Abstract
European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence 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 the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes 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 for N. 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 pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene 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 expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.
Highlights
Conventional end-point PCR demonstrated that the primer sets for amplification of the actin, mips, S8 and 18s ribosomal RNA adenine methylase transferase (18sAMT) sequences were specific and generated amplification products of the expected size only when template from N. ditissima was used (Fig 1 and S1 Fig)
RT-qPCR using the validated reference genes will enable a finer dissection of the temporal expression patterns of candidate genes in future studies to enable prioritisation of targets for functional characterisation
Gene expression analysis of three N. ditissima candidate virulence genes (g4542, g5809 and g7123) provided evidence of significant up-regulation during apple infection, making them good candidates for further functional characterisation to elucidate their role in N. ditissima pathogenicity
Summary
Over time a canker develops that can girdle the trunk or branch, causing the death of any distal shoots [7]. This disease occurs predominantly during wet seasons when dispersal of ascospores and conidia, and infection in orchards is facilitated [8]. Ascospores, produced in red perithecia, can be observed within a year after initial canker formation. These two-celled spores can be expelled from the perithecium and wind-dispersed, or exuded as a white-cream sticky mass and splash dispersed, during high humidity periods [9]. Regardless of spore type, spore dispersal, germination and infection are highly facilitated by rainfall
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