JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4432-4440 Vol. 172, No. 8

0021-9193/90/084432-09$02.00/0Copyright © 1990, American Society for Microbiology

Overexpression of virDI and virD2 Genes inAgrobacterium tumefaciens Enhances T-Complex

Formation and Plant TransformationK. WANG,t A. HERRERA-ESTRELLA, AND M. VAN MONTAGU*

Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Ghent, Belgium

Received 25 January 1990/Accepted 14 May 1990

The VirDl and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of theAgrobactenium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We havedetermined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti

plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673,1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7,and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was

absolutely essential for its endonuclease activity. When extra copies of the virDI and virD2 genes were presentin an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules couldbe detected at early stages of vir induction. Such strains possessed the ability to transform plants with higherefficiency.

Agrobacterium tumefaciens is the etiological agent ofplant crown gall disease. It was demonstrated that the crowngalls are produced as the result of transfer of a segment ofDNA, the transferred DNA (T-DNA), from the tumor-inducing (Ti) plasmid of the bacterium to the plant nucleargenome (13, 31). The T-DNA is delimited by imperfect25-base-pair (bp) direct repeats (termed border sequences).The right 25-bp border sequence is crucial in cis for directingefficient T-DNA transfer into the plant nuclear genome (23,40). Another set of elements responsible for the transferprocess reside outside of the T-DNA, in the 35-kbp virulence(vir) region of the Ti plasmid. The vir region is composed ofseven operons (virA, virB, virC, virD, virE, virF, and virG)(30). Except for the virA gene, which is constitutivelyexpressed in A. tumefaciens, the other six operons areinduced in response to small phenolic compounds, such asacetosyringone (AS), excreted by wounded plant cells (3,33). Activation of vir gene expression results in the genera-tion of site-specific nicks within the bottom strand of allT-DNA border repeats and a linear single-stranded DNAmolecule (the T-strand) corresponding to the bottom strandof the T-DNA region (1, 34, 41, 44). The polarity of T-strandssuggests that they are produced in a right-to-left direction,utilizing border nicks as initiation and termination sites.Both nicking and T-strand synthesis require two polypep-tides encoded by the 5' half of the virD locus (virDI andvirD2) (35, 44).The T-strand is considered the intermediate that is trans-

ferred from Agrobacterium spp. to the plant cell. Recentwork has provided more evidence to support this hypothe-sis. First, it has been shown that the virE2 gene encodes asingle-stranded DNA-binding protein which interacts withthe T-strand nonspecifically (6-8, 14). As the transfer inter-mediate, the DNA molecule must be protected from exo-and endonucleolytic degradation during the whole transferprocess, until it integrates into the plant nuclear genome.

* Corresponding author.t Present address: Research Department, Garst Seed Company,

Slater, IA 50244.

The fact that Agrobacterium has evolved a single-strandedDNA-binding protein specifically under vir control supportsthe argument that the T-strand may be the intermediatemolecule. Second, in addition to the endonuclease activity ofthe VirDl and VirD2 proteins, the VirD2 protein was foundto be firmly associated with the 5' end of the T-strand (theT-complex) (16, 18, 42, 45). The asymmetrical binding of theVirD2 protein to the T-strand (only at the 5' end) maytherefore confer its property of polarity as an intermediate inthe transfer process.

If the T-complex were the true transfer intermediate, onecould expect a direct correlation between the expression ofthe virDI and virD2 genes, the production of the T-strand,and tumor formation on plants. In this work, we show thatwhen extra copies of virDi and virD2 are present in Agro-bacterium strains containing a wild-type Ti plasmid, in-creased amounts of T-strand can be detected soon after ASinduction. In addition, increased expression of virDI andvirD2 leads to elevated tumorigenicity on plants.Due to the inconsistencies between the sequences re-

ported for the virD genes from different Agrobacteriumspecies (10, 15, 17, 20) and our own results, we present ournucleotide sequence for the virD gene of the nopalineplasmid pTiC58. The discrepancies between our sequenceand the previously reported one (15) are discussed. Theamino acid comparison of the VirD2 proteins from differentAgrobacterium species shows that its N-terminus but not theC-terminus is highly conserved. The C-terminal 50% ofVirD2 can be deleted or replaced without affecting itsendonuclease activity in Escherichia coli. Although theC-terminal domains of VirD2 proteins are highly divergent,they display similar hydropathy profiles. This suggests thatthis domain is involved in the steps subsequent to bordercleavage and T-strand synthesis.

MATERIALS AND METHODSBacterial strains and plasmids. E. coli MC1061 (4) was

used as the host for all cloning and T-strand assays. PlasmidpUC18 (43) was used as the vector for the various subclones.pGA472' (2) is a plasmid with a broad-host-range origin of

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replication which contains the right and left border frag-ments of nopaline T-DNA. pGV910 (Van den Eede et al.,unpublished data) is a plasmid containing the origin ofreplication from the broad-host-range plasmid pVS1 (19).pND1, pND2, pND3, and pND4 (16) are pUC18 derivativescontaining various parts of the nopaline virD genes.pGV3850 (46) is a nopaline Ti plasmid mutant in which all theoncogenic functions of the T-DNA were substituted bypBR322. pGV2301 (J.-P. Hernalsteens, unpublished data) isa derivative of the nopaline plasmid pTiC58 carrying thekanamycin resistance gene from transposon Tn9O3.Plasmid constructions. Figure 3A illustrates the subclones

of the virD gene in the pUC18 vector. Plasmid pND5 wasconstructed by introduction of a 1,556-bp EcoRV fragmentfrom pND1 into the SmaI site of pUC18. Plasmid pND6 wasconstructed by deletion of a 108-bp XmaIII (coordinate 1696)to EcoRI (coordinate 1804) fragment from pND3.A 3.8-kilobase (kb) SacIl-BamHI fragment containing the

origin of replication from pVS1 was obtained from pGV910and ligated to the corresponding sites of pND1 to generatepVND1. The same fragment was introduced into the NdeIsite of pND3 to generate pVND3. Plasmid pVND3-Km wasconstructed as follows. A 1.5-kb EcoRI-NdeI fragment car-rying the neomycin phosphotransferase II (nptII) gene wasisolated from plasmid pLKM91 (gift from J. Botterman),which was derived from pKm9 (27). This fragment wasligated into the EcoRI site of pVND3 to generate a VirD2-NPTII fusion protein.

Nucleotide sequence analysis. Nucleotide sequence wasdetermined by the chain-termination method (28) after sub-cloning of the virD operon into plasmid pUC18. Reagents forDNA sequencing were from P-L Biochemicals. The com-puter program Genalign (Software; Intelligenetics, Inc.) wasused for homology searching of proteins. The program Pepwas used for hydrophobicity searching (prediction based onthe Hopp and Wood index).

T-strand and border nick assays. The assays for theT-strand and the border nick in E. coli and A. tumefacienswere done as described previously (16).NPTHI assays. The neomycin phosphotransferase II

(NPTII) activity of AS-induced Agrobacterium cells wasassayed by the method of Platt and Yang (25). The NPTIIactivity of the VirD2-NPTII fusion protein in E. coli carryingpVND3-Km was examined by native polyacrylamide gelelectrophoresis (PAGE) as described by Van den Broeck etal. (38).

Virulence assays on plants. Bacterial inoculations wereperformed on various plants as described by Wang et al. (39)for tobacco (Nicotiana tabacum W38) and for potato (Sola-num tuberosum cv. Bintje), by Joos et al. (21) for kalanchoe(Kalanchoe daigremontiana), and by Peralta et al. (24) forcarrot (Daucus carota).

RESULTS

Nucleotide sequence of the virD locus. Plasmid pND1 (16)contains a 3.7-kb Sall-KpnI fragment overlapping the virDlocus of nopaline Ti plasmid pTiC58 (9). Figure 1 shows thenucleotide sequencing strategy with pND1 and the resultingsequence. The first three open reading frames (ORFs) en-coded three polypeptides of 16.13, 49.65, and 21.35 kilodal-tons (kDa). The fourth open reading frame was only partiallysequenced. This sequence was reported previously byHagiya et al. (15). However, a comparison of our sequencewith their data showed that there were differences at 28positions. For example, Hagiya et al. (15) suggested that

ORF1 encoded a polypeptide comprised of 109 amino acids,while we deduced that it should encode a polypeptide of 147amino acids. This difference is due to two missing guanidineresidues (coordinates 963 and 966, Fig. 1B) at correspondingpositions 470 and 473 in the sequence of Hagiya et al. (15).The 147-amino-acid VirDl deduced from our data was 93%homologous with the VirDl proteins of Agrobacteriumrhizogenes pRiA4b and the octopine Ti plasmid pTiA6NC(both 149 amino acids in length) (17, 20, 44).

Similar nucleotide discrepancies occurred in ORF3. Thisregion was 597 bp long and encoded a polypeptide of 199amino acids (Fig. 1B). The corresponding region determinedby Hagiya et al. (15) was designated ORF4 and found to becapable of encoding a polypeptide of 273 amino acids.Sequence comparison revealed that this difference was dueto a guanidine residue missing (coordinate 3008, Fig. 1B) atcorresponding position 2503 in the sequence of Hagiya et al.(15). The VirD3 proteins had low homology between Agro-bacterium species (17, 26). Neither molecular size nor aminoacid composition was similar between octopine pTiA6NCand A. rhizogenes pRiA4b (17). Nevertheless, the VirD3protein of 199 amino acids deduced by our data had a masssimilar to that of the VirD3 protein of octopine pTiA6NC(201 amino acids [26]) and had 31% homology overall (datanot shown).Our sequencing data show that ORF2 of the virD locus

was 1,338 bp in size and encoded a polypeptide of 49.7 kDa.This result does not agree with the earlier report that twopolypeptides of 15 and 29 kDa should be produced from thisregion (15). Eighteen differences at the nucleotide level,including base insertions or deletions, have been found inthis 1,338-bp-long sequence when it was compared with thecorresponding sequence of Hagiya et al. (15). However, ourdata are consistent with the reported virD2 sequences fromthe octopine strain (20, 44) and from A. rhizogenes (17). Inboth cases, VirD2 was found to encode a polypeptide of 48kDa. Moreover, a 56-kDa protein was detected in E. colimaxicells when a VirD2 antibody was used (10). Our workprovided a similar result. An XmnI fragment (correspondingto positions 1119 to 2000, Fig. 1B) carrying part of the virD2gene was cloned behind the T7 RNA polymerase promoter(36). This fragment produced a truncated polypeptide of 38kDa when expressed in E. coli (data not shown), which is inagreement with the predicted polypeptide of 33 kDa fromthis fragment. No polypeptide of 15 kDa was detected fromthe corresponding region (positions 1125 to 1553). Based onthese data, we conclude that the nopaline VirD2 proteinconsists of 446 amino acids with a molecular mass of 49.7kDa.

Function analysis of the virD2 gene. A comparison of aminoacid sequences of the VirD2 proteins from pTiC58,pTiA6NC, and pRiA4b revealed high homology throughoutthe N-terminal half (90% identity at residues 1 to 203), whilethe C-terminal half was completely divergent (Fig. 2). Adeletion of 50% of the VirD2 protein at its C-terminal part(up to the EcoRI site in pND3, Fig. 3A) did not affectborder-nicking or T-strand-binding capacity in E. coli (Fig.3B, lane 1) (9, 16).To localize the domain essential for nicking and binding

activity of the VirD2 protein, more deletions were made.Two constructs, pND6 and pND5, carrying 3' deletions ofvirD2 up to the XmaIII and EcoRV sites (Fig. 3A), respec-tively, were introduced into an E. coli strain which harboredborder-carrying plasmid pGA472' (2). E. coli cells containingeach of the constructs and pGA472' were lysed by sodiumdodecyl sulfate (SDS). DNAs from the phenol-aqueous

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N NS H B K

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BGTCGACAATG CTT?CGtT ATCGLOCCO GTTAACATOG TCGOGGATCA ACAGTSS GOTOOCA 70

TGLTCGTGT? GTTGAGTTCG CTCGLAA CSCG?ATC OOCAOGOCA TAATCAAATC CTTOGAGTTC 140

GGCGTCCT TAOOCCT CAAOGGTC CATTTCCTCG OCOGTAGA CTTCOCLGAL OGAOCCTAG 210

GTATTGCTOC GLAGOOCO?TT TCTTTCClT CGCCTTAGTG GTCOGTTTTC ATCANGTCC AAGACA 280

ATCGTTTCC GCACTOCA AAGOCOC AAAGACCAT AGTCTG CTTTC0T CTCCTCTT 350

GAAGAOCA AATGTCAGAA GTTTCATGTC CTATOCCTT StTTTGTG AAXOCGAAGt GYGYCTTLozC 420

TTTTATTTGT GTGTATGATTtTOCGATAAT TCATAAGTAA TGTAGTAMTT A nTGATT ATATTTCAAT 490

TTTATTGTAA TATAATTTCA ATTGTAATAA TATAAAATA AATATCtM ATGTGTTCTT GATTTCGTTT 560

TGTATATGOC TAGATTCA TCTOCCACCA CGAOGAATG CTOCGCOG WAAGTTCAG ATCTTTCT 630

ORF1CTTCTAT0OL GGALACT ATG TCG CM GOC AG? AGO ACC TC AGT GiC ATT GCC

MET S-r Gln Gly Sr Arg Pro Thr Ser Ser Asp Ile Ala

GTC AAC CAG COC GM TOC CC AAG tT GCA OWC TTC LAG GtC GCC AGT ACC CG 740Va1 Ann Gln Arg Glu Cys Val Lys Val Glu Gly Phe Lys Val Val St Thr Arg

TLA AG TCG GCA TA? GAO AG? TTT TCT CAT CAG OCA COC tTG CTG GMC CTC 794

Lou Arg Sot Ala Glu Tyr Glu Ser Phb S-r His Gln Al Arg Leu Leu Gly Leu

TCC GAC AGC ATG GMC ATA COG G?T 0C CDC CCC ATT GOt QOCmTT CTT GLA 848Ser Asp Sar MfT Ala I Arg Val Ala Va Arg Ag Ile Gly Gly Phe Leu Glu

ATC GAC OCA GAO ACT CC? CAT AGO ATG GAO OCC ATA CTA CAA tCC ATA GL ACA 90211e Asp Ala Glu Thr Arg His Arg MET Glu Ala Ile Leu Gln SOr Il Gly Thr

CTC TCA AOC LAC A? GCC GC CTG CTA ?C? 0CC TAT 0CC GAL AL? CCG AOC A?G 956

Leu Sor SOr Ann I11 Ala Ala Leu Lou Ser Ala Tyr Ala Glu Ann Pro Thr MET

GAT TTCG AG OCT tTO CCL OCT GAL CCT ATC 0CC ttC MAL tCT TTC OCT GAC 1010Asp Leu Glu Al Leu Arg Ala Glu Arg II* Ala Phe Gly Lys Sor The Ala Asp

CTC GAC GGC TTG CTC CC? TCC A?? TTG ?CC GTA tCA COG CCG COO ATC GAC OGt 1064Lou Asp Gly Lu Leu Arg Sor Ile Leu Ser Val Scr Arg Arg Arg Ile Asp Gly

TGC TCG CTC CTG AMA GAOCC TTG TAO CACTGACTA OCACTTOC OGOLATAT 1121

Cyn Sor Leu Leu Lys Asp Ala LeuORF2

TCG ACT CCC GA? CGA OCT CAL ATC ACT CCC ATT GMG CCCGA GOC A 1175ME? Pro Asp Arg Ala Gln Va1 Ie Ile Arg Ile Val Pro Gly Gly 0ly Thr

MG ACC CT? CM CM AT? ATC ALT CAG ?TG GAG TA? CTA ?CC COG LAO GGC AGO 1229Lys Thr Lou Gln Gln Ile O1l Ann Gln Leu Glu ?yr Leu Ser Arg Lys Gly Arg

CTG GAG CTC CAG CCt TCA GCC CG CAT CTC GA? ASTCTC CTC CCL COG GA? CM 1283Lou Glu Leu Gln AMg S-r Al Atg Sis Leu Asp Ie Pro Leu Pro Pro Asp Gln

ATC CAC GAL CT? 0ccCC AOQCTOO GC CM AG CTG ACT TA? GAC GAL 133711l His Glu Leu Ala Arg S-r Trp Val Gln Glu Thr Gly Thr Tyr Asp Glu aer

CAG CCA GAC GAG GAL AGO CAL CAG GAG tSG ACC ACC CA? ACT ACT OTA AC tC 1391Gln Pro Asp Glu Arg Gln Gln Glu Leu Thr Thr Sis IIle Ie Val 5tr The

CCC 0CC G0T AOC AGC CAG GTA G OCT TAT GCC AGC COG GA TOO OCA 1445

Pro Ali Gly Thr Ser Gln Val Ala Ala Tyr Ala Ala SOr Arg Glu Trp Ala Ala

GAG A6C CT? G00 tCA QOC OCA QQC GMG GOC CG TAC LAC TA CTt ACC CC TtC 1499Giu IM? Phe Gly Sr Gly Ala Gly Gly 0ly Arg Tyr Ann Tyr Lou Thr Ala Fle

CAC ATC GAT WCCAC CAC CCA CAT CTG CAT CtC GtC GTC ALT CCG CCC GAL CT? 1553His Iie Asp Arg Asp lis Pro Sis Leu Sis Va1 Va1 Val Ann AMg Arg Glu Leu

TTA OG CAO OOC TOG CTCG AG ATA TC? CCO COC CA? C CM CTC MT TAC 1607Lenu Hin Gly Trp Leu Lys l Ser Ag Arg Nis Pro Gln Leu Lsn Tyr Anp

OCC CSC COC ATA LAG ATC QCC C ACT TCL CT? CC? CTW? AT 0CC CC GA? 1661Ale Leu Arg Ie Lys ME? Ala Glu Ie Sr Leu Arg lis G1y I* Ala Lou LAp

CG AOC CGA CCA OCA GA CUT WC ATC ACX GAG COG ATC ACT TAT OCC CM 1715Ala Ser Arg Arg Ala Clu Arg Gly Ile Thr Clu rg Pro Ile Ter Tyr Ala Cln

TA? C CO CT? GAG C GAG CAG OCT CCC CM ACC WT TCC C GACO GA? 1769Tyr Arg AMg Leu Clu Arg Clu Gln le Arg CLn Ie* Arg Pbe Gl Asp Ala Lsp

TTC GA CAG TCC TC =CC CM OGA GA? CA? CL GAA ?TC AGC CM CCT ?TC GA? 1923

Leu Giu Gln Set 5er Pro Gln Gly Anp Sin Pro Clu PbT Sr Gln Pro Pbe Asp

ACA TCC CCA CT? GM OCA TCC OM OOC CC GAG GAC 6C CC? CO C 1577

Thr Sor Pro PTh Gln Ala Sr Ala G1y Gly Pro Glu Lsp OC? Pro AMg Pro LA

AL? CCO CAG W TCG CM GSS CA? CSC CG*C CCLA OCT 00T OCC AG LA 1931Asn Arg Gln Lar Gil 5cr Gn V.1 Nie Leu Gin Glu Pro Ala Gly Val 5cr Asn

00C TCC C?? CCO GTT QCA STT Gl A0 GW CCC CT? OCT CMA CCA 19650lu Ala 01y Va1 Lou Vl Arg Va1 Ale Lou Glu Tir Clu Arg Leu Ala Gin Pro

TCC OTT TCC CAM AT? CTC OCC GAC GAO A?A AGC GM TC? TSCC CUT GT? 2039Pbe Va1 5cr Glu Tir IIS Len Ala Amp Asp SIe 017 5cr Gly 5r Ser Arg Val

GAG QQC GOG AGC OCA LAO aC ACT = GA? AT? CCT CCC OCA OCL 2093

Ala Clu y1rg Val Glu 5cr Ala An Arg Tir Pro Lsp lIe Pro Arg Ale Ala

ACT QC OCT AC CAC AOL CAC CCO CAG CCGC?T OCA ASCC CC 2147

Thr Glu Ala Ala Tir Sis Tir Thr s A p ArM Gin g Mrg Ala Lys rg Pro

CA? GA? CGA GAC C00 ACG?C OCA AMA CCt ACO TTG CA GMC 6TC 2201Sis ALp LAp Amp 01y 0ly Pro Sr Gly Ala Lys Arg V.1 Tir Ln Gilu Gly le

GMC O? CAG CA C 0CC OC CM CAG CGA GOC AG? AGT WOC = TTL 2255Ala Va1 01y Pro Gl Arg Thr Ala Gly Glu GIn Ap Gly Scr Scr 01y Pro Leu

CM C CM QOCT 0A ACC?TC CMC CCA TCC CCA ca AC0QCC ACG tC COC 0CC 2309Glu Arg GLn Ala 01y Tier Br Arg Pro 5cr Pro Pro Thr Ala Tier Tr rg Ale

AQC ACC OCA ACC OAT CCA TT STCC OCT AOL CC CAC CTC CAG CAA COM A" 0c 23635cr Tir Ala Thr Amp 5cr Leu 5cr Ala Thr Ale Nie Leu Ga Gln rg Arg 01y

CTC CTT TCA LAG CC?CT CC? GA? GA? CA? 0GA CMA G ACG CM C AM 2417Val Leu 5cr Ly Arg Pro Arg Glu Asp Amp Asp Gly Glu Pro 5cr Glu rg Lye

CGCC GAGA OAT GA CCC A LAG CC? 000 QC AL? AO WA TAGLAOA 2472AMg Glu AMg sp Clu Arg Sr Lye Asp Gly Arg Gly 1y An Arg Arg

ORF3OACCI AT0 OCA MT CM GAG TCC ACC AC6 CAC TA? 0CC 0G0 TT CC? 0TG 2530

IfT Ala Aa Gilu Glu Phe Thr Arg His Tyr Ala ?rp Pro V.1 Pro V.1

OCT TCC AAT GA? CM 0W CC? A6C 0CCcGCO CA CCc CAG OCA CAA CCA 2564Ale Sr Aso Asp Glu Gly Arg 01y Thr Al Arg I1* Pro IIe Gin Ala ClG Ser

ACC OCT 06A CMA -AC CWO GAC ACT SCC OTC CCA ACC QS TTCC CCA 2638le Va1 Ala 01y Glu ALp 01y Arg Asp Thr 5cr Val Pro bTr Ala Ln 5cr Arg

CO CCL AT? CAT CLC GMC GTC CM CMAA ACO TCO QC ACG QOC 06A 2692

Pro Pro ls Gilu Asp MT Pro Sis Gly Vl Giln 0lu Tbr 5cr Ala 5cr 01y Gly

CGA CTG CCA?GCCCWCTGCM GA? SCC TA ATCC W CCLA ATA TOC CM 2746Arg Leu 01y Ala Ale Arg Leu Arg Amp 5cr Val le Pro Pro 01y Ile Sr Clu

GM CaC AC GAC CTA TOC WCA AT? TTC COG AMA AMA AGC 0G? TCC SC cc ACC 2800Al Arg Thr Asp Leu 5cr Ala Ile Leu Arg Lys Lys Sr Gly Scr he ArMg Thr

00T 6T0 CAG TAT CTC CC? OG CT? CO CM MT TT? CA? AMACM GAC AGO 2854Gin Tyr Leu Arg 01y Leu Glu AMg Glu Ao DTe Asp Lye Gn ALp AMg

GC 0CC AG? 0cC Te CCLA GA? TTA AGT OCc AUG OC ATA AAG CCl CO CCC CM 2906Glu Ala 5cr Ala Leu Pro Asp Leu Sr Al Arg G1y Ile Lys Arg Pro Arg Glu

AT? GAG TAT =OC AT OCA AGC 00A TA CC ATA AAGacC GAGOCOC T?A 2962

l1e Glu Tyr Pro Sly An Ala 5cr Leu Thr le Lye Ara GI Asp Leu

GOC ATA 6C AAT ACT 6CC TCO WCA TCC? TCC GeseAAC CM G CCC OCA 3016

Gly Ile Clu Ie Amn Tbr Ie Sr Ale 5cr 5or Pro Val Arg Gly Arg Ala

TTC 0CC Cm CTC OCA AGO CC CC 06A 6CC COT GCA CMA S00 CCA CC 6CC 3070fTe Va Clu Len Ala rg Ag Ala Gly Thr 01y Arg Val CGi Cy& 5cr Ala Ile

OOC A" TAG ;LC;AGTCCOCCAGG AAGTTAC GTTTCCCfiL_C TTTCCT?CTCs 313901y Arg L.O- RF4

CTATTCAGOC CCAOTCCCO AATOTTCG GCGATACST 0OCAGG CAG CCAGTCLAO 3209

ACCATTTCCT CACCT AG7T7GT?W ATT766 CAC TAT CTTGCCC 3279

AAOCCGA TCTTTCATT ACOTAACA CCCCMATCC GAAOCTGAG OCLAGTCCC ATAGCCCAOA 3349

CLOAAACT CT?M TCAGCAGT CCWC GAAAOW6 00OG?OC T00C L13419

ATCCT?O? CCTCGA CCTTT1X OCGC0CCAG AGTTACCAC AAAA CTTCCT 3'e9

CAA OG ACICC0ACCA TG? TCCCC C7CCAOM C 3559

00cCCAC TT?CC?A cACAGCAGCTTTC C OC0TCCG 3 2

CA "W GGTIIA C SAACTG 00T 3662

FIG. 1. Nucleotide sequence of virD operon of pTiC58 (GenBank accession no. M33673). (A) Sequencing strategy. Sequencing was donein both directions (as indicated by the arrows) starting from the chosen restriction enzyme sites. B, BamHI; Bg, BgIIl; E, EcoRI; H, HindIll;K, KpnI; N, NruI; S, Sall. ORF, Open reading frame. Numbers correspond to those assigned previously (9) to the fragments obtained uponrestriction of plasmid pTiC58 with the corresponding enzymes. (B) Nucleotide sequence and deduced amino acid sequence.

4434 WANG ET AL. J. BACTERIOL.

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virDl-virD2 OVEREXPRESSION IN A. TUMEFACIENS 4435

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pTiC58 301 GSSrYAEgRVesANrtpDiPRaaTeaAtHTThdRQRRaKRPhbDDD-t`,EpRiA4b 293 GSppVsdvRagnANadsDlPR stvartTdysQRwSKRPrDDDeCrLEGmAVGPEAnAGErDsrgdPvappAeTSRPSslqdmaRpNTATD

pTiA6NC 381 PvSA sigteqpeaspKRPRdrhDWlggRXRARgnRrdDGUR GtpT iC58 401 sLSAtaHLqQRRvLSKRPRedDDEPSERXReRDeRSkDGRGGNMpRiA4b 390 PLaAsgHLeQRRGtLSKRPRveDDDGEPSERKRARDdSqDGGRQNn

FIG. 2. Comparison of amino acid sequence of VirD2 proteins from octopine pTiA6NC (44), nopaline pTiC58, and A. rhizogenes pRiA4b(17). The amino acids in the shaded areas are identical. Double dots indicate functionally conservative amino acid substitutions. Single dotsindicate that two of three amino acid substitutions are functionally equivalent. Nonconservative differences are indicated by lowercase letters.

interphase and aqueous phase after phenol extraction wereexamined for the presence of T-strand molecules (16). SinceVirD2 was demonstrated to bind firmly (covalently) at the 5'end of the T-strand molecule, the T-strand-VirD2 proteincomplex (T-complex) was expected to be present only in thephenol-aqueous interphase (Fig. 3B, lanes 1 and 4) (16).Figure 3B shows that bacteria carrying plasmid pND6 orpND5 no longer produced T-complex (Fig. 3A, lanes 2 and3), indicating that the 108-bp DNA sequence between theXmaIII and EcoRI sites of virD2 is essential for generatingthe T-complex. Since these constructs have lost part of the

VirD2 conserved region, it suggests that most, if not all, ofthe N-terminal domain is functionally involved in nickingand T-strand formation, while the C-terminal domain is notrequired for the enzymatic activity.

It has been shown that the 3-galactosidase gene fused tovir genes can be regulated under the virA and virG system(31). We have used a similar approach to characterize generegulation but used the nptII gene (27). The constructpVND3-Km carries a chimeric virD2 gene with the nptIIgene translationally fused in the middle of the reading frame(see Materials and Methods). This plasmid was tested for its

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FIG. 3. Functional analysis of virD2 in E. coli. (A) Various subclones of virDI and virD2 in plasmid pUC18. Boxes represent ORF1, ORF2,ORF3, and part of ORF4 of the virD operon. Numbers above restriction enzyme sites indicate their coordinates. B, BamHI; E, EcoRI; ES,EcoRV; P, PstI; S, Sal; X, XmaIII. (B) Autoradiography of T-strand assay in E. coli. E. coli strains containing pGA472' and various virDsubclones were lysed by SDS and phenol extracted. DNAs from the phenol-aqueous interphase (int) or aqueous phase (aq) were treated withpronase and precipitated. After agarose electrophoresis, DNA samples were blotted without denaturation and hybridized with 32P-labeledplasmid pGA472'.

pTiA6NC

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pRiA4b

pTiA6NC 101

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Aa b B

_4S0 Np .X:

:0..

FIG. 4. VirD2-NPTII fusion. (A) T-strand assay in E. coli carry-

ing various plasmids. All manipulations are described in the legend

to Fig. 3B. Lanes: a, pND3 plus pGA472'; b, pVND3-Km plus

pGA472'; c, pGA472'. (B) Native PAGE for detecting the VirD2-

NPTII (VD2-NPT) fusion protein. Lanes: 1, pVND3-Km; 2, control

strain carrying wild-type nptII. (C) NPTII assay of A. tumefaciens.

-AS, Cells incubated without AS; +AS, cells incubated with AS.

ability to generate T-strands in E. coli (Fig. 4A). Neomycin

phosphotransferase activity was assayed by native PAGE as

described previously (38). Figure 4B shows that the fusion

product had a different mobility in native polyacrylamide

gels. The same plasmid was introduced into an Agrobacte-

rium strain carrying a wild-type Ti plasmid, pGV2301 (J.-P.

Hernalsteens, unpublished data). As can be seen in Fig. 4C,

the enzymatic activity of the nptII gene was increased about

10-fold when A. tumefaciens(pVND3-Km pGV2301) was

incubated with AS (Fig. 4C). These data suggest that the

VirD2-NPTII fusion protein is being regulated by the virA

and virG system, as previous studies showed for a virD-lacZfusion (31). The fact that the VirD2-NPT1I fusion protein is

active in T-strand production provides further evidence that

the C-terminal part of the protein is not essential for this

function. The C-terminus can be replaced by a totally

different peptide without disturbing its activity. On the other

hand, introduction of this construct into A. tumefaciensallowed us to monitor AS induction with a system different

from the widely used vir-lacZ fusions. In this case, the level

of induction of the different vir genes can be studied withoutinterference, due to the well-known stability of the LacZ

protein (37).

Overexpression of virDi and virD2 increases T-strand pro-

duction in A. tumefaciens at early times after induction. In

previous work we had observed that subclones of the virD

gene in a pUC18 vector resulted in a higher yield of T-

complex molecules than when cloned in a pBR322 vector in

E. coli (16). Since both virD clones contained the virD

promoter, the difference in yield is probably due to thehigher copy number of the pUC18 vector. The T-complexmolecule has been hypothesized to be a transfer intermedi-

ate; therefore, it was interesting to investigate whetherincreasing the copy number of the virD gene would lead to

increased amounts of T-strand in A. tumefaciens and, in

turn, increased oncogenicity on plants.

A 4-kb fragment containing a broad-host-range origin ofreplication from pVS1 (19) was introduced into pND2 (seeMaterials and Methods). This new construct, pVND1, car-rying virDi and 50% of virD2, was mobilized into A. tume-faciens pGV3850 (46). Agrobacterium strains carryingpVND1 and pGV3850 thus harbored six to seven extracopies of the virDi and virD2 genes in addition to theirwild-type virD gene. Total DNA from AS-induced Agrobac-terium was digested with HindIII and subjected to alkalinegel electrophoresis (22). Under these conditions, the nickedborder fragments generated by the endonuclease activityupon HindIll digestion could be strand separated. Theseparated molecules were blotted onto a nitrocellulose mem-brane in 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 Msodium citrate) and hybridized with the 32P-labeled fragmentHindIII-23 containing the right border.

Figure SB shows the effect on generation of border nicksand T-strands at different time points after AS induction. At4 h after AS induction, border nick products and T-strandmolecules were already detected in the strain carryingpVND1 and pGV3850 (Fig. SB, lane 2; Fig. SC, lane 2), whilelittle or no nick products and few T-strands were detected inthe strain carrying pGV3850 alone (Fig. SB, lane 1; Fig. SC,lane 1). A similar effect was observed after 8 h of ASinduction (Fig. SB, lanes 3 and 4; Fig. SC, lanes 3 and 4).However, after 16 h of AS induction, this enhancing effect ofextra copies of virDI and virD2 on nick and T-strandproduction in the strain harboring pVND1 and pGV3850 wasno longer significant in comparison with that in the straincarrying pGV3850 alone (Fig. 5B and C, lanes 5, 6, 7, and 8).When incubated in the absence of AS, neither strain pro-duced T-strands (Fig. SC, lanes 9 and 10). This indicates thateven though the rate of induction was increased, the finalnumber of nick products and T-strand molecules reached thesame level in both A. tumefaciens strains.

Overproduction of VirDl and VirD2 proteins in A. tume-faciens elevates transformation efficiency on plants. PlasmidpVND1 was also introduced into A. tumefaciens carryingpGV2301. This strain harbors a wild-type nopaline Ti plas-mid (J.-P. Hernalsteens, unpublished data). The effect ofoverproduction of VirDl and VirD2 on plant transformationwas examined by assaying tumorigenicity. Tobacco leafdisks, kalanchoe stems, carrot root slices, and potato tuberdisks were chosen as plant material for Agrobacteriuminfection.The responses of kalanchoe stems to the strain carrying

pVND1 and pGV2301 could be observed a few days earlierthan those to the strain carrying only pGV2301. The size oftumors caused by the strain carrying pVND1 and pGV2301was significantly larger than that caused by the strain carry-ing pGV2301. For example, after 19 days of infection,tumors caused by A. tumefaciens(pVND1 pGV2301) wereabout 8 mm in diameter, while tumors caused by A.tumefaciens(pGV2301) were about 3 mm in diameter. More-over, more roots were generated on the infection sites of A.tumefaciens(pVNDI pGV2301) (Fig. 6A). A similar effectoccurred on carrot root slices (Fig. 6B). More tumors wereobserved on slices infected by A. tumefaciens(pVND1pGV2301).To obtain quantitative data for comparison of transforma-

tion efficiency, an assay system on potato tuber disks wasused. Uniformly prepared potato tuber disks made by pre-viously established procedures (39), were inoculated withequal amounts bacterial cells, and the number of individualtumors was then scored after 14 days of infection. Theresults are presented in Table 1. The strain carrying

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6.5HI --IH10

L. , ,L01 1

3.5 3

B 4hr 8hr 16hr 24hra b a b d b a b

1 2 3 4 5 6 7

C (-AS)4 hr 8hr 16hr 24hr 24hrab a b a b a b a b

1 2 3 4 5 6 7 8 9 10

FIG. 5. Kinetics of the generation of border nicks and T-strands.(A) Possible T-DNA border structure of pGV3850 (46) T-DNA afterAS induction. The vertical bars represent HindIII restriction sites.HindIII-10 is a 6.5-kb Hindlll fragment containing the left bordersequence, and HindIII-23 is a 3.3-kb HindlIl fragment containingthe right border sequence. The T-DNA (dotted line) is from pBR322in this case. Arrows represent the border nicks at the bottom strandof T-DNA after AS induction. Solid circles represent the bindingproteins at the 5' ends of nicked products. (B) Autoradiography ofalkaline gel electrophoretic analysis. Bacteria were collected after 4,8, 16, and 24 h of incubation with AS. Total agrobacterial DNA wasextracted by the pronase treatment procedure described before (16).DNAs were digested with HindIlI, separated by alkaline gel elec-trophoresis, and blotted in 20x SSC. Fragment HindIII-23 was usedas the probe. The solid triangle indicates HindIII-23; the opentriangles indicate nick products (sizes shown in kilobases). Thearrowhead indicates T-strand molecules. Lanes: a, strain carryingpGV3850; b, strain carrying pVND1 plus pGV3850. Note that allfragments indicated here are single stranded. Other bands areprobably the result of cross-hybridization of contaminating pBR322sequences in the probe, since the HindIII-23 fragment was sub-cloned in a pBR322 vector. (C) T-strand assay in Agrobacteriumcells. All sample manipulations and the nondenaturing gel systemhave been described before (16). 32P-labeled plasmid pBR322 wasused as a probe. -AS, Cells incubated without AS. Lanes: a, straincarrying pGV3850; b, strain carrying pVND1 and pGV3850.

pGV2301 produced on average 71.3 tumors per disk (30 diskswere assayed). The strain carrying pVND1 and pGV2301produced on average 102.7 tumors per disk (32 disks wereassayed).

Since the data were derived from a large sample size, theprobability that the VirDl- and VirD2-overproducing straincarrying pVND1 and pGV2301 is different from the straincarrying only wild-type pGV2301 in its tumor-forming abilitycould be assessed statistically. As shown in Table 1, a t valueof 3.78 indicates that the probability that this difference isdue to chance is less than 0.001, suggesting that the twostrains have a real discrepancy in their ability to form tumorson potato tuber disks.

FIG. 6. Virulence assays on plants. For each panel, the topshows the result with a strain carrying pGV2301 only, and thebottom shows the result with a strain carrying pVND1 pluspGV2301. (A) Inoculations on stems of kalanchoe. (B) Inoculationson root slices of carrots.

While the elevated transformation efficiency of the straincarrying pVND1 and pGV2301 was observed on kalanchoe,carrot, and potato, this difference was not significant whentobacco leaf disks were used as an assay system (data notshown). This is actually consistent with the previous obser-vation that the potato system is more sensitive in detectingdifferences in tumorigenicity between strains (39).

DISCUSSION

The nucleotide sequence of the virD locus from thenopaline-type Ti plasmid pTiC58 was determined in thiswork. In contrast to the previous report on the same DNAfragment (15), our analysis predicts that the first three openreading frames of this locus could produce three polypep-tides of 16.1, 49.7, and 21.4 kDa.VirDl proteins have been found to have high homology

(93%) between Agrobacterium species (K. Wang, unpub-lished data). This protein is absolutely needed for catalyzingsite-specific and strand-specific cleavage at the T-DNAborder sequences with the VirD2 protein (16, 18, 35, 44).Recently, it has been reported that the VirDl protein pos-sesses topoisomerase activity capable of converting super-coiled DNA (form I) to relaxed DNA (form IV) (12). Thefunction of VirD3 is not yet known. This protein is notconserved, since there is only 31% homology between theequivalent proteins of octopine and nopaline Ti plasmids (K.Wang, unpublished data). Deletion of the virD3 gene has noeffect on T-complex formation in E. coli (10, 16). However,the need for the virD3 gene for tumorigenicity has yet to bedetermined.The virD2 gene encodes a protein which is involved in the

AS-induced activity for nicking at the T-DNA border se-

quences and T-strand generation (35, 44). This protein is alsofound to bind firmly (covalently?) to the 5' end of theT-strand (16, 18, 42, 45). The striking feature of this protein,in contrast to many other vir-encoded peptides, is theevolutionary conservation of the amino acid sequencethroughout the N-terminal half of the protein (Fig. 2). Ninetypercent homology was found between residues 1 and 203(45% of the total sequence) of VirD2 from nopaline, oc-topine, and A. rhizogenes Ti plasmids, whereas the remain-ing 55% of the sequence had only 26% homology. Twenty-seven residues in the first 203 amino acids were different

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TABLE 1. Oncogenicity of A. tumefaciens carrying various plasmids on potato tuber diskSa

No. of tumors No. of tumors No. of tumorsDisk no. pVND1 + Disk no. pN1+Disk no. VD+pGV2301 pGV2301 pGV2301 pVN2301 pGV231 pVND2301

1 48 84 11 86 91 21 38 1392 40 67 12 96 120 22 85 963 61 56 13 86 99 23 145 464 57 87 14 78 107 24 112 1025 49 61 15 55 121 25 113 936 31 95 16 65 140 26 91 887 28 103 17 64 143 27 104 568 65 93 18 80 73 28 106 699 35 214 19 49 60 29 55 17010 88 162 20 66 116 30 63 117

a Mean (± SD) number of tumors per disk: pGV2301, 71.3 ± 27.9; pVND1 plus pGV2301, 102.7 ± 36.6; to.001, 3.78.

between bacterial strains. Among these differences, 10 res-idues were conserved substitutions, e.g., arginine for lysine,valine for leucine, alanine for threonine, and aspartic acid forglutamic acid. The conserved N-terminal 50% of VirD2 isessential for the activity of border-nicking and T-strandbinding, since a deletion located at the end of the first203-amino-acid domain (Fig. 3A, pND6) abolished this ac-tivity completely (Fig. 3). However, deletions at the variableregion of the C-terminal 50% (residues 227 to 447) do notaffect T-complex formation in E. coli (10, 16).The role of the C-terminal half is not quite understood. A

computer homology search indicated that while this domainwas poorly conserved, all VirD2 proteins displayed a verysimilar hydropathy profile in this region (Fig. 7). Geneticstudies showed that when the C-terminus of VirD2 wasreplaced by ,-galactosidase (30) or NPTII protein up toamino acid 227 (this work), T-strand formation neverthelessoccurred (35) (Fig. 4A). Moreover, the N-terminal half of theprotein was sufficient to stimulate tumor formation by A.tumefaciens (Table 1). On the other hand, an Agrobacteriumstrain harboring a lacZ insertion mutation in the virD2sequence encoding the C-terminus was avirulent (30).

It has been observed that there are conserved amino acidclusters at the C-terminus which resemble the consensusnuclear localization signal found in different nuclear proteins(5, 32). In fact, in addition to the two potential nucleus-targeting signals at its C-terminus (clusters KRPR at posi-tions 417 to 420 and KRXR at positions 431 to 434, Fig. 2),a third putative signal sequence was found at positions 31 to34 (RKGR). A truncated VirD2 (the first 292 amino acids)containing only this putative signal from the N-terminal halfwas able to act as a nucleus-targeting sequence in plants (A.Herrera-Estrella, M. Van Montagu, and K. Wang, unpub-lished data). Therefore, the remaining two postulated signalsat the C-terminal region could act as auxiliary nucleus-targeting signals which enhance the efficiency of transport,as shown in animal systems (11). That a lacZ insertionmutation in the C-terminus-encoding part of virD2 wasavirulent may be the result of a change of the tertiarystructure around the insertion, interfering with possibleprotein-protein interactions required at a later stage in theT-DNA transfer process.

Overexpression of the virDi and virD2 genes in A. tume-faciens accelerates both border cleavage and T-strand for-mation. After 4 h of AS induction, more T-strands wereproduced in a strain harboring pVND1 and pGV3850 than ina strain harboring pGV3850 alone. The amount of bordernick products was also increased by the presence of morecopies of virD. While nick products were only seen after 8 h

ofAS induction in the strain carrying pGV3850, border nickscould be detected after 4 h of incubation with AS in the straincarrying pVND1 and pGV3850.A temporal difference in the generation of T-strand and the

border nick products was also observed. A few hours afterincubation of A. tumefaciens with AS, T-strand formationcould already be detected, but not border nick products (Fig.SB, lanes 1 and 2). This delay in production of border nickscould be explained by a DNA synthesis displacement mech-anism at the T-DNA. The transfer process is initiated bycleavage of the bottom strand on the T-DNA borders byVirD proteins. The second step is the displacement of the 5'to the 3' nicked border, producing a single-stranded mole-cule, T-strand. The nicks at the bottom strand of T-DNAborders are simultaneously filled by the replication systemwith the T-DNA top strand as the template. Thus, althoughthe border cleavages presumably occur prior to the forma-tion of T-strand, nick products are detected later thanT-strand production in an induced bacterial population.

It is interesting that the enhancing effect on the generationof T-complex by overexpression of virDi and virD2 in A.tumefaciens can only be observed at early hours of ASinduction. This suggests that the generation of T-complex isregulated. When the amount of substrate, the T-DNA bordersequences, is limiting, increasing the amount of the VirDland VirD2 proteins accelerates T-strand formation, but thetotal amount of T-strand is not elevated. It is unlikely thatthe increased amount of T-complex was degraded in bacte-rial cells, since it is known that the T-strand is linked firmlyby VirD2 at its 5' end and covered entirely by VirE2 proteins(6, 29). Such molecules would not be easily attacked bynucleases. Another possibility is that excess T-complex istransported out of the bacteria. However, there is no evi-dence of T-complex being secreted (34). In addition, suc-cessful transfer of T-DNA needs cell interaction betweenbacterium and plant. The third possibility is that the amountof VirD proteins is regulated in an induced bacterial cell.Excess VirD proteins are eventually degraded when theborder sequences as substrates are limiting.The transformation efficiency was elevated on kalanchoe,

carrot, and potato, which were infected by an Agrobacte-rium strain carrying extra copies of the virDI and virD2genes. This was probably correlated with the increasedT-complex production soon after the virD genes were in-duced. When more VirD proteins were available in the cell,the reaction between VirD proteins and the T-DNA bordersequences accelerated, and T-complex molecules were gen-erated earlier. As a putative transfer intermediate, T-com-plex might therefore enhance plant transformation effi-

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virDl-virD2 OVEREXPRESSION IN A. TUMEFACIENS 4439

1.

- 1.

I

pTiA6NC

100 200 300 400

pTiC58 A

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IpII /I \lllVlJIL1 IJLirA. IJ .sL Li J . . .]

1.0

100 200 300 400

pRiA4b

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100 200 300 400

FIG. 7. Hydropathy profile of VirD2 proteins of octopine(pTiA6NC), nopaline (pTiC58), and A. rhizogenes (pRiA46) plas-mids. Regions above the horizontal axis indicate hydrophilicity,whereas those below the axis indicate hydrophobicity.

ciency. Further work is needed to determine whether plantstransformed by VirD-overproducing strains contain moreT-DNA insertions than those transformed by the wild-typeA. tumefaciens.

ACKNOWLEDGMENTS

We thank A. Caplan and M. Holsters for discussions and criticalreading of the manuscript, R. Villarroel and J. Gielen for DNA

sequencing, Z.-M. Chen for plant work, J. Coppieters for computeranalysis, and M. De Cock, S. Van Gijsegem, and V. Vermaercke forpreparation of the manuscript.K.W. and A.H.E. are indebted to the CEC for a training grant.

This work was supported by a grant from the Services of the PrimeMinister (UIAP 12.0C01.87).

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Nester. 1987. Processing of the T-DNA ofAgrobacterium tume-faciens generates border nicks and linear, single-stranded T-DNA. J. Bacteriol. 169:1046-1055.

2. An, G., B. D. Watson, S. Stachel, M. P. Gordon, and E. W.Nester. 1985. New cloning vehicles for transformation of higherplants. EMBO J. 4:277-284.

3. Bolton, G. W., E. W. Nester, and M. P. Gordon. 1986. Plantphenolic compounds induce expression of the Agrobacteriumtumefaciens loci needed for virulence. Science 232:983-985.

4. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of genecontrol signals by DNA fusion and cloning in Escherichia coli.J. Mol. Biol. 138:179-207.

5. Chelsky, D., R. Ralph, and G. Jonak. 1989. Sequence require-ments for synthetic peptide-mediated translocation to the nu-cleus. Mol. Cell. Biol. 9:2487-2492.

6. Christie, P. J., J. E. Ward, S. C. Winans, and E. W. Nester.1988. The Agrobacterium tumefaciens virE2 gene product is asingle-stranded DNA-binding protein that associates with T-DNA. J. Bacteriol. 170:2659-2667.

7. Citovsky, V., G. De Vos, and P. Zambryski. 1988. Single-stranded DNA-binding protein encoded by the virE locus ofAgrobacterium tumefaciens. Science 240:501-504.

8. Das, A. 1988. Agrobacterium tumefaciens virE operon encodesa single-stranded DNA-binding protein. Proc. Natl. Acad. Sci.USA 85:2909-2913.

9. Depicker, A., M. De Wilde, G. De Vos, R. De Vos, M. VanMontagu, and J. Scheli. 1980. Molecular cloning of overlappingsegments of the nopaline Ti-plasmid pTiC58 as a means torestriction endonuclease mapping. Plasmid 3:193-211.

10. De Vos, G., and P. Zambryski. 1989. Expression of Agrobacte-rium nopaline-specific VirDl, VirD2, and VirCl proteins andtheir requirement for T-strand production in E. coli. Mol.Plant-Microbe Interactions 2:43-52.

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ni L J \I WIW%,VV,.t:...11V IIIIIII r l 7W, ', I |Pjry I I' T~v~ UW

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