Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8 - PubMed (original) (raw)

Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8

R B Kumar et al. J Bacteriol. 2001 Jun.

Abstract

The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10. A second mutant VirB8(S87L) is defective in interaction with VirB9.

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Figures

FIG. 1

FIG. 1

Phenotype of the virB8 mutants. The virB8 mutants were tested for the ability to complement a deletion in virB8. The mutants in A. tumefaciens PC1008 (ΔB8) were used to infect K. daigremontiana leaves and scored for tumor formation 3 weeks after infection. A subset of the mutants listed on Table 1 is shown. The numbers indicate the mutant number. Plasmid pAD1433 or its derivative harbors virB8 or the mutant. A348, wild-type strain; −, uninfected wound site.

FIG. 2

FIG. 2

Effect of the virB8 mutations on protein stability. The level of VirB8 and its mutants were monitored by Western blot assays using purified VirB8 antibodies (16). Lanes 1, 2, and 13, uninduced A348, induced A348, and PC1008/pAD1433, respectively; lanes 3 to 12, virB8 mutants 10, 13, 17, 19, 21, 24, 26, _virB8_S87L, _virB8_T192M, and _virB8_R107A, respectively.

FIG. 3

FIG. 3

Role of VirB8 arginine-107 in DNA transfer to plants. An arginine at position 107 of VirB8 was changed to alanine by site-specific mutagenesis (17). The mutants were tested by complementation assays. wt, wild-type virB8; R107A, _virB8_R107A.

FIG. 4

FIG. 4

Identification of amino acids responsible for the avirulent phenotype of the double mutants. Mutations that led to single-amino-acid substitutions in virB8 were introduced by site-specific mutagenesis, and the mutants were introduced into A. tumefaciens PC1008. The resultant strains were used to infect K. daigremontiana leaves. The strains used for infection were A. tumefaciens A348 (A348), PC1008 (ΔB8), and PC1008 harboring a plasmid that expresses wild-type virB8 (wt), _virB8_V52I (V52I), _virB8_S87L (S87L), _virB8_V189M (V189M), or _virB_T192M (T192M).

FIG. 5

FIG. 5

Dominant-recessive phenotype of the virB8 mutants. A plasmid expressing virB8 or its mutant was introduced into A. tumefaciens A348, and the resultant strains were used to infect K. daigremontiana leaves.

FIG. 6

FIG. 6

Interaction of the VirB8 mutants with VirB8, VirB9, and VirB10. The interaction of VirB8 (wt) and its mutant with VirB8 (B8), VirB9 (B9), and VirB10 (B10) was monitored by the two-hybrid assay in yeast as described previously (10). A blue colony color indicates a positive interaction.

FIG. 7

FIG. 7

Conservation of glycine-78 and sequences around arginine-107 in the VirB8 homologs. The amino acid sequence of a segment of A. tumefaciens VirB8 and its homologs is shown. Residues identical to the A. tumefaciens VirB8 sequence are shown as dots. Sequences that exhibited a high degree of conservation are boxed. Glycine-78, alanine-100, and arginine-107 are shown in boldface. The numbers on the left indicate the position of the first amino acid residue shown in the figure. The gaps were introduced to achieve maximum homology.

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References

    1. Albright L M, Yanofsky M F, Leroux B, Ma D Q, Nester E W. Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol. 1987;169:1046–1055. - PMC - PubMed
    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. 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. Berger B, Christie P. Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes. J Bacteriol. 1994;176:3646–3660. - PMC - PubMed

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