Host repair protein TDP2 cleaves VirD2 from Agrobacterium T-DNA complexes

When Agrobacterium tumefaciens genetically transforms a plant cell, it delivers a single-stranded DNA segment, the T-DNA, as a covalent complex with the virulence protein VirD2. This complex must ultimately be integrated into the plant genome, but the protein attached to the DNA 5′ end blocks the ligation step, so VirD2 has to be removed before integration can be completed.

Researchers had proposed that TDP2, tyrosine phosphodiesterase 2, a protein known to resolve protein-DNA adducts, might provide the pathway for cleaving VirD2 from the T-complex, but this function had never been proven directly. To test it, the team developed a simple technique to generate fluorescently labeled VirD2-ssDNA complexes in vitro, establishing a tractable experimental system for studying VirD2 removal from synthetic T-complexes.

Using this system, the researchers mapped the DNA substrate requirements for VirD2-mediated formation of these complexes and then asked whether TDP2 could act on them. They found that TDP2 from both mammalian and plant sources specifically cleaved VirD2 from the synthetic T-complexes.

The activity was dependent on magnesium ions and required a conserved histidine residue in the C-terminal region of TDP2, indicating that the reaction follows the catalytic mechanism expected for this repair enzyme.

The authors note that these results strongly support the hypothesis that plant TDP2 can remove VirD2 from the T-DNA before its integration into the plant genome. Because the work was performed with synthetic complexes in vitro, it demonstrates biochemical capability rather than direct observation of the process inside plant cells, but it provides the first direct evidence that TDP2 can perform this cleavage on VirD2-T-DNA complexes.

Subject of Research: Removal of the Agrobacterium VirD2 protein from T-DNA complexes by the DNA repair protein TDP2

Article Title: Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2

Article References: Lacroix, B., & Citovsky, V. (2026). Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2. PLOS Pathogens, 22(10), e1014750. https://doi.org/10.1371/journal.ppat.1014750

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014750

Keywords: Agrobacterium, T-DNA, VirD2, TDP2, DNA repair, protein-DNA adducts, genetic transformation, plant genome integration, tyrosine phosphodiesterase 2, in vitro biochemistry, Removal, synthetic