T-DNA integration in plants results from polymerase-θ-mediated DNA repair

Abstract

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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Figure 1: Pol-θ-deficient plants are refractory to T-DNA integration via floral dip transformation.
Figure 2: Pol θ is required for root transformation-mediated Arabidopsis transgenesis.
Figure 3: Filler DNA reflects Pol-θ-dependent extension of minimally paired T-DNA–Arabidopsis genome molecules.

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Acknowledgements

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.).

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M.v.K. and M.T. conceived the study and designed the research with critical input from S.d.P. and P.J.J.H., who also provided infrastructure. M.v.K., S.d.P. and R.R. performed the experiments. R.v.S. performed bioinformatic analyses. All authors contributed to the interpretation of the results and to the manuscript, which was written by M.v.K. and M.T.

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Correspondence to Marcel Tijsterman.

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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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