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Agrobacterium-mediated Transformation ConceptBY: Lorato Lekgari | Category: Others | Submitted: 2010-07-15 17:15:12
Agrobacterium, a gram-negative bacterium, is used in plants biotechnology in plant transformation, commonly referred to as Agrobacterium-mediated transformation. This bacteria causes crown gall or hairy roots in plants by transferring a segment of DNA, referred to as the T-DNA region, from the tumor inducing (Ti) or hairy root inducing (Ri) plasmid which the Agrobacterium harbours. Agrobacterium tumefaciens carries the Ti plasmid, whereas the Ri plasmid is found in Agrobacterium rhizogenes. The bacterium infects the plants through an already existing wound on the plant. This result in the cells of the plant, that are infected, in multiplying at a very high rate due to hormones, auxin and cytokinin that the Agrobacterium produces in the host's cells. In biotechnology, in order to engineer a plant that will have desired traits, the bacteria is disarmed by replacing the tumor inducing T-DNA region of the Ti or Ri plasmid with a gene of interest before the bacteria containing this gene of interest is mobilised into the plant cells. By studying the way in which Agrobacterium infects plants, scientists were able to study the way in which the Agrobacterium perceives signals from a wounded plant, resulting on the bacteria moving into the exposed cells of the plant.
The infection of the plant happens in a series of steps and the genes involved in these steps have been identified. Firstly the Agrobacterium attaches to the host cells through bacterium and host cell ligand-receptor interaction that occurs due to phenolics, mainly acetosyringone, which the Agrobacterium perceives. This attachment leads to the activation of the Vir proteins in the bacteria. Virulence (Vir) proteins are the ones responsible for the production of the T-DNA region of the plasmid and its delivery into the host plant's cell. The named Vir proteins are VirA, VirB, VirC, VirD, VirE, VirF and VirG. The first Vir protein that act after attachment is VirA, located on the bacterial transmembrane, to which acetosyringone binds, thus it acts as the first step of signal perception and transduction leading to the transphosphorylation of the VirG protein. This VirA/VirG complex activity activates the other Vir proteins. VirD1/D2 then generate a mobile copy of the T-DNA, which gets cleaved by VirD2 and coated by the single stranded DNA binding protein VirE and then its transported from the bacterium to the host plant's cell by the VirB/VirD4 protein complex. The VirE protein coat protects the T-DNA from being degraded by both the bacterial and the host plant cell's nucleases. After reaching the plant cells, the T-DNA mature complex has to travel to the nucleus, where the host plant's DNA is located. This brings in the action of proteins in the host to mobilise the single stranded mature protein coated T-DNA complex, to the nucleus in the cell.
In the Host Plant's Cell
The plant VirE2-interacting protein (VIP1) and the bacterial functional homolog VirE3, interacts with the VirE2 coating the T-DNA, adapting the VirE2 coating the T-DNA to the host plant's karyopherin alpha involved in the nuclear-import machinery in the host, to be transported to the nucleus. Upon reaching the nucleus, VirF interact with VIP1 and ASK1 (homolog of the yeast SKP1 protein in the SCF box), resulting in proteolytic degradation the coating around the T-DNA so that it can integrate with the host's DNA. After the integration, the T-DNA then becomes a part of the infected cell's DNA, thus the T-DNA is replicated as the plant's genomic DNA divides resulting in multiple copies.
This is true also when the T-DNA is replaced by a gene of interest which will then impart the desired characters to the plant's cells. And as plant cells are able to regenerate into whole plant, expressing the total genetic potential of the parent plant or cell, a concept referred to as totipotency, the cells now with the gene of interest are cultured in growth media to regenerate plants whose genome contain the gene imparted by the Agrobacterium and thus the desired traits. Research to fully understand the function and action of all the proteins involved is still ongoing and with more insight, Agrobacterium-mediated transformation will get even more improved to achieve higher rates of plant transformation.
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