Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system

Abstract

CHS15 is one of a family of bean genes encoding chalcone synthase, which catalyzes the first reaction in a branch pathway of phenylpropanoid biosynthesis for the production of flavonoid pigments and UV protectants and isoflavonoid-derived phytoalexins. The functional architecture of the CHS15 promoter was dissected by a novel homologous plant in vitro transcription initiation system in which whole-cell and nuclear extracts from suspension-cultured soybean cells direct accurate and efficient RNA polymerase II-mediated transcription from an immobilized promoter template. Authentic transcription from the CHS15 promoter template was also observed with whole-cell extracts from suspension-cultured cells of bean, tobacco, and the monocot rice, and the soybean whole-cell extract transcribed several other immobilized promoter templates. Hence, this procedure may be of general use in the study of plant gene regulation mechanisms in vitro. Assay of the effects of depletion of the soybean whole-cell extract by preincubation with small regions of the CHS15 promoter or defined cis elements showed that trans factors that bind to G-box (CACGTG, -74 to -69) and H-box (CCTACC, -61 to -56 and -121 to -126) cis elements, respectively, make major contributions to the transcription of the CHS15 promoter in vitro. Both cis element/trans factor interactions in combination are required for maximal activity. Delineation of these functional cis element/trans factor interactions in vitro provides the basis for study of the mechanisms underlying developmental expression of CHS15 in pigmented petal cells established by G-box and H-box combinatorial interactions, and for characterization of the terminal steps of the signal pathway for stress induction of the phytoalexin defense response.