Data_Sheet_1_High-Throughput Sequencing Facilitates Characterization of a “Forgotten” Plant Virus: The Case of a Henbane Mosaic Virus Infecting Tomato.docx

High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.

高通量测序技术极大地拓展了植物病毒研究及诊断的边界，不仅促进了新病毒或罕见病毒株及其基因组序列的快速发现，而且推动了植物病毒学的深入研究。在本研究中，我们运用高通量测序技术发现了一种感染番茄的新病毒——颠茄花叶病毒（Potyvirus，Potyviridae科），该病毒首次于20世纪初在英国栽培的颠茄中发现。在斯洛文尼亚采集的一株表现出严重坏死症状、病因未知的番茄植株样本中，通过小RNA和核糖体RNA去富集的总RNA高通量测序分析，揭示了样本中存在马铃薯病毒M（Carlavirus，Betaflexiviridae科）、南方番茄病毒（Amalgavirus，Amalgamaviridae科）和颠茄花叶病毒的混合感染。通过组装测序读段，成功获得了颠茄花叶病毒的完整基因组序列。通过在选定的测试植物上重新接种感染材料，成功分离出颠茄花叶病毒，并进行了寄主范围分析，证实该病毒对多种植物物种具有致病性。由于公共数据库中元数据有限，病毒分离物的分类鉴定最初为假设性。因此，在后续研究中，我们利用小RNA测序技术确定了来自不同病毒收藏所获取的四株历史病毒分离物的基因组序列。利用这些新的序列信息进行的系统发育分析，使我们能够将颠茄花叶病毒分类定位为辣椒花叶病毒系统发育群内的Potyvirus属成员，并确定了新番茄分离株与历史分离株之间的关系，表明该病毒至少存在四种可能的毒株。颠茄花叶病毒在番茄中的首次发现及其对这一寄主的致病性研究，对于植物保护和商业番茄生产具有重要意义。由于该病毒最初存在于混合感染中，且其全基因组尚未测序，因此在常规诊断中可能被忽视。本研究证实了高通量测序技术与经典植物病毒鉴定方法相结合，适用于识别和系统发育分类那些基因组序列数据稀缺或未知的隐秘病毒和历史病毒分离株。