Polθ-mediated T-DNA integration is a conserved mechanism across plant species

Agrobacterium-mediated transformation remains the cornerstone of plant biotechnology, providing a versatile system for stable gene delivery and crop improvement. In Arabidopsis thaliana, DNA polymerase theta (Polθ) has been identified as a central factor mediating T-DNA integration via theta-mediated end joining (TMEJ). Yet, whether this mechanism and its absolute requirement represent a model-specific phenomenon or a general principle across crops has been unresolved. Here, using genetic analysis in rice and high-resolution footprint analyses in rice and soybean, complemented with data from other species, we demonstrate that Polθ-mediated T-DNA integration is conserved across angiosperms. Integration consistently follows a shared logic: capture of T-DNA's left border depends predominantly on TMEJ, while the right border end can be resolved by both TMEJ and canonical non-homologous end joining (cNHEJ). This unifying framework establishes that mechanistic insights concerning transgenesis translate well from Arabidopsis to crop species, providing a predictive foundation for plant transformation and genome engineering.