Module 3: Broad Title: Plant Genetic Engineering and Production of Transgenic Plants

Lecture 25: Agrobacterium Mediated Gene Transfer


3. Organization of T-DNA

The transfer DNA (T-DNA) is the transferred DNA of the tumour inducing plasmid (pTi) of some Agrobacterium species of bacteria. T-DNA has both its side 24 kb direct repeat border sequence and contains the gene for tumor / hairy root induction and also for opines biosynthesis (Figure 25.2). pTi has three genes, two of these genes (iaaM and iaaH) encode enzymes which together convert tryptophane in to IAA (Indol-3-acetic acid) a type of auxin. If these two genes are deleted then shooty crown gall will produce. Therefore, the locus was earlier called ‘shooty locus' and the genes were designated as tms 1 (tumour with shoots) and tms 2. The third gene, ipt, encodes an enzyme which produces Zeatin-type cytokinin isopentenyl adenine. The deletion of ipt, causes rooty crown galls and the region was earlier designated as ‘rooty locus' and denoted by tmr (tumour having roots). In addition to these, another locus called tml and the deletion of which results in large tumours. Besides, T-DNA also contains genes involved in opine biosysnthesis which are located near the right border of T-DNA.

Figure 25.2: Nopaline type Ti plasmid T-DNA (Arrows indicating the direction of transcription and                        number indicates the transcriptional unit)

4. T-DNA transfer and integration

The steps involved in T-DNA transfer and integration in to the plant genome are explained in Figure 25.3.

 

Figure 25.3: T-DNA transfer and integration

Wounded plant cell releases phenolics substances and sugars (1); which are sensed by vir A, vir A activates vir G, vir G induces expression of vir gene of Ti-plasmid (2); vir gene produce all the vir -protein (3); vir D1 and vir D2 are involve in ssT-DNA production from Ti-plasmid and its export (4) and (5); the ssT-DNA (with associated vir D1 and vir D2) with vir E2 are exported through transfer apparatus vir B (6); in plant cell, T-DNA coated with vir E2 (7); various plant proteins influence the transfer of T-DNA + vir D1 + vir D2 + vir E2 complex and integration of T-DNA to plant nuclear DNA(8). (LB= left border; RB= Right border; pTi = Ti plasmid, NPC = nuclear pore complex)