Transient expression assays using GUS constructs and fluorometric detection for analysis of T-DNA transfer

Abstract

The genetic transfer of DNA from Agrobacterium tumefaciens to the chromosome of a plant cell is one of the foundations for plant genetic engineering. Details of this transformation process are reviewed in [1]. Briefly, A. tumefaciens initiates transfer of T-DNA from its Ti plasmid in response to phenolic signals produced by wounded plants. The plant phenolics trigger induction of bacterial plasmid-encoded virulence (vir) genes that, in turn, mediate T-DNA transfer. The specific functions of many of the vir genes and proteins have been identified. For example, VirA is a membrane-bound receptor that transduces the phenolic signal to VirG, which is probably activated by phosphorylation. VirG transcriptionally activates the other vir genes. VirD proteins make site-specific nicks at the T-DNA border regions, and VirD2 remains bound at the 5’ end of the T-DNA strand (single-stranded T-DNA molecule). Once nicked, the T-strand is thought to be displaced from the plasmid as a replacement strand is synthesized. The displaced single-stranded DNA is coated with VirE2 protein to form the T-complex. Transport of the T-complex through the bacterial membrane probably occurs via a channel composed of VirB proteins. It has been postulated that this T-complex is transferred to the plant cell. Once inside the plant cell, the T-complex may be guided into the nucleus via nuclear targeting sequences present on the VirD2 and VirE2 proteins. In the nucleus, the T-DNA integrates randomly into the plant chromosome.