Volume 6, Issue 6, Pages 1814-1829 (November 2013) A New β-Estradiol-Inducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5  Kathrin Schlücking, Kai H. Edel, Philipp Köster, Maria M. Drerup, Christian Eckert, Leonie Steinhorst, Rainer Waadt, Oliver Batistič, Jörg Kudla  Molecular Plant  Volume 6, Issue 6, Pages 1814-1829 (November 2013) DOI: 10.1093/mp/sst065 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Maps of Inducible Expression Cassettes Inserted in pUC18 or pGPTVII Vector Backbone. Expression of the optimized inducible cassette is either driven by P16ΔS or SUPERR. Sequence of the sXVE fusion protein is followed by a 3’UTR, pea rbcS E9, and LexA operator sequence. The minimal –46 35S promoter drives target gene expression. The transcription is stopped by the Nos-terminator. Plasmids containing GFP, mCherry, GUS, StrepII, or HA tag are available. Depending on the orientation of the reporter gene or tag, the multiple cloning sites in the plasmids are slightly different. Restriction sites flanking the promoter, expression cassette, and the Nos-terminator allow convenient exchange of all elements. All expression cassettes are available in pGPTVII or pUC18 backbone. pGPTVII backbones have a Basta resistance in plants; GFPC and GFPN construct are also available with Hygromycin or Kanamycin resistance. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Concentration Series of β-Estradiol-Induced GFP Expression. (A) Leaves of N. benthamiana transiently transformed with P16ΔS:sXVE:GFPC and SUPERR:sXVE:GFPC, treated with β-estradiol concentrations from 0.01 μM to 100 μM to induce GFP expression. Controls were mock-treated. (B) Western blots and immunodetection with a GFP-specific antibody of transformed leaves shown in (A). β-estradiol concentrations are indicated. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Time Course of Induced GFP Expression. (A) Leaves of N. benthamiana, transiently transformed with P16ΔS:sXVE:GFPC and treated with 100 μM β-estradiol or mock, were analyzed over a time course of 5 d. (B) The same procedure as in (A) but with SUPERR:sXVE:GFPC-infiltrated leaves. (C) Constitutively expressing 35S:GFPC control. (D) Western Blots and immunodetection with a GFP-specific antibody. dpin, days post induction; dpi, days post infiltration. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression of GFPN:RBOHF Protein under Different Promoters. Leaves of N. benthamiana were infiltrated with Agrobacteria harboring different constructs containing GFPN:RBOHF under control of different promoters. (A) 35S:GFPN:RBOHF. (B) G10-90:GFPN:RBOHF:pER8. (C) SUPERR:sXVE:GFPN:RBOHF. Pictures were taken 3 dpin. Scale bars represent 50 μm. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Induced Expression of GFP in Transiently Transformed Arabidopsis Mesophyll Protoplasts. Transiently transformed protoplasts were treated with 5 μM β-estradiol (A, B, left) or mock (A, B, right) and analyzed 15h after the treatment. Scale bars represent 7.5 μm. (A) P16ΔS:sXVE:GFPC:pUC. (B) SUPERR:sXVE:GFPC:pUC. (C) Constitutive active control 35S:GFP:pUC, fluorescence (left) and bright field (right). Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Stable Expression of Inducible Constructs in Arabidopsis. GUS activity in with P16ΔS:sXVE:GUS (A) or SUPERR:sXVE:GUS (B) transformed Arabidopsis plants treated with 5 μM β-estradiol for 24h or mock-treated. Stable mCherry expression of Arabidopsis plants, transformed with SUPPER:sXVE:mCherry (C, D, E), induced with 5 μM β-estradiol. Images of roots (C, D) and epidermal guard cells (E). Fluorescence (left), bright field (right). Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Expression of GFP Driven by UBQ10 Promoter in Stably Transformed Arabidopsis Plants. (A) Seven-day-old seedlings were transferred to media containing 5 μM β-estradiol and induced for 24h. Images were taken from cotyledones and roots. (B) Images of leaves from 21-day-old plants sprayed with 100 μM β-estradiol and induced for 48h. (C) Images of leaves (left) and a petal (right) from 42-day-old plants sprayed with 100 μM β-estradiol and induced for 48h. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 Recruitment of CIPK5 from the Cytoplasm to the Tonoplast after Induced Expression of CBL3. (A) Localization of constitutively expressed GFPN:CIPK5 and GFP (left) and co-infiltrated with inducible CBL3:mCherry before β-estradiol treatment (right). (B) Overlay images of GFPN:CIPK5 and GFP with CBL3:mCherryC at 1 dpin (left). Overlay of fluorescence was analyzed by scanning fluorescence intensities at regions of interest indicated by white lines within the pictures (right). Scale bars represent 20 μm. Molecular Plant 2013 6, 1814-1829DOI: (10.1093/mp/sst065) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions