Agrobacterium- A Natural Genetic Engineer

Agrobacterium tumefaciens & Agrobacterium rhizogenes are plant pathogen, gram negative bacteria. First evidence indicating these bacteria as causative agent of the crown gall goes back to 1907 reported by Smith & Townsend. Virulent strains of A. tumefaciens & A. rhizogenes when interact with susceptible dicotyledonous plant cells, induces diseases known as crown gall & hairy roots, respectively. These contain a large megaplasmid (more than 200kb) which plays a key role in tumor & hairy root, for this reason it was named Ti plasmid or Ri plasmid.

The part of this plasmid which is transferred to plant genome is called T-DNA and is flanked by 25bp sequences. Any DNA sequence placed between these flanking sequences will be transferred to the plant genome and because of this reason it is used as vector. This vector is very efficient and natural way of transformation but is limited to plants

There is chv E gene in the genome of the bacteria which is responsible for the sugar enhancement of vir gene induction & bacterial chemotaxis

A tumefaciens T-DNA transfer process

The process of gene transfer from A. tumefaciens to the plant genome involves several important steps, these steps are:

        1) Colonization of Bacteria

This is the first & essential step in tumor or root induction & it takes place when Agrobacterium is attached to the plant cell surface. A chromosomal 20kb att locus contains the genes required for bacterium attachment to the plant cell & is involved in cell surface protein

       2) Induction of bacterial virulence system 

The T-DNA transfer is mediated by products encoded by the 30-40kb vir region of the Ti plasmid. This region is composed by at least six essential operons (vir A, vir B, vir C, vir D, vir E, vir G) & two non- essential (vir F, vir H);vir A, vir G & vir F have only one gene; vir E, vir C & vir H have two genes; vir D has four genes; vir B has eleven genes.

virA & vir G are responsible for activating the transcription of the other vir genes. They code for Vir A & Vir G protein and this together is called “Vir A-Vir G” system. 

Vir A is sensor protein that detects signal molecules, mainly small phenolic compounds released from wounded plants. The signals for Vir A activation include acidic pH, phenolic compounds such as acetosyringone & certain class of monosaccharides which acts synergistically with phenolic compounds. 

Activated Vir A has the capacity to transfer its phosphate to a conserved aspartate residue of the cytoplasmic DNA binding protein Vir G. The activation of vir system also depends on external factors like temperature & pH. 

      3) Formation of T-DNA transfer complex 

Activation of vir gene produces the generation of single-stranded(ss) molecules representing the copy of bottom T-DNA strand. Any DNA placed between T-DNA borders will be transferred to the plant cells as single strand DNA & integrated into the plant genome. 

The protein Vir D1 & Vir D2 recognizes the T-DNA border sequences & nick (endonucleases activity), after endonucleotic cleavage, Vir D2 remains covalently attached to the 5’- end of the ss-T-DNA strand. There is enhancer sequence next to the right border which is recognized by Vir C1 protein. 

     4)T-DNA transfer 

T-DNA must pass through plant cell wall & cellular spaces. The ssT-DNA –Vir D2 complex is coated by 69KaD Vir E2 protein, which is a single strand DNA binding protein and contains two nuclear location signals which helps in integration of T-DNA into plant genome. ssT-DNA-Vir D2 coated with Vir E2 protein, this whole complex or association prevents the attack of nucleases & , in addition extends the ssT-DNA strand reducing its diameter to approximately 2nm, making the translocation easier. 

The protein Vir E2  contains two plant nuclear location signals and Vir D2 has one. Vir E1 also helps in export of “T-DNA –VirD2-VirE2” complex with help of Vir D4. Vir B operons helps in translocation of T-DNa by making T-DNA transfer apparatus which is similar to conjugation system. Vir B2 & Vir B11 are essential for DNA transfer apparatus while B1 has a lesser contribution to this process. 

Two accessory vir operons are vir F & vir H. The role of virF has been identied as assistance in nuclear targeting, vir H operon consist of two genes that code for Vir H1 & Vir H2 protein. These Vir protein are not essential but could enhance the transfer efficiency, detoxifying contain plant compounds that can effect bacterial growth. 

    5)Integration of T-DNA into plant genome