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.