Next Generation Sequencing of grapevine wood pathogen infection in the presence of a root biocontrol agent.. Next Generation Sequencing of grapevine wood pathogen infection in the presence of a root biocontrol agent.

The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from grapevine wood infected by a fungal pathogen in the presence of a root biological control agent. One of the goals was to obtain molecular data about the fungus pathogen (Phaeomoniella chlamydospora) during grapevine wood infection. Grapevine pathogen-infected wood mRNA profiles of 2-month-old plantlets (14 days post infection) were generated by deep sequencing, in triplicate, using Illumina Hiseq2500. The sequence reads that passed quality filters were analyzed by TopHat followed by Cufflinks. qRTaPCR validation was performed using SYBR Green assays. Using an optimized data analysis workflow, we mapped sequence reads to the grapevine genome (build IGGP 12x) and identified pathogen transcripts. RNAseq analyses, using a ribosomal RNA depletion technology for library preparation, provided identification of genes expressed by P. chlamydospora during infection: as for genes related to effector biosynthesis enzymes, carbohydrate-active enzymes and transcription regulators involved in known regulation pathways in fungi. Insights about P. oligandrum modulation of grapevine infection by this pathogen were also found. Our study represents the first detailed analysis of grapevine wood infection by a fungal pathogen generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive evaluation of mRNA content within grapevine wood tissue. We conclude that RNA-seq based transcriptome characterization would permit the dissection of complex biologic interactions.

本研究旨在对比存在根系生物防治剂时，被真菌病原菌侵染的葡萄木质部的下一代测序（Next-Generation Sequencing, NGS）转录组谱分析（RNA-seq）数据。其中一项研究目标为获取葡萄木质部侵染过程中，该真菌病原菌（Phaeomoniella chlamydospora）的分子数据。本研究以接种病原菌14天的2月龄葡萄幼苗的侵染木质部为材料，采用Illumina Hiseq2500平台进行三次生物学重复的深度测序，获得其mRNA转录组谱。通过质量过滤的测序读段先经TopHat比对，再使用Cufflinks进行分析。采用SYBR Green法完成了qRTaPCR验证。本研究采用优化后的数据分析流程，将测序读段比对至葡萄基因组（版本IGGP 12x），并成功鉴定出病原菌的转录本。本次RNA-seq分析采用核糖体RNA去除技术进行文库构建，成功鉴定出P. chlamydospora在侵染过程中表达的基因，包括与效应因子生物合成酶、碳水化合物活性酶以及参与真菌已知调控通路的转录调控因子相关的基因。本研究还揭示了P. oligandrum对该病原菌侵染葡萄过程的调控相关见解。本研究为首次利用RNA-seq技术对真菌病原菌侵染葡萄木质部的过程开展的详细转录组分析。本文报道的优化数据分析流程可为转录组表达谱的比较研究提供参考框架。研究结果表明，NGS可全面评估葡萄木质部组织内的mRNA表达情况。综上，基于RNA-seq的转录组表征研究可解析复杂的生物互作过程。