2 Genetic modification of plant cells

2.1 Crown gall disease: genetic engineering in nature

A. tumefaciens causes crown gall disease in a wide range of dicotyledonous plants. (Dicotyledonous plants, are also known as dicots, have broad leaves with branching veins. An example would be a broad leaved tree like an oak. Narrow leaved plants with parallel grains such as grasses are known as monocotyledonous plant or monocots.) The infection normally occurs at the site of a wound in the plant. The disease gains its name from the large tumour-like swellings, or galls, that occur on the stem, branches or roots of the plant. (Tumour induction is specific to these plants and is unrelated to gene-induced tumour formation in animals.) The galls often occur at the crown of the plant, the point where the main roots join the stem (Figure 2). During an infection, the bacterium transfers part of its DNA into the plant's cells. The DNA becomes integrated into the plant's genome, causing the production of galls and changes in cell metabolism.

Figure 2: A crown gall on the trunk of an eastern red cedar, Juniperus virginiana. Unusually, in this instance the bacterium has infected a monocot. (The soil has been removed from the roots.)

A. tumefaciens can be modified to allow foreign genes to be incorporated into the genome of plant cells. In order to understand the processes involved, it is important to understand how ‘natural’ infection occurs.

Most of the genes involved in crown gall disease are not borne on the chromosome of A. tumefaciens but on a plasmid, termed the Ti (tumour-inducing) plasmid. A plasmid is a circle of DNA separate from the chromosome, capable of replicating independently in the cell and of being transferred from one bacterial cell to another.

The Ti plasmid is large, between 200 and 800 kb in size. However, a relatively small (12–24 kb) region of the Ti plasmid, called the transfer DNA (T-DNA), is integrated into a host plant chromosome during the infection process. This region is indicated in Figures 3 and 4; it contains the genes coding for both gall formation and for the synthesis of opines. Opines are modified amino acids. They are synthesised by plant cells within the crown gall and provide a source of carbon (and sometimes nitrogen) for A. tumefaciens, but cannot be used by the plant itself. Essentially, the bacteria hijack the biochemical machinery of the plant cells, using them to generate a food source that only it can utilise. You may notice in Figure 4 that the genes encoding bacterial enzymes used in opine catabolism (i.e. its breakdown) are also present in the Ti plasmid, but they are located outside the T-region.

The genes responsible for the transfer of the T-DNA into the host are also located outside the T-DNA region itself. These genes make up the virulence region and they encode proteins that facilitate the transfer of the T-DNA, and its integration into the plant cell's genome.

An overview of the events in crown gall formation is given in Figure 3.

Figure 3: How A. tumefaciens genetically transforms plants. A. tumefaciens contains a tumour-inducing (Ti) plasmid, which contains both virulence (vir) genes and a transfer-DNA (T-DNA) region. The bacterium attaches to a plant cell, and the T-DNA and Vir proteins are transferred to the plant through a transport channel. Inside the plant cell, the Vir proteins promote the integration of the T-DNA into the plant genome.

Figure 4: An enlarged representation of the Ti plasmid. The T-DNA has left and right borders at its extremities and includes genes that produce tumours and opines. Outside the T-DNA is the virulence region. This is a cluster of genes that encode proteins that facilitate the transfer of the T-DNA into the host. The origin of DNA replication (ORI) is a sequence specific to A. tumefaciens at which DNA copying starts, allowing the plasmid to be copied within the bacterium. Three positions, A, B and C, are marked for use in the question in Section 2.2.