Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous plant species when the bacteria infect wounded tissue (18). The tumor-inducing (Ti) plasmid carries genes essential for tumorigenesis (63,72). The transferred DNA (T-DNA) portion of the Ti plasmid enters plant cells and integrates into nuclear DNA (8,9,74), in which expression of certain T-DNA genes leads to tumorous growth (2,5,32,46,59). Virulence (vir) genes necessary for T-DNA transmission (transfer and integration) lie elsewhere on the Ti plasmid (24, 55), and wounded plants release phenolic compounds that induce vir expression (54).Border sequences define the T-DNA ends (42,43,48,65). T-DNA transfer begins when the VirD2 endonuclease nicks the right-hand border sequence (66, 78) and attaches to the 5Ј end of the nicked DNA strand (22,27,30,67,79). Strand displacement continues leftward (to the nicked left-hand border sequence), generating linear VirD2-bound T strands (3,33,56), which the bacteria appear to export into plant cells (58, 80). VirE2 single-stranded DNA-binding (SSB) protein (11-13, 16, 26, 47) may also accompany T strands into plant cells. Both VirE2 and VirD2 contain plant nuclear localization signals and probably play important roles inside infected plant cells (14,28,31,51,62).Export of DNA and proteins from A. tumefaciens into plant cells depends on membrane-associated proteins encoded by the virB operon (10,23,34,49,50,60,61,(68)(69)(70)(71)) (for reviews, see references 29 and 81) and the virD4 gene (33, 38). The VirB proteins are similar to pertussis toxin liberation (Ptl) proteins of Bordetella pertussis that mediate export of pertussis toxin (15,73) and to proteins that facilitate conjugal transfer of IncP␣ plasmid RP4 (Trb proteins) (35) and IncN plasmid pKM101 (Tra proteins) (44). VirD4 protein has similarity to TraG, another protein required for conjugal transfer of RP4 (35). Thus, Agrobacterium proteins essential for tumorigenesis appear to facilitate export of both proteins and DNA into plant cells by using pathways that operate in other bacteria.VirE2 plays an important role inside plant cells but not inside Agrobacterium cells. T strands (56), which are widely accepted as intermediates of T-DNA transfer (45,75,81), accumulate to wild-type levels in virE2 mutants (57, 64), showing that VirE2 does not stabilize T strands inside bacterial cells. In addition, virE2 mutants can transfer T strands into plant cells (80), albeit with unknown efficiency, proving that VirE2 is not essential for export of T strands. Transgenic tobacco plants that produce VirE2 protein are susceptible to transformation by a virE mutant (14), indicating that VirE2 is necessary inside plant cells.