TY - JOUR
T1 - Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana
AU - Pereman, Idan
AU - Melamed-Bessudo, Cathy
AU - Dahan-Meir, Tal
AU - Herz, Elad
AU - Elbaum, Michael
AU - Levy, Avraham A.
N1 - We would like thank Yaron Shav-Tal for the LacOx256 construct and Anthonius Matzke for the pBC-35S-RL plasmid. Funding This work was supported by the TRACTAR (Tracking and Targeting a T-DNA Vector for Precise Engineering of Plant Genomes) ERC grant (Project 268646) to Avraham A. Levy and Michael Elbaum. Author contributions - conceptualization, A.A.L. and M.E.; methodology, I.P. and C.M.-B.; investigation, I.P. and C.M.-B.; resources, I.P., C.M.-B., T.D.-M. and E.H.; writing—original draft preparation, I.P.; writing—review and editing, I.P., C.M.-B., T.D.-M., E.H., M.E. and A.A.L.; supervision, M.E. and A.A.L.; project administration, I.P. and C.M.-B.; funding acquisition, M.E. and A.A.L. This article belongs to the Special Issue DNA Damage and Repair in Plants)
PY - 2019/12/9
Y1 - 2019/12/9
N2 - 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.
AB - 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.
U2 - https://doi.org/10.3390/ijms20246209
DO - https://doi.org/10.3390/ijms20246209
M3 - مقالة
SN - 1661-6596
VL - 20
JO - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
IS - 24
M1 - 6209
ER -