Agrobacterium Mediated Transformation

Agrobacterium tumefaciens is a soil borne, gram negative bacterium. It causes the crown gall disease in dicots via a unique mechanism of DNA transfer and integration of the transferred DNA (T-DNA) into the host-plant genome. This unique feature has established Agrobacterium tumefaciens as a Nature’s own genetic engineer.

Crown gall formation depends on the presence of a plasmid known as Ti (Tumour inducing) plasmid. Ti plasmids contain one or multiple T-region which is defined by T-DNA border sequences. The border sequences serve as the target for the VirD1/ VirD2 endonuclease and also serve as the attachment site for VirD2 protein. T-DNA is a part of Ti plasmid which is actually transferred from the bacterium into the plant cell where is becomes integrated into the genome of the plant. T-DNA carries genes that encode proteins involved in hormone (auxin and cytokinin) biosynthesis and biosynthesis of novel plant metabolites called opines and agropines. The production of auxin and cytokinin causes the plant cell to proliferate and so form the gall. These proliferating cells also produce opine and agropines which are used by Agrobacterium as its sole carbon and energy source.

T-DNA transfer and Integration

Signal recognition by Agrobacterium

The transformation process begins following the wounding of the plant which causes release of signals such as phenolics and sugars and is perceived by Agrobacterium. These signals activate the bacterial virulence (vir) genes located at the Ti plasmid.  

Attachment to Plant cells

Agrobacterim are chemically attracted to the site of wounding and colonise the wound site. Chromosomal virulence genes chv genes) are involved in the attachment of the bacterial cells to the plant cells. Attachment involves initial attachment via a polysaccharide and then by a mesh of cellulose fibre produced by the bacterium.

Generation of T-DNA transfer complex

A DNA segment (25-bp) is excised from the lower strand of Ti-plasmid by the VirD2/VirD1 endonucleae complex, producing a single stranded T-DNA. VirD2 remains attached to the 5’ end of the T-DNA and guide the T-complex through the bacterial export complex. This immature polar T-complex and several other Vir proteins (like VirE2 VirE3, VirF, and VirD5) are exported into the plant cell cytoplasm via a Type IV secretion system (T4SS), composed of VirD4 proteins and 11 VirB proteins.

Increasing evidence suggests that the VirD2-conjugated T-strand (immature T-complex) and VirE2, along with other effector proteins are exported independently to the host plant cell’s cytoplasm. Once inside the plant cell cytoplasm, mature T-complex formation begins with the association of the VirD2-conjugated T-strand with the VirE2 molecules. The T complex is then imported into the host cell nucleus. The nuclear pore imposes several physical and molecular restrictions on the nuclear import of the T-complex, and the bacteria overcomes these barriers by using the host protein karyopherin α, which interacts with the bacterial VirD2 protein, and leads to directed translocation of the T-complex through the nuclear pore complex(NPC) into the karyoplasm. Though VirE2 is essential for the translocation, it doesnot interact directly with the host nuclear-import machinery. However, it interacts with VirE2-interacting protein 1 (VIP1), which functions as an adaptor between the host nuclear import machinery and the host cell chromatin and VirE2. A recent report shows that VIP1 is a target protein for an Agrobacterium induced MAPK, a possible mechanism by which Agrobacterium induces and uses the defense signaling of the cell to deliver its T-complex into the host cel nucleus.

Inside the nucleus, VirF associates with the T-complex via its binding to VIP1 and recruits the host cell ASK1 and cullin proteins, forming the SCFVirF complex, which activates proteasomal degradation of VIP1 and VirE2, leading to uncoating of the T-strand.

The uncoated T-strand can undergo partial degradation in the nucleus and then becomes converted to a double stranded form. Integration of T-DNA occurs by non-homologous recombination (NHR). In the NHR pathway DNA-protein complex binds to the free double stranded ends of the T-DNA and is directed to DSBs formed in the host genome. The integrating double stranded T-DNA molecules are ligated to the ends of the DSB via the activity of plant DNA ligases.

In this way Agrobacterium uses host cellular mechanisms and pathways for infection. More specifically, the transfer and integration of T-DNA into the host genome requires interaction with the host nucleus at different levels.