Subcellular mapping of ToBRFV movement protein in Nicotiana glutinosa via confocal laser scanning microscopy and agroinfiltration

Movement proteins (MPs) are essential for plant virus spread, mediating cell-to-cell transport through plasmodesmal connections. A plethora of studies have been reported for molecular characterization of Tobamovirus fructirugosum (tomato brown rugose fruit virus, ToBRFV) and its interactions with the host plants, however subcellular localization of the ToBRFV-movement protein (ToBRFV-MP) has not been reported in any host to date. Here, we investigated the subcellular localization of 266 amino acid long ToBRFV-MP in Nicotiana glutinosa using a transient expression system. The expression construct comprising N-terminal cMyc-tagged MP fused to cyan fluorescent protein (ToBRFV-MP-CFP) at its C-terminus via a five-amino acid linker sequence (GGGGS), and driven by CaMV 35S promotor was cloned into pCAMBIA2300 binary vector for the expression of recombinant fusion protein, and designated as pCAM-ToBRFV-MP-CFP. This construct was introduced into Agrobacterium tumefaciens LBA4404 for in planta expression through standard agroinfiltration assay. The expression and integrity of about 59 kDa fusion protein was confirmed in Western blot analysis using anti-cMyc tag antibody. The localization of MP-CFP fusion protein to inner cell periphery was suggested by the cyan fluorescence, in a distinct punctate pattern, under confocal laser scanning microscopy. Subsequent aniline blue staining demonstrated the co-localization of MP-CFP fluorescence alongside the plasmodesmata-associated callose deposits, supporting plasmodesmal localization of movement protein. These findings demonstrated the first experimental evidence for the plasmodesmata-associated localization of the ToBRFV-MP, and was in support of its proposed role in inter- and intracellular movement. By establishing this transient expression system, we have created conditions for analyzing the behavior of viral movement proteins in diverse plant species and gained insight into their role in the trafficking of viruses through plasmodesmata.