Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions

Abstract

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

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

“…In contrast, it is reported that VirD2 localizes at one pole in most of the cells in previous studies, in which the expression of FLAG-tagged VirD2 was under the virB promoter control (21). Our time-course study on GFP-VirD2 localization indicated that the discrepancy is likely due to the difference in gene expression levels.…”

“…In addition, the polar positioning of T-complex facilitated by VirC1 can be also helpful for the recruitment of T-complex to the T4SS apparatus (21). VirC1 antibody immunoprecipitated both T-strand and Ti-plasmid (21), suggesting that VirC1 binds to VirD2 during relaxosome assembly and remains bound during transit to the T4SS apparatus. On the other hand, VBP1 antibody immunoprecipitated only T-strand (Fig.…”

“…tumefaciens VirB/D4 T4SS is archetypal; it can transfer both DNA and protein substrates to various eukaryotic cells (1,2,4,5). Recently, it was reported that VirC1 can spatially coordinate the early conjugative DNA transfer reactions, including the recruitment of T-complex to cell poles (21). However, VirC1 recruits T-complex to the cell poles independently of the T4SS components (VirD4 substrate receptor and VirB channel subunits) (21); it is not clear how the VirD2-VirD4 interaction is mediated.…”

“…This is consistent with our observation that the GFP-VirD2 fusion could not complement a virD2 Ϫ mutation in the tumorigenesis assay (data not shown). Nevertheless, the GFP-VirD2 fusion was useful to study VirD2 localization, because its localization was similar to the FLAG-tagged VirD2 that is fully functional for T-DNA transfer (21).…”

Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

“…VirC1 specifically binds to overdrive, and disruption of virC1 results in a loss of virulence similar to deletion of overdrive, suggesting that VirC1 functions through binding to overdrive (27,28). VirC1 belongs to the ParA/MinD ATPase superfamily containing a deviant Walker A box that is required for border nicking and for localizing the T-DNA to the cell pole (29,30). VirC1 also interacts with all other known T-DNA-processing proteins, including VirD1, VirD2, and VirC2, and thus it was proposed that VirC1 nucleates relaxosome assembly at T-DNA borders and spatially positions the T-DNA at the cell pole (29).…”