The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double-Strand Break Repair-Based Insertion Mechanism.
Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host's DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34,000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double-strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.
Center for Biotechnology & Department of Biology, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Nils
N
Kleinboelting
Center for Biotechnology & Department of Biology, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Gunnar
G
Huep
Center for Biotechnology & Department of Biology, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Ingo
I
Appelhagen
Center for Biotechnology & Department of Biology, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany.
Prisca
P
Viehoever
Department of Medicine IV, University Hospital Freiburg, Berliner Allee 29, 79110 Freiburg, Germany.
Yong
Y
Li
Center for Biotechnology & Department of Biology, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany. Electronic address: bernd.weisshaar@uni-bielefeld.de.
Bernd
B
Weisshaar
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics* / Q000382:microbiology; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant; D017384:Sequence Deletion; D057345:Sequence Inversion
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA
Agrobacterium tumefaciens; Arabidopsis thaliana; T-DNA integration; double-strand break repair