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Plant and Cell Physiology, 1996, Vol. 37, No. 6 847-854
© 1996

Subtractive Hybridization between cDNAs from Untreated and AMO-1618-Treated Cultures of Gibberella fujikuroi1,2

James Christian Jennings3, Jo Ann Banks and Ronald Charles Coolbaugh

Department of Botany & Plant Pathology, Purdue University West Lafayette, Indiana 47907, U.S.A.

The gibberellin (GA) biosynthetic pathway includes four apparent cytochrome P450-mediated steps that convert kaurene to 7{alpha}-hydroxykaurenoic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7{alpha}-hydroxykaurenoic acid, is mediated by kaurenoic acid hydroxylase. This reaction can be catalyzed in vitro by microsomal preparations from the fungus Gibberella fujikuroi (Saw.) Wr. and monitored by HPLC. Cultures grown in the presence of 84 µM AMO-1618 (an inhibitor of kaurene synthesis) had reduced levels of GA3 in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. However, the level of hydroxylase activity from AMO-1618-treated cultures could be induced several-fold by growing cultures in the presence of 350 µM kaurene. Since transcripts related to GA biosynthesis might be decreased in AMO-1618-treated cultures, a subtractive hybridization procedure was used to enrich cDNA fragments corresponding to messages that are more abundant in untreated than treated cultures. A fungal cDNA library was screened with the subtraction products and a clone was isolated that corresponds to two down-regulated transcripts in AMO-1618-treated cultures. This cDNA does not encode a cytochrome P450 but may be associated with GA biosynthesis.

1Supported by DowElanco and USDA/CSRS grant 92-34190-6941.

2Journal Paper No. 14912 of the Purdue University Agricultural Experiment Station.

3Present address: Weed Science Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705, U.S.A.

(Received February 21, 1996; Accepted June 26, 1996)
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