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Agrobacterium T‐ DNA integration into the plant genome can occur without the activity of key non‐homologous end‐joining proteins
Author(s) -
Park SoYon,
Vaghchhipawala Zarir,
Vasudevan Balaji,
Lee LanYing,
Shen Yunjia,
Singer Kamy,
Waterworth Wanda M.,
Zhang Zhanyuan J.,
West Christopher E.,
Mysore Kirankumar S.,
Gelvin Stanton B.
Publication year - 2015
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12779
Subject(s) - biology , ku80 , dna ligase , non homologous end joining , agrobacterium , microbiology and biotechnology , mutant , transformation (genetics) , genetics , dna repair , dna , gene , dna binding protein , transcription factor
Summary 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 KU 70, KU 80, ARTEMIS , DNA ‐ PK cs, DNA ligase IV ( LIG 4), Ataxia telangiectasia mutated ( ATM ), and ATM ‐ and Rad3‐related ( ATR ), play an important role in ‘classical’ (c) NHEJ . Other proteins, including histone H1 ( HON 1), XRCC 1, and PARP 1, 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 KU 70 , KU 80 , and the plant‐specific DNA LIGASE VI ( LIG 6 ) 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.