syn-tasiRnas targeting the coat protein of potato virus Y confer antiviral resistance in <i>Nicotiana benthamiana</i>

Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.反式作用小干扰RNA（trans-acting small interfering RNAs，tasiRNAs）是一类长度为21核苷酸的相位性小干扰RNA（phased siRNAs），其生物合成路径为：在微小RNA（microRNA，miRNA）靶位点处，经Argonaute（AGO）蛋白催化切割的RNA产物通过RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase，RDR）生成双链RNA底物，随后由类Dicer核酸酶（Dicer-like，DCL）连续催化切割产生。目前已有研究通过人工合成获得靶向病毒RNA的合成型tasiRNAs（syn-tasiRNAs），此类分子可赋予植物病毒抗性。本研究通过将TAS1c基因的5条内源siRNA替换为马铃薯Y病毒（potato virus Y，PVY）基因组外壳蛋白（coat protein，CP）区域的210 bp片段，设计得到靶向PVY侵染的syn-tasiRNAs。结果显示，在本氏烟草（Nicotiana benthamiana，N. benthamiana）中瞬时表达syn-tasiR-CPpvy2可赋予抗病毒抗性，表现为无PVY侵染症状且病毒积累量显著降低，表明syn-tasiR-CPpvy2可精准靶向并沉默PVY的CP基因，有效抑制病毒侵染。syn-tasiR-CPpvy1处理的植株症状有所减轻，病毒积累量亦有所下降；而表达syn-tasiR-CPpvy3的植株则出现严重的PVY侵染症状，病毒积累量与对照组无显著差异。本研究还通过单一前体构建了同时靶向PVY和马铃薯X病毒（potato virus X，PVX）的syn-tasiR-CPpvy/pvx。在本氏烟草中瞬时表达syn-tasiR-CPpvy/pvx3与syn-tasiR-CPpvy/pvx5后，植株可同时抵御PVY与PVX的侵染。上述结果表明，工程化改造的syn-tasiRNAs不仅可特异性诱导针对单一靶标病毒的抗病毒抗性，还可被设计为多靶点沉默策略以靶向不同病毒，从而有效防控植物的复合病毒侵染。