Extended Data Fig. 2: Experimental validation of the genetic association between SE and MTC in developmental processes, modulation of gene expression, and protein-protein interactions.
From: SERRATE drives phase separation behaviours to regulate m6A modification and miRNA biogenesis
a, Design of artificial miRNAs (mta-a, -b, mtb, and fip37). The sequence alignment of the artificial miRNAs (in red) with their target sequences (in blue). Be noted: two independent artificial-miRNA lines for MTB and FIP37 were generated and validated. One elite line for each was used for further studies. b – d, RT-qPCR assays showed that the amount of MTA, MTB and FIP37 transcripts was largely reduced in knockdown lines of mta (b), mtb (c), and fip37 (d) vs Col-0 and se-2, respectively. Data are mean ± s.d. of three independent experiments. P values, one-way ANOVA analysis with Dunnett’s multiple comparisons test. For (b), p values for relative expression of MTA at the se-2, mta-a, and mta-b vs Col-0 are 0.23, < 0.0001, and < 0.0001, respectively. For (c), p values for relative expression of MTB at the se-2 and mtb vs Col-0 are 0.98 and 0.0013, respectively. For (d), p values for relative expression of FIP37 at the se-2 and fip37 vs Col-0 are 0.99 and 0.0010, respectively. e, f, Western blots showed the reduced MTA (e) and MTB (f) proteins in their amiR-KD lines. Endogenous proteins were detected by indicated antibodies, respectively. HSP70 was a loading control. g, Knockdown lines of mta, mtb, and fip37 displayed developmental defects in seedlings. The photos were taken of 10-day-old seedlings, with Col-0 and se-2 serving as controls. Scale bars, 1 cm. h, Y2H screening showed that neither DCL1 nor HYL1 directly interact with m6A writers. 1:5 serial dilutions are shown. -LT, lacking Leu and Trp; -LTHA, lacking Leu, Trp, His and Ade. i, LCI assays in Nicotiana Benthamiana demonstrated that both MTB and FIP37 can mediate the interaction between MTA and SE. CPM: Count per minute j – l, Co-IP assays validated the interaction between SE and MTC in plants. Proteins were extracted from transgenic plants of p35S::Flag-MTA (j), p35S::Flag-MTB (k), and p35S::Flag-4xMyc-FIP37 (l) transgenic plants, respectively. Lysates were then supplied with or without 50 μg/mL of RNase A prior to IP with an anti-FLAG antibody. SE was detected via a specific anti-SE antibody. HSP70 serves as a negative control. m, BiFC assays validated the interactions between SE and MTC in Arabidopsis mesophyll protoplast. PAG1 serves as a positive control. and showed similar results. Scale bar, 10 μm. At least three independent experiments were performed (e, f, i, j, k, and l), ten transgenic plants exhibited were photographed (g), ten independent colonies (h) and ten independent protoplasts for each interaction were tested (m), and representative images are shown.
