the results leading to non-selected T-DNA-integra-
deficiency can be overcome through ectopic overexpression
tion events could not be verified, as sequence analysis was
of heterologous importin isoforms (Bhattacharjee et al,
presented only and transgenic plants could not be recovered
2008).
because of the special experimental design. O’Malley et al
A possible explanation for the differences in reported VirE2
(2007) developed a novel PCR-based method for high
localisation might be found in the stress-dependent subcel-
throughput sequencing the T-DNA/genomic DNA junction
lular translocation of the VirE2-interacting protein VIP1.
of 150 000 T-DNA insertional mutants. The analysis of this
Stress-triggered phosphorylation of VIP1 mediates VIP1 nu-
library should provide detailed information on the sequence
clear localisation and virulence presumably by directing
requirements for T-DNA integration at a large scale.
VirE2 to the nucleus (Djamei et al, 2007). Thus, under stress
Two VirE2-interacting proteins, VIP1 and VIP2, have
conditions such as those occurring in cell bombardment,
been isolated and characterised. Most data on VIP1 originate
phosphorylated VIP1 may pull the otherwise cytoplasmic
from experiments in tobacco cells (Tzfira et al, 2001, 2002).
VirE2 into the nucleus.
Elevated levels of VIP1 enhance Agrobacterium susceptibility
VIP1 interacts with VirE2 and IMPa-1. Tzfira et al (2002)
and transformation efficiency, as shown by larger calli
found interaction between VIP1 and IMPa-1, but not between
formed in infected VIP1 overexpressing plants (Tzfira et al,
VirE2 with IMPa-1. They, therefore, suggested that VIP1 may
2002). Further interaction experiments led to the suggestion
serve as an adaptor molecule to facilitate the import of VirE2-
that VIP1 functions as a bridge between VirE2 and the
bound T-strands into the nucleus. Bhattacharjee et al (2008),
plant importin a-1 (IMPa-1), thereby facilitating nuclear
however, found that VirE2 can directly bind all tested im-
import of VirE2 and its associated T-DNA (Tzfira et al,
portin to isoforms (IMPa-1, -2, -3, -4, -7, and -9) in Y2H and
2002).

6 The EMBO Journal VOL 00 | NO 00 | 2010 & 2010 European Molecular Biology Organization
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

BiFC interaction studies. Combining own results and those of In addition to rad5, some radiation-sensitive Arabidopsis
Tzfira et al (2002), Bhattacharjee et al (2008) proposed that ecotypes are also recalcitrant to Agrobacterium-mediated
VirE2 may use several cellular mechanisms for nuclear im- transformation. By examining almost 40 Arabidopsis eco-
port, thereby creating additional opportunities for T-complex types for susceptibility to root transformation by
entry into the nucleus. This explanation seems plausible, Agrobacterium, ecotype UE-1 was found to be both slightly
particularly when considering that MPK3, the VIP1-nuclear- radiation-hypersensitive and transformation-deficient (Nam
targeting kinase, is only transiently (5–15 min) activated on et al, 1997). Further testing of the rat mutants revealed that 5
Agrobacterium contact (Djamei et al, 2007). VIP1-indepen- of the initial 21 rat mutants were integration deficient, as
dent nuclear translocation of VirE2 may, therefore, be parti- indicated by high transient, but low stable transformation
cularly relevant for securing T-DNA-complex entry over a efficiency. One of these mutants, rat5, contains an insertion
prolonged period. Moreover, as VIP1, irrespective of its in the 30 untranslated region of a histone H2A gene (Mysore
phosphorylation status, can bind IMPa-1 (Djamei and et al, 2000). Although highly recalcitrant to stable transfor-
Pitzschke, unpublished) and VirE2 (Lacroix et al, 2008), it mation by root inoculation, rat5 is efficiently transformed by
may still assist VirE2 nucelar translocation indirectly by flower vacuum infiltration. These results suggest that some
guiding VirE2 to the nuclear periphery. Direct VirE2–IMPa-4 factor(s) required for efficient transformation are present in
interaction would then accomplish nuclear VirE2 import. the female gametophyte, but absent in root somatic tissue. As
This assumption is in accordance with the results from Lee rat5 plants can be complemented to transformation profi-
et al (2008). Particle bombardment in onion cells revealed ciency with the wild-type RAT5 histone H2A gene, the rat5
VirE2–IMPa-1 protein complexes around the nucleus, but mutant is haplo-insufficient (dosage-dependent). The func-
VirE2–IMPa-4 complexes exclusively within the nucleus. tion of histones in T-DNA transformation is further empha-
sised in a recent study by Tenea et al (2009), who tested the
effect of overexpression of several histones on Arabidopsis
Functions of plant proteins in T-DNA
transformation and transgene expression. After transfection,
integration transgene DNA was found to accumulate more rapidly in
As T-DNA must interact with chromatin to integrate into plant histone HTA1-overexpressing plants. The authors proposed
chromosomal DNA, it is likely that altering chromatin con- that enhanced Agrobacterium-mediated transformation
formation will affect T-DNA integration. Forward and reverse through histone overexpression is due to the protection of
genetic approaches have been carried out to determine which incoming transgene DNA during the initial stages of transfor-
chromatin proteins are important for transformation (Zhu mation. The main mechanism by which histones confer
et al, 2003; Crane and Gelvin, 2007). In this way, mutants susceptibility seems to be conserved, as overexpression of
were identified in or near various histone genes, histone Arabidopsis HTA1 can not only enhance transformation
acetyltransferase genes, histone deacetylase genes as rat efficiency in Arabidopsis (Yi et al, 2002, 2006), but also in
mutants. Moreover, 340 stable Arabidopsis RNAi mutant rice (Zheng et al, 2009).
lines were screened for rat phenotypes. These lines com- Mutations in fas1 and fas2, encoding two subunits of the
prised 109 chromatin genes of 15 gene families, including chromatin assembly factor CAF1, show greatly increased
bromodomain and chromodomain proteins, chromatin remo- frequencies of homologous recombination and T-DNA inte-
delling complexes, DNA methyltransferases, global transcrip- gration (Endo et al, 2006). Studies on the Arabidopsis protein
tion factors, histone acetyltransferases, histone deacetylases, KU80 further stress the active participation of the host’s
histone H1, methyl-binding-domain proteins, MAR-binding repair machinery in T-DNA integration. KU80, an important
filament-like proteins, nucleosome assembly factors, and protein in the non-homologous end-joining complex (Jeggo
SET-domain proteins. Silencing of 24 chromatin genes repro- et al, 1999), directly binds to double-stranded T-DNA inter-
ducibly resulted in some level of decreased transformation mediates (Li et al, 2005b), which are rapidly converted from
susceptibility. As T-DNA integrates into the plant genome by T-strands early in the infection process and are essential
illegitimate recombination (Mayerhofer et al, 1991), plants intermediates of T-DNA integration (reviewed in Tzfira
deficient in DNA repair and recombination may be deficient et al, 2004a, b). Ku80 mutants are defective in T-DNA inte-
in T-DNA integration. Such DNA metabolism mutants are gration, but not in transient T-DNA expression, whereas KU80
likely to be hypersensitive to DNA-damaging agents such as overexpression results in increased susceptibility to
UV and radiation and DNA-damaging drugs such as bleomy- Agrobacterium infection and increased resistance to DNA-
cin. Sonti et al (1995) investigated a number of radiation- damaging agents (Li et al, 2005b). The function of KU80
sensitive Arabidopsis mutants for transient and stable during the transformation of germ-line cells, however, is still
Agrobacterium-mediated transformation. Among these, debatable. Ku80 has been reported to be both required
uvh1 and rad5 mutants seemed to be resistant to stable, but (Friesner and Britt, 2003) and dispensable (Gallego et al,
not to transient transformation, as assessed by formation of 2003) for T-DNA integration.
kanamycin-resistant calli. However, an in-depth analysis by
Nam et al (1998) confirmed stable transformation deficiency
Agrobacterium and the plant innate
only for rad5, but not uvh1 mutants. Although the latter did
form less calli on kanamycin selection medium, tumour
immune response
growth on non-selective medium as well as stable phosphi- Plant factors involved in Agrobacterium perception
notricin resistance were similar to wild type. Furthermore, Agrobacterium-attacked plants do not simply come to terms
results from these authors also suggest RAD5 to be involved with their fate. Similar to other pathogens, Agrobacterium is
in some step before T-DNA integration, such as T-DNA sensed as an invader and triggers the ‘innate immune
transfer or nuclear targeting (Nam et al, 1998). response’, characterised by the expression of defence genes

& 2010 European Molecular Biology Organization The EMBO Journal VOL 00 | NO 00 | 2010 7
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

and accumulation of reactive oxygen species (reviewed in of flg22 resulted in abolishment of GUS expression in wild
Pitzschke et al, 2009a). This reaction is achieved through the type and efr mutants, whereas co-injection of elf18 only
perception of pathogen-associated molecular patterns abolished GUS expression in wild type, but not in efr.
(PAMPs) by specific receptors. Although several PAMPs N. benthamiana, an easily transformed plant species, does
have been isolated, only few receptors are yet identified. not have an EF-Tu recognition system. However, transgenic
Putative plant receptors for Agrobacterium include a vitro- N. benthamiana plants expressing Arabidopsis EFR are cap-
nectin-like protein (Wagner and Matthysse, 1992), a rhicad- able of inducing elf18-triggered defence responses. Together,
hesin-binding protein (Swart et al, 1994), and several these observations lead to the conclusion that EFR-mediated
VirB2-interacting proteins (Hwang and Gelvin, 2004; re- EF-Tu perception restricts Agrobacterium-mediated transfor-
viewed in Citovsky et al, 2007). A recent in-depth study by mation (Zipfel et al, 2006).
Clauce-Coupel et al (2008) on vitronectins strongly indicates A recent study sheds light on the molecular mechanism
that this group of proteins is unlikely to act as receptors for that links EFR receptor activation to intracellular signal
site-specific Agrobacterium attachment. transduction and stresses differences between flg22- and
The most intensively investigated PAMP is flagellin, a elf18-triggered limitation of Agrobacterium transformation
highly conserved bacterial protein. In Arabidopsis, it is (Ishikawa, 2009). Arabidopsis mutants affected in the
perceived by the receptor protein FLS2, a leucine-rich repeat G-protein b-subunit (agb1-2) show significantly reduced
receptor such as kinase (LRR-RLK). On perception of flagellin ROS production on flg22 or elf18 treatment, whereas stress-
or its derived highly conserved 22 amino-acid peptide, flg22, triggered MAPK activation (analysed by immunoblotting with
FLS2 becomes activated and initiates a phospho-relay-based an antibody recognising active MPK3 and MPK6) is appar-
signal transduction through the MAPK cascade MEKK1- ently not affected. Moreover, these mutants are impaired in
MKK1/2-MPK4 (Qiu et al, 2008; Pitzschke et al, 2009a, b). the elf18- but not in flg22-triggered immunity against
Subsequently, the MPK4-activated transcription factor Agrobacterium. Therefore, a function of AGB1 as positive
WRKY33 contributes to the defence-related transcriptional regulator integrating flagellin and EF-Tu perception into ROS
re-programming (Petersen et al, 2008). production, and specifically in EF-Tu signalling to limit
The flagellin proteins of Agrobacterium and of symbiotic Agrobacterium transformation, has been proposed.
bacteria (rhizobia) are distinct from those of most other
microbes in that they are not recognised and do not trigger
Gene expression re-programming
a defence response, implying that other PAMP-receptor pairs
are responsible for recognition of these organisms. Indeed, a in response to Agrobacterium
prominent Agrobacterium PAMP, the elongation factor EF-Tu, Agrobacterium attack leads to a major re-programming of
has been identified. Although highly conserved in all prokar- gene expression in plants. Already in the pre-microarray-era,
yotes, Agrobacterium EF-Tu is fully active as an elicitor large-scale expression analyses (cDNA-AFLP) have revealed
(Kunze et al, 2004). Interestingly, despite their chemical that many Agrobacterium-induced genes are related to plant
dissimilarity, flg22 and EF-Tu share several characteristics. defence and to general stress responses (Ditt et al, 2001).
Both PAMPs inhibit seedling growth and activate a common Using different Agrobacterium strains, Veena et al (2003)
set of signalling events and defence responses, while acting could show that the transfer of T-DNA and Vir proteins can
with no apparent synergy (Zipfel et al, 2006). These modulate the expression of plant genes in tobacco cell
responses include MAPK activation, alkanisation of the med- culture. The authors concluded that T-DNA and Vir protein
ium, and an oxidative burst. Moreover, a microarray analysis transfer acts as suppressors of the defence response. Later, it
of the response of Arabidopsis to flg22 and the EF-Tu-derived was found that an attachment-deficient Agrobacterium mu-
peptide elf18 revealed a clear correlation of differential gene tant hyper-induced defence-related genes in Ageratum con-
expression, whereas no apparent flg22 or elf18-specific yzoides cell culture. Interestingly, also non-pathogenic
subsets of genes were identified. A surprisingly high number Escherichia coli triggered such hyper-induction (Ditt et al,
of RLK-encoding genes (100 of 610) were found to be rapidly 2006), and it was concluded that Agrobacterium can dampen
induced by these PAMPs (Zipfel et al, 2006). plant defence in an attachment-dependent manner (Ditt et al,
A targeted reverse genetics approach has led to the identi- 2006). The authors also observed a strong variability in
fication of a receptor kinase essential for EF-Tu perception, transformation efficiency between individual experimental
EFR1. EFR1, similar to FLS2, is a member of the LRR-RLK series, and further analyses revealed that enhanced basal
protein family. The important function of LRR-RLKs in defence gene expression correlates with resistance to
plant-microbe sensing has already become evident for root Agrobacterium transformation. These findings correlate
nodule symbiosis. Lotus japonicus mutants affected in the with the observation that co-injection of plants with
LRR-RLK SYMRK (symbiosis receptor kinase) fail to engage Agrobacterium plus flg22 or elf18 elicitors abolishes transfor-
in symbiosis (Stracke et al, 2002). Likewise, Arabidopsis mation (Zipfel et al, 2006). One factor that may explain the
mutants lacking fls2 show no response to flg22 treatment reported negative correlation between transformation effi-
(Asai et al, 2002). The efr mutants are insensitive to elf18, ciency and stress status may be salicylic acid (SA). This
while showing a normal flg22 response (Zipfel et al, 2006) plant hormone is an important signal in regulating the plant
and an otherwise normal phenotype. The striking feature of response to pathogens. It accumulates in local and systemic
efr mutants is their high susceptibility to Agrobacterium tissues of stress-exposed plants and induces expression of
infection, as shown by the enhanced expression of a pathogenesis-related genes. A study by Yuan et al (2007) now
T-DNA-harboured reporter gene (GUS) on transient transfor- implicates SA in the repression of the Vir regulon, the
mation of seedlings. On pre-treatment with flg22, an increase attenuation of the function of the VirA kinase as well as in
of receptor-binding sites for EF-Tu was observed. Co-injection the degradation of an Agrobacterium quormone. Accordingly,

8 The EMBO Journal VOL 00 | NO 00 | 2010 & 2010 European Molecular Biology Organization
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

plant mutants overproducing SA were recalcitrant to tumour above) and the host protein VIP1. Agrobacterium thus does
formation. These finding are further supported by Anand et al not only abuse VIP1 as such for delivering the VirE2/T-DNA
(2008). In summary, from the bacterial point of view, for into the host cell nucleus, but also actively manipulates the
maximal transformation efficiency, a minimally stressed state subcellular localisation of this plant protein. As this was not
of host cells seems desirable. Reciprocally, plants may evade enough, once nuclear transfer of the T-DNA complex has
Agrobacterium infection if their defence system is in an been accomplished, T-DNA integration into the plant genome
alerted state. is likely to be achieved through abuse of yet another manip-
A kinetic study on the response of tobacco BY2 cell ulation of the host cell machinery. Tzfira et al (2004a, b)
cultures to various Agrobacterium strains, including T-DNA suggest that nuclear VirF protein may mediate the degrada-
and Vir protein transfer-incompetent strains, helped to dis- tion of the T-DNA complex through the plant proteasome,
sect the stress versus transformation efficiency ambivalence resulting in the release of the T-DNA, which subsequently can
and allowed to distinguish general transcriptional responses integrate into the host genome, the final step of stable
resulting from attachment or proximity of Agrobacterium transformation.
near BY2 cells from transformation-specific responses in- As not only Agrobacterium, but also flg22, triggers MPK3
duced by the transfer of T-DNA and/or Vir proteins into activation and rapid nuclear accumulation of VIP1 (Djamei
plant cells (Veena et al, 2003). Agrobacterium triggers an et al, 2007), the question arises which function the stress-
early (3–12 h) induction of stress genes, whose expression is dependent cytoplasmic-nuclear translocation of VIP1 has in
subsequently repressed by transfer-competent strains. the plant response to other stresses. We have recently shown
Concomitant with the decline of this general defence, that VIP1 is a functional bZIP transcription factor, which
T-DNA and Vir protein transfer occurs. In contrast, Vir binds to a novel DNA regulatory motif, VIP1 response
transfer-deficient strains trigger a second wave (after 24 h) element (VRE) (Pitzschke et al, 2009c). VREs are overrepre-
of defence gene expression and fail to transfer T-DNA. sented in the promoter regions of stress-responsive genes.
Through their cDNA macroarray, Veena Jiang et al (2003) VIP1 binds in vivo to the promoter of a stress-responsive
also revealed that several histone genes were more strongly transcription factor, MYB44, under conditions that activate
induced by transfer-competent strains than transfer-deficient the MPK3 pathway. The mpk3 mutants are impaired in stress-
strains. Together with the observed constitutive repression of triggered induction of VIP1 target genes, whereas VIP1 over-
numerous histone genes in the vip2 mutant, which are expressing plants show constitutively elevated transcript
affected in stable, but not in transient transformation levels. From these observations, a function of VIP1 as a
(Anand et al, 2007), these findings emphasise the importance mediator of MPK3-mediated stress gene modulation was
of histones in Agrobacterium transformation and indicate that derived. How does the function of VIP1 as activator of the
an elevated pool of histones is required for facilitating T-DNA defence responses correlate with the finding that VIP1-over-
integration into the host genome. expression results in enhanced transformation efficiency
The mechanisms of transcriptional re-programming of (Tzfira et al, 2002)? A plausible explanation is that the higher
Agrobacterium-infected tissue and the extent to which bacter- transformation rate is achieved through a more efficient
ial factors contribute to this re-programming are still mostly nuclear transfer of the T-DNA complex, as more VirE2-T-
unknown. On the basis of the findings that VirE3 has trans- DNA molecules can be ‘piggy-packed’ by VIP1. The degrada-
activating activity in yeast, localises to plant nuclei, and that tion of the T-DNA complex and VIP1 through the action of
VirE3 can bind pBrp, a general plant-specific transcription VirF and the plant proteasome serves both the release of the
factor, a function of VirE3 as potential plant transcriptional T-DNA and the prevention of VIP1-induced defence gene
activator mediating the expression of tumour development- expression. According to this model, not VirF, VirD2, or the
specific genes was proposed (Garcia-Rodriguez et al, 2006). fidelity of the plant proteasomal machinery, but VIP1 phos-
phorylation/localisation and the presumably directly corre-
lated nuclear import of VirE2/T-DNA might be the limiting
Defence versus transformation—how
factors in transformation.
to evade the innate immune response Owing to its dual function as a stress-responsive transcrip-
From the above sections, it becomes apparent that a major tion factor on the one hand and as T-DNA/VirE2 shuttle on
barrier for Agrobacterium infection is the defence response the other hand, VIP1 may be one of the factors that ‘tips the
triggered in the host cell. Once plant defence can be blocked, scales’, that is it decides between successful transformation
reduced, or circumvented, much higher transformation effi- and failure of transformation because of elevated basal stress
ciencies can be achieved. Even more, in at least one aspect, levels. As MPK3 is very sensitive to numerous stresses, its
Agrobacterium have learnt to turn the tables in that they can target protein, VIP1, can potentially be phosphorylated,
even benefit from being recognised as a pathogen. Similar to translocate to the nucleus and induce stress gene expression
numerous other microbes, Agrobacterium triggers the activa- equally easily. The battle between Agrobacterium and plant,
tion of MAPKs, primarily MPK3, MPK4, and MPK6 (Djamei therefore, is to ‘compete’ for one of the two VIP1 functions.
et al, 2007). Y2H experiments, in vivo interaction, and kinase
assays have shown that VIP1, the VirE2-interacting protein 1,
interacts with and becomes specifically phosphorylated by
Conclusions/summary
MPK3 (Djamei et al, 2007). Stress-triggered phosphorylation The transformation of plants by Agrobacterium is a complex
of VIP1 results in the rapid translocation of this protein from process that involves multiple steps and the concerted action
the cytoplasm to the nucleus. In contrast to VirD2, which of both microbial and host factors. As evidenced by a still
passes into the host nucleus on its own, VirE2 needs the increasing number of reports on novel plant–Agrobacterium
assistance of both importin a (Bhattacharjee et al, 2008, see protein interactions, much is still to be learnt to understand

& 2010 European Molecular Biology Organization The EMBO Journal VOL 00 | NO 00 | 2010 9
 Agrobacterium-induced tumour formation in plants by plant transformation
A Pitzschke and H Hirt

the entire transformation process in detail. The main recent and to speed up the progress in generating stress-resistant
progress includes the characterisation of Vir-interacting pro- plants.
teins in vivo, the function of histones in stable integration and
the elucidation of surprisingly smart strategies used by Acknowledgements
Agrobacterium to circumvent or even abuse the plant defence
The work was supported by grants of the University of Vienna and
system. Clearly, the in-depth study and molecular analysis of the Austrian Science Foundation.
the plant–Agrobacterium interaction will not only add to our
understanding of pathogen strategies for host infection, but Conflict of interest
harbour also the potential to render plants transformable that
are otherwise recalcitrant to Agrobacterium transformation The authors declare that they have no conflict of interest.

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