Kikkert M, van Poelwijk F, Storms M, Kassies W, Bloksma H, van Lent J, Kormelink R, Goldbach R
Department of Virology, Wageningen Agricultural University, The Netherlands.
J Gen Virol. 1997 Jul;78 ( Pt 7):1755-63. doi: 10.1099/0022-1317-78-7-1755.
A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.
建立并测试了一种用于研究番茄斑萎病毒（TSWV）感染的植物原生质体系统。使用聚乙二醇介导的高感染性TSWV颗粒接种，通常50%或更多的黄花烟草原生质体被感染。在这些细胞中，接种后16小时（p.i.）可检测到病毒RNA和病毒蛋白合成，并且至少持续到接种后90小时。病毒结构蛋白[核蛋白（N）和包膜糖蛋白G1和G2]以及非结构病毒蛋白NSs和NSm积累到足以进行检测和免疫细胞分析的量。在矮牵牛叶片上进行的局部病斑试验和电子显微镜分析证实产生了成熟的、有感染性的病毒颗粒，这强调了在该系统中完成了完整感染周期的结论。用TSWV颗粒接种豇豆原生质体后，获得了相当比例的感染细胞以及相当数量的NSs、NSm和N蛋白，但检测到的病毒糖蛋白量比黄花烟草原生质体中的少得多，子代病毒颗粒也较少。基于黄花烟草的原生质体系统，现在已经有了一个强大的同步单细胞感染系统，可用于更精确地体内研究番茄斑萎病毒感染过程中发生的过程。
