Mechanism of T-DNA to transfer Short Notes | Biotechnology

In this article we will discuss about:- 1. What Is T-DNA? 2. Features of Ti Plasmid 3. Mechanism of T-DNA to transfer 4. Effcts of T-DNA on The Plant and 5. Application of T-DNA

1. What Is T-DNA?

• T DNA stands for transfer DNA 
• It derives its name from the fact that the bacterium transfers this DNA fragment into the host plant's nuclear DNA genome. 
• It is a part of Ti plasmid (Tumor inducer plasmid) of the Agrobacterium tumefaciens
• Agrobacterium tumefaciens is a phytopathogenic, gram- negative, non sporing, motile bacillus.
• It is found in the rhizosphere
• It causes crown gall disease in plants, a disease of dicotyledonous plants characterized by a tumorous phenotype.

2. Features of Ti Plasmid:

• It is responsible for crown gall disease 
• It has 200 kb and It has 8 genes (vir, shi, roi, nos, noc, ocs, occ and ori V) 
• A part of Ti-plasmid, T DNA, integrates into the plant chromosomal DNA 
• The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

3. Mechanism of T-DNA to transfer:

• A.Tumefaciens is attracted towards wound site by chemotaxis  The wound induces the production of phenolic compounds such as acetosyringone  The lipopolysaccharides aid in attachment of A. Tumefaciens.
• Ti plasmid code for chemotactic receptors. The processing of T-DNA transfer is mediated by the vir (virulence) region. The vir gene encode a set of proteins responsible for the excision, transfer and integration of the T-DNA into the plant nuclear genome: VirB, VirC, VirD, VirE. Vir A and vir G proteins constitute a two component regulatory system that controls vir gene expression. VirA detects the phenolic compounds, acetosyringone, resulting in autophosphorylation.
• The autophosphorylated VirA phosphorylates VirG which leads to the activation of vir gene and this one get expressed.  After vir gene activation, T strand, a single stranded copy of T DNA is produced: 
• Two VirD proteins, i.e VirD1 &VirD2, recognize the 25 bp border sequence and produce a ss endonucleolytic cleavage in the bottom end of each border These nicks are used as initiation and termination sites for T strand production 
• VirC locus then produces 2 proteins, VirC1 and VirC2 which promotes generation of multiple copies per cell of T strand. 

• Then VirD1 is detached from T strand whereas VirD2 remains tightly bound on the 5’end to ensure that in subsequent steps, the 5’ end is the leading end.  To prevent T strand degradation by endonucleases during its journey to the plant nucleus, VirE locus produce VirE2 ss nucleic acid-binding protein
• It binds tightly on T-strand which results in the unfolding of the ssDNA, i.e T-strand, thus reducing its diameter to 2nm facilitating its transfer through membrane channels.  The T-strand along with VirD2 & VirE2 is called as T complex 
• The T complex exit the bacterial cell membranes and cross the plant cell membranes and enter the plant cell.  Once inside the cell, T complex targets the plant nucleus and cross the nuclear membrane where it integrates into the plant DNA. The transport of T Complex is mediated by VirB-VirD4 transporter complex. 
• Once inside the plant, T complex is led by VirD2 and VirE2 interacting proteins along with VirF towards the nucleus. 
• In the nucleus, VirF with the interacting proteins unbind from the t complex. Then the right border initiates integration of tDNA region while VirD2 and VirE2 are unbinding from the T strand.

4. Effcts of T-DNA on The Plant

• T-DNA disorganize the proliferation of cells in the plant 
• It reprograms the cell to grow into a tumor and produce a unique source food for the bacteria 
• The synthesis of the plant hormones auxin and cytokinin enables the plant cell to grow unc ontrollably, thus forming the crown gall tumors typically. 
• The morphology of the galls is controlled by the loci in T-DNA: 
• Tml causes large tumors  Tmr induces tumor in roots 
• Tms induces tumor in shoots

5. Application of T-DNA

T-DNA transfer capabilities are used in genetic engineering to introduce foreign genes into desired plants: the tumor-promoting and opine- synthesis genes of T-DNA are removed and replaced by genes of interest. Such plants are then called transgenic plants.