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Background  Real-time RT-PCR has become a common and robust technique to detect and quantify low-abundance mRNA expression and is a prefered tool when examining fungal gene expression in infected host tissues. However, correct evaluation of gene expression data requires accurate and reliable normalization against a reference transcript. Thus, the identification of reference genes with stable expression during different conditions is of paramount importance. Here, we present a study where in vitro and  in planta  experiments were used to validate the expression stability of reference gene candidates of  Puccinia helianthi  Schw . , an obligate pathogen that causes rust in sunflower ( Helianthus annuus ).    Results  Eleven reference genes of  P. helianthi  were validated at different growth stages. Excel-based software geNorm, BestKeeper and NormFinder were used to evaluate the reference gene transcript stabilities. Of eleven reference gene candidates tested, three were stably expressed in urediniospores, germinating growth stage and  in planta . Two of these genes ( UBC ,  EF2 ), encoding ubiquitin-conjugating enzyme and elongation factor 2, proved to be the most stable set of reference genes under the experimental conditions used.    Conclusion  We found that  UBC  and  EF2  are suitable candidates for for the standardization of gene expression studies in the plant pathogen  P. helianthi  and potentially other related pathogens.

Selection of reference genes for quantitative real-time PCR normalization in the plant pathogen Puccinia helianthi Schw.