Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1.
Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.
Vergunst
Annette C
AC
Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. vergunst@rulbim.leidenuniv.nl
van Lier
Miranda C M
MC
den Dulk-Ras
Amke
A
Hooykaas
Paul J J
PJ
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D019426:Integrases / Q000235:genetics; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010941:Plant Tumors / Q000235:genetics / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport / Q000235:genetics; D017384:Sequence Deletion; D014170:Transformation, Genetic / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007473:Ion Channels; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D014764:Viral Proteins; D037521:Virulence Factors; C051582:virE1 protein, Agrobacterium tumefaciens; C051583:virE2 protein, Agrobacterium; C045073:Cre recombinase; D019426:Integrases