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

Abstract

Department of Medical Biotehcnology, Soonchunhyang University, Asan 336-745, Korea(Received on October 25, 2011; Accepted on November 2, 2011)The filamentous fungus Fusarium graminearum is animportant cereal pathogen. Although quantitative real-time PCR (qRT-PCR) is commonly used to analyze theexpression of important fungal genes, no detailedvalidation of reference genes for the normalization ofqRT-PCR data has been performed in this fungus.Here, we evaluated 15 candidate genes as references,including those previously described as housekeepinggenes and those selected from the whole transcriptomesequencing data. By a combination of three statisticalalgorithms (BestKeeper, geNorm, and NormFinder),the variation in the expression of these genes wasassessed under different culture conditions that favoredmycelial growth, sexual development, and trichothecenemycotoxin production. When favoring mycelial growth,GzFLO and GzUBH expression were most stable incomplete medium. Both EF1A and GzRPS16 expressionwere relatively stable under all conditions on carrotagar, including mycelial growth and the subsequentperithecial induction stage. These two genes were alsomost stable during trichothecene production. For thecombined data set, GzUBH and EF1A were selected asthe most stable. Thus, these genes are suitable referencegenes for accurate normalization of qRT-PCR data forgene expression analyses of F. graminearum and otherrelated fungi. Keywords : Fusarium graminearum, gene expression,quantitative real-time PCR, reference genesQuantitative real-time PCR (qRT-PCR) is the most sensi-tive and specific method for quantifying mRNA expressionlevels of individual target genes of interest. qRT-PCRexpression analysis has several advantages over otherconventional methods, but requires data normalization withappropriate reference gene(s) whose expression should beunaffected throughout the study conditions (Anderson etal., 2004; Bustin et al., 2004; Gutierrez et al., 2008). Severalhousekeeping genes involved in basic cellular functions,such as ribosomal genes, and those encoding actin, beta-tubulin (BTUB), translation elongation factor (EF1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) havebeen used as reference genes for qRT-PCR in humans andother model organisms. However, several studies havedemonstrated that some of these traditional housekeepinggenes are unsuitable references because their expressionlevels vary under certain conditions (Czechowski et al.,2005; Dheda et al., 2004; Lee et al., 2002; Suzuki et al.,2000). Therefore, it is necessary to evaluate candidatereference genes for qRT-PCR expression analysis underspecific experimental conditions in the organism to bestudied. The filamentous fungus Fusarium graminearum (telo-morph: Gibberella zeae) is an important cereal pathogen,and produces mycotoxins that are harmful to humans andanimals (Desjardins and Proctor, 2007; McMullen et al.,1997). Complete genome sequencing of F. graminearum(Cuomo et al., 2007) has allowed for genome-wide expre-ssion profiling using DNA microarrays under variousdevelopmental and physiological conditions, such as my-celial growth (Guldener et al., 2006) sexual development(Hallen et al., 2007), germination (Seong et al., 2008) andpathogenesis (Guldener et al., 2006; Stephens et al., 2008).Although the microarray data in these studies were notvalidated by qRT-PCR, it has become the most commonmethod to obtain specific expression patterns of genesidentified from gene expression profiles (Chen et al., 2011;Liu et al., 2011; Pandolfi et al., 2010) or functional studies(Lee et al., 2009; Lysoe et al., 2009). However, theexpression stabilities of the housekeeping genes used asreferences for qRT-PCR analysis in even these studies havenot been properly validated. The objective of this study was to identify F. gramine-arum reference genes suitable for normalization of qRT-PCR data under different conditions such as mycelialgrowth, sexual development, and mycotoxin production.We selected 15 candidate references, including the commonhousekeeping genes used in previous studies (Chen et al.,2011; Lee et al., 2009; Liu et al., 2011; Lysoe et al., 2009;Pandolfi et al., 2010) and a new set of genes selected from