Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana

Idan Pereman, Cathy Melamed-Bessudo, Tal Dahan-Meir, Elad Herz, Michael Elbaum, Avraham A. Levy

Research output: Contribution to journalArticlepeer-review

Abstract

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

Original languageEnglish
Article number6209
Number of pages10
JournalINTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume20
Issue number24
DOIs
StatePublished - 9 Dec 2019

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Spectroscopy
  • Catalysis
  • Inorganic Chemistry
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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