Activities of virE1 and the VirE1 Secretion Chaperone in Export of the Multifunctional VirE2 Effector via an Agrobacterium Type IV Secretion Pathway

Abstract

Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance. Deletion of virE1 did not affect transcription but decreased translation of virE2, as shown by analysis of lacZ transcriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in cis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans with virE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant. Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficient virE2 translation in the context of expression from the native P virE promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.Agrobacterium tumefaciens transfers at least three macromolecular substrates, oncogenic T-DNA, VirE2 single-stranded DNA-binding protein (SSB), and VirF protein, to plant cells during the course of infection (8). Substrate transfer is mediated by a type IV secretion system assembled from the products of the ϳ9.5-kbp virB operon and the virD4 gene (29). This transfer system is a bona fide conjugation apparatus, as suggested by its ancestral relatedness to transfer systems (Tra) of several broad-host-range plasmids and as demonstrated by its ability to transfer the mobilizable IncQ plasmid RSF1010 to bacterial and plant recipient cells. In recent years, other type IV systems have been shown to contribute to the virulence of several mammalian pathogens. The pathogens utilizing type IV systems during infection include Helicobacter pylori (12), Bordetella pertussis (3), Brucella spp. (31, 42), Bartonella henselae (35,40), and Legionella pneumophila (47). A type IV system of H. pylori exports CagA to the cytosol of mamm...

Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1

“…1A; Deng et al, 1999). In fact, we were unable to obtain A. tumefaciens strains carrying plasmids expressing Cre::VirE2 in the absence of virE1 in cis from the virE or virF promoter (data not shown), probably due to the role that virE1 plays to stabilize Cre::VirE2 as it does VirE2 (Zhao et al, 2001). Therefore, we were unable to assay Cre::VirE2 protein transport in the absence of VirE1.…”

“…Besides, the gene encoding the chaperone is located in the same operon as the gene encoding its cognate substrate. VirE2 is unstable in the absence of VirE1 (Deng et al, 1999;Zhao et al, 2001; this study). VirE1 has been implicated in posttranslational stabilization of VirE2 and maintaining VirE2 in a translocation competent state, by preventing premature interaction of VirE2 with T-DNA in the bacterial cell, membrane insertion, and/or VirE2/VirE2 interaction (Deng et al, 1999;Dumas et al, 2001;Zhao et al, 2001).…”

“…Only one of these transported substrates, VirE2, is accompanied by a chaperone molecule called VirE1 (Deng et al, 1999;Sundberg and Ream, 1999;Zhao et al, 2001). VirE1 has the structural characteristics of a molecular chaperone (Wattiau et al, 1996), being a small, acidic protein with a dimerization domain.…”

“…It has been shown that VirE1 plays a prominent role in the export process of VirE2 by preventing aggregation, enhancing stability, and therefore likely maintaining VirE2 in a (unfolded) transport competent state (Deng et al, 1999;Sundberg and Ream, 1999;Zhao et al, 2001). Dumas et al (2001) suggested that VirE2 might form a transmembrane channel in the plant cytoplasmic membrane to facilitate transport of ssDNA.…”

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners

“…In Agrobacterium, VirE2 is coexpressed with its specific chaperone VirE1 (21)(22)(23). VirE1 is also essential for virulence, although it is not thought to be secreted to the plant.…”