Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

Abstract

European canker, caused by the necrotrophic fungal phytopathogenNeonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis ofN.ditissimavirulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in theN.ditissima-apple pathosystem has been published to date. In this study, eightN.ditissimagenes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific forN.ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenicN.ditissimaisolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated thatactinandmyo-inositol-1-phosphate synthase(mips), or their combination, could be utilised as the most suitable reference genes for normalisation ofN.ditissimagene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expressionin plantacompared toin vitrowith expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved inN.ditissimapathogenicity and are priority candidates for further functional characterization.