The role of post-Golgi transport pathways and sorting motifs in the plasmodesmal targeting of the movement protein (MP) of Ourmia melon virus (OuMV)

Natali Ozber; Paolo Margaria; Charles T. Anderson; Massimo Turina; Cristina Rosa

benthamiana protoplasts (Fig. 1A) . To identify the nature of the endosomal compartments in which the MP resides, we transiently co-expressed mCherry-fused organelle marker proteins (Geldner et al., 2009) , VTI12 (a TGN marker; Uemura et al., 2004) , and RHA1 (a MVB/PVC marker; Sohn et al., 2003) , with GFP:MPwt along with RdRp and CP in N. benthamiana leaf epidermal cells. We also applied two well-known inhibitors of vesicle trafficking, brefeldin A (BFA; an ARF GEF inhibitor) and Wortmannin (Wm; a phosphatidylinositol-3 and -4 kinase inhibitor), to these cells to dissect the pathways through which MP is trafficked. mCherry:VTI12 showed the expected punctate pattern of TGN that overlapped with GFP:MPwt, as shown in Fig. 1B , suggesting that MP is recruited to TGN. While 81-89% of MP co-localized with the TGN marker, the co-localization of TGN marker with MP was 49-68%, suggesting that not all mCherry:VTI12-labeled TGN compartments were occupied by MP. We next examined the effect of BFA on GFP:MPwt at the TGN. It has been previously reported that 5-10 μM concentrations of BFA promote dissociation of TGN into small vesicular compartments in tobacco BY-2 cells (Ito et al., 2017) . Hence, we treated N. benthamiana leaves expressing both mCherry:VTI12 and GFP:MPwt with 10 µM BFA. Upon BFA treatment, both mCherry:VTI12 and GFP:MPwt dispersed into the cytoplasm. While GFP:MPwt dissociated into smaller punctate structures, some of these structures did not completely co-localize with the TGN marker ( Fig 1B, lower panel) . When we increased the concentration of BFA to 50 μM, we observed BFA-induced GFP:MPwt aggregates in N. benthamiana epidermal cells (not . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/724716 doi: bioRxiv preprint shown). We hypothesize that formation of these aggregates were associated with the toxicity of BFA rather than direct effect of BFA on the trafficking pathways, and we used the lower concentration in all the further experiments.