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J Bacteriol, January 1998, p. 430-434, Vol. 180, No. 2
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of Agrobacterium virB Genes in
Transfer of T Complexes and RSF1010
Karla Jean
Fullner*
Department of Microbiology, University of
Washington, Seattle, Washington 98195-7242
Received 22 August 1997/Accepted 31 October 1997
 |
ABSTRACT |
Nonpolar virB mutants of Agrobacterium
tumefaciens were tested for RSF1010 mobilization and
extracellular complementation. virB2 to virB11
were essential for transfer in both assays. virB1 was
essential only for high frequency transfer of RSF1010 and VirE2.
Coordinated transfer of a preassembled T complex is supported by these
data and competition studies.
| |
TEXT |
During infection of dicotyledonous
plants, Agrobacterium tumefaciens transfers a segment of its
Ti plasmid, the transferred DNA (T-DNA), into the plant cell. The
transfer intermediate, which is termed the T strand, is composed of the
single-stranded T-DNA covalently attached to the protein VirD2
(23). At least 7 of the 11 genes of the virB
operon and virD4 are essential for transfer of T strands
(5, 15, 16, 27). These genes, along with virE1,
are also essential for transfer of the virE2-encoded
single-stranded binding protein into plant cells (5, 8, 15,
25). VirE2 is essential for tumorigenesis because it coats the T
strand, protecting it from degradation and assisting in its
translocation of the T-DNA to the plant cell nucleus (10, 21,
28). Two models have been proposed for the simultaneous transfer
of T strands and VirE2. The first model proposes that VirE2 binds to
the T strand within A. tumefaciens and that a preassembled T
complex is then transferred by a virB
operon-virD4-encoded complex to the plant cell
(29). The second model proposes that VirE2 and T strands are
transferred via separate virB-virD4-encoded channels and
that the T complex is assembled in the plant cell cytoplasm (5,
25).
In addition to transfer of T strands and VirE2 protein, the conjugative
plasmid RSF1010 can be transferred by A. tumefaciens either
to plant cells or to other agrobacteria (3, 6). Transfer of
RSF1010 to either recipient is also dependent on at least two of the
virB genes and virD4 (3, 15).
Recently, studies of RSF1010 mobilization led to the demonstration that
the 11 genes of the virB operon are also essential for the
synthesis of thin, brittle pili (13, 14). In the present
study, the connection between genes required for pilus assembly and
transfer events was further established by characterizing nonpolar
mutations in each of the 11 virB genes for function in the
transfer of RSF1010 and in extracellular complementation.
Nonpolar virB mutants.
Some of the nonpolar
virB operon mutants used in this study were generated by
transposon mutagenesis of pKJF78, a derivative of pUC19 carrying the
entire virB operon from pTiA6 as a 10.4-kb NdeI-XhoI fragment. Mutagenesis with
Tn5virB (11) was done as described by de Bruijn
and Lupski (12). The exact sites of transposon insertions on
pKJF78 (Table 1) were mapped and
sequenced, and the transposons were transferred onto pTiA6 by marker
exchange (7). Additional nonpolar virB operon
mutants were obtained from P. Christie (University of Texas at Houston)
or from A. Binns (University of Pennsylvania). All mutants used were
first confirmed to be avirulent by infection of Kalanchöe
diagremontiana and for nonpolarity by trans
complementation with plasmids pPC925, pPC933, pPC961, pPC975, and
pPC9103 (4) obtained from P. Christie; pED52 and pED37
(11) obtained from A. Binns; and pKJF101 (18) and
pKJF30 (15) (data not shown).
virB2 to virB11 and virD4: a
core complex for pilus assembly and transfer of three substrates.
It has previously been established that each of the 10 genes
virB2 to virB11 is essential for both
tumorigenesis and pilus assembly (4, 13). If pili are
essential for all transfer processes, then these 10 genes should also
be essential for transfer of RSF1010, VirE2, and T-DNA.
To assay RSF1010 mobilization, a derivative of RSF1010 conferring
resistance to only gentamicin was created by removing the
kanamycin
resistance gene from pML122 (
17) by digestion with
SalI and religating the plasmid. This plasmid, pML122
Km,
was
mobilized into strains bearing nonpolar mutations in genes
virB2 to
virB11. The resulting strains were
tested for their abilities
to mobilize pML122
Km to
A. tumefaciens UIA143 at 19°C and pH
5.3 as previously described
(
14). In three separate experiments,
all strains with
nonpolar mutations in the genes
virB2 to
virB11 did not transfer pML122
Km (Table
1). Thus, these 10 genes encode
proteins which are absolutely essential for the conjugative transfer
of
RSF1010. These data expand on the results of two previous studies
which
showed that
virB4,
virB11, and
virD4
are each required for
conjugative transfer (
3,
15).
A. tumefaciens strains with mutations in the
virE
operon have the unusual property of having their virulence restored
when
coinfected with a T-DNA-defective but
virE2+ strain (
19). This phenomenon,
which is known as extracellular
complementation, is believed to occur
because the T strand and
VirE2 protein, when transferred separately,
can associate within
the plant cell cytoplasm to create a complex that
can move into
the nucleus (
10). Previously, it had been
shown that
virD4 and
seven genes of the
virB
operon,
virB4 to
virB6 and
virB8 to
virB11,
are required by both the donor of the T strand and
the donor of
VirE2 protein to achieve extracellular complementation
(
5,
8,
15). To extend the characterization of the genetic
requirements
for extracellular complementation, cells with mutations in
10
genes,
virB2 to
virB11, were each tested for
their abilities to
complement extracellularly either the
virE2 mutant At12516 (
14)
or the T-DNA-defective
mutant At10002 (
20). As expected, coinfection
of At12516
with At10002 resulted in the formation of tumors on
K. diagremontiana (Fig.
1 and
2). However, none of the strains
with
nonpolar mutations in
virB2 to
virB11 restored
tumorigenesis
to either At12516 (Fig.
1) or At10002 (Fig.
2). In one
assay,
severely attenuated tumors developed when a
virB2 or
virB5 mutant
served as the donor of VirE2 protein (Fig.
1).
The formation of
these tumors was not reproducible, and tumors arose
only when
dense cell inocula were applied.
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FIG. 1.
virB mutants as donors of VirE2 protein
during extracellular complementation. Overnight cultures were diluted
to an optical density at 600 nm of 2.0. A 5-µl volume of a 1:1
mixture (2 × 107 cells) of At12516 and the indicated
virB mutants was inoculated onto a leaf surface wound
created by scratching with an 18-gauge needle. The photographs were
taken 30 days postinfection.
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FIG. 2.
virB mutants as donors of T strands during
extracellular complementation. Overnight cultures were diluted to an
optical density at 600 nm of 2.0. A 5-µl volume of a 1:1 mixture
(2 × 107 cells) of At10002 and the indicated
virB mutants was inoculated onto a leaf surface wound
created by scratching with an 18-gauge needle. The photographs were
taken 30 days postinfection.
|
|
These and previous studies showed that
virB2 to
virB11 and
virD4 are essential for transfer of T
strands, VirE2 protein, and
the plasmid RSF1010. Apparently, none of
these genes encodes a
protein which specifically functions in the
movement of one but
not the other substrate. Further, among the genes
virB2 to
virB11,
there are no differences in the
genes which function for transfer
to plants versus bacteria, indicating
that none of these genes
encodes a plant-specific adhesin. Taken
together, these observations
suggested that
virB2 to
virB11 and
virD4 produce a core transfer
complex
at the
A. tumefaciens membrane. This complex is essential
both for assembly of the pilus, which establishes contact with
either a
plant or a bacterium, and for the subsequent transfer
of all three
substrates: T strands, RSF1010, and VirE2.
Deletion of virB1 leads to a low level of efficiency of
RSF1010 transfer.
Recent data indicate that VirB1 may facilitate
contact with target plant cells, either directly or by participating in
pilus assembly. A processed form of VirB1, VirB1*, can be sheared from intact cells, suggesting that this protein functions on the exterior of
the bacterium (1). Other studies have shown that the N
terminus of VirB1 is homologous to lysozyme (2, 18),
suggesting a role in modifying the bacterial cell wall to ease assembly
of the pilus or transfer structure. Consistent with these proposed roles, A348B1, a strain bearing a deletion of virB1, does
not make pili observable by electron microscopy (13);
however, this strain does induce attenuated tumors (4). To
further examine the role of VirB1 in transfer, A348B1 was tested for
function in transfer assays.
In the RSF1010 mobilization assay, A348
B1 mobilized pML122
Km to
recipient
A. tumefaciens UIA143 in only 3 of 15 assays.
In
the 3 positive cases, the transfer frequency was on the order
of
10
6, compared to 10
3 for the wild-type
strain A348. This result demonstrated that
loss of VirB1 does have a
major effect on transfer. If VirB1 is
required for enhancing cell
contacts, it is apparently also important
for bacterium-to-bacterium
contacts.
Deletion of virB1 is more detrimental to VirE2 protein
transfer than to T-DNA transfer during extracellular
complementation.
To further examine the effect of the
virB1 deletion on transfer to plant cells, the effect of the
mutation on extracellular complementation was tested. Since A348B1
is partially virulent on K. diagremontiana (2),
first either its T-DNA processing genes or its virE2 gene
was deleted from it to generate avirulent VirE2 protein and T-strand
donors, respectively. These deletions were generated by electroporating
either pKS124 (20) or pKJF82 (14) into A348B1,
followed by screening for double homologous recombination as previously
described for the construction of At10002 (20) and At12516
(14). Southern analysis (22) and Western blotting
(15) with antibodies against VirE2, VirD2, and VirD4
confirmed that the resulting strains had the correct Ti plasmid
arrangements and produced the appropriate proteins (data not shown).
The resulting strains, At10002B1 (virB1 and defective for
T strand) and At12516B1 (virB1 virE2), were completely avirulent on K. diagremontiana (Fig.
3E and F).
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FIG. 3.
virB1 mutants in extracellular
complementation. Overnight cultures were diluted to an optical density
at 600 nm of 0.2. Strains were either mixed 1:1 for coinfections or
diluted 1:1 with MG/L (7) broth for single infections.
Wounds were created with an 18-gauge needle, and 5 µl (2 × 106 cells) of the indicated strains was inoculated. The
photograph was taken 22 days postinfection. Inoculated A. tumefaciens strains included At12516 with At10002 (A),
At12516B1 with At10002B1 (B), At12516 with At10002B1 (C),
At12516B1 with At10002 (D), At12516B1 (E), and At10002B1
(F).
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|
Coinfection of At10002
B1 with At12516
B1 (Fig.
3B) generated
highly attenuated tumors, demonstrating that VirB1 is important
for
transfer of either VirE2 or T strands during extracellular
complementation. Interestingly, coinfection of At12516
B1 with
At10002 resulted in a tumor (Fig.
3D), while coinfection of
At10002
B1
with At12516 produced highly attenuated tumors (Fig.
3C).
These
data indicated that loss of
virB1 was more detrimental
to VirE2
protein transfer than to T-strand transfer during
extracellular
complementation.
A differential pattern of T-strand transfer compared to VirE2 protein
transfer during extracellular complementation would
be expected if
VirB1 is required for improving cell contacts,
and, thereby, for
increasing the overall frequency of transfer
events. A strain with a
poor overall transfer efficiency would
be less likely to provide the
600 individual molecules of VirE2
protein necessary to coat a 20-kb T
strand within the cytoplasm
of the plant cell (
9). However,
the transfer efficiency is
probably sufficient for a single transfer
event required to donate
a T strand during coinfection.
These results contradict other studies which concluded that infection
of plant cells normally occurs by separate transfer
of VirE2 and T
strands (
5,
25). If this were the case, A348
B1
could not
induce even attenuated tumors because VirE2 protein
transfer would be
severely hampered, just as it is during extracellular
complementation.
Thus, in order for A348
B1 to induce attenuated
tumors, it must
transfer both T strands and VirE2 protein in a
single transfer event,
as does a T strand during extracellular
complementation.
Competition studies support a model for preassociation of the T
complex.
Further evidence supporting the preassociation of VirE2
with T strands and their transfer as a single event was found in
competition assays. Strain At10002 does not make T strands due to a
deletion of the T-DNA processing genes virD1 and
virD2 (20). When assayed for RSF1010 mobilization
between bacteria, this mutant had a slight but reproducible reduction
in conjugation frequency (Table 2). As
the deletion in At10002 altered the genetic organization of the
virD operon, it is possible that reduced synthesis of the essential protein VirD4 caused the observed decrease in the transfer frequency. However, Western blotting showed that the steady-state levels of VirD4 protein were similar in wild-type A348 and the mutant
At10002, demonstrating that synthesis of VirD4 is not affected by the
upstream deletion in At10002 (data not shown).
Competition studies have shown that transfer of VirE2 protein to plant
cells is inhibited by the presence of RSF1010 (
5).
Based on
this finding, it is possible that the inhibition of RSF1010
transfer
from strain At10002 was due to the presence of VirE2
protein. If this
is the case, deletion of the
virE2 gene from
At10002 should
relieve the inhibition of pML122 transfer and restore
transfer to
wild-type levels. To test this possibility, the
virE2 gene
was deleted from At10002 as described above for the construction
of
At12516
B1. The resulting strain, At10005, mobilized pML122
to the
A. tumefaciens recipient UIA143 at a frequency similar
to
that measured for A348 (Table
2). Thus, elimination of
virE2 from At10002 promoted pML122 transfer.
These data show that VirE2 protein can inhibit RSF1010 mobilization
between bacteria in the absence of T strands. However,
the
virE2 deletion mutant At12516 showed little increase in the
frequency of RSF1010 mobilization compared to wild-type A348 (Table
2).
Thus, the inhibitory effect of VirE2 protein on RSF1010 mobilization
occurs primarily when T strands are absent.
These observations are consistent with models that favor preassociation
of the T strand and the VirE2 protein within the bacterium.
In the
presence of T strands, free VirE2 protein would be sequestered
away
from the transfer complex, allowing uninhibited transfer
of RSF1010
between bacteria.
However, these findings seem to contradict the work of Binns et al.
(
5), who concluded that RSF1010 entry into plant cells
was
dependent on
virE2. In concordance with their results, the
particular
virE2 mutant used in their studies does not
mobilize
RSF1010 between bacteria (data not shown). However, this
defect
was attributed to the fact that this particular mutant produces
a VirE2::
-galactosidase fusion protein (
5)
since a deletion
of
virE2 had no effect on transfer (Table
2). The results presented
here thus concur with the earlier findings of
Buchanan-Wollaston
et al. (
6), who demonstrated that
transfer of RSF1010 into
plant cells is not dependent on
virE2. These data are further
consistent with the
observation of Binns et al. (
5) that overexpression
of
VirE2 in a wild-type background inhibited RSF1010 transfer
to plant
cells by threefold (
5).
Summary.
These studies have shown that VirB2 to VirB11, along
with VirD4, comprise a core transfer complex which is required for both pilus assembly and transfer events. This core complex functions to
transfer three diverse substrates without regard for the nature of the
recipient. Differential transfer was noted in the absence of VirB1, in
agreement with its proposed role in facilitating cell contacts.
Recently, virB1 to virB11 and virD4
were shown to be the only Ti plasmid genes essential for pilus assembly
and transfer (13). However, it is possible that unidentified
chromosomally encoded proteins are essential either as integral members
of the transfer complex or as accessory proteins required for assembly of the pilus and transfer structures.
Although it was clear that RSF1010, T strands, and VirE2 utilized the
same transfer complex, questions remain as to whether
the T strand and
VirE2 associate within the bacterium or within
the plant cytoplasm. The
data presented here on extracellular
complementation of
virB1 mutants and competition of RSF1010 and
VirE2 for the
transfer complex support a model for the coordinated
movement of T
strands and VirE2 protein into plant cells as a
single, preassembled T
complex.
| |
ACKNOWLEDGMENTS |
I thank Peter Christie and Andrew Binns for providing nonpolar
virB mutants and complementing plasmids; Denis Bougarel and Lin Lee for their technical assistance; and Joe Don Heath, Trevor Charles, Kathryn Stephens, Wanyin Deng, and Eugene Nester for their
helpful suggestions.
This work was supported by Public Health Service National Research
Service award 5T32 GM07270-21 from the National Institute of General
Medical Sciences and by the University of Washington Graduate School
Committee for Plant-Molecular Integration and Function.
| |
FOOTNOTES |
*
Present address: Department of Microbiology and
Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Bldg.
D1, Rm. 408, Boston, MA 02115. Phone: (617) 432-0832. Fax: (617)
738-7664. E-mail: kfullner{at}warren.med.harvard.edu.
| |
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J Bacteriol, January 1998, p. 430-434, Vol. 180, No. 2
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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