Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice

Abstract

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.

Gene targeting in plants: 25 years later

“…It has been shown recently that these difficulties may be caused by the existence of independent end-joining pathways in plant cells (Charbonnel et al, 2011). A mild reduction in T-DNA transformation efficiencies was reported when single NHEJ factors were knocked out, but no GT experiments have yet been published concerning this issue (Friesner and Britt, 2003;Nishizawa-Yokoi et al, 2012).…”

Advances in New Technology for Targeted Modification of Plant Genomes

“…The fact that overexpression of the rice protein OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance intrachromosomal HR was taken as an indication that these proteins might, in fact, be involved in end resection in plants ( Kwon et al 2012 ). Indeed, Arabidopsis plants with a defi cit of RECQ4A show some defi ciency in both the SSA and SDSA pathways.…”

“…Not only the induction of DSBs but also modulation of the recombination and/or repair machinery might hamper the functional characterization of the endogenous gene of interest as well as the molecular breeding of crop plants, because alteration of the recombination and/or repair machinery may result in unexpected or pleiotropic phenotypes. For example, suppression of Ku70 involved in NHEJ processes in rice led to increased HR frequency, and its null mutants exhibited severely retarded growth phenotypes (Nishizawa-Yokoi et al 2012 ). In this respect, the approaches to enrich gene-targeted recombinants by effective selection systems appear to be less problematic and more straightforward than induction of DSBs or modulation of the recombination and/or The positive selection marker hpt gene was removed by the site-specifi c Cre recombinase repair machinery, because no materials other than homologous segments to be recombined with target sequences are necessary to be introduced into the plant cells, although an effi cient and large-scale transformation procedure needs to be developed (see below).…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…tumefaciens (Friesner and Britt, 2003;Gallego et al, 2003). However, in other studies, contradictory data indicated that Arabidopsis ku80 or ku70 (At1g16970) mutant plants and rice plant lines downregulated in ku70, ku80, or lig4 showed different transformation responses (van Attikum et al, 2001;Friesner and Britt, 2003;Gallego et al, 2003;Li et al, 2005b;Jia et al, 2012;Nishizawa-Yokoi et al, 2012;Vaghchhipawala et al, 2012;Mestiri et al, 2014;Park et al, 2015). These discrepancies might be the result of different techniques and different plant tissues used to examine transformation efficiency, or they may reveal more complex and redundant pathways for T-DNA integration mechanisms during A. tumefaciens infections (Tzfira et al, 2004a;Citovsky et al, 2007;Gelvin, 2010aGelvin, , 2010bMagori and Citovsky, 2012;Lacroix and Citovsky, 2013).…”

“…Integration and/ or expression of T-DNA AtLIG4 (DNA ligase IV) At5g57160 Ziemienowicz et al, 2000;Friesner and Britt, 2003;van Attikum et al, 2003;Zhu et al, 2003a;Nishizawa-Yokoi et al, 2012;Park et al, 2015KU80 At1g48050 van Attikum et al, 2001Friesner and Britt, 2003;Gallego et al, 2003;Li et al, 2005b;Nishizawa-Yokoi et al, 2012;Jia et al, 2012;Mestiri et al, 2014;Park et al, 2015KU70 At1g16970 van Attikum et al, 2001Li et al, 2005b;Nishizawa-Yokoi et al, 2012;Jia et al, 2012;Mestiri et al, 2014;Park et al, 2015 MRE11 (meiotic recombination 11) At5g54260 van Attikum et al, 2001;Jia et al, 2012; XRCC1 (homolog of X-ray repair cross complementing 1) At1g80420 Mestiri et al, 2014;Park et al, 2015 XRCC2 (homolog of X-ray repair cross complementing 2) At5g64520 Mestiri et al, 2014;Park et al, 2015 XRCC4 (homolog of X-ray repair cross complementing 4) At3g23100 Vaghchhipawala et al, 2012;Park et al, 2015 XPF/RAD1/UVH1 (ultraviolet hypersensitive 1) At5g41150 Nam et al, 1998;Mestiri et al, 2014;Park et al, 2015 PARP1 (poly(ADP-ribose) polymerases 1) At2g31320 Jia et al, 2012;Park et al, 2015 HTA1 (histone H2A) At5g54640 Nam et al, 1999;Mysore et al, 2000a, b;Yi et al, 2002Yi et al, , 2006…”