Non-Homologous End-Joining Proteins Limit T-DNA Integration into the Plant Genome and Facilitate Transgene Expression

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 “classical” (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a “backup” (b)NHEJ process. 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, lig4, hon1, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methlyation, suggesting that the integrated transgenes were silent. 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.