Real-Time Quantitative Expression Studies of the Zearalenone Biosynthetic Gene Cluster in Fusarium graminearum

Son

et al. 2010

Appl Environ Microbiol

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 concentrationdependent 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.

Section: Vol 76 2010 Zearalenone-inducible Promoter 3091mentioning

confidence: 73%

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

Son

et al. 2010

Appl Environ Microbiol

The Plant Pathology Journal

“…In most F. graminearum studies, only single housekeeping gene, such as actin, BTUB, EF1A, EF1B, or 18S rRNA, is used as an internal control without in-depth evaluation (Chen et al, 2011;Lee et al, 2009;Liu et al, 2011;Pandolfi et al, 2010). Lysøe et al (Lysøe et al, 2009) evaluated three housekeeping genes (BTUB, EF1A, and UBC) using BestKeeper, but did not provide detailed data.…”

Section: Discussionmentioning

confidence: 99%

“…Complete genome sequencing of F. graminearum (Cuomo et al, 2007) has allowed for genome-wide expression profiling using DNA microarrays under various developmental and physiological conditions, such as mycelial growth (Güldener et al, 2006) sexual development (Hallen et al, 2007), germination (Seong et al, 2008) and pathogenesis (Güldener et al, 2006;Stephens et al, 2008). Although the microarray data in these studies were not validated by qRT-PCR, it has become the most common method to obtain specific expression patterns of genes identified from gene expression profiles (Chen et al, 2011;Liu et al, 2011;Pandolfi et al, 2010) or functional studies (Lee et al, 2009;Lysøe et al, 2009). However, the expression stabilities of the housekeeping genes used as references for qRT-PCR analysis in even these studies have not been properly validated.…”

mentioning

confidence: 99%

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Evaluation of Potential Reference Genes for Quantitative RT-PCR Analysis in Fusarium graminearum under Different Culture Conditions

Kim¹,

Yun²

2011

The filamentous fungus Fusarium graminearum is an important cereal pathogen. Although quantitative realtime PCR (qRT-PCR) is commonly used to analyze the expression of important fungal genes, no detailed validation of reference genes for the normalization of qRT-PCR data has been performed in this fungus. Here, we evaluated 15 candidate genes as references, including those previously described as housekeeping genes and those selected from the whole transcriptome sequencing data. By a combination of three statistical algorithms (BestKeeper, geNorm, and NormFinder), the variation in the expression of these genes was assessed under different culture conditions that favored mycelial growth, sexual development, and trichothecene mycotoxin production. When favoring mycelial growth, GzFLO and GzUBH expression were most stable in complete medium. Both EF1A and GzRPS16 expression were relatively stable under all conditions on carrot agar, including mycelial growth and the subsequent perithecial induction stage. These two genes were also most stable during trichothecene production. For the combined data set, GzUBH and EF1A were selected as the most stable. Thus, these genes are suitable reference genes for accurate normalization of qRT-PCR data for gene expression analyses of F. graminearum and other related fungi.

“…The molecular structure of ZEA resembles that of the mammalian hormone 17β-estradiol, thus consumption of the toxin by mammals stimulates hypoestrogenic responses and can result in infertility and has also been linked to cancer [4,8]. Limited information about the biological roles of ZEA is available, although it has been speculated that ZEA has functions in binding and activation of the K + channel β subunit, involved in a signal transduction pathway [9]. ZEA has been shown to possess antifungal properties propounding the hypothesis that ZEA is synthesized to increase competitiveness with other fungi inhabiting the same niche [10].…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic profiling to identify genes involved in Fusarium mycotoxin Deoxynivalenol and Zearalenone tolerance in the mycoparasitic fungus Clonostachys rosea

et al. 2014

BackgroundClonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin that exhibits antifungal growth. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades ZEA. However, the molecular basis of resistance to DON in C. rosea is not understood. We have exploited a genome-wide transcriptomic approach to identify genes induced by DON and ZEA in order to investigate the molecular basis of mycotoxin resistance C. rosea.ResultsWe generated DON- and ZEA-induced cDNA libraries based on suppression subtractive hybridization. A total of 443 and 446 sequenced clones (corresponding to 58 and 65 genes) from the DON- and ZEA-induced library, respectively, were analysed. DON-induced transcripts represented genes encoding metabolic enzymes such as cytochrome P450, cytochrome c oxidase and stress response proteins. In contrast, transcripts encoding the ZEA-detoxifying enzyme ZHD101 and those encoding a number of ATP-Binding Cassette (ABC) transporter transcripts were highly frequent in the ZEA-induced library. Subsequent bioinformatics analysis predicted that all transcripts with similarity to ABC transporters could be ascribed to only 2 ABC transporters genes, and phylogenetic analysis of the predicted ABC transporters suggested that they belong to group G (pleiotropic drug transporters) of the fungal ABC transporter gene family. This is the first report suggesting involvement of ABC transporters in ZEA tolerance. Expression patterns of a selected set of DON- and ZEA-induced genes were validated by the use of quantitative RT-PCR after exposure to the toxins. The qRT-PCR results obtained confirm the expression patterns suggested from the EST redundancy data.ConclusionThe present study identifies a number of transcripts encoding proteins that are potentially involved in conferring resistance to DON and ZEA in the mycoparasitic fungus C. rosea. Whilst metabolic readjustment is potentially the key to withstanding DON, the fungus produces ZHD101 to detoxify ZEA and ABC transporters to transport ZEA or its degradation products out from the fungal cell.