Bioprotection, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
PLoS One. 2020 Nov 13;15(11):e0238157. doi: 10.1371/journal.pone.0238157. eCollection 2020.
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.
欧洲溃疡病，由坏死型真菌病原菌 Neonectria ditissima 引起，是全球范围内对苹果危害最大的疾病之一。目前对 N. ditissima 毒力的分子基础知之甚少。鉴定在感染过程中表达上调的基因，这些基因可能与毒力有关，是理解这一问题的第一步。反转录定量实时 PCR（RT-qPCR）可用于鉴定这些候选毒力基因，但依赖于使用参考基因对相对基因表达数据进行归一化。然而，迄今为止，尚未有报道专门针对在 N. ditissima-苹果病理系统中选择合适的真菌参考基因。在这项研究中，选择了 8 个 N. ditissima 基因作为候选 RT-qPCR 参考基因进行基因表达分析。设计用于扩增这些基因区域的引物子集（6 个）对来自其他真菌病原体的模板没有扩增产物，这些病原体存在于苹果园中。这六个引物组的扩增效率令人满意，范围从 81.8 到 107.53%。使用 geNorm、NormFinder 和 BestKeeper 统计算法分析当高度致病的 N. ditissima 分离株在 10 种培养条件下培养时的表达稳定性表明，肌动蛋白和肌醇-1-磷酸合酶（mips）或它们的组合可用作归一化 N. ditissima 基因表达的最合适参考基因。作为一个测试案例，这些参考基因用于研究三个候选毒力基因在感染时间过程中的表达。所有这三个基因都与真菌效应子基因具有相似的特征，与体外相比，在植物体内的表达上调，在接种后五到六周时达到峰值（wpi）。因此，这三个基因很可能与 N. ditissima 的致病性有关，是进一步功能表征的优先候选基因。
