A PROTOPLAST SYSTEM FOR STUDYING TOMATO SPOTTED WILT VIRUS-INFECTION

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 th e structural viral proteins [nucleoprotein (N) and the envelope glycop roteins G1 and G2] and the nonstructural viral proteins NSs and NSm ac cumulated to amounts sufficient for detection and immunocytological an alysis. Local lesion tests on petunia leaves and electron microscopica l analysis confirmed the production of mature, infectious virus partic les, underlining the conclusion that a full infection cycle was comple ted in this system. Upon inoculation of unguiculata (cowpea) protoplas ts with particles, comparable proportions of infected cells and amount s of NSs, NSm and N protein were obtained, but much lower amounts of v iral glycoproteins were detected than in N. rustica protoplasts, and p rogeny virus particles were less abundant. With the N. rustica-based p rotoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the proc esses occurring during tospovirus infection.