T-DNA-Genome junctions form early after infection and are influenced by the chromatin state of the host genome

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown

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

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

Mechanistic models for modification of methylation status of targeted alleles by GT.

<p>Methylated cytosines are indicated by grey lollipops. The GT vector is denoted by a red line, with a red asterisk illustrating the genetic perturbation introduced by the vector. If gap enlargement occurs following DSB induction (pathway on the left), the methylation template is lost and the targeted allele loses its methylation. However, if GT occurs via strand assimilation (pathway on the right) or via DSB induction but without gap enlargement (pathway in the middle), the methylation pattern of the WT can be restored upon maintenance methylation.</p

Methylation changes at the targeted <i>PPOX</i> locus in lines TGT-2 and TGT-3.

<p>(<b>A</b>) The methylation landscape in TGT-2 and TGT-3 lines (upper and lower graphs, respectively). Blue circles, triangles and squares correspond to WT, T2 and T3 generations, respectively. Dotted lines mark the CG positions at which the methylation level had changed relative to WT. (<b>B</b>) CG positions that lost methylation stability in the two targeted lines. CG position is denoted beneath each bar. The numbers above each bar denote the level of methylation as fraction.</p

Ectopic Gene Targeting (EGT)-derived duplication of the <i>PROHIBITIN1</i> gene and subsequent changes in DNA methylation.

<p>(<b>A</b>) Schematic presentation of the WT <i>CRUCIFERIN3</i> locus, including the upstream <i>PROHIBITIN1</i> gene. Methylated cytosines are marked by vertical lines whose heights are proportional to the level of methylation (Cokus, Feng et al. 2008; Lister, O'Malley et al. 2008). The region analyzed by bisulfite-sequencing is highlighted in yellow. (<b>B</b>) The GT vector, containing sequences homologous to the target (dark blue) as well as the mRFP fluorescent marker (red), invades the <i>CRUCIFERIN3</i> target and primes DNA synthesis (dark blue dotted arrow), copying the <i>PROHIBITIN1</i> sequence. (<b>C</b>) and (<b>D</b>) Genetic and epigenetic consequences of EGT: (<b>C</b>) Methylation status at the endogenous copy, in T2 (upper scheme) and T3 (lower scheme) generations after GT. The red vertical line marks the newly methylated CG at position 18. (<b>D</b>) The vector including the <i>PROHIBITIN1</i> fragment that had integrated into chromosome 1 (green dotted lines) creating gene duplication. The methylation status of this duplicated copy is shown, in T2 (upper scheme) and T3 (lower scheme) generations after GT. Grey bars: the vector RB (right border) sequences.</p

Loss of CG methylation at the targeted <i>PPOX</i> locus in the TGT-1 line.

<p>Cytosine methylation data obtained from bisulfite-sequencing of the PPOX fragment in the targeted line TGT-1 and two WT lines from ecotypes <i>Columbia (Col)</i> and <i>Wassilewskija</i> (Ws). Each circle represents a cytosine residue, either methylated (full circle) or non-methylated (empty circle). Cytosines are color-coded by their sequence context: red for CG, blue for CHG and green for CHH (H is C, T or A). Each row represents an independent clone. The numbers at the top of the columns indicate the position in base pairs, relative to the first base in this fragment. The second SNP introduced by the GT vector, is shown by an asterisk.</p