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Agrobacterium
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| Agrobacterium tumefaciens
(Smith and Townsend 1907) Conn 1942
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Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause 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 genetic engineering.
The genus Agrobacterium is quite heterogeneous. Recent taxonomic studies have reclassified all of the Agrobacterium species into new genera, such as Ahrensia, Pseudorhodobacter, Ruegeria, and Stappia, but most species have been controversially reclassified as Rhizobium species.
Plant pathogen
The large growths on these roots are galls induced by Agrobacterium sp.
Agrobacterium 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 conjugative transfer of a DNA segment (T-DNA) from the bacterial tumour-inducing (Ti) plasmid. The closely related species, Agrobacterium 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, Agrobacterium tumefaciens, Agrobacterium rhizogenes, and Agrobacterium vitis. Strains within Agrobacterium tumefaciens and Agrobacterium rhizogenes are known to be able to harbour either a Ti or Ri-plasmid, whilst strains of Agrobacterium vitis, 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. Some environmental strains of Agrobacterium possess neither a Ti nor Ri-plasmid. These strains are avirulent.
The plasmid T-DNA is integrated semi-randomly into the genome of the host cell,
and the tumor morphology 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 biosynthesis of the plant hormones, auxin and cytokinins, and for the biosynthesis of opines, providing a carbon and nitrogen source for the bacteria that most other micro-organisms can't use, giving Agrobacterium a selective advantage.
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).
In humans
Although generally seen as an infection in plants, Agrobacterium can be responsible for opportunistic infections in humans with weakened immune systems,
but has not been shown to be a primary pathogen in otherwise healthy
individuals. One of the earliest associations of human disease caused by
Agrobacterium radiobacter was reported by Dr. J. R. Cain in Scotland (1988). A later study suggested that Agrobacterium
attaches to and genetically transforms several types of human cells by
integrating its T-DNA into the human cell genome. The study was
conducted using cultured human tissue and did not draw any conclusions
regarding related biological activity in nature.
Uses in biotechnology
The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, 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. The genes to be introduced into the plant are cloned into a plant transformation vector that contains 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.
Plant (S. chacoense) transformed using Agrobacterium. Transformed cells start forming calluses on the side of the leaf pieces
Transformation with Agrobacterium can be achieved in multiple ways. Protoplasts or alternatively leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. In agroinfiltration the Agrobacterium
may be injected directly into the leaf tissue of a plant. This method
transforms only cells in immediate contact with the bacteria, and
results in transient expression of plasmid DNA.
Agroinfiltration is commonly used to transform tobacco (Nicotiana). A common transformation protocol for Arabidopsis is the floral dip method: inflorescence are dipped in a suspension of Agrobacterium, and the bacterium transforms the germline cells that make the female gametes. The seeds
can then be screened for antibiotic resistance (or another marker of
interest), and plants that have not integrated the plasmid DNA will die
when exposed to the correct condition of antibiotic.
Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.
Agrobacterium is listed as being the vector of genetic material that was transferred to these USA GMOs:
- Soybean
- Cotton
- Corn
- Sugar Beet
- Alfalfa
- Wheat
- Rapeseed Oil (Canola)
- Creeping bentgrass (for animal feed)
- Rice (Golden Rice)
Genomics
The sequencing of the genomes of several species of Agrobacterium has permitted the study of the evolutionary history of these organisms and has provided information on the genes and systems involved in pathogenesis, biological control and symbiosis. One important finding is the possibility that chromosomes are evolving from plasmids
in many of these bacteria. Another discovery is that the diverse
chromosomal structures in this group appear to be capable of supporting
both symbiotic and pathogenic lifestyles. The availability of the genome
sequences of Agrobacterium species will continue to increase,
resulting in substantial insights into the function and evolutionary
history of this group of plant-associated microbes.
History
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).
