dsRNAs composed of multiple functionally characterized, efficient siRNAs provide effective protection against Cucumber mosaic virus

In response to viral infection of a plant, Dicer-like enzymes (DCLs) recognize viral double-stranded RNA and process it into a large number of small interfering RNAs (siRNAs). However, only a few of these siRNAs actually interfere with viral replication by forming a functional RNA-induced silencing complex (RISC). The activity of a certain siRNA depends mainly on its efficient loading into an antiviral Argonaute (AGO) protein, the core component of RISC, and on the accessibility of its complementary target site within the viral RNA. Here, we used a novel in vitro screening approach to identify functionally effective siRNAs (esiRNAs) targeting Cucumber mosaic virus (CMV, strain Fny). The approach is essentially based on a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), which exhibits DCL activity and facilitates the assembly of active RISCs with an in vitro translated AGO protein of choice. We exposed double-stranded versions of CMV RNA 2 and RNA 3 to BYL to generate a pool of viral siRNAs. Total RNA was isolated from the reactions and DCL-generated CMV siRNAs were identified by small RNA-seq. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated from the pool by FLAG-AGO immunoprecipitation (AGO-IP) and again analyzed by small RNA-seq. The antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo. Finally, double-stranded RNAs composed of multiple esiRNA sequences were designed. The DCL-mediated processing of these edsRNAs into siRNAs in BYL was analyzed by small RNA-seq and the antiviral activity was characterized in vitro and in vivo.

当植物遭受病毒侵染时，类Dicer酶（DCLs）可识别病毒双链RNA，并将其切割加工为大量小干扰RNA（siRNAs）。然而，其中仅有少数siRNA能够通过组装具有功能的RNA诱导沉默复合体（RISC），实际发挥干扰病毒复制的作用。单条siRNA的活性主要取决于两点：一是能否高效装载至RISC的核心组分——抗病毒Argonaute（AGO）蛋白，二是其互补靶位点在病毒RNA内的可及性。本研究采用一种新型体外筛选方法，鉴定出靶向黄瓜花叶病毒（CMV，Fny株系）的功能有效siRNA（esiRNA）。该方法核心依托烟草BY-2原生质体的细胞质提取物（BY-2裂解液，BYL），该提取物具备DCL活性，且可与体外翻译的目标AGO蛋白组装为活性RISC。研究人员将黄瓜花叶病毒RNA2与RNA3的双链形式加入BYL中，以制备病毒siRNA库。从反应体系中分离总RNA，并通过小RNA测序鉴定经DCL切割产生的CMV siRNA。在另一组实验中，通过FLAG标记AGO免疫沉淀（AGO-IP）从该库中分离与AGO1/RISC或AGO2/RISC结合的siRNA，并再次通过小RNA测序进行分析。对与AGO蛋白具有高亲和力的siRNA，分别在体外与体内验证其抗病毒活性。最终，研究人员设计了包含多条esiRNA序列的双链RNA。通过小RNA测序分析此类工程化双链RNA（edsRNAs）在BYL中经DCL介导切割为siRNA的过程，并在体外与体内验证其抗病毒活性。