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. 2017 Oct 20;7:13712. doi: 10.1038/s41598-017-14146-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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TH1 acts as a transcriptional repressor in the transient expression assay. (a) Transcriptional activation assay in yeast cell. Yeast co-transformants were incubated on the selective medium (SD/-Ade/-His/-Leu/-Trp plus X-α-Gal) at 30 °C for 3 d. If TH1 is a transcription activator, the β-Galactosidase reporter gene will be expressed and the filter will be stained blue in the Colony-lift Filter Assay. pGADT7-RecT & pGBKT7-53 (positive control); pGADT7-RecT & pGBKT7-Lam (negative control). (b) Constructs used in transient expression assay in rice leaf protoplasts. Six different effector plasmids were made by fusing TH1, TH1^S2-89 with activator domain VP16, repressor domain EAR or the GAL4 DNA binding domain (GAL4-DB). The reporter plasmid Pro35S-GAL4:LUC contains a CaMV 35 S promoter, 5 × GAL4 binding site, a TATA box in front of the firefly luciferase (LUC) gene and a nopaline synthase terminator. The p35S: REN plasmid expressing the Renilla luciferase was used as an internal control. (c) Relative luciferase activities in rice leaf protoplasts using TH1, TH1^s2-89, VP16, TH1-VP16, TH1^s2-89-VP16, EAR, TH1-EAR and TH1^s2-89-EAR as effector compared with GAL4-DB control. Error bars indicate SE (n = 3). Double asterisks indicate significant difference at P < 0.01 by Student’s t test.