Summary auto-generated
Researchers developed a plant protoplast system to study tomato spotted wilt virus (TSWV) infection in single cells. Using polyethylene glycol-mediated inoculation, they infected Nicotiana rustica and Vigna unguiculata (cowpea) protoplasts with purified TSWV particles, achieving infection rates of 50% or higher. In N. rustica protoplasts, viral RNA and protein synthesis became detectable at 16 hours post-inoculation and continued for at least 90 hours. Both structural proteins (nucleoprotein and envelope glycoproteins) and nonstructural proteins (NSs and NSm) accumulated in sufficient quantities. Electron microscopy and local lesion tests on petunia leaves confirmed production of mature, infectious virus particles, indicating complete infection cycles. In V. unguiculata protoplasts, infection efficiency was lower, with reduced viral glycoprotein production and fewer enveloped particles. The N. rustica-based system provides a synchronized, single-cell infection model for detailed studies of tospovirus molecular biology, allowing investigation of infection processes without the complexity of whole-plant systems.
Key findings
- N. rustica protoplasts achieved 50% or higher infection rates with TSWV using PEG-mediated inoculation, with viral protein and RNA synthesis beginning at 16 hours post-inoculation
- All major TSWV proteins (structural: N, G1, G2; nonstructural: NSs, NSm) were produced in infected N. rustica protoplasts, and mature infectious virus particles formed as confirmed by electron microscopy and local lesion assays
- V. unguiculata protoplasts showed lower infection efficiency with markedly reduced glycoprotein synthesis and fewer enveloped particles compared to N. rustica
- The protoplast system provides synchronized single-cell infection allowing detailed analysis of viral replication, protein localization, and maturation processes
- NSm protein correctly localized to tubular structures at the cell surface despite absence of plasmodesmata, confirming proper viral protein targeting even in protoplasts
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Abstract
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.