Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure

Abstract

Plant cell transformation by Agrobacteriwun Iwefaciens involves the transfer of a single-stranded DNAprotein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VwrB proteins were detected in the membrane fractions only. Three of these, VWrB8, VirB9, and VWrBlO, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VwrB proteins, VirBi, VWrB4, and VWrB1I, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VwrB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions. Agrobacterium tumefaciens has the unique ability to transfer DNA to plant cells. In nature, the transfer and expression of a specific fragment of DNA (T-DNA) cause a neoplastic growth on the plant host. In the laboratory, this DNA transfer phenomenon has been exploited by using disarmed vectors to introduce new genes into plants. The T-DNA and the components necessary for packaging and transfer of the single-stranded T-DNA (T-strand) (45) are encoded by the tumor-inducing (Ti) plasmid in A. tumefaciens. The virulence (vir) region contains four operons (virA, virG, virD, and virB) that encode proteins required for virulence on all hosts (44). Three additional operons (virE, virC, and virH) encode proteins that are required only on some hosts (25,44).The transfer of DNA from the bacterium to the plant cell involves at least seven recognizable steps. Of the bacterial components necessary for DNA transfer, only the first step is thought to be mediated by chromosomally encoded functions (reviewed in reference 5). The remaining steps involve proteins that are encoded by the vir region on the Ti plasmid (reviewed in reference 59). The seven steps are as follows: (i) the bacterium binds to a specific component of the plant cell surface; (ii) a bacterial membrane protein (VirA) recognizes signals released by a wounded plant cell and transduces the signal v...

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…It localizes extracellularly to the pilus (Schmidt-Eisenlohr et al, 1999a,b), but is also found in cytoplasm and in inner-membrane fractions of pili-producing bacteria (Thorstenson et al, 1993). However, since VirB5 mutants can be complemented extracellularly (Winans and Walker, 1985), the protein's functions are probably only required on the cell surface.…”

“…VirB9 of A. tumefaciens is present in the inner and the outer membranes, with the majority of the protein co-localizing with VirB8 and VirB10 at discrete sites in the outer membrane (Fernandez et al, 1996a;Kumar et al, 2000;Shirasu and Kado, 1993;Thorstenson et al, 1993). Additionally, VirB9 forms disulWde-bridged complexes with outer-membrane-associated VirB7 that have a stabilizing eVect on VirB10 (Beaupré et al, 1997).…”

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

“…Effect of virB11 merodiploids on biological activity. T-DNA transport is thought to be catalyzed by a protein complex composed of virB and virD4 gene products in the membranes of A. tumefaciens (57,65). Since VirB11 localizes to the inner membrane, it may function in the protein complex to transfer T-DNA to plant cells (12,57,64).…”

“…The two potential nucleotide-binding proteins in the virB operon, VirB4 and VirB11, are both absolutely required for virulence (7,20,51,54). Both proteins are also localized to the inner membrane of A. tumefaciens even though VirB11 does not contain an N-terminal export sequence or long hydrophobic stretches of amino acids (12,57,65). VirB4 localizes to the membrane fraction of induced A. tumefaciens even when the Ti plasmid is absent from the cell (7).…”

“…virD4 and the virB operon are not required for any of the early steps such as attachment, induction, or T-DNA processing but are absolutely essential for tumor formation (7,20,51,54,66). VirD4, VirB1, VirB2, VirB3, VirB4, VirB5, VirB8, VirB9, VirB10, and VirB11 have all been localized to the membrane fraction by immunological techniques (12,41,50,51,57,58,67). These proteins could form a pilus-like structure in the membrane that is similar to those involved in conjugation of plasmids between gram-negative bacteria (26,72).…”