aminotriazole) supplemented with 10 mM PCB. The plates were incubated for 3 d in 1 mmol m22 s21 red light (Pfr) or 13 mmol m22 s21 far-red light (Pr). As a control, equal amounts of overnight cultures were spotted onto non-selective (L–W–) plates without PCB. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. Found at: doi:10.1371/journal.pgen.1000143.s003 (0.4 MB TIF) Figure S4 FHY1 does not protect phyA-NLS-GFP from degradation in the nucleus. (A) Total protein extracts were prepared from seedlings expressing phyA-NLS-GFP in wild-type (FHY1) or fhy1 mutant background. The seedlings were grown for 4 days in the dark (Dark) or irradiated for 1 day with far-red light (15 mmol m22 s21) after 3 days in the dark (FR). The protein extracts were separated by SDS-PAGE and used for immuno- blotting with antibodies specific for phyA or DET3 (loading control). (B) phyA-NLS-GFP levels in FHY1 and fhy1-1 back- ground were quantified using quantitative western blot analysis. The seedlings were grown as described in (A) and the mean value +/2 SEM of biological triplicates is indicated. FHY1 phyA-NLS- GFP; phyA-211 FHY1 PPHYA:PHYA-NLS-GFP (Col6Ler). fhy1 phyA- NLS-GFP; phyA-211 fhy1-1 PPHYA:PHYA-NLS-GFP (Col6Ler). Found at: doi:10.1371/journal.pgen.1000143.s004 (0.11 MB TIF) Figure S5 Reversible interaction of FHY1/FHL and phyA. (A) explains how the yeast two hybrid b-galactosidase activity assay in (B) was done. (B) Yeast (strain Y187) was transformed with plasmids encoding AD-FHY1/phyA-BD (FHY1) or AD-FHL/ phyA-BD (FHL). Overnight cultures supplemented with 10 mM PCB were grown in nonselective medium in the dark. The cultures were then irradiated for 5 min with 12 mmol m22 s21 red light and incubated in the dark for another 240 min before measuring the b-galactosidase activity. Immediately (0 min), 60 min, 120 min or 240 min after the red light pulse a 5 min far-red light pulse (13 mmol m22 s21) was given. Error bars indicate the SEM (n = 3). MU, Miller units. Found at: doi:10.1371/journal.pgen.1000143.s005 (0.01 MB TIF) Figure S6 Nuclear import model explaining FHY1 dependent phyA nuclear accumulation in far-red light. In seedlings irradiated with FR only a minor fraction of the phyA molecules is in the References 1. Casal JJ, Luccioni LG, Oliverio KA, Boccalandro HE (2003) Light, phytochrome signalling and photomorphogenesis in Arabidopsis. Photochem Photobiol Sci 2: 625–636. 2. Mathews S (2005) Phytochrome evolution in green and nongreen plants. J Hered 96: 197–204. 3. Chen M, Chory J, Fankhauser C (2004) Light signal transduction in higher plants. Annu Rev Genet 38: 87–117. 4. Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58: 21–45. 5. Lin C, Shalitin D (2003) Cryptochrome structure and signal transduction. Annu Rev Plant Biol 54: 469–496. 6. Scha¨fer E, Nagy F, eds (2006) Photomorphogenesis in Plants and Bacteria. 3 ed. Dordrecht: Springer. 7. 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FHY1 Mediates phyA Nuclear Import active Pfr from (#,2%). Upon binding of PfrA to FHY1 the PfrA- FHY1 complex is transported into the nucleus using the NLS of FHY1 and the general nuclear import machinery. Once in the nucleus most of the transported PfrA-FHY1 complexes will dissociate in FR into PrA and free FHY1. Free FHY1 will recycle to the cytosol and be available for further import cycles. In contrast, PrA and PfrA are trapped in the nucleus because they are i) too big to exit the nucleus by diffusion and ii) not actively exported into the cytosol. How FHY1 recycling works and if dissociation of the phyA-FHY1 complex is essential for initiation of downstream signaling remains unknown. Found at: doi:10.1371/journal.pgen.1000143.s006 (0.58 MB TIF) Table S1 List of accession numbers. The table shows the accession numbers of the sequences used for the alignment in Figure 1A as well as the databases, in which the sequences were found. GenBank (NCBI): http://www.ncbi.nlm.nih.gov/sites/ entrez?db = nucleotide. JGI (Joint Genome Institute): http:// genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html. MAtDB v2.0 (Arabidopsis Genome Database): http://mips.gsf.de/proj/plant/ jsf/athal/. The Gene Index Project: http://biocomp.dfci.harvard. edu/tgi/plant.html. Found at: doi:10.1371/journal.pgen.1000143.s007 (0.04 MB DOC) Experimental procedures. at: doi:10.1371/journal.pgen.1000143.s008 (0.03 MB Text S1 Found DOC) Acknowledgments We thank Martine Trevisan for technical support and Karin Schumacher (University of Tu¨ bingen, Germany) for antibodies against DET3. Furthermore, we are grateful to Akira Nagatani (University of Kyoto, Japan) for sharing unpublished information and for providing antibodies against phyA. Author Contributions Conceived and designed the experiments: TG JJC ES AH CF. Performed the experiments: TG FS AT DD JJC AH CF. Analyzed the data: TG FS DD JJC ES AH CF. Wrote the paper: AH CF. 13. 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