Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3.
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.
Schrammeijer
Barbara
B
Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
den Dulk-Ras
Amke
A
Vergunst
Annette C
AC
Jurado Jácome
Esmeralda
E
Hooykaas
Paul J J
PJ
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019426:Integrases / Q000235:genetics; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics; D037521:Virulence Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007473:Ion Channels; C089368:OSA protein, Shigella flexneri; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D037521:Virulence Factors; C051583:virE2 protein, Agrobacterium; C045073:Cre recombinase; D019426:Integrases