title: Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins.
abstract: Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (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 thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, 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 (uidA) 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, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.
authors: Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA; Plant Transformation Core Facility, University of Missouri, Columbia, MO, 65211, USA; Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, 24061, USA.
authors: So-Yon
authors: SY
authors: Park
authors: Zarir
authors: Z
authors: Vaghchhipawala
authors: Balaji
authors: B
authors: Vasudevan
authors: Lan-Ying
authors: LY
authors: Lee
authors: Yunjia
authors: Y
authors: Shen
authors: Kamy
authors: K
authors: Singer
authors: Wanda M
authors: WM
authors: Waterworth
authors: Zhanyuan J
authors: ZJ
authors: Zhang
authors: Christopher E
authors: CE
authors: West
authors: Kirankumar S
authors: KS
authors: Mysore
authors: Stanton B
authors: SB
authors: Gelvin
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D059766:DNA End-Joining Repair; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics*; D016254:Mutagenesis, Insertional; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA
keywords: Agrobacterium; Arabidopsis thaliana; Nicotiana benthamiana; T-DNA integration; non-homologous end joining; plant transformation