Mutagenesis of the Agrobacterium VirE2 single-stranded DNA-binding protein identifies regions required for self-association and interaction with VirE1 and a permissive site for hybrid protein construction

Abstract

The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation. By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.

FIG. 1

VirE2 self-association and interaction with VirE1 as determined with the yeast two-hybrid assay (2, 4). At least 75 yeast colonies per quadrant were replica plated onto nitrocellulose and assayed for β-galactosidase activity. Strains carrying pXZbE2 (BD::E2) and pXZaE1 (AD::E1) or pXZaE2 (AD::E2) displayed high levels of β-galactosidase activity.

FIG. 2

VirE2 self-association in A. tumefaciens as determined by immunoprecipitation analysis. Immunoprecipitates were electrophoresed through SDS–12.5% (lanes 1 to 7) or SDS–10% (lanes 8 to 11) polyacrylamide gels, and blots were developed with anti-VirE2 (top panel) or anti-GFP (bottom panel) antiserum as previously described (25). Material was precipitated from extracts of wild-type A348 (lane 1) and A348(pCSKB100) (lane 2) with anti-VirE2 antiserum. Material was precipitated from extracts of A348(pCSKB100) (lane 3), A348(pXZB43) (lane 4), LBA4404(pXZB43) (lane 5), KE1(pXZB43) (lane 6), and KE1(pXZB426) (lane 7) with anti-GFP antiserum. Material was precipitated from extracts of wild-type A348 (lane 8), A348(pXZB761) (lane 9), KE1(pXZB761) (lane 10), and KE1(pXZ427) (lane 11) with anti-i31 peptide antiserum. The various forms of VirE2 are indicated at the right. Some proteolyis of VirE2::GFP is evident. The heavy staining band at ∼45 kDa is due to immunoreactivity of immunoglobulin G (IgG) heavy chain present in the immunoprecipitates. M, molecular mass markers from Bio-Rad (top panel) or Gibco-BRL (bottom panel), with sizes (in kilodaltons) indicated at left.

FIG. 3

VirE2 interaction with a functional VirE1::GFP hybrid protein in A. tumefaciens as determined by immunoprecipitation analysis. Immunoprecipitates were electrophoresed through SDS–12.5% polyacrylamide gels, and blots were developed with anti-VirE2 antiserum and then redeveloped with anti-GFP antiserum. Material was precipitated from KE1(pXZB168) (lane 1), A348 (lane 2), A348(pXZB168) (lane 3), LBA4404(pXZB168) (lane 4), KE1(pXZB169) (lane 5), and PC1000(pXZB168) (lane 6) with anti-VirE2 antiserum. The positions of VirE2 and VirE1::GFP are indicated at the right. The heavy staining band at ∼45 kDa is due to immunoreactivity of immunoglobulin G heavy chain present in the immunoprecipitates. M, molecular mass markers from Gibco-BRL, with sizes (in kilodaltons) indicated at the left.

FIG. 4

Deletion analysis to define sequence requirements for VirE2 self-association and interaction with VirE1. Derivatives of VirE2 fused to the GAL4 binding domain in plasmid pAS are depicted schematically at the left. The structure of VirE2 (533 amino acids), with the positions of the two nuclear localization sequences (NLS1 and NLS2) and restriction sites used for subcloning, is presented at the top. Some restriction fragments were subcloned from the pXZ7XX plasmids listed that carry virE2::i31 alleles, as described in the text. Restriction sites are as follows: B, BamHI; M, MluI; Bg, BglII; Ev, EcoRV; Sc, SacI; H, HindIII; Sp, SspI; and S, SalI. At right are β-galactosidase activities of patched yeast colonies carrying the pXZbE2 plasmids listed with either pXZaE2 (AD::E2) or pXZaE1 (AD::E1). +, β-galactosidase activity evident; −, background levels of β-galactosidase activity. Activities of two representative, independently transformed colony patches are shown.

FIG. 5

i31 peptide insertion analysis to define sequence requirements for VirE2 self-association and interaction with VirE1. VirE2::i31 derivatives fused to that GAL4 binding domain in plasmid pAS2 are depicted schematically at the left. Sites of i31 insertions are denoted by small vertical lines. The structure of VirE2 (533 amino acids), with the positions of its NLSs, are shown at the top. At right are β-galactosidase activities of patched yeast colonies carrying one of the pXZbE2 plasmids listed with either pXZaE2 (AD::E2) or pXZaE1 (AD::E1). +, β-galactosidase activity evident; −, background levels of β-galactosidase activity. Activities of two representative, independently transformed colony patches are shown.

FIG. 6

Promoter and virE1 sequence requirements for synthesis of functional VirE2. The P_virB, P_lac, and P_virE promoters were used to drive expression of virE2 or virE1 and virE2, and the P_lac and P_virE promoters were used to drive expression of virE1 containing a translation stop signal at codon 6 and all of virE2. At12516 cells carrying these plasmids were assayed for VirE2 protein abundance by immunoblot analysis and for virulence by inoculation on K. daigremontiana. Samples of protein extracts normalized for total protein content on a per-cell equivalent were electrophoresed through SDS–12.5% polyacrylamide gels, and blots were developed with anti-VirE2 antiserum. The strains used were A348 (lane 1), At12516(pPCB731) (lane 2), At12516(pPCB732) (lane 3), At12516(pXZB235) (lane 4), At12516(pXZB27) (lane 5), At12516 (lane 6), A348 (lane 7), At12516(pXZB237) (lane 8), At12516(pXZB46) (lane 9), At12516(pXZB236) (lane 10), and At12516(pXZB27) (lane 11). Virulence assays of each strain show oncogenic proliferation clearly distinguishable from sites of inoculation that did not proliferate. Results of virulence assays of At12516 cells harboring these plasmids are summarized at the right.

FIG. 7

Steady-state abundance and functionality of VirE2::i31 insertion mutants in A. tumefaciens. A348 and At12516 cells carrying these plasmids were assayed for VirE2 protein abundance by immunoblot analysis. (A) Blots were prepared as described in the legend to Fig. 6 with protein samples from plasmid-carrying A348 (top panel) or At12516 (bottom panel) cells. Strains used were as follows: wild type, A348 (top panel) and At12516(pXZB27) (bottom panel); and ΔE2, At12516 (top and bottom panels); the numbers refer to the VirE2 residue immediately preceding the i31 insertion. The small arrow denotes the position of native VirE2. The large arrowhead denotes VirE2::i31 mutants, which migrated to different positions in SDS-polyacrylamide gels. (B) The functionality of the i31 insertion mutants was assessed by inoculation of strains on K. daigremontiana leaves (left leaf, A348 merodiploids coexpressing wild-type virE2 and the indicated virE2::i31 allele; right leaf, At12516 expressing the indicated virE2::i31 allele). The allele for VirE2.39::i31 restored virulence of At12516 cells to wild-type levels. The tumor at the top left was induced by inoculation with wild-type A348, and the tumor at the bottom right was induced by inoculation with At12516(pXZ27). The wound site at the top right shows the avirulence of At12516.