Single-Stranded DNA Binding Protein Encoded by the virE Locus of Agrobacterium tumefaciens

155

150

Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified. We characterized an Arabidopsis mutant generated by T-DNA insertional mutagenesis, rat5, that is resistant to Agrobacterium root transformation. rat5 contains two copies of T-DNA integrated as a tandem direct repeat into the 3 untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence. Transient and stable ␤-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and rat5 Arabidopsis plants indicated that the rat5 mutant is deficient in T-DNA integration. We complemented the rat5 mutation by expressing the RAT5 histone H2A gene in the mutant plant. Overexpression of RAT5 in wild-type plants increased Agrobacterium transformation efficiency. Furthermore, transient expression of a RAT5 gene from the incoming T-DNA was sufficient to complement the rat5 mutant and to increase the transformation efficiency of wild-type Arabidopsis plants.T-DNA transformation ͉ haplo-insufficiency

Section: T-dna Transformation ͉ Haplo-insufficiencymentioning

confidence: 99%

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

Mysore

Nam

Gelvin

2000

155

150

“…VirE2 is a nonsequence specific single-stranded DNA-binding protein which presumably coats the T-DNA and protects it from host nucleases (7). VirE2 functions primarily in plants because plants expressing VirE2 can be successfully infected by A. tumefaciens lacking VirE2 (8). Both VirD2 and VirE2 possess functional nuclear localization signals that guide the T-DNA to the plant cell nucleus (9,10).…”

mentioning

confidence: 99%

Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB

Chen

Nester

2000

The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid. We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins. Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2::green fluorescent protein construct were not detected in the above fraction. Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2. The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19°C as compared with 28°C. When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced. However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm. Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB. Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system. virulence protein export ͉ secretion system

“…VirD2 cleaves the bottom strand of the T-DNA at the two border sequences to generate singlestranded (ss) T-strand (8,9) and remains covalently associated with the 5Ј end of the T-strand (10). VirE2 is a nonspecific ssDNAbinding protein that can coat the length of the T-strand in vitro (11).…”

mentioning

confidence: 99%

Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus

Guo

Jin

Sun

et al. 2007

Bacterial type IV secretion system (T4SS) belongs to a growing class of evolutionarily conserved transporters that translocate DNA and proteins into a wide variety of organisms including bacterial and eukaryotic cells. Archetypal is the Agrobacterium tumefaciens VirB/D4 T4SS that transfers oncogenic T-DNA to various eukaryotic cells, which is transferred as a nucleoprotein T-complex with VirD2 as the pilot protein. As a derivative of plasmid conjugation systems, the VirB/D4 T4SS can also transfer certain mobilizable plasmids and bacterial proteins like VirE2 and VirF, although it is unknown how the membrane-bound T4SS recruits different transfer substrates. Here, we show that a cytoplasmic VirD2-binding protein (VBP) is involved in the recruitment of the T-complex to the energizing components of the T4SS, including VirD4, VirB4, and VirB11. VBP is also important for the recruitment of a conjugative plasmid to a different transfer system independent of VirB/D4. These data indicate that VBP functions as a previously unrecognized recruiting protein that helps couple nucleoprotein substrates to the appropriate transport sites for conjugative DNA transfers. VBP has three functionally redundant homologs, and similar homologs can be found in different bacterial genomes, suggesting a previously uncharacterized class of proteins involved in conjugative DNA transfers.bacterial type IV secretion system ͉ T-DNA ͉ virulence