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agrobacteria and plant transformation

Scientists have known since 1907 that agrobacteria cause tumor formation and other horticultural abnormalities in plants, but it was not until the 1970's that they discovered that agrobacteria induce these changes by incorporating part of their own genetic material into the plant genome. After binding to plant cells, agrobacteria use genes encoded on a tumor-inducing (Ti) plasmid separate from the bacterial chromosome to transfer a segment of DNA into the plant. These segments are then expressed by the plant to produce hormones that stimulate uncontrolled growth and induce tumor formation and to produce opines, amino acid derivatives which serve as a source of carbon and nitrogen for the agrobacteria.

The Ti plasmid contains four main elements: the transfer DNA (T-DNA) region which is transformed into the plant cell, the virulence (vir) region which is responsible for incorporating the T-DNA into the plant genome, genes responsible for opine catabolism, and genes responsible for transfer of the plasmid during bacterial conjugation. Genes on the bacterial chromosome facilitate binding of the agrobacterium to the plant cell surface, triggering activation of the vir region to initiate transformation.

In this activation, the constitutively expressed VirA transmembrane protein activates the cytoplasmic VirG, which acts as a transcription factor for the VirD operon. VirD1 and VirD2 then cut out the bottom strand of T-DNA from the Ti plasmid, with VirD2 clinging to the 5' end to prevent degradation and to orient the single strand of T-DNA, and this complex is tranported from the agrobacterium cell to the plant cell, where it is coated by the protein expressed by VirE2. VirD2 and VirE2 help direct the complex to the plant nucleus. In the nucleus the T-DNA is incorporated into the plant genome via illegitimate recombination.

Because all material in the T-DNA region is incorporated into the plant genome as a block, and because none of agrobacteria's natural T-DNA is used to drive the transformation itself (it is only expressed once in the plant), we can take advantage of agrobacteria's transformation mechanism by replacing the agrobacteria's natural T-DNA with our own constructs. This strategy forms the basis for agrobacterium-mediated transformation. For the iGarden, we modified an existing system graciously provided by The Arabidopsis Information Resource which splits the Ti Plasmid into two parts - one vector to contain the T-DNA, and one to contain the virulence genes. We inserted our completed plant constructs into the T-DNA plasmid, transformed into agrobacteria containing the virulence plasmid, and transformed Arabidopsis via flower-dipping with the construct-containing agrobacteria.

Sources:
"Agrobacterium-mediated transformation - Overview." www.patentlens.net
de la Riva, Gustavo. "Agrobacterium tumefaciens: a natural tool for plant transformation." Electronic Journal of Biotechnology 1.3 (1998)
The Arabidopsis Information Resource www.arabidopsis.org