Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in...

Full description

Saved in:
Bibliographic Details
Published in:Plant physiology (Bethesda) 2003-11, Vol.133 (3), p.956-965
Main Authors: Mary-Dell M. Chilton, Que, Qiudeng
Format: Article
Language:English
Subjects:
Agrobacterium tumefaciens
Base Sequence
Deoxyribonucleic acid
DNA
DNA, Bacterial - genetics
DNA, Plant - chemistry
DNA, Plant - genetics
DNA, Plant - metabolism
Endodeoxyribonucleases - genetics
Endodeoxyribonucleases - metabolism
Focus Issue: The Agrobacterium-Plant Cell Interaction
Gene Targeting - methods
Genetic loci
Genetic vectors
Genome, Plant
Genomes
Genomics
Introns
Molecular Sequence Data
Nicotiana - genetics
Nicotiana - microbiology
Nicotiana tabacum
Plant cells
Plant genomes
Plant Tumors - genetics
Plant Tumors - microbiology
Plants
Plants, Genetically Modified
Polymerase chain reaction
Rhizobium - genetics
Rhizobium - growth & development
Sequence Analysis, DNA
Sequence Homology, Nucleic Acid
Transformation, Genetic - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.103.026104