Fig. 2: RDM16 forms condensates in vivo in response to heat stress.
a Diagram of RDM16. Top, functional domains of RDM16. CC1, splicing factor domain present in the CC1-like family. Prp3, pre-mRNA processing factor 3. DUF, domain of unknown function. Bottom, potential intrinsically disordered regions (IDRs) of RDM16, as analyzed by the Predictor of Natural Disordered Regions online tool (PONDR; http://www.pondr.com/). The black lines with asterisk and the cyan numbers underneath indicate the IDR and its average score. b, d and h Confocal microscopy of root cells of RDM16:RDM16-GFP (b, d) and overexpressing RDM16-GFP (RDM16-GFP OE) (h) transgenic seedlings that were treated as indicated. Scale bars, 10 μm (b, d) or 5 μm (h). c, e Quantitative analysis of the RDM16-GFP fluorescent spots in (b, d). n = 239, 247, and 221 for 22 °C, 37 °C and recovery (c); n = 234, 248, and 212 for 22 °C, 45 °C and recovery (e). The number on the right represents the number of granules per cell. f, g and i Representative micrographs and quantification of fluorescence recovery after photobleaching (FRAP) of RDM16-GFP granules after treatment as indicated. The arrowheads indicate the condensate targeted for photobleaching. Scale bars, 5 μm. n = 5 (f), n = 6 (g) or n = 7 (i). j, k Representative micrographs and quantification of RDM16-GFP in protoplasts before and during heat treatment at 37 °C. Scale bars, 5 μm. n = 90, 122, and 88 for 0-, 15-, and 30-min treatments at 37 °C, respectively. The boxes represent the interquartile range, with the middle line defining the median. The lines extending from the quartiles of the box are called ‘whiskers’ and show the maximum and minimum values. In (c, e and k), different letters indicate significant differences by non-parametric version of one-way ANOVA: Kruskal–Wallis test followed by Dunn’s multiple comparisons test (P < 0.05). In (f, g and i), data are means ± SD. The maximum recovery fraction (Rmax) and half-time of recovery (t1/2) was calculated from the logarithmic curve and the best-fit values were generated by GraphPad. All experiments were performed at least three times with similar results.
