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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The two most abundant transcripts derived from TR‐DNA within plant cells transformed by an octopine strain of Agrobacterium tumefaciens arise from divergent transcription, both originating within an ˜500 bp section of the T‐DNA. Using a combination of subcloning and exonuclease digestion, a 479‐bp DNA fragment, directly flanked by the initiation codons for the two adjacent open reading frames, was isolated. The resulting DNA fragment was fused, in both orientations, to the neomycin phosphotransferase (NPT II) gene of the transposon Tn5 prior to introduction into Nicotiana tabacum cells via the Ti plasmid. The intergenic fragment was found to initiate expression of the NPT II gene in either orientation as assayed by kanamycin resistance of the transformed plant tissue as well as by enzymatic assay of the NPT II gene product. The plasmids described here are potential selection‐expression vectors for plant systems.

SummaryStilbene synthase genes (STS) have previously been used to enhance disease resistance in plants. In order to study the effects of modified STS expression patterns in plants, heterologous promoters were fused to an STS gene and the chimeric genes were transferred to tobacco. Very high constitutive expression of STS mediated by a duplicated upstream region of the 35S RNA promoter from CaMV affected secondary biosynthetic pathways. STS overexpression caused altered flower pigmentation and male sterility, probably due to competition between the introduced STS and the endogenous chalcone synthase for the substrates 4-coumaroyl CoA and malonyl CoA. Furthermore, tobacco plants with tapetum-specific STS expression were male-sterUe. Thus, STS genes are promising tools in strategies for engineering altered flower colours and the development of a novel hybrid seed system.