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

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

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

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

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