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

doi:10.3390/ijms20246209

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

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 language	English
Article number	6209
Number of pages	10
Journal	International Journal of Molecular Sciences
Volume	20
Issue number	24
DOIs	https://doi.org/10.3390/ijms20246209
Publication status	Published - 9 Dec 2019

Funding

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)

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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title = "Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana",

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.",

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year = "2019",

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AU - Pereman, Idan

AU - Melamed-Bessudo, Cathy

AU - Dahan-Meir, Tal

AU - Herz, Elad

AU - Elbaum, Michael

AU - Levy, Avraham A.

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.

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

Abstract

Funding

All Science Journal Classification (ASJC) codes

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