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

A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for 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 (receivedfor review April 28, 1992)

ABSTRACT The T-DNA portion of the Agrobacterium 1C) (28), and one of them (Fig. 1A) mediates nuclear trans- tumefaciens tumor-inducing (Ti) plasmid integrates into plant port when fused to 8-glucuronidase or /3-galactosidase and nuclear DNA. Direct repeats define the T-DNA ends; transfer synthesized in tobacco cells (29, 30). Proteins enter nuclei by begins when the VirD2 endonuclease produces a site-specific ATP-dependent active transport through nuclear pores (31). nick in the right-hand border repeat and attaches to the 5' end Nuclear import depends upon NLSs, short regions rich in of the nicked strand. Subsequent events generate linear snge- basic amino acids; many NLSs, including those in VirD2 and stranded VirD2-bound DNA molecules that include the entire Xenopus nucleoplasmin, are bipartite sequences that contain T-DNA (T-strands). VirD2 protein contains a nuclear localiza- two interdependent basic domains, both needed for full tion signal (NLS) near the C terminus and may direct bound activity (28) (Fig. 1). Receptors, called NLS binding proteins, T-strands to plant nuclei. We constructed mutations in virD2 recognize NLSs and direct NLS-containing proteins to nu- and showed that the NLS was important for tumorigenesis, clear pores where transport into nuclei occurs (31). Because although T-strand production occurred normally in its ab- VirD2 protein contains at least one NLS (29, 30), it may pilot sence. A tobacco etch virus NLS, substituted for the VirD2 covalently bound T-strands into host nuclei (14, 32); how- NLS, restored tumor-inducing activity. Amino acids (the ever, the importance of the NLS (Fig. 1A) for tumorigenesis omega sequence) at the C terminus of VirD2, outside the NLS remains untested. The NLS in octopine-type VirD2 protein and the endonuclease domain, contributed s fcantly to (KRPRDRHDGELGGRKRAR; Fig. 1A) lies near the C tumorigenesis, suggesting that VirD2 may serve a third im- terminus (aa 396-413; see Fig. 2), which is important for portant function in T-DNA transfer. virulence: a mutant strain lacking only the last two amino acids of VirD2 exhibits very weak virulence, and removal of Agrobacterium tumefaciens causes crown gall tumors on the final 26 aa, including half of the NLS, abolishes tumor- many plants when the bacteria infect wounded tissue (1). The igenesis (26). We performed a detailed genetic analysis and Ti plasmid carries genes essential for tumorigenesis. Trans- discovered that precise deletion of the basic amino acids in ferred DNA (T-DNA) enters plant cells and integrates into the NLS near the C terminus of VirD2 reduced tumor nuclear DNA (2) where expression of certain T-DNA genes induction to about 60% of control values and that an NLS leads to tumorous growth (1). Virulence genes (vir) necessary from tobacco etch virus (TEV) could compensate in part for for T-DNA transfer lie elsewhere on the Ti plasmid; wounded loss of the VirD2 NLS. We also found that a second region, plants produce phenolic compounds that induce vir expres- the omega sequence, at the C terminus of VirD2 played a sion (3). T-DNA transmission requires, in cis, the right-hand major role in tumorigenesis; deletion of the omega sequence 25-base-pair (bp) border sequence, and deletions removing it reduced tumor induction to just 3% of wild type. abolish tumorigenesis (4-6). Loss of a nearby sequence, called overdrive, reduces tumorigenesis several hundredfold (7). The endonuclease encoded by virDI and virD2 nicks the METHODS bottom strand of each border sequence at a specific site (8, Bacteriology. Escherichia coli strains used were SK1592 9), and VirD2 protein attaches covalently at the 5' end of the (thi supE endA sbcBJ5 hsdR4) (33), SF800 (polAl thy gyrA) nicked DNA strand (10-14). Subsequent events displace (34), and CJ236 (dut-J ung-) thi-J relAl pCJ105) (35). A. linear single-stranded DNAs composed of the bottom strand tumefaciens strains of the T-DNA (T-strands) (15-17). VirE2 single-strand DNA (Table 1) contain derivatives of the binding protein binds cooperatively to T-strands (18-23). A. octopine-type plasmid pTiA6NC in the C58 chromosomal tumefaciens probably transfers T-DNA into plant cells via background (36). To select drug-resistant bacteria, we used T-strand intermediates (24). ampicillin (50 Ag/ml) or kanamycin (25 ,Ag/ml) in L-agar or VirD2 contains at least two functional domains. The N-ter- L-broth (33) for E. coli and carbenicillin (100 ,ug/ml), genta- minal 262 amino acids of VirD2 [424 amino acids (aa) total] micin (50 .&g/ml), or kanamycin (100 ,ug/ml) in AB minimal perform border nicking and attachment and suffice for agar or YEP broth (36) for A. tumefaciens. Homogenotiza- T-strand production (10), but mutations at the C terminus tions were performed as described (36). abolish or severely attenuate tumorigenesis (25, 26). The Deletions. A. tumefaciens strain WR1715 harbored a Ti N-terminal endonuclease domains of VirD2 proteins from plasmid with 70% of virD2 deleted (aa 94-388). To construct three different strains show 85% sequence conservation, but this deletion, we isolated the virD operon as a 5783-bp Sst elsewhere they share only short stretches of similarity (27). I-Bgl II fragment from pVK225 (37) and inserted it into Among the short conserved regions, two resemble the Xe- pBS-Bgl (38). Cleavage with Kpn I followed by ligation nopus nucleoplasmin nuclear localization signal (NLS) (Fig. Abbreviations: T-DNA, transferred DNA; NLS, nuclear localization signal; SV40, simian virus 40; T antigen, large tumor antigen; aa, The publication costs of this article were defrayed in part by page charge amino acid(s); TEV, tobacco etch virus; T-strands, linear single- payment. This article must therefore be hereby marked "advertisement" stranded DNAs composed of the bottom strand of the T-DNA. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11837 Downloaded by guest on October 1, 2021 11838 Genetics: Shurvinton et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A Agrobacterium octopine-type VirD2 IWPRDRHDGELGGR13AR (396-413) Agrobacterium nopaline-type VirD2 KRPREDDDGEPSERRER (417-434) Agrobacterium rhizogenes VirD2 KRPRVEDDGEPSERKRAR (406-423)

B Agrobacterium octopine-type VirD2 KRRNDEEAGPSGANRRGLK (338-356) Agrobacterium nopaline-type VirD2 XRPHDDDGGPSGAUVTLE (339-357) Agrobacterium rhizogenes VirD2 KRPRDDDEGPSGAKVRLE (328-346)

C Xenopus nucleoplasmin KPAATKKAGQA1aCLD (155-172)

D SV40 virus large T antigen 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 ofamino acids shown. removed 885 bp (295 codons) from virD2 to produce pWR209 Table 1. Virulence of strains with mutations in virD2 (Al; Fig. 2). We joined pWR2O9 and broad-host-range plas- virD2 Relative mid pVK100 (37) at their Bgi II sites to form pWR211. We Strain allele Disks Tumors perdisk P virulence transformed pWR211 into A. tumefaciens strain MX304 (25) Group a containing a virD2::Tn3-lac gene and isolated a carbenicillin- WR1753 + 110 13.6 ± 11.3 100 sensitive homogenote (WR171S) in which Al replaced the WR1811 A3 113 12.6 ± 10.2 0.3 93 transposon in the Ti plasmid. Southern analysis (33) con- WR1749 A2 107 0.57 ± 0.89 <0.001 4.2 firmed the structure of this Ti plasmid. Group b We tested all mutations in WR1715, the virD2 null mutant. WR1753 + 130 24.8 ± 14.5 100 Plasmids containing the virD promoter, virDI, and wild-type WR1749 A2 132 0.24 ± 0.49 1 or mutant alleles of virD2 in pUC18 (33) were inserted into WR1777 sub#1 131 3.7 ± 3.1 <0.001 15 pVK100 at the EcoPJ site and transformed into WR1715. The Group c virD2+ pVK100 derivative (WR1753; Table 1) restored full WR1753 + 261 15.1 ± 11.1 100 virulence to WR1715. WR1769 A4 266 12.2 ± 8.7 <0.001 81 To remove the NLS near the C terminus of VirD2 WR1753 + 443 18.3 ± 14.2 100 (KRPRDRHDGELGGRKRAR; aa 396-413), we used Nru I WR1768 A5 510 12.5 ± 10.5 <0.001 68 sites (Fig. 2) to remove 135 bp (codons 373-417, including the WR1753 + 701 16.1 ± 15.2 100 NLS) producing A2 (Fig. 2). To create virD2A3, we deleted WR1766 A4+5 582 9.5 ± 10.4 <0.001 59 60 bp (codons 373-392) from the leftmost Nru I site rightward Group d to the HindIII site upstream ofthe KRPR codons (Fig. 2). The WR1753 + 377 15.5 ± 16.4 100 end produced by HindIII cleavage was made blunt by Kle- WR1828 A9 387 10.8 ± 11.7 <0.001 69 now DNA polymerase I. A3 did not remove the NLS (Fig. 2). WR1830 A4+5+9 239 9.9 ± 11.2 0.2 63 We constructed A4 (codons 396-399), A5 (codons 410-413), Group e and A9 (codons 338-356) (Fig. 2) by oligonucleotide-directed WR1753 + 51 10.2 ± 7.2 100 mutagenesis (35). WR1814 ins#1 50 9.4 ± 6.3 0.4 93 Insertions. We inserted a 12-bp oligonucleotide, composed WR1813 ins#2 49 10.1 ± 9.1 0.9 99 of two Xho I sites, into virD2 at Nru I or Tha I sites. Each WR1812 ins#3 48 5.1 ± 3.4 <0.001 50 insertion added four serine to virD2 WR1815 ins#4 49 4.2 ± 3.9 <0.001 41 codons at the positions Group f indicated (Fig. 2). WR1753 + 159 9.7 ± 8.4 100 Substitultions. Sub#1 contained a 228-bp Nru I fragment, WR1826 sub#2 213 0.32 ± 0.68 <0.001 3.3 which encodes the NLS in TEV NIa protease (39), inserted Group g into the Nru I site created by A2 (Fig. 2). Sub#3 and sub#4 WR1753 + 46 26.5 ± 13.1 100 had 33-bp and 72-bp oligonucleotides, respectively, inserted WR1749 A2 46 0.35 ± 0.63 1.3 into the same Nru I site; these DNAs encoded NLSs (un- WR1816 sub#3 46 0.09 ± 0.28 <0.001 0.33 derlined) and kinase sites (overlined) from simian virus 40 WR1753 + 36 20.9 ± 15.5 100 (SV40) large tumor antigen (T antigen) (sub#3; VSTPPKK- WR1749 A2 39 0.38 ± 0.8 1.8 KRKV; ref. 40) or Xenopus nucleoplasmin (sub#4; VSPP- WR1817 sub#4 77 0.12 ± 0.32 <0.001 0.55 RAVKRPAATKKAGOAKKKKV; ref. 28). The valine res- Group h idues at each end of sub#4 and at the N terminus of sub#3 WR1753 + 117 19.4 ± 15.9 100 do not normally flank the NLSs. Sub#5 and sub#6 resulted WR1766 A4+5 118 12.7 ± 9.8 <0.001 65 from insertion of a 27-bp oligonucleotide encoding the SV40 WR1821 sub#5 115 7.5 ± 6.3 <0.001 39 T antigen NLS (underlined) (APKKKRKVR; ref. 41) into an WR1753 + 63 9.5 ± 6.5 100 Sst II site created by A5 (Fig. 2). The same oligonucleotide in WR1768 A5 64 7.9 ± 4.9 0.01 83 the opposite orientation produced sub#7 (encoding GP- WR1825 sub#6 63 5.2 ± 3.2 <0.001 54 CASSSAR). WR1829 sub#7 63 8.1 ± 5.4 Verification of Mutatio. Sequences of altered regions of <0.001 85 virD2 were verified by the dideoxynucleotide chain- The groups (a-h) indicate strains tested on the same tubers. termination method (33). We mapped insertion mutations by Student's t test indicated whether two strains differed significantly. P values refer to comparisons with the strain immediately above restriction analysis. within each group, except for group e, where all comparisons are T-Strand Assays. We cultured bacteria overnight at 28°C in with WR1753. P < 0.01 indicates that the two strains differ signifi- M9 minimal medium (pH 5.4) containing 100 ,uM acetosyrin- cantly. P > 0.05 indicates that the data do not prove a significant gone (3). After lysis in 1.25% sarcosyl and Pronase (2.5 difference, and the strains probably exhibit equivalent virulence. All mg/ml), we extracted with phenol and chloroform and pre- strains carry virD2Al on the Ti plasmid. Virulence is expressed as a cipitated nucleic acids with ethanol (15). We digested the percentage of that of WR1753. sub, Substitution; ins, insertion. DNA with EcoRI, which cleaves single-stranded DNA, sub- Downloaded by guest on October 1, 2021 Genetics: Shurvinton et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11839

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

rsKRr RkgLk KRPR RKRaR

Kp Nr Nr endonuclease Kp Hi 262 aa 162 aa if conserved insert 1 insert 2 inserts 3 & 4 (276) (372) (417 & 421) 7Tumors: A1 (94-388) 0 42 (373-417) 1% 43 (373-392) _ 90 44 (396-399) 1 80 AS (41 0-41 3) 1 70 A4+5 I I 60 49 (338-356) M 60 A9+4+5 M I sub #2 (418-421) 0 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 depict sequences removed by deletions. Numbers in parentheses indicate amino acid positions. Virulence on potato (Tumors) is expressed as a percentage of that of virD2+ strain WR1753. Uppercase letters indicate amino acids conserved in three VirD2 proteins, and lowercase letters are nonconserved amino acids found in octopine-type VirD2. jected 10 ,ug of nucleic acids to agarose gel electrophoresis, exhibited severely reduced virulence, producing tumors that and transferred single-stranded DNA from the native gel to weighed an average ofonly 16% as much as those induced by nitrocellulose by blotting (15). We detected bound T-strand wild-type strain WR1753 (Fig. 4 B and C). Similar mutations DNA by hybridization with a radiolabeled T-DNA restriction in animal NLSs destroy their function (28, 41). The fact that fragment. elimination of the C-terminal NLS did not abolish tumori- Virulence Assays. We inoculated strains with different genesis suggests that the proteins bound to T-strands may virD2 alleles onto tubers from potato cultivar Desiree and contain more than one NLS. stems of tobacco cv. Xanthi-nc as described (4, 42). a) a1) cc0 U) cc- RESULTS cQ 0~Q_ 0 An Additional virD2 Nul Allele. Extant null mutations in C4 virD2 result from transposon most of which elim- N N cNl insertions, 0. O C inate expression of genes downstream in the virD operon. .I_ Two nonpolar insertions in virD2 permit expression of virD3 > > and virD4 (25, 26), but regulation of these genes may not be normal. We created virD2lI (Fig. 2), which does not shift the translational reading frame, to eliminate VirD2 activity with- out affecting expression ofother genes. The strain (WR1715) containing this mutation in its Ti plasmid was avirulent and did not produce T-strands. Wild-type copies of virDi and virD2 complemented virD2AJ, restoring tumorigenesis (WR1753; Table 1, group a) and T-strand accumulation (Fig. 3). Tumorigenesis requires virD4; thus, Al did not affect expression of virD4. Nuclear llzation Sequences in VirD2. The C-terminal NLS ofVirD2 directs f-glucuronidase and f-galactosidase to plant nuclei (29, 30), but its role in T-DNA transfer has not been tested. To assess the importance of the NLS for tumorigenesis, we deleted 45 codons from virD2, including those that encode the NLS (A2; Fig. 2). This mutation reduced virulence to 1-4% ofwild type (compare WR1749 to WR1753; Table 1, groups a and b; Fig. 4A) but did not affect production of protein-bound T-strands (Fig. 3). The bipartite NLS contains two basic domains, KRPR and FIG. 3. T-strand accumulation. Nondenatured transfer of DNA RKRAR (Fig. 2). We created deletions that removed KRPR detects only single-stranded molecules. Lanes 1 and 2, T-strand or KRAR and A5; aa 396-399 and accumulation in strain WR1749 (virD2A2); lanes 3 and 4, strain (A4 410-413; Fig. 2). These WR1753 (virD2+). DNA samples were prepared with (lanes 1 and 3) mutations caused modest (20-30%o) reductions in virulence or without (lanes 2 and 4) Pronase in the lysis buffer. T-strands that on potato tuber discs (WR1768 and WR1769; Table 1, group remained covalently bound to VirD2 were extracted into the aque- c); even a double mutant lacking both sequences (WR1766) ous/phenol interphase (lanes 2 and 4). Denatured transfer ofEcoRI- exhibited only a 35-40%)o reduction in virulence (Table 1, digested DNAs indicated that recovery and loading ofTi plasmid was groups c and h). However, on tobacco stems, WR1766 equivalent for each sample. Downloaded by guest on October 1, 2021 11840 Ge'netics: Shurvinton et A Proc. NatL Acad. Sci. USA 89 (1992)

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

B virD2 (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 the The resemblance between the VirD2 and Xenopus nucle- Xenopus nucleoplasmin NLS (Fig. 1C). This region contains oplasmin NLSs suggested that animal NLSs might function two stretches of basic amino acids (KRR and RKGLK; aa in plants. Also, the NLS from SV40 T antigen, when fused to 338-340 and 352-356) separated by a conserved sequence ,3-glucuronidase or phage T7 RNA polymerase, mediates (GPSGA; aa 346-350) predicted to form a bend (Fig. 1B). transport of these proteins into nuclei of plant cells (43, 44). Deletion of this region (A9; aa 338-356; Fig. 2) reduced We tested whether NLSs from Xenopus nucleoplasmin or virulence only 30%o on potato (WR1828; Table 1, group d). SV40 T antigen could substitute for the VirD2 NLS. Both the Thus, removal of the inferred (KRRNDEEAGPSGANRK- T antigen (in WR1816) and nucleoplasmin (in WR1817) NLSs GLK) or previously identified (KRPRDRHDGELGGRK- nearly abolished the residual virulence associated with the RAR) NLSs caused similar phenotypes. Removal of both virD2A mutation (Table 1, group g). The SV40 NLS reduced sequences (WR1830; Table 1, group d) did not decrease the intermediate-level virulence of strains containing virulence further. virD2A4+S (WR1821) and virD2A5 (WR1825) by 2-fold, but Omega, a Third Domain in VirD2. To determine the im- an inverted copy of the SV40 codons (in WR1829) had no portance of amino acids outside the NLS, we created A3, effect (Table 1, group h). Thus, animal and viral NLSs which removed 20 aa (373-392) upstream of KRPR (aa interfered with T-DNA transfer despite their similarity to the 396-399) (Fig. 3) but did not affect virulence (WR1811; Table VirD2 NLS. 1, group a). A2 and A3 shared a common left endpoint differing only in the additional 25 aa (393-417) removed by A2. Therefore, removal of these 25 aa (which included the DISCUSSION NLS) caused the 25- to 100-fold decrease in virulence (Table Others proposed that VirD2 protein may pilot T-strands into 1, groups a and b). However, deletions of just KRPR and nuclei of plant cells because it contains sequences that KRAR (A4 and AS; Fig. 2), which inactivate the NLS, resemble animal NLSs and forms a covalent bond with reduced virulence only slightly on potato. Thus, A2 must T-strand DNA (14, 32). In support of this hypothesis, VirD2 affect another functional domain. To locate this domain, we contains a C-terminal NLS that mediates nuclear transport constructed mutations downstream of the NLS (Fig. 2). when fused to other proteins (29, 30). The phenotypes of Insertion offour serine residues (after aa 421) into a sequence virD2A2 and virD2A3 showed that 25 aa that include the NLS (DGRGG; aa 419-423; Fig. 2) at the C terminus (ins#4) were very important for tumorigenesis but did not affect reduced tumorigenesis to 40-50o of wild type (WR1815; T-strand production. Because VirD2A2 protein retained en- Table 1, group e) as did an insertion (ins#3) four codons donuclease activity, we infer that the mutant protein re- upstream (WR1812; Table 1, group e). Insertion offour serine mained stable and did not undergo global changes in confor- residues into other sites (ins#1 and ins#2; after aa 276 and mation. An NLS from TEV was able to substitute for the 372) in virD2 had no effect on function (WR1813 and WR1814; C-terminal NLS. Thus, NLS sequences in VirD2 played Table 1, group e). We introduced ins#3 and ins#4 into the significant roles in T-DNA transfer. same virD2 gene and used the Xho I sites to delete the Nuclear transport of T-DNA did not depend solely on the intervening four codons (DDGR), creating substitution 2 C-terminal NLS in VirD2. Deletion of the sequences KRPR (Fig. 2). This substitution reduced virulence to 3% of wild and KRAR diminished virulence substantially on tobacco type (WR1826; Table 1, group f). Thus, the C-terminal region stems but only slightly on potato tubers, even though basic of VirD2 serves a second function in addition to nuclear residues are essential for activity of an NLS (31). Thus, the targeting; we call this domain omega (DGRGG). C-terminal NLS was important but not essential for tumor- Alternative NLS Sequences. To determine whether a known igenesis. Elimination of a second potential NLS near the C plant NLS could substitute for the NLS in VirD2, we terminus of VirD2 (A9), even in combination with the dele- replaced the 45 aa missing in VirD2A2 with a 72-aa NLS from tions of KRPR (A4) and KRAR (AS), failed to reduce viru- TEV NIa protease. This NLS includes two essential clusters lence further. (9 and 11 aa) rich in basic residues (39) but differs completely Mutations that affect the efficiency of T-DNA transfer in primary sequence from the C-terminal NLS of VirD2. The often alter the host range of A. tumefaciens (45-47). Genes TEV NLS increased virulence by 15- to 19-fold (compare that affect T-strand levels directly (virC) (48, 49), or indirectly WR1777 to WR1749; Table 1, group b; Fig. 4A) but did not (virA and virG) (50), influence host range and virulence restore tumorigenesis completely. (45-47). Plant species may differ in the threshold levels of Downloaded by guest on October 1, 2021 Genetics: Shurvinton et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11841

T-DNA transfer necessary to cause appreciable tumorigen- 7. Peralta, E. G., Hellmiss, R. & Ream, W. (1986) EMBO J. 5, esis. This phenomenon may explain why tobacco stems were 1137-1142. 8. Wang, K., Stachel, S. E., Timmerman, B., Van Montagu, M. & more sensitive to loss of the VirD2 NLS than potato tubers. Zambryski, P. (1987) Science 235, 587-591. Herrera-Estrella etal. (51) reported that the first 292 amino 9. Yanofsky, M. F., Porter, S. G., Young, C., Albright, L. M., Gor- acids of VirD2, the endonuclease domain, also contain an don, M. P. & Nester, E. W. (1986) Cell 47, 471-477. NLS. They suggested that aa 32-35 (RKGK), which include 10. Ward, E. R. & Barnes, W. M. (1988) Science 242, 927-930. may transport 11. Young, C. & Nester, E. W. (1988) J. Bacteriol. 170, 3367-3374. three basic residues, mediate nuclear of 12. Howard, E. A., Winsor, B. A., De Vos, G. & Zambryski, P. (1989) T-DNA. We converted one lysine to glutamic acid (RKGK to Proc. Natl. Acad. Sci. USA 86, 4017-4021. REGK) without diminishing tumor induction (data not 13. Herrera-Estrella, A., Chen, Z., Van Montagu, M. & Wang, K. shown). This base change also did not reduce the virulence (1988) EMBO J. 7, 4055-4062. of a strain (with M4, A5, and A9 mutations) lacking the 14. Durrenberger, F., Crameri, A., Hohn, B. & Koukolikova-Nicola, Z. C-terminal NLS. Thus, the RKGK sequence appears unim- (1989) Proc. Nail. Acad. Sci. USA 86, 9154-9158. 15. Stachel, S. E., Timmerman, B. & Zambryski, P. (1986) Nature portant for nuclear import of T-DNA. (London) 322, 706-712. VirE2 single-stranded binding protein, which contains two 16. Jayaswal, R. K., Veluthambi, K., Gelvin, S. B. & Slightom, J. L. NLSs (52), may target T-DNA to host nuclei in the absence (1987) J. Bacteriol. 169, 5035-5045. of the NLS in VirD2. A T-strand DNA 15 kilobases long can 17. Albright, L. M., Yanofsky, M. F., Leroux, B., Ma, D. & Nester, accommodate 500 molecules of VirE2 (22) but only one of E. W. (1987) J. Bacteriol. 169, 1046-1055. VirD2. It seems likely that the VirE2 NLSs play an important 18. Christie, P. J., Ward, J. E., Winans, S. C. & Nester, E. W. (1988) J. Bacteriol. 170, 2584-2591. role in nuclear transport of T-strands. 19. Das, A. (1988) Proc. Nail. Acad. Sci. USA 85, 2909-2913. The omega sequence (DGRGG), at the C terminus of VirD2, 20. Sen, P., Pazour, G. J., Anderson, D. & Das, A. (1989) J. Bacteriol. was much more important for tumorigenesis than basic resi- 171, 2573-2580. dues in the adjacent NLS (KRPR and KRAR). Sub#2, which 21. Gieti, C., Koukolikova-Nicola, Z. & Hohn, B. (1987) Proc. Nail. removed the sequence DDGR, reduced virulence on potato Acad. Sci. USA 84, 9006-9010. whereas of KRPR and KRAR caused only a 22. Citovsky, V., De Vos, G. & Zambryski, P. (1988) Science 240, 30-fold, deletion 501-504. 2-fold decrease. Omega may facilitate correct protein folding 23. Citovsky, V., Wong, M. L. & Zambryski, P. (1989) Proc. Natl. and thereby indirectly affect nuclear targeting, or it may serve Acad. Sci. USA 86, 1193-1197. another function important for tumorigenesis. For example, 24. Stachel, S. E. & Zambryski, P. (1986) Cell 47, 155-157. omega may interact with other Vir proteins that export 25. Stachel, S. E. & Nester, E. W. (1986) EMBO J. 5, 1445-1454. T-DNA-VirD2 complexes from bacteria. 26. Steck, T. R., Lin, T. S. & Kado, C. I. (1990) Nucleic Acids Res. 18, 6953-6958. Despite the resemblance between animal NLSs and the 27. Wang, K., Herrera-Estrella, A. & Van Montagu, M. (1990) J. C-terminal NLS in VirD2, NLSs from animals diminished the Bacteriol. 172, 4432-4440. ability of VirD2 to promote T-DNA transfer. The decrease in 28. Robbins, J., Dilworth, S. M., Laskey, R. A. & Dingwall, C. (1991) virulence was significant but not large (2- to 4-fold). The SV40 Cell 64, 615-623. T-antigen NLS has directed two different proteins to plant 29. Howard, E. A., Zupan, J. R., Citovsky, V. & Zambryski, P. C. nuclei (43, 44); thus, nuclear transport of large T-DNA- (1992) Cell 68, 109-118. 30. Tinland, B., Koukolikova-Nicola, Z., Hall, M. N. & Hohn, B. protein complexes may differ from import of individual (1992) Proc. Natl. Acad. Sci. USA 89, 7442-7446. proteins. The reduction in virulence may indicate that the 31. Silver, P. A. (1991) Cell 64, 489-497. SV40 and Xenopus NLSs, when placed in VirD2, interact 32. Stachel, S. E. & Zambryski, P. C. (1989) Nature (London) 340, with NLS binding proteins in the host but do not efficiently 190-191. enter nuclei. In contrast, a genuine plant NLS (from TEV) 33. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Clon- for the native VirD2 sequence. The ing (Cold Spring Harbor Lab., Cold Spring Harbor, New York). substituted efficiently 34. Heffron, F., Bedinger, P., Champoux, J. J. & Falkow, S. (1977) TEV NLS did not restore full activity to virD2A2, probably Proc. Nadl. Acad. Sci. USA 74, 702-706. because A2 not only removes the C-terminal NLS but also 35. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods may affect the activity of the adjacent omega sequence. The Enzymol. 154, 367-382. TEV NLS likely compensated for loss of the native NLS, 36. Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., although a targeting signal that normally mediates import of Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153. 37. Knauf, V. C. & Nester, E. W. (1982) Plasmid 8, 45-54. a single protein may not be fully capable of transporting a 38. Gombart, A. F., Blissard, G. W. & Rohrmann, G. F. (1989) J. Gen. large T-DNA-protein complex. Nuclear localization of Virol. 70, 1815-1828. VirD2 has been well documented (29, 30); however, direct 39. Carrington, J. C., Freed, D. D. & Leinicke, A. J. (1991) Plant Cell evidence for its involvement in tumor induction has now been 3, 953-962. demonstrated here. 40. Rihs, H. P. & Peters, R. (1989) EMBO J. 8, 1479-1484. 41. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984) Cell 39, 499-509. We thank John Hays, Priscilla Dombek, and Ann Hagemann for 42. Shurvinton, C. E. & Ream, W. (1991) J. Bacteriol. 173, 5558-5563. critiques of the manuscript and Jim Carrington, Patricia Zambryski, 43. van der Krol, A. R. & Chua, N. H. (1991) Plant Cell 3, 667-675. and Vitaly Citovsky for communicating data prior to publication. 44. Lassner, M. W., Jones, A., Daubert, S. & Comai, L. (1991) Plant Grants from the National Institutes of Health (RO1 GM38294 and Mol. Biol. 17, 229-234. BRSG RR07079) supported this research; W.R. received support from 45. Jin, S., Komari, T., Gordon, M. P. & Nester, E. W. (1987) J. National Institutes of Health Grant RCDA A100838. This is Oregon Bacteriol. 169, 4417-4425. State University Agricultural Experiment Station paper 9956. 46. Leroux, B., Yanofsky, M. F., Winans, S. C., Ward, J. E., Ziegler, S. F. & Nester, E. W. (1987) EMBO J. 6, 849-856. 1. Winans, S. C. (1992) Annu. Rev. Microbiol. 56, 12-31. 47. Yanofsky, M. F., Lowe, B., Montoya, A. L., Rubin, R. A., Krul, 2. Chilton, M. D., Drummond, M. H., Merlo, D. J., Sciaky, D., W., Gordon, M. P. & Nester, E. W. (1985) Mol. Gen. Genet. 201, Montoya, A. L., Gordon, M. P. & Nester, E. W. (1977) Cell 11, 237-246. 263-271. 48. Toro, N., Datta, A., Yanofsky, M. & Nester, E. (1988) Proc. Nail. 3. Stachel, S. E., Messens, E., Van Montagu, M. & Zambryski, P. Acad. Sci. USA 85, 8558-8562. (1985) Nature (London) 318, 624-629. 49. De Vos, G. & Zambryski, P. (1989) Mol. Plant-Microbe Interact. 2, 4. Peralta, E. G. & Ream, L. W. (1985) Proc. Nail. Acad. Sci. USA 43-52. 82, 5112-5116. 50. Stachel, S. E. & Zambryski, P. (1986) Cell 46, 325-333. 5. Shaw, C. H., Watson, M. & Carter, G. (1984) Nucleic Acids Res. 51. Herrera-Estrelia, A., Van Montagu, M. & Wang, K. (1990) Proc. 12, 6031-6041. Natl. Acad. Sci. USA 87, 9534-9537. 6. Wang, K., Herrera-Estrella, L., Van Montagu, M. & Zambryski, P. 52. Citovsky, V., Zupan, J., Warnick, D. & Zambryski, P. (1992) (1984) Cell 38, 455-462. Science 256, 1802-1805. Downloaded by guest on October 1, 2021