The most familiar transformation, Agrobacterium, is derived from the tumor-inducing mechanism of the soil bacterium Agrobacterium tumefaciens. Agrobacterium tumefaciens is the causal agent of crown gall disease. The utility of these bacteria has developed from an understanding of the molecular basis of the disease symptoms of tissue hyperplasia, namely, the transfer of DNA from the bacterium to the plant nuclear genome.
Only a relatively small discrete portion of the plasmid is transferred to the plant cells during the tumor formation process and this region is now familiarly known as the T-DNA (transferred DNA).
The types of morphological effects observed in the transformed plant tissues are controlled by the complement of genes that are transferred. Plasmids that confer the tumor-inducing ability on Agrobacterium strains have become known as Ti (tumor-inducing) plasmids. The plasmids controlling the hairy root morphology are commonly called the Ri (root-inducing) plasmids.
Ti and Ri plasmids from several strains of Agrobacterium have been well characterized by a variety of physical and genetic techniques. For example, some of the regions contain genes which are essential for gene transfer. Therefore, the T-DNA regions from various Ti and Ri plasmids appear to be mobilized to plant cells by a conserved mechanism.
The genes which are located in the T-DNA region contain the necessary structural features for expression by the plant. The key elements of the T-DNA are the 25-base pair imperfect direct repeats present at the boundaries of the T-DNA. Most plant/T-DNA junction sequences which were isolated from transformed cells occur within or near the border sequences.
T-DNA transfer begins with the introduction of the bacteria into a plant wound. Wounding is necessary for the synthesis of compounds by the plant which induce the expression of the vir genes. Three additional conditions have also been found to be conducive to T-DNA transfer: a low pH medium, low phosphate, and the proper sugar source. Within the T-DNA plasmid, there is a number of vir genes:
VirA – It is a membrane-associated protein which monitors the presence of the induction compounds. It is constiutively expressed with VirG and is involved in mediating the induction of the other genes.
VirC – It is not absolutely required for T-DNA transfer. It contains VirC1 protein which appears to mediate the enhancer effect of the overdrive sequence. It is another vir gene product that has been shown to have DNA binding activity. It binds specifically to the overdrive sequence of the octopine-type A6 T-DNA.
-- They are found within the bottom strand of the
25 base pair (bp) border. The virD
codes for at least two polypeptides called VirDI
and VirDII. VirDI
has been shown to
possess helicase activity and is also required for
T-strand formation. VirDII has
been shown to be required for the nicking activity and single-strand synthesis
and codes an endonuclease activity. Its
protein can be found covalently attached to the 3’ end of the T-strand which protect
the T-DNA from exonucleolytic degradation. It is also used to direct a fusion protein to the plant cell
VirE – It behaves as a single-stranded DNA binding protein. However, it is not absolutely required for T-DNA transfer. It may provide protection to the T-strand intermediate during the transfer process with VirDII. VirE appears to be a nonspecific single-stranded binding protein that coats the T-strand, presumably affording protection from endonucleolytic catalysis.
VirG – It belongs to a class of positive regulatory genes in bacteria which link gene induction to different external stimuli. It is constiutively expressed with VirA. VirG binds specifically to upstream regions of, at least, some of the vir genes.
With the help of virus in the T-DNA, the building up of the desired genes can be well-developed.