Application of Bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin.

Bruchins, mono and bis (3-hydroxypropanoate) esters of long chain alpha,omega-diols, are a recently discovered class of insect elicitors that stimulate cell division and neoplasm formation when applied to pods of peas and certain other legumes. Differential display analysis resulted in the identification of an mRNA whose level was increased by the application of Bruchin B to pea pods. The corresponding amplification product was cloned and sequenced and a full length cDNA sequence was obtained. This cDNA and the gene from which it was derived were assigned the name CYP93C18 based upon sequence similarities to the cytochrome P450 mono-oxygenase CYP93C subfamily, which contains isoflavone synthase genes from legumes. RNA gel blots and quantitative RT-PCR demonstrated that expression of CYP93C18 increased within 8 h of bruchin treatment to a maximum of 100-200-fold of the level in untreated pods, and then declined. The up-regulation of CYP93C18 was followed by an increase in the level of the isoflavone phytoalexin, pisatin. Pisatin was detectable in the bruchin-treated pods after 16 h and reached a maximum between 32 h and 64 h. This, the first report of induction of phytoalexin biosynthesis by an insect elicitor, suggests that Bruchin B not only stimulates neoplasm formation, but also activates other plant defence responses.