2.2 Using A. tumefaciens to genetically modify plant cells
Genetic engineers have capitalised on the fact that part of the DNA from the Ti plasmid of A. tumefaciens is integrated into the plant genome during the infection process. Ti plasmids can be isolated and a foreign gene spliced in at an appropriate point, making it possible to transfer the novel gene into the plant.
In Figure 4, three positions on the Ti plasmid are marked by letters A, B and C. Which would be the best place to insert the foreign gene?
Position B is evidently no use since this part of the plasmid is not transferred during infection. Position A looks attractive but although the genes in this region facilitate the gene transfer they are not themselves transferred. This leaves position C; only the T-DNA is integrated into the plant genome, so the foreign gene would need to be inserted somewhere in this region.
The principle underlying the use of the Ti plasmid as a vector for plant transformation is that any gene placed between the left and right border sequences (i.e. within the T-DNA region, see Figure 4) will be transferred into the infected plant cell. However the Ti plasmid is rather large and, as such, difficult to manipulate. Special procedures have been devised that allow the use of a much smaller 'artificial' Ti plasmids. We will describe one such procedure, the binary vector system.
An artificial Ti plasmid (Figure 5a) is generated that contains the gene we wish to transfer and a plant selectable marker gene (such as one for resistance to the antibiotic kanamycin) between the left and right borders from the T-DNA region. We will return to the role of the kanamycin resistance gene below, but what you need to know here is that its purpose is to allow us to detect whether plant cells have taken up the foreign gene.
The commonly accepted definition of an antibiotic is that it is a chemical which kills or inhibits the growth of bacteria. You are probably familiar with the use of antibiotics to treat bacterial infections. A more precise definition would be that antibiotics are substances produced naturally by various organisms (usually bacteria, fungi or plants) in order to limit the growth of, or kill other organisms. The organisms affected are usually, but not always, bacteria. Some antibiotics, like kanamycin, are toxic to plant cells; other antibiotics, like streptomycin are not. In fact streptomycin is used to minimise losses from certain bacterial diseases of apples and pears.
It is important that the artificial Ti plasmid contains origins of replication so that it can be copied both in A. tumefaciens and in E. coli. Most of the manipulations required to modify the Ti plasmid are carried out in E. coli as indicated in Figure 5.
What features of a normal Ti plasmid (Figure 4) are missing from the artificial Ti plasmid (Figure 5a)?
The tumour-producing genes, the virulence region and the genes coding for opine synthesis and catabolism.
Which of these features is essential to allow transfer and integration of the genes in the T-DNA region?
Only the virulence region is necessary. The tumour-producing genes and the genes related to opine synthesis and catabolism are not required. We do not want the modified plants to produce galls, or for the transgenic plants to synthesise opines - it would utilise valuable resources.
So, having constructed the artificial Ti plasmid, we now need a technique that allows the features of the virulence region to be present in the A. tumefaciens. Using the binary vector system, this problem is solved by including the virulence region (vir) in a second plasmid. This is called a disarmed Ti plasmid (see Figure 5b) because the entire T-region has been removed. This is often referred to as a helper vector and we will use this simpler term.
The artificial Ti plasmid is transferred from E. coli to A. tumefaciens containing a helper vector (Figure 5b) via a process known as conjugation - a term used to describe direct transfer of genetic material from one bacterium to another.
The modified A. tumefaciens containing both the artificial Ti plasmid and the helper vector is then used to infect the target plant cells. On infection, the virulence genes are activated and the DNA between the left- and right-hand borders of the artificial Ti plasmid is transferred to a plant chromosome. The full process is summarised in Figure 5.