Abstract
BackgroundReal-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).ResultsEleven 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.ConclusionWe 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.
Highlights
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
Because of the intrinsic difficulties in genome manipulation in P. helianthi, we focused on gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR)
None of the other primer pairs gave amplicons in the non-infected leaves, confirming the absence of P. helianthi in these controls. This pair of primers cannot be used as reference gene for Real time PCR to study rust gene expression
Summary
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. 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). Sunflower rust caused by the Basidiomycete Puccinia helianthi is one of the most destructive disease in major sunflower producing areas worldwide. It is common and widespread in China, occurs annually on cultivated sunflower (Helianthus annuus L.) and naturalized wild annual species. This obligate biotrophic fungus has several developmental stages varying in form and function, all within the sunflower host. The study of expression patterns of key genes involved in an interaction between P. helianthi and sunflower at the molecular level would help in the breeding of resistant sunflower cultivars
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