The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another - PubMed (original) (raw)

The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another

A Das et al. J Bacteriol. 2000 Feb.

Abstract

The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.

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Figures

FIG. 1

FIG. 1

Interactions of VirB7, VirB8, VirB9, and VirB10. Interaction of VirB proteins was studied by the yeast two-hybrid assay (16). Yeast strains harboring the appropriate plasmids were streaked on solid medium containing X-Gal. A positive interaction is manifested by the blue colony color phenotype. The interactions of VirB7 (B7), VirB8 (B8), VirB9 (B9), and VirB10 (B10) with one another were investigated. One interacting protein is indicated on the left column and the partner proteins are indicated under the colony. For control (C) experiments, the activator vector was introduced into a strain containing the LexA-VirB8, -VirB9, or -VirB10 fusion.

FIG. 2

FIG. 2

In vitro analysis of the interactions of VirB10. Interaction of VirB10 with VirB8, VirB9, and VirB10 was monitored by the GST-pulldown assay (2). Purified histidine-tagged VirB10 was incubated with GST or a GST fusion protein immobilized on glutathione-Sepharose. Binding of His-VirB10 was determined by analysis of bound proteins by SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis using anti-VirB10 antibodies. Lanes 1 to 4, protein bound to immobilized GST, GST-VirB10, GST-VirB8, and GST-VirB9, respectively; lanes 5 to 8, the corresponding unbound proteins; lane 9, immobilized GST-VirB10 incubated with buffer only. The higher-molecular-weight band in lane 2 is the GST-VirB10 fusion protein. The His-VirB10 protein is indicated by the arrowhead.

FIG. 3

FIG. 3

Delineation of interaction domains of VirB8, VirB9, and VirB10. Interactions of VirB8 (B), VirB9 (C), VirB10 (D), and their derivatives with VirB8, VirB9, and VirB10 were investigated by the two-hybrid assay. (A) A linear representation of the VirB proteins and their derivatives used for fusion construction. Fusions containing the periplasmic domain (F), an N-terminal fragment (N), a central fragment (M), and a C-terminal fragment (C) were tested for interaction with the three proteins. The solid box indicates a hydrophobic region.

FIG. 4

FIG. 4

Phenotype of a virB8 mutant. The effect of alteration of VirB8 serine 87 to leucine on DNA transfer was monitored by complementation assays (A). Agrobacterium sp. strain A348ΔB8 (ΔB8) harboring plasmid pAD1433 containing wild-type (wt) virB8 or its derivative pAD1433S87L containing virB8S87L (S87L) was used to infect Kalanchöe leaves. Tumor formation was monitored 3 weeks after infection. A348, Agrobacterium sp. strain A348 that harbors the octopine Ti-plasmid pTiA6. (B) Interaction of VirB8S87L with VirB8, VirB9, and VirB10 was studied by the yeast two-hybrid assays. A blue colony color phenotype indicates a positive interaction. Row 1, VirB8; row 2, VirB8S87L.

FIG. 5

FIG. 5

A model for the T-DNA transport pore. Individual VirB proteins are identified by numbers. The T pilus composed of VirB2 can form a channel through the outer membrane.

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References

    1. Anderson L B, Hertzel A V, Das A. Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci USA. 1996;93:8889–8894. - PMC - PubMed
    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: Greene and Wiley-Interscience; 1993.
    1. Baron C, Thorstenson Y R, Zambryski P C. The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol. 1997;179:1211–1218. - PMC - PubMed
    1. Beaupre C E, Bohne J, Dale E M, Binns A N. Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. J Bacteriol. 1997;179:78–89. - PMC - PubMed
    1. Beijersbergen A, Dulk-Ras A D, Schilperoort R A, Hooykaas P J. Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science. 1992;256:1324–1327. - PubMed

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