Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3

Abstract

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.

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium

“…In this respect, the A. tumefaciens VirB͞D4 T4SS is an appealing focus for study because it transfers both DNA molecules into host cells, and, independent of these DNA molecules, the effector proteins VirE2, VirE3, and VirF (13,26).…”

“…A. tumefaciens strain LBA1100 (25), containing octopine type pTiB6 with a complete virulence (vir) region but lacking T-DNA and transfer functions, was used for most transport experiments with A. thaliana root explants. Selection conditions of the strains and methods for introduction of plasmids are described in detail elsewhere (26). In some experiments, LBA2587, a derivative of LBA1100 with a precise deletion of virD4 (27), was used.…”

“…To create site-directed mutants of Cre::virF⌬42N (pSDM3155) (13,26), we used the protocol described by Sawano and Miyawaki (28). The Cre::VirF⌬42N fusion protein was chosen for mutagenesis studies because it was the most efficiently transferred Cre::Vir protein analyzed (13).…”

“…Further details of the cloning procedures, creation of targeted Cre fusions, sequences of oligonucleotides, plasmid, and DNA sources can be found in Supporting Materials and Methods and Tables 2 and 3 which are published as supporting information on the PNAS web site. DNA sequences, encoding the C-terminal 19 aa of VirF, 10 aa of VirF, 50 aa of VirD1, VirD2, VirD3, VirD5, Ysa, Atu6154, and MobA, as well as full-length VirD2, were translationally fused to cre in plasmid pSDM3197 (26). All cre control and cre-vir genes used in this study are expressed from the A. tumefaciens virF promoter sequence, and the chimeric proteins contain an N-terminally located simian virus 40 nuclear localization signal sequence to ensure nuclear targeting after Vir-mediated translocation into host cells.…”

The versatile bacterial type IV secretion systems

“…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] .…”