Plant CellPhysiol. 37(6): 782-789 (1996)
JSPP © 1996
Transient Gene Expression in Plant Cells Mediated by Agrobacterium
tumefaciens: Application for the Analysis of Virulence Loci
Yasushi Yoshioka 1 , Yoshito Takahashi1 \ Ken Matsuoka 2 , Kenzo Nakamura2, Jun Koizumi 1 ,
Mineo Kojima 3 and Yasunori Machida 15
1
2
3
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan
Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464-01 Japan
Department of Applied Biology, Shinshu University, Tsuneta 3-15-1, Ueda, Nagano, 386 Japan
A simple system is described for detection of the transfer of T-DNA from Agrobacterium cells to suspension-cultured tobacco BY-2 cells. A modified reporter gene for figlucuronidase (GUS) that contained an intron sequence
was introduced into the T-DNA region such that the GUS
protein could be synthesized in plant cells only after transfer of the T-DNA to plant nuclei. When BY-2 cells were
co-cultured with Agrobacterium cells that contained the
modified reporter gene, transient synthesis of GUS protein
was observed between 36 and 48 h after the onset of
co-culture. The level of GUS activity reached a plateau
within as little as 48 h. This temporal profile of GUS activation suggests that the transient activity might have been
due to expression of the GUS gene in the T-DNA that had
been transferred to the plant nuclei but had not yet been integrated into the plant chromosomes. Levels of transient
GUS activity were also examined with various vir mutants
of Agrobacterium and in a mutant with an altered chromosomal acvB gene, the gene for a protein that has been postulated to function outside bacterial cells. During co-culture
with virB, virD2, virD4 and acvB mutants, GUS activity remained at background levels, and the GUS activity in the
case of the virE2 mutant was thirty-fold lower than with
the wild type. On the basis of these results, we discuss the
roles of these genes during infection by Agrobacterium of
plant cells.
Key words: Agrobacterium tumefaciens — T-DNA transfer — Ti plasmid — Transient gene expression — vir genes.
Agrobacterium tumefaciens harboring the Ti plasmid
induces formation of crown gall tumors on a wide variety
of dicotyledonous plants. Upon infection of plants, the
Abbreviations: GUS, 0-glucuronidase; T-DNA, transferred
DNA; NLSs, nuclear localization signals.
4
Present address: Biochemistry Laboratory, Kanebo Ltd., 5-3-28
Kotobuki-cho, Odawara, Kanagawa, 250 Japan.
5
Corresponding author: Yasunori Machida, Department of Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya,
464-01 Japan. Tel: 81-52-789-2502, Fax: 81-52-789-2966. e-mail:
[email protected].
transferred DNA (T-DNA) is processed in bacterial cells
and transferred to plant cells, where it is integrated into
plant nuclear DNA. Such processes require products encoded in the vir region of the Ti plasmid (for reviews, see Kado
1991, Zambryski 1992, Hooykaas and Beijersbergen 1994).
The vir region of the octopine Ti plasmid contains seven
complementation loci (virA-virF; Melchers et al. 1990).
Most vir genes (B, C, D and E) are expressed in response to
plant signal compounds when Agrobacterium cells invade
plants (Stachel et al. 1986a, Cangelosi et al. 1990, Shimoda
et al. 1990) and their encoded products are thought to be involved in various early steps in the infection by Agrobacterium.
The VirDl and VirD2 proteins are involved in the generation of single-stranded T-DNA molecules (called Tstrands) in Agrobacterium (Stachel et al. 1986b) and their
transfer to plant cells (Tinland et al. 1994, Yusibov et al.
1994). The VirD2 protein introduces specific nicks in
the border sequences of T-DNA (Yanofsky et al. 1986,
Albright et al. 1987, Pansegrau et al. 1993) and it is covalently linked to the 5' ends of T-strands (Ward et al. 1988,
Herrera-Estrella et al. 1988, Young and Nester 1988, Diirrenberger et al. 1989, Tinland and Hohn 1995). The VirE2
protein has the ability to bind to single-stranded DNA in a
non-sequence-specific manner (Gietl et al. 1987, Das 1988,
Christie et al. 1988, Citovsky et al. 1988), although the virE
locus is essential only for strong virulence (Rossi et al.
1996). Both VirD2 and VirE2 have nuclear localization
signals (NLSs) and are localized in plant nuclei after they
have been synthesized in plant cells (Herrera-Estrella et al.
1990, Howard et al. 1992, Tinland et al. 1992, Citovsky et
al. 1992). Protein/T-DNA complexes, which are composed
of the T-strand and VirD2, are therefore thought to
migrate to plant cells and to be targeted to the nuclei
(Tinland and Hohn 1995). The results of recent genetic
analysis by Sundberg et al. (1996) suggest that VirE2 can
move to plant cells without the transfer of T-DNA. VirE2
has also been proposed to bind to the T-strand and to protect it against nucleolytic attack in plant cells (Rossi et al.
1996). The movement of these proteins from Agrobacterium cells to plant cells and nuclei has not, however, been
confirmed.
The vir region encodes a number of proteins that have
782
Transient gene expression and virulence loci
been predicted to be localized in the inner and outer membranes or the periplasmic space of Agrobacterium cells.
Such localization has also been demonstrated experimentally for VirD4 (Okamoto et al. 1991) and for some VirB
proteins (Ward et al. 1988, Christie et al. 1989, Kuldau
et al. 1990, Berger and Christie 1993, Shirasu et al. 1994,
Beijersbergen et al. 1994). Therefore, these proteins are
thought to participate in the transfer of T-DNA at the surface of bacterial cells. Although the functions of most of
these proteins remain to be defined, it has been proposed
that common mechanisms are involved in bacterial conjugation and in the transfer of T-DNA to plant cells (Lessl and
Lanka 1994).
A number of chromosomal genes have been described
that affect virulence (Hooykaas and Beijersbergen 1994). A
mutation in one such gene, acvB, does not impair the ability of the mutant Agrobacterium to attach to carrot cells
(Wirawan et al. 1993) nor does it interfere with the generation of T-strands (unpublished data of Wirawan and Kojima), an indication that this mutation affects processes subsequent to the generation of the T-strands.
In contrast to the recent progress made in the elucidation of the molecular events that occur in Agrobacterium
cells, little is known about processes such as the transfer of
T-DNA to the plant cells and their nuclei, and the eventual
integration of the T-DNA into chromosomes. In attempt to
examine aspects of the transfer of T-DNA, the expression
of /?-glucuronidase (GUS) in plant cells after infection by
Agrobacterium has been exploited as a sensitive assay system and, from the results of such experiments, possible
roles of Vir proteins have been proposed (KoukolikovaNicola et al. 1993, Rossi et al. 1993, 1996). No evidence
has, however, been reported to indicate that the GUS activity detected previously was, in fact, due to the integrationindependent expression of the reporter gene for GUS.
In order to examine the reliability of the above-described assay system for studying steps in the transfer of TDNA, we investigated the kinetics of the appearance of
GUS protein and its activity after the infection of suspension-cultured tobacco cells with Agrobacterium cells that
carried an intron-containing gene for GUS in the T-DNA.
Our results revealed the transient synthesis of the GUS protein at the early stages of infection, which is probably induced independently of the integration of T-DNA into the
chromosomal DNA of the plant cells.
Materials and Methods
Tobacco BY-2 cells—Suspension cultures of the BY-2 cell line
of tobacco (Kato et al. 1972) were maintained in modified Linsmaier and Skoog medium (Linsmaier and Skoog 1965, Onouchi
et al. 1991) with 0.2 mg of 2,4-D per liter, with weekly dilution.
Bacteria—Agrobacterium tumefaciens C58ClCm harboring
pTiB6S3trac, EHA101 harboring pEHAlOl, A208 harboring
pTiT37, C58 harboring pTiC58 and A348 harboring pTiA6 have
783
been described previously (Petit et al. 1978, Sciaky et al. 1978,
Hood et al. 1986, Stachel and Nester 1986). A. tumefaciens A348mx226, A348mx238, A348mx307, A348mx355, and A348mx341
harbor pTiA6 with insertion of the transposon Tn3-HoHol in the
virA, virB, virD3, virD4, and virE2 gene, respectively (Stachel and
Nester 1986). Although the insertion in A. tumefaciens A348mx307 was previously mapped in virD4 (Porter et al. 1987), our
analysis of the nucleotide sequence indicated that it was located in
virD3 (our unpublished data). A. tumefaciens A348mx304 and
A348mx334 harbor pTiA6 with insertion of the transposon TniHoHol in the carboxy-terminal portion and the amino-terminal
portion of virD2, respectively (Stachel and Nester 1986).
A. tumefaciens Bl 19 is an acvB mutant of A208 that was isolated
by mutagenesis with transposon TnJ (Wirawan et al. 1993).
Plasmids—The gene for hygromycin phosphotransferase
under control of the 35S promoter of cauliflower mosaic virus was
inserted between the GUS gene and left border of the T-DNA
of pIG121 (Ohta et al. 1990) to generate pIG121-HM. Plasmid
pAO416VG was constructed by inserting the 4.5-kilobase-pair
(kb) So/I fragment of pTOK9 (Jin et al. 1987) that covered the
region from virG to virC of pEHAlOl into the Sail site of a mini
Ri plasmid (Nishiguchi et al. 1987). To generate pVDP1234G,
which contained the entire portion of the virD operon of pTiA6,
as well as the region from virG to virC of pEHAlOl, a 6.35-kb
Sacl-BamHl fragment containing the entire virD operon of pTiA6
was treated with T4 DNA polymerase to blunt the ends. This fragment was inserted into the Xbal site of pAO416VG that had been
treated with T4 DNA polymerase to blunt the ends. Plasmid
pVDP34G was generated by inserting the DNA segment that contained the virD promoter and the virD3 and virD4 genes into
pAO416VG: a 3.98-kb Kpnl-BamHl fragment of pTiA6 containing the virD3 and virD4 genes was treated with T4 DNA polymerase to blunt the ends. This fragment was ligated with a 0.59-kb
Sacl-Smal fragment of pTLA6 that contained the promoter of the
virD operon. The ligated fragment was blunt-ended with T4 DNA
polymerase and then inserted into the Xbal site of pAO416VG
that had been treated with T4 DNA polymerase. To generate
pVDP4G, which contained the virD promoter fused to virD4, the
Rsal-BamHl fragment that covered the coding region of virD4
and the Sacl-Smal fragment that included the virD promoter were
first introduced between the BamHl and Sad cloning sites in
pUC18 by treatment with T4 DNA ligase, then the Sacl-BamHl
fragment that contained the promoter-fused virD4 was bluntended with T4 DNA polymerase and inserted into the Xbal site of
pAO416VG as described above.
Enzymes—Enzymes were purchased from Toyobo (Osaka,
Japan), Takara Shuzo Co., Ltd. (Shiga, Japan) and New England
Biolabs (MA, U.S.A.).
Co-culture of BY-2 cells with Agrobacterium—BY-2 cells
were co-cultured with Agrobacterium tumefaciens as described by
An (1985). Stable transformants were selected by monitoring resistance to 200 fig ml"' kanamycin. Agrobacterium cells were cultured until the optical density at 600 nm reached 1.5. The culture
was centrifuged and then cells were concentrated 10-fold in LB
medium. One hundred /il of this culture were mixed with 4 ml of a
suspension culture of BY-2 cells that had been grown for 3 days in
the fresh medium for BY-2 cells. The mixture was incubated at
26°C in darkness for various periods of time. In the experiment
with vir mutants of the A348 strain, the mixture was incubated at
22°C because the activity of yS-glucuronidase (GUS) in BY-2 cells
that had been co-cultured at 22°C was approximately 10-fold
higher than the activity in BY-2 cells that had been co-cultured at
26°C.
784
Transient gene expression and virulence loci
Assay of GUS activity—A fluorometric assay for GUS activity was performed by the procedure of Jefferson et al. (1986). BY2 cells were collected at various times after the start of co-culture
with Agrobacterium and disrupted by sonication (three 30-s
pulses). After removal of cell debris by centrifugation, each
supernatant was recovered and used for the determination of
the concentration of protein and the activity of GUS by the
fluorometric assay.
Determination of the rate of synthesis of GUS protein—An
aliquot of cells from the co-culture was washed with LS medium
and incubated for 30 min in LS medium that contained 50n% ml"'
chloramphenicol. Cells were then labeled with 2.8 MBq of [35S]
Protein-Labeling Mix (ICN, U.S.A.) for 30 min and the reaction
was quenched by chilling. Cells were disrupted by sonication and
GUS protein was immunoprecipitated with antiserum against /?glucuronidase (Clontech, CA, U.S.A.). Precipitated proteins were
fractionated on an SDS-polyacrylamide gel and the radioactivity
of "S in the region that corresponded to the band of GUS protein
was measured with a Bio-imaging Analyzer (Fuji-BAS2000,
Tokyo, Japan). The values presented in Figure IB were obtained
by subtracting a background value that was the average of the
radioactivity in four regions where no bands were detected.
Induction of vir genes—The expression of vir genes was induced as described by Shimoda et al. (1993).
Results
Transient expression of a T-DNA gene upon infection
by Agrobacterium of BY-2 cells—Plasmid pIG121-HM
was used for detection of the activity of yS-glucuronidase
(GUS) that was synthesized specifically after the T-DNA
that contained a GUS construct had been transferred to
plant cells. The T-DNA in this plasmid included a gene for
GUS with an intron sequence in its coding region, fused to
the 35S promoter of cauliflower mosaic virus (referred to as
intron-GUS), such that active GUS could not be synthesized in bacterial cells (data not shown). Active GUS protein encoded by this construct could only be synthesized in
plant cells after transfer of the gene to plant nuclei.
Plasmid pIG121-HM was introduced into the various
strains of Agrobacterium strains listed in Figure 1. Cells of
these strains were co-cultured with the BY-2 line of tobacco
cells. Figure 1A shows that GUS activity was detected
in the BY-2 cells 48 h after the start of co-cultivation with
all strains examined, although levels of activity differed
among the strains. Levels of GUS activity were increased
only slightly by an additional 48 h of co-cultivation. The
highest GUS activity was found in the case of co-cultivation with strain EHA101, which carried the disarmed supervirulent plasmid pEHAlOl; the lowest GUS activity was
found in the case of co-cultivation of strain A348 with BY2 cells.
We measured the rate of synthesis of GUS protein by
a pulse-labeling method, as described in Materials and
Methods. Figure IB shows that the rate of synthesis of
GUS protein was maximal between 36 h and 42 h after the
onset of co-culture and then decreased rapidly.
As shown in Figure IB, a lag time of 24 h was observed before the induction of synthesis of GUS protein.
This lag time might include the time required for induction
B
400
2,000
S
Q.
300-
200-
In
100-
CO
o
C58C1Cm(pTlB6S3tra)
A348(DT1AB>
20
40
60
80
Time (hours)
100
20
40
60
80
Time (hours)
Fig. 1 (A) Kinetics of changes in levels of GUS activity after the start of co-culture of BY-2 cells with strains of Agrobacterium that carried pIG121-HM (the intron-GUS gene).
Co-culture and measurements of GUS activity were performed as described in Materials
and Methods. Each activity is the average of results from four experiments.
(B) Changes in the rate of synthesis of GUS protein after
the start of co-culture of BY-2 cells with EHA101, which carried pIG121-HM.
The procedure is described in Materials and Methods.
Each value, which was determined with a Bio-imaging Analyzer, is the average of results from two experiments.
Transient gene expression and virulence loci
785
Table 1 Comparison of the transient activity of GUS with the frequency of transformation by various strains of
Agrobacterium
Strain
Ti plasmid
EHA101
pEHAlOl
A208
pTiT37
C58
pTiC58
C58ClCm
pTiB6S3tra
A348
pTiA6
Frequency of transformation"
(number of Km' calli (0.1 ml culture)"1)
-pIG121-HM
+ pIG121-HM
c
494 ±203
229± 19
0
86 ± 75
25± 8
15± 1
0
0
0
0
GUS activity*
(pmol MU min"' (mg protein)"1)
+PIG121-HM -PIG121-HM
338 ±202
164± 55
58 ± 42
19± 9.3
12± 2.6
1.5 ±0.70
1.1 ±1.3
0.80±0.10
0.73±0.16
1.6 ±0.74
" Frequencies represent means of three independent experimental determinations (±SEM).
* Activities were measured 72 h after the start of co-culture and represent means of four independent experimental determinations
(±SEM).
of vir genes, for generation of T-strands, for transfer of
these molecules from bacterial cells to plant nuclei and for
synthesis of the active GUS enzyme. We examined whether
induction of vir genes prior to the onset of co-culture might
decrease the lag time. Agrobacterium EHA101 cells harboring pIG121-HM were treated with 10 jiM acetosyringone
and 10 mM glucose for 24 h to induce vir genes and then
these cells were co-cultured with BY-2 cells, and GUS activity was determined. In spite of pre-induction, the profiles
of activation of GUS were unchanged: GUS activity was
first detected at 36 h and reached a plateau value at 48 h
(data not shown). Thus, the time needed for induction of
the expression of vir genes and for accumulation of the Tstrand in bacterial cells might not be a major factor responsible for the lag time.
We determined the frequency of transformation of
BY-2 cells by various strains of Agrobacterium. As shown
in Table 1, there appeared to be a parallel correlation between the level of GUS activity and the frequency of transformation of the BY-2 cells. Therefore, the efficiency of
transformation of BY-2 cells by Agrobacterium can be
predicted from the results of this simple transient expression assay.
Examination of vir mutants of Agrobacterium—We
examined levels of GUS activity when BY-2 cells were cocultured with various vir mutants of Agrobacterium. The
series of vir mutants examined were, however, derived from
strain A348, which was found to be very inefficient in terms
of expression of GUS activity in infected tobacco cells
(Fig. 1, Table 1). To overcome this problem, we introduced
Table 2 Effects of vir mutations on the transient activity of GUS
Strain
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
A348/pAO416VG, pIG121-HM
A348/pIG121-HM
A348/pAO416VG
A348mx226/pAO416VG, pIG121-HM
A348mx238/pAO416VG, pIG121-HM
A348mx355/pAO416VG, pIG121-HM
A348mx341/pAO416VG, pIG121-HM
A348mx334/pVDP34G, pIG121-HM
A348mx304/pVDP34G, pIG121-HM
A348mx334/pVDP1234G, pIG121-HM
A348mx304/pVDP1234G, pIG121-HM
A348mx307/pVDP4G, pIG121-HM
A348mx307/pVDP34G, pIG121-HM
Relevant genotype
Wild type
Wild type
Wild type
vir A "
virBJ- "
virD4~
virE2~~
v/r£>2"(N-ter)/v/>£>3 + , 4+
virD2-(C-tei)/virD3+, 4+
virD2~/virDl+, 2+, 3+, 4+
virD2~/virDl+, 2+, 3+, 4+
virD3~/virD4+
virD3-/virD3+, 4+
GUS activity"
(pmol MU min" 1 (mg protein)"1)
340 ±72
8.18± 1.9
2.33± 0.35
1.59± 0.28
l.62± 0.92
l.25± 0.38
1 1.9 ± 2.4
l.36± 0.39
l.30± 0.24
279 ±64
16.7 ± 1.8
184 ±59
175 ±35
" Activities were measured 72 h after the start of co-culture and represent means of four independent experimental determinations
(±SEM).
* It is unknown whether or not downstream genes might have been expressed.
786
Transient gene expression and virulence loci
pAO416VG, which contained the DNA segment that extended from the virG to the virC region of the Ti plasmid
pEHAlOl of a supervirulent strain, into A348 cells. This
DNA segment has been shown to enhance levels of acetosyringone-induced expression of vir genes (Jin et al. 1987). As
shown in Table 2, the activity of GUS increased 40-fold
when pAO416VG was introduced into cells of the A348
strain that harbored pIG121-HM. Use of this plasmid in
the vir mutants derived from A348 allowed us to obtain
reliable data on the activity of GUS in BY-2 cells. In addition, our results indicate that the level of GUS activity was
strongly correlated with the level of expression of vir genes
in Agrobacterium cells.
We introduced pAO416VG and pIG121-HM into the
vir A mutant (A348mx226), the virB mutant (A348mx238;
the insertion in the middle of virB operon), the virD4 mutant (A348mx355), and the virE2 mutant (A348mx341) that
had been generated by the insertion of Tn5-HoHol, which
contains the coding sequence of the lacZ gene. We co-cultured BY-2 cells with each of these mutants. Table 2 summarizes the activities of GUS detected 72 h after the start of
co-culture. When BY-2 cells were co-cultured with the
virA, virB or virD4 mutants, GUS activities were at background levels. Significant activity was detected in the BY-2
cells that had been co-cultured with the virE2 mutant, although the level of activity was 30-fold lower than with the
wild type.
We examined two mutants with lesions in the virD2
gene. In one mutant, A348mx334, TnJ-HoHol was inserted in the coding region for the amino-terminal portion
of VirD2; in the other mutant, A348mx3O4, the insertion was in the coding region for the carboxy-terminal portion of VirD2. We introduced pVDP34G (a derivative of
pAO416VG) that contained virD3 and virD4 genes, and
pIG121-HM into these virD2 strains, since the expression
of the virD3 and virD4 genes was not detected in these
virD2 mutants, probably as a consequence of the polar
effect of the insertion of TnJ-HoHol (data not shown).
The expression of virD4 genes in the strains that carried
pVDP34G was confirmed by Western blot analysis with an
antibody against the VirD4 protein (data not shown).
When BY-2 cells were co-cultured with these virD2 mutants, GUS activities were at background levels (lines 8 and
9 in Table 2).
To examine the capacity of complementation of a
wild-type virD2 gene, we introduced pVDP1234G (a derivative of pAO416VG) that contained the entire virD operon
into each virD2 mutant. The level of expression of GUS increased significantly when BY-2 cells were co-cultured with
the virD2 mutant, A348mx334, that harbored pIG121-HM
and pVDP1234G (line 10 in Table 2). In the case of A348mx304, by contrast, the level of GUS activity did not markedly increase: it was still 17-fold lower than GUS activity
observed with A348mx334 that carried pVDP1234G (lines
Table 3 Transient activity of GUS in BY-2 cells after coculture for 72 h with the acvB mutant
Strain
Genotype
GUS activity"
(pmolMUmin" 1
(mg protein)"1)
A208/pIG121-HM
Wild type
127.0±26.5
B119/pIG121-HM
acvB
1.2± 0.3
° Activities were measured 72 h after the start of co-culture and
represent means of four independent experimental determinations
(±SEM).
10 and 11 in Table 2).
We also examined the effect of the virD3 mutation by
introducing pVDP4G, which contained a virD4 gene, or
pVDP34G (see above) into the A348mx307 strain that had
an insertion in the virD3 gene. As shown in Table 2 (lines
12 and 13), the same levels of GUS activities were found
when either strain was used. These results indicate that the
VirD3 protein is not required for the transfer of T-DNA.
The data are consistent with previous observations that this
protein is not necessary for the tumorigenicity of Agrobacterium (Hirayama and Oka 1990, Koukolikovd-Nicola et
al. 1993).
Examination of a chromosomal vir gene, acvB—We
introduced pIG121-HM into the B119 strain, which contained an insertion in the acvB gene in its chromosome
(Wirawan et al. 1993) and into the A208 strain, an isogenic
wild-type line of Agrobacterium, and co-cultured BY-2
cells with each strain. As shown in Table 3, the level of
GUS expression observed with Bl 19 that harbored pIG121HM was similar to the background level 72 h after the start
of co-culture.
Discussion
The results of our pulse-labeling experiments showed
that the rate of synthesis of GUS protein in the BY-2 cells
increased markedly between 24 h and 36 h after the onset
of co-culture of the tobacco cells with Agrobacterium cells
that carried the intron-GUS construct and then decreased
rapidly. Such transient synthesis cannot be explained by
assuming that GUS activity was due to the expression of
the intron-GUS gene that is stably resident in tobacco chromosomes. It seems most likely that the observed GUS activity was due to GUS protein that was transiently synthesized
from T-DNA containing intron-GUS that had been transferred from Agrobacterium cells to tobacco nuclei but had
not yet been integrated into the chromosomal DNA. There
are two possible explanations for the relatively high level of
transient expression of GUS. (1) A large number of T-
Transient gene expression and virulence loci
strand molecules might have been transferred from bacterial cells to many plant cells and successively transported to
nuclei, but only a limited number of the T-strand molecules
among those transported might have been successfully integrated into plant chromosomes. A large amount of GUS
protein might have been synthesized from the intron-GUS
gene in such extra-chromosomal T-DNA molecules before
integration. (2) Transcription of the intron-GUS gene in
the extra-chromosomal T-DNA might have been much
more efficient than that of the integrated form in chromosomes, and this difference could have caused the high rate
of transient synthesis of GUS protein. Although the molecular basis of such transient synthesis is unknown, the transient expression of GUS was probably due to the presence
of extra-chromosomal T-DNA prior to its integration because the rate of synthesis of GUS protein after integration
into plant chromosomes should be constant.
In our experiments, no expression of the intron-GUS
gene in BY-2 cells was detected with virB and virD4 mutants (Table 1). Since the virB mutant (A348mx238) in
the present investigation had an insertion of Tni-HoHol in
virB7 and produced a VirB7-LacZ fusion protein (Stachel
and Nester 1986, Ward et al. 1990), the VirB7 and/or some
VirB proteins encoded downstream of virB7 could not be
synthesized by this virB mutant. The processing of T-DNA
occurs normally in the virD4 mutant (Yanofsky et al. 1986,
Yamamoto et al. 1987, Veluthambi et al. 1987, Stachel et
al. 1987) and in this virB mutant (our unpublished data),
when each is incubated with acetosyringone or a plant exudate. Therefore, the results of the present study suggest
that the vir genes discussed above function in the transfer
of T-DNA from the bacterial cells to the plant nuclei at the
bacterial membrane. The present results cannot rule out
the possibility that the T-DNA might be transferred from
the virD4 and virB mutants to plant cells but the mutant
Vir proteins might have prevent active GUS protein from
being synthesized in the plant cells. However, this possibility seems to be unlikely because the VirD4 protein
(Okamoto et al. 1991) and some of the VirB proteins
(Christie et al. 1989, Ward et al. 1990, Kuldan et al. 1990)
have been shown to be localized at the bacterial membrane.
Only a low level of GUS activity was found in BY-2
cells that had been co-cultured with the virE2 mutant. In
the virE2 mutant, the processing of T-DNA normally
occurs (Yanofsky et al. 1986, Yamamoto et al. 1987,
Veluthambi et al. 1987, Stachel et al. 1987). The amount of
single-stranded T-DNA molecules that were detected in the
cytoplasm of the plant protoplasts was, however, reduced
when the tobacco cells were co-cultivated with a virE2 mutant as compared to co-cultivation with the wild type
(Yusibov et al. 1994). Recently, it was reported that the
VirE2 protein is transferred to plant cells independently of
the transfer of T-DNA, via the function of the VirEl protein (Sundberg et al. 1996). The VirE2 protein has been
787
also shown to function in protecting the entire singlestranded T-DNA against nucleolytic attack in plant cells
(Rossi et al. 1996). Such nucleolytic degradation of the intron-GUS gene in BY-2 cells might account for the reduction in GUS activity observed when these cells were co-cultured with the virE2 mutant.
The results of our experiment with one of virD2
mutants support the hypothesis that the VirD2 protein functions in the transfer of T-DNA to plant nuclei, as well as
making nick in the border sequences of T-DNAs and acting
in the formation of T-strands. We used two mutants, one
(A348mx334) with insertion of the transposon TnJ-HoHol
in the middle of the virD2 cistron and the other (A348mx304) with insertion in the region close to the 3' end of the
cistron (Porter et al. 1987). Both mutants can produce
VirD2-LacZ fusion proteins (Stachel and Nester 1986) that
retain the amino-terminal monopartite NLS of VirD2 but
not the carboxy-terminal bipartite NLS. When BY-2 cells
were co-cultured with either virD2 mutant, no GUS activity
was detected (lines 8 and 9 in Table 2). Experiments reported previously showed that nicking of border and formation of T-strand can be induced in the A348mx304
mutant but not in the A348mx334 mutant, even though
both mutants are non-tumorigenic (Yanofsky et al. 1986,
Stachel et al. 1987). A similar observation was also made
with virD2 mutants of another type of Ti plasmid (Steck et
al. 1990). Therefore, the mutant VirD2 protein that is synthesized in A348mx304 cells seems to retain at least partial
activity for nicking. Nevertheless, as shown in Table 2,
A348mx3O4 was unable to induce the activity of GUS in
plant nuclei. When only the carboxy-terminal NLS was
deleted from the VirD2 protein, a similar reduction in the
transient expression of GUS was observed (Rossi et al.
1993). These results, together with the observation that
VirD2 can bind to the 5' end of the T-strand, suggest that
the VirD2 protein functions at a certain step during transfer of the T-strand, as well as in production of the Tstrand: it might serve as a pilot protein for transport of TDNA to plant nuclei, as predicted previously (Tinland and
Hohn 1995). Alternatively, the T-strand that contained the
intron-GUS gene might have been transferred from the
A348mx304 mutant to the BY-2 nuclei but the mutant
VirD2 protein from this strain might have inhibited expression of the intron-GUS gene in the nuclei. If the mutant
VirD2 protein were to bind to the T-strand, such an inhibitory role of the mutant VirD2 protein would be feasible
(see below).
With regard to the function of VirD2, it is worth
noting that, although the ability of the A348mx341 strain
to induce GUS activity was apparently restored by introducing the entire virD region (Table 2, line 10), the ability of
the A348mx304 strain was only partially restored by the
same virD construct (Table 2, line 11). The mutant VirD2
protein generated in A348mx304 cells might have interfered
788
Transient gene expression and virulence loci
with the action of the wild-type VirD2 protein. A possible
explanation for this dominant mutation is that the mutant
VirD2 protein might have a higher binding affinity for the
5-end of T-strands than the wild-type VirD2. The complex
containing the T-strand and the mutant VirD2 might be inactive, either for transfer of the T-strand to plant nuclei or
for expression of the intron-GUS gene, even if the T-DNA/
mutant-VirD2 complex were transferred to the plant
nuclei.
The authors thank Michael Ronemus (Yale University) for
critical reading of this manuscript. This research was supported in
part by a Grant-in-Aid for General Scientific Research (B) (to
Y.M.) and a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Science and Culture of
Japan (to Y.Y.).
References
Albright, L.M., Yanofsky, M.F., Leroux, B., Ma, D. and Nester, E.W.
(1987) Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J. Bacteriol. 169:
1046-1055.
An, G . (1985) High-efficiency transformation of cultured tobacco cells.
Plant Physiol. 79: 568-570.
Beijersbergen, A., Smith, S.J. and Hooykaas, P.J.J. (1994) Localization
and topology of VirB proteins of Agrobacterium tumefaciens. Plasmid
32: 212-218.
Berger, B. and Christie, P.J. (1993) The. Agrobacterium tumefaciens virD4
gene product is an essential virulence protein requiring an intact
nucleoside triphosphate-binding domain. J. Bacteriol. 175: 1723-1734.
Cangelosi, G.A., Ankenbauer. R.G. and Nester, E.W. (1990) Sugars induce the Agrobacterium virulence genes through a periplasmic binding
protein and a transmembrane signal protein. Proc. Natl. Acad. Sci. USA
87: 6708-6712.
Christie, P.J., Ward, J.E., Winans, S.C. and Nester, E.W. (1988) The
Agrobacterium tumefaciens virE2 gene product is a single-strandedDNA-binding protein that associates with T-DNA. / . Bacteriol. 170:
2659-2667.
Christie, P.J., Ward, J.E., Jr., Gordon, M.P. and Nester, E.W. (1989) A
gene required for transfer of T-DNA to plants encodes an ATPase with
autophosphorylating activity. Proc. Natl. Acad. Sci. USA 86: 96779681.
Citovsky, V., De Vos, G. and Zambryski, P. (1988) Single-stranded DNAbinding protein encoded by the virE locus of Agrobacterium tumefaciens. Science 240: 501-504.
Citovsky, V., Zupan, J., Warnick, D. and Zambryski, P. (1992) Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 256:
1802-1805.
Das, A. (1988) Agrobacterium tumefaciens virE operon encodes a singlestranded DNA-binding protein. Proc. Natl. Acad. Sci. USA 85: 29092913.
Diirrenberger, F., Crameri, A., Hohn, B. and Koukolikova-Nicola, Z.
(1989) Covalently bound VirD2 protein of Agrobacterium tumefaciens
protects the T-DNA from exonucleolytic degradation. Proc. Natl. Acad.
Sci. USA 86: 9154-9158.
Gietl, C , Koukolikova-Nicola, Z. and Hohn, B. (1987) Mobilization of TDNA from Agrobacterium to plant cells involves a protein that binds
single-stranded DNA. Proc. Natl. Acad. Sci. USA 84: 9006-9010.
Herrera-Estrella, A., Chen, Z., Van Montagu, M. and Wang, K. (1988)
VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA-protein complex at the 5' terminus of T-strand
molecules. EMBO J. 7: 4055-4062.
Herrera-Estrella, A., Van Montagu, M. and Wang, K. (1990) A bacterial
peptide acting as a plant nuclear targeting signal: the amino-terminal por-
tion of Agrobacterium VirD2 protein directs a /J-galactosidase fusion
protein into tobacco nuclei. Proc. Natl. Acad. Sci. USA 87: 9534-9537.
Hirayama, T. and Oka, A. (1990) The virE and virD3 genes are nonessential for induction of hairy roots on plants by Agrobacterium
rhizogenes A4. Bull. Inst. Chem. Res., Kyoto Univ. 67: 227-238.
Hood, E.E., Helmer, G.L., Fraley, R.T. and Chilton, M.-D. (1986) The
hypervirulence of Agrobacterium tumefaciens A281 is encoded in a
region of pTiBo542 outside of T-DNA. J. Bacteriol. 168: 1291-1301.
Hooykaas, P.J.J. and Beijersbergen, A.G.M. (1994) The virulence system
of Agrobacterium tumefaciens. Annu. Rev. Phytopathol. 32: 157-179.
Howard, E.A., Zupan, J.R., Citovsky, V. and Zambryski, P.C. (1992)
The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in
plant cells. CW/68: 109-118.
Jefferson, R.A., Burgess, S.M. and Hirsh, D. (1986)/?-Glucuronidase from
Escherichia coli as a gene-fusion marker. Proc. Natl. Acad. Sci. USA 83:
8447-8451.
Jin, S., Komari, T., Gordon, M.P. and Nester, E.W. (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens
A281. /. Bacteriol. 169: 4417-4425.
Kado, C.I. (1991) Molecular mechanisms of crown gall tumorigenesis.
Critic. Rev. Plant Sci. 10: 1-32.
Kato, K., Matsumoto, T., Koiwai, A., Mizusaki, S;, Nishida, K.,
Noguchi, M. and Tamaki, E. (1972) Liquid suspension culture of tobacco cells. In Fermentation Technology Today. Edited by Terui, G. pp.
689-695. Society of Fermetation Technology, Osaka, Japan.
Kuldau, G.A., De Vos, G., Owen, J., McCaffrey, G. and Zambryski, P.
(1990) The virB operon of Agrobacterium tumefaciens pTiC58 encodes
11 open reading frames. Mol. Gen. Genet. 221: 256-266.
Koukolfkova-Nicola, Z., Raineri, D., Stephens, K., Ramos, C , Tinland,
B. and Nester, E.W. (1993) Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of
T-DNA into the plant cell nucleus and in T-DNA integration. / . Bacteriol. 175: 723-731.
Lessl, M. and Lanka, E. (1994) Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell 77: 321-324.
Linsmaier, E.M. and Skoog, F. (1965) Organic growth factor requirements
of tobacco tissue cultures. Physiol. Plant. 18: 100-127.
Melchers, L.S., Maroney, M.J., den Dulk-Ras, A., Thompson, D.V., van
Vuuren, H.A.J., Schilperoort, R.A. and Hooykaas, P.J.J. (1990) Octopine and nopaline strains of Agrobacterium tumefaciens differ in
virulence; molecular characterization of the virF locus. Plant Mol. Biol.
14: 249-259.
Nishiguchi, R., Takanami, M. and Oka, A. (1987) Characterization and sequence determination of the replicator region in the hairy-root-inducing
plasmid pRiA4b. Mol. Gen. Genet. 206: 1-8.
Ohta, S., Mita, S., Hattori, T. and Nakamura, K. (1990) Construction and
expression in tobacco of a /?-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol. 31: 805813.
Okamoto, S., Toyoda-Yamamoto, A., Ito, K., Takebe, I. and Machida,
Y. (1991) Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol. Gen. Genet. 228: 24-32.
Onouchi, H., Yokoi, K., Machida, C , Matsuzaki, H., Oshima, Y., Matsuoka, K., Nakamura, K. and Machida, Y. (1991) Operation of an efficient
site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucl. Acids Res. 19: 6373-6378.
Pansegrau, W., Schoumacher, F., Hohn, B. and Lanka, E. (1993) Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of
Agrobacterium tumefaciens Ti plasmid: analogy to bacterial conjugation. Proc. Natl. Acad. Sci. USA 90: 11538-11542.
Petit, A., Tempe, J., Kerr, A., Holsters, M., Van Montagu, M. and Schell,
J. (1978) Substrate induction of conjugative activity of Agrobacterium
tumefaciens Ti plasmids. Nature (London) 271: 570-571.
Porter, S.G., Yanofsky, M.F. and Nester, E.W. (1987) Molecular characterization of the virD operon from Agrobacterium tumefaciens. Nucl.
Acids Res. 15: 7503-7517.
Rossi, L., Hohn, B. and Tinland, B. (1993) The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for
transfer of T-DNA to plant. Mol. Gen. Genet. 239: 345-353.
Transient gene expression and virulence loci
Rossi, L., Hohn, B. and Tinland, B. (1996) Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of
Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 93: 126-130.
Sciaky, D., Montoya, A.L. and Chilton, M.D. (1978) Fingerprints of Agrobacterium Ti plasmids. Plasmid 1: 238-253.
Shimoda, N., Toyoda-Yamamoto, A., Nagamine, J., Usami, S., Katayama, M., Sakagami, Y. and Machida, Y. (1990) Control of expression
of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc. Natl. Acad. Sci. USA 87: 6684-6688.
Shimoda, N., Toyoda-Yamamoto, A., Aoki, S. and Machida, Y. (1993)
Genetic evidence for an interaction between the VirA sensor protein and
the ChvE sugar-binding protein of Agrobacterium. J. Biol. Chem. 268:
26552-26558.
Shirasu, K., Koukolfkova-Nicola, Z., Hohn, B. and Kado, C.I. (1994) An
inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity
and similarities to conjugative transfer genes. Mol. Microbiol. 11: 581—
588.
Stachel, S.E. and Nester, E.W. (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium
tumefaciens. EMBO J. 5: 1445-1454.
Stachel, S.E., Nester, E.W. and Zambryski, P.C. (1986a) A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Natl.
Acad. Sci. USA 83: 379-383.
Stachel, S.E., Timmerman, B. and Zambryski, P. (1986b) Generation of
single-stranded T-DNA molecules during the initial stages of T-DNA
transfer from Agrobacterium tumefaciens to plant cells. Nature (London) 322: 706-712.
Stachel, S.E., Timmerman, B. and Zambryski, P. (1987) Activation of
Agrobacterium tumefaciens vir gene expression generates multiple
single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' VJ>D gene products. EMBO J. 6: 857-863.
Steck, T.R., Lin, T.S. and Kado, C.I. (1990) VirD2 gene product from the
nopaline plasmid pTiC58 has at least two activities required for
virulence. Nucl. Acids Res. 18: 6953-6958.
Sundberg, C , Meek, L., Carrol, K., Das, A. and Ream, W. (1996) VirEl
protein mediates export of the single-stranded DNA-binding protein
VirE2 from Agrobacterium tumefaciens into plant cells. /. Bacteriol.
178: 1207-1212.
Tinland, B. and Hohn, B. (1995) Recombination between prokaryotic and
789
eukaryotic DNA: integration of Agrobacterium tumefaciens T-DNA
into the plant genome. In Genetic Engineering, Principles and Methods.
Edited by Setlow, J.K. Vol. 17. pp. 209-229. Plenum, New York.
Tinland, B., Hohn, B. and Puchta, H. (1994) Agrobacterium tumefaciens
transfers single-stranded transferred DNA (T-DNA) into the plant cell
nucleus. Proc. Natl. Acad. Sci. USA 91: 8000-8004.
Tinland, B., KoukoUkova-Nicola, Z., Hall, M.N. and Hohn, B. (1992)
The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals. Proc. Natl. Acad. Sci. USA 89: 7442-7446.
Veluthambi, K., Jayaswal, R.K. and Gelvin, S.B. (1987) Virulence genes
A, G and D mediate the double-stranded border cleavage of T-DNA
from the Agrobacterium Ti plasmid. Proc. Natl. Acad. Sci. USA 84:
1881-1885.
Ward, E.R. and Barnes, W.M. (1988) VirD2 protein of Agrobacterium
tumefaciens is very tightly linked to the 5' end of T-strand DNA. Science
242: 927-930.
Ward. J.E., Akiyoshi, D.E., Regier, D., Datta, A., Gordon, M.P. and
Nester, E.W. (1988) Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. /. Biol. Chem. 263: 5804-5814.
Ward, J.E., Dale, E.M., Nester, E.W. and Binns, A.N. (1990) Identification of a VirB 10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J. Bacteriol. 172: 5200-5210.
Wirawan, I.G.P., Kang, H.W. and Kojima, M. (1993) Isolation and characterization of a new chromosomal virulence gene of Agrobacterium
tumefaciens. J. Bacteriol. 175: 3208-3212.
Yamamoto, A., Iwahashi, M., Yanofsky, M.F., Nester, E.W., Takebe, I.
and Machida, Y. (1987) The promoter proximal region in the virD locus
of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA. Mol. Gen. Genet. 206: 174-177.
Yanofsky, M.F., Porter, S.G., Young, C , Albright, L.M., Gordon, M.P.
and Nester, E.W. (1986) The virD operon of Agrobacterium tumefaciens
encodes a site-specific endonuclease. Cell 47: 471-477.
Young, C. and Nester, E.W. (1988) Association of the VirD2 protein with
the 5' end of T strands in Agrobacterium tumefaciens. J. Bacteriol. 170:
3367-3374.
Yusibov, V.M., Steck, T.R., Gupta, V. and Gelvin, S.B. (1994) Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc. Natl. Acad. Sci. USA 91: 2994-2998.
Zambryski, P.C. (1992) Chronicles from the Agrobacterium-p\ant cell
DNA transfer story. Annu. Rev. Plant Mol. Biol. 43: 465-490.
(Received February 6, 1996; Accepted June 17, 1996)