Transcript Figure 1
Figure S1 a cmSP165/166 severe b MIM159 (mir159a) Figure S1: Detailed phenotype of severe cmSP165/166 and MIM159 plants. Severe cmSP165/166 (15x) plants have characteristic trumpet- and cup-shaped (white arrows) leaves, as well as fasciated stems and flowers. Figure S2 PHB REV 0.25 * 0.2 0.15 * 0.1 * * 0.05 * * Relative RNA levels 0 PHV 0.015 * 0.01 * 0.005 * * 0 miR166 10 8 * * * cmSP165 S * cmSP165 M 2 MIM165 M 4 MIM165 W 6 WT 0 Figure S2: Analysis of target transcript and miRNA levels in cmSP165/166 (15x) and MIM165/166 plants. qRT-PCR analysis of PHB, REV, PHV and miR166 levels in MIM165/166 and cmSP165/166 (15x) primary transformants. Transcript levels were normalized to CYCLOPHILIN and miRNA levels were normalized to snoR101. RNA was extracted from 4-weekold primary transformants using tissue from whole plants. Measurements are the average of three technical replicates with error bars representing the SEM. Asterisks mark statistically significant changes compared to wildtype. Figure S3 cmSP165/166 miR165: 3’-CCCCCUACUUCGGACCAGGCU-5’ miR166: 3’-CCCCUUACUUCGGACCAGGCU-5’ cmSP165/166: 5’-ggGGGAUGAAcgCUGGUCCGa-3’ MIM165/166 miR165: miR166: MIM165/166: 3’-CCCCCUACUUC---GGACCAGGCU-5’ 3’-CCCCUUACUUC---GGACCAGGCU-5’ 5’-GGGGAAUGAATctaCCUGGUCCGA-3’ STTM165/166-48 miR165 3’-CCCCCUACUUC---GGACCAGGCU-5’ 5’-GGGGGAUGAAGctaCCUGGUCCGA-3’ STTM165 miR166 3’-CCCCUUACUUC---GGACCAGGCU-5’ 5’-GGGGAAUGAAGctaCCUGGUCCGA-3’ STTM166 Figure S3: Nucleotide sequences of cmSP165/166, MIM165/166 and STTM165/166. Underlined nucleotides mark mismatches between the miRNA and the respective construct. Red nucleotides in MIM165/166 and STTM165/166-48 mark the nucleotides opposite position 11 of the miRNA. Table S1 ∆G (kcal/mol) ∆G % cmSP165/166 -31.3 83% MIM165/166 -26.9 71% STTM165/166-48 -35.5 94% cmSP159 -26.3 79% MIM159/STTM159 -28.8 86% MIM319 -26.5 80% MIM319 -29.3 75% MIM159 -24.3 62% MIM172 -26.4 84% MIM172cs -21.0 67% miR166a miR159a miR319a/b miR172a Table S1: Free energy calculations of the interaction between cmSPs, MIMs, STTMs and their corresponding miRNAs. Free energy (∆G) values are shown in kcal/mol and as percentage compared to a perfect match with the respective miRNA. ∆G values were calculated using the DINAmelt Web Server (http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting). Figure S4a STTM159-48 STTM156-48 STTM165/166-48 STTM172-48 Figure S4b cmSP159 cmSP156 cmSP165/166 cmSP172 Figure S4c MIM159 MIM165/166 MIM172 miRNA target site accessibility MIM156 Target MIMIC ∆Gopen (kcal/mol) MIM159 3.75 MIM165/166 6.06 MIM156 2.15 MIM172 6.17 Figure S4: Prediction of RNA secondary structure and miRNA target site accessibility of different decoy constructs. RNA secondary structures of STTMs (Figure S4a), cmSPs (Figure S4b) and MIMs (Figure S4c) were predicted using the Vienna RNAfold web server (http://rna.tbi.univie.ac.at/). The heat map indicates base-pair probabilities from low (blue) to high (red). Sequences encircled in black mark the miRNA binding site. The miRNA target site accessibility of MIMs (Figure S4c) was calculated as the energy required for un-pairing all target site nucleotides (ΔGopen) using the Vienna RNAup web server (http://rna.tbi.univie.ac.at/). The target site was defined as the miRNA binding site plus 17 nt upstream and 13 nt downstream, which has been determined as optimal window in Kertesz et al., 2007, and is used by the psRNATarget server (Dai and Zhao, 2011).