Agrobacterium is a genus of bacteria that causes tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for plant improvement by genetic engineering.

Taxonomic note: The Agrobacterium genus is quite heterogeneous. Recent taxonomic studies have reclassified all of the Agrobacterium species in to new genera, such as Ruegeria, Pseudorhodobacter and Stappia, but most species have been reclassified as Rhizobium species.

Agrobacterium as a plant pathogen

The large growths on these roots are galls induced by Agrobacterium sp.

A. tumefaciens causes crown-gall disease in plants. The disease is characterised by a tumour like growth or gall on the infected plant, often at the junction between the root and the shoot. Tumors are incited by the transfer of a DNA (T-DNA) segment from the bacterial tumour-inducing (Ti) plasmid. The closely related species, A. rhizogenes, induces root tumors, and carries the distinct Ri (root-inducing) plasmid. Although the taxonomy of Agrobacterium is currently under revision it can be generalised that 3 biovars exist within the genus, A. tumefaciens or biovar 1, A. rhizogenes or biovar 2, and A. vitis or biovar 3. Strains within biovars 1 and 2 are known to be able to harbour either a Ti or Ri-plasmid, whilst strains of biovar 3, generally restricted to grapevines, can harbour a Ti-plasmid. Non-Agrobacterium strains have been isolated from environmental samples which harbour a Ri-plasmid whilst laboratory studies have shown that non-Agrobacterium strains can also harbour a Ti-plasmid. Many environmental strains of Agrobacterium do not possess either a Ti or Ri-plasmid. These strains are avirulent.

The plasmid T-DNA is integrated semi-randomly into the genome of the host cell (Francis and Spiker, 2005. Plant Journal. 41(3): 464.), and the virulence (vir) genes on the T-DNA are expressed, causing the formation of a gall. The T-DNA carries genes for the biosynthetic enzymes for the production of unusual amino acids, typically octopine or nopaline. It also carries genes for the biosyntheis of the plant hormones, auxin and cytokinins. By altering the hormone balance in the plant cell, the division of those cells cannot be controlled by the plant, and tumors form. The ratio of auxin to cytokinin produced by the tumor genes determines the morphology of the tumor (root-like, disorganized or shoot-like).

Agrobacterium in biotechnology

The ability of Agrobacterium to transfer genes to plants has been exploited for genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes, the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants. A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome (1983).

The genes to be introduced into the plant are cloned into the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.

Agroinfiltration is a method in plant biology to induce transient expression of genes in a plant or to produce a desired protein. In the method a suspension of Agrobacterium is injected into a plant leaf, where it transfers the desired gene to plant cells. The benefit of agroinfiltration when compared to traditional plant transformation is speed and convenience.

First step of the protocol is to introduce a gene of interest to a strain of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a buffer solution. This solution is then placed in a syringe (without a needle). The tip of the syringe is pressed against the underside of a leaf while simultaneosly applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incission made to the underside of the leaf.

Once inside the leaf the Agrobacterium transforms the gene of interest to a portion of the plant cells. The gene is then transiently expressed. The plant can be monitored for a possible effect in the phenotype, subjected to experimental conditions or harvested and used for purification of protein of interest. Many plants can be transformed by this method, but the most common ones are Nicotiana benthamiana and Nicotiana tabacum'.'

Plant (S. chacoense) transformed using Agrobacterium. Transformed cells start forming calluses on the side the leaf pieces

Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in an Agrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance, and plants that have not integrated the plasmid DNA will die.

Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.

See also

• Agroinfiltration
• Marc Van Montagu

External links

• Current taxonomy of Agrobacterium species, and new Rhizobium names