SummaryEvidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive.Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70,OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA.Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70,OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents.Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.