Volume 41, Issue 5, Pages 527-539.e5 (June 2017) The Protein Phosphatase 4 and SMEK1 Complex Dephosphorylates HYL1 to Promote miRNA Biogenesis by Antagonizing the MAPK Cascade in Arabidopsis  Chuanbin Su, Ziwei Li, Jinping Cheng, Lei Li, Songxiao Zhong, Li Liu, Yun Zheng, Binglian Zheng  Developmental Cell  Volume 41, Issue 5, Pages 527-539.e5 (June 2017) DOI: 10.1016/j.devcel.2017.05.008 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 SMEK1 Is Required for miRNA Biogenesis in Plants (A) Morphological phenotypes of Col-0, smek1, rescued smek1, and hyl1-2 plants. 4-week-old (Col-0, SMEK1-GFP smek1-1, and hyl1-2) and 6-week old (smek1) seedlings are shown as indicated. SMEK1-GFP was driven by the endogenous SMEK1 promoter, and introduced into smek1-1. Scale bars, 1 mm. (B) SMEK1 showing the PH domain (orange), the SMK-1 domain (green), and the Armadillo fold (red). The SMK-1 domain overlaps a small region (yellow) of the Armadillo fold. (C) Northern blotting of miRNAs in Col-0, smek1, and rescued smek1 plants. The left two panels show the results from inflorescences of Col-0 and smek1 mutants. The right panel shows the results from 10-day-old seedlings of Col-0, smek1-1, and SMEK1-GFP smek1-1. U6 was used as a loading control. The numbers indicate the relative abundance of miRNAs and represent the mean of three biological repeats. (D) miRNA sequencing analysis in inflorescences of Col-0 and smek1 mutants. The normalized abundances of miRNAs were calculated as reads per ten million (RPTM), and log2-transformed ratios of smek1/Col-0 were plotted. Each circle represents one miRNA. The dashed line indicates mean values. See also Figure S1. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 HYL1 Is Degraded in smek1 Mutants (A and B) Western blotting to determine HYL1 levels in 7-day-old seedlings of Col-0, smek1, and SMEK1-OE (35S::SMEK1-FLAG). Actin and Hsc70 served as loading controls. The numbers indicate the relative abundance of HYL1 and represent the mean of three repeats. The light-gray arrow indicates a non-specific band or the SMEK1-FLAG band. The dark-gray and black arrows indicate two SMEK1 bands. (C) Western blotting to determine YFP-HYL1 (HYL1p::YFP-HYL1) levels in seedlings of Col-0, smek1-1, and se-1. Actin served as a loading control. The numbers indicate the relative abundance of YFP-HYL1 and represent the mean of three biological repeats. (D) Visualization of YFP-HYL1 signals in root cells of Col-0 and smek1-1 from the elongation zone. Scale bars, 10 μm. (E) The cell-free protein decay assay using GST-HYL1 incubated with Col-0 or smek1-1 lysates. The levels of GST-HYL1 were determined with anti-GST antibody. The experiments were reproducible in three biological repeats. (F) The cell-free protein decay assay using MBP-HYL1-D1D2 (only two dsRBD of HYL1) incubated with Col-0 or smek1-1 lysates. The levels of MBP-HYL1-D1D2 were determined with anti-MBP antibody. (G) Statistical analysis of decay rate of MBP-HYL1-D1D2 in (F). The abundance of MBP-HYL1-D1D2 at 0.5 hr, 1 hr, and 2 hr was calculated relative to that at 0 hr. Data are presented as mean ± SE; n = 3. See also Figure S2. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 SMEK1 Interacts with HYL1 and Co-localizes with HYL1 (A) BiFC analysis between SMEK1 and HYL1. The interaction of paired proteins results in purple chemiluminescence (purple in image). More than five leaves were examined for each pair, and a graph is shown. (B) HYL1-FLAG pulls down SMEK1-His. Protein precipitates were analyzed by western blotting using anti-FLAG and anti-His antibodies. 1% input proteins were used for SMEK1-His and MBP-His, and 10% input proteins were used for HYL1-FLAG. (C) Reciprocal CoIP between SMEK1 and HYL1. GFP-Trap beads were used to immunoprecipitate HYL1 in 10-day-old seedlings of HYL1p::YFP-HYL1 transgenic plants, and anti-SMEK1 antibody was used to detect SMEK1. FLAG-M2 beads were used to immunoprecipitate SMEK1 in 10-day-old seedlings of 35S::SMEK1-FLAG transgenic plants, and anti-HYL1 antibody was used to detect HYL1. The red arrow indicates a non-specific band. The green and purple arrows indicate two SMEK1 bands. (D) Nuclear localization of GFP-tagged SMEK1 driven by the endogenous promoter in root epidermal cells. In 30% of nuclei, SMEK1 localized in discrete nuclear foci, indicated by red arrows. (E) Co-localization of SMEK1 and HYL1 in nuclear bodies of root cells in SMEK1p::SMEK1-RFP and HYL1p::HYL1-YFP doubly transgenic plants. The white arrows indicate the signals of dicing body for SMEK1 and HYL1. Scale bars, 10 μm. See also Figure S3. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Dephosphorylation of HYL1 Increases Its Stability (A) HYL1 abundance after treatment with PD98059 and Calyculin A. Total protein from 10-day-old seedling grown on medium with (+) or without (−) 100 μM of PD98059 or 1 μM of Calyculin A was detected by western blotting. The numbers indicate the relative levels of YFP-HYL1 or HYL1 and represent the mean of three biological replicates. (B) The cell-free protein decay assay using GST-HYL1 incubated with lysates of 10-day-old Col-0 seedlings with the addition of Mock (DMSO), 100 μM PD98059, or 1 μM Calyculin A. The levels of GST-HYL1 were determined with anti-GST antibody. (C) Statistical analysis of decay rate of GST-HYL1 in (B). The relative levels of GST-HYL1 were calculated by image analysis and plotted versus the level in the initial sample (0 min). Data are presented as mean ± SE; n = 3. (D) Western blotting of HYL1 accumulation in 10-day-old seedlings from Col-0 and CPL1-OE (35S::CPL1-YFP) (numbers [#] indicate individual transgenic lines). Anti-YFP antibody was used to detect CPL1-YFP. Actin was used as a loading control. The numbers indicate relative levels of HYL1 and represent the mean of two biological repeats. (E) The cell-free protein decay assay to determine the decay patterns of the wild-type HYL1 (WT), the phospho-less HYL1 (SΔA), and the phosphomimic HYL1 (SΔD). Lysates from transiently expressed various versions of 35S::HYL1-YFP N. benthamiana leaves. Hsc70 was used as a loading control. (F) Statistical analysis of decay rate of HYL1 in (E). The relative levels of different versions of HYL1 were calculated by image analysis and plotted versus the level in the initial sample (0 hr). Data are presented as mean ± SE; n = 3. See also Figure S4. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 SMEK1 Inhibits the Activation of the MAPK Cascade (A) Western blotting to determine HYL1 levels in 10-day-old seedlings of Col-0, mpk3, and mpk6. Actin served as a loading control. The numbers indicate the abundance of HYL1 and represent the mean of three biological repeats. (B) Western blots to determine HYL1 levels in Col-0 and NtMEK2-FLAG transgenic plants. Total proteins of 10-day-old seedlings treated with DEX or Mock (DMSO) as the indicated times were analyzed. Actin served as a loading control. The numbers indicate the relative abundance of HYL1 and represent the mean of three repeats. Anti-FLAG antibody was used to detect MEK2 levels. (C) Analysis of MPK activation in 10-day-old seedlings of Col-0 and smek1-1. Phosphorylated MPK6 (gray arrow) and MPK3 (black arrow) were detected by western blotting with the anti-phospho ERK1/2 antibody. Hsc70 served as a loading control. The results represent one of three biological replicates. (D) In vitro kinase assay analysis of MEK2. Commercially dephosphorylated MBP was incubated with the immunoprecipitates from 10-day-old seedlings of NtMEK2-FLAG transgenic plants (induced by DEX for 24 hr) with or without the immunoprecipitates from 10-day-old seedlings of SMEK1-FLAG transgenic plants. The 10-day-old seedlings of Col-0 crude extracts were used as a negative control. An anti-phosphothreonine antibody was used to detect phosphorylated MBP. Rep1 and Rep2 represent results from two biological replicates. See also Figure S5. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 SMEK1 Assembles a Functional PP4 that Dephosphorylates HYL1 (A) CoIP between PPX1, PPX2, and SMEK1. Plasmids with different combinations of PPX1-YFP, PPX2-YFP, and SMEK1-FLAG were co-expressed in N. benthamiana. Anti-GFP and anti-FLAG antibodies were used to detect PPX and SMEK1, respectively. (B) Co-localization of SMEK1 and PPX1 or PPX2 in nuclear bodies. Plasmids with PPX1-RFP or PPX2-RFP and SMEK1-YFP were co-expressed in N. benthamiana. Leaves were observed under fluorescence microscopy. Scale bars, 10 μm. (C) In vitro phosphatase assay to determine the phosphatase activity of the PP4 and SMEK1 complex. Immunoprecipitates from transiently expressing the vector (harboring the FLAG tag alone) were used as a negative control. The bottom panel indicates the immunoprecipitated PP4 subunits by western blotting. Data are presented as mean ± SE; n = 3. (D) In vitro phosphatase assay to test HYL1 as a substrate of the PP4 and SMEK1 complex. The assay was done as in (C), except that the commercial peptide was replaced with two serine-phosphopeptides or a tyrosine-phosphopeptide of HYL1. Data are presented as mean ± SE; n = 3. (E) Phosphoprotein mobility-shift assay using Phostag in 7-day-old seedlings of Col-0 and smek1 mutants. Hypo- and hyperphosphorylated HYL1 forms are indicated as P− and P+, respectively. Ponceau S staining of Rubisco was used as the loading control. The intensity of the bands was measured with Tanon-5200 Gel Image System (Version 4.2.5) software and calculated as the ratio of the hypo-/hyperphosphorylated forms. Total proteins were analyzed by SDS-PAGE without Phostag to show reduced accumulation of total YFP-HYL1 in smek1 mutants (bottom panel). (F) Partial rescue of smek1-1 phenotypes by increasing dephosphorylated HYL1. 8-week-old plants and leaves of smek1-1 harboring 35S::HYL1 (WT), phospho-less 35S::HYL1 (SΔA), and phosphomimic 35S::HYL1 (SΔD) are shown. See also Figure S6. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 The Protein Level of SMEK1 Is Induced by ABA (A) Greening response of Col-0, smek1-1, 35S::SMEK1-FLAG (SMEK1-OE), and hyl1-2 under ABA treatment. Seeds were germinated on Murashige and Skoog medium with indicated ABA. Pictures were taken 10 days after planting. (B) Western blotting to determine MPK activation with (+) or without (−) ABA treatment in various genotypes. Phosphorylated MPK6 and MPK3 were detected with the anti-phospho ERK1/2 antibody. Seven-day-old seedlings were treated with 50 μM ABA (DMSO was used as the Mock control) for 30 min, and total proteins were extracted for western blotting. Anti-MPK3 and anti-HYL1 antibodies were used to detect total amounts of MPK3 and HYL1, respectively. Hsc70 was the loading control. Data are presented as mean ± SE; n = 3. (C) Western blotting to determine the protein levels of SMEK1 with (+) or without (−) ABA treatment. 7-day-old seedlings of Col-0 were treated with the control (−) or 100 μM ABA (+) for 30 min, and three biological replicates were performed. smek1-1 was used as the negative control of anti-SMEK1 antibody. The red arrow indicates a non-specific band. The green and purple arrows indicate two SMEK1 bands. Hsc70 was the loading control. (D) Western blotting to determine the protein levels of SMEK1-FLAG with (+) or without (−) ABA treatment. 7-day-old seedlings of 35S::SMEK1-FLAG transgenic plants were treated with the control (−) or 100 μM ABA (+) for 30 min, and three biological replicates were performed. Anti-FLAG antibody was used to detect SMEK1-FLAG (asterisks). Hsc70 was the loading control. See also Figure S7. Developmental Cell 2017 41, 527-539.e5DOI: (10.1016/j.devcel.2017.05.008) Copyright © 2017 Elsevier Inc. Terms and Conditions