Five questions on the cell-to-cell movement of Orthotospoviruses

Fig. 1. TSWV NSm forms distinct tubule in protoplast. Expression of NSm in tobacco protoplasts (B), Mock inoculated with water (C). Adapted with permission from Lewandowski and Adkins, 2005.

Fig. 2. GBNV NSm has membrane remodelling ability in vitro and in vivo. Transmission electron Micrograph showing the interaction of refolded NSm with liposomes to form larger size of liposomes and the site of liposome fusion (shown by arrow) can be observed (A) Liposome alone, (B) Refolded NSm alone (C) Liposomes incubated with refolded NSm. (D) and (E) confocal images of N. benthamiana leaves expressing ERGFP (D) and ERGFP along with NSm. (Adapted from Singh et al., 2014 and Singh and Savithri, 2015).

Fig. 3. TSWV NSm localize to ER membrane at well defined places forming a structure similar to vesicle (A) Expression of NSm-YFP, white arrow indicates the structure similar to vesicle, (B) Expression of ER Marker mCherry-HDEL. (C) Overlay image (A) and (B) (© 2016 Feng et al. ) (Adapted from Feng et al, 2016).

Fig. 4. C-terminal coiled coil domain is responsible for ER localization of NSm.Subcellular localization of GBNV–NSm and its deletion mutants in N. benthamiana(Adapted from (Singh and Savithri, 2015) (A): GFP–NSm (B) GFP–NSm NΔ50 localizes to PD. The deletion of C-terminus region abrogates the localization of NSm to PD as shown (C) localization of GFP-NSm CΔ40. The C-terminal 79 amino acid possessing coiled coil domain form vesicle like structure near cell membrane as shown in(D) GFP-Nsm C79 aa.

Fig. 5. GBNV NSm has Amphipathic helix. Helical wheel projection of the α-helix from GBNV NSm protein in the C-terminal region, N-terminus region of Amphiphysin and Endophilin and coiled coil domain of NSm. Amphipathic helix was prepared by server Heliquest (https://heliquest.ipmc.cnrs.fr/). Red, acidic amino acid; blue, basic amino acid; yellow, aliphatic amino acid; purple and pink, polar amino acid prepared (A) C-terminus of NSm (C) and (D) Amphipathic helix of amhiphysin and endophilin by (B) coiled coil domain present at C-terminal region (Image adapted from Singh et al., 2014). Coiled coil domans are involved in membrane remodelling.

Fig. 6. Tospoviruses use ER membrane system to transport NSm to PD. (Adapted from Singh and Savithri, 2015). Localization of GFP–NSm at PD (A), The localization of GFP NSm when the N. benthamiana leaves infiltrated with GFP:NSm construct are treated with BFA 10μg/ml (B) and 25μg/ml(C).

Fig. 7. (A) Domain mapping of Tospoviral NSm. The N-terminal unfolded region associates with N protein, The residues100–150 are involved in conferring resistnace and in interaction with SW-5b and C-terminal coiled coil domain associates with ER network and remodels it to vesicles (B) The probable mechanism of cell to cell movement of tospoviruses.