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 Rossi

a

a

Friedrich Miecher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland

b

Institute 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

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 aswell as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes wasfast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For importof short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lackingits C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 moleculeswere imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, theNLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute forVirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 di-rects 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 ofnuclei represents an essential aspect of the life of eukaryoticcells. Substrates for such transport include proteins, nucleicacids, and protein–nucleic acid complexes. The nuclear traf-fic of nucleic acids is most likely mediated by nuclear importand export signals present on proteins associated with thenucleic acids. Examples include nuclear–cytoplasmic trans-port of small nuclear RNA particles, export of mRNA (re-viewed in Nakielny et al., 1997), and nuclear transport ofviral RNA or DNA. The T-DNA of Agrobacterium presents aparticularly interesting and unique import substrate. ThisDNA is transferred, possibly in a conjugation-like mode, tothe plant cell, where it is transported into the nucleus andintegrated in chromosomal DNA (reviewed in Lartey andCitovsky, 1997; Zupan and Zambryski, 1997; Rossi et al., 1998;Hansen and Chilton, 1999; Gelvin, 2000). Agrobacterium iswidely used as an efficient and precise DNA delivery systemfor plants; therefore, analysis of T-DNA import is importantnot only in its own right but also because it may help to fur-ther enhance the system’s utility. Transfer and integration of

T-DNA (reviewed in Tinland and Hohn, 1995; Sheng andCitovsky, 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 tu-mor-inducing (Ti) plasmid as well as on the bacterialchromosome. Two 25-bp imperfect direct repeats, termedborder sequences, define the T-DNA. In the presence of theVirD1 protein, VirD2 nicks the border sequence in a site-and strand-specific manner and covalently attaches to the5

9

end of the nicked DNA. The nicked DNA is thought to bedisplaced from the plasmid to yield single-stranded T-DNA.This single-stranded T-DNA–VirD2 complex is transferred tothe plant cell and is coated with the single-stranded DNA(ssDNA) binding protein VirE2, forming the so-called T-DNAcomplex. The T-DNA complex enters the nucleus, and theT-DNA is finally integrated into the plant cell genome.

Like the transport of other nucleic acids, nuclear import ofT-DNA is also expected to depend on proteins accompany-ing it to the plant cell. Proteins imported into the nucleusgenerally contain motifs composed of one or two stretchesof basic amino acids. These motifs are termed nuclear local-ization signals (NLSs) and are recognized by the nuclear im-port machinery. The import process can be divided into twosteps: importin-mediated, NLS-dependent docking of theprotein at the nuclear pore, and RanGTP-dependent trans-location through the channel of the nuclear pore complex(NPC) (reviewed in Görlich, 1997; Nakielny and Dreyfuss,

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.

370 The Plant Cell

1999). Both VirD2 and VirE2 contain NLSs that are functionalin targeting reporter proteins into plant nuclei (Herrera-Estrella et al., 1990; Citovsky et al., 1992, 1994; Howard etal., 1992; Tinland et al., 1992). VirD2 contains two NLSs, amonopartite NLS in the N-terminal part of the protein and abipartite NLS in the C-terminal part of the protein. The N-ter-minal NLS of VirD2 appeared not to be required for T-DNAtransfer, because mutations in this sequence had no effecton T-DNA transfer efficiency (Rossi et al., 1993). In contrast,the C-terminal NLS of VirD2 has been shown to be required forefficient transfer of the bacterial T-DNA to the plant nucleus(Shurvinton et al., 1992; Rossi et al., 1993; Narasimhulu etal., 1996; Mysore et al., 1998). Although the VirE2 proteinwas found to be required for T-DNA transfer (Citovsky et al.,1992, 1994; Rossi et al., 1996), the role of its NLSs in nu-clear import of the T-DNA could not be evaluated. Point mu-tations or partial deletions of the C- or N-terminal NLSsabolished or modified the ssDNA binding properties of theprotein (Citovsky et al., 1992, 1994; Dombek and Ream,1997). In microinjection experiments, it was determined thatVirE2 mediates the nuclear uptake of ssDNA into plant cells,but the role of VirD2 in this process was not studied (Zupanet al., 1996). Here, we describe a direct test of the involve-ment of both VirD2 and VirE2 in plant cell nuclear import.

A direct test involves the use of artificially produced T-DNAcomplexes in combination with in vitro nuclear import sys-tems. In previous studies, T-DNA complexes were reconsti-tuted from fluorescently labeled ssDNA and purified VirD2and VirE2 and tested in a mammalian in vitro nuclear importsystem (Ziemienowicz et al., 1999). Such systems wereshown to be dependent on cytosolic factors such as import-ins

a

and

b

, Ran, and pp15 (Adam et al., 1991; Moore andBlobel, 1992; Görlich and Laskey, 1995). The import of T-DNAcomplexes was shown to depend on both VirD2 and VirE2(Ziemienowicz et al., 1999). The activity of VirD2, covalentlyattached to the 5

9

terminus of T-DNA, was dependent onthe C-terminal NLS. In this work, we studied the mechanismof import of T-DNA complexes in tobacco in vitro nuclearimport systems. In these systems, the import of various pro-tein substrates was shown to be NLS dependent, rapid, sat-urable, and inhibited by GTP

g

S, but it did not requireexternally added importins and other cytosolic factors(Hicks et al., 1996; Merkle et al., 1996; Merkle and Nagy,1997). These findings are consistent with the observationthat Arabidopsis importin

a

localizes mainly at the nuclearmembrane (Smith et al., 1997).

We found that both virulence proteins, VirD2 and VirE2,are required for efficient import of the T-DNA complex intoplant nuclei. According to the model proposed here, it is theC-terminal NLS of the VirD2 protein that recognizes the nu-clear import machinery. For complete translocation of theT-DNA complex through the pore, the VirE2 protein is alsoessential. VirE2 function in the nuclear import of T-DNAcould be substituted by another ssDNA binding protein,RecA. These findings provide new insights into the mecha-nism of the nuclear import of large DNA–protein complexes.

RESULTS

To study directly the mechanism of nuclear import of the T-DNAcomplex during Agrobacterium-mediated plant transformation,we produced the T-DNA complex in vitro. Complexes consist-ing of ssDNA covalently attached to VirD2 and coated by VirE2were tested for in vitro import into plant nuclei.

Reconstruction of the T-DNA Complex in Vitro

The artificial T-DNA complex was prepared using rho-damine-labeled ssDNA and purified VirD2 and VirE2 pro-teins (for details, see Ziemienowicz et al., 1999). VirD2 is asite-specific cleaving-joining enzyme that nicks the lowerstrand of the Ti plasmid at the border sequences (Yanofskyet al., 1986; Stachel et al., 1987). The cleavage activity ofVirD2 could be reproduced in vitro using single-stranded oli-gonucleotides or denatured double-stranded DNA contain-ing a border sequence (Pansegrau et al., 1993; Jasper et al.,1994). We tested the efficiency of the cutting reaction onrhodamine-labeled ssDNAs (25, 250, and 1000 nucleotideslong) containing the border sequence. The cleavage wasprecise and efficient (95, 88, and 73% of the 25-, 250-, and1000-nucleotide ssDNAs were cleaved, respectively), andVirD2 remained covalently attached to the 5

9

termini of thecleaved border sequences (data not shown; see Pansegrauet al., 1993). Cleavage by VirD2 lacking the C-terminal NLSwas comparable in efficiency and precision to cleavage bythe corresponding wild-type protein.

It has been shown that VirE2 binds to ssDNA in a cooper-ative manner (Citovsky et al., 1989; Sen et al., 1989; Zupanet al., 1996). The cooperativity of the binding of our purifiedVirE2 protein to ssDNA was tested in a gel shift assay, asdescribed previously (Ziemienowicz et al., 1999). Fluores-cently labeled VirE2 behaved like unlabeled protein (data notshown). To prepare a “complete” artificial T-DNA complex,rhodamine-labeled or unlabeled ssDNA was cleaved byVirD2, followed by incubation with an excess of unlabeled orfluorescently labeled VirE2.

The contribution of virulence proteins to the import ofT-DNA complexes was tested in a plant in vitro protein im-port system (Merkle et al., 1996). The import of fluorescentlylabeled substrates into evacuolated permeabilized tobaccoBY-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 ana-lyzed the nuclear import of labeled virulence proteins VirD2and VirE2. Both proteins contain NLSs that are functional invivo 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 thenuclei; fluorescently labeled VirD2, VirD2

D

NLS (mutantVirD2 lacking the C-terminal NLS due to an NruI deletion butstill containing the N-terminal NLS), and VirE2 were found tolocalize to the plant nuclei (Figure 1 and Table 1). LabeledBSA linked to the NLS of simian virus 40 (SV40) large T anti-gen (BSA-NLS) was also found in the nuclei, whereas la-beled BSA without NLS remained in the cytoplasm (data notshown; Merkle et al., 1996). The kinetics of the import ofboth VirD2 and VirE2 was comparable to that of the modelsubstrate (data not shown). These experiments demonstratethat the import system used accurately reflects the intracel-lular distribution of virulence proteins and that it can be usedto analyze the import of the T-DNA complex.

Nuclear Import of a Short Oligonucleotide–VirD2 Complex Requires the C-Terminal NLS of theVirD2 Protein

We used both wild-type VirD2 and VirD2

D

NLS to produceimport substrates by reacting the proteins with fluorescentlylabeled 25-nucleotide oligonucleotides containing the rightborder sequence of pTiA6. The oligonucleotide itself wasnot transported actively to the nuclei (Figure 2A; Table 1). Incontrast, the short VirD2wt–ssDNA complex was activelyimported and accumulated rapidly in the nuclei of permeabi-lized plant cells (Figure 2B). The short ssDNA complexedwith the mutant VirD2

D

NLS protein was import deficient anddid not accumulate in the plant nuclei (Figure 2C). Thus, theVirD2 protein can import a small covalently attached oligo-nucleotide to the plant nucleus. Moreover, this import wasabsolutely 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 didnot accumulate in plant nuclei (Figure 3A and Table 1). In con-trast to the experiments described above showing VirD2-dependent nuclear translocation of 25-nucleotide ssDNA,VirD2 covalently attached to the 250-nucleotide DNA was notable to translocate it to the nucleus (Figure 3B). Only whenthe VirD2wt–ssDNA complex was coated with VirE2 was im-port of the protein–DNA complex into the plant nuclei efficient(Figure 3C) and fast (occurring with a kinetics similar to that ofthe proteins tested alone: 90% of import in 10 min, 99 to100% in 20 min). The characteristics of the nuclear import of1-kb T-DNA complexes did not differ from those of com-plexes formed with a 250-nucleotide ssDNA (Table 1). Thenuclear import of both T-DNA complexes was dependenton the presence of VirE2 and the C-terminal NLS of VirD2,because efficient import of the VirD2

D

NLS–ssDNA–VirE2complexes 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 VirD2DNLS.(C) Localization of VirE2.Cy3.5-labeled proteins (red fluorescence) were incubated with perme-abilized tobacco protoplasts for 20 min at room temperature in thedark. The nuclei were stained with SYTO 13 dye (green fluorescence).Fluorescently labeled components are indicated by asterisks.

372 The Plant Cell

Because the import of longer ssDNAs required the VirE2protein, we also tested whether VirE2 was sufficient for theimport of long complexes. Import of the 250-nucleotide or1-kb ssDNA complexed with VirE2 alone was not detected(Figure 3E; Table 1). These results allow the conclusion thatthe targeting/import function of the VirD2 NLS is a necessaryprerequisite for nuclear entrance of VirE2-coated ssDNA (seebelow) and that this coating is essential for the completetranslocation 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 ofprotein molecules resemble each other mechanistically, wetested known competitors and inhibitors of protein import.The nuclear import of proteins in the plant in vitro systemhas been shown to be inhibited by the nonhydrolyzable ana-log GTP

g

S but not by wheat germ agglutinin (WGA), whichblocks the nuclear import of proteins in mammalian systems

(Hicks and Raikhel, 1995; Merkle et al., 1996). Consistentwith these data, import of the VirD2wt–ssDNA–VirE2 com-plex into the plant nuclei was inhibited only in the presenceof GTP

g

S (Table 2) and not in the presence of WGA (Figure3F and Table 2). In addition, import of the complete T-DNAcomplex could be inhibited by an excess of unlabeled BSA-NLS competitor, whereas BSA alone was not able to blockT-DNA complex nuclear transport (Table 2). These resultssuggest that the classic protein import pathway is used totransport 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 intothe plant nucleus suggested that the NLSs of VirE2 are notavailable to the import machinery when the protein is boundto ssDNA (see above). To analyze the fate of VirE2 in thelocalization of the various complexes, we performed thecomplementary experiment in which we monitored the dis-tribution of fluorescently labeled VirE2 in the plant in vitroimport system. Although the protein by itself moved to thenucleus (Figure 1C), it remained in the cytoplasm whenbound to an oligonucleotide of 38 (Figure 4A and Table 1) or250 (Table 1) nucleotides. These data confirm the resultsobserved with a DNA-labeled complex. In addition, they im-ply that binding to DNA actually sequesters the NLSs ofVirE2 or otherwise makes them unavailable to the importmachinery.

The experiments described so far demonstrated that 250-nucleotide and longer ssDNAs efficiently enter plant nucleionly when complexed to VirD2 and VirE2. VirD2 most likelyremains attached to the T-DNA upon translocation to thenucleus, because it still has a function in integration (Tinlandet al., 1995; Mysore et al., 1998). To analyze the fate of theVirE2 protein molecules as part of the T-DNA complex, weproduced a complex from unlabeled 1-kb ssDNA covalentlyattached to VirD2 and reacted it with fluorescently labeledVirE2. Any unreacted VirE2 was bound to an excess of38-nucleotide oligonucleotide, yielding a complex that couldnot be imported (see above). A VirE2-derived signal wasfound in the nucleus only when VirD2wt was complexedwith T-DNA and VirE2; when the mutant VirD2

D

NLS proteinwas part of the complex, the labeled VirE2 protein mole-cules remained excluded (Figures 4B and 4C and Table 1).These data confirm that the VirE2 protein entered into thenuclei as part of the T-DNA complex and was not strippedoff 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 inthe nuclear import of T-DNA, we tested other ssDNA bind-

Table 1.

Summary of Nuclear Import Experiments Using Complete Import Systems

a

Substrate Nuclear Import

VirD2wt protein*

1

VirD2

D

NLS protein*

1

VirE2 protein*

1

25-nt oligonucleotide*

2

VirD2wt–25-nt oligonucleotide*

1

VirD2

D

NLS–25-nt oligonucleotide*

2

250-nt ssDNA*

2

VirD2wt–250-nt ssDNA*

2

VirE2–250-nt ssDNA*

2

VirD2wt–250-nt ssDNA*–VirE2

1

VirD2

D

NLS–250-nt ssDNA*–VirE2

2

1-kb ssDNA*

2

VirD2wt–1-kb ssDNA*

2

VirE2–1-kb ssDNA*

2

VirD2wt–1-kb ssDNA*–VirE2

1

VirD2

D

NLS–1-kb ssDNA*–VirE2

2

VirE2*–250-nt ssDNA

2

VirE2*–38-nt ssDNA

2

VirD2wt–1-kb ssDNA–VirE2*

1

38-ntoligonucleotide

1

VirD2

D

NLS–1-kb ssDNA–VirE2*

1

38-ntoligonucleotide

2

a

The import of different substrates into the plant nuclei was per-formed in vitro, as described in Methods. Each experiment was re-peated at least three times, and for each sample, at least 100 nucleiwere scored. Asterisks indicate fluorescently labeled substrates. nt,nucleotides; (

1

), 99 to 100% of nuclei showing red fluorescence;(

2

), 0 to 1% of nuclei showing red fluorescence.

VirD2/VirE2–Mediated Nuclear Import of T-DNA 373

ing proteins that do not contain NLSs for their ability to sub-stitute for the function of VirE2. First, we tested thelocalization of the

Escherichia coli

ssDNA binding proteinsSSB and RecA. Whereas the SSB protein was found not toenter the plant nuclei, RecA localized both outside and in-side of the plant nuclei (Table 3). This transfer of RecA intoplant nuclei occurred in an importin-independent and likelypassive manner, because (1) RecA does not possess anymotif resembling known NLSs, (2) it is a small protein (38kD) that is expected to pass freely through the channel ofthe NPC, and (3) transfer of RecA into plant nuclei was notaffected by the import inhibitor GTP

g

S or the import com-petitor BSA-NLS (Table 3). This is in agreement with previ-ous findings that the nuclear targeting of RecA was stronglyenhanced when the protein was fused to an NLS, whereasunmodified RecA localized poorly to the nuclei of RecA-expressing transgenic tobacco cells (Reiss et al., 1996). Wetested the ability of the SSB and RecA proteins to substitutefor the function of VirE2 in nuclear import of the T-DNA

complex. Import of ssDNA–protein complexes into plant nu-clei occurred only when RecA but not SSB was used inplace of VirE2 (Table 3). Import of the VirD2–ssDNA–RecAcomplex was dependent on the C-terminal NLS of VirD2(Table 3), as was observed for VirD2–ssDNA–VirE2 com-plexes (Table 1).

RecA Cannot Substitute for VirE2 in Efficient T-DNA Transfer to Tobacco Plants

Efficient T-DNA transfer by Agrobacterium lacking VirE2was reestablished by expressing VirE2 in the target plantcells (Citovsky et al., 1992). Because RecA can substitutefor VirE2 in the entry of T-DNA into the nucleus (see above),transgenic plants expressing RecA or nt-RecA (RecA withthe NLS of the SV40 large T antigen) were tested for theirability to complement the Agrobacterium strain lackingVirE2. The T-DNA transfer efficiency was calculated based

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 VirD2DNLS.The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). The plant nuclear import assay was performed usingrhodamine-labeled oligonucleotide (red fluorescence), as described in Methods. Fluorescently labeled components are indicated by asterisks.

374 The Plant Cell

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) VirD2DNLS–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 com-plexes into plant nuclei was performed as described in Methods. DNA was labeled with rhodamine (red fluorescence). Fluorescently labeledcomponents are indicated by asterisks.

VirD2/VirE2–Mediated Nuclear Import of T-DNA 375

on the activity of the

b

-glucuronidase gene (

uidA

) present onT-DNA, as measured by the number of blue spots on to-bacco seedlings. The efficiency of T-DNA transfer from the

virE2

2

Agrobacterium strain into seedlings of wild-type SR1tobacco and plantlets transgenic for VirE2 or RecA wascompared with the efficiency of a wild-type Agrobacteriumstrain. As expected, VirE2 transgenic plants were found tocomplement the strain lacking VirE2 for efficient T-DNAtransfer (data not shown). However, the efficiency of T-DNAtransfer by an Agrobacterium strain lacking VirE2 to trans-genic plants expressing RecA or nt-RecA was found to besimilar to the transfer efficiency to wild-type SR1 plants (Table4). Thus, RecA transgenic plants are not able to complementthe absence of VirE2 in Agrobacterium. This finding suggestsan activity of VirE2 that goes beyond nuclear import.

DISCUSSION

Agrobacterium is a plant pathogen that naturally is able totransfer 10- to 20-kb DNA to the nucleus of the plant cell.Even transfer and integration of T-DNAs as long as 150 and170 kb have been demonstrated (Miranda et al., 1992;Hamilton et al., 1996). According to a model for the struc-ture of the VirE2–T-DNA complex (Citovsky et al., 1997), 20-and 170-kb DNA represent complexes 1.4 and 12

m

m long,respectively. These sizes have to be compared with the di-mensions of the NPC through which the T-DNA complexhas to travel: 120 nm in diameter and 100 nm in depth (re-viewed in Moroianu, 1997). Transfer of T-DNA to the nucleustherefore poses logistic problems similar to those of translo-cation of long mRNAs into the cytoplasm; in both cases, theNPC has to be recognized by one extremity of the molecule,and the translocation of a long molecule has to be executedto completion. We set out to explore the cell’s ability to per-mit nuclear passage of complex structures. For this effort,we reconstructed ssDNA–VirD2–VirE2 complexes and ana-lyzed in detail their import into plant nuclei. We found thatthe T-DNA complex was imported efficiently into the nucleiof permeabilized tobacco cells.

In living cells, many nucleic acids move into and out of thenucleus. These include cellular RNAs such as tRNA, smallnuclear RNA, and spliced mRNA as well as viral RNAs andDNAs. In general, transport of these nucleic acids seems tobe mediated by proteins (reviewed in Nakielny et al., 1997;Nakielny and Dreyfuss, 1999). For instance, small nuclearRNAs are exported to the cytoplasm, where they are com-plexed with proteins into small nuclear RNA particles thatthen use the m3G cap and Sm protein for reimport into thenucleus (Fischer et al., 1991; Michaud and Goldfarb, 1992;Marshallsay and Lührmann, 1994; Palacios et al., 1997). Theuptake of influenza virus RNA has been suggested to bemediated by its nucleoprotein (O’Neill et al., 1995), and hu-man immunodeficiency virus-1 viral DNA is presumablytransported to the nucleus via NLSs of the Gag matrix and

Vpr proteins in the form of a nucleoprotein preintegrationcomplex (Goldfarb, 1995; Stevenson, 1996).

In a manner similar to that of cellular and viral nucleic ac-ids that have to pass the nuclear membrane, the T-DNA ofAgrobacterium also uses proteins for its nuclear entry. Thetwo bacterial proteins VirD2 and VirE2 are associated with theT-DNA, travel to the plant cell during Agrobacterium-medi-ated plant transformation, and are known to contain func-tional NLSs. In the experiments reported here, both proteinslocalized to the nuclei of permeabilized tobacco cells, sug-gesting that their NLSs are functional in protein import inthis system. This is consistent with previous findings of thelocalization of both VirD2 and VirE2 proteins in plant nucleiin vivo (Citovsky et al., 1992, 1994; Rossi et al., 1993).

To reconstruct the T-DNA complex, we attached VirD2enzymatically to the 5

9

ends of ssDNAs of different lengths,and these complexes were coated with VirE2. This artificialT-DNA complex is currently the best possible approximationof the native complex, although we cannot exclude the pos-sibility that other proteins of bacterial and/or plant origin arealso involved in vivo.

Function of VirD2 in the Nuclear Import of ssDNA

The VirD2 protein alone was sufficient to transfer shortsingle-stranded oligonucleotides to tobacco nuclei. Thisfunction was strictly dependent on the presence of the C-ter-minal NLS of the VirD2 protein. These results are consistentwith genetic data reporting that only the C-terminal NLS ofthis protein is necessary for the efficient transformation ofplants (Shurvinton et al., 1992; Rossi et al., 1993). Interest-ingly, in several experimental systems, deletions of theC-terminal NLS did not lead to a complete block of virulence

Table 2.

Characteristics of the Nuclear Import of Proteins and the T-DNA Complex

a

SubstrateInhibitors/Competitors Nuclear Import

VirD2*, VirE2* WGA

1

VirD2*, VirE2* GTP

g

S

2

VirD2* BSA-NLS

2

VirD2* BSA

1

VirD2wt–1-kb ssDNA*–VirE2 WGA

1

VirD2wt–1-kb ssDNA*–VirE2 GTP

g

S

2

VirD2wt–1-kb ssDNA*–VirE2 BSA-NLS

2

VirD2wt–1-kb ssDNA*–VirE2 BSA

1

a

The import assay of fluorescently labeled proteins (VirD2*, VirE2*)and T-DNA complexes was performed in vitro using plant nuclei, asdescribed in Methods. Each experiment was repeated at least threetimes, and for each sample, at least 100 nuclei were scored. Aster-isks indicate fluorescently labeled substrates. (

1

), 99 to 100% of nu-clei showing red fluorescence; (

2

), 0 to 1% of nuclei showing redfluorescence.

376 The Plant Cell

(Shurvinton et al., 1992; Mysore et al., 1998), whereas in oursystem, the NLS was absolutely required for nuclear importof the T-DNA complex. These differences may be explainedin part by the different NLS mutants used. In addition, theimport system used here is independent of cell divisions,whereas dividing cells may allow the entry of proteins lack-ing NLSs into newly forming nuclei.

Our data also reveal that the N-terminal NLS by itselfcould not mediate the transport of the T-DNA complex tothe nucleus, although this NLS is functional in transportingthe DNA-free form of VirD2 (Herrera-Estrella et al., 1990;Tinland et al., 1992; Rossi et al., 1993) (Figure 2B). This find-ing indicates that the N-terminal NLS of VirD2 is maskedwhen the protein is covalently bound to DNA; indeed, the ty-rosine 29 with which VirD2 is bound to DNA is only a fewamino 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 (seeabove). However, rapid and efficient nuclear import of long(250 and 1000 nucleotides) ssDNAs required the additionalpresence of the VirE2 protein. Neither of the two proteinsalone led to the efficient import of long ssDNAs. These re-sults and those discussed above lead to the proposal thatVirD2 pilots the complex to the nuclear pore and initiatesimport, whereas VirE2 is required to ensure the translocationof the entire T-DNA complex. Therefore, VirE2 may haveseveral roles that are not mutually exclusive. Its importancemay be attributed to its NLSs; it may allow specific interac-tions between the NPC and the T-DNA complex, and it mayexert its function by shaping the T-DNA complex into a formcompetent for translocation through a nuclear pore.

In our experiments, the VirE2 protein, although it was ableto enter the plant nucleus as free protein, was excludedfrom the nucleus when bound to ssDNA. These results indi-cate that the NLSs of VirE2 are inaccessible to the nuclearimport machinery when the protein is bound to DNA. In-deed, analyses of VirE2 revealed a partial overlap of thessDNA binding domain or the cooperativity domain with theNLSs (Citovsky et al., 1992, 1994; Dombek and Ream,1997). This is consistent with the notion that DNA/RNAbinding domains of the majority of nuclear proteins are com-posed of basic amino acids and therefore resemble and canfunction as NLSs (LaCasse and Lefebvre, 1995). Another

Figure 4.

Localization of the VirE2 Protein Associated with ssDNA inPlant Nuclei.

(A)

A 38-nucleotide oligonucleotide reacted with labeled VirE2.

(B)

VirD2wt–ssDNA–VirE2-labeled complex quenched with excessof 38-nucleotide oligonucleotide.

(C)

VirD2

D

NLS–ssDNA–VirE2-labeled complex quenched with ex-cess of 38-nucleotide oligonucleotide.In vitro nuclear import of different complexes of Cy3.5-labeled VirE2(red fluorescence) with ssDNAs was performed as described inMethods. The positions of the nuclei are indicated by staining withSYTO 13 dye (green fluorescence). Fluorescently labeled compo-nents are indicated by asterisks.

VirD2/VirE2–Mediated Nuclear Import of T-DNA 377

important aspect of the function of VirE2 in the nuclear im-port of the T-DNA complex was revealed by our search forother ssDNA binding proteins that may replace VirE2 in thisprocess. Although the RecA protein does not contain a se-quence resembling known NLSs, it could substitute for VirE2in the T-DNA complex during nuclear targeting. The abilityof the NLS lacking the RecA protein to function in the nu-clear import of ssDNA–protein complexes suggests againthat 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 func-tion of the VirE2 protein in the uptake of ssDNA into plantnuclei. The nuclear accumulation of fluorescently labeledssDNA microinjected into the stamen hairs of

Tradescantiavirginiana

was found to depend on binding of VirE2 to thessDNA (Zupan et al., 1996). Import of the ssDNA–VirE2complex into the nucleus of stamen hairs of

T. virginiana

wasinhibited by WGA, in contrast to the lack of inhibition byWGA in the plant in vitro nuclear import systems describedto date (Hicks et al., 1996; Merkle et al., 1996) and in our ex-periments. We deliberately tested VirD2-independent nu-clear import of ssDNA–VirE2 complexes, but we detectedonly very inefficient import after drastic extension of the re-action time (from 20 min to 2 hr). This finding may reflect thelow residual but VirE2-dependent T-DNA transfer observedin vivo in the absence of the C-terminal NLS of VirD2 (Rossiet al., 1996), although other explanations are also possible.This low activity is probably not important for the process ofAgrobacterium-mediated plant transformation. To resolvethis issue, microinjection experiments using complexes

consisting of ssDNA, VirE2, and VirD2 may have to be per-formed.

Studies of VirE2 function in several animal systems haveled to the discovery of interesting differences. Therefore, itcannot be excluded that transfer of T-DNA complexesthrough NPCs of different animal nuclei follows slightly dif-ferent rules. ssDNA–VirE2 complexes tested for import intothe nuclei of permeabilized HeLa cells were found to be ex-cluded from them (Ziemienowicz et al., 1999). On the otherhand, ssDNA–VirE2 complexes microinjected into Drosoph-ila embryonic cells and Xenopus oocytes were found to lo-calize in the nuclei (Guralnick et al., 1996). However, theNLSs of the VirE2 protein used in the latter experimentswere modified slightly to resemble the NLS of nucleoplas-min (Guralnick et al., 1996), whereas the VirE2 protein usedin experiments with permeabilized HeLa and tobacco cellscontained unmodified NLS sequences.

Alternately (or in addition), VirE2 may actively participatein the translocation of the T-DNA complex by specific inter-actions inside the channel of the NPC. Inside this channel,the translocation substrate has to travel a distance of

z

100nm. The proteins seem to pass through a number of importintermediate points in the channel, but until now the mech-anism of the actual translocation has remained obscure(Görlich, 1997; Nakielny and Dreyfuss, 1999). It is possiblethat VirE2 could interact with those uncharacterized inter-mediates and favor the translocation of the huge T-DNAcomplex into the nucleus. However, these specific interac-tions 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 nu-clear pore. The VirE2 protein coat may impose a specificstructure onto the otherwise unstructured but chargedssDNA, thereby allowing translocation through the NPC chan-nel. Electron microscopy data suggest that VirE2 packagesssDNA into semirigid and hollow cylindrical filaments havinga telephone cord–like coiled structure that potentially can be

Table 3.

RecA but Not SSB Can Substitute for VirE2 in T-DNA Nuclear Import

a

SubstrateInhibitors/Competitors Nuclear Import

SSB*

2

RecA*

1

/

2

RecA* GTP

g

S

1

/

2

RecA* BSA-NLS

1

/

2

VirD2wt–1-kb ssDNA*–VirE2

1

VirD2wt–1-kb ssDNA*–RecA 1

VirD2DNLS–1-kb ssDNA*–RecA 2

VirD2wt–1-kb ssDNA*–SSB 2

VirD2wt–1-kb ssDNA*–RecA GTPgS 2

VirD2wt–1-kb ssDNA*–RecA BSA-NLS 2

a The import assay of fluorescently labeled proteins and T-DNA com-plexes was performed in vitro using plant nuclei, as described inMethods. Each experiment was repeated at least three times, andfor each sample, at least 100 nuclei were scored. Asterisks indicatefluorescently labeled substrates. (1), 99 to 100% of nuclei showingred fluorescence; (2), 0 to 1% of nuclei showing red fluorescence;(1/2), red fluorescence was distributed equally outside and insidethe nucleus.

Table 4. T-DNA Transfer to Wild-Type and Transgenic VirE2 and RecA Plantsa

Plant Transfer Efficiency (%)

SR1 0.018RecA 0.01160.0553nt-RecA 0.03260.0368VirE2 100

a A complementation experiment with an Agrobacterium strain lackingthe virE2 gene and wild-type (SR1) or transgenic (VirE2, RecA, or nt-RecA) tobacco seedlings was performed, as described in Methods.Complementation efficiency was determined by comparing the T-DNAtransfer efficiency of the Agrobacterium strain lacking VirE2 with thetransfer efficiency of the wild-type strain. Efficient complementationwas 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 bind-ing to it (the DNA length is extended by 50%) (Flory et al.,1984; Nishinaka et al., 1997), leading to the formation oflong filaments of DNA–protein complexes that are only 5.2nm (Flory and Radding, 1982) or 9.3 nm (Flory et al., 1984)wide. Both values are ,10 nm (the diameter of the NPCchannel), and the former value is similar to that calculatedfor ssDNA–VirE2 complexes (4.4 nm) in the extended stage(that is, before folding into more complexed helical filamentsof 12.6 nm; Citovsky et al., 1997). In contrast, upon bindingto ssDNA, SSB condenses it, leading to the formation ofcompact filaments of nucleosome-like structure with a widthof 12 nm (Chrysogelos and Griffith, 1982) that may preventpassage of the DNA–protein complex through the 10-nm-wideNPC channel.

Although the requirement for the VirE2 protein in the nu-clear import of ssDNA is well documented, it was not clearwhether the VirE2 protein molecules bound to the DNA ac-tually enter the nucleus or are stripped off upon entranceinto the NPC channel. The experiments reported here, how-ever, unequivocally demonstrate that VirE2 enters plant nu-

clei together with the T-DNA complex in a strictly VirD2-dependent fashion. In the nucleus, VirE2 and VirD2 take onnew roles in the transformation process, as “chaperone” toprotect the DNA from nucleolytic attack (VirE2; Rossi et al.,1996) and as an aid in the integration of complete T-DNAunits into the plant genome (VirD2; Tinland et al., 1995;Mysore et al., 1998).

Lack of VirE2 in an Agrobacterium strain can be comple-mented by its expression in the plant cell, indicating that thisprotein performs its function inside the recipient plant cell.However, the function of the VirE2 protein in the plant cell isnot limited to the nuclear import of T-DNA. We performedcomplementation tests using a VirE22 Agrobacterium strainfor transformation of wild-type or transgenic tobacco seed-lings expressing either VirE2 or RecA. Although RecA wasable to replace VirE2 in nuclear import of T-DNA complexes,it was unable to substitute for VirE2 in the efficient transfor-mation of tobacco plants. This finding indicates that VirE2 isnot only essential for the transfer of large T-DNA moleculesinto the nucleus but also required in a step preceding or fol-lowing nuclear import, such as movement of the complexinside 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 blacktriangles. See text for more details.

VirD2/VirE2–Mediated Nuclear Import of T-DNA 379

nuclear pore, or integration of the T-DNA into the plantgenome.

Import of T-DNA Complexes into the Nuclei of Tobacco Cells Is Mediated by the Classic Nuclear Import Pathway

In our experiments, the import of the T-DNA complex intoplant nuclei was as specific as the import into HeLa cell nu-clei reported previously (Ziemienowicz et al., 1999). Interest-ingly, import into mammalian nuclei was mediated by theclassic NLS-dependent mechanism, because it required im-portin a (Rch1; Ziemienowicz et al., 1999), which was shownto bind to all tested NLS substrates (Miyamoto et al., 1997).Because the import of the T-DNA complex into plant nucleiwas inhibited by BSA coupled to an NLS and by GTPgS, animportin-dependent pathway is likely to be used to importthe complex into plant nuclei as well. Currently, several im-portins a can be found in Arabidopsis databases, but onlytwo of them were characterized. One, AtKAPa, recognizesthe C-terminal but not the N-terminal NLS of VirD2 (Ballasand Citovsky, 1997); the other recognizes three classes ofimport signals (Smith et al., 1997). The former importin maybe involved in the import of the T-DNA complex in plants,whereas the latter one may be involved in the nuclear tar-geting of VirD2 devoid of its C-terminal NLS by interactionwith its N-terminal NLS, an activity that is probably irrelevantbiologically. Thus, Agrobacterium seems to have “learned”to abuse the classic nuclear protein import pathway to effi-ciently ferry large DNA molecules into the plant nucleus.

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 complexinto plant nuclei (Figure 5). The T-DNA in the plant cell oc-curs in a covalent complex with the VirD2 protein, coveredby VirE2. The VirD2 protein pilots the T-DNA complex to thenuclear pore using its C-terminal NLS, which is recognizedby the NLS receptor importin a. The T-DNA complex boundto importin a then docks to the NPC via importin b, and the59 terminus of the T-DNA is directed toward the nuclearpore channel, where translocation is initiated. This is analo-gous to the nuclear export of the Balbiani ring RNA particle,which leaves the nucleus with the 59 end in the lead (Daneholt,1997). During translocation, the RNA particle moves throughthe channel in an extended state. For translocation to com-pletion of a long T-DNA complex through the nuclear porechannel, VirE2 is required. VirE2, by its cooperative bindingto ssDNA, covers its negative charges and creates a struc-ture that enables translocation through the nuclear pore.Such a structure, which electron microscopy reveals toresemble a telephone cord, can be extended upon passageof the T-DNA complex through the nuclear pore channel(Citovsky et al., 1997). Alternately (or in addition), VirE2 binds

to nuclear import intermediates inside the pore to facilitatetranslocation.

METHODS

Cloning of virE2 into the Expression Vector pET3a

The plasmid pSW108 (Winans et al., 1987) contains an XhoI fragmentfrom pTiA6 encompassing the virE operon promoter and the openreading frames of VirE1 and VirE2. A StuI-SmaI fragment frompSW108 was inserted in the HincII site of pUC18, resulting in theplasmid pUCSS. An NdeI restriction site was introduced into the firstATG (underlined) of the virE2 gene by polymerase chain reaction(PCR) using primers p1 (59-ATCGTAGCCTGCAGAGTCATATGG-ATCTTTCTGGCAATGAGAAATCC-39) and p2 (59-GTTTGATAAAAG-ATCTCTGTGCC-39) and plasmid pSW108 as a template. The PCRproduct was cut with PstI and BglII and inserted into pUCSS previ-ously cut with the same restriction enzymes, yielding the plasmidpUCE2. An NdeI-BamHI fragment from plasmid pUCE2 was insertedin plasmid pET3a (Studier et al., 1990) previously digested with thesame enzymes, resulting in plasmid pETE2.

Overexpression and Purification of VirE2

The plasmid pETE2 was introduced into Escherichia coli strainBL21(DE3) (Studier et al., 1990) by electroporation. The transformedcells were grown in 500 mL of Luria-Bertani medium containing 100mg/mL ampicillin at 378C with shaking. At OD600 5 0.5 to 1, expres-sion was induced by the addition of isopropyl-b-D-thiogalactoside toa final concentration of 1 mM. Shaking was continued for 5 hr. Thecells were then pelleted by centrifugation at 4000g at 48C for 30 min.The pellet was resuspended in 50 mL of ice-cold lysis buffer A1 (50mM Tris-HCl, pH 8.5, 50 mM NaCl, 5 mM EDTA, 0.1 mM phenylmeth-ylsulfonyl fluoride [PMSF], 1 mM DTT, 0.5 mg/mL lysozyme, and0.1% Tween 20). After 1 hr of incubation on ice, the lysis mixture wascentrifuged at 20,000g for 10 min at 48C. The pellet was resuspendedin 25 mL of buffer B1 (50 mM Tris-HCl, pH 8.5, 1 M NaCl, 5 mMEDTA, 0.1 mM PMSF, and 1 mM DTT) containing 1% Tween 20, in-cubated for 30 min on ice, and centrifuged at 20,000g for 10 min. Thepellet was washed in the same volume of buffer B1 containing 0.1%Tween 20. After centrifugation (20,000g for 10 min), the VirE2 proteinwas solubilized in 50 mL of buffer C1 (25 mM NaOAc, pH 5.0, 0.1 mMPMSF, 1 mM DTT, and 6 M urea) and incubated for 30 min on ice.

The supernatant fluid obtained after centrifugation (20,000g for 10min) represented the inclusion body fraction of proteins containingmainly VirE2. This fraction was applied to a cation exchange column(EconoPacS; Bio-Rad) equilibrated in buffer C1. Bound proteinswere eluted with a linear NaCl gradient (0 to 1 M). VirE2 peak frac-tions were dialyzed overnight against buffer D1 (50 mM Tris-HCl, pH8.5, 100 mM NaCl, and 0.05% Tween 20). After centrifugation(20,000g for 10 min), the supernatant solution was applied to a sin-gle-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 gra-dient (0 to 1 M) in the same buffer. The VirE2 peak fractions were di-alyzed overnight against buffer E1, centrifuged (20,000g for 10 min)to remove insoluble material, frozen in liquid nitrogen in small ali-quots, and stored at a concentration of 50 to 100 ng/mL at 2808C.

380 The Plant Cell

ssDNA Binding Assay

The purified VirE2 protein as well as the RecA protein (New EnglandBioLabs, Bioconcept, Allschwil, Switzerland) were tested for theirss-DNA binding properties, as described previously (Citovsky et al.,1989; Ziemienowicz et al., 1999). Nearly the same amount of VirE2(0.2 mg) or RecA (0.28 mg) was required to cover completely 1 ng of250-nucleotide ssDNA.

Production of Fluorescently Labeled ssDNA

The 25-nucleotide oligonucleotide 59-CAACGGTATATATCCTGC-CAGTCAG-39, containing 20 nucleotides of the pTiA6 border sequence(underlined), was labeled with tetramethylrhodamine-6–dUTP atthe 39 end using terminal transferase (Boehringer Mannheim), ac-cording to the protocol provided by the supplier. Rhodamine-labeled1-kb DNA was produced by PCR amplification, as described else-where (Ziemienowicz et al., 1999), using two primers, each contain-ing 20 nucleotides of the pTiA6 border sequence. For production of250-nucleotide ssDNA, primers p3 (59-CCACGGTATATATCCTGCCAG-GGTATTTCACACCGCATATGG-39) and p5 (59- CCACGGTATATATCC-TGCCAGGTAGAGAATTATGC-39) were used.

Site-Specific ssDNA Cleavage Activity of VirD2

For overexpression of the VirD2 wild-type and VirD2DNLS mu-tant proteins, the previously described plasmids pFSVirD2 andpFSVirD2NruI were used (Tinland et al., 1994; Ziemienowicz et al.,1999). In the plasmid pFSVirD2NruI, a sequence localized betweentwo NruI sites within the virD2 gene was deleted. This deletion re-sulted in a mutant VirD2 lacking the C-terminal nuclear localizationsignal (NLS) and 29 amino acids surrounding it. For simplicity, in thisreport, this mutation is called VirD2DNLS. VirD2wt and VirD2DNLSmutant proteins were purified as described previously (Pansegrau etal., 1993). The efficiency of the cutting reaction by VirD2 andVirD2DNLS was tested on rhodamine-labeled 250-nucleotide and1-kb ssDNAs and on rhodamine-labeled 25-nucleotide oligonucle-otide, as described (Ziemienowicz et al., 1999). The efficiencies werecomparable.

Preparation of Fluorescently Labeled Proteins

Proteins were labeled directly with Cy3.5 reagent (from the Cy3.5monoclonal antibody labeling kit; Amersham-Pharmacia) using thesame protocol recommended by the supplier for labeling of mono-clonal antibodies.

Formation of the VirD2–ssDNA–VirE2 Complex

For the formation of the VirD2–ssDNA–VirE2 complex, 0.15 mg of un-labeled or rhodamine-labeled 1-kb ssDNA (or 40 ng of 250-nucle-otide ssDNA or 10 pmol of 25-nucleotide oligonucleotide) wasreacted with 7.2 mg of VirD2wt or VirD2DNLS protein in TNM buffer(20 mM Tris-HCl, pH 8.8, 50 mM NaCl, and 5 mM MgCl2) for 1 hr at378C (15 mL final volume). The full complex was made by adding 0.5mg of unlabeled or fluorescently labeled VirE2 and incubating themixture (20 mL 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 com-plexes, an in vitro system of permeabilized evacuolated protoplastsderived from tobacco (Nicotiana tabacum) BY2 suspension-culturedcells was used as described previously (Merkle et al., 1996). To 20 mLof permeabilized plant cells, 0.2 mg of VirD2 or VirE2 protein or 4 mLof T-DNA complex with or without VirD2wt or VirD2DNLS and/orVirE2 protein was added. In quenching experiments, 4 mL of formedcomplex was incubated on ice for 15 min with 585 pmol of a 38-nucle-otide oligonucleotide (59-GGTATATATCCTGCCAGGGTATTTCAC-ACCGCATATGG-39) before the import reaction. For inhibitionexperiments, wheat germ agglutinin (WGA) (Sigma; final concentra-tion 0.5 mg/mL) or GTPgS (final concentration 8 mM) was added tothe reaction mixture, followed by incubation for 20 min at room tem-perature before the addition of the complex. The import reaction wasperformed for 20 min at room temperature in the dark. For the com-petition experiments, nuclei diluted 10-fold (in import buffer withoutTriton X-100) were preincubated with 58 mg of unlabeled BSA linkedto the nuclear localization signal of simian virus 40 (SV40) large T an-tigen (BSA-NLS) or BSA for 5 min on ice and then incubated with 0.2mg of fluorescently labeled BSA-NLS or VirD2 or 0.4 mL of the com-plex for 5 min on ice. Nuclei were visualized by staining with SYTO 13(Molecular Probes, Eugene, OR) at a final concentration of 0.5 mM.Localization of fluorescently labeled samples was analyzed by con-focal microscopy using a fluorescein isothiocyanate filter for the dyeand a tetramethyl rodamine isothiocyanate filter for rhodamine- orCy3.5-labeled samples. No transmission was observed betweenthese two channels. Signal detection (sensitivity) and processing ofthe scanned images were identical within each experimental series.

Transgenic Tobacco Plants Expressing the VirE2 orRecA Protein

SR1 tobacco lines G64/2 and G63/19 transgenic for the recA geneand for the SV40 large T antigen NLS sequence fused to the 59 end ofthe recA gene (nt-recA) were described previously (Reiss et al.,1996). SR1 E3 tobacco plants contain the virE2 gene of the nopalinestrain of Agrobacterium tumefaciens under the control of the 35Spromoter 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(z0.1% of the total plant protein).

Complementation Assay

Agrobacterium strains GV3101(pPM6000, pTd33), GV3101(pPM6000K,pTd33) containing a deletion of z70% of the virD2 gene, andGV3101(pPM6000E, pTd33) containing a deletion of the virE2 openreading frame were used for transient transformation of tobaccoseedlings, as described elsewhere (Rossi et al., 1993, 1996). The ef-ficiency of T-DNA transfer by the GV3101(pPM6000E, pTd33) Agro-bacterium strain to young tobacco seedlings of SR1 plants andtransgenic plants expressing RecA, nt-RecA, and VirE2 was mea-sured as described previously (Rossi et al., 1993). In this test, the ac-tivity of b-glucuronidase encoded by the uidA gene present in theT-DNA of plasmid pTd33 (Tinland et al., 1995) represents a measure

VirD2/VirE2–Mediated Nuclear Import of T-DNA 381

of the concentration of T-DNA molecules arriving in the plant nu-cleus. T-DNA transfer efficiency was evaluated by counting the num-ber of blue spots that formed in 50 seedlings after incubation with5-bromo-4-chloro-3-indolyl-b-D-glucuronide, the substrate for b-gluc-uronidase. This number was compared with that for wild-type strainGV3101(pPM6000, pTd33) used at different concentrations. Dilutionof agrobacterial strains used for inoculations was done with thetransfer-defective strain GV3101(pPM6000K, pTd33) (Rossi et al.,1993).

ACKNOWLEDGMENTS

We thank Christopher Marshallsay for donation of fluorescently la-beled BSA-NLS, Serge Kocher for help with the confocal micro-scope, and Véronique Gloeckler for excellent technical assistance.We thank Dr. B. Reiss for donation of transgenic plants expressingRecA and nt-RecA, and Dr. H. Steinbiss (Max-Planck-Institute, Co-logne, Germany) for donation of transgenic plants expressing VirE2.We also thank Drs. Pawel Pelczar and Philippe Crouzet for criticalreading of the manuscript.

Received August 15, 2000; accepted December 1, 2000.

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DOI 10.1105/tpc.13.2.369 2001;13;369-383Plant Cell

Alicja Ziemienowicz, Thomas Merkle, Fabrice Schoumacher, Barbara Hohn and Luca RossiProteins

Import of Agrobacterium T-DNA into Plant Nuclei: Two Distinct Functions of VirD2 and VirE2

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