Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groundnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.

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Sendai virus (150 haemagglutinating units/10(6) cells) stimulates rat thymocytes incubated in medium containing 5 mM-glucose at 37 degrees C to produce luminol-dependent chemiluminescence and a simultaneous increase in O2 consumption of 40%. Stimulation of thymocytes with Sendai virus is accompanied by reduction of exogenous acetylated ferricytochrome c, which is inhibited by superoxide dismutase, and the quantitative conversion of ferricyanide to ferrocyanide, which is not. Replacement of air in the gas space with N2 inhibits the chemiluminescent response by 97% but does not prevent the virus-stimulated reduction of ferricyanide. The non-permeant ferricyanide anion (2 mM) also inhibits the chemiluminescent response to Sendai virus, its accompanying 'extra' O2 uptake and the reduction of acetylated ferricytochrome c without affecting the basal respiration of the cells. Thymocytes in which the basal O2 consumption has been stimulated maximally with dinitrophenol (10 microM) or inhibited completely with antimycin A (0.1 microM) respond to Sendai virus with an additional increment of ferricyanide-inhibitable O2 consumption. The chemiluminescent response to virus is not inhibited by concentrations of antimycin A that block the basal respiration completely. We suggest that a portion of the increased O2 uptake induced by Sendai virus is involved in the non-mitochondrial reduction of O2 to O2- at the cell surface where the non-permeant ferricyanide anion inhibits O2-. formation by acting as an alternative high-affinity electron acceptor to O2.

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