Proc. Natl. Acad. Sci. USAVol. 89, pp. 11837-11841, December 1992Genetics

A nuclear localization signal and the C-terminal omega sequence inthe Agrobacterium tumefaciens VirD2 endonuclease are importantfor tumor formation

(DNA transfer/plant tumors/nuclear transport/crown gall)

CLAIRE E. SHURVINTON, LARRY HODGES, AND WALT REAM*Department of Agricultural Chemistry and Program in Molecular Biology, Oregon State University, Corvallis, OR 97331-6502

Communicated by Mary-Dell Chilton, August 10, 1992 (received for review April 28, 1992)

ABSTRACT The T-DNA portion of the Agrobacteriumtumefaciens tumor-inducing (Ti) plasmid integrates into plantnuclear DNA. Direct repeats define the T-DNA ends; transferbegins when the VirD2 endonuclease produces a site-specificnick in the right-hand border repeat and attaches to the 5' endof the nicked strand. Subsequent events generate linear snge-stranded VirD2-bound DNA molecules that include the entireT-DNA (T-strands). VirD2 protein contains a nuclear localiza-tion signal (NLS) near the C terminus and may direct boundT-strands to plant nuclei. We constructed mutations in virD2and showed that the NLS was important for tumorigenesis,although T-strand production occurred normally in its ab-sence. A tobacco etch virus NLS, substituted for the VirD2NLS, restored tumor-inducing activity. Amino acids (theomega sequence) at the C terminus of VirD2, outside the NLSand the endonuclease domain, contributed s fcantly totumorigenesis, suggesting that VirD2 may serve a third im-portant function in T-DNA transfer.

Agrobacterium tumefaciens causes crown gall tumors onmany plants when the bacteria infect wounded tissue (1). TheTi plasmid carries genes essential for tumorigenesis. Trans-ferred DNA (T-DNA) enters plant cells and integrates intonuclear DNA (2) where expression of certain T-DNA genesleads to tumorous growth (1). Virulence genes (vir) necessaryforT-DNA transfer lie elsewhere on the Ti plasmid; woundedplants produce phenolic compounds that induce vir expres-sion (3). T-DNA transmission requires, in cis, the right-hand25-base-pair (bp) border sequence, and deletions removing itabolish tumorigenesis (4-6). Loss of a nearby sequence,called overdrive, reduces tumorigenesis several hundredfold(7). The endonuclease encoded by virDI and virD2 nicks thebottom strand of each border sequence at a specific site (8,9), and VirD2 protein attaches covalently at the 5' end of thenicked DNA strand (10-14). Subsequent events displacelinear single-stranded DNAs composed of the bottom strandof the T-DNA (T-strands) (15-17). VirE2 single-strand DNAbinding protein binds cooperatively to T-strands (18-23). A.tumefaciens probably transfers T-DNA into plant cells viaT-strand intermediates (24).VirD2 contains at least two functional domains. The N-ter-

minal 262 amino acids of VirD2 [424 amino acids (aa) total]perform border nicking and attachment and suffice forT-strand production (10), but mutations at the C terminusabolish or severely attenuate tumorigenesis (25, 26). TheN-terminal endonuclease domains of VirD2 proteins fromthree different strains show 85% sequence conservation, butelsewhere they share only short stretches of similarity (27).Among the short conserved regions, two resemble the Xe-nopus nucleoplasmin nuclear localization signal (NLS) (Fig.

1C) (28), and one of them (Fig. 1A) mediates nuclear trans-port when fused to 8-glucuronidase or /3-galactosidase andsynthesized in tobacco cells (29, 30). Proteins enter nuclei byATP-dependent active transport through nuclear pores (31).Nuclear import depends upon NLSs, short regions rich inbasic amino acids; many NLSs, including those in VirD2 andXenopus nucleoplasmin, are bipartite sequences that containtwo interdependent basic domains, both needed for fullactivity (28) (Fig. 1). Receptors, called NLS binding proteins,recognize NLSs and direct NLS-containing proteins to nu-clear pores where transport into nuclei occurs (31). BecauseVirD2 protein contains at least one NLS (29, 30), it may pilotcovalently bound T-strands into host nuclei (14, 32); how-ever, the importance of the NLS (Fig. 1A) for tumorigenesisremains untested. The NLS in octopine-type VirD2 protein(KRPRDRHDGELGGRKRAR; Fig. 1A) lies near the Cterminus (aa 396-413; see Fig. 2), which is important forvirulence: a mutant strain lacking only the last two aminoacids of VirD2 exhibits very weak virulence, and removal ofthe final 26 aa, including half of the NLS, abolishes tumor-igenesis (26). We performed a detailed genetic analysis anddiscovered that precise deletion of the basic amino acids inthe NLS near the C terminus of VirD2 reduced tumorinduction to about 60% of control values and that an NLSfrom tobacco etch virus (TEV) could compensate in part forloss of the VirD2 NLS. We also found that a second region,the omega sequence, at the C terminus of VirD2 played amajor role in tumorigenesis; deletion of the omega sequencereduced tumor induction to just 3% of wild type.

METHODSBacteriology. Escherichia coli strains used were SK1592

(thi supE endA sbcBJ5 hsdR4) (33), SF800 (polAl thy gyrA)(34), and CJ236 (dut-J ung-) thi-J relAl pCJ105) (35). A.tumefaciens strains (Table 1) contain derivatives of theoctopine-type plasmid pTiA6NC in the C58 chromosomalbackground (36). To select drug-resistant bacteria, we usedampicillin (50 Ag/ml) or kanamycin (25 ,Ag/ml) in L-agar orL-broth (33) for E. coli and carbenicillin (100 ,ug/ml), genta-micin (50 .&g/ml), or kanamycin (100 ,ug/ml) in AB minimalagar or YEP broth (36) for A. tumefaciens. Homogenotiza-tions were performed as described (36).

Deletions. A. tumefaciens strain WR1715 harbored a Tiplasmid with 70% of virD2 deleted (aa 94-388). To constructthis deletion, we isolated the virD operon as a 5783-bp SstI-Bgl II fragment from pVK225 (37) and inserted it intopBS-Bgl (38). Cleavage with Kpn I followed by ligation

Abbreviations: T-DNA, transferred DNA; NLS, nuclear localizationsignal; SV40, simian virus 40; T antigen, large tumor antigen; aa,amino acid(s); TEV, tobacco etch virus; T-strands, linear single-stranded DNAs composed of the bottom strand of the T-DNA.*To whom reprint requests should be addressed.

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A Agrobacterium octopine-type VirD2Agrobacterium nopaline-type VirD2Agrobacterium rhizogenes VirD2

B Agrobacterium octopine-type VirD2Agrobacterium nopaline-type VirD2Agrobacterium rhizogenes VirD2

C Xenopus nucleoplasmin

D SV40 virus large T antigen

IWPRDRHDGELGGR13ARKRPREDDDGEPSERRERKRPRVEDDGEPSERKRAR

(396-413)(417-434)(406-423)

KRRNDEEAGPSGANRRGLK (338-356)XRPHDDDGGPSGAUVTLE (339-357)KRPRDDDEGPSGAKVRLE (328-346)

KPAATKKAGQA1aCLD (155-172)

PKV (126-132)

FIG. 1. NLSs from A. tumefaciens, Xenopus, and SV40. Bold typeface denotes basic residues presumed important for nuclear targeting.Plant NLSs have been previously identified (A) or inferred based on sequence similarity (B). Numbers in parentheses indicate positions ofaminoacids shown.

Table 1. Virulence of strains with mutations in virD2virD2 Relative

Strain allele Disks Tumors perdisk P virulenceGroup a

WR1753 + 110 13.6 ± 11.3 100WR1811 A3 113 12.6 ± 10.2 0.3 93WR1749 A2 107 0.57 ± 0.89 <0.001 4.2

Group bWR1753 + 130 24.8 ± 14.5 100WR1749 A2 132 0.24 ± 0.49 1WR1777 sub#1 131 3.7 ± 3.1 <0.001 15

Group cWR1753 + 261 15.1 ± 11.1 100WR1769 A4 266 12.2 ± 8.7 <0.001 81WR1753 + 443 18.3 ± 14.2 100WR1768 A5 510 12.5 ± 10.5 <0.001 68WR1753 + 701 16.1 ± 15.2 100WR1766 A4+5 582 9.5 ± 10.4 <0.001 59

Group dWR1753 + 377 15.5 ± 16.4 100WR1828 A9 387 10.8 ± 11.7 <0.001 69WR1830 A4+5+9 239 9.9 ± 11.2 0.2 63

Group eWR1753 + 51 10.2 ± 7.2 100WR1814 ins#1 50 9.4 ± 6.3 0.4 93WR1813 ins#2 49 10.1 ± 9.1 0.9 99WR1812 ins#3 48 5.1 ± 3.4 <0.001 50WR1815 ins#4 49 4.2 ± 3.9 <0.001 41

Group fWR1753 + 159 9.7 ± 8.4 100WR1826 sub#2 213 0.32 ± 0.68 <0.001 3.3

Group gWR1753 + 46 26.5 ± 13.1 100WR1749 A2 46 0.35 ± 0.63 1.3WR1816 sub#3 46 0.09 ± 0.28 <0.001 0.33WR1753 + 36 20.9 ± 15.5 100WR1749 A2 39 0.38 ± 0.8 1.8WR1817 sub#4 77 0.12 ± 0.32 <0.001 0.55

Group hWR1753 + 117 19.4 ± 15.9 100WR1766 A4+5 118 12.7 ± 9.8 <0.001 65WR1821 sub#5 115 7.5 ± 6.3 <0.001 39WR1753 + 63 9.5 ± 6.5 100WR1768 A5 64 7.9 ± 4.9 0.01 83WR1825 sub#6 63 5.2 ± 3.2 <0.001 54WR1829 sub#7 63 8.1 ± 5.4 <0.001 85The groups (a-h) indicate strains tested on the same tubers.

Student's t test indicated whether two strains differed significantly.P values refer to comparisons with the strain immediately abovewithin each group, except for group e, where all comparisons arewith WR1753. P < 0.01 indicates that the two strains differ signifi-cantly. P > 0.05 indicates that the data do not prove a significantdifference, and the strains probably exhibit equivalent virulence. Allstrains carry virD2Al on the Ti plasmid. Virulence is expressed as apercentage of that of WR1753. sub, Substitution; ins, insertion.

removed 885 bp (295 codons) from virD2 to produce pWR209(Al; Fig. 2). We joined pWR2O9 and broad-host-range plas-mid pVK100 (37) at their Bgi II sites to form pWR211. Wetransformed pWR211 into A. tumefaciens strain MX304 (25)containing a virD2::Tn3-lac gene and isolated a carbenicillin-sensitive homogenote (WR171S) in which Al replaced thetransposon in the Ti plasmid. Southern analysis (33) con-firmed the structure of this Ti plasmid.We tested all mutations in WR1715, the virD2 null mutant.

Plasmids containing the virD promoter, virDI, and wild-typeor mutant alleles of virD2 in pUC18 (33) were inserted intopVK100 at the EcoPJ site and transformed into WR1715. ThevirD2+ pVK100 derivative (WR1753; Table 1) restored fullvirulence to WR1715.To remove the NLS near the C terminus of VirD2

(KRPRDRHDGELGGRKRAR; aa 396-413), we used Nru Isites (Fig. 2) to remove 135 bp (codons 373-417, including theNLS) producing A2 (Fig. 2). To create virD2A3, we deleted60 bp (codons 373-392) from the leftmost Nru I site rightwardto the HindIII site upstream ofthe KRPRcodons (Fig. 2). Theend produced by HindIII cleavage was made blunt by Kle-nowDNA polymerase I. A3 did not remove the NLS (Fig. 2).We constructed A4 (codons 396-399), A5 (codons 410-413),and A9 (codons 338-356) (Fig. 2) by oligonucleotide-directedmutagenesis (35).

Insertions. We inserted a 12-bp oligonucleotide, composedof two Xho I sites, into virD2 at Nru I or Tha I sites. Eachinsertion added four serine codons to virD2 at the positionsindicated (Fig. 2).

Substitultions. Sub#1 contained a 228-bp Nru I fragment,which encodes the NLS in TEV NIa protease (39), insertedinto the Nru I site created by A2 (Fig. 2). Sub#3 and sub#4had 33-bp and 72-bp oligonucleotides, respectively, insertedinto the same Nru I site; these DNAs encoded NLSs (un-derlined) and kinase sites (overlined) from simian virus 40(SV40) large tumor antigen (T antigen) (sub#3; VSTPPKK-KRKV; ref. 40) or Xenopus nucleoplasmin (sub#4; VSPP-RAVKRPAATKKAGOAKKKKV; ref. 28). The valine res-idues at each end of sub#4 and at the N terminus of sub#3do not normally flank the NLSs. Sub#5 and sub#6 resultedfrom insertion of a 27-bp oligonucleotide encoding the SV40T antigen NLS (underlined) (APKKKRKVR; ref. 41) into anSst II site created by A5 (Fig. 2). The same oligonucleotide inthe opposite orientation produced sub#7 (encoding GP-CASSSAR).

Verification of Mutatio. Sequences of altered regions ofvirD2 were verified by the dideoxynucleotide chain-termination method (33). We mapped insertion mutations byrestriction analysis.

T-Strand Assays. We cultured bacteria overnight at 28°C inM9 minimal medium (pH 5.4) containing 100 ,uM acetosyrin-gone (3). After lysis in 1.25% sarcosyl and Pronase (2.5mg/ml), we extracted with phenol and chloroform and pre-cipitated nucleic acids with ethanol (15). We digested theDNA with EcoRI, which cleaves single-stranded DNA, sub-

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338 356 373 392 396 399 410 413 417 421 424| ~ ~ ~~~~~ ~~~~~~~~I

rsKRrnDeeaGPSGAnRkgLkaaqVdsEAnvGEqDtrddsfnkAaDpvsAsigteqpeaspKRPRdrhnDGEIggRKRaRgfnRrdDGRGGGt

rsKRr RkgLk KRPR RKRaR

Kp Nr Nr

endonuclease Kp Hi262 aa 162 aa

conserved insert 1(276)

ifinsert 2 inserts 3 & 4(372) (417 & 421 )

A1 (94-388)

42 (373-417)43 (373-392) _

44 (396-399) 1AS (41 0-41 3) 1

A4+5 I I49 (338-356) M

A9+4+5 M Isub #2 (418-421) 0

Tumors:0

1%

90

80706060

FIG. 2. Mutations in virD2. Sites used for deletions are shown; Hi, Kp, and Nr denote HindIll, Kpn I, and Nru I sites. Black bars depictsequences removed by deletions. Numbers in parentheses indicate amino acid positions. Virulence on potato (Tumors) is expressed as apercentage of that of virD2+ strain WR1753. Uppercase letters indicate amino acids conserved in three VirD2 proteins, and lowercase lettersare nonconserved amino acids found in octopine-type VirD2.

jected 10 ,ug of nucleic acids to agarose gel electrophoresis,and transferred single-stranded DNA from the native gel tonitrocellulose by blotting (15). We detected bound T-strandDNA by hybridization with a radiolabeled T-DNA restrictionfragment.

Virulence Assays. We inoculated strains with differentvirD2 alleles onto tubers from potato cultivar Desiree andstems of tobacco cv. Xanthi-nc as described (4, 42).

RESULTSAn Additional virD2 Nul Allele. Extant null mutations in

virD2 result from transposon insertions, most of which elim-inate expression of genes downstream in the virD operon.Two nonpolar insertions in virD2 permit expression of virD3and virD4 (25, 26), but regulation of these genes may not benormal. We created virD2lI (Fig. 2), which does not shift thetranslational reading frame, to eliminate VirD2 activity with-out affecting expression of other genes. The strain (WR1715)containing this mutation in its Ti plasmid was avirulent anddid not produce T-strands. Wild-type copies of virDi andvirD2 complemented virD2AJ, restoring tumorigenesis(WR1753; Table 1, group a) and T-strand accumulation (Fig.3). Tumorigenesis requires virD4; thus, Al did not affectexpression of virD4.

Nuclear llzation Sequences in VirD2. The C-terminalNLS ofVirD2 directs f-glucuronidase and f-galactosidase toplant nuclei (29, 30), but its role in T-DNA transfer has notbeen tested. To assess the importance of the NLS fortumorigenesis, we deleted 45 codons from virD2, includingthose that encode the NLS (A2; Fig. 2). This mutationreduced virulence to 1-4% of wild type (compare WR1749 toWR1753; Table 1, groups a and b; Fig. 4A) but did not affectproduction of protein-bound T-strands (Fig. 3).The bipartite NLS contains two basic domains, KRPR and

RKRAR (Fig. 2). We created deletions that removed KRPRor KRAR (A4 and A5; aa 396-399 and 410-413; Fig. 2). Thesemutations caused modest (20-30%o) reductions in virulenceon potato tuber discs (WR1768 and WR1769; Table 1, groupc); even a double mutant lacking both sequences (WR1766)exhibited only a 35-40%)o reduction in virulence (Table 1,groups c and h). However, on tobacco stems, WR1766

exhibited severely reduced virulence, producing tumors thatweighed an average of only 16% as much as those induced bywild-type strain WR1753 (Fig. 4 B and C). Similar mutationsin animal NLSs destroy their function (28, 41). The fact thatelimination of the C-terminal NLS did not abolish tumori-genesis suggests that the proteins bound to T-strands maycontain more than one NLS.

a)

0cc0~Q_

NC40..I_

a1)U)cc-cQ0

N cNlO C> >

FIG. 3. T-strand accumulation. Nondenatured transfer of DNAdetects only single-stranded molecules. Lanes 1 and 2, T-strandaccumulation in strain WR1749 (virD2A2); lanes 3 and 4, strainWR1753 (virD2+). DNA samples were prepared with (lanes 1 and 3)or without (lanes 2 and 4) Pronase in the lysis buffer. T-strands thatremained covalently bound to VirD2 were extracted into the aque-ous/phenol interphase (lanes 2 and 4). Denatured transfer ofEcoRI-digested DNAs indicated that recovery and loading ofTi plasmid wasequivalent for each sample.

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A virD2 + (WR1753) virD2X2 (WR1749) virD2,%2 +-TEV NLS (WR1 777j%

L''HIvirD2 (WR1753) C virD2A4+A5 (WR1766)

FIG. 4. Tumors on potato disks (A) and tobacco stems (B and C).

VirD2 contains a second region (Fig. 1B) resembling theXenopus nucleoplasmin NLS (Fig. 1C). This region containstwo stretches of basic amino acids (KRR and RKGLK; aa338-340 and 352-356) separated by a conserved sequence(GPSGA; aa 346-350) predicted to form a bend (Fig. 1B).Deletion of this region (A9; aa 338-356; Fig. 2) reducedvirulence only 30%o on potato (WR1828; Table 1, group d).Thus, removal of the inferred (KRRNDEEAGPSGANRK-GLK) or previously identified (KRPRDRHDGELGGRK-RAR) NLSs caused similar phenotypes. Removal of bothsequences (WR1830; Table 1, group d) did not decreasevirulence further.Omega, a Third Domain in VirD2. To determine the im-

portance of amino acids outside the NLS, we created A3,which removed 20 aa (373-392) upstream of KRPR (aa396-399) (Fig. 3) but did not affect virulence (WR1811; Table1, group a). A2 and A3 shared a common left endpointdiffering only in the additional 25 aa (393-417) removed byA2. Therefore, removal of these 25 aa (which included theNLS) caused the 25- to 100-fold decrease in virulence (Table1, groups a and b). However, deletions of just KRPR andKRAR (A4 and AS; Fig. 2), which inactivate the NLS,reduced virulence only slightly on potato. Thus, A2 mustaffect another functional domain. To locate this domain, weconstructed mutations downstream of the NLS (Fig. 2).Insertion offour serine residues (after aa 421) into a sequence(DGRGG; aa 419-423; Fig. 2) at the C terminus (ins#4)reduced tumorigenesis to 40-50o of wild type (WR1815;Table 1, group e) as did an insertion (ins#3) four codonsupstream (WR1812; Table 1, group e). Insertion offour serineresidues into other sites (ins#1 and ins#2; after aa 276 and372) in virD2 had no effect on function (WR1813 and WR1814;Table 1, group e). We introduced ins#3 and ins#4 into thesame virD2 gene and used the Xho I sites to delete theintervening four codons (DDGR), creating substitution 2(Fig. 2). This substitution reduced virulence to 3% of wildtype (WR1826; Table 1, group f). Thus, the C-terminal regionof VirD2 serves a second function in addition to nucleartargeting; we call this domain omega (DGRGG).

Alternative NLS Sequences. To determine whether a knownplant NLS could substitute for the NLS in VirD2, wereplaced the 45 aa missing in VirD2A2 with a 72-aa NLS fromTEV NIa protease. This NLS includes two essential clusters(9 and 11 aa) rich in basic residues (39) but differs completelyin primary sequence from the C-terminal NLS of VirD2. TheTEV NLS increased virulence by 15- to 19-fold (compareWR1777 to WR1749; Table 1, group b; Fig. 4A) but did notrestore tumorigenesis completely.

The resemblance between the VirD2 and Xenopus nucle-oplasmin NLSs suggested that animal NLSs might functionin plants. Also, the NLS from SV40 T antigen, when fused to,3-glucuronidase or phage T7 RNA polymerase, mediatestransport of these proteins into nuclei of plant cells (43, 44).We tested whether NLSs from Xenopus nucleoplasmin orSV40 T antigen could substitute for the VirD2 NLS. Both theT antigen (in WR1816) and nucleoplasmin (in WR1817) NLSsnearly abolished the residual virulence associated with thevirD2A mutation (Table 1, group g). The SV40 NLS reducedthe intermediate-level virulence of strains containingvirD2A4+S (WR1821) and virD2A5 (WR1825) by 2-fold, butan inverted copy of the SV40 codons (in WR1829) had noeffect (Table 1, group h). Thus, animal and viral NLSsinterfered with T-DNA transfer despite their similarity to theVirD2 NLS.

DISCUSSIONOthers proposed that VirD2 protein may pilot T-strands intonuclei of plant cells because it contains sequences thatresemble animal NLSs and forms a covalent bond withT-strand DNA (14, 32). In support of this hypothesis, VirD2contains a C-terminal NLS that mediates nuclear transportwhen fused to other proteins (29, 30). The phenotypes ofvirD2A2 and virD2A3 showed that 25 aa that include the NLSwere very important for tumorigenesis but did not affectT-strand production. Because VirD2A2 protein retained en-donuclease activity, we infer that the mutant protein re-mained stable and did not undergo global changes in confor-mation. An NLS from TEV was able to substitute for theC-terminal NLS. Thus, NLS sequences in VirD2 playedsignificant roles in T-DNA transfer.Nuclear transport of T-DNA did not depend solely on the

C-terminal NLS in VirD2. Deletion of the sequences KRPRand KRAR diminished virulence substantially on tobaccostems but only slightly on potato tubers, even though basicresidues are essential for activity of an NLS (31). Thus, theC-terminal NLS was important but not essential for tumor-igenesis. Elimination of a second potential NLS near the Cterminus of VirD2 (A9), even in combination with the dele-tions of KRPR (A4) and KRAR (AS), failed to reduce viru-lence further.

Mutations that affect the efficiency of T-DNA transferoften alter the host range of A. tumefaciens (45-47). Genesthat affect T-strand levels directly (virC) (48, 49), or indirectly(virA and virG) (50), influence host range and virulence(45-47). Plant species may differ in the threshold levels of

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T-DNA transfer necessary to cause appreciable tumorigen-esis. This phenomenon may explain why tobacco stems weremore sensitive to loss of the VirD2 NLS than potato tubers.

Herrera-Estrella etal. (51) reported that the first 292 aminoacids of VirD2, the endonuclease domain, also contain anNLS. They suggested that aa 32-35 (RKGK), which includethree basic residues, may mediate nuclear transport ofT-DNA. We converted one lysine to glutamic acid (RKGK toREGK) without diminishing tumor induction (data notshown). This base change also did not reduce the virulenceof a strain (with M4, A5, and A9 mutations) lacking theC-terminal NLS. Thus, the RKGK sequence appears unim-portant for nuclear import of T-DNA.VirE2 single-stranded binding protein, which contains two

NLSs (52), may target T-DNA to host nuclei in the absenceof the NLS in VirD2. A T-strand DNA 15 kilobases long can

accommodate 500 molecules of VirE2 (22) but only one ofVirD2. It seems likely that the VirE2 NLSs play an importantrole in nuclear transport of T-strands.The omega sequence (DGRGG), at the C terminus of VirD2,

was much more important for tumorigenesis than basic resi-dues in the adjacent NLS (KRPR and KRAR). Sub#2, whichremoved the sequence DDGR, reduced virulence on potato30-fold, whereas deletion of KRPR and KRAR caused only a

2-fold decrease. Omega may facilitate correct protein foldingand thereby indirectly affect nuclear targeting, or it may serve

another function important for tumorigenesis. For example,omega may interact with other Vir proteins that exportT-DNA-VirD2 complexes from bacteria.

Despite the resemblance between animal NLSs and theC-terminal NLS in VirD2, NLSs from animals diminished theability of VirD2 to promote T-DNA transfer. The decrease invirulence was significant but not large (2- to 4-fold). The SV40T-antigen NLS has directed two different proteins to plantnuclei (43, 44); thus, nuclear transport of large T-DNA-protein complexes may differ from import of individualproteins. The reduction in virulence may indicate that theSV40 and Xenopus NLSs, when placed in VirD2, interactwith NLS binding proteins in the host but do not efficientlyenter nuclei. In contrast, a genuine plant NLS (from TEV)substituted efficiently for the native VirD2 sequence. TheTEV NLS did not restore full activity to virD2A2, probablybecause A2 not only removes the C-terminal NLS but alsomay affect the activity of the adjacent omega sequence. TheTEV NLS likely compensated for loss of the native NLS,although a targeting signal that normally mediates import ofa single protein may not be fully capable of transporting a

large T-DNA-protein complex. Nuclear localization ofVirD2 has been well documented (29, 30); however, directevidence for its involvement in tumor induction has now beendemonstrated here.

We thank John Hays, Priscilla Dombek, and Ann Hagemann forcritiques of the manuscript and Jim Carrington, Patricia Zambryski,and Vitaly Citovsky for communicating data prior to publication.Grants from the National Institutes of Health (RO1 GM38294 andBRSG RR07079) supported this research; W.R. received support fromNational Institutes of Health Grant RCDA A100838. This is OregonState University Agricultural Experiment Station paper 9956.

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Genetics: Shurvinton et al.

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