Fig. S1. Accumulation of Vir proteins in A348 and virC mutant strains induced with acetosyringone (AS) for vir gene expression. Strains: A348, wild-type strain; virD2, Mx311; virC1, Mx365 (polar on downstream virC2); virC1(C1), Mx365(pKAB187) producing VirC1; virC1(C2), Mx365(pKA114) producing VirC2; virC1(C1,C2), Mx365(pKAB188) producing VirC1 and VirC2; virC1(C1KQ,C2), Mx365(pKAB190) producing VirC1K15Q and VirC2; virC2, Mx364; virC2(C2), Mx364(pKA114) producing VirC2. Cells were induced for 6 or 24 h, or incubated for 24 h in the absence of AS (*, lane 2), and total cellular proteins were loaded on SDS-polyacrylamide gels on a per cell equivalent basis. Antibodies to VirC1 (C1), VirD2 (D2), VirB9 (B9), and the constitutively synthesized, chromosomally-encoded ChvE protein, were used to detect the respective proteins by immunostaining. M, Molecular mass markers, with corresponding sizes in kilodaltons at the left. M A348virD2C1 virC1 D2 ChvE B9 C2 624 virC2 C1C2 246 C1KQ, C C2 6 C *

Fig. S2. Detection of T-strand interactions with T4S channel subunits, as monitored with the TrIP assay (Cascales and Christie, 2004). Antibodies to VirD4, VirB11, VirB6, VirB8, VirB2, and VirB9 co-precipitated the respective proteins and the T-strand transfer intermediate from extracts of WT strain A348, but not the virD2 relaxase mutant, which is defective in T-DNA processing. The antibodies also precipitated T-strand from virC mutants lacking one or both of the virC genes (virC1, Mx365 which is polar on virC2 expression; virC2, Mx364) even though these mutants generate low levels of the transfer intermediate. (+), T-DNA amplification product detected by agarose gel electrophoresis; (-), no detectable amplification product. The results indicate that VirC proteins are not required for translocation of the T-DNA transfer intermediate through the VirB/D4 T4S channel. Reference: Cascales E, Christie PJ (2004) Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304: Strains T-DNA transfer to: D4B11B6B8B2B A348 virD2 virC1 virC

36 WT  virB virD2 M A C1 D1 D2 D C2 FL C1, C2 FL C1, C2 FL C1, C2 FL KQ, C2 FL 36 WT-Ti + D4 M C1 C2 FL 30 C1KQ D4 54 C1, C2 FL C1, C2 FL B Fig. S3. Co-immunoprecipitation of Vir proteins with VirC1. Immunoprecipitates recovered with anti-VirC1 antibodies were assayed for the presence of the Vir proteins listed at the right. Strains: Panel A: A348(C1,C2 FL ), A348(pKAB192) producing both VirC1 and FLAG-VirC2 (C2 FL ) from an IncP replicon;  virB operon mutant, PC1000;  virB(C1,C2 FL ), PC1000(pKAB192) producing VirC1 and C2 FL from an IncP replicon; virD2, Mx311 which is polar on virD3,virD4 and virD5 expression; virD2(C1,C2 FL ), Mx311(pKAB192) producing VirC1 and C2 FL. Panel B: A348(C1,C2 FL ), A348(pKAB192) producing both VirC1 and C2 FL from an IncP replicon; -Ti + D4, strain KA2002 (a derivative of strain A136 which lacks the Ti plasmid and has virA and virG introduced into the chromosome) plus pKA21 producing VirD4 (Atmakuri et al., 2003); KA2002(pKA21) was engineered to produce VirC1 and VirC2 variants by transformation with the following plasmids: C1 (VirC1 from pKAB187); KQ (VirC1K15Q from pKAB5189); C1,C2 FL (VirC1 and VirC2 FL from pKAB192), KQ,C2 FL (VirC1K15Q and VirC2 FL from pKAB193). M, Molecular mass markers with sizes in kilodaltons listed at the left. The results in panel A indicate that VirC1 interacts directly or indirectly with VirC2, VirD1, VirD2, and VirD4. Furthermore, a presumptive complex of VirC1, VirC2, and VirD1 forms independently of VirD2 relaxase and, therefore, T-strand processing. The results in panel B indicate that VirC1 forms complex(es) with VirC2 and VirD4 independently of other Ti-encoded proteins. Reference: Atmakuri K, Ding Z, Christie PJ (2003) VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol Microbiol 49:

DICFluor VirC2 FL Fig. S4. Localization of VirC1 and VirC2 at the same cell pole as monitored by immunofluorescence microscopy (IFM). Strains: Top panels - Mx365(pKAB192) producing VirC1 and FLAG-VirC2; Middle panels - Mx365(pKAB193) producing VirC1K15Q and FLAG- VirC2; Bottom panels - Mx365(pKAB220) producing VirC1K15E. Cells were induced with acetosyringone for h and analyzed by IFM. VirC1, VirC1K15Q, VirC1K15E were detected with Alexa fluor R 488 goat-anti-rabbit IgG as the secondary antibody (green, left panels); VirC2 FL was detected with Rhodamine Red TM -X goat anti-mouse IgG (red, right panels) as the secondary antibody. (DIC) Nomarski microscopy; Fluor, fluorescence microscopy. VirC1KE VirC1KQ VirC1 VirC2 FL

virC1 (C1KQ-GFP) A348 (C1-GFP) A348(GFP) virC1 (C1-GFP) DICFluor A348 (C2-GFP) virC1(GFP) virC1 (C2-GFP) FluorDIC Fig. S5. Polar localization of VirC1 and VirC2 proteins fused to the green fluorescent protein (GFP). A348 and virC1 mutant (Mx365) cells producing VirC1- GFP or VirC1KQ-GFP (left panels) or VirC2-GFP (right panels). Fusion proteins were produced from the following plasmids: VirC1-GFP (pKAB58); VirC1K15Q- GFP (pKAB110); VirC2-GFP (pKA115); GFP control (pZDB69). Cells were photographed 3 h after acetosyringone induction; Fluor (fluorescence microscopy), DIC (Nomarski microscopy). About 1000 cells of each strain were examined and nearly all producing the VirC-GFP fusion proteins (99%) displayed unipolar foci; by contrast, all cells of the control strain A348(GFP) producing GFP exhibited uniform fluorescence.

C1, C2 C1C2KQ A348 virC1virC2 + D2 FL D2 FL 54 Fig. S6. Immunodetection of FLAG-tagged VirD2 in A348 and virC mutant strains 18 h after acetosyringone induction. Strains: A348, WT strain; A348(D2 FL ), A348(pKA196) producing VirD2 FL ; A348(D2 FL,C1,C2), A348(pKA196, pKAB188) producing VirD2 FL, VirC1 and VirC2; virC1(D2 FL ), Mx365(pKA196) producing VirD2 FL ; virC1(D2 FL,C1), Mx365(pKA196, pKAB187) producing VirD2 FL and VirC1; virC1(D2 FL,C1KQ), Mx365(pKA196, pKAB189) producing VirD2 FL and VirC1K15Q; virC1(D2 FL,C2), Mx365(pKA196, pKA114) producing VirD2 FL and VirC2; virC2(D2 FL ), Mx364(pKA196) producing VirD2 FL. M, Molecular mass markers, with sizes in kilodaltons listed at the left. M

V I L I I L A S K S K E VirC1 A6 VISKLISKILEA T I L V A L G S K S E E VirC1 C58 TISKLVSEALEG K G K R G G F L L F MinD E.coli KGFLKRLFGG R I I I S K E Y G A Soj E.coli RIEYIRGASK 1 Fig.S7. Helical wheel diagrams of the C-terminal 12 aminoacids (aa) (shown below helical wheel diagrams) of Agrobacterium tumefaciens VirC1 from the pTiA6 and pTiC58 plasmids, and 10 aa of Escherichia coli Soj and MinD (Hu and Lutkenhaus, 2003) ATPases. Red circles indicate hydrophobic residues; blue indicate hydrophilic residues. Circle diameters, with corresponding aa’s indicated, gradually decrease in size as the helix is read from top to bottom. Helices were developed with the HelixWheel determination tool (Marcel Turcotte, University of Ottowa (ExPASy Proteomics tools)). VirC1 and MinD proteins, but not Soj, show potential C-terminal amphipathic helices. Reference: Hu Z, Lutkenhaus J (2003) A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum. Mol Microbiol 47:

Cell Pole B8 B6 B4/B11 B2/B5 D4 B10 B7/B9 D2 C1 3’ D1 D2 C2 P P C1 LB RB D2 C1 D2 C1 D2 C1 D2 C1 D1 Fig. S8. Proposed model for generation of T-DNA transfer intermediate and its recruitment to the polar VirB/D4 type IV secretion (T4S) machine. The A. tumefaciens VirB/D4 T4S apparatus is shown as a transenvelope organelle that mediates the passage of the ssDNA transfer intermediate. The relaxosome complex consisting of VirC1, VirC2, VirD1, and VirD2 relaxase binds to the polar-localized Ti-plasmid at the Right Border (RB) and flanking overdrive sequence (not shown), generating a ssDNA transfer intermediate. The processed T-strand covalently bound at its 5’ end with VirD2 relaxase, in concert with VirC1 accumulates in the cytoplasm and at the cytoplasmic membrane. VirC1 recruits the transfer intermediated to the VirD4 substrate receptor by a mechanism dependent on NTP energy for subsequent translocation through the secretion apparatus. Membrane-bound “P” is a factor(s) that mediates polar accumulation of VirC1 and VirC2 proteins.