Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

RNA沉默是一种序列特异性的基因调控机制，在植物中同时兼具抗病毒功能。为拮抗抗病毒RNA沉默，病毒已进化出RNA沉默抑制子（RNA silencing suppressors, RSS）。针对番茄斑萎病毒属（tospovirus）病毒，其非结构蛋白NSs已被鉴定为该类抑制子。尽管已有研究证实番茄斑萎病毒（tomato spotted wilt virus, TSWV）的NSs蛋白可结合长链与短链双链RNA（dsRNA），但目前关于其抑制RNA沉默非细胞自主组分的能力，相关研究仍较为有限。本研究选取代表3个不同番茄斑萎病毒属物种的TSWV、花生环斑病毒（groundnut ringspot virus, GRSV）以及番茄黄环病毒（tomato yellow ring virus, TYRV）的NSs蛋白，探究其抑制局部与系统性沉默的能力与强度。我们建立了一套可量化本氏烟（Nicotiana benthamiana）16C株系中GFP沉默程度的实验体系，以实现不同RNA沉默抑制子相对活性的比较。实验结果显示，三种番茄斑萎病毒属病毒的NSs蛋白均可抑制局部沉默与系统性沉默。出乎意料的是，尽管TYRV的NSs蛋白表达量更低，但其对系统性RNA沉默的抑制效果却与TSWV及GRSV的NSs蛋白相当。利用上述建立的实验体系，我们进一步分析了一系列携带预测RNA结合结构域突变的TSWV NSs基因构建体，以及来自TSWV 160和171株系（Tsw抗病基因的抗病突破株系）的NSs蛋白抑制系统性GFP沉默的能力。结果表明，NSs蛋白可通过另一种作用模式实现RNA沉默抑制，该模式作用于小干扰RNA（siRNAs）的生物发生及其封存过程的下游阶段。最后，我们结合NSs蛋白结合长、短链dsRNA的特性，以及近期针对TSWV NSs蛋白开展的、用于定位RSS活性及触发Tsw介导抗病性所需结构域的突变筛选研究，对上述实验结果展开了讨论。