Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice.
Evidence 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.
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.
Ayako
A
Nishizawa-Yokoi
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.|Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan.
Satoko
S
Nonaka
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.
Hiroaki
H
Saika
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.|Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, 244-0813, Japan.
Yong-Ik
YI
Kwon
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.|Institute for Environmental Science and Technology, Saitama University, Shimo-Okubo 255, Sakura-ku, Saitama-shi, 338-8570, Japan.
Keishi
K
Osakabe
Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.|Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, 244-0813, Japan.
Seiichi
S
Toki
D060054:Agrobacterium / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D010940:Plant Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins; D004265:DNA Helicases; D011088:DNA Ligases; D000072481:DNA Ligase ATP