The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.

No abstract

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.

The virA gene of Agrobacterium tumefaciens encodes an inner membrane that mediates the transcriptional activation of virulence genes in response to plant signal molecules. We report here a functional analysis of the N‐terminal, C‐terminal and periplasmic domains of VirA in transmembrane signalling. First, we show that VirA has a transmembrane topology by analysis of the alkaline phosphatase activities, determined by several virA‐phoA gene fusions. Second, we report here the construction of several virA‐tar chimeric genes, in which the 3′‐coding region of virA is conserved to study transmembrane signalling, as well as the construction of a set of virA deletion mutations. Results of analyses of vir induction behaviour and tumour inducing abilities of agrobacteria carrying these mutant genes do not support existing models for the chemoreceptor function of the VirA periplasmic domain. We demonstrate that the periplasmic domain of VirA can be either replaced by a corresponding region of the E.coli chemosensory protein Tar or even totally deleted from VirA without a loss of function. Here, we present a model of VirA which involves a receptor function for the second membrane‐spanning domain and an intracellular signalling function for the cytoplasmic domain of VirA. In addition, we show that VirA plays a role in determining the sensitivity for pH and temperature in acetosyringone‐mediated vir induction, and we propose a role for the VirA periplasmic domain in detection of the external pH conditions.

The VirA protein of Agrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediated vir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.