There are many bacteria, fungi and viruses which may infect a plant and cause dis-ease, fungi being one of the major causes of plant disease. Most micro-organisms will not be pathogens for a particular plant as the manifestation of disease requires some quite specific reactions and responses between host and infectious agent. Infection elicits numerous responses some of which may be quite complicated, and so has been the centre of some fairly intensive research. The result of this has been the identification and isolation of plant genes involved in resistance to pathogens and pathogen virulence genes. Manipulation of these to reduce the level of environmentally damaging chemicals to protect crop plants is an active area of research and the manipulation of these genes is discussed in the previouly. This section is restricted to two examples, one bacterium and one virus, which are chosen because of their relevance to plant genetic engineering described. The bacterium is Agrobacterium tumefaciens and the virus is Cauliflower Mosaic Virus.
A feature of infection caused by this Gram positive organism, is a tumour-like growth, seen as a crown gall in plants. This is the consequence of injection by the bacterium of a small piece of DNA which carries on it genes which code for opines which encourage further invasion of the plant by the bacterium, and for plant growth hormones including auxin whose activity stimulates plant growth thus producing the characteristic tumour. It is interesting to note this example of gene transfer from prokaryote to eukaryote occurring with moderate frequency in nature. The genes coding the information required for the insertion of this small piece of DNA including the insertion sequence itself, are carried on a plasmid, called Ti plasmid whose structure is shown in Figure 9.4. The process of infection is stimulated by exudate from a plant which has been wounded by some means not necessarily the result of infection, for example, by frost damage. The vir genes are activated leading to nicks being introduced at the borders of the T-DNA leading to the release of one of the strands of the double-stranded DNA. A copy is made of the remaining strand by the usual methods of DNA synthesis and repair thus restoring the status quo of the Ti plasmid. The single-stranded piece of T-DNA is free to be transferred into the plant cell through a wound site and on into the plant cell nucleus. Here the complementary strand is synthesised in the normal way using the plant cell enzymes and the resulting double-stranded DNA integrates into the plant genome. The T-DNA comprises three genes including those for plant hormones and opines, as mentioned above.
Once the T-DNA is integrated into the host plant cell genome, these genes may be expressed leading to the establishment of crown gall disease. Agrobacteriumtumefaciens then contains a very effective delivery system for bacterial genes intoa plant cell; a natural process which is utilised by genetic engineers to introduce ‘foreign’ genes into plants.
The study of plant viruses has lagged behind that of animal or bacterial viruses due to difficulties in their culture and isolation. While some may be grown in isolated plant cultures, many require the whole plant and some also require an insect intermediate either simply as a means of transmission between plants, or additionally as a site for virus replication. Transfer of viruses between plants by insects is the most common means, especially by insects which penetrate and suck plants, aphids being an example. However, there are other routes such as transfer by plant parts through infected seed, tubers or pollen. Other agents are those able to penetrate plant tissue which include soil nematodes and parasitic fungi. The genetic material of most plant viruses is RNA, either double or single stranded. Caulimovirus, or more commonly, Cauliflower Mosaic Virus is unusual in having a DNA genome. This has proven very fortuitous for genetic engineer-ing as it possesses two very strong constitutive promoters, the 35S and the 19S; for an explanation of the term ‘promoter’. Since these promoters originated from a plant virus, any construct made with the intention of expressing the ‘foreign’ gene in a plant has a higher likelihood of the signals being recog-nised by the transcription machinery of the plant than if they were derived from, for example, bacterial promoters. These promoters have proved very successful and particularly the 35S, often designated in publications as ‘35S CaMV’, has become almost the archetype promoter to drive the expression of ‘foreign’ genes in plants. However, while genetic engineering, building on these and other natural abilities, may permit novel tech-nological approaches to emerge, which have major potential relevance to the environmental sphere, currently they are of somewhat limited commercial appli-cation. At present, there seems to be much more scope, at least in practical terms, for the bundling of existing technologies into treatment trains, or the re-entry of post-processed biological material into the chain of commercial utility.
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