Transgene integration in aspen: structures of integration sites and mechanism of T‐DNA integration

Abstract

SummaryTo obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results con®rm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identi®ed in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strandannealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

“…The right T-DNA borders of our events exhibit an average of 5.7 bp resection from the VirD2 nicking site in the RB repeat. This value is within the range reported for T-DNA inserts in earlier studies: 5.7 bp in the study of Mayerhofer et al (1991), 3.9 bp in the work of Gheysen et al (1991), and 3.7 bp in transgenic aspen (Populus tremula; Kumar and Fladung, 2002). Our events exhibited an average of a 41.5-bp deletion at the LB, again within the range observed by others: 33.8 bp by Gheysen et al (1991), 24.6 bp by Mayerhofer et al (1991), and 6.7 bp in the aspen study (Kumar and Fladung, 2002).…”

“…The simplest way to account for the remarkable preservation of the 3Ј extension of the target I-CeuI cut site in so many of our targeted events is to propose that these ends initiate the repair of the DS break by synthesis-dependent strand annealing (SDSA) mechanism of NHEJ, a model proposed for T-DNA integration by earlier investigators (Salomon and Puchta, 1998;Kumar and Fladung, 2002). According to this model, one of the 3Ј ends of the genomic cut site (possibly further exposed by 5Ј exonuclease activity on the sister strand) serves as primer for repair synthesis along the T-strand.…”

“…Mayerhofer et al (1991) found an average of 47.3 bp (with a range of 29-73 bp), whereas Gheysen et al (1991) reported 18.6 bp (with a range of 13-28 bp) for Arabidopsis transformants. More recently, Kumar and Fladung (2002) analyzed 10 aspen transformants and found target deletions averaging 88.3 bp (with a range of 0-570 bp). Thus, our events appear to fall within the range of random insertion events in the extent of target site deletion, as well as the loss of donor DNA at left and RBs, as summarized above.…”

T-DNA Integration in Arabidopsis Chromosomes. Presence and Origin of Filler DNA Sequences

“…Also, T-DNA integration has been studied as an example of NHEJ. Short regions of microhomology, insertions of filler DNA, and small deletions have been reported as features of T-DNA/plant DNA junctions (Matsumoto et al, 1990;Gheysen et al, 1991;Mayerhofer et al, 1991;Tinland, 1996;Fladung, 1999;Kumar and Fladung, 2002;Meza et al, 2002). More direct NHEJ analysis in plants involved the characterization of naturally occurring deletions in plant genomes (Ralston et al, 1988;Wessler et al, 1990) and the sequencing of newly formed junctions at repaired DSBs (Gorbunova and Levy, 1997;Salomon and Puchta, 1998).…”

Next generation biotechnology: how sophisticated constructs lead to further insights and new approaches towards biotechnology’s demands

“…The integration site of transgenic sequences into the host genome, mediated by Agrobacterium is supposed to be favorably in AT-rich regions, as it was shown for tobacco (Gheysen et al 1987), Arabidopsis (Mayerhofer et al 1991), rice (Takano et al 1997) and poplar (Kumar & Fladung 2002). Despite this, the expression of genes in those regions may still differ greatly from one transgenic line to another.…”