Research project

Understanding plant transgenesis

How is Pol θ responsible for T-DNA integration, and how do other DNA double-strand break repair pathways interact with Pol θ? How may we manipulate T-DNA integration to stimulate error-free integration at a predetermined genomic site?

Duration: 2017 - 2020
Contact: Marcel Tijsterman
Funding: NWO
Partners: Leiden University Medical Center (LUMC), Genome stability group

Unravelling the molecular mechanisms that underlie plant genetic modification.

In nature, Agrobacterium tumefaciens genetically modifies plants by inserting T-DNA, containing tumor-inducing genes, into their genomes, causing crown gall disease. In the laboratory, tumor-inducing genes can be replaced by any desired sequence, making genetic transformation by Agrobacterium the most popular tool to create genetically modified plants.

T-DNA integration in Arabidopsis roots: wild type Arabidopsis roots have successfully integrated T-DNA into their genome, while teb-5 Polymerase θ-deficient plants are unable to integrate T-DNA into their genome — **T-DNA integration in Arabidopsis roots:** wild type Arabidopsis roots have successfully integrated T-DNA into their genome, while teb-5 Polymerase θ-deficient plants are unable to integrate T-DNA into their genome

In 2016, we discovered, in collaboration with the Genome Stability Group at the LUMC, that T-DNA integration requires Polymerase Theta (Pol θ). DNA double-strand break repair by Pol θ captures T-DNA into genomic breaks. Random integration events, which constitute almost all events, are created by Pol θ. Random integration of T-DNA heavily impacts the function of integrated T-DNA, and impacts the genomic environment. Therefore, it represents a safety risk in genetically modified plants.

**Model for T-DNA integration:** A T-DNA is captures into a double-strand break in the genome of a plant by Polymerase θ-mediated End-Joining. A double-strand break may capture a single T-DNA, but tandem integrations are more common.

Current work aims to unravel the context in which Pol θ operates in T-DNA integration. We study interactions between Pol θ and other double-strand break repair pathways and their effects on the frequency of T-DNA integration, the molecular outcome of integration events, and explore possibilities to steer the T-DNA integration process towards error-free gene targeting.

Van Kregten et al., T-DNA integration in plants results from polymerase-θ-mediated DNA repair, Nat. Plants, 2016