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.

M24 24 24 24 24 246 6 6 6 6A348 virD2 C1

virC1

D2

ChvE

B9

C26 24

virC2C1C2

246

C1KQ,C2

246 246C2

6 C1

54

36

54

36

*

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: 1170-1173

StrainsT-DNA transfer to:

D4 B11 B6 B8 B2 B9

+ + + ++ +A348

virD2

virC1

virC2

- - - - --+ + + ++ +

+ + + ++ +

36

WT virB virD2

M

A

C1

D1

D2

D4

21

54

C2FL30

54

C1,C2FL

C1,C2FL

C1,C2FL

KQ,C2FL

36

WT -Ti + D4

M

C1

C2FL30

C1 KQ

D454

C1,C2FL

C1,C2FL

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,C2FL), A348(pKAB192)

producing both VirC1 and FLAG-VirC2 (C2FL) from an IncP replicon; virB

operon mutant, PC1000; virB(C1,C2FL), PC1000(pKAB192) producing VirC1

and C2FL from an IncP replicon; virD2, Mx311 which is polar on virD3,virD4 and

virD5 expression; virD2(C1,C2FL), Mx311(pKAB192) producing VirC1 and C2FL.

Panel B: A348(C1,C2FL), A348(pKAB192) producing both VirC1 and C2FL 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,C2FL (VirC1

and VirC2FL from pKAB192), KQ,C2FL (VirC1K15Q and VirC2FL 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: 1699-1713

DICFluor Fluor

VirC2FL

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 16-18 h and analyzed by IFM. VirC1, VirC1K15Q, VirC1K15E were

detected with Alexa fluorR 488 goat-anti-rabbit IgG as the secondary antibody (green, left panels);

VirC2FL was detected with Rhodamine RedTM-X goat anti-mouse IgG (red, right panels) as the

secondary antibody. (DIC) Nomarski microscopy; Fluor, fluorescence microscopy.

VirC1KE

VirC1KQ

VirC1

VirC2FL

virC1 (C1KQ-GFP)

A348 (C1-GFP)

A348(GFP)

virC1 (C1-GFP)

DICFluorA348

(C2-GFP)

virC1(GFP)

virC1 (C2-GFP)

Fluor DIC

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 C1 C2KQ

A348 virC1 virC2

+ D2FL

D2FL54

Fig. S6. Immunodetection of FLAG-tagged VirD2 in A348 and virC mutant strains 18 h

after acetosyringone induction. Strains: A348, WT strain; A348(D2FL), A348(pKA196)

producing VirD2FL; A348(D2FL,C1,C2), A348(pKA196, pKAB188) producing VirD2FL,

VirC1 and VirC2; virC1(D2FL), Mx365(pKA196) producing VirD2FL; virC1(D2FL,C1),

Mx365(pKA196, pKAB187) producing VirD2FL and VirC1; virC1(D2FL,C1KQ),

Mx365(pKA196, pKAB189) producing VirD2FL and VirC1K15Q; virC1(D2FL,C2),

Mx365(pKA196, pKA114) producing VirD2FL and VirC2; virC2(D2FL), Mx364(pKA196)

producing VirD2FL. M, Molecular mass markers, with sizes in kilodaltons listed at the left.

M

1

3

4

7

8

11

12

2

5

6

9

10

V

I

L

I

I

L

A

S

K

S

K

E VirC1A6

VISKLISKILEA

1

3

4

7

8

11

12

2

5

6

9

10

T

I

L

V

A

L

G

S

K

S

E

E VirC1C58

TISKLVSEALEG

1

3

4

7

8

2

5

6

9

10

K

G

K

R

G

GF

L

L

F

MinDE.coli

KGFLKRLFGG

3

4

7

8

2

5

6

9

10

R

I

I

I

S

KE

Y

G

A

SojE.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: 345-355

Cell Pole

B8

B6B4/B11

B2/B5

D4

B1

0

B

7/B

9

B1

0

B

7/B

9

D2

C1

3’

D1D2

C2

P P

C1

LB

RB

D2

C1

D2

C1

D2C1

D2

C1

D1

D1

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.