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.

Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicago sativa

“…In both of these reports, the expression of Zeolin and γ-Zein (optimized with N.tabacum) in N.tabacum was 1.23 and 1.86 times higher than M.sativa, respectively. Additionally, the number of transformed t-DNA (Pereman et al 2019) into the nucleus and the effect of timing on the degradation of foreign t-DNA (Gelvin 2021) could be another possible explanation for the difference in expression among constructs and plants.…”

Genomic consequences associated with Agrobacterium‐mediated transformation of plants

“…Two recent studies have investigated the number of transferred T-strands in transient tobacco systems. Firstly, Pereman et al used a dsDNA labeling technique to estimate that 5-8 dsT-DNA loci were present in each nucleus following transfer with a single vector (Pereman et al, 2019). Secondly, it was recently estimated that >20 unique T-DNAs from different cells could be inserted into a plant cell if Agrobacterium culture density and plant tissue used are optimized (Carlson et al, 2023).…”