”Classical” Non-Homologous End-Joining Proteins Limit T-DNA Integration into the Plant Genome

Non-homologous end-joining (NHEJ), a double-strand (ds) DNA break repair process, is the major model proposed for Agrobacterium T-DNA integration into the plant genome. Several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA Ligase IV, ATM, and ATR, play an important role in NHEJ. We examined transient and stable transformation frequencies of Arabidopsis roots mutant for these and other genes. Mutants in KU70, KU80, ARTEMIS, and the plant-specific DNA LIGASE VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, or lig4 mutants. We hypothesize that plants mutant in several NHEJ genes show increased T-DNA integration because delayed dsDNA break-repair provides greater opportunity for T-DNA integration.