Formation of complex extrachromosomal T-DNA structures in Agrobacterium-infected plants

AUTHORS Kamy Singer, Yoel M. Shiboleth, Jianming Li, Tzvi Tzfira.

ADMIN(S) Kamy Singer , Yoel Shiboleth

ABSTRACT Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during Agrobacterium -mediated transformation are still poorly understood. Here we show that complex extrachromosomal T-DNA structures form in Agrobacterium -infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta . We recovered circular T- DNA molecules (T-circles) using a novel plasmid rescue method. Sequencing analysis of the T- circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end-joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown “filler” sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant’s DNA double-strand break repair machinery. This can lead to circularization, integration, or formation of extrachromosomal complex T-DNA structures that subsequently may integrate.

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA

Plant Physiology July 2012 pp.112.200212

ADDITIONAL INFORMATION

DNA Extraction Protocol

Agroinfiltration Protocol

U of M MCDB

KEY WORDS Agrobacterium; T-DNA integration; Genetic Transformation

SPAPER ID 2

Author summary

A common method for delivering a foreign gene to a plant is to place it within a “T-DNA”, a DNA region in the genome of the unique bacterium named Agrobacterium tumefaciens. Agrobacterium then transfers the T-DNA into the nucleus of a plant cell where the T-DNA may subsequently integrate into the plant genome. Although Agrobacterium-mediated genetic transformation of plant cells has been studied extensively, how T-DNAs integrate in the plant genome is largely unknown. While previous studies focused on T-DNA molecules after they integrated into the plant genome, we studied “free-floating” DNA molecules inside the plant nucleus in order to learn about the structure of T-DNAs before their integration.

Using a new method, we captured the T-DNAs from plant leaves a few days after infiltrating them with Agrobacterium. We found that T-DNAs and other DNA molecules “floating” in the plant nucleus are joined to each other. This explains why transgenic plants often contain unpredictable DNA sequences. Our system which enables the capture of “free-floating” DNA fragments in plants can also be exploited to study how plants recognize and repair foreign or damaged pieces of DNA.