Lin Pin-Chun, Hu Wen-Chi, Lee Shu-Chuan, Chen Ying-Lan, Lee Chi-Ying, Chen Yet-Ran, Liu Li-Yu Daisy, Chen Po-Yen, Lin Shih-Shun, Chang Ya-Chun
1 Department of Plant Pathology and Microbiology, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei, Taiwan;
2 Institute of Biotechnology, National Taiwan University, 81, Chang-Xing St., Taipei, Taiwan;
Mol Plant Microbe Interact. 2015 Jun;28(6):711-26. doi: 10.1094/MPMI-08-14-0246-R. Epub 2015 May 29.
The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.
齿瓣兰环斑病毒（ORSV）衣壳蛋白（CP）第100位的谷氨酸（E(100)）在本氏烟草的病毒长距离移动中起重要作用。具有谷氨酸到丙氨酸替换的ORSV(E100A)突变体在本氏烟草中表现出系统感染性丧失。透射电子显微镜和尺寸排阻色谱分析表明，E(100)对于CP-CP相互作用和病毒粒子组装至关重要。为了鉴定ORSV触发或应答基因以及与CP相互作用的蛋白（CP-IP），本研究采用了基于下一代测序和蛋白质组学分析的综合组学方法。分析了本氏烟草健康叶片和受ORSV感染叶片的全转录组，并利用基因信息创建了本氏烟草蛋白质数据库，用于质谱分析后的蛋白质鉴定。综合组学方法鉴定出了几种与ORSV CP(WT)相互作用的假定宿主蛋白，它们被归类为光系统亚基、防御相关蛋白和细胞分裂成分。分别通过半定量逆转录聚合酶链反应和体外结合试验检测了这些CP-IP的表达模式和与CP的相互作用，以验证计算机模拟数据。在这些蛋白中，与CP(WT)相比，本氏烟草的一种蛋白酶抑制剂（NbPI2）与CP(E100A)高度相关，并且NbPI1和NbPI2在受ORSV感染的植物中被高度诱导。（通过烟草脆裂病毒诱导的基因沉默系统）沉默NbPI1和NbPI2的植物在ORSV感染方面没有表现出差异。因此，NbPI1和NbPI2是否在植物免疫中发挥作用需要进一步研究。总之，综合组学方法为鉴定ORSV CP-IP提供了大量有价值的信息，这些CP-IP将有助于我们了解这种病毒的移动以及植物-病毒相互作用。
