Dispatch
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Plant transformation: A pilus in Agrobacterium T-DNA transfer
Christian Baron and Patricia C. Zambryski
Agrobacterium tumefaciens transfers a protein–DNA
complex to plant cells in a process similar to bacterial
conjugation; the mechanism of transfer is beginning to
be unravelled by biochemical, genetic and electron
microscopic studies.
Figure 1
Address: Department of Plant and Microbial Biology, University of
California at Berkeley, 111 Koshland Hall, Berkeley, California 94720,
USA.
Current Biology 1996, Vol 6 No 12:1567–1569
© Current Biology Ltd ISSN 0960-9822
Analysis of conjugative DNA transfer of the F episome
between Escherichia coli cells was a major focus of bacterial
genetics in the 1940s and 1950s, and contributed to our
understanding of circular bacterial chromosomes and
genetic recombination. Electron microscopy revealed an F
episome-dependent sex pilus in donor (male) strains only,
leading researchers to propose the pilus might be a
channel for injection of DNA into recipient (female)
strains. DNA transfer through the pilus was never demonstrated, however, and the currently held view of pilus
function is that it mediates contact formation between bacteria; close juxtaposition of the two connected cells is
brought about by pilus retraction. The DNA-transfer
channel is then formed from other components of the
transfer apparatus. The mechanics of transfer have still not
been elucidated, but the importance of pilus-mediated
conjugative exchange remains clear: for example, transfer
of plasmids carrying drug-resistance determinants between
pathogenic bacteria causes major problems in health care.
Furthermore, adhesive pili have proved to be virulence
factors of various human and animal pathogenic bacteria.
Until recently, pili had not been ascribed a role in plant
pathogen systems. A new study, however, suggests that
tumor induction on plants after infection with Agrobacterium tumefaciens may depend on a pilus structure [1].
Pathogenicity of A. tumefaciens depends on the transfer
into plant cells of a segment of DNA, known as the T
DNA, from the bacterial tumor-inducing (Ti) plasmid, and
the subsequent integration of the T-DNA into the host
genome. T-DNA-encoded enzymes produce phytohormones and the consequent hormone imbalance causes
tumor formation (Fig. 1). As many steps in this process of
genetic transformation have been worked out in detail,
they are only briefly reviewed here; we shall then focus on
recent insights into the DNA-transfer process.
Agrobacterium infects plants at wound sites and specific phenolic compounds and sugars in the acidic plant exudates
Transfer of oncogenic T-DNA from A. tumefaciens causes tumor
formation on plants. The photograph shows tumor development on an
infected Arabidopsis thaliana plant three weeks after wounding and
infection by A. tumefaciens.
induce the bacterial virulence (vir) system. Vir proteins are
responsible for displacing an up-to 20 kilobase long piece of
single-stranded DNA — the T-strand — from the T-DNA
region. One of the enzymes involved in T-strand production, VirD2, remains covalently bound at its 5′ end, and the
entire T-strand is coated with a non-sequence-specific
single-strand-binding protein, VirE2. Transfer of this
protein–DNA complex — the T-complex — through two
bacterial membranes into the plant cytoplasm requires
eleven VirB proteins and VirD4, and this transfer process is
currently the focus of intense study. Once it reaches the
plant cytoplasm, nuclear localization sequences in VirD2
and VirE2 target the T-complex into the nucleus, where
the DNA integrates into the plant genome.
Even before molecular details of Vir protein action were
known, it was suggested that Agrobacterium-mediated
genetic transformation of plant cells may resemble bacterial
conjugation [2]. A clearer picture emerged when sequence
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Current Biology 1996, Vol 6 No 12
analysis of the virB operon, which is required for the transfer step, predicted that most of the encoded proteins are
either membrane-associated or exported into the bacterial
periplasm, and several groups have since confirmed these
predicted locations [3]. This led to the hypothesis that the
VirB proteins might form a channel for transfer of Tcomplex from the bacterium into the plant cytoplasm.
Strong support for this idea came from sequence analysis of
gene products required for the conjugative transfer of plasmids from different incompatibility groups (IncP, IncN and
IncW) and those required for the secretion of pertussis
toxin, which show strong homologies to VirB proteins [4,5].
Thus, transfer of protein–DNA complexes between organisms and secretion of certain protein toxins may use a
similar mechanism.
Suggestive evidence has now been reported in support of
the suggested similarity between plant transformation
and bacterial conjugation. Fullner et al. [1] first compared
vir-induced and non-induced Agrobacterium cells by transmission electron microscopy, and found thin brittle pili
specifically on the induced cells. Next, they surveyed a
set of vir mutants to identify the Vir proteins involved in
the synthesis of these structures. They found that VirA,
VirG, VirD4 and VirB1 through VirB11 are absolutely
required for pilus formation. VirA and VirG constitute the
two-component regulatory system required for induction
of the vir regulon, and, as the other twelve proteins are
those required for T-complex transfer to plant cells, it
can be assumed that the pilus structure plays a role in
this process. By analogy to bacterial conjugation, the Vir
pilus may bind plant cells and mediate an intimate
contact between donor and recipient that is followed by
T-complex transfer.
An immediate question raised by this discovery is what is
the composition of the pilus? On the basis of the similarity
in sequence between VirB2 and the TraA propilin of the F
episome, and the similar amino-terminal processing reactions of both proteins, it was suggested that VirB2 may be
a component of the Agrobacterium pilus [6]. Another candidate pilus component is VirB5: TraC, the VirB5 homolog
in the IncN plasmid conjugation system, can complement
a traC deletion strain when supplied externally, which is
consistent with its being a pilus component [7]. Our own
results bring a third candidate into play [8]. VirB1 undergoes proteolytic processing, and large amounts of the
carboxy-terminal processing product, VirB1*, are recovered from the supernatant of Agrobacterium cells induced
in liquid culture. This finding is in accord with a pilus
component, because the brittle structure of pili is susceptible to breaking by mechanical shearing forces, and pilus
fragments would then accumulate in liquid-culture media.
With three candidate proteins in the line-up, it will only
be a matter of time until biochemical isolation or immunoelectron microscopy reveals the pilus constituents.
Eleven VirB proteins and VirD4 assemble the pilus, some
of them forming its structural components. Antisera
against all twelve proteins have been generated, and deletion mutants for each gene are available to make this
macromolecular transfer process amenable to a thorough
analysis. However, we are only beginning to understand
how the putative transmembrane structure transfers the
T-complex into plant cells, and which roles individual proteins play. The essential VirD4, VirB4 and VirB11 proteins contain nucleotide-binding motifs and may therefore
supply energy for the transfer process. Recent results have
assigned a possible enzymatic function to the amino terminus of VirB1, the sequence of which shows significant
similarity to the active site of transglycosylases and
lysozymes [9]. Transglycosylases are involved in synthesis
and rearrangement of the bacterial cell wall, and this led to
the hypothesis that VirB1 may aid in assembly of the
transmembrane complex by local lysis of the murein cell
wall. Support for this hypothesis came from mutagenesis
of the putative active site residues of VirB1 [9], which partially abolished its function, but the enzymatic activity has
not yet been directly demonstrated.
Whereas roles for individual VirB proteins have yet to be
defined, three groups [10–12] have described an interaction between VirB7 and VirB9 that is required for stabilization of the putative transmembrane complex.
Amino-terminal fatty-acid modification tethers VirB7 to
the outer membrane, and VirB7 binds VirB9 via a covalent
disulfide bridge. Deletion of the virB7 gene leads to loss of
VirB9 and a concomitant decrease in the steady-state levels
of several other VirB proteins. Such stabilizing effects are
indicative of protein–protein interactions between components of macromolecular structures, suggesting that VirB9
and VirB7 may form an assembly center for the T-complex
transfer machinery (Fig. 2).
Future research will address the question of whether the
pilus mediates contact formation with the plant cell, and if
so whether this contact transmits a signal triggering
channel formation and T-complex transfer. The Ti
plasmid virulence system can transfer genetic information
to a wide variety of different plant species, other bacteria
and even yeast. It will be exiting to investigate how
Agrobacterium can promiscuously interact with such a wide
variety of hosts, and whether pilus-mediated contact
formation always plays a role.
The problem of how a macromolecular protein–DNA
complex is transferred through two bacterial membranes
and the plant cytoplasmic membrane is equally challenging.
Which part of the T-complex — VirD2, VirE2 or DNA — is
recognized by which component of the transfer machinery?
Do the bacteria poke a hole through the plant membrane,
and if so which components of the VirB complex fulfill this
function? Finally, does the plant cell have a receptor for
Dispatch
Figure 2
Pilus
(VirB1*, VirB2, VirB5?)
VirB1*
Outer
membrane
VirB7 S
S VirB7
S
VirB9
S
VirB9
Murein
cell wall
?
VirB1
Outer
Inner
membrane
membrane
Cytoplasm
VirD4
Vir
VirB4 D2
VirB11
NTP
NTP
NTP
T-complex
VirE2
© 1996 Current Biology
A model of the transmembrane VirB complex, which may form a
channel for T-complex transfer from A. tumefaciens to plant cells. The
following functions have been assigned to individual VirB proteins:
the amino terminus of VirB1 may locally lyse the murein cell wall to
facilitate assembly of the transmembrane channel; the VirB1 processing
product VirB1* (orange circle) is partially localized extracellularly and
may be a pilus component; VirB7 forms a disulfide-linked heterodimer
with VirB9 and confers outer membrane attachment; the VirB7–VirB9
heterodimer stabilizes the transmembrane VirB channel; NTPase
activities of VirD4, VirB11 and VirB4 may supply energy for complex
assembly and/or T-complex transfer. VirB1*, VirB2 and VirB5 may be
structural components of the pilus.
VirB-channel docking or T-complex transfer? The Agrobacterium–plant-cell interaction has become a model system for
studying bacterial conjugation and macromolecular secretion in general, and discoveries to be made in future are
likely to shed important light on related processes in different organisms.
Acknowledgements
Research on the Agrobacterium–plant cell interaction in our laboratory is
supported by NSF grant IBN-9507782.
References
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