Single insertion lines were selected by determining the kanr/kans ratio in T2. Homozygous progeny of two representative single insertion lines for each construct were used for further studies. pphyA40-phyA (contains PPHYA:PHYA-CFP:TerRbcS) is a T-DNA vector derived from pCHF40-phyA (contains P35S:PHYA- CFP:TerRbcS) and was used to generate plants expressing PHYA promoter driven phyA-CFP. pphyA40-phyA and pCHF40-phyA were obtained as described for pphyA30-phyA and pCHF30-phyA but contain ECFP (Clontech) instead of EYFP [26]. pCHF70-, pCHF72- and pCHF73-FHY1 167–202 are T-DNA vectors used to generate plants expressing CaMV 35S promoter driven YFP-FHY1 CT, NLS-YFP-FHY1 CT (artificial FHY1) and NES-YFP-FHY1 CT. Details regarding cloning of these constructs can be found in Text S1. pCHF70-, pCHF72- and pCHF73-FHY1 167–202 were used for Agrobacterium-mediated transformation of Ler and fhy1-1 (only pCHF72-FHY1 167–202), pphyA40-phyA for transformation of phyA-201 [57]. Transgenic plants were selected on soil using BASTA (AgrEvo). Unless indicated otherwise, homozygous progeny of single insertion lines (1:3 segregation of the selection marker) were used for the experiments. Lines co-expressing either NLS- or NES-YFP-FHY1 CT and phyA-CFP were obtained by genetic crossing of Ler P35S:NLS/ NES-YFP-FHY1 CT and phyA-201 PPHYA:PHYA-CFP (Ler ecotype). The F1 generation was used for microscopic analysis. The phyA-211 fhy1-1 plants expressing phyA-NLS-GFP were obtained by crossing phyA-211 PPHYA:PHYA-NLS-GFP5 (Col ecotype) into fhy1-1 (Ler ecotype) background. In F2 siblings were selected that were homozygous for the transgene and phyA-211 and either wild-type (i. e. phyA-211 FHY1 PPHYA:PHYA-NLS-GFP5, in Col6Ler background) or mutant (i. e. phyA-211 fhy1-1 PPHYA: PHYA-NLS-GFP5, in Col6Ler background) for FHY1. In all experiments with phyA-211 fhy1-1 PPHYA:PHYA-GFP5 the phyA- 211 FHY1 PPHYA:PHYA-NLS-GFP5 in Col6Ler background was used as wild-type control. phyA-211 fhy3-1 PPHYA:PHYA-NLS-GFP plants were obtained by crossing phyA-211 PPHYA:PHYA-NLS-GFP (Col ecotype) into fhy3-1 (Col ecotype) background. In F2 seedlings homozygous for phyA- 211, fhy3-1 and the transgene were selected. Plant Material The Columbia (Col-0) and Landsberg erecta (Ler) ecotype of A. thaliana were used as wild type. phyA-211 [58], cop1-4 [59] and fhy3- 1 [60,61] are in Col while fhy1-1 [61,62] and phyA-201 [58] are in Ler. phyA-211 PPHYA:PHYA-GFP5 (A-GFP1), phyA-211 fhy1-1 PPHYA:PHYA-GFP5, phyA-211 fhy3-1 PPHYA:PHYA-GFP5 and fhy1- 1 P35S:YFP-FHY1 were previously described [27]. A second phyA- 211 PPHYA:PHYA-GFP5 line (A-GFP2) which was obtained during the screen described previously [27] was used because its phyA- GFP protein level is close to the phyA-NLS-GFP protein level in the lines we obtained. Hypocotyl Length, Anthocyanin Accumulation Measurements of hypocotyl length in continuous FR light and anthocyanin accumulation were performed as described [63]. For hypocotyl length seedlings were grown on half-strength MS, 0.7% agar while for anthocyanin accumulation seedlings were grown on half-strength MS, 0.7% agar supplemented with 1.5% sucrose. The VLFR of hypocotyl elongation and its transition to the HIR was investigated essentially as described [64]. Briefly, chilled seeds were exposed to red light for 6 h followed by 18 h of incubation in darkness before transfer to pulses of FR (3 min) given at different dark intervals (117 min, 57 min, 27 min or 0 min = continuous FR). Hypocotyl length was measured to the nearest 0.5 mm after FHY1 Mediates phyA Nuclear Import 3 d of treatment and is expressed relative to dark controls. Data are means and SE of at least 11 replicate boxes (10 seedlings per box). Microscopy Microscopic analyses in Figures 2A–D and 4A–D were performed with a Leica DM 600B equipped with Leica LTR6000 laser (software LAS, Leica Application Suite) using GFP and DAPI filter sets and a 206 air objective. 3-day-old dark- grown seedlings were directly observed under the microscope (dark condition). For light conditions, 3 day-old-dark-grown seedlings were pretreated for 10 min with white light before they were observed under the microscope. For microscopic analyses in Figures 1C and 1D, 2E and 2F, 4E– H and 7B a Zeiss Axioscope 2 equipped with a 636 oil-immersion objective and GFP, YFP and CFP specific filter sets was used. The seedlings used for microscopy were grown as described in the figure legends. Materials and Methods for Figures S1–S6 can be found in Text S1. Supporting Information Figure S1 phyA protein levels in our transgenic lines. Col, phyA- 211 as well as phyA-211 seedlings expressing PPHYA:PHYA-NLS, PPHYA:PHYA-NLS-GFP or PPHYA:PHYA-GFP (i.e. the lines used in this study) were grown in the dark. After 4 days total protein was extracted and separated by SDS-PAGE. Quantitative western blot analysis was used to measure the phyA levels. The mean value +/2 SEM of biological triplicates is indicated. Found at: doi:10.1371/journal.pgen.1000143.s001 (0.05 MB TIF) Figure S2 Cytoplasmically localized FHY1 CT induces a dominant negative phenotype. (A) Morphology of seedlings expressing FHY1 CT. Wild-type (Ler), fhy1-1 and phyA-201 seedlings as well as transgenic lines expressing different FHY1 167–202 ( = FHY1 CT) constructs were grown for 5 days in the dark or in far-red light (15 mmol m22 s21). #2590, #2607, #2619, #2638, #2643; Ler P35S:YFP-FHY1 167–202 (Ler YF CT). #4520, #4527; Ler P35S:NLS-YFP-FHY1 167–202 (Ler NLS-YF CT). #4578, #4597; Ler P35S:NES-YFP-FHY1 167–202 (Ler NES- YF CT). (B) Protein levels in seedlings expressing FHY1 CT. Wild- type (Ler), fhy1-1 and phyA-201 seedlings as well as the transgenic lines shown in (A) were grown for 4 days in the dark. Total protein was extracted and analyzed by SDS-PAGE and immunoblotting. phyA and (NLS-/NES-) YFP-FHY1 CT were detected using polyclonal antibodies specific for the N-terminal half of Arabi- dopsis phyA and GFP, respectively. The amido black stained PVDF membranes are shown as loading controls (15 mg total protein per lane). Found at: doi:10.1371/journal.pgen.1000143.s002 (1.39 MB TIF) Figure S3 FHY1 interacts with importin alpha. (A) Pull down experiment for FHY1 and importin alpha. In vitro synthesized 35S- labeled importin alpha was incubated for 2 hours with recombi- nant GST-FHY1-H6, GST-FHY1 DNLS-H6 and GST-H6 (nonbinding control) bound to GSH sepharose. After washing, the sepharose beads were incubated with SDS-PAGE sample buffer for elution. The samples were separated by SDS-PAGE and transferred onto a PVDF membrane. A phosphorimager was used for signal detection. Lane 1 contains 4% of the input used in lanes 2–4. Both the autoradiogram (top) and the Amido Black-stained membrane are shown. (B) FHY1 DNLS normally interacts with phyA. Yeast (strain AH109) was transformed with the indicated plasmids. A 5 ml aliquot of overnight cultures was spotted onto selective synthetic dropout plates (L–W–H–, containing 1 mM 3- PLoS Genetics | www.plosgenetics.org 10 August 2008 | Volume 4 | Issue 8 | e1000143