Fig. 4: 14-3-3 proteins repress FD condensation and enhance DNA binding.

a,b, Confocal images of SAM cells of gFD::mVenus-FD; fd-3 wild-type (no. 22; a) or T282A (no. 9; b) plants. mVenus–FD is shown in green and cell walls (blue) and nuclei (magenta) were stained with Direct Red 23 and DAPI, respectively. Scale bars, 20 μm. c,d, 2.5D images of cells from e (c) and f (d); bars indicate pixel value of signal intensity. e,f, Left, magnified views of cells in a (e) and b (f); scale bars, 2 μm. Right, individual cell nuclei; scale bars, 1 μm. White lines indicate representative regions that were analysed in g. g, The signal intensity variation (maximum – minimum/total signal intensity) for DAPI, mVenus–FD and mVenus–FD(T282A) pixel point distribution. h, AlphaFold-predicted structure of FD. Colours indicate model confidence (pLDDT). i, Top, protein domain structure of FD. Bottom, predictions of disordered regions by PrDOS algorithms³⁶. j, Left, phase diagram of mScarlet1, mScarlet1–FD(T282E) and mScarlet1–FD(T282A) droplets. Right, schematic of protein fusion used for in vitro phase separation assay. Scale bars, 10 μm. k, Fluorescence recovery after photobleaching of mScarlet1–FD(T282E) droplets. The photobleaching pulse was applied at t = 0. Scale bar, 2.5 μm. l, Time course of recovery after photobleaching of mScarlet1–FD(T282E) droplets. Data are mean ± s.d. (n = 13). m, Fusion of droplets containing mScarlet1–FD(T282E) in in vitro phase separation assay. Scale bar, 2.5 μm. Data in j–m are representative of three independent experiments. n, Amplicons for ChIP–qPCR analysis in o and quantitative PCR with reverse transcription (RT–qPCR) in p. o, ChIP–qPCR showing FD enrichment in target gene regulatory or transcribed regions in FD::3HA3Flag-FD; fd-3 and FD::3HA3Flag-FD^T282A; fd-3 plants. p, RT–qPCR of FD and FD target gene mRNAs in FD::3HA3Flag-FD; fd-3 and FD::3HA3Flag-FD^T282A; fd-3 plants. All values are normalized to ACTIN2. Data in o,p are mean ± s.e.m. of three independent biological replicates. Statistical significance was determined by pairwise one-sided t-test.