SUBTRACTIVE HYBRIDIZATION BETWEEN CDNAS FROM UNTREATED AND AMO-1618-TREATED CULTURES OF GIBBERELLA-FUJIKUROI

The gibberellin (GA) biosynthetic pathway includes four apparent cytoc hrome P450-mediated steps that convert kaurene to 7 alpha-hydroxykaure noic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7 alpha-hydroxykaurenoic acid, is mediated by kaurenoic acid hydro xylase. This reaction can be catalyzed in vitro by microsomal preparat ions from the fungus Gibberella fujikuroi (Saw.) Wr. and monitored by HPLC. Cultures grown in the presence of 84 mu M AMO-1618 (an inhibitor of kaurene synthesis) had reduced levels of GA(3) in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. However, the level of hydroxylase activity from AMO-1618-treated cultures coul d be induced several-fold by growing cultures in the presence of 350 m u M kaurene. Since transcripts related to GA biosynthesis might be dec reased in AMO-1618-treated cultures, a subtractive hybridization proce dure was used to enrich cDNA fragments corresponding to messages that are more abundant in untreated than treated cultures. A fungal cDNA li brary was screened with the subtraction products and a clone was isola ted that corresponds to two down-regulated transcripts in AMO-1618-tre ated cultures. This cDNA does not encode a cytochrome P450 but may be associated with GA biosynthesis.