Download Polar location and functional domains of the Agrobacterium

Molecular Microbiology (2002) 43(6), 1523–1532
Polar location and functional domains of the
Agrobacterium tumefaciens DNA transfer protein VirD4
Renu B. Kumar1 and Anath Das1,2*
1
Department of Biochemistry, Molecular Biology and
Biophysics, and 2Plant Molecular Genetics Institute,
University of Minnesota, 1479 Gortner Avenue, St Paul,
MN 55108, USA.
Summary
Agrobacterium tumefaciens VirD4 is essential for
DNA transfer to plants. VirD4 presumably functions
as a coupling factor that facilitates communication
between a substrate and the transport pore. To serve
as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study,
we observed that several constituents of the transport apparatus localize to the cell membranes. In
this study, we demonstrate that VirD4 has a unique
cellular location. In immunofluorescence microscopy,
cells probed with anti-VirD4 antibodies had foci of
fluorescence primarily at the cell poles, indicating
that VirD4 localizes to the cell pole. Polar location of
VirD4 was not dependent on T-DNA processing, the
formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large
cytoplasmic region contains a nucleotide-binding
domain. To investigate the role of these domains in
DNA transfer, we introduced mutations in virD4 and
studied the effect of a mutation on substrate transfer.
A deletion of most of the periplasmic domain as well
as the alterations of glycine 151 to serine and lysine
152 to alanine led to the complete loss of DNA
transfer, indicating that both domains are essential
for substrate transfer. Subcellular localization of the
mutant proteins indicated that both the periplasmic
and the nucleotide-binding domains are required for
polar localization of VirD4. The periplasmic domain
mutant VirD4D36–61 was distributed throughout the cell
membrane, whereas the nucleotide binding site
mutant VirD4G151S localized to sites other than the
cell poles. Polar location of VirD4 suggests a role for
the cell pole in DNA transfer.
Accepted 5 December, 2001. *For correspondence. E-mail
[email protected]; Tel. (+1) 612 624 3239; Fax (+1) 612 625 5780.
© 2002 Blackwell Science Ltd
Introduction
The transfer of macromolecules across membranes
and kingdoms is an essential biological process. Type IV
transport is used by bacteria to deliver macromolecules
to prokaryotic and eukaryotic cells (Covacci et al., 1999;
Christie, 2001). Agrobacterium tumefaciens uses type IV
transport to transfer DNA to plant cells. Escherichia coli
transfers plasmids by conjugation by the type IV mechanism. Human and animal pathogens, e.g. Helicobacter
pylori, Bordetella pertussis, Brucella suis and Rickettsia
prowazekii, use a similar method to deliver pathogenesisrelated effector proteins and other molecules. DNA transfer from Agrobacterium to plants causes the crown gall
tumour disease (Zupan et al., 2000). The transferred
(T-)DNA and the genes essential for its transfer, the
virulence (vir) genes, are encoded within the large Ti(tumour-inducing) plasmid. DNA transfer requires the
virA, virB, virD, virE and virG genes (Stachel and Nester,
1986). The Vir proteins are primarily responsible for the
processing of the DNA, its transfer to the plant cell and
its integration into the plant nuclear genome. The T-DNA
exits the bacterium through a transport apparatus composed primarily of the VirB proteins. VirB2 and VirB5 form
a T-pilus that presumably promotes host–recipient interaction (Fullner et al., 1996; Lai and Kado, 1998; SchmidtEisenlohr et al., 1999). The T-pilus is found primarily at
one cell pole (Lai et al., 2000). Five proteins, VirB6–
VirB10, are postulated to be the core constituents of the
transport apparatus (Das and Xie, 2000). Most of these
proteins interact with one another and form various
protein complexes (Anderson et al., 1996; Spudich et al.,
1996; Baron et al., 1997; Beaupre et al., 1997; Das et al.,
1997; Das and Xie, 2000). Subcellular localization studies
have indicated that three VirB proteins localize to a few
sites on the bacterial membranes (Kumar et al., 2000).
These sites may represent the transport apparatus.
The VirD proteins catalyse processing of the T-DNA and
its transfer. VirDl and VirD2 introduce a nick at the T-DNA
border sequences producing the single-stranded T-strand
DNA composed of the bottom strand of the T-DNA (Zupan
et al., 2000). VirD3 is not required for tumour formation
(Vogel and Das, 1992). VirD4, an essential DNA transfer
protein, is postulated to mediate communication between
a substrate and the transport pore (Cabezon et al., 1997).
VirD4 is an inner membrane protein with two membranespanning domains near the N-terminus, and both the N-
1524 R. B. Kumar and A. Das
VirD4 by immunofluorescence (IF) and immunoelectron
(IE) microscopy. In IFM, wild-type A. tumefaciens labelled
with anti-VirD4 antibodies had a punctated appearance
(Fig. 1). The bacteria had a single or few foci of high fluorescence on the membranes, indicating that VirD4 localized to very few sites (Fig. 1C). In bacteria with a single
or two foci of fluorescence, the protein invariably localized
to the cell poles. Quantitative analysis indicated that about
95% of the foci localized to the cell poles (Table 3). About
50% of the cells exhibited multiple foci of fluorescence. In
these bacteria, the protein localized to other areas of the
membranes including the poles. Strains with mutations in
the vir genes were analysed to study the requirement of
T-DNA processing and transport apparatus assembly
in the polar localization of VirD4. Neither T-strand DNA
synthesis nor the formation of a transport apparatus is
required for targeting VirD4 to the cell poles, because a
non-polar deletion in virD2 and a deletion in virB had no
effect on VirD4 localization (Fig. 1E and F). In bacteria
that expressed only virD4, the protein localized to the cell
pole, indicating that no other Vir protein is essential for its
proper subcellular localization (Fig. 1G).
In IEM, gold particles representing VirD4 localized to
the membranes and were found in clusters (Fig. 1 and
Table 1). Clustering of gold particles suggests complex
formation. Proteins of the bacterial chemoreceptor complex and the T-DNA transport apparatus exhibit a similar
pattern in IEM (Maddock and Shapiro, 1993; Kumar et al.,
2000). About 85% of gold particles localized to the membranes, and ª 70% of those were in the clusters, suggesting that VirD4 forms a protein complex (Table 1).
High fluorescence at the VirD4 localization sites in IFM
(Fig. 1) is consistent with this hypothesis.
and C-terminus of the protein are cytoplasmic (Okamoto
et al., 1991; Das and Xie, 1998). Amino acid residues
13–30 and 68–86 comprise the membrane-spanning
domains. Near the N-terminus of the large cytoplasmic
domain, there is a Gly-x-Gly-x-Gly-xn-Lys (x = any
residue) sequence at residues 151–174 that bears weak
resemblance to the Walker A box sequence, a sequence
motif found conserved in nucleotide-binding proteins
(Balzer et al., 1994). The role of this sequence in T-DNA
transfer is not known.
The VirD4 and VirB proteins are essential for DNA
transfer (Beijersbergen et al., 1992). These proteins are
found conserved in the type IV transport family (Lessl
et al., 1992; Christie, 2001). Like VirD4, its homologues
in the E. coli plasmids F, RP4 and R388 are essential for
conjugal transfer of plasmid DNA (Balzer et al., 1994).
These proteins can functionally substitute for one another
in the transfer of only a subset of conjugal plasmids
(Cabezon et al., 1994; Hamilton et al., 2000). Two regions
of the VirD4 homologues that correspond to motifs A and
B of the Walker box have the highest sequence conservation. These motifs of the RP4 homologue TraG are
essential for the conjugal transfer of the plasmid (Balzer
et al., 1994). The C-terminal one-quarter of the proteins
exhibits low sequence similarity. Recent analysis of the
structure of the cytoplasmic domain of R388 TrwB, a
VirD4 homologue, indicated that it is a hexamer with a
central 20 A0 channel that can function as a pore at the
inner membrane (Gomis-Ruth et al., 2001). The role as a
coupling protein and/or a pore-forming function of the
VirD4 family of proteins will require them to localize near
the transport apparatus. In the present study, we determined the subcellular location of VirD4 and identified its
essential domains. We demonstrate that VirD4 localizes
to the cell pole and that two domains are essential for
targeting it to the proper site on the cell membrane.
Functional domains of VirD4
VirD4 is a bitopic membrane protein with a short periplasmic domain near its N-terminus (Das and Xie, 1998). The
cytoplasmic region of VirD4 contains a Gly-x-Gly-x-Gly
nucleotide-binding motif. To determine the role of the
nucleotide-binding motif in DNA transfer, we introduced
mutations in virD4 that led to a change in the conserved
amino acids. Three residues, glycine at position 151,
lysine 152 and lysine 174, were mutated to serine, alanine
and threonine respectively. These changes are conservative and are expected to have no major effect on protein
structure (Bordo and Argos, 1991). Each mutant was
Results
VirD4 localizes to the cell pole
Transport through the T-DNA transport apparatus will
require co-operation between VirD4 and the apparatus.
The process will be facilitated by proximal cellular location of the two components. Our previous studies demonstrated that the VirB proteins forms a protein complex
at the bacterial membranes (Kumar et al., 2000). In the
present study, we determined the subcellular location of
Strain
vir gene
expressed
Total no. of gold
particles (no. of cells)
No. of particles
on membranes (%)
No. of particles
in clusters (%)
AD709
A348
virD4
virD4, virB1–11
234 (40)
302 (62)
197 (84)
277 (91)
136 (69)
206 (74)
Table 1. Subcellular localization of VirD4 by
immunoelectron microscopy.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
Polar location of Agrobacterium VirD4 1525
Fig. 1. Subcellular localization of VirD4. Subcellular location of VirD4 was determined by immunofluorescence (A–G) and immunoelectron
(H and I) microscopy as described previously (Kumar et al., 2000). An arrowhead indicates the location of VirD4.
A–C and H. Agrobacterium A348.
D. A348DD. E. WR1715. F. A348DB.
G and I. AD709.
AD709 expresses only the virD4 gene. A348DD, A348DB and WR1715 have a deletion in virD, virB and a non-polar deletion in virD2
respectively.
A. Phase-contrast microscopy.
B. Fluorescence microscopy.
C–G. Composites of phase and fluorescence images.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
1526 R. B. Kumar and A. Das
spacer. A large deletion of the vir gene regulator VirA did
not affect its ability to sense plant phenolics (Melchers
et al., 1989). By site-specific mutagenesis, we introduced
a deletion of 26 residues in the periplasmic domain,
amino acids 36–61, and studied the effect of the deletion on VirD4 function by complementation assays. The
virD4D36–61 mutant failed to complement the null mutation
in virD4 (Fig. 2A), indicating that the periplasmic domain
is essential for VirD4 function. Another region of interest
is the C-terminal end of VirD4. Sequence comparison with
the homologues indicated that this is the most divergent
region. We speculated that a low homology suggests that
this region probably confers a specificity function. This
property will make the C-terminal end indispensable. To
test the requirement of the extreme C-terminus in VirD4
function, we introduced a stop codon at residue 553. The
mutant will synthesize a truncated protein that lacks the
C-terminal 104 amino acids. In complementation experiments, the mutant virD4L553X failed to complement the
virD4 null mutant, indicating that the truncated protein is
non-functional.
Fig. 2. A. Phenotype of the virD4 mutants. The effect of a mutation
in virD4 on T-DNA transfer was monitored by complementation
assays. Kalanchöe diagremontiana leaves were infected with the
indicated strains, and tumour formation was assessed about
4 weeks after infection. Strains used: A136, –Ti plasmid; A348,
+pTiA6; wt, G151S, K152A, K174T, L553X and D36-61 –At12506
harbouring a plasmid that expresses wild-type virD4 or its mutant.
B. Effect of a mutation on the stability of VirD4. Total proteins of A.
tumefaciens strains expressing the virD4 alleles were separated by
SDS–PAGE, transferred to a nitrocellulose membrane and probed
with anti-VirD4 antibodies. Lane 1, uninduced A. tumefaciens A348;
lanes 2–7, induced A. tumefaciens At12506 expressing wild-type
virD4 or its mutants.
introduced into the virD4 null mutant A. tumefaciens
At12506 (Fullner et al., 1994), and its ability to complement the virD4 mutation was tested by tumour formation
assays. Wild-type virD4, as expected, complemented
the mutation (Fig. 2A). None of the mutants, virD4G151S,
virD4K152A and virD4Kl74T, complemented the virD4 null
mutant, indicating that glycine 151, lysine 152 and lysine
174 are essential for VirD4 function. As these residues
are likely to be involved in nucleotide binding, nucleotide
binding (and/or hydrolysis) by VirD4 is probably required
for DNA transfer.
The short (residues ª 31–67) periplasmic domain of
VirD4 can have a structural role, i.e. it serves as a spacer
between the two membrane-spanning domains or is required for an additional function. To study its role in DNA
transfer, we introduced a deletion in the periplasmic
domain and studied the effect of the mutation on VirD4
function. The length of the region is expected to be
amenable to alteration if its sole function is to serve as a
Alteration of lysine 174 and the loss of the C-terminal
end affect protein stability
The loss of function of the virD4 mutants described above
could result from the effect of a mutation on protein function or protein stability. To study whether a mutation
affects protein stability, Western blot assays were used
(Anderson et al., 1996). The purified anti-VirD4 antibodies recognized a single protein band in the induced wildtype A. tumefaciens A348 (Fig. 2B, lane 2). Analysis of
the mutants by Western blot assays indicated that three
mutations, G151S, D36-61 and K152T, do not affect
protein stability because strains expressing the mutants
accumulated the mutant protein at a level comparable
with that of the wild-type protein (Fig. 2B, lanes 3, 6
and 7). The other two mutant proteins, VirD4K174T and
VirD4L553X, were unstable, indicating that the mutations
destabilized the protein (Fig. 2B, lanes 4 and 5). The labile
nature of VirD4K174T is somewhat surprising, as the equivalent lysine in one VirD4 homologue tolerated a similar
substitution (Balzer et al., 1994).
VirD4 mutants are defective in VirE2 transfer
The A. tumefaciens T-DNA transport apparatus can transport both DNA and protein (Beijersbergen et al., 1992;
Vergunst et al., 2000). One protein substrate translocated
through the transport apparatus is VirE2. We studied
whether the VirD4 domains essential for DNA transfer
are also essential for VirE2 transfer. The in planta complementation assay of Otten et al. (1984) was used to
monitor VirE2 transfer. None of the virD4 mutants com© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
Polar location of Agrobacterium VirD4 1527
Table 2. Effect of a mutation in virD4 on VirE2 transfer.
Strain VirE2 donor
(VirD4 phenotype)
T-DNA donor
Tumour formation
PC2760 (wt)
–
PC2760 (wt)
AD 1011 (G151S)
AD1012 (K152A)
AD1015 (D36–61)
AD1016 (wt)
–
MX358
MX358
MX358
MX358
MX358
MX358
–
–
+
–
–
–
+
VirE2 transfer was monitored by in planta complementation assays
(Otten et al., 1984). –, no tumour; +, tumour.
plemented the virE2 mutant, indicating that all mutations
in virD4 abolished the ability to transfer VirE2 to plants
(Table 2). In dominance assays, all mutants had a recessive phenotype.
The C-terminal end encodes a VirD4–VirD4
interaction domain
Subcellular localization studies suggested that VirD4
probably forms an oligomeric complex. The formation
of the complex will require VirD4 to be involved in homotypic interactions. Therefore, VirD4 should contain selfinteraction domains. We tested this hypothesis using the
two-hybrid assay in yeast (Das and Xie, 2000). Owing to
its large size (656 amino acids), VirD4 was divided into
three smaller overlapping proteins, and interaction
between each segment was monitored by the two-hybrid
assay. No VirD4–VirD4 interaction was observed with
fusions containing the N-terminal (N) and the central (M)
regions of the protein. A polypeptide that contained the Cterminal domain (CTD) was functional in the interaction,
indicating that the CTD of VirD4 encodes an interaction
domain (Fig. 3). A yeast cell expressing a CTD–activator
fusion and a CTD–LexA fusion activated expression of the
reporter lacZ gene, producing a blue colony colour phenotype on indicator plates containing Xgal. In control
Fig. 3. Identification of the VirD4 interaction domains. VirD4–VirD4
interaction was studied by the yeast two-hybrid assay (Das and
Xie, 2000). VirD4 was divided into three overlapping segments: the
N-terminal, middle and C–terminal segments. Interactions between
the N-terminal (N–N), middle (M–M) and C-terminal (C–C)
segments were studied. Cells expressing the vector plasmids (V),
the CTD–LexA fusion and the activator vector (C+Act) and the
CTD–activator fusion and the LexA vector (C+LexA) are shown
as controls.
experiments, yeast expressing a CTD fusion and the
reciprocal non-fusion protein (LexA/activator) exhibited a
white colony colour phenotype.
Two domains are required for targeting VirD4
to the cell pole
To study whether an avirulent mutation is affected in VirD4
localization, we determined the subcellular location of the
mutants by IFM (Fig. 4). Two mutants failed to target
VirD4 to the cell poles. The VirD4D36–61 protein failed to
form foci of fluorescence and was distributed randomly
across the cell membrane (Fig. 4A). The subcellular distribution of the mutant protein is similar to that of alkaline
phosphatase reported previously (Kumar et al., 2000). A
second mutant, virD4G151S, was inefficient in targeting
the protein to the cell pole (Fig. 4B). Cells expressing
VirD4G151S had foci of high fluorescence; however, these
foci were found at sites other than the cell poles at a reasonable frequency (arrowhead). About 68% of the foci
localized to sites other than the cell pole (Table 3). In con-
Fig. 4. Subcellular location of the VirD4 mutants. Subcellular location of VirD4D36–61 (A), VirD4G151S (B) and VirD4K152A (C) were
determined by immunofluorescence microscopy. An arrowhead identifies mislocalized VirD4.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
1528 R. B. Kumar and A. Das
virD4
expressed
No. of cells
analysed
No. of cells with 1–2
foci (total no. of foci)
No. of cells with
polar foci (no.a)
Percentage
of polar focib
Wild type
G151S
K152A
171
103
99
81 (122)
49 (68)
50 (69)
75 (116)
9 (22)
37 (56)
95
32
81
Table 3. Effect of mutations in virD4 on polar
location of the protein.
a. No. of polar foci includes count of foci in cells with exclusively polar foci and cells with both
polar and non-polar foci.
b. Wild type versus G151S and wild type versus K152A have P-values of 2.2 ¥ 10–16 and
4.6 ¥ 10–3 respectively. A value <0.05 indicates that a difference is statistically significant.
trast, in bacteria expressing wild-type VirD4, 95% of the
foci localized to the poles. As the G151S mutation maps
to the nucleotide-binding domain, the mutant is probably
defective in ATP utilization. These results suggest that
targeting of VirD4 to the cell pole may be an energyrequiring process. The third mutant virD4K152A, a mutation
in the nucleotide binding site motif, had a near wild-type
phenotype. The mutant protein was found primarily at the
cell pole, although targeting to other areas of the membrane was observed at a very low frequency (Fig. 4C,
arrowhead). The mutant probably has a low ATP utilization activity that is nearly sufficient for its targeting to the
cell poles but is insufficient for DNA transfer. An alternative hypothesis is that the G151S mutation affects another
property of the protein essential for its proper subcellular
localization.
Discussion
This study reports the subcellular location of the A.
tumefaciens VirD4 protein and the identification of two of
its essential functional domains. The periplasmic domain
and the nucleotide-binding domain are required for the
substrate transfer function of VirD4. Two other nucleotidebinding proteins, VirB4 and VirB11, are also essential
for T-DNA transfer (Christie, 2001). The requirement of
multiple nucleotide-binding proteins in T-DNA transfer
suggests that several steps in the DNA translocation
pathway are energy-dependent processes requiring the
participation of a specific protein(s). The present study
demonstrates that the short periplasmic domain of VirD4
is essential for VirD4 function. A deletion of most of the
domain led to the complete loss of VirD4 activity. The
deletion is unlikely to affect the membrane topology of
the protein, because it does not affect sequences immediately adjacent to the hydrophobic domains. A similar
analysis of the A. tumefaciens VirA protein indicated
that the periplasmic domain does not play a role in sensing vir gene inducers (Melchers et al., 1989).
Subcellular localization by IFM demonstrated that VirD4
localizes to the cell poles. These studies also suggest that
VirD4 forms a large oligomeric complex. The formation of
foci of fluorescence in IFM and the clustering of gold particles in IEM are suggestive of oligomerization. A similar
analysis of the mutants indicated that both the periplasmic and the nucleotide-binding domains are essential for
the proper subcellular localization of VirD4. The avirulent
phenotype of the mutants suggests a requirement for
polar location of VirD4 in DNA transfer. These studies indicated that the primary sequence of VirD4 encodes information necessary for its proper subcellular localization.
Other proteins, e.g. Pseudomonas aeruginosa histidine
kinase PilS, are known to encode information on polar
localization within the primary sequence (Boyd, 2000).
The phenotype of the two mutants suggests a mechanism for the polar localization of VirD4. Random distribution of VirD4D36–61 throughout the cell membranes
indicates that the periplasmic domain is essential for the
targeting of VirD4 to the cell poles. The lack of foci of fluorescence in the mutant suggests that the protein is probably defective in oligomerization as well. Defects in both
oligomerization and polar localization suggest a two-step
mechanism for the targeting of VirD4 to a cell pole. The
first step is oligomerization of VirD4, which is manifested
as foci of fluorescence in IFM. The second step is the targeting of an oligomer to the cell pole. VirD4D36–61 is defective in oligomer formation and therefore cannot localize
to the cell pole. As protein–protein interaction is required
for oligomerization, the periplasmic domain probably
encodes an interaction domain that supports the formation of higher oligomers of VirD4. The other interaction
domain we identified, the C-terminal interaction domain
(Fig. 3), is probably involved in forming a small oligomer,
e.g. a dimer. The phenotype of a second mutant,
VirD4G151S, is consistent with the proposed model for
VirD4 localization and provides new information on the
mechanism. VirD4G151S contains the periplasmic domain
and would be able to form the higher oligomer. In IFM,
this mutant exhibited foci of fluorescence, and not an
overall fluorescence. The mutant, however, failed to
localize exclusively to the cell pole. As the mutation is
expected to affect nucleotide binding, these results
suggest that the second step, the targeting of the oligomer
to the cell pole, may be an energy-dependent process.
Two bacterial proteins, Listeria monocytogenes IscA and
Shigella flexneri ActA, localize to a single pole and use
different mechanisms for their unipolar localization
(Lybarger and Maddock, 2001).
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
Polar location of Agrobacterium VirD4 1529
The polar location of VirD4 raises interesting questions
on the location of the T-DNA transport apparatus. The
VirD4 and VirB complexes act together in the transport of
substrates across the transport apparatus. To serve their
role in DNA transfer, VirD4 and the VirB protein complex
need to be adjacent to, if not overlapping, one another.
Therefore, a transport apparatus assembled at a cell pole
can mediate substrate transfer. Three constituents of the
transport apparatus, VirB8, VirB9 and VirB10, localized to
sites on the cell membrane including the poles (Kumar
et al., 2000). Although the VirB proteins were found at
several sites, VirD4 was more localized. One explanation
for the difference is that all complexes contain both VirD4
and the VirB proteins. A difference in the quality of the
antibodies (titre and/or specificity) affected the sensitivity
of the detection system. A second possibility is that there
are two types of complexes, a VirD4–VirB complex
that transports substrate from the cytoplasm and a VirB
complex that transports periplasmic and membrane
proteins. A relatively lower level of VirD4 in the cell will
enable only a subset of the VirB complexes to associate
with VirD4. The excess VirB complexes are used for the
transport of the pilin protein from the membrane to the site
of pilus assembly in a virD4-independent manner. Two
studies suggest that the VirB proteins without VirD4 can
form a functional transport apparatus (Craig-Mylius and
Weiss, 1999; Lai et al., 2000). The formation of the T-pilus
does not require VirD4 but is absolutely dependent on the
VirB proteins, and a VirD4 homologue is not necessary
for the export of pertussis toxin in B. pertussis. Both VirB2
and the pertussis toxin subunits are transported across
the cytoplasmic membrane in a Sec-dependent process.
Transport of these substrates can bypass the requirement
for VirD4. VirD4 may be the additional necessary component when vir-dependent transport of a cytoplasmic
substrate is required, e.g. DNA, VirD2, VirE2 and VirF.
We postulate that a polar VirD4–VirB complex functions
in substrate transfer from the cytoplasm. Co-localization
studies suggesting that both VirD4 and a transport apparatus protein VirB8 localize to the cell pole support the
existence of a polar VirD4–VirB complex (R. B. Kumar,
unpublished results). The polar location of the VirD4–VirB
complexes explains how bacteria can deliver a substrate
to the plant cell. Attachment of bacteria to plant cells is
essential for the transfer of T-DNA, and bacteria attaches
to the plant cell in a polar manner (Matthysse, 1987). The
assembly of the transport apparatus at the cell pole will
therefore be the most efficient mechanism for substrate
transfer. Proteins essential for critical biological processes, e.g. cell division, chromosome partitioning and
cell cycle control in bacteria, have been localized to the
cell poles (Lybarger and Maddock, 2001). Substrate
transfer by the type IV transport mechanism is another
process that probably uses the cell pole.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1523–1532
Experimental procedures
Strains and plasmids
Strains and plasmids used in this study are listed in Table 4.
Mutagenesis of VirD4
Mutations in virD4 were introduced by deoxyoligonucleotidedirected site-specific mutagenesis using single-stranded
pAD1373 DNA as a template (Das and Xie, 2000). To facilitate screening of the mutants, a new restriction site was
introduced at the site of mutation whenever possible. The
mutation and the mutagenic oligonucleotide used were as
follows (base change underlined): glycine 151 to serine,
dACGCGTGCTAGCAAAGGCG; lysine 152 to alanine,
dGCGTGCTGGCGCCGGCGTCGGCATC; lysine 174 to
threonine, dCCCTCGACGTTACGGGAGAATTGTT; and
deletion of residues 36–61, dTTTCGCCGGGGTACCGTC
TTCTGG. All mutations were confirmed by DNA sequence
analysis. Plasmids pAD1373 and the mutant derivatives were
linearized with the restriction endonuclease EcoICRI and
cloned into the filled-in HindIII site of plasmid pTJS75 to construct the wide-host-range derivatives. Plasmids were introduced into A. tumefaciens At12506 for complementation
assays (Das and Xie, 2000). Transfer of VirE2 was monitored
by in planta complementation assays (Otten et al., 1984).
Proteins were analysed by Western blot assays after
SDS–PAGE (Anderson et al., 1996).
Subcellular localization
Subcellular location of VirD4 and its mutants was determined
by immunofluorescence and immunoelectron microscopy
as described previously (Kumar et al., 2000). Wild-type
Agrobacterium A348 and its derivatives were grown in AB
minimal medium. Bacteria were induced by growth in
AB Mes, pH 5.8, medium in the presence of 100 mM
acetosyringone.
Antibodies
Antibodies against VirD4 were raised in rabbits using a
histidine-tagged VirD4 fusion protein as an antigen. The
antibodies were purified by affinity chromatography on immobilized fusion protein–affigel columns.
VirD4–VirD4 interaction
Interaction of VirD4 was monitored by the yeast two-hybrid
assay (Das and Xie, 2000). The large cytoplasmic region of
VirD4 was divided into three overlapping regions (leucine
87–glycine 321, proline 264–arginine 501 and proline
461–end) and used for the analysis of protein–protein interaction by the yeast two-hybrid assay. Genes encoding the
desired coding region were amplified by polymerase chain
reaction (PCR) and cloned into both pJK202 and pJG4-5. The
plasmids were introduced into yeast AD842 and tested for
interaction by monitoring reporter lacZ gene expression.
1530 R. B. Kumar and A. Das
Table 4. Strains and plasmids.
Strain or plasmid
Relevant characteristics
Source or reference
recA1 endA1 hsdR17(rk–mk+) supE44 thi-1 gyrA
relA1 l– f80d/lacZDM15(DlacZYA-argF)U169
Laboratory stock
A. tumefaciens
A136
A348
A348DB
A348DD
At12506
WR1715
MX358
PC2760
AD709
AD1011
AD1012
AD1013
AD1014
AD1015
AD1016
C58 heat cured of pTiC58
A136 containing pTiA6
A348 with a deletion in pTiA6 virB
A348 with a deletion in pTiA6 virD
A348 virD4:tn3hoho1
A348 with a non-polar deletion in pTiA6 virD2
A348 virE2:tn3hoho1
Ach5 with a deletion of pTiAch5 T-DNA
A136 with plasmid pAD1356
At12506 carrying pAD1678
At12506 carrying pAD1679
At12506 carrying pAD1680
At12506 carrying pAD1681
At12506 carrying pAD1682
At12506 carrying pAD1686
Laboratory stock
Laboratory stock
Anderson et al. (1996)
Vogel and Das (1992)
Fullner et al. (1994)
Shurvinton et al. (1992)
Stachel and Nester (1986)
Beijersbergen et al. (1992)
Das and Xie (1998)
This study
This study
This study
This study
This study
This study
Saccharomyces cerevisiae
EGY48
AD842
ura3 his3 trp1 LexAop-leu2
EGY48 containing pSH18-34
Finley and Brent (1995)
Das and Xie (2000)
A virDp–virD4 chimeric gene in a colE1 vector
Plasmid pAD1373 in wide-host-range vector pTJS75
Plasmid pAD1373G151S in pTJS75
Plasmid pAD1373K152 A in pTJS75
Plasmid pAD1373K174T in pTJS75
Plasmid pAD1373L553X in pTJS75
Plasmid pAD1373D36-61 in pTJS75
2.3 kb PstI–EcoRI fragment into the PstI site of
plasmid pRSET C, expresses a his-tagged fusion
protein containing residues 111–656 of VirD4
Cloning vector for the construction of LexA fusions
Cloning vector for the construction of activator fusions
A plasmid containing Gal1-LexAop-lacZ, a reporter
gene in yeast
0.7 kb EcoRI fragment encoding Leu-87/Gly-327
of VirD4 obtained by PCR amplification in pJG4-5
(activator–VirD4 N-terminal fusion)
0.65 kb EcoRI fragment encoding Pro-461/end of
VirD4 obtained by PCR amplification in pJG4-5
(activator–VirD4 C-terminal fusion)
0.72 kb XhoI fragment encoding Pro-264/Arg-501
of VirD4 obtained by PCR amplification in pJG4-5
(activator–VirD4 middle fusion)
0.7 kb EcoRI fragment of pAD1580
in pJK202 (LexA–VirD4N fusion)
0.72 kb XhoI fragment of pAD1592
in pJK202 (LexA–VirD4M fusion)
0.65 kb EcoRI fragment of pAD1582
in pJK202 (LexA–VirD4C fusion)
Das and Xie (1998)
This study
This study
This study
This study
This study
This study
This study
E. coli
DH5a
Plasmids
pAD1373
pAD1686
pAD1678
pAD1679
pAD1680
pAD1681
pAD1682
pTJ16
pJK202
pJG4-5
pSH18-34
pAD1580
pAD1582
pAD1592
pAD1693
pAD1694
pAD1695
Das and Xie (2000)
Finley and Brent (1995)
Finley and Brent (1995)
This study
This study
This study
This study
This study
This study
Acknowledgements
References
We thank T. Johnson for generating VirD4 antibodies, P. Judd
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analysis, M. Sanders and G. Ahlstrand for assistance with
microscopy, Drs E. Nester and W. Ream for Agrobacterium
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reading of an earlier version of the manuscript. This work was
supported by a grant from the National Science Foundation
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