Table_1_Gene Validation and Remodelling Using Proteogenomics of Phytophthora cinnamomi, the Causal Agent of Dieback.XLSX

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen.

肉桂疫霉（Phytophthora cinnamomi）是一种致病性卵菌（oomycete），可在全球范围内的多种自然生态系统及诸多农业重要作物中引发植物枯梢病。目前，其注释版草图基因组序列已通过JGI Mycocosm数据库公开发布，该序列预估包含26131个基因模型。本研究利用肉桂疫霉的可溶性菌丝蛋白、细胞外分泌蛋白组（分泌组，secretome）以及游动孢子蛋白，通过校正基因注释、发掘新基因来优化该物种的基因组注释。
研究团队将多样化的亚蛋白质组数据集整合至自主构建的蛋白质基因组学（proteogenomics）分析流程中，成功优化了肉桂疫霉的基因组注释结果。实验采用液相色谱-质谱联用法（Liquid chromatography mass spectrometry）获取高置信度肽段，并将其与注释版基因组以及自主生成的6框翻译（6-frame translation）序列进行谱图匹配。
通过谱图匹配，共验证了2764个草图基因组中的原有基因注释。借助蛋白质基因组学分析流程，研究团队利用质谱数据对肉桂疫霉基因组进行校正，成功鉴定出23个全新的基因模型以及60个经过修正的基因特征，结果表明可检测蛋白质组中存在3%的错误注释比例。上述新发现的基因特征通过全肽段支持证据进行了进一步验证，同时辅以基因本体论（Gene Ontology）分析、功能结构域鉴定等功能基因组学实验进行验证。
本研究证实，将质谱谱图数据与蛋白质基因组学分析流程相结合，可有效优化重要植物病原菌的基因组注释，并发掘被遗漏的基因。本研究首次将质谱谱图数据应用于卵菌病原菌的基因组校正与手动注释工作中。