Enhanced Agrobacterium‐mediated transformation revealed attenuation of exogenous plasmid DNA installation in recipient bacteria by exonuclease VII and SbcCD

• Published in: Genes to cells : devoted to molecular & cellular mechanisms Vol. 25; no. 10; pp. 663 - 674
• Main Authors: Kiyokawa, Kazuya, Ohmine, Yuta, Yunoki, Kazuya, Yamamoto, Shinji, Moriguchi, Kazuki, Suzuki, Katsunori
• Format: Journal Article
• Language: English
• Published: Tokyo Wiley Subscription Services, Inc 01.10.2020
• Subjects: Agrobacterium; Complementary DNA; conjugation; Deoxyribonucleic acid; DNA; DNA biosynthesis; Exonuclease; horizontal DNA transfer; Mutants; Nuclease; Relaxase; type IV secretion system; T‐DNA transfer
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1111/gtc.12802

In DNA transfer via type IV secretion system (T4SS), relaxase enzyme releases linear ssDNA in donor cells and recircularizes in recipient cells. Using VirB/D4 T4SS, Agrobacterium cells can transfer an IncQ‐type plasmid depending on Mob relaxase and a model T‐DNA plasmid depending on VirD2 relaxase. Mobilization to Escherichia coli of the former plasmid is much more efficient than that of the latter, whereas an entirely reverse relationship is observed in transfer to yeast. These data suggest that either plasmid recircularization or conversion of ssDNA to dsDNA in the recipient bacterial cells is a rate‐limiting step of the transfer. In this study, we examined involvement of exonuclease genes in the plasmid acceptability. By the VirD2‐dependent T‐DNA plasmid, E. coli sbcDΔ and sbcCΔ mutant strains produced threefold more exconjugants, and a sbcDΔ xseAΔ mutant strain yielded eightfold more exconjugants than their wild‐type strain. In contrast to the enhancing effect on the VirD2‐mediated transfer, the mutations exhibited a subtle effect on the Mob‐mediated transfer. These results support our working hypothesis that VirD2 can transport its substrate ssDNA efficiently to recipient cells and that recipient nucleases degrade the ssDNA because VirD2 has some defect(s) in the circularization and completion of complementary DNA synthesis. Agrobacterium‐mediated plant transformation (AMT) machinery can disseminate DNAs to a wide range of eukaryotes including human and yeast cells, though it is weak at introducing plasmid DNAs to bacteria. This paper revealed enhancement of plasmid DNA acceptability in E. coli AMT by some exonuclease mutations. The notion highlights the function of DNA relaxases in plasmid DNA recircularization and completion of DNA synthesis and gives a clue to widen the AMT host range much more as well as to control the proliferation of multiple‐drug resistance organisms.