Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown1. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA–plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3′ end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer–template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.
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We thank B. Klemann and A. den Dulk-Ras for technical assistance and P. Lindenburg for photography. M.T is supported by grants from the European Research Council (203379, DSBrepair), the European Commission (DDResponse) and ZonMW/NGI-horizon. This work was also sponsored by the Royal Academy of Sciences (Academy Professorship award to P.J.J.H.).
The authors declare no competing financial interests.
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van Kregten, M., de Pater, S., Romeijn, R. et al. T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nature Plants 2, 16164 (2016). https://doi.org/10.1038/nplants.2016.164
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