Agrobacterium Tumifacience
Compiled by: Hairul Anuar bin Sobikin
Agrobacterium tumefaciens is a ubiquitous soil borne pathogen responsible for Crown Gall disease , affecting many higher species of plant. The pathogen is a problem for agriculture all over the world. DNA transfer from Agrobacterium tumefaciens to eukaryotic cells is the only known example of interkingdom DNA transfer .
A. tumefaciens is most well known for its ability to integrate a small part of the Ti plasmid into the host plant genome, which causes the plant cells to become cancer cells and produce specific compounds called Opines, which the bacterium utilise as a carbon source. This property means that many textbooks class A. Tumefaciens as a genetic parasite, since the bacterium redirects the metabolic activities of the plant to produce compounds specific to the bacterium. It is this process which gives A. Tumefaciens its potential to be used as a tool for plant transformation.
Agrobacterium tumefaciens is a gram negative, motile, rod shaped bacterium which is non sporing, and is closely related to the N-fixing rhizobium bacteria which form root nodules on leguminous plants. The bacterium is surrounded by a small number of peritricious flagella. Virulent bacteria contain one or more large plasmids, one of which carry the genes for tumor induction and is known as the Ti (tumour inducing) plasmid. The Ti plasmid also contains the genes that determine the host range and the symptoms, which the infection will produce. Without this Ti plasmid, the bacterium is described as being non virulent and will not be able to cause disease on the plant
This organism inhabits the soil in many regions of the world. It often congregates around the roots of plants, trees, and shrubs to take advantage of nutrients which may leak from the root system to sustain it. As long as a plant remains healthy, Agrobacterium tumefaciens should not be a problem, as the surface layers of the roots will keep the bacteria out.
The Agrobacterium injects a plasmid (naked circular DNA) into the host (in this case tomato) cells. The idea that the cell cycle could be regulated by chemicals was inspired by the relationship between Agrobacterium tumefaciens (a bacterium) and its host plants. An infection with this bacterium caused a rapidly growing tumor to develop in just about any tissue of suitable host species. This fact indicated that the cells of higher plants are totipotent (capable of becoming meristematic...changing its developmental fate). This tumor was a lump of undifferentiated (having no particular fate) cells. Once infected, the lump could be cured of its infection by either heat shock or by antibiotics and the tumor would continue to grow in a tumor form.
The Agrobacterium injects a plasmid (naked circular DNA) into the host (in this case tomato) cells. This plasmid is called the Ti (Tumor inducing) plasmid. This piece of prokaryotic DNA has two segments of DNA called the "left border" and the "right border" with genes in between. These "borders" permit recombination of the genes into the host genome. The genes turn on cytokinin synthesis!
This, in turn results in the development of a "crown gall" tumor on the plant. The fact that the plant can be cured of the bacteria later and the tumor continues to thrive, simply demonstrates that the Ti DNA has become a permanent addition to the host genome. The bacteria may be killed, but the genes remain in the cells.
This realization of course provided the smart researcher with a useful tool for transforming plant cells with foreign DNA. Anytime a scientist wants to insert an engineered gene into a plant cell, the gene simply has to be put between the borders of the Ti plasmid (probably along with an antibiotic resistance gene for selection purposes) and let the Agrobacterium inject the gene into the cells for incorporation in the genome. Of course we are interested in making a transgenic whole plant, so we cut out the cytokinin synthesis gene and replace it with the gene of our research interest. Since the cytokinin over-production genes are absent, the cells can develop into whole transgenic plants (thanks to totipotency of plant cells!). This process is briefly outlined below.
The relative amounts of cytokinins and auxins can regulate the differentiation that can occur from transformed cells. So when it is time to regenerate whole plants from engineered cells, the relative ratio of these two hormones regulates what develops. Here you can see an array of cytokinin concentrations and auxin concentrations on callus growth. The no hormone control is in the lower left corner.
When a plant is injured, however, it allows the bacteria to enter, setting up an opportunity for the bacteria to colonize and cause crown gall disease. The bacteria themselves are actually not responsible; rather, the tumor is caused by a plasmid produced by the bacteria. This plasmid is known as a tumor inducing (Ti) plasmid, referencing the fact that it carries DNA which will cause tumors to develop. When bacteria are stripped of the Ti plasmid, they are still functional.
For plants infected with Agrobacterium tumefaciens, there may not be much to be done. The plants can be pruned to remove the growth, and measures such as soil sterilization can also help. In some cases, it may be necessary to fully remove plants and their roots and to sterilize the soil to start from scratch. Since it can take several years to bring production up to previous levels, this measure is often avoided, if possible, to cut down on losses caused by infection.
Crown Gall Disease
Crown Gall disease is a common plant pathogen, affecting over 600 types of plants. The disease mostly affects monocotyledonous species, such as woody and herbaceous plants and can be identified by the appearance of tumors or galls of varying size and shape on the lower stem and main roots of the plant. Crown Gall disease can affect many commercially important and valuable crops such as Grapes, Rice and Sugar Beet.
Crown Galls first appear as small, white, soft protrusions, initially found at the base of the plant stem. As the tumors enlarge, the surface takes on a mottled dark brown appearance due to the death and decay of the peripheral cells. The tumor usually appears either as a swelling of the plant tissue, or as a separate mass of tissue close to the plant surface, joined only by a narrow neck of tissue. Tumors can either be soft and spongy and may crumble on touch, or can be hard and appear knobby or knotty. Some tumors can reach up to 30cm in diameter; though 5-10cm is more common. The tumors may rot away in the autumn, only to re-appear again the following spring.
When infected with the bacterium, plants may also become stunted, produce small chlorotic leaves, and are more susceptible to extreme environmental conditions such as winter cold and wind.Plant disease is currently a major problem facing the developed world. Currently as much as 30% of the yearly total production of food crops is lost due to plant disease. At the current time, there is enough food produced to feed the population, but only just. If the predicted population increase over the next 2-3 decades take place, it will be necessary to increase food production to meet demand. As such a large proportion of crops are lost to plant pathogens each year, there is currently much interest in developing strategies to increase plants natural resistance to pathogenic attack. A. tumefaciens can remain dormant in the soil over winter, and can live saprophytically for many years.
The diagram below gives some of the applications for plant transformation technology. 
Usefulness as a gene delivery system
Agrobacterium tumefaciens is seen as such a useful gene delivery system because it is able to carry any gene of interest within the T-complex, and insert the gene into the target plants DNA with a high degree of success. The reason for this is because unlike other mobile genetic elements such as transposons and retroviruses, the T-DNA strand does not encode functions required for movement and integration of the DNA. Therefore the T-DNA strand can be replaced by a gene of interest which will be inserted automatically into the host plant nucleus with a high degree of success and with little human intervention. This process is usually much more efficient than traditional methods of genetic modification. Follow the link below to the Purdue Agricultural Biotechnology Website, to see an animated demonstration of how A. tumefaciens can be used to genetically modify plants.
A. tumefaciens mediated transformation is relatively efficient for many species, and a low copy number of intact, unmonified transgenes are frequently integrated successfully into the plant genome. However, transformation of many crop species has, in the past, been relatively ineficient, although recent advances in transformation technology is set to change that.
There are however a few species of dicotyledonous plants and most species of monocotyledonous which are recalcitrant to transformation by A. tumefaciens. Ke et al. (2001) investigated whether synthesis at a high level of a T-strand DNA intermediate could improve the transformation efficiency of plants. It was found that a mutation in the gene regulator virG & VirGN54D when combined to produce a strain producing high level of T-strand DNA did indeed have a positive effect on the efficiency of transformation.
Despite the many recent developments in the world of plant genetic manipulation, A. tumefaciens still remains a major method of choice for transforming plant cells, despite the development of sophisticated alternative gene transfer methods. Work is still ongoing to try and improve our understanding of the gene transfer mechanism. A number of economically important cereals have now been transformed using A. tumefaciens (Newell, 2000), working alongside other, more traditional gene transfer methods.
Applications of the Technology
Agrobacterium tumefaciens may prove to be the breakthrough needed in order to successfully insert foreign DNA into plant genomes for genetic modification. Bacterial vectors such as Eschericia colihave already been used successfully as vectors in microbiology (Kikkert et al., 1999) ; this same technology can now be applied to the field of botany. Several different plant species have already been successfully transformed, including Lettuce (Curtis, 1995), Rice (Hiei, 1997) and Tomato (Tzfira et al., 2002). This proves that direct gene transfer methods are no longer the only avenue of approach for transforming important crop plants (Newell, 2000). One of the main reasons for favouring transformation by A. tumefaciens is that it allows delivery of a well defined piece of DNA into the plant genome, although the success rate is not 100% (Gheysen et al., 1998). There are however some valid arguments against the validity of A. tumefaciens mediated transformation.Conclusion
Since the identification of Agrobacterium tumefaciens as the causative agent of Crown Gall disease, the interaction of this species with the host plant has been of great fascination to many botanists. However, it was nut until recently when it was apparant just how useful A. tumefaciens could be as a gene delivery system. During the last 15 years, improvements in biotechnology have come a long way since the realisation that plants can be genetically modified to give desirable phoentypic variations. Now that we are able to make transgenic plants, the main questions facing plant scientists are how to regulate gene expression, how can transformation be made more efficient and consistent, and perhaps most importantly, what are the environmental implications of this technology.
One of the main drawbacks of A. tumefaciens is its inability to effectively transform many monocotyledons, although current research by Ke et al. (2001) suggest that genetically engineered "supervirulent" strains may be effective in transforming many different plant species.Important problems facing plant transformation which still remain to be solved include regulation of the DNA integration, and achieving the holy grail of plant transformation technology, that is targeted gene disruption and gene replacement by homologous recombination. Recent reports of efficient targeting in Arabidopsis thaliana suggest that this breakthrough is closer than we might think (Gelvin, 1998).
It seems probable that Agrobacterium mediated transfer techniques will soon be extended to other recalcitrant species of commercially important plants as soon as the methodologies are optimized.
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