M. Storms

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 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.

Expression of the movement protein of Tomato spotted wilt virus in its insect vector Frankliniella occidentalis

Summary. Tomato spotted wilt virus (TSWV) is able to infect both its botanical hosts and its insect vector (thrips). In plant tissue the NSM protein of TSWV functions as viral movement protein (MP), aggregating into plasmodesma-penetrating tubules to establish cell-to-cell movement. As upon heterologous expression NSM was able to form similar tubules on the surface of insect (Spodoptera frugiperda) cells, we have now investigated the expression and cellular manifestation of this protein in infected thrips tissue. It is shown that NSM, though detectably expressed in both the L2 larval and adult thrips stages, does not aggregate into tubules, indicating that this requirement is associated to its function as MP in plants, and raising the question if NSM has a function at all during the insect life cycle of TSWV.