Fig. 4: SP reduces D-body assembly through its IDR1.

a Confocal images of N. benthamiana leaf cells co-expressing SEb–YFP, DCL1–YFP, or HYL1–CFP with SP, SP mutants (muSP1), or control proteins (myc). White arrows indicate punctate structures in the nuclei. White arrows highlight punctate structures in the nuclei. Scale bars, 5 μm. b Quantification of nuclear puncta in (a). Data represent means ± SD (n = 3 biological replicates). Statistical significance was assessed using a two-sided unpaired Student’s t test. c Pull-down experiments using His–SEb, MBP–HYL1, and GST–SP at increasing molar ratios. Input and pull-down samples were analyzed by immunoblotting with anti-His, anti-GST, and anti-MBP antibodies. d MST curves showing binding affinities of SEb and HYL1 in the presence of GST–SP at varying concentrations. Excitation power 60%, MST power 40%, all labeled proteins were at 50 nM. The 95% confidence intervals of the Kd are as follows: HYL1 + His-SEb: ~ 0.15–2.00 μM and HYL1 + His-SEb + GST-SP: ~ 1.92–7.23 μM. e Confocal images of N. benthamiana leaf cells co-expressing SEb–YFP and HYL1–CFP with SP or SP mutants (SPΔIDR1, NLCD-SPΔIDR1). White arrows highlight nuclear puncta. Scale bars, 5 μm. f LCI assay detecting interactions between SEb and HYL1 in N. benthamiana leaves co-expressing SP or its IDR1 mutants (SPΔIDR1, NLCD-SPΔIDR1). g MST binding curves comparing the effects of SP and its variants on the binding affinity between SEb and HYL1. Excitation power 3%, MST power 40%, all labeled proteins were at 1 μM. The 95% confidence intervals of the Kd are as follows: HYL1 + SEb-MBP + His-SP: ~ 2.68–17.65 μM, HYL1 + SEb-MBP + His-NLCD-SPΔIDR1: ~ 4.07–23.63 μM and HYL1 + SEb-MBP + His-SPΔIDR1: ~ 0.92–19.95 μM. h Confocal images showing in vitro droplet formation of fluorescently tagged SEb–mCherry and HYL1–CFP under different co-incubated conditions with SP or its variants (SPΔIDR1, NLCD-SPΔIDR1). Scale bars, 50 μm. The experiments were repeated three times with similar results (a, c, e, f, h). Data represent the mean ± SD (n  =  3 independent titrations) (d, g).