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

Leaf sections were collected from agroinfiltrated leaves and upper uninoculated leaves, and homogenized in 2x SDS-PAGE buffer. Extracted proteins were separated in SDS polyacrylamide gels (4% stacking gel, 10% separation gel; Laemmli and Favre, 1973) . The proteins were transferred to PVDF membranes (MilliporeSigma) for western . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 blot analysis. OuMV MP was detected using a non-commercial anti-MP (A314) antiserum as described in Rastgou et al., 2009, followed The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 punctate structures (arrowheads) with FM4-64. The extent of colocalization was quantified using the PSC plugin in ImageJ. rp, linear Pearson correlation coefficient; rs, nonlinear Spearman's rank correlation coefficient. Images represent single optical planes. Scale bars, 15 μm, 10 μm (magnified region). (B-D) N. benthamiana epidermal cells expressing GFP:MPwt (green) with RdRp and CP, and organelle markers (magenta), (B) mCherry:VTI12 (TGN/EE), (C) mCherry:RHA1 (MVB/PVC) and (D) Man49:mCherry (Golgi). Cells were treated with 10 μM BFA (B and D, bottom panel) or 33 μM wortmannin (C, bottom panel) for 1h. White arrowheads indicate colocalized spots, green and magenta arrowheads denote individual GFP:MPwt and mCherry:VTI12 spots respectively. Images were taken at 66 hpi. Panels represent maximum intensity Z projections. A selected region was analyzed to show the intensity profile generated by ImageJ. Scale bars, 5 μm.