Supporting Information

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Fig. 5. Alignment of NEO, PHY, and PHOT proteins. Identical residues in all or two among three canonical phytochromes or phototropins are colored with red or blue, respectively. Red, blue, and orange lines enclose the phytochrome sensor, LOV and serine/threonine kinase domains, respectively. Introns are indicated by gray lines. A blue circle indicates a conserved cysteine residue to ligate the bilin chromophore or to be indispensable for the LOV/FMN photocycle. Note that MsNEO1 LOV1 has this cysteine residue but does not bind FMN in vitro (pale blue circle). Alignment was made with CLUSTALW.

Fig. 6. Gene structure of MsNEO genes. Rectangles and connecting black lines indicate exons and introns, respectively. Red, blue, and yellow regions indicate the phytochrome sensor, the LOV domain, and serine/threonine kinase domains, respectively. Gray portions indicate 5'- or 3'-untranslated regions. Introns whose positions are equivalent in each gene are connected with thin black lines (gray lines for introns between MsNEO2 and MsPHOTA genes).

Fig. 7. Alignment of LOV domains of neochromes and phototropins. The cysteine residue necessary for FMN-cysteinyl adduct formation is shown in red. FMN-interacting residues revealed by the AcNEO1 LOV2 crystal structure are marked blue. Residues forming salt bridges are green. Residues similar but not identical to the AcPHY3 LOV2 domain are pale. Arrows indicate amino acid substitutions known to impair FMN-binding or to disrupt the photocycle. Residues in MsNEO expected to disrupt FMN-binding or photocycle are in black boxes.

Movie 1. Transient expression of MsNEO1 can rescue the Adiantum neo mutant rap2. Prothallial cells of the rap2 mutant of Adiantum capillus-veneris were bombarded with P-35S-driven MsNEO2 and GFP genes. Transformed cells were identified by their GFP⁺ fluorescence phenotype. Chloroplast movement was induced by 20-mm microbeam irradiation in a transformed cell (left cell) and in a neighboring untransformed cell. Cells were observed under infrared light and photographed at 1-min intervals, while the video was assembled at 30 images per sec. The position and initiation of red light irradiation, or the transition from red light to blue light, is indicated as a flashed white band. Dark-adapted plants were irradiated with red light at 0.55 mmol·m^–2·s^–1 for 3 h, then with blue light at 0.38 mmol·m^–2·s^–1 for an additional 90 min.

Supporting Methods

Cloning of Mougeotia scalaris Photoreceptor Genes. Total RNA and genomic DNA were extracted by using modified cetyltrimethylammonium bromide methods as described in ref. 1. To obtain Mougeotia PHOT cDNA, RT-PCR with the degenerate primers (1) 5'-GGIATHGAYYTIGCIACIACIYTIGARMG-3' and 5'-GCDATRTAYTCYTCIGTICCIACRAAISWRTT-3' was performed, and the amplified PCR products were subcloned into pGEM-T Easy (Promega) and sequenced. MsNEO2 gene PCR products were amplified from genomic DNA with the degenerate primers 5'-CAYGGITGYCAYIYICARTAYATG-3' and 5'-GCDATRTAYTCYTCIGTICCIACRAA-3'. RACE-PCR was performed according to the manufacturer’s protocol (Invitrogen). For 5'-RACE, first-strand cDNA was synthesized by using a gene-specific primer 5'-1 (MsNEO1, 5'-GCCACCTTCTCTTCAAAACG-3'; MsNEO2, 5'-CTTCCTTCTTCTGCCTCTGT-3'; MsPHOTA, 5'-ATCCCAGTTCTCAGTGTTGC-3'; MsPHOTB, 5'-GCAGAATCTCAGCAGCATAG-3'), followed by 5'-2 (MsNEO1, 5'-ATGGTCCACTGCCTCTCCTC-3'; MsNEO2, 5'-GGCTCCACGTCATCAGGTTG-3'; MsPHOTA, 5'-TCGTCCTTATTTCCTGTGTT-3'; MsPHOTB, 5'-TCTGCAACGGAGGAATGGAT-3') and 5'-3 (MsNEO1, 5'-CGGAGGTGGAAGTGTTGTCT-3'; MsNEO2, 5'-GTTCAATCCCGAGCAAGTCT-3'; MsPHOTA, 5'-TGTCTGTGTCCTTTCCTTGC-3'; MsPHOTB, 5'-GTGTCCTGGTCTGTATCTGG-3') in nested reactions. For 3'-RACE, cDNA was amplified with 3'-1 (MsNEO1, 5'-ACACGAGGGAGGAGCACATC-3'; MsNEO2, 5'-GGACGATGGGAAGAAGAGGA-3'; MsPHOTA, 5'-TGAAGAAGATGGGGACAGAG-3'; MsPHOTB, 5'-CCAAACAAGTGAAGACAACG-3') followed by 3'-2 (MsNEO1, 5'-CGTTTTGAAGAGAAGGTGGC-3'; MsNEO2, 5'-CGAATTGCATCAGAGAGGGA-3'; MsPHOTA, 5'-AGAAACAGAAACGGCTCAAG-3'; MsPHOTB, 5'-AAGTTTTAGGGCTGGTGGAC-3') in nested reactions. To determine genomic DNA sequences of MsNEO1 and MsNEO2, genes were amplified by using four primer pairs, d6-5'-6, d8-5'-4, d3-5'-1, and d1-u1 (d6, 5'-AAGCATTTCGCATCCCTAGT-3'; 5'-6, 5'-GCCAGAGAGTCAGTGCAGAG-3'; d8, 5'-CGCGTGGGAAGCCGAATGAT-3'; 5'-4, 5'-CGAGTCGACTTTTCCTTCAG-3'; d3, 5'-CTTTCTGGGACCTTGTGACC-3'; d1, 5'-CCATCAAGCCTCTCGGAAAT-3'; u1, 5'-TGCCCACACCTAACAGACCT-3') for MsNEO1, and F-u2 and d3-R (F, 5'-GTCAGTGTTCGCTCCGTCAT-3'; u2, 5'-AGAGCATTGCCGTCCTTGTC-3'; d3, 5'-TCACGGATGCCACCCAAGAG-3'; R, 5'-TGCAATCAAACCCGACATCA-3') for MsNEO2. MsPHY1 gene and also cDNA were amplified by using two primers (F, 5'-ATGTCGTCCTCTAAGCGGTC-3'; R, 5'-CTACTTCATGCTGGCCTGGT-3') and were sequenced. MsPHOTA and MsPHOTB genes were amplified with a pair of primers, d1-u1 and d1 SalI-u1 NotI, respectively (d1, 5'-TTCCACGACCCAGTTTTCAC-3'; u1, 5'-CAAAACCGGACAATACAAAA-3'; d1 SalI, 5'-GTCGACATGAATTCGCCGCTATCGCC-3'; u1 NotI, 5'-GCGGCCGCTCAAAGTCCAAGACACAGT-3'). In all cases, two or three independent clones were sequenced.

1. Kagawa, T., Kasahara, M., Abe, T., Yoshida, S. & Wada, M. (2004) Plant Cell Physiol. 45, 416–426.