Holeva Maria C, Sklavounos Athanasios, Rajeswaran Rajendran, Pooggin Mikhail M, Voloudakis Andreas E
Laboratory of Bacteriology, Scientific Directorate of Phytopathology, Benaki Phytopathological Institute, 14561 Kifissia, Greece.
Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, 11855 Athens, Greece.
Plants (Basel). 2021 May 12;10(5):963. doi: 10.3390/plants10050963.
(CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant-virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant . Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.
黄瓜花叶病毒（CMV）是一种具有全球分布的破坏性植物病毒，是已知植物病毒中寄主范围最广的，也是用于理解植物 - 病毒相互作用的模式植物病毒。自从发现RNA干扰（RNAi）作为一种主要的抗病毒防御机制以来，基于RNAi的技术已被开发用于植物抗病毒病害保护。在植物和动物中，RNAi的一个关键触发因素是双链RNA（dsRNA），它由Dicer和类Dicer（DCL）家族蛋白加工成小干扰RNA（siRNA）。在本研究中，CMV外壳蛋白（CP）和2b基因的dsRNA在体外和体内产生，并应用于代表系统茄科寄主的烟草植物以及局部寄主植物上。两种dsRNA处理均保护植物免受CMV的局部和系统感染，但不能抵御无关病毒的感染，证实了抗病毒RNAi的序列特异性。当dsRNA与CMV接种同时或在接种前四天应用时，抗病毒RNAi有效，但在接种后四天无效。体内产生的dsRNA比体外产生的更有效；在用体内dsRNA处理时，dsRNA - CP比dsRNA - 2b更有效，而在体外dsRNA处理时效果相反。对体内dsRNA - CP处理和未处理的烟草植物的小RNA进行Illumina测序表明，对系统叶片中CMV感染的干扰与病毒衍生的21和22核苷酸（nt）siRNA的积累大幅减少相吻合，这些siRNA可能分别由烟草DCL4和DCL2产生。虽然21 - nt类病毒siRNA在未处理的植物中占主导地位，但在dsRNA处理的植物中21 - nt和22 - nt类siRNA以几乎相等（但较低）的水平积累，这表明dsRNA处理可能会增强DCL2的活性。综上所述，我们的研究结果证实了局部应用dsRNA对植物抗病毒保护的有效性，并为抗病毒RNAi的机制提供了更多的见解。
