Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA

Many studies of secondary metabolite localization and activity have been motivated in large part by the desire to learn whether or not these substances indeed play a major role in resistance. Evidence has been obtained that at least some secondary metabolites do have such a role. Results of a variety of informative studies will be reviewed. Localization of metabolite accumulation has been investigated by two general strategies: (i) extraction and analysis of metabolite content of thin tissue sections, and (ii) microscopical observation of histochemical staining or autofluorescence. With care, both of these approaches can yield estimates of the metabolite concentrations that contact the pathogen. Antimicrobial activities of the metabolites are assessed by measurement of pathogens' germination or growth in vitro in the presence and absence of the metabolite. A problem in interpreting results of such tests is that sensitivity of the pathogen to the metabolite is probably influenced by the different environmental conditions in planta. However, experimental estimates of secondary metabolite concentrations at infection sites have usually been far in excess of the concentrations needed to arrest pathogen growth in vitro.

Whether secondary metabolites have effective activity during plant-pathogen interactions has in a few cases been tested by genetic modification of plants: biosynthesis of metabolites has been suppressed, biosynthetic flux has been diverted to other products, or biosynthesis has been enhanced by overexpression of genes coding for rate-limiting enzymes. The effects of these alterations in secondary metabolism upon disease resistance have usually, but not always, indicated important roles for the metabolites.

Since some plant host resistance systems are more effective than others, it appears that the defenses of some plant species might be enhanced by metabolic engineering. The potential benefits of engineering plants to produce new metabolites, perhaps with genes from other species, will first be explored by assaying in vitro the activities of new metabolites against the host plants' pathogens. The prospect of engineering foreign metabolites into plants should spur inquiry into the regulation of location and timing of secondary metabolite biosynthesis. The constitutive promoters which have been serviceable for regulation of first-generation transgenic crops will not be adequate for secondary metabolite engineering, since these substances are generally toxic to humans and livestock, and some are also toxic to the plants which produce them. For such non-specifically toxic metabolites, it will be desirable to regulate the transgenes which encode their biosynthesis in ways that will be effective in the recipient plants, but not excessive. If biosynthetic genes are to be moved from one plant species to another, we can anticipate types of information about each species that will be useful. About the donor species, it would be well to learn the compartmentation and timing requirements of enzymes and intermediate metabolites of the biosynthetic pathway. In the recipient species, the transgenes should be regulated to deploy the pathway similarly. The transgenes will also need to be put under control of promoter systems which are responsive to the recipient's own stress or developmental signals. An integrated use of methods of biochemistry, cell biology and molecular genetics will reveal to us the intricate and economical mechanisms by which highly successful plant species defend themselves and may enable us to reconstruct them on the foreign battlegrounds of other plant/pathogen systems.

The authors' research has been supported by the National Science Foundation, the NRI Competitive Grants Program/USDA, and the Oklahoma Agricultural Experiment Station.