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.

3The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).Most of the genes required for tumorigenesis are found on large extrachromosomal elements called Ti plasmids. Indeed, transfer of Ti plasmids into certain nonpathogenic bacteria converts them into tumorigenic pathogens (43). Ti plasmids are generally referred to by the types of opines whose catabolism they direct (see below). However, this nomenclature is becoming less satisfactory as we discover that all known Ti plasmids direct the catabolism of more than one opine and that opine catabolic genes are found in a variety of combinations in different plasmids. The Ti plasmids pTiA6NC, pTi15955, pTiAch5, pTiR10, and pTiB6S3, which are widely considered to be functionally identical, are generally referred to as octopine-type Ti plasmids (or, less frequently, octopine, mannityl opine-type Ti plasmids). The DNA sequencing of these plasmids was initiated almost 20 years ago (21) and was recently completed in our three laboratories. The resulting 194,140-nucleotide sequence is a composite assembly of sequences from all of the plasmids listed above. The close similarity of these plasmids is exemplified by the sequence of a 42-kb segment of the vir regions of pTiA6NC and pTi15955. These sequences differ at only one base, and this polymorphism is silent at the amino acid level. We have no evidence for polymorphisms elsewhere except for a large deletion that is unique to pTiA6NC (Fig. 1). The restriction map deduced from this sequence agrees almost perfectly with the published restriction map of pTiAch5 (25). All known and suspected genes are depicted in Fig. 1, and their demonstrated or putative functions are described in Table 1. The DNA sequence of this Ti plasmid provides a useful framework to review the roles of this plasmid in the biology of plant infection and colonization.This Ti plasmid contains 155 open readin...

No abstract

Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in AGROBACTERIUM: Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF.

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.