Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens

Abstract

Tumours are induced by Agrobacterium tumefaciens on a variety of plants. The virulence determinants of A. tumefaciens reside on a large tumour-inducing (Ti) plasmid. This plasmid carries two regions essential for tumour induction, namely the T region and the Vir region. During infection the T region is transferred to the plant cell, where it becomes stably integrated in one of the host chromosomes as T-DNA. Expression of T-DNA leads to the production of the plant hormones auxin and cytokinin, as well as to the synthesis of specific amino-acid derivatives termed opines. Agrobacterium strains are classified according to the types of opines produced by the tumours they induce. The Vir region contains genes that are expressed in the bacterium and are required for T-DNA transfer to plant cells, and several other genes that affect the efficiency of transfer and the host range. Vir regions from different Ti plasmids may vary slightly in the genes they contain: for instance, the virF gene, which is present in the Vir-region of octopine Ti plasmids, is absent from nopaline Ti plasmids. Mutation of the virF gene leads to a weakened virulence of octopine strains on tomato and Nicotiana glauca (shrub tobacco). Nopaline strains are strongly attenuated in N. glauca compared with octopine strains because of the absence of the virF virulence gene from the Ti plasmid in nopaline strains. The virF gene product may be transferred to and be active in plant cells. Here we isolate transgenic N. glauca plants in which the virF coding sequence is expressed using the cauliflower mosaic virus 35S promoter. The presence of the VirF protein converts the non-host N. glauca into a host for tumour formation by A. tumefaciens nopaline strains and octopine virF mutants. Our results indicate that certain virulence gene products such as the VirF protein may be transferred to plant cells during tumour induction, where they function as mediators of T-DNA transfer.

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

“…Cascales and Christie (14) recently demonstrated, in an elegant study using a T-DNA immunoprecipitation assay, that the coupling protein VirD4 is the first component of the T4SS to interact with the T-DNA͞VirD2 transfer intermediate. The VirE2 protein interacts at the cell poles of the bacterium with VirD4 (20), strongly supporting the model that VirD4 is the cytoplasmic component of the T4SS that sorts not only nucleoprotein complexes but also the effector proteins for translocation and that T4SS are actually committed protein translocation systems (21,22). Translocation of the effector proteins VirF, VirE2, and VirE3 is mediated by a C-terminal transport signal (13,20,23,24).…”

“…According to a current model, T4SSs are dedicated protein translocation machines that have evolved to transfer also nucleoprotein complexes (4,21). The relaxase protein that is covalently bound to the 5Ј end of the ssDNA molecule is thought to possess the T4SS transport signal and thus act as a pilot for the complex.…”

Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins

“…This list includes DNA apparently of any length as long as it contains a recognition site for processing by the VirD2 endonuclease (34), the non-self-transmissible IncQ plasmid RSF1010, most likely also as a single-stranded DNA-protein particle (4, 9, 52), the VirE2 single-stranded DNA-binding protein (7,14,35), and VirF, which functions as an important virulence factor for infection of certain plant species (40). Second, recent studies have shown that the T-complex transporter delivers DNA to many dicotyledonous and monocotyledonous plant species as well as to agrobacteria (3,25) and yeast (10,11,36) recipient cells.…”

The Role of the Ubiquitin-Proteasome System in Agrobacterium tumefaciens-Mediated Genetic Transformation of Plants

“…The host factor VBF may be the key to understanding why A. tumefaciens strains lacking the virF gene can still induce tumors on some plant species (Hooykaas et al, 1984;Melchers et al, 1990;Jarchow et al, 1991;Regensburg-Tuïnk and Hooykaas, 1993). In such plants, A. tumefaciens may exploit a host VBF-like protein during infection as an alternative to VirF.…”

“…As a subunit of SCF, VirF targets at least VIP1 and its associated VirE2 for proteasomal degradation . This VirF function appears important for the infection process, as mutations in the virF gene substantially reduce infection efficiency in many, but not all, hosts (Melchers et al, 1990;Regensburg-Tuïnk and Hooykaas, 1993;Schrammeijer et al, 2001). …”