VirE2, a Type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens

Abstract

SummaryAgrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens -a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) -and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens . Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH 2 -terminal membranespanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB -encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.

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

“…Pili are produced both on the lateral sides and on the poles of a bacterium, although in case of the A. tumefaciens T-pili they are more abundantly found at cell poles . Likewise, the Mpf transport apparatus of A. tumefaciens (VirB/VirD4) localizes to the bacterial poles (Atmakuri et al, 2003;Judd et al, 2005;Kumar and Das, 2002). This diVerence of the A. tumefaciens VirB/VirD4 system versus bacterial conjugation systems is also reXected in the observed Fig.…”

“…This indicates that substrate recognition by the CP primarily occurs via interactions with protein components of the transfer-competent protein/DNA-intermediate. By analogy, the CP of A. tumefaciens, VirD4, interacts with the protein substrate VirE2 (Atmakuri et al, 2003), which is secreted into plant cells independent of T-DNA transfer (Vergunst et al, 2000). The interaction is mediated by the C-terminal domain of VirE2 (Atmakuri et al, 2003).…”

The versatile bacterial type IV secretion systems

“…Cytological evidence favouring this role was recently presented. In A. tumefaciens, the VirD4 CP localizes at the cell poles 37 or it can be artificially localized at the mid-cell 38,39 , so marking distinct cellular positions for monitoring the recruitment of substrates tagged with green fluorescent protein (GFP). Indeed, VirD4 was shown to recruit GFP fused to the VirE2 effector protein, and further cytological and biochemical studies established that the interaction occurs independently of any requirement for Mpf proteins, the T-strand or even VirE1, a secretion CHAPERONE for VirE2 (REF.…”

“…Indeed, VirD4 was shown to recruit GFP fused to the VirE2 effector protein, and further cytological and biochemical studies established that the interaction occurs independently of any requirement for Mpf proteins, the T-strand or even VirE1, a secretion CHAPERONE for VirE2 (REF. 39). Of further interest, VirD4 was shown to interact with the carboxy-terminal region of VirE2, adding to evidence that SECRETION SIGNALS are localized at the carboxyl termini of VirE2 and other protein substrates of the VirB/D4 T4S system [39][40][41][42] .…”

“…39). Of further interest, VirD4 was shown to interact with the carboxy-terminal region of VirE2, adding to evidence that SECRETION SIGNALS are localized at the carboxyl termini of VirE2 and other protein substrates of the VirB/D4 T4S system [39][40][41][42] .…”