Journal Article

Subtractive Hybridization between cDNAs from Untreated and AMO-1618-Treated Cultures of <i>Gibberella fujikuroi</i><sup>1</sup><sup>2</sup>

James Christian Jennings, Jo Ann Banks and Ronald Charles Coolbaugh

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 37, issue 6, pages 847-854
Published in print September 1996 | ISSN: 0032-0781
e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029021
Subtractive Hybridization between cDNAs from Untreated and AMO-1618-Treated Cultures of Gibberella fujikuroi12

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The gibberellin (GA) biosynthetic pathway includes four apparent cytochrome P450-mediated steps that convert kaurene to 7α-hydroxykaurenoic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7α-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.

Keywords: AMO-1618; Cytochrome P450; Gibberella fujikuroi; Gibberellin biosynthesis; Kaurenoic acid

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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