Volume 4, Issue 3, Pages 546-555 (May 2011) A High-Throughput Screening System for Arabidopsis Transcription Factors and Its Application to Med25-Dependent Transcriptional Regulation  Ou Bin , Yin Kang-Quan , Liu Sai-Nan , Yang Yan , Gu Tren , Wing Hui Jennifer Man , Zhang Li , Miao Jin , Kondou Youichi , Matsui Minami , Gu Hong-Ya , Qu Li-Jia   Molecular Plant  Volume 4, Issue 3, Pages 546-555 (May 2011) DOI: 10.1093/mp/ssr002 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Scheme of Mating-Based Screening Method. All pDEST22/pDEST22m–TFs were transformed independently to Y187 strain in 96-well plates to generate yeast TFs library. AH109 strain expressing bait protein or YM4271 strain containing bait DNA was mated to Y187 TFs library in 96-well plates. After mating, the cultures were 10-fold diluted and plated on selective solid medium plates. At the steps labeled with *, a replicator (V&P Scientific) can be used to minimize the time cost. Molecular Plant 2011 4, 546-555DOI: (10.1093/mp/ssr002) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Validation of the TFs Library and Mating-Based Screening Methods. (A) Screening with NPR1 as bait in an Y2H. The strong NPR1–TGA3 interaction could be detected on most stringent selective medium SD-Leu-Trp-His-Ade. The weak NPR1–TGA2 interaction could be only detected on less stringent selective medium SD-Leu-Trp-His. F8, TGA3; H1: TGA2. Yeast growth on the non-selective medium SD-Leu-Trp showed the mating efficiency. (B) Screening with CCA1 promoter as bait in an Y1H. The interaction of CHE and CCA1 promoter could be detected on medium with a different concentration of 3-AT. E5, CHE. Yeast growth on the non-selective medium SD-Trp-His showed the mating efficiency. Molecular Plant 2011 4, 546-555DOI: (10.1093/mp/ssr002) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Identification of TFs that Interact with Med25 or Bind to the GCC-Box of PDF1.2 Promoter. (A) Identifying Med25-interacting TFs. All positive clones were diluted to 10−2, 10−3, and 10−4, and then plotted on non-selective SD-Leu-Trp and selective SD-Leu-Trp-His plates. AD, Gal4 activation domain; BD, Gal4 DNA binding domain. (B) Identifying TFs binding to the GCC-box of PDF1.2 promoter. All positive clones were diluted to 10−2, 10−3, and 10−4, and then plotted on non-selective SD-Trp-His and selective SD-Trp-His (25 mM 3-AT) plates. AD, Gal4 activation domain. Molecular Plant 2011 4, 546-555DOI: (10.1093/mp/ssr002) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 In Vitro Confirmation of the Interactions of TFs with Med25 and with GCC-Box of PDF1.2 Promoter. (A) GST pull-down: immunoblot with anti-MBP antibody showed MBP–Med25ACID could be pulled down by GST–At3g23220, GST–At3g23240, and GST–At4g18450, but not by the control GST. (B) BiFC assay of MED25 interaction with At3g23220, At3g23240, and At4g18450 in vivo. GFP signal was detected in Nicotiana benthamiana leaves co-infiltrated with Agrobacterium co-expressing C–Med25 and At3g23220, C–Med25 and At3g23240, and C–Med25 and At4g18450. DAPI staining indicates the location of nuclei. Merged images show co-localization of GFP and DAPI signals. (C) EMSA showed that GST–At3g23220, GST–At3g23240, and GST–At4g18450 bound to GCC-box of PDF1.2 promoter. Competition assays with unlabeled probes showed the specific binding. GST was used as a control. Molecular Plant 2011 4, 546-555DOI: (10.1093/mp/ssr002) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 A Proposed Model for Med25-Dependent Transcriptional Regulation. Three AP2-EREBP family TFs (At3g23220, At3g23240, At4g18450) act downstream of the JA signaling pathway. They bind to the GCC-box of PDF1.2 promoter, and then transmit the transcription message to RNA polymerase II through Mediator via interaction with Med25 subunit. GTFs, general transcription factors. Molecular Plant 2011 4, 546-555DOI: (10.1093/mp/ssr002) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions