title: Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.
abstract: Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.
authors.affiliation: Institute of Biology, Leiden University, Sylviusweg 72, Leiden, 2333 BE, The Netherlands.
authors.forename: Philippe A
authors.initials: PA
authors.lastname: Sakalis
authors.forename: G Paul H
authors.initials: GP
authors.lastname: van Heusden
authors.forename: Paul J J
authors.initials: PJ
authors.lastname: Hooykaas
mesh_terms: D016960:Agrobacterium tumefaciens / Q000502:physiology / Q000648:ultrastructure*; D017360:Arabidopsis / Q000382:microbiology* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D058947:Bacterial Secretion Systems / Q000502:physiology*; D003199:Computer Systems; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007473:Ion Channels / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D008870:Microtubules / Q000382:microbiology / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011523:Protoplasts; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure; D014026:Nicotiana / Q000382:microbiology* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007473:Ion Channels; D008164:Luminescent Proteins; D010446:Peptide Fragments; C051583:virE2 protein, Agrobacterium; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: Agrobacterium tumefaciens; BiFC; Saccharomyces cerevisiae; VirE2.; protein translocation; split GFP; type 4 secretion system