Fig. 1: SRBSDV P6 undergoes LLPS in vivo and in vitro.
a Subcellular localization of SRBSDV P6 in N. benthamiana leaf cells at 2 days post agroinfiltration (dpa). GFP alone was used as the control. Scale bar, 20 μm. b Subcellular localization of SRBSDV P6 in rice protoplasts at 12 h post transfection. GFP alone was used as a control. White arrows indicate droplets. Scale bar, 5 μm. c Disorder profiles of P6 were determined using multiple computational predictors, including: (i) VL-XT, XL1-XT, and CAN-XT from the Predictor of Natural Disordered Regions (PONDR; http://www.pondr.com/), and (ii) IUPred-long and IUPred-short from the Intrinsically Unstructured Protein Predictor (IUPred3; https://iupred3.elte.hu/). Thick black lines indicate IDRs. d FRAP assay showing the GFP-P6-formed droplets in the cytoplasm of N. benthamiana leaf cells. White circles indicate the photobleached areas. 0 s–40 s indicate the times after photobleaching pulse. Scale bar, 10 μm. e Recovery curve of the photobleached GFP-P6 droplets in the cytoplasm. The data are the means ± SD (n = 12 droplets). f The fusion between two GFP-P6-formed droplets in cells was captured through Confocal Time-lapse Microscopy. Scale bar, 10 μm. g An in vitro droplet formation assay using different concentrations of purified prokaryotically expressed RFP-P6. Scale bar, 10 μm. h In vitro formation of RFP-P6 droplets in different concentrations of KCl. Scale bar, 10 μm. i FRAP assay showing the recovery of a photobleached RFP-P6 droplet in vitro. Scale bar, 1 μm. j Recovery curve of photobleached RFP-P6 droplets in vitro. The data are the means ± SD (n = 12 droplets). k Confocal images showing the phase separation of RFP-P6△IDR3 and RFP-IDR3 in vitro. Scale bar, 10 μm. l FRAP assay showing the recovery of the photobleached RFP-IDR3 droplets in vitro. Scale bar, 1 μm. m Recovery curve of the photobleached RFP-IDR3 droplets. The data are the means ± SD (n = 12 droplets). Experiments in (a, b, g, h, and k) were repeated three times with similar results.
