Inducible viral inoculation system with cultured plant cells facilitates a biochemical approach for virus-induced RNA silencing.
An inducible virus infection system was demonstrated to be an efficient protein expression system for inducing synchronous virus vector multiplication in suspension-cultured plant cells. A GFP-tagged tomato mosaic virus (ToMV-GFP) derivative that has a defect in its 130 K protein, a silencing suppressor of ToMV, was synchronously infected to tobacco BY2 cultured cells using this system. In the infection-induced cells, viral RNA was degraded rapidly, and a cytosol extract prepared from the infected cells showed RNA degradation activity specific for ToMV- or GFP-related sequences. In lysate prepared from cells infected by ToMV-GFP carrying the wild-type 130 K protein, sequence-specific RNA degradation activity was suppressed, although siRNA derived from the virus was generated. Furthermore, the 130 K protein interfered with 3'-end methylation of siRNA. The inducible virus infection system may provide a method for biochemical analysis of antiviral RNA silencing and silencing suppression by ToMV.
Tamai
Atsushi
A
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-machi, Ishikawa, Japan. atamai30@ishikawa-pu.ac.jp
Dohi
Koji
K
Mori
Masasi
M
Meshi
Tetsuo
T
Ishikawa
Masayuki
M
D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D034622:RNA Interference; D020871:RNA Stability; D018749:RNA, Plant / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D012367:RNA, Viral / Q000037:antagonists & inhibitors*; D013194:Staining and Labeling / Q000379:methods; D014026:Nicotiana / Q000821:virology*; D017898:Tobamovirus / Q000235:genetics / Q000254:growth & development*; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication
D018749:RNA, Plant; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins