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

Sequence motifs associated with T-DNA–genome junctions sites.

<p>The motifs CACCAC (P-value = 1e-147, HOMER, E-value = 1.7e-234, MEME) and A-rich (P-value < 1e-21, HOMER, E-value = 2.3e-483, MEME) were associated with T-DNA–genome junction sites.</p

T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome - Table 1

<p>T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome</p> - Table

Association of genomic features with T-DNA-genome junctions under selective and non-selective conditions.

<p>A–The genomic distribution of unselected and selected T-DNA–genome junctions across chromosome 4. The numbers of T-DNA–genome junctions (circles, and smoothed blue line) do not show a correlation with the distribution of transposons (TE, red line) and promoters (green line). T-DNA integrations under selective conditions (orange line) correlates with genes/promoters [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>]. B- The portion of each genomic feature: TE (red), genes (purple), promoters (green) and the remaining regions (other, grey). The portion of genomic features is represented across all the genome (genome wide) and according to the number of T-DNA–genome junctions: without selection (Unselected) and with selection (Selected- data from Alonso et al., 2003 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>]). Selected events [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>] show an enrichment in promoters (χ² test, compared to unselected events, p = 2.88E-24) and a decrease in TE regions (χ2 test, compared to unselected events, p = 0.005).</p

Epigenetic modifications around T-DNA–genome junctions.

<p>The analysis was performed separately in pericentric (grey background) and in remaining (distal) chromosomal regions. The up and downstream regions to T-DNA–genome junctions (represented as the 0 bp) are shown on the X axis. The Y axis represents the arbitrary level of the epigenetic markers. Blue squares–unselected T-DNA–genome junctions. Orange triangle–integrations under selective conditions [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>]. Black circles–control, random genomic positions.–. A- CG methylation. B–Nucleosome occupancy. C- H3K27me3 modification.</p

Experimental design–<i>Arabidopsis</i> roots were infected with <i>A</i>. <i>tumefaciens</i>.

<p>DNA was extracted at 0, 6 and 24 hours post infection. Extracted DNA was digested with 3 restriction enzymes: <i>Eco</i>RI (RI), <i>Hind</i>III (H3) and <i>Xba</i>I (Xb). An adapter was ligated to the overhang end of the digested DNA. T-DNA-genomic junctions were amplified using three different primers from within the T-DNA and one primer from the adapter (primers—black arrows, LB–left border, RB–right border). Amplicons were sequenced using high throughput sequencing. Adapter to adapter products were reduced as detailed in O’Malley et al. 2007 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref027" target="_blank">27</a>].</p