Yaegashi H, Yamatsuta T, Takahashi T, Li C, Isogai M, Kobori T, Ohki S, Yoshikawa N
The United Graduate School of Agricultural Sciences, Iwate University, Iwate, Morioka, Japan.
Arch Virol. 2007;152(10):1839-49. doi: 10.1007/s00705-007-1011-4. Epub 2007 Jun 30.
Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.
表达绿色荧光蛋白的苹果潜隐球形病毒（GFP-ALSV）用于分析在表达GFP的烟草植株（GFP-烟草）中病毒诱导的基因沉默（VIGS）。在接种了GFP-ALSV的GFP-烟草中，接种后5天（dpi）接种叶片上出现小黑点，然后扩展，最终在12 dpi后覆盖叶片的整个区域。第12片真叶以上的上部叶片的大部分荧光在21 dpi时消失。因此，GFP-ALSV感染有效地触发了烟草植株中转基因（GFP基因）的VIGS。对GFP沉默叶片的分析表明，病毒RNA和蛋白质在大多数GFP mRNA已被降解的所有叶片中积累。在能够轻松检测到GFP mRNA的siRNA的样品中未检测到源自ALSV-RNAs的siRNAs。直接组织印迹分析表明，GFP-ALSV的传播总是先于GFP-烟草受感染叶片中VIGS的诱导。使用本氏烟草16c品系进行的GFP叶斑试验表明，三种衣壳蛋白之一的Vp20是一种干扰系统沉默的沉默抑制因子。
