Development of a Conditional Gene Expression System Using a Zearalenone-Inducible Promoter for the Ascomycete Fungus Gibberella zeae

摘要

The ascomycete fungus Gibberella zeae is an important plant pathogen that causes fusarium head blight on small grains. Molecular studies of this fungus have been performed extensively to uncover the biological mechanisms related to pathogenicity, toxin production, and sexual reproduction. Molecular methods, such as targeted gene deletion, gene overexpression, and gene fusion to green fluorescent protein (GFP), are relatively easy to perform with this fungus; however, conditional expression systems have not been developed. The purpose of this study was to identify a promoter that could be induced by zearalenone (ZEA) for the development of a conditional expression system in G. zeae. Through microarray analysis, we isolated one zearalenone response gene (ZEAR) whose expression was increased more than 50 times after ZEA treatment. Northern blot analysis showed that the ZEAR transcript dramatically increased after 1 h of ZEA treatment. To determine the utility of the ZEAR promoter, called Pzear, in a conditional expression system, we transformed a Pzear::GFP fusion construct into G. zeae. Our data showed a ZEA concentration-dependent increase in GFP expression. We also replaced the promoter of G. zeae metE (GzmetE), an essential gene for methionine biosynthesis, with the Pzear promoter. The growth of the Pzear-GzmetE mutant on minimal medium was dependent on the ZEA concentration supplemented in the medium and showed that GzMetE expression was induced by ZEA. This study is the first report of an inducible promoter in G. zeae. Our system will be useful for the characterization of essential gene functions in this fungus through differential and ZEA-dependent gene expression. In addition, the Pzear promoter may be applicable as a biosensor for the detection of ZEA contamination in agricultural products.