Download Import of Agrobacterium T-DNA into Plant Nuclei: Two

The Plant Cell, Vol. 13, 369–383, February 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
Import of Agrobacterium T-DNA into Plant Nuclei: Two Distinct
Functions of VirD2 and VirE2 Proteins
Alicja Ziemienowicz,a,1 Thomas Merkle,b Fabrice Schoumacher,c Barbara Hohn,a,2 and Luca Rossia
a Friedrich
Miecher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland
für Biologie II, Zellbiologie, Universität Freiburg, D-79104 Freiburg, Germany
c University Women’s Clinic, Biochemistry/Endocrinology Unit, Department of Research, Schanzenstrasse 46, CH-4031
Basel, Switzerland
b Institute
To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as
well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was
fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import
of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking
its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules
were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the
NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for
VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore,
by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.
INTRODUCTION
Regulated and efficient molecular trafficking into and out of
nuclei represents an essential aspect of the life of eukaryotic
cells. Substrates for such transport include proteins, nucleic
acids, and protein–nucleic acid complexes. The nuclear traffic of nucleic acids is most likely mediated by nuclear import
and export signals present on proteins associated with the
nucleic acids. Examples include nuclear–cytoplasmic transport of small nuclear RNA particles, export of mRNA (reviewed in Nakielny et al., 1997), and nuclear transport of
viral RNA or DNA. The T-DNA of Agrobacterium presents a
particularly interesting and unique import substrate. This
DNA is transferred, possibly in a conjugation-like mode, to
the plant cell, where it is transported into the nucleus and
integrated in chromosomal DNA (reviewed in Lartey and
Citovsky, 1997; Zupan and Zambryski, 1997; Rossi et al., 1998;
Hansen and Chilton, 1999; Gelvin, 2000). Agrobacterium is
widely used as an efficient and precise DNA delivery system
for plants; therefore, analysis of T-DNA import is important
not only in its own right but also because it may help to further enhance the system’s utility. Transfer and integration of
1 Current address: Department of Biotechnology, University of Gdansk,
Kladki 24, 80-822 Gdansk, Poland.
2 To whom correspondence should be addressed. E-mail hohnba1@
fmi.ch; fax 41-61-697-3976.
T-DNA (reviewed in Tinland and Hohn, 1995; Sheng and
Citovsky, 1996; Tinland, 1996; Zupan and Zambryski, 1997;
de la Cruz and Lanka, 1998; Rossi et al., 1998; Gelvin, 2000)
requires bacterial virulence (Vir) proteins encoded on the tumor-inducing (Ti) plasmid as well as on the bacterial
chromosome. Two 25-bp imperfect direct repeats, termed
border sequences, define the T-DNA. In the presence of the
VirD1 protein, VirD2 nicks the border sequence in a siteand strand-specific manner and covalently attaches to the
5⬘ end of the nicked DNA. The nicked DNA is thought to be
displaced from the plasmid to yield single-stranded T-DNA.
This single-stranded T-DNA–VirD2 complex is transferred to
the plant cell and is coated with the single-stranded DNA
(ssDNA) binding protein VirE2, forming the so-called T-DNA
complex. The T-DNA complex enters the nucleus, and the
T-DNA is finally integrated into the plant cell genome.
Like the transport of other nucleic acids, nuclear import of
T-DNA is also expected to depend on proteins accompanying it to the plant cell. Proteins imported into the nucleus
generally contain motifs composed of one or two stretches
of basic amino acids. These motifs are termed nuclear localization signals (NLSs) and are recognized by the nuclear import machinery. The import process can be divided into two
steps: importin-mediated, NLS-dependent docking of the
protein at the nuclear pore, and RanGTP-dependent translocation through the channel of the nuclear pore complex
(NPC) (reviewed in Görlich, 1997; Nakielny and Dreyfuss,
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1999). Both VirD2 and VirE2 contain NLSs that are functional
in targeting reporter proteins into plant nuclei (HerreraEstrella et al., 1990; Citovsky et al., 1992, 1994; Howard et
al., 1992; Tinland et al., 1992). VirD2 contains two NLSs, a
monopartite NLS in the N-terminal part of the protein and a
bipartite NLS in the C-terminal part of the protein. The N-terminal NLS of VirD2 appeared not to be required for T-DNA
transfer, because mutations in this sequence had no effect
on T-DNA transfer efficiency (Rossi et al., 1993). In contrast,
the C-terminal NLS of VirD2 has been shown to be required for
efficient transfer of the bacterial T-DNA to the plant nucleus
(Shurvinton et al., 1992; Rossi et al., 1993; Narasimhulu et
al., 1996; Mysore et al., 1998). Although the VirE2 protein
was found to be required for T-DNA transfer (Citovsky et al.,
1992, 1994; Rossi et al., 1996), the role of its NLSs in nuclear import of the T-DNA could not be evaluated. Point mutations or partial deletions of the C- or N-terminal NLSs
abolished or modified the ssDNA binding properties of the
protein (Citovsky et al., 1992, 1994; Dombek and Ream,
1997). In microinjection experiments, it was determined that
VirE2 mediates the nuclear uptake of ssDNA into plant cells,
but the role of VirD2 in this process was not studied (Zupan
et al., 1996). Here, we describe a direct test of the involvement of both VirD2 and VirE2 in plant cell nuclear import.
A direct test involves the use of artificially produced T-DNA
complexes in combination with in vitro nuclear import systems. In previous studies, T-DNA complexes were reconstituted from fluorescently labeled ssDNA and purified VirD2
and VirE2 and tested in a mammalian in vitro nuclear import
system (Ziemienowicz et al., 1999). Such systems were
shown to be dependent on cytosolic factors such as importins ␣ and ␤, Ran, and pp15 (Adam et al., 1991; Moore and
Blobel, 1992; Görlich and Laskey, 1995). The import of T-DNA
complexes was shown to depend on both VirD2 and VirE2
(Ziemienowicz et al., 1999). The activity of VirD2, covalently
attached to the 5⬘ terminus of T-DNA, was dependent on
the C-terminal NLS. In this work, we studied the mechanism
of import of T-DNA complexes in tobacco in vitro nuclear
import systems. In these systems, the import of various protein substrates was shown to be NLS dependent, rapid, saturable, and inhibited by GTP␥S, but it did not require
externally added importins and other cytosolic factors
(Hicks et al., 1996; Merkle et al., 1996; Merkle and Nagy,
1997). These findings are consistent with the observation
that Arabidopsis importin ␣ localizes mainly at the nuclear
membrane (Smith et al., 1997).
We found that both virulence proteins, VirD2 and VirE2,
are required for efficient import of the T-DNA complex into
plant nuclei. According to the model proposed here, it is the
C-terminal NLS of the VirD2 protein that recognizes the nuclear import machinery. For complete translocation of the
T-DNA complex through the pore, the VirE2 protein is also
essential. VirE2 function in the nuclear import of T-DNA
could be substituted by another ssDNA binding protein,
RecA. These findings provide new insights into the mechanism of the nuclear import of large DNA–protein complexes.
RESULTS
To study directly the mechanism of nuclear import of the T-DNA
complex during Agrobacterium-mediated plant transformation,
we produced the T-DNA complex in vitro. Complexes consisting of ssDNA covalently attached to VirD2 and coated by VirE2
were tested for in vitro import into plant nuclei.
Reconstruction of the T-DNA Complex in Vitro
The artificial T-DNA complex was prepared using rhodamine-labeled ssDNA and purified VirD2 and VirE2 proteins (for details, see Ziemienowicz et al., 1999). VirD2 is a
site-specific cleaving-joining enzyme that nicks the lower
strand of the Ti plasmid at the border sequences (Yanofsky
et al., 1986; Stachel et al., 1987). The cleavage activity of
VirD2 could be reproduced in vitro using single-stranded oligonucleotides or denatured double-stranded DNA containing a border sequence (Pansegrau et al., 1993; Jasper et al.,
1994). We tested the efficiency of the cutting reaction on
rhodamine-labeled ssDNAs (25, 250, and 1000 nucleotides
long) containing the border sequence. The cleavage was
precise and efficient (95, 88, and 73% of the 25-, 250-, and
1000-nucleotide ssDNAs were cleaved, respectively), and
VirD2 remained covalently attached to the 5 ⬘ termini of the
cleaved border sequences (data not shown; see Pansegrau
et al., 1993). Cleavage by VirD2 lacking the C-terminal NLS
was comparable in efficiency and precision to cleavage by
the corresponding wild-type protein.
It has been shown that VirE2 binds to ssDNA in a cooperative manner (Citovsky et al., 1989; Sen et al., 1989; Zupan
et al., 1996). The cooperativity of the binding of our purified
VirE2 protein to ssDNA was tested in a gel shift assay, as
described previously (Ziemienowicz et al., 1999). Fluorescently labeled VirE2 behaved like unlabeled protein (data not
shown). To prepare a “complete” artificial T-DNA complex,
rhodamine-labeled or unlabeled ssDNA was cleaved by
VirD2, followed by incubation with an excess of unlabeled or
fluorescently labeled VirE2.
The contribution of virulence proteins to the import of
T-DNA complexes was tested in a plant in vitro protein import system (Merkle et al., 1996). The import of fluorescently
labeled substrates into evacuolated permeabilized tobacco
BY-2 cells was monitored using confocal microscopy.
VirD2 and VirE2 Are Imported into Plant Nuclei in Vitro
To test this import system for our purposes, we first analyzed the nuclear import of labeled virulence proteins VirD2
and VirE2. Both proteins contain NLSs that are functional in
vivo in targeting reporter proteins to the plant cell nucleus
(Herrera-Estrella et al., 1990; Citovsky et al., 1992, 1994;
Howard et al., 1992; Tinland et al., 1992). Also in the in vitro
VirD2/VirE2–Mediated Nuclear Import of T-DNA
371
import system, the virulence proteins accumulated in the
nuclei; fluorescently labeled VirD2, VirD2 ⌬NLS (mutant
VirD2 lacking the C-terminal NLS due to an NruI deletion but
still containing the N-terminal NLS), and VirE2 were found to
localize to the plant nuclei (Figure 1 and Table 1). Labeled
BSA linked to the NLS of simian virus 40 (SV40) large T antigen (BSA-NLS) was also found in the nuclei, whereas labeled BSA without NLS remained in the cytoplasm (data not
shown; Merkle et al., 1996). The kinetics of the import of
both VirD2 and VirE2 was comparable to that of the model
substrate (data not shown). These experiments demonstrate
that the import system used accurately reflects the intracellular distribution of virulence proteins and that it can be used
to analyze the import of the T-DNA complex.
Nuclear Import of a Short Oligonucleotide–VirD2
Complex Requires the C-Terminal NLS of the
VirD2 Protein
We used both wild-type VirD2 and VirD2 ⌬NLS to produce
import substrates by reacting the proteins with fluorescently
labeled 25-nucleotide oligonucleotides containing the right
border sequence of pTiA6. The oligonucleotide itself was
not transported actively to the nuclei (Figure 2A; Table 1). In
contrast, the short VirD2wt–ssDNA complex was actively
imported and accumulated rapidly in the nuclei of permeabilized plant cells (Figure 2B). The short ssDNA complexed
with the mutant VirD2⌬NLS protein was import deficient and
did not accumulate in the plant nuclei (Figure 2C). Thus, the
VirD2 protein can import a small covalently attached oligonucleotide to the plant nucleus. Moreover, this import was
absolutely dependent on the C-terminal NLS of VirD2.
Plant Nuclear Import of 250- and 1000-Nucleotide ssDNA
Requires an NLS Containing VirD2 as Well as VirE2
A 250-nucleotide, fluorescently labeled ssDNA on its own did
not accumulate in plant nuclei (Figure 3A and Table 1). In contrast to the experiments described above showing VirD2dependent nuclear translocation of 25-nucleotide ssDNA,
VirD2 covalently attached to the 250-nucleotide DNA was not
able to translocate it to the nucleus (Figure 3B). Only when
the VirD2wt–ssDNA complex was coated with VirE2 was import of the protein–DNA complex into the plant nuclei efficient
(Figure 3C) and fast (occurring with a kinetics similar to that of
the proteins tested alone: 90% of import in 10 min, 99 to
100% in 20 min). The characteristics of the nuclear import of
1-kb T-DNA complexes did not differ from those of complexes formed with a 250-nucleotide ssDNA (Table 1). The
nuclear import of both T-DNA complexes was dependent
on the presence of VirE2 and the C-terminal NLS of VirD2,
because efficient import of the VirD2 ⌬NLS–ssDNA–VirE2
complexes could not be detected (Figure 3D and Table 1).
Figure 1. Localization of VirD2 and VirE2 in Plant Nuclei.
(A) Localization of VirD2.
(B) Localization of mutant VirD2⌬NLS.
(C) Localization of VirE2.
Cy3.5-labeled proteins (red fluorescence) were incubated with permeabilized tobacco protoplasts for 20 min at room temperature in the
dark. The nuclei were stained with SYTO 13 dye (green fluorescence).
Fluorescently labeled components are indicated by asterisks.
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Table 1. Summary of Nuclear Import Experiments Using Complete
Import Systemsa
Substrate
Nuclear Import
VirD2wt protein*
VirD2⌬NLS protein*
VirE2 protein*
25-nt oligonucleotide*
VirD2wt–25-nt oligonucleotide*
VirD2⌬NLS–25-nt oligonucleotide*
250-nt ssDNA*
VirD2wt–250-nt ssDNA*
VirE2–250-nt ssDNA*
VirD2wt–250-nt ssDNA*–VirE2
VirD2⌬NLS–250-nt ssDNA*–VirE2
1-kb ssDNA*
VirD2wt–1-kb ssDNA*
VirE2–1-kb ssDNA*
VirD2wt–1-kb ssDNA*–VirE2
VirD2⌬NLS–1-kb ssDNA*–VirE2
VirE2*–250-nt ssDNA
VirE2*–38-nt ssDNA
VirD2wt–1-kb ssDNA–VirE2*⫹38-nt
oligonucleotide
VirD2⌬NLS–1-kb ssDNA–VirE2*⫹38-nt
oligonucleotide
⫹
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫺
a The import of different substrates into the plant nuclei was performed in vitro, as described in Methods. Each experiment was repeated at least three times, and for each sample, at least 100 nuclei
were scored. Asterisks indicate fluorescently labeled substrates. nt,
nucleotides; (⫹), 99 to 100% of nuclei showing red fluorescence;
(⫺), 0 to 1% of nuclei showing red fluorescence.
Because the import of longer ssDNAs required the VirE2
protein, we also tested whether VirE2 was sufficient for the
import of long complexes. Import of the 250-nucleotide or
1-kb ssDNA complexed with VirE2 alone was not detected
(Figure 3E; Table 1). These results allow the conclusion that
the targeting/import function of the VirD2 NLS is a necessary
prerequisite for nuclear entrance of VirE2-coated ssDNA (see
below) and that this coating is essential for the complete
translocation of larger DNA molecules through the NPC.
Import of the VirD2–T-DNA–VirE2 Complex into Plant
Cell Nuclei Follows the Rules of Protein Import into
Nuclei of Plant Cells
To determine whether T-DNA complex import and import of
protein molecules resemble each other mechanistically, we
tested known competitors and inhibitors of protein import.
The nuclear import of proteins in the plant in vitro system
has been shown to be inhibited by the nonhydrolyzable analog GTP␥S but not by wheat germ agglutinin (WGA), which
blocks the nuclear import of proteins in mammalian systems
(Hicks and Raikhel, 1995; Merkle et al., 1996). Consistent
with these data, import of the VirD2wt–ssDNA–VirE2 complex into the plant nuclei was inhibited only in the presence
of GTP␥S (Table 2) and not in the presence of WGA (Figure
3F and Table 2). In addition, import of the complete T-DNA
complex could be inhibited by an excess of unlabeled BSANLS competitor, whereas BSA alone was not able to block
T-DNA complex nuclear transport (Table 2). These results
suggest that the classic protein import pathway is used to
transport the T-DNA complex to plant nuclei.
VirE2 Bound to ssDNA Is Unable to Translocate to the
Nucleus without the Action of the VirD2 NLS
The inability of the VirE2 protein alone to import ssDNA into
the plant nucleus suggested that the NLSs of VirE2 are not
available to the import machinery when the protein is bound
to ssDNA (see above). To analyze the fate of VirE2 in the
localization of the various complexes, we performed the
complementary experiment in which we monitored the distribution of fluorescently labeled VirE2 in the plant in vitro
import system. Although the protein by itself moved to the
nucleus (Figure 1C), it remained in the cytoplasm when
bound to an oligonucleotide of 38 (Figure 4A and Table 1) or
250 (Table 1) nucleotides. These data confirm the results
observed with a DNA-labeled complex. In addition, they imply that binding to DNA actually sequesters the NLSs of
VirE2 or otherwise makes them unavailable to the import
machinery.
The experiments described so far demonstrated that 250nucleotide and longer ssDNAs efficiently enter plant nuclei
only when complexed to VirD2 and VirE2. VirD2 most likely
remains attached to the T-DNA upon translocation to the
nucleus, because it still has a function in integration (Tinland
et al., 1995; Mysore et al., 1998). To analyze the fate of the
VirE2 protein molecules as part of the T-DNA complex, we
produced a complex from unlabeled 1-kb ssDNA covalently
attached to VirD2 and reacted it with fluorescently labeled
VirE2. Any unreacted VirE2 was bound to an excess of
38-nucleotide oligonucleotide, yielding a complex that could
not be imported (see above). A VirE2-derived signal was
found in the nucleus only when VirD2wt was complexed
with T-DNA and VirE2; when the mutant VirD2 ⌬NLS protein
was part of the complex, the labeled VirE2 protein molecules remained excluded (Figures 4B and 4C and Table 1).
These data confirm that the VirE2 protein entered into the
nuclei as part of the T-DNA complex and was not stripped
off in the cytoplasm upon nuclear entry.
RecA but Not SSB Can Substitute for VirE2 Function in
the Nuclear Import of T-DNA
To further evaluate the need for NLSs of the VirE2 protein in
the nuclear import of T-DNA, we tested other ssDNA bind-
VirD2/VirE2–Mediated Nuclear Import of T-DNA
373
Figure 2. Import of a 25-Nucleotide Oligonucleotide into Plant Nuclei Requires the Functional NLS of the VirD2 Protein.
(A) Oligonucleotide alone.
(B) Oligonucleotide bound to VirD2wt.
(C) Oligonucleotide bound to mutant VirD2⌬NLS.
The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). The plant nuclear import assay was performed using
rhodamine-labeled oligonucleotide (red fluorescence), as described in Methods. Fluorescently labeled components are indicated by asterisks.
ing proteins that do not contain NLSs for their ability to substitute for the function of VirE2. First, we tested the
localization of the Escherichia coli ssDNA binding proteins
SSB and RecA. Whereas the SSB protein was found not to
enter the plant nuclei, RecA localized both outside and inside of the plant nuclei (Table 3). This transfer of RecA into
plant nuclei occurred in an importin-independent and likely
passive manner, because (1) RecA does not possess any
motif resembling known NLSs, (2) it is a small protein (38
kD) that is expected to pass freely through the channel of
the NPC, and (3) transfer of RecA into plant nuclei was not
affected by the import inhibitor GTP ␥S or the import competitor BSA-NLS (Table 3). This is in agreement with previous findings that the nuclear targeting of RecA was strongly
enhanced when the protein was fused to an NLS, whereas
unmodified RecA localized poorly to the nuclei of RecAexpressing transgenic tobacco cells (Reiss et al., 1996). We
tested the ability of the SSB and RecA proteins to substitute
for the function of VirE2 in nuclear import of the T-DNA
complex. Import of ssDNA–protein complexes into plant nuclei occurred only when RecA but not SSB was used in
place of VirE2 (Table 3). Import of the VirD2–ssDNA–RecA
complex was dependent on the C-terminal NLS of VirD2
(Table 3), as was observed for VirD2–ssDNA–VirE2 complexes (Table 1).
RecA Cannot Substitute for VirE2 in Efficient T-DNA
Transfer to Tobacco Plants
Efficient T-DNA transfer by Agrobacterium lacking VirE2
was reestablished by expressing VirE2 in the target plant
cells (Citovsky et al., 1992). Because RecA can substitute
for VirE2 in the entry of T-DNA into the nucleus (see above),
transgenic plants expressing RecA or nt-RecA (RecA with
the NLS of the SV40 large T antigen) were tested for their
ability to complement the Agrobacterium strain lacking
VirE2. The T-DNA transfer efficiency was calculated based
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Figure 3. Import of a 250-Nucleotide ssDNA into Plant Nuclei Requires Both VirD2 and VirE2 Proteins.
(A) ssDNA alone.
(B) VirD2wt–ssDNA complex.
(C) VirD2wt–ssDNA–VirE2 complex.
(D) VirD2⌬NLS–ssDNA–VirE2 complex.
(E) VirE2–ssDNA complex.
(F) VirD2wt–ssDNA–VirE2 complex and wheat germ agglutinin (WGA; final concentration 0.5 mg/mL).
The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). In vitro import of different ssDNA–protein complexes into plant nuclei was performed as described in Methods. DNA was labeled with rhodamine (red fluorescence). Fluorescently labeled
components are indicated by asterisks.
VirD2/VirE2–Mediated Nuclear Import of T-DNA
on the activity of the ␤-glucuronidase gene (uidA) present on
T-DNA, as measured by the number of blue spots on tobacco seedlings. The efficiency of T-DNA transfer from the
virE2⫺ Agrobacterium strain into seedlings of wild-type SR1
tobacco and plantlets transgenic for VirE2 or RecA was
compared with the efficiency of a wild-type Agrobacterium
strain. As expected, VirE2 transgenic plants were found to
complement the strain lacking VirE2 for efficient T-DNA
transfer (data not shown). However, the efficiency of T-DNA
transfer by an Agrobacterium strain lacking VirE2 to transgenic plants expressing RecA or nt-RecA was found to be
similar to the transfer efficiency to wild-type SR1 plants (Table
4). Thus, RecA transgenic plants are not able to complement
the absence of VirE2 in Agrobacterium. This finding suggests
an activity of VirE2 that goes beyond nuclear import.
DISCUSSION
Agrobacterium is a plant pathogen that naturally is able to
transfer 10- to 20-kb DNA to the nucleus of the plant cell.
Even transfer and integration of T-DNAs as long as 150 and
170 kb have been demonstrated (Miranda et al., 1992;
Hamilton et al., 1996). According to a model for the structure of the VirE2–T-DNA complex (Citovsky et al., 1997), 20and 170-kb DNA represent complexes 1.4 and 12 ␮m long,
respectively. These sizes have to be compared with the dimensions of the NPC through which the T-DNA complex
has to travel: 120 nm in diameter and 100 nm in depth (reviewed in Moroianu, 1997). Transfer of T-DNA to the nucleus
therefore poses logistic problems similar to those of translocation of long mRNAs into the cytoplasm; in both cases, the
NPC has to be recognized by one extremity of the molecule,
and the translocation of a long molecule has to be executed
to completion. We set out to explore the cell’s ability to permit nuclear passage of complex structures. For this effort,
we reconstructed ssDNA–VirD2–VirE2 complexes and analyzed in detail their import into plant nuclei. We found that
the T-DNA complex was imported efficiently into the nuclei
of permeabilized tobacco cells.
In living cells, many nucleic acids move into and out of the
nucleus. These include cellular RNAs such as tRNA, small
nuclear RNA, and spliced mRNA as well as viral RNAs and
DNAs. In general, transport of these nucleic acids seems to
be mediated by proteins (reviewed in Nakielny et al., 1997;
Nakielny and Dreyfuss, 1999). For instance, small nuclear
RNAs are exported to the cytoplasm, where they are complexed with proteins into small nuclear RNA particles that
then use the m3G cap and Sm protein for reimport into the
nucleus (Fischer et al., 1991; Michaud and Goldfarb, 1992;
Marshallsay and Lührmann, 1994; Palacios et al., 1997). The
uptake of influenza virus RNA has been suggested to be
mediated by its nucleoprotein (O’Neill et al., 1995), and human immunodeficiency virus-1 viral DNA is presumably
transported to the nucleus via NLSs of the Gag matrix and
375
Table 2. Characteristics of the Nuclear Import of Proteins and the
T-DNA Complexa
Substrate
Inhibitors/
Competitors
Nuclear Import
VirD2*, VirE2*
VirD2*, VirE2*
VirD2*
VirD2*
VirD2wt–1-kb ssDNA*–VirE2
VirD2wt–1-kb ssDNA*–VirE2
VirD2wt–1-kb ssDNA*–VirE2
VirD2wt–1-kb ssDNA*–VirE2
WGA
GTP␥S
BSA-NLS
BSA
WGA
GTP␥S
BSA-NLS
BSA
⫹
⫺
⫺
⫹
⫹
⫺
⫺
⫹
a The import assay of fluorescently labeled proteins (VirD2*, VirE2*)
and T-DNA complexes was performed in vitro using plant nuclei, as
described in Methods. Each experiment was repeated at least three
times, and for each sample, at least 100 nuclei were scored. Asterisks indicate fluorescently labeled substrates. (⫹), 99 to 100% of nuclei showing red fluorescence; (⫺), 0 to 1% of nuclei showing red
fluorescence.
Vpr proteins in the form of a nucleoprotein preintegration
complex (Goldfarb, 1995; Stevenson, 1996).
In a manner similar to that of cellular and viral nucleic acids that have to pass the nuclear membrane, the T-DNA of
Agrobacterium also uses proteins for its nuclear entry. The
two bacterial proteins VirD2 and VirE2 are associated with the
T-DNA, travel to the plant cell during Agrobacterium-mediated plant transformation, and are known to contain functional NLSs. In the experiments reported here, both proteins
localized to the nuclei of permeabilized tobacco cells, suggesting that their NLSs are functional in protein import in
this system. This is consistent with previous findings of the
localization of both VirD2 and VirE2 proteins in plant nuclei
in vivo (Citovsky et al., 1992, 1994; Rossi et al., 1993).
To reconstruct the T-DNA complex, we attached VirD2
enzymatically to the 5⬘ ends of ssDNAs of different lengths,
and these complexes were coated with VirE2. This artificial
T-DNA complex is currently the best possible approximation
of the native complex, although we cannot exclude the possibility that other proteins of bacterial and/or plant origin are
also involved in vivo.
Function of VirD2 in the Nuclear Import of ssDNA
The VirD2 protein alone was sufficient to transfer short
single-stranded oligonucleotides to tobacco nuclei. This
function was strictly dependent on the presence of the C-terminal NLS of the VirD2 protein. These results are consistent
with genetic data reporting that only the C-terminal NLS of
this protein is necessary for the efficient transformation of
plants (Shurvinton et al., 1992; Rossi et al., 1993). Interestingly, in several experimental systems, deletions of the
C-terminal NLS did not lead to a complete block of virulence
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The Plant Cell
(Shurvinton et al., 1992; Mysore et al., 1998), whereas in our
system, the NLS was absolutely required for nuclear import
of the T-DNA complex. These differences may be explained
in part by the different NLS mutants used. In addition, the
import system used here is independent of cell divisions,
whereas dividing cells may allow the entry of proteins lacking NLSs into newly forming nuclei.
Our data also reveal that the N-terminal NLS by itself
could not mediate the transport of the T-DNA complex to
the nucleus, although this NLS is functional in transporting
the DNA-free form of VirD2 (Herrera-Estrella et al., 1990;
Tinland et al., 1992; Rossi et al., 1993) (Figure 2B). This finding indicates that the N-terminal NLS of VirD2 is masked
when the protein is covalently bound to DNA; indeed, the tyrosine 29 with which VirD2 is bound to DNA is only a few
amino acids away from the N-terminal NLS.
Function of VirE2 in the Nuclear Import of ssDNA
Import of a 25-mer oligonucleotide required VirD2 only (see
above). However, rapid and efficient nuclear import of long
(250 and 1000 nucleotides) ssDNAs required the additional
presence of the VirE2 protein. Neither of the two proteins
alone led to the efficient import of long ssDNAs. These results and those discussed above lead to the proposal that
VirD2 pilots the complex to the nuclear pore and initiates
import, whereas VirE2 is required to ensure the translocation
of the entire T-DNA complex. Therefore, VirE2 may have
several roles that are not mutually exclusive. Its importance
may be attributed to its NLSs; it may allow specific interactions between the NPC and the T-DNA complex, and it may
exert its function by shaping the T-DNA complex into a form
competent for translocation through a nuclear pore.
In our experiments, the VirE2 protein, although it was able
to enter the plant nucleus as free protein, was excluded
from the nucleus when bound to ssDNA. These results indicate that the NLSs of VirE2 are inaccessible to the nuclear
import machinery when the protein is bound to DNA. Indeed, analyses of VirE2 revealed a partial overlap of the
ssDNA binding domain or the cooperativity domain with the
NLSs (Citovsky et al., 1992, 1994; Dombek and Ream,
1997). This is consistent with the notion that DNA/RNA
binding domains of the majority of nuclear proteins are composed of basic amino acids and therefore resemble and can
function as NLSs (LaCasse and Lefebvre, 1995). Another
Figure 4. Localization of the VirE2 Protein Associated with ssDNA in
Plant Nuclei.
(A) A 38-nucleotide oligonucleotide reacted with labeled VirE2.
(B) VirD2wt–ssDNA–VirE2-labeled complex quenched with excess
of 38-nucleotide oligonucleotide.
(C) VirD2⌬NLS–ssDNA–VirE2-labeled complex quenched with excess of 38-nucleotide oligonucleotide.
In vitro nuclear import of different complexes of Cy3.5-labeled VirE2
(red fluorescence) with ssDNAs was performed as described in
Methods. The positions of the nuclei are indicated by staining with
SYTO 13 dye (green fluorescence). Fluorescently labeled components are indicated by asterisks.
VirD2/VirE2–Mediated Nuclear Import of T-DNA
Table 3. RecA but Not SSB Can Substitute for VirE2 in T-DNA
Nuclear Importa
Substrate
SSB*
RecA*
RecA*
RecA*
VirD2wt–1-kb ssDNA*–VirE2
VirD2wt–1-kb ssDNA*–RecA
VirD2⌬NLS–1-kb ssDNA*–RecA
VirD2wt–1-kb ssDNA*–SSB
VirD2wt–1-kb ssDNA*–RecA
VirD2wt–1-kb ssDNA*–RecA
Inhibitors/
Competitors
GTP␥S
BSA-NLS
GTP␥S
BSA-NLS
Nuclear Import
⫺
⫹/⫺
⫹/⫺
⫹/⫺
⫹
⫹
⫺
⫺
⫺
⫺
a The import assay of fluorescently labeled proteins and T-DNA complexes was performed in vitro using plant nuclei, as described in
Methods. Each experiment was repeated at least three times, and
for each sample, at least 100 nuclei were scored. Asterisks indicate
fluorescently labeled substrates. (⫹), 99 to 100% of nuclei showing
red fluorescence; (⫺), 0 to 1% of nuclei showing red fluorescence;
(⫹/⫺), red fluorescence was distributed equally outside and inside
the nucleus.
important aspect of the function of VirE2 in the nuclear import of the T-DNA complex was revealed by our search for
other ssDNA binding proteins that may replace VirE2 in this
process. Although the RecA protein does not contain a sequence resembling known NLSs, it could substitute for VirE2
in the T-DNA complex during nuclear targeting. The ability
of the NLS lacking the RecA protein to function in the nuclear import of ssDNA–protein complexes suggests again
that the function of the agrobacterial ssDNA binding protein
(VirE2) does not seem to involve its NLSs.
Our results do not support a report describing the function of the VirE2 protein in the uptake of ssDNA into plant
nuclei. The nuclear accumulation of fluorescently labeled
ssDNA microinjected into the stamen hairs of Tradescantia
virginiana was found to depend on binding of VirE2 to the
ssDNA (Zupan et al., 1996). Import of the ssDNA–VirE2
complex into the nucleus of stamen hairs of T. virginiana was
inhibited by WGA, in contrast to the lack of inhibition by
WGA in the plant in vitro nuclear import systems described
to date (Hicks et al., 1996; Merkle et al., 1996) and in our experiments. We deliberately tested VirD2-independent nuclear import of ssDNA–VirE2 complexes, but we detected
only very inefficient import after drastic extension of the reaction time (from 20 min to 2 hr). This finding may reflect the
low residual but VirE2-dependent T-DNA transfer observed
in vivo in the absence of the C-terminal NLS of VirD2 (Rossi
et al., 1996), although other explanations are also possible.
This low activity is probably not important for the process of
Agrobacterium-mediated plant transformation. To resolve
this issue, microinjection experiments using complexes
377
consisting of ssDNA, VirE2, and VirD2 may have to be performed.
Studies of VirE2 function in several animal systems have
led to the discovery of interesting differences. Therefore, it
cannot be excluded that transfer of T-DNA complexes
through NPCs of different animal nuclei follows slightly different rules. ssDNA–VirE2 complexes tested for import into
the nuclei of permeabilized HeLa cells were found to be excluded from them (Ziemienowicz et al., 1999). On the other
hand, ssDNA–VirE2 complexes microinjected into Drosophila embryonic cells and Xenopus oocytes were found to localize in the nuclei (Guralnick et al., 1996). However, the
NLSs of the VirE2 protein used in the latter experiments
were modified slightly to resemble the NLS of nucleoplasmin (Guralnick et al., 1996), whereas the VirE2 protein used
in experiments with permeabilized HeLa and tobacco cells
contained unmodified NLS sequences.
Alternately (or in addition), VirE2 may actively participate
in the translocation of the T-DNA complex by specific interactions inside the channel of the NPC. Inside this channel,
the translocation substrate has to travel a distance of ⵑ100
nm. The proteins seem to pass through a number of import
intermediate points in the channel, but until now the mechanism of the actual translocation has remained obscure
(Görlich, 1997; Nakielny and Dreyfuss, 1999). It is possible
that VirE2 could interact with those uncharacterized intermediates and favor the translocation of the huge T-DNA
complex into the nucleus. However, these specific interactions would not be expected for the RecA protein.
ssDNA alone forms complex secondary structures
(Chrysogelos and Griffith, 1982; Flory and Radding, 1982)
that may hinder its passage through the channel of the nuclear pore. The VirE2 protein coat may impose a specific
structure onto the otherwise unstructured but charged
ssDNA, thereby allowing translocation through the NPC channel. Electron microscopy data suggest that VirE2 packages
ssDNA into semirigid and hollow cylindrical filaments having
a telephone cord–like coiled structure that potentially can be
Table 4. T-DNA Transfer to Wild-Type and Transgenic VirE2 and
RecA Plantsa
Plant
Transfer Efficiency (%)
SR1
RecA
nt-RecA
VirE2
0.018
0.011⫾0.0553
0.032⫾0.0368
100
a A complementation experiment with an Agrobacterium strain lacking
the virE2 gene and wild-type (SR1) or transgenic (VirE2, RecA, or ntRecA) tobacco seedlings was performed, as described in Methods.
Complementation efficiency was determined by comparing the T-DNA
transfer efficiency of the Agrobacterium strain lacking VirE2 with the
transfer efficiency of the wild-type strain. Efficient complementation
was obtained only with the VirE2 transgenic plants (set to 100%).
378
The Plant Cell
extended upon passage through the nuclear pore channel
(Citovsky et al., 1997). RecA stretches the DNA upon binding to it (the DNA length is extended by 50%) (Flory et al.,
1984; Nishinaka et al., 1997), leading to the formation of
long filaments of DNA–protein complexes that are only 5.2
nm (Flory and Radding, 1982) or 9.3 nm (Flory et al., 1984)
wide. Both values are ⬍10 nm (the diameter of the NPC
channel), and the former value is similar to that calculated
for ssDNA–VirE2 complexes (4.4 nm) in the extended stage
(that is, before folding into more complexed helical filaments
of 12.6 nm; Citovsky et al., 1997). In contrast, upon binding
to ssDNA, SSB condenses it, leading to the formation of
compact filaments of nucleosome-like structure with a width
of 12 nm (Chrysogelos and Griffith, 1982) that may prevent
passage of the DNA–protein complex through the 10-nm-wide
NPC channel.
Although the requirement for the VirE2 protein in the nuclear import of ssDNA is well documented, it was not clear
whether the VirE2 protein molecules bound to the DNA actually enter the nucleus or are stripped off upon entrance
into the NPC channel. The experiments reported here, however, unequivocally demonstrate that VirE2 enters plant nu-
clei together with the T-DNA complex in a strictly VirD2dependent fashion. In the nucleus, VirE2 and VirD2 take on
new roles in the transformation process, as “chaperone” to
protect the DNA from nucleolytic attack (VirE2; Rossi et al.,
1996) and as an aid in the integration of complete T-DNA
units into the plant genome (VirD2; Tinland et al., 1995;
Mysore et al., 1998).
Lack of VirE2 in an Agrobacterium strain can be complemented by its expression in the plant cell, indicating that this
protein performs its function inside the recipient plant cell.
However, the function of the VirE2 protein in the plant cell is
not limited to the nuclear import of T-DNA. We performed
complementation tests using a VirE2 ⫺ Agrobacterium strain
for transformation of wild-type or transgenic tobacco seedlings expressing either VirE2 or RecA. Although RecA was
able to replace VirE2 in nuclear import of T-DNA complexes,
it was unable to substitute for VirE2 in the efficient transformation of tobacco plants. This finding indicates that VirE2 is
not only essential for the transfer of large T-DNA molecules
into the nucleus but also required in a step preceding or following nuclear import, such as movement of the complex
inside the plant cell, transport of the T-DNA complex to the
Figure 5. Model for the Nuclear Import of the T-DNA Complex.
(A) Docking of the VirD2–T-DNA–VirE2 complex at the nuclear pore.
(B) and (C) Passage through the nuclear pore channel.
The nuclear import machinery and NPC are simplified in this model. Sizes are not to scale. Nuclear import intermediates are indicated by black
triangles. See text for more details.
VirD2/VirE2–Mediated Nuclear Import of T-DNA
379
nuclear pore, or integration of the T-DNA into the plant
genome.
to nuclear import intermediates inside the pore to facilitate
translocation.
Import of T-DNA Complexes into the Nuclei of Tobacco
Cells Is Mediated by the Classic Nuclear Import Pathway
METHODS
In our experiments, the import of the T-DNA complex into
plant nuclei was as specific as the import into HeLa cell nuclei reported previously (Ziemienowicz et al., 1999). Interestingly, import into mammalian nuclei was mediated by the
classic NLS-dependent mechanism, because it required importin ␣ (Rch1; Ziemienowicz et al., 1999), which was shown
to bind to all tested NLS substrates (Miyamoto et al., 1997).
Because the import of the T-DNA complex into plant nuclei
was inhibited by BSA coupled to an NLS and by GTP␥S, an
importin-dependent pathway is likely to be used to import
the complex into plant nuclei as well. Currently, several importins ␣ can be found in Arabidopsis databases, but only
two of them were characterized. One, AtKAP␣, recognizes
the C-terminal but not the N-terminal NLS of VirD2 (Ballas
and Citovsky, 1997); the other recognizes three classes of
import signals (Smith et al., 1997). The former importin may
be involved in the import of the T-DNA complex in plants,
whereas the latter one may be involved in the nuclear targeting of VirD2 devoid of its C-terminal NLS by interaction
with its N-terminal NLS, an activity that is probably irrelevant
biologically. Thus, Agrobacterium seems to have “learned”
to abuse the classic nuclear protein import pathway to efficiently ferry large DNA molecules into the plant nucleus.
Cloning of virE2 into the Expression Vector pET3a
A Model for Nuclear Import of the T-DNA Complex
On the basis of our new data and published observations,
we propose a model for the uptake of the T-DNA complex
into plant nuclei (Figure 5). The T-DNA in the plant cell occurs in a covalent complex with the VirD2 protein, covered
by VirE2. The VirD2 protein pilots the T-DNA complex to the
nuclear pore using its C-terminal NLS, which is recognized
by the NLS receptor importin ␣. The T-DNA complex bound
to importin ␣ then docks to the NPC via importin ␤, and the
5⬘ terminus of the T-DNA is directed toward the nuclear
pore channel, where translocation is initiated. This is analogous to the nuclear export of the Balbiani ring RNA particle,
which leaves the nucleus with the 5⬘ end in the lead (Daneholt,
1997). During translocation, the RNA particle moves through
the channel in an extended state. For translocation to completion of a long T-DNA complex through the nuclear pore
channel, VirE2 is required. VirE2, by its cooperative binding
to ssDNA, covers its negative charges and creates a structure that enables translocation through the nuclear pore.
Such a structure, which electron microscopy reveals to
resemble a telephone cord, can be extended upon passage
of the T-DNA complex through the nuclear pore channel
(Citovsky et al., 1997). Alternately (or in addition), VirE2 binds
The plasmid pSW108 (Winans et al., 1987) contains an XhoI fragment
from pTiA6 encompassing the virE operon promoter and the open
reading frames of VirE1 and VirE2. A StuI-SmaI fragment from
pSW108 was inserted in the HincII site of pUC18, resulting in the
plasmid pUCSS. An NdeI restriction site was introduced into the first
ATG (underlined) of the virE2 gene by polymerase chain reaction
(PCR) using primers p1 (5⬘-ATCGTAGCCTGCAGAGTCATATGGATCTTTCTGGCAATGAGAAATCC-3⬘) and p2 (5⬘-GTTTGATAAAAGATCTCTGTGCC-3⬘) and plasmid pSW108 as a template. The PCR
product was cut with PstI and BglII and inserted into pUCSS previously cut with the same restriction enzymes, yielding the plasmid
pUCE2. An NdeI-BamHI fragment from plasmid pUCE2 was inserted
in plasmid pET3a (Studier et al., 1990) previously digested with the
same enzymes, resulting in plasmid pETE2.
Overexpression and Purification of VirE2
The plasmid pETE2 was introduced into Escherichia coli strain
BL21(DE3) (Studier et al., 1990) by electroporation. The transformed
cells were grown in 500 mL of Luria-Bertani medium containing 100
␮g/mL ampicillin at 37⬚C with shaking. At OD600 ⫽ 0.5 to 1, expression was induced by the addition of isopropyl-␤-D-thiogalactoside to
a final concentration of 1 mM. Shaking was continued for 5 hr. The
cells were then pelleted by centrifugation at 4000g at 4⬚C for 30 min.
The pellet was resuspended in 50 mL of ice-cold lysis buffer A1 (50
mM Tris-HCl, pH 8.5, 50 mM NaCl, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM DTT, 0.5 mg/mL lysozyme, and
0.1% Tween 20). After 1 hr of incubation on ice, the lysis mixture was
centrifuged at 20,000g for 10 min at 4⬚C. The pellet was resuspended
in 25 mL of buffer B1 (50 mM Tris-HCl, pH 8.5, 1 M NaCl, 5 mM
EDTA, 0.1 mM PMSF, and 1 mM DTT) containing 1% Tween 20, incubated for 30 min on ice, and centrifuged at 20,000g for 10 min. The
pellet was washed in the same volume of buffer B1 containing 0.1%
Tween 20. After centrifugation (20,000g for 10 min), the VirE2 protein
was solubilized in 50 mL of buffer C1 (25 mM NaOAc, pH 5.0, 0.1 mM
PMSF, 1 mM DTT, and 6 M urea) and incubated for 30 min on ice.
The supernatant fluid obtained after centrifugation (20,000g for 10
min) represented the inclusion body fraction of proteins containing
mainly VirE2. This fraction was applied to a cation exchange column
(EconoPacS; Bio-Rad) equilibrated in buffer C1. Bound proteins
were eluted with a linear NaCl gradient (0 to 1 M). VirE2 peak fractions were dialyzed overnight against buffer D1 (50 mM Tris-HCl, pH
8.5, 100 mM NaCl, and 0.05% Tween 20). After centrifugation
(20,000g for 10 min), the supernatant solution was applied to a single-stranded DNA (ssDNA) agarose column (Amersham-Pharmacia,
Dübendorf, Switzerland) equilibrated with buffer E1 (50 mM Tris-HCl,
pH 8.5, and 100 mM NaCl). VirE2 was eluted with a linear MgCl2 gradient (0 to 1 M) in the same buffer. The VirE2 peak fractions were dialyzed overnight against buffer E1, centrifuged (20,000g for 10 min)
to remove insoluble material, frozen in liquid nitrogen in small aliquots, and stored at a concentration of 50 to 100 ng/mL at ⫺80⬚C.
380
The Plant Cell
ssDNA Binding Assay
The purified VirE2 protein as well as the RecA protein (New England
BioLabs, Bioconcept, Allschwil, Switzerland) were tested for their
ss-DNA binding properties, as described previously (Citovsky et al.,
1989; Ziemienowicz et al., 1999). Nearly the same amount of VirE2
(0.2 ␮g) or RecA (0.28 ␮g) was required to cover completely 1 ng of
250-nucleotide ssDNA.
Production of Fluorescently Labeled ssDNA
The 25-nucleotide oligonucleotide 5⬘-CAACGGTATATATCCTGCCAGTCAG-3⬘, containing 20 nucleotides of the pTiA6 border sequence
(underlined), was labeled with tetramethylrhodamine-6–dUTP at
the 3⬘ end using terminal transferase (Boehringer Mannheim), according to the protocol provided by the supplier. Rhodamine-labeled
1-kb DNA was produced by PCR amplification, as described elsewhere (Ziemienowicz et al., 1999), using two primers, each containing 20 nucleotides of the pTiA6 border sequence. For production of
250-nucleotide ssDNA, primers p3 (5⬘-CCACGGTATATATCCTGCCAGGGTATTTCACACCGCATATGG-3⬘) and p5 (5⬘- CCACGGTATATATCCTGCCAGGTAGAGAATTATGC-3⬘) were used.
Site-Specific ssDNA Cleavage Activity of VirD2
For overexpression of the VirD2 wild-type and VirD2⌬NLS mutant proteins, the previously described plasmids pFSVirD2 and
pFSVirD2NruI were used (Tinland et al., 1994; Ziemienowicz et al.,
1999). In the plasmid pFSVirD2NruI, a sequence localized between
two NruI sites within the virD2 gene was deleted. This deletion resulted in a mutant VirD2 lacking the C-terminal nuclear localization
signal (NLS) and 29 amino acids surrounding it. For simplicity, in this
report, this mutation is called VirD2⌬NLS. VirD2wt and VirD2⌬NLS
mutant proteins were purified as described previously (Pansegrau et
al., 1993). The efficiency of the cutting reaction by VirD2 and
VirD2⌬NLS was tested on rhodamine-labeled 250-nucleotide and
1-kb ssDNAs and on rhodamine-labeled 25-nucleotide oligonucleotide, as described (Ziemienowicz et al., 1999). The efficiencies were
comparable.
Preparation of Fluorescently Labeled Proteins
Proteins were labeled directly with Cy3.5 reagent (from the Cy3.5
monoclonal antibody labeling kit; Amersham-Pharmacia) using the
same protocol recommended by the supplier for labeling of monoclonal antibodies.
Formation of the VirD2–ssDNA–VirE2 Complex
For the formation of the VirD2–ssDNA–VirE2 complex, 0.15 ␮g of unlabeled or rhodamine-labeled 1-kb ssDNA (or 40 ng of 250-nucleotide ssDNA or 10 pmol of 25-nucleotide oligonucleotide) was
reacted with 7.2 ␮g of VirD2wt or VirD2⌬NLS protein in TNM buffer
(20 mM Tris-HCl, pH 8.8, 50 mM NaCl, and 5 mM MgCl2) for 1 hr at
37⬚C (15 ␮L final volume). The full complex was made by adding 0.5
␮g of unlabeled or fluorescently labeled VirE2 and incubating the
mixture (20 ␮L final volume) for 30 min on ice.
Assay of in Vitro Nuclear Import in Permeabilized Evacuolated
BY2 Protoplasts
To test the nuclear import of virulence proteins and T-DNA complexes, an in vitro system of permeabilized evacuolated protoplasts
derived from tobacco (Nicotiana tabacum) BY2 suspension-cultured
cells was used as described previously (Merkle et al., 1996). To 20 ␮L
of permeabilized plant cells, 0.2 ␮g of VirD2 or VirE2 protein or 4 ␮L
of T-DNA complex with or without VirD2wt or VirD2⌬NLS and/or
VirE2 protein was added. In quenching experiments, 4 ␮L of formed
complex was incubated on ice for 15 min with 585 pmol of a 38-nucleotide oligonucleotide (5⬘-GGTATATATCCTGCCAGGGTATTTCACACCGCATATGG-3⬘) before the import reaction. For inhibition
experiments, wheat germ agglutinin (WGA) (Sigma; final concentration 0.5 mg/mL) or GTP␥S (final concentration 8 mM) was added to
the reaction mixture, followed by incubation for 20 min at room temperature before the addition of the complex. The import reaction was
performed for 20 min at room temperature in the dark. For the competition experiments, nuclei diluted 10-fold (in import buffer without
Triton X-100) were preincubated with 58 ␮g of unlabeled BSA linked
to the nuclear localization signal of simian virus 40 (SV40) large T antigen (BSA-NLS) or BSA for 5 min on ice and then incubated with 0.2
␮g of fluorescently labeled BSA-NLS or VirD2 or 0.4 ␮L of the complex for 5 min on ice. Nuclei were visualized by staining with SYTO 13
(Molecular Probes, Eugene, OR) at a final concentration of 0.5 ␮M.
Localization of fluorescently labeled samples was analyzed by confocal microscopy using a fluorescein isothiocyanate filter for the dye
and a tetramethyl rodamine isothiocyanate filter for rhodamine- or
Cy3.5-labeled samples. No transmission was observed between
these two channels. Signal detection (sensitivity) and processing of
the scanned images were identical within each experimental series.
Transgenic Tobacco Plants Expressing the VirE2 or
RecA Protein
SR1 tobacco lines G64/2 and G63/19 transgenic for the recA gene
and for the SV40 large T antigen NLS sequence fused to the 5⬘ end of
the recA gene (nt-recA) were described previously (Reiss et al.,
1996). SR1 E3 tobacco plants contain the virE2 gene of the nopaline
strain of Agrobacterium tumefaciens under the control of the 35S
promoter and the polyadenylation signal of cauliflower mosaic virus
(H. Steinbiss, unpublished data). The amount of the RecA, nt-RecA,
and VirE2 protein produced in G64, G63, and E3 plants was similar
(ⵑ0.1% of the total plant protein).
Complementation Assay
Agrobacterium strains GV3101(pPM6000, pTd33), GV3101(pPM6000K,
pTd33) containing a deletion of ⵑ70% of the virD2 gene, and
GV3101(pPM6000E, pTd33) containing a deletion of the virE2 open
reading frame were used for transient transformation of tobacco
seedlings, as described elsewhere (Rossi et al., 1993, 1996). The efficiency of T-DNA transfer by the GV3101(pPM6000E, pTd33) Agrobacterium strain to young tobacco seedlings of SR1 plants and
transgenic plants expressing RecA, nt-RecA, and VirE2 was measured as described previously (Rossi et al., 1993). In this test, the activity of ␤-glucuronidase encoded by the uidA gene present in the
T-DNA of plasmid pTd33 (Tinland et al., 1995) represents a measure
VirD2/VirE2–Mediated Nuclear Import of T-DNA
of the concentration of T-DNA molecules arriving in the plant nucleus. T-DNA transfer efficiency was evaluated by counting the number of blue spots that formed in 50 seedlings after incubation with
5-bromo-4-chloro-3-indolyl-␤-D-glucuronide, the substrate for ␤-glucuronidase. This number was compared with that for wild-type strain
GV3101(pPM6000, pTd33) used at different concentrations. Dilution
of agrobacterial strains used for inoculations was done with the
transfer-defective strain GV3101(pPM6000K, pTd33) (Rossi et al.,
1993).
ACKNOWLEDGMENTS
We thank Christopher Marshallsay for donation of fluorescently labeled BSA-NLS, Serge Kocher for help with the confocal microscope, and Véronique Gloeckler for excellent technical assistance.
We thank Dr. B. Reiss for donation of transgenic plants expressing
RecA and nt-RecA, and Dr. H. Steinbiss (Max-Planck-Institute, Cologne, Germany) for donation of transgenic plants expressing VirE2.
We also thank Drs. Pawel Pelczar and Philippe Crouzet for critical
reading of the manuscript.
Received August 15, 2000; accepted December 1, 2000.
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Import of Agrobacterium T-DNA into Plant Nuclei: Two Distinct Functions of VirD2 and VirE2
Proteins
Alicja Ziemienowicz, Thomas Merkle, Fabrice Schoumacher, Barbara Hohn and Luca Rossi
Plant Cell 2001;13;369-383
DOI 10.1105/tpc.13.2.369
This information is current as of August 3, 2017
References
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