History And Morphology Of Agrobacterium Biology Essay

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This review aims to give a comprehensive description of the morphology of the genus Agrobacterium, as well as outlining the milestones that have been reached in determining the characteristics of this unique and fascinating bacterium. Research over the past century has led to the knowledge we have today and means that we have been able to employ the techniques used by Agrobacterium for our own gain. Agrobacterium's ability to instigate horizontal gene transfer between kingdoms means that it has proven invaluable as a vector in genetic studies, allowing the insertion of exogenous genes into a host organism.

The genus Agrobacterium was first proposed in 1942(Conn, 1942) to better classify a group of bacterium which had previously been observed as the cause of gall tumours in plants(Smith and Townsend, 1907). Strong comparisons were drawn between these gall tumours and cancerous tumours seen in the Animalian kingdom. It was soon noted(White and Braun, 1943) that the bacterium did not need to be present for an infection to persist and so research was carried out with the aim of determining the cause of infection in bacteria-free tissue. Evidence was given against the presence of a tumefacient virus, instead it was stated that the tumour cells themselves had become "disease-producing agents"; something later proven to be accurate. The paper's closing statement was that it may be possible to detail the processes by which this cell transformation occurs and use this knowledge in the fight against what the author terms "the cancer problem".

Around the time of the creation of the genus it was noted that the nutrition requirements of the Agrobacterium are very different to that of any other bacterium(Starr, 1946). The ability of certain species of Agrobacterium to survive on nitrogen-rich organic and inorganic sources was proven through extensive experimentation(H. E. Sagen, 1934).

The influences of other factors upon Agrobacterium virility have been the subject of much study. This included studies into soil moisture content(Riker, 1926), temperature(Braun, 1947) and other factors, all of which led to the optimisation of conditions so that the accuracy of further experiments in the field could be enhanced.

One of the reasons for the exceptional virility of Agrobacterium is its ability to stimulate the production of compounds called opines. The first studies into the synthesis of novel compounds for catabolism by crown gall cells were in the mid-50s and identified the breakdown of amino acids(Loiret, 1956), particularly arginine(Morel, 1956), as the source. Opines are utilised by the Agrobacterium as a source of carbon and nitrogen for metabolism, giving them a competitive advantage over other organisms(Dessaux et al., 1988). At the time the compounds were not identified as opines, but since this time over 20 different types of opines have been identified(Dessaux et al., 1988). Gall tumours contain different combinations of these 20 types according to the strain of Agrobacterium and the nature of the Ti plasmid within it (see Dessaux et al., 1988 for references).

Braun in 1947 introduced the concept of the possibility of a "tumour-inducing principle". It was not known at the time what this could be and in 1965 it was proposed that there is some form of physical entity that is transferred to the host to initiate transformation(Kern, 1965). Consequentially much research was conducted into finding out what this was, including the investigation of various sterile bacterial fractions(Gribnau and Veldstra, 1969), growth inducing substances(GALSKY and LIPPINCOTT, 1969), nuclear DNA and substantial work into a viral route of infection. There were a number of hybridization studies that proved that gall tumour formation was initiated by the transfer of genes from Agrobacterium to host (Milo and Sahai Srivastava, 1969, Schilperoort et al., 1967), however the exacting nature of the Ti principle was not determined until the mid-70s when a section of extra-nuclear DNA called a Ti plasmid(Zaenen et al., 1974, Watson et al., 1975) was proposed.

Agrobacterium's potential for use in genetic studies was uncovered in 1983. Schell and his team had previously noted the ability of Agrobacterium to act as a natural vector for the transgenesis of hosts(Schell et al., 1979) and so proposed and tested the ability of species in the genus to transfer genes of choice(Schell et al., 1983). Others in the field had also noted the potential of the Ti plasmid as a vector, however Schell was the first to use it successfully; without destroying the host cells' capacity to regenerate.

host range

Extensive recent studies have been conducted in order to investigate the range of hosts available to the Agrobacterim. One of the most prominent of these was conducted on behalf of the New York Botanical Garden(Cleene and Ley, 1976) and featured a comprehensive list of 643 species found to be susceptible to Agrobacterial infection; this was out of 1193 total tested. This included not only dicotyledonous plants, previously stated by some to be the only domain susceptible, but also some monocot species. They did not find that any fungi or other cell types were susceptible, however much more recent research has found that many more cell types can be at risk in the correct conditions. It has been suggested that transfer of T-DNA into non-plant species could be possible due to similarities between the mechanism by which Agrobacterium transforms a host and intracellular pathogens present in the mammals Brucella spp. and Legionella pneumophila(Christie, 2004); a type IV secretion system. Agrobacterium have also been found able to transform species such as yeast(Bundock et al., 1995), fungi(de Groot et al., 1998), and even cultured HeLa human cells(Kunik et al., 2001).

An explanation that has been offered for the comparatively lower susceptibility of monocots is a difference in the cell membrane proteins; attachment and transfer are both prevented(Zupan and Zambryski, 1995). Some monocot's may also have inhibitors which make cells less susceptible to both attachment and transfer(Sahi et al., 1990).


Vir gene identification/classification

Binary vs cointegrate? Flavour-saver?

Plant Wound Reactions

Before the Agrobacterium can undergo transformation of a host it must first identify one that is both wounded and suitable. When a plant is wounded in some way it exudes a myriad of compounds(Smith, 1911), many of which identify it to the Agrobacterium as a suitable host for infection. Two of these signal molecules, acetosyringone and a-hydroxyacetosyringone, are phenolics have been identified to specifically activate the expression of the entire vir region of the plant at high concentrations and cause chemoattraction at low concentrations(Shaw et al., 1986). They hereby facilitate the recognition of susceptible cells(Stachel et al., 1985). It has been suggested that the synthesis of these molecules occurs by the degradation and/or repair of cell wall-associated lignin(Baron and Zambryski, 1995) and is coded for in the Ti plasmid(Ashby et al., 1987); a seemingly logical proposition.

Braun in 1948 stated that there is a "window of competence" in host plants: a time period in which young plants are most susceptible to infection. More recent research has explained this by stating that T-DNA integration occurs at the point of cell division (Binns and Thomashow, 1988) or during crown gall tumorigenesis(Morel G., 1970), the former of which occurs at a greater rate in young plants.

Flavonoid inducers have also been identified as wounding signal molecules. They are known to be the instigator of symbiotic relationships between legumes and Rhizobia but could also aid Agrobacterial infection(Redmond JW, 1986).

As well as exudate release, the wounding event also triggers the expression of a large number of defense-related genes (Lamb, 1987). These include genes coding for coding for phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase(Lamb, 1987), chitinase(Hedrick et al., 1988), and hydroxyproline-rich glycoproteins(Melan et al., 1993). FUNCTION OF THESE?

An additional but crucial hormone released by plant wounding is jasmonic acid and its derivative methyl jasmonate. This hormone plays a vital role in plant growth and development (Farmer, 1994, Sembdner and Parthier, 1993), as well as wounding; levels are significantly increased post-wounding(Albrecht et al., 1993). Research has also been conducted by the exogenous addition of jasmonates(Farmer et al., 1992). This was found to trigger expression of defense related proteins and subsequent accumulation of secondary metabolites(Gundlach et al., 1992); a mimicry of the wounding reaction and so detectable by Agrobacterium.

Host-Agrobacterium interactions

Following the recognition of a suitable host by the mechanisms described Agrobacterium must travel to the host and undergo T-DNA transfer. As previously mentioned, Agrobacterium travel up the concentration gradient of two specific phenolics towards a host; proven by the creation of a mutant deficient in chemotaxis to root exudates(Hawes, 1988). They also, however, are proven to be attracted to carbohydrates(Loake et al., 1988) by a highly sensitive system which is stated to suggest a role in Agrobacterium ecology.

Chemotaxis (Mao et al., 2003) is the mechanism by which Agrobacterium can travel to the host for cell to cell contact and DNA transfer(Shaw, 1991), driven by attraction to wound exudates. When the concentration of phenolics becomes sufficiently high the genes which code for a membrane sensor protein (VirA) and a cytoplasmic response regulator protein (VirG) are specifically activated. It has also been shown that, in low concentrations of acetosyringone, monosaccharides such as galactose or glucose can also induce expression(Cangelosi et al., 1990). As well as detection of phenolics, these two genes also cause activation of the remaining Vir genes(Fuqua and Winans, 1994), leading to infection.

After the Agrobacterium has travelled towards a potential host it must attach to a cell at the wound site(Lippincott and Lippincott, 1969), which occurs at a similar time to the activation of the virulence region. Cellulose filaments are synthesized by Agrobacterium, binding it to the host(Matthyssc, 1986). Genes for attachment such as chvA, chvB, pscA and att are located chromosomally(Ziemienowicz, 2001).

genes of the plasmid

The Ti plasmid contains the majority of genes required by the Agrobacterium for the tumorigenesis and transformation of a suitable plant host. The plasmid has been mapped into a number of different regions, the most prolific of which is the virulence region, containing at least 6 operons(Schrammeijer et al., 2000) - 8 according to some academics(Vogel and Das, 1992). The operons are VirABCDEFGH, although VirF and VirH are omitted by some. Vir's A, B, D and G have been shown by testing of their mutants to be vital for host transformation, with the remaining genes that were tested (virC and virE) shown to increase the efficiency of T-DNA transfer(Stachel and Nester, 1986). Each of the Vir operons has a number of polypeptides that are encoded within, for example VirD codes for polypeptides VirD1 to VirD5.

Also located externally of the T-DNA are genes coding for T-DNA transfer, conjugation of genes between bacterium, as well as opine catabolism(Zupan and Zambryski, 1995). There is also an Origin of Replication which is the site at which replication can be initiated.

The T-DNA (transfer-DNA) is the section of the plasmid which is transferred into the host cell nucleus and causes transformation of the cell. This region contains two types of gene, both of which are expressed by the plant host. The first type of gene codes for tumour formation, as well as the production of auxin and cytokinins. The second type code for opine synthesis by gall tumour cells.



One of the main reasons for the high virulence of the Agrobacterium is its ability to conjugate: transfer DNA between individuals. In the late 60s Kerr developed a technique of transferring oncogenicity from virulent to avirulent strains, although it was not until 6 years later that it was uncovered that this occurred by the Ti plasmids(Watson et al., 1975). Further work around this time confirmed this, and also showed that the opines octopine and nopaline can induce this (Genetello et al., 1977). More recently it has been suggested that this could play a key role in cell density sensing(Fuqua and Winans, 1994). This latter paper further investigated the role of Ti-plasmid based genes in conjugation, finding that octopine stimulates the secretion of Agrobacterium autoinducer (AAI) by gene donors; proven by the effect of its exogenous addition.

t-dna: tracking the journey

A T-complex is created…

Once the Agrobacterium has attached to the host cell the passage of the T-DNA from the Ti plasmid to its integration into the genome of the plant cell can be assigned to two steps(Tinland, 1996). The first step is a bacterial cell step and involves the preparation of the T-DNA for exportation into the plant; formation of a T-complex. This T-complex consists of a single strand of T-DNA with a VirD2 protein at the 5' end and a coating of VirE2 proteins(Howard and Citovsky, 1990). The VirE2 protein coat envelopes the T-DNA and prevents degradation once outside of the bacterial cell.

…transferred from bacteria to plant cell…

A type IV secretion mechanism is then created by 11 different polypeptides coded for by the VirB gene(Salmond, 1994) as well as virD4. The mechanism consists of a filamentous pilus first proposed in 1996, although it was unclear at the time whether or not the T-complex was larger than the lumen of the pillus thus preventing transfer through it. It was later shown (Fullner et al., 1996) that the pilus, coded for by virB2, was directly involved in transfer, along with a transporter complex. The main role of the pilus is to sense the contact and binding to a plant cell and so initate the transporter complex.

…transported into the plant cell nucleus…

The second step involved in T-complex transfer occurs in the plant cell and concerns movement of the T-DNA into the plant's nucleus. In order for the T-complex to pass into the cell nuclear pores they must enlarge as the complex is usually larger(Citovsky et al., 1997) than the pores through which it must pass(Forbes, 1992). The VirD2 protein is an endonuclease which acts to guide the T-complex through nuclear pores in the plant cell as well as it's much earlier role of cutting the T-DNA from the 24bp border(Barker et al., 1983) sequence to initiate T-strand synthesis, aided by VirD1.

Once inside the plant cell the T-complex (ssDNA, virE2 and virD2) forms a helical structure (Citovsky et al., 1997). Its transport into the nucleus is believed to be instigated by the attached proteins rather than the ssDNA; it is possible to remove and replace the ssDNA and still observe full integration. Nuclear localization signals (NLSs) and importins are contained within the virE2 and virD2 proteins are vital for import into the nucleus, particularly the C-terminal NLS on the virD2(Rossi et al., 1993). It has been proposed that the virE2 protein has a very different role. Rather than coding for a specific protein or allowing creation of a complex it may simply change the shape of the previously unstructured ssDNA allowing its passage through the nuclear pore. There have also been indicators given that the classic importin-based import pathway may be important(Ziemienowicz, 2001).

…and finally integrated into the plant DNA.

This final step of the journey of T-DNA from plasmid to plant is probably the most elusive. It is known that illegitimate integration occurs(Gheysen et al., 1991), however knowledge of the details of the mechanisms therein is largely speculative. Modern research conducted into this area has largely been centred upon the role of the VirD2 protein; a protein of the virulence region of T-DNA proposed to act as a DNA ligase.

Work has also been done into the targeting of the T-DNA into the plant genome. It was suggested by a number of studies that the exogenous DNA was targeted to gene-rich euchromatic regions of the genome, however a serious problem with the experimental technique used in these studies has been noted and so the research repeated


Vir gene expression (see plan).

Functions of the different vir's.