Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2
- 1Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, United States of America
Abstract
During plant genetic transformation, Agrobacterium transfers to its host cell a single-stranded segment of DNA (T-DNA), as a covalent complex with the virulence protein VirD2, which is ultimately integrated into the plant genome. The covalently bound VirD2 molecule is an obstacle to the ligation of the T-DNA 5’-end to host genomic DNA; therefore, VirD2 must be cleaved from the VirD2-T-DNA complex (T-complex) before the integration is completed. An interesting idea was raised that TDP2 (tyrosine phosphodiesterase 2), a protein known to resolve protein-DNA adducts, could represent a pathway for VirD2 removal from the T-complex; however, this function has not been proven directly. Here, we developed a simple technique to generate fluorescently labeled VirD2-ssDNA complexes in vitro and explored the DNA substrate requirements for the VirD2-mediated formation of these complexes, establishing a tractable experimental system for studying VirD2 removal from synthetic T-complexes. Using this system, we demonstrated that TDP2, from mammalian or plant origin, can specifically cleave VirD2 from these synthetic T-complexes; this activity was Mg2+-dependent and required a conserved C-terminal histidine residue of TDP2. Our results strongly support the hypothesis that plant TDP2 can remove VirD2 from the T-DNA before its integration into the plant genome. Author summary: Agrobacterium tumefaciens and related species are fascinating pathogenic bacteria that can genetically transform plants by transferring and integrating a segment of their own DNA into the genome of their host cells. This extraordinary ability has been harnessed as an essential tool for research and biotechnology. The transferred DNA (T-DNA) is exported from the bacterial cell to its host cell nucleus as a complex (named the T-complex) comprised of a single-stranded DNA segment covalently attached to the effector protein VirD2; it is thus necessary that VirD2 is cleaved from the T-DNA to allow its ligation to the genome DNA. Here, we generated fluorescently labeled artificial T-complexes and developed a system suitable for in vitro study of VirD2 removal from T-complex. Next, we demonstrated that purified TDP2 is indeed able to cleave precisely VirD2 from the imported T-DNA strand. Our findings provide new insights into the mechanism of Agrobacterium T-complex processing prior to T-DNA integration, and highlight how the activity of host plant factors can be recruited to facilitate Agrobacterium infection process.
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BibTeX
@article{lacroix2026removal,
title = {{Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2}},
author = {Benoît Lacroix and Vitaly Citovsky},
journal = {PLOS Pathogens},
year = {2026},
month = oct,
doi = {10.1371/journal.ppat.1014750},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014750},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Removal of VirD2 from a synthetic T-DNA complex of Agrobacterium by the host DNA repair protein TDP2
AU - Benoît Lacroix
AU - Vitaly Citovsky
PY - 2026
DA - 2026/10/26
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014750
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014750
AB - During plant genetic transformation, Agrobacterium transfers to its host cell a single-stranded segment of DNA (T-DNA), as a covalent complex with the virulence protein VirD2, which is ultimately integrated into the plant genome. The covalently bound VirD2 molecule is an obstacle to the ligation of the T-DNA 5’-end to host genomic DNA; therefore, VirD2 must be cleaved from the VirD2-T-DNA complex (T-complex) before the integration is completed. An interesting idea was raised that TDP2 (tyrosine phosphodiesterase 2), a protein known to resolve protein-DNA adducts, could represent a pathway for VirD2 removal from the T-complex; however, this function has not been proven directly. Here, we developed a simple technique to generate fluorescently labeled VirD2-ssDNA complexes in vitro and explored the DNA substrate requirements for the VirD2-mediated formation of these complexes, establishing a tractable experimental system for studying VirD2 removal from synthetic T-complexes. Using this system, we demonstrated that TDP2, from mammalian or plant origin, can specifically cleave VirD2 from these synthetic T-complexes; this activity was Mg2+-dependent and required a conserved C-terminal histidine residue of TDP2. Our results strongly support the hypothesis that plant TDP2 can remove VirD2 from the T-DNA before its integration into the plant genome. Author summary: Agrobacterium tumefaciens and related species are fascinating pathogenic bacteria that can genetically transform plants by transferring and integrating a segment of their own DNA into the genome of their host cells. This extraordinary ability has been harnessed as an essential tool for research and biotechnology. The transferred DNA (T-DNA) is exported from the bacterial cell to its host cell nucleus as a complex (named the T-complex) comprised of a single-stranded DNA segment covalently attached to the effector protein VirD2; it is thus necessary that VirD2 is cleaved from the T-DNA to allow its ligation to the genome DNA. Here, we generated fluorescently labeled artificial T-complexes and developed a system suitable for in vitro study of VirD2 removal from T-complex. Next, we demonstrated that purified TDP2 is indeed able to cleave precisely VirD2 from the imported T-DNA strand. Our findings provide new insights into the mechanism of Agrobacterium T-complex processing prior to T-DNA integration, and highlight how the activity of host plant factors can be recruited to facilitate Agrobacterium infection process.
SN - 1553-7374
SN - 1553-7366
ER -