Agrobacterium
tumefaciens

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Research

Contents

Initial Research
Wood & Braun, (1942)
Kado et al., (1984)
Current Research

Initial Research

As Agrobacterium is such a common plant pathogen, it has been widely studied for many years. Records date back to 1897 when DelDott & Cavara first isolated a bacterium from tumours on infected grape plants. Smith & Townsend, (1907) were the first to discover that plants could be infected using a needle dipped in culture medium. This discovery led to the important conclusion that the bacterium requires a wound site in the plant in order for it to enter and induce a tumorous response. This is the reason why it is present in many soil samples, yet relatively few plants are affected. Jensen (1910), found that he could successfully graft tumours from sugar beet crop onto red beet. The tumours grew in the absence of the bacterium.

Plant tumours don't normally kill the plant, but Crown Gall disease can be fatal to the plant if the tumours become too enlarged. When examined under a microscope, the tumours can be seen to develop very small shoots, and are classified as either Teratomas, or Teratomata.

Wood & Braun, (1942)

• Crown Galls have simple nutrient requirements;
  • Sucrose.
  • Salts.
  • Vitamins.
• Non-transformed tissues require auxins & cytokinins.
• Tumours produce specific compounds calles Opines, using an arginine precursor.
• Opines;
  • Nitrogen base.
  • Contain a Carbon source.
  • Some have a closed ring structure.
  • Several different types, most prevalent;
    • Octapine.
    • Nopaline.
• Opines therefore act as a Carbon and Nitrogen source for the bacterium.

Kado et al., (1984)

• Discovered that Auxin (IAA) concentration is 200-500 times higher in Crown Galls than in ordinary plant tissues.
• Friable, unorganised tumours have;
  • 10 times more IAA than compact tumours.
  • 20 times more IAA than teratoma tumours.
• Nicotiana tabacum Crown Galls were found to contain 1,620 times more cytokinin than non-transformed plant tissues. Also noted that;
  • Accumulation of Potassium and Phosphorous is more rapid in the galls.
  • Octapine gall can utilise lactose as a Carbon source.
• Also found that the shoots produced on the surface of galls will actually grow and produce plants if they are grafted onto another rootstock.

Current Research

A number of scientists are currently interested in being able to combine the best characteristics of A. tumefaciens mediated transformation (high efficiency, low copy number and stable integration) , with the species independant transformation characteristics found whilst using Particle Bombardment.

Work by Hansen & Chilton (1996) has uncovered a novel approach for transformation, termed 'Agrolistic' transformation. This technology utilises three plasmids, two separately containing the restriction proteins VirD1 and VirD2, under control of a CaMV promotor. The third plasmid contained the target DNA, incorporating a Neomycin Phosphotransferase (NPTII) gene. The plasmids were then delivered to tobacco cells using direct DNA uptake methods. The theory behind this method is that if the VirD1 and VirD2 proteins could be expressed in the plant, they could nick the T-DNA at the border sequences, which the plant would then integrate into its own genome. Sequence analysis revealed that NPTII was indeed being expressed in the host cells, but also that the entire plasmid had in some cases been integrated into the plant genome. The authors claimed that this technology could be used to transform any tissue that was successful to transformation by bombardment, and set out to show this by successfully transforming maize (Hansen et al., 1997).

The T-DNA and the virulence (vir) region are two distinct regions of all Ti plasmids which are necessary for plant transformation by A. tumefaciens. The binary Ti vectors (able to replicate in Eschrichia coli and A. Tumefaciens) utilised by A. tumefaciens contain an antibiotic resistance gene for selection, whilst some of the more versatile Ti plasmids contain a series of lacZ restriction sites which allow complementation based screening for recombinant plasmids (Norrander et al., 1983). Hellens et al. (2000), set out to try and design a new binary Ti vector system which would overcome some of the current difficulties related to the current plasmids, such as low recombination frequency, interchangable selector markers and the difficulty experienced in transfering large fragments of DNA.

The new vector developed by Hellens et al. (2000) was termed the pGreen Ti vector, and overcame the above difficulties by having a reduced plasmid size, transformation selection flexibility and an extensive multiple cloning site. The pGreen Ti vector should also be easily adaptable to future improvements in transformation technology. However, the pGreen vector is unable to replicate in Agrobacterium without the helper plasmid, pSoup, being present, thus improving the biological safety of the plasmid by preventing it from replicating throughout the gene pool. More detailed information can be found at the pGreen internet site.

Further interesting discoveries have been made by Tzfira et al. (2002), who found that overexpression of Arabidopsis VIP1 in tobacco plants made them more susceptible to stable genetic transformation by A. tumefaciens, probably due to increased nuclear import of the T-DNA.

Research into the mechanism of transfer between the bacterium and the host cell by Dumas et al., (2001) uncovered much information about the role of the VirE2 protein in forming membrane channels. VirE2 interacts with lipids to form a transmembrane channel with a high conductance value, which has been shown to be specific for ssDNA. However, there are still, at the time of writing, many questions which remain unanswered, such as how the pore can open to allow the T-DNA complex through, yet still retain the integrity of the plant cell.

To date, much of the research taking place has focused on increasing the transformation efficiency, largely by concentrating on factors within the Agrobacterium cell itself. This has been achieved either by intriducing multiple copies of various vir genes, or by optimizing tissue culture and innoculation techniques (Tzfira et al., (2002). This proves that there is still a large scope for improvement of the new technology before we can fully appreciate its potential usefulness as a gene delivery system.