Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.

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

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.

The repair of G-quadruplex-induced DNA damage.

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.

Agrobacterium-mediated T-DNA transfer and integration by minimal VirD2 consisting of the relaxase domain and a type IV secretion system translocation signal.

The VirD2 protein of Agrobacterium tumefaciens is essential for processing and transport of the T-DNA. It has at least three functional domains: a relaxase domain at the N terminus, a bipartite nuclear localization signal (NLS), and a sequence called omega at the C terminus. We confirm here that deletions of the C-terminal part of VirD2 led to lack of transfer of T-DNA but, for the first time, we report that virulence is restored when these truncations are supplemented at the C terminus by a short translocation signal from the VirF protein. The lack of virulence of C-terminal deletions suggests that the C-terminal part contains all or part of the translocation signal of VirD2. Using a novel series of mutant VirD2 proteins, the C-terminal half of VirD2 was further investigated. We demonstrate that the C-terminal 40 amino acids of VirD2, which include the NLS and omega, contain all or part of the translocation domain necessary for transport of VirD2 into plant cells, while another element is present in the middle of the protein. The finding that a type IV secretion system transport signal at the C terminus of VirD2 is necessary for virulence provides evidence for the role of VirD2 as a pilot protein driving translocation of the T-strand.