Recent studies showed, that ozone-induced gene expression occurs via at least two different signalling mechanisms that are ethylene-dependent (fi-1,3-glucanases) and ethylene-independent independent (stilbene synthase). To identify transacting factors involved in ozone-induced gene expression we analyzed a 150 bp PCR fragment of an ozone-responsive promoter segment of the grape vine resveratrol synthase gene (Vst1) in combination of a cDNA library prepared from ozone-treated tobacco plants, using the yeast one-hybrid screening system. Two cDNA clones that encode WRKY binding proteins were isolated by this screening technique. The open reading frame of NtWRKY10 and NtWRKYll showed an identity of 93.5 % and the deduced amino acid sequence an identity of 89.3 %. According to the WRKY domain classification in Arabidopsis, both proteins belong to subgroup II. Comparison with known tobacco WRKY proteins indicate that WRKY10 and WRKY11 belong to a new class of tobacco WRKY transcription factors. Electrophoretic mobility shift assays (EMSA) of yeast extracts, containing the WRKY fusion protein showed a weak binding to the radioaclively labelled 150 bp ozone-responsive Vst1 fragment. These results are consistent with an involvement of WRKY proteins in ozone-induced phytoalexin gene expression.
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