T-DNA

All the information on this page has been obtained from Ziemienowicz (2001).

The soil bacterium Agrobacterium tumefaciens, is a plant pathogen responsible for tumour induction on dicotyledonous plants due to its ability to transfer DNA to the plant cell. In biotechnology, this ability to transfer DNA, is widely used for the transformation of plants. During tumour induction, Agrobacterium attaches onto plant cells and then transfers part of its DNA to some of these cells. The transferred DNA (T-DNA) which is found on a large Ti (tumour inducing) plasmid (Figure 1 below), is modified within the bacterium and is transferred to the plant where it becomes integrated into the plant genome.

Figure 1: the Ti Plasmid (click to enlarge)

Proteins which are encoded by the virulence (vir) region of the Ti plasmid regulate the T-DNA modification and transfer. Phenolic compounds that are derived from a wounded plant cell induce expression of the vir region genes. Virulence proteins recognise 25bp imperfect direct repeats (the border sequences) that define the T-DNA. In the presence of VirD1 protein, VirD2 cleaves the border sequence in a site- and strand-specific manner and as a result becomes covalently attached to the 5' end of the nicked DNA. A single-stranded T-DNA is produced when the nicked DNA is released from the plasmid. The T-DNA-VirD2 complex and the VirE2 protein are believed to be transformed the plant through a pilus-like structure containing VirB and VirD4 proteins.

In the plant cell, the T-DNA is coated with the single-stranded DNA-binding protein, VirE2. The complex that comprises of the T-DNA bound to the binding protein then moves into the nucleus and is integrated into the nuclear genome. Expression of genes located on the T-DNA leads to the formation of proteins involved with the production of auxins and cytokynines. These plant hormones cause the tumourous phenotype that is characterised by the ability of the plant cells to proliferate limitlessly and autonomously, even in the absence of added phytohormones.

Crown gall tumours are characterised by the production of opines (derivatives of amino-acids). The biosynthesis of opines is catalysed by opine synthase enzymes, which are encoded by the T-DNA. Opines are formed in the tumours can be metabolised by the tumourigenic agrobacteria, by not by most other soil organisms. Thus, Agrobacterium creates for itself a favourable niche by genetic modification plant cells.

Figure 2: A simplified representation of the journey T-DNA makes from the Agrobacterium to the plant cell (click to enlarge). LB and RB, left border and right border repeats, respectively; NPC, nuclear pore complex.

The ability of Agrobacterium to transfer a fragment of its own DNA to the plant cell provides a powerful tool for plant biotechnology and therefore Agrobacterium-mediated DNA transfer is one of the most commonly used techniques of plant transformation. Moreover, the relative ease with which Agrobacterium can be used to affect change in a wide range of hosts has allowed scientists from around the world to apply this technology to specific problems allowing the safe and precise manipulation of plants as research tools (Binns; 2002).

The insertion of a known segment of DNA into a gene of interest has been an invaluable research tool for a variety of systems such as Arabidopsis. The unique feature of foreign DNA as a mutagen is that the inserted sequence not only creates a mutation but also 'tags' the affected gene, facilitating its isolation. Transposons, short mobile genetic elements that can move from one site to another within a genome, were the first type of insertional mutagen to be used for this purpose. Given that Arabidopsis does not contain endogenous active transposons, the integration of T-DNA provides an alternative system by which insertional mutagenesis can be carried out. T-DNA, including any sequences