1. Supporting Figure 3. Identification of prenylated coumarins using mass spectrometry in N. benthamiana transiently expressing PcPT. Leaves were first infiltrated with agrobacteria transformed with the PcPT-containing plasmid, and 72 h post inoculation, a second infiltration was done with umbelliferone. Finally, 24 h later, phenolic compounds were extracted and analyzed. A) MS Chromatogram of [M+H] + parent ions corresponding to two compounds, P1 and P2, with a molecular weight of M=230 (m/z = 231) and retention time at 20.27 min and 24.74 min, respectively. B) MS 2 analysis of P1, and C) that of P2. Both molecules display the same fragmentation pattern with a characteristic fragmentation of a C-prenylation (∆neutral loss = 56). D) MS 2 analysis of DMS, and E) osthenol as standard molecules. Relative abundance 100 80 60 40 20 0 05101520253035 P2 20,27 P1 24,74 A Time (min) Relative abundance 100 50 0 100 50 0 100 50 0 100 50 0 60100140180220 60100140180220 60100140180220 60100140180220 m/z 174.86175.08 175.07 B D C E

2. Clone nameC1C2C3C4C5 ∆CtND Clone nameP1P2P3P4P5P6 ∆Ct4.291.341.395.253.234.95 Supporting Figure 4. Real-time PCR to detect PcPT in Ruta graveolens. (A)and (C) Representative amplification curves of PcPT and 16S ribosomal RNA in PcPT transformant of R. graveolens and the control plants, respectively. (B) and (D)  Ct values of independent clones of R. graveolens and the control plants, respectively. A B C D 16S PcPT 16S PcPT

3. 0 20 40 60 0 4812 16 mAU ( at 330 nm ) 0 20 40 60 0 4812 16 mAU ( at 330 nm ) 0 20 40 60 0 4812 16 mAU ( at 330 nm ) Umbelliferone 4-coumarate Retention time ( min ) Standards RgC2’H EV Supporting Figure 5. 4-Coumaroyl CoA 2’-hydroxylase (C2’H) assay in the double transformant of PcPT and RgC2’H in N. benthamiana leaves. Supernatant of ultracentrifugation of the cell-free extract was used as crude enzyme (Mizutani et al., 2012). Enzymatic reactions were carried out for 1 h, and reaction products were measured by HPLC analysis. EV (empty vector) indicates the negative control using pBIN-GW instead of pBIN-PcPT and pBIN-RgC2’H. Arrowhead indicates the enzyme reaction product, umbelliferone (20 ± 1 nmol/mg protein/h). Values are means ± SD of triplicate experiments.

4. 0 10 20 30 01020304050 mAU ( at 333 nm) 0 10 20 30 01020304050 mAU ( at 333 nm) 38394041 - 10 - 6 - 2 0 10 20 30 01020304050 mAU ( at 333 nm) Retention time ( min ) Umbelliferone Osthenol DMS 38394041 - 10 - 6 - 2 Standards Full assay Heat denatured Retention time ( min ) mAU ( at 333 nm ) Supplementary Figure 6. Native umbelliferone dimethylallyltransferase activities in parsley. Umbelliferone dimethylallyltransferase assay was performed using the microsomal fraction prepared from parsley leaves as the crude enzyme. After the incubation for 16 h, the reaction mixture was analyzed by HPLC. Inserts are enclosed chromatogram between 38-41 min. Black and white arrowheads indicate the peaks of DMS (16.1 ± 0.2 nmol/mg protein) and osthenol (0.366 ± 0.006 nmol/mg protein), respectively, and the ratio of 6- and 8-prenylation is ca. 100 : 2. No activity was seen in the heat-denatured microsome. Values are means ± SD of triplicate experiments.

5. Supporting Figure 7. Chemotaxonomy of furanocoumarin occurence in higher plants. The phylogenetic tree was adapted from Nelson et al (2004). Apiaceae Moraceae Apiaceae + Fabaceae Rutaceae Linear and Angular Furanocoumarin Linear Furanocoumarin Gymnosperms Amborella Laurales Liliopsida Ranunculales Caryophyllales Santalales Berberidopsidales Myrothamnaceae Gunneraceae Saxifragales Crossomatales Gerianales Myrtales Celestrales Malpighiales Oxalidales Zygophylales Cucurbitales Fagales Brassicales Malvales Cornales Ericales Garryales Gentianales Lamiales Solanales Aquifoliales Asterales Dipsicales Rosales Fabales Sapindales Apiales