Supporting Information Méheust et al. 10.1073/pnas.1517551113 S-Gene Expression Analysis extracellular domain. This novel protein is found only in green Given that the analysis using dozens of algal and protist genomic algae and plants, and may play a role in responding to salt stress. datasets showed that homologs of all of the S genes are expressed, For S genes present in the diatom Phaeodactylum tricornutum, we asked whether some of them might be differentially expressed we used RNA-seq data from ref. 30 that compared cultures in response to stress. This question was motivated by two ob- grown under control and nitrogen (N)-depleted conditions. This servations: (i) The composite genes entered eukaryote nuclear analysis showed that out of six families present in this alga (2, 1, genomes via primary endosymbiosis, and therefore they may still 28, 20, 35, 61), three are differentially expressed under N stress retain ancient cyanobacterial functions even when fused to novel (28, 35, 61). One of these is family 28, which encodes an domains, and (ii) many of the S genes are redox enzymes or N-terminal bacterium-derived, calcium-sensing EF-hand domain encode domains involved in redox regulation, and therefore their fused, intriguingly, to a cyanobacterium-derived region with sim- roles may involve sensing and/or responding to cellular stress ilarity to the plastid inner-membrane proten import component resulting from the oxygen-evolving photosynthetic organelle. To Tic20, which acts as a translocon channel. This fused protein may + address this issue, we inspected RNA-seq data from organisms use Ca2 as a signal for protein import, and is found in several that encoded particular S genes that had either been generated stramenopile (brown algal) species. The homolog in P. tricornutum under conditions of cellular stress or spanned the light–dark (NCBI gi:219117465) is significantly down-regulated (P = 2.57e-23) transition. For genes present in Chlamydomonas reinhardtii,we under N depletion. used data from ref. 53 that compared triplicate transcript data Finally, for S genes present in Arabidopsis thaliana, we used from this alga grown in standard Tris-acetate-phosphate (TAP) medium (54) or TAP with the addition of 200 mM NaCl. The RNA-seq data reflecting light-dependent DE in seedlings, coty- composite gene families 10, 24, and 18 showed significant dif- ledons, and roots (14). This analysis showed that four distinct ferential expression (DE) under salt stress [up-regulation in both S-gene families have DE in plant tissues in the presence of light cases (P = 4.14e-4, P = 0.0268, and P = 0.0077, respectively)]. (14, 19, 1, 18). One of the gene families showing DE is family 1, These genes encode bacterial–cyanobacterial domain fusions which is broadly distributed in algae and plants and is composed that are involved in stress responses (i.e., rhodanese domain in of a cyanobacterium-derived N-terminal hydrolase domain fused 10) and in preventing protein misfolding (i.e., DnaJ/Hsp40 do- to a non-cyanobacterium-derived LPLAT family (lysophospho- main at the N terminus of 24). Interestingly, the DnaJ domain is lipid acyltransferases) domain. This gene was significantly up- fused to an upstream SCP superfamily region that likely forms an regulated in all three plant tissues in the presence of light. Méheust et al. www.pnas.org/cgi/content/short/1517551113 1of5 Méheust et al. Gloeochaete.witrockiana.SAG46_84 Cparadoxa Cyanidioschyzon.merolae.strain.10D Porphyridium.aerugineum.SAG.1380.2 Cmerolae Streptophyta Rhodella.maculata.CCMP736 carragheen Gsulphuraria Haptophyta Erythrolobus.australicus.CCMP3124 Rhodosorus.marinus.UTEX.LB.2760 www.pnas.org/cgi/content/short/1517551113 Ppurpureum Madagascaria.erythrocladiodes.CCMP3234 Bacillariophyta Erythrolobus.madagascarensis.CCMP3276 Timspurckia.oligopyrenoides.CCMP3278 Compsopogon.coeruleus.SAG.36.94 Galdieria.sulphuraria Glaucophyta Rhodosorus.marinus.CCMP769 Ostreococcus.tauri Vcarteri Euglenozoa Picocystis.salinarum.CCMP1897 Micromonas.sp..RCC299 Mpusilla Otauri Dinophyta Chlorella.variabilis Creinhardtii Auxenochlorella.protothecoides Csubellipsoidea Ochrophyta hollow.green.seaweed Micromonas.sp.NEPCC29 Micromonas.sp.CCMP2099 Chlorophyta Cvariabilis Dunaliella.tertiolecta.CCMP1320 Pyramimonas.parkeae.CCMP726 Ostreococcus.lucimarinus.CCE9901 Cryptophyta Coccomyxa.subellipsoidea.C.169 Micromonas.sp.RCC472 Tetraselmis.striata.LANL1001 Chlamydomonas.reinhardtii Unknown Bathycoccus.prasinos shepherd.s.purse Pedicularis.kerneri Cercozoa Manoao.colensoi Zamia.kickxii long.grained.rice Cycas.media.subsp..media Rhodophyta Dacrydium.nausoriense gray.rockcress Zamia.integrifolia chickpea Castilleja.rubicundula Triphysaria.pusilla Gossypium.arboreum Pherosphaera.fitzgeraldii Ceratozamia.mirandae Encephalartos.hildebrandtii Chorispora.tenella Eutrema.halophilum Podocarpus.spathoides Eutrema.salsugineum Dontostemon.senilis Dacrydium.guillauminii Podocarpus.salignus Encephalartos.concinnus Encephalartos.caffer Encephalartos.sclavoi Encephalartos.aemulans Glycine.soja Pedicularis.kansuensis Encephalartos.cycadifolius Nicotiana.tomentosiformis Encephalartos.dyerianus Fig. S1. Selaginella.moellendorffii Encephalartos.gratus Boschniakia.himalaica muskmelon Pieris.nana Sterigmostemum.violaceum Altenstein.s.bread.tree Taxonomic distribution of the 67 S genes discovered in our study. The taxonomic distribution of the data is shown with black boxes indicating a presenc Tozzia.alpina Cordylanthus.ramosus Encephalartos.woodii Zamia.pumila Podocarpus.lucienii Cycas.litoralis Zamia.hymenophyllidia Euphrasia.collina Orobanche.californica Nicotiana.benthamiana Physcomitrella.patens Encephalartos.trispinosus common.sunflower wood.tobacco Euphrasia.alsa sweet.orange Japanese.rice Encephalartos.whitelockii Populus.euphratica distributionofthe67S-genefamilies Taxonomic Macrozamia.fraseri Encephalartos.aplanatus Eruca.vesicaria Japanese.apricot malo.sina Pedicularis.foliosa Boschniakia.rossica Cycas.chamberlainii Encephalartos.princeps Castilleja.exserta Afrocarpus.mannii Saccharum.hybrid.cultivar.R570 Podocarpus.aristulatus Solanum.demissum Strobilanthes.attenuata African.oil.palm Aptosimum.pumilum Encephalartos.nubimontanus Pedicularis.tuberosa castor.bean Nicotiana.attenuata Citrus.clementina Muricaria.prostrata Fragaria.vesca.subsp..vesca sugar.beet Podocarpus.guatemalensis Eucalyptus.grandis Encephalartos.kisambo Brassica.juncea wine.grape Zamia.pygmaea Zamia.acuminata Castilleja.tenuis Transcriptomes + Transcriptomes Podocarpus.cunninghamii Robeschia.schimperii Jatropha.curcas barrel.medic Ceratozamia.morettii Encephalartos.cupidus foxtail.millet Orobanche.ludoviciana Macrozamia.stenomera Nelumbo.nucifera Encephalartos.dolomiticus bugle apple Podocarpus.lawrencei Castilleja.miniata Cleome.hassleriana soybean Podocarpus.totara Zamia.neurophyllidia Lecomtella.madagascariensis spotted.monkey.flower Lepidozamia.peroffskyana Encephalartos.lehmannii Encephalartos.ituriensis Genome dataset Phaseolus.vulgaris Pyrus.x.bretschneideri Ceratozamia.mixeorum Encephalartos.longifolius Cycas.xipholepis Encephalartos.senticosus Encephalartos.transvenosus field.mustard Cycas.maconochiei Encephalartos.pterogonus Triticum.urartu Encephalartos.eugene.maraisii Afrocarpus.falcatus Glechoma.hederacea Cycas.calcicola Encephalartos.munchii Retrophyllum.comptonii Rheum.australe Morus.notabilis Zamia.portoricensis Madagascar.periwinkle Oryza.officinalis Encephalartos.lanatus + Encephalartos.lebomboensis Capsella.rubella Arabidopsis.lyrata.subsp..lyrata NCBI RefSeq sesame wild.Malaysian.banana Ceratozamia.miqueliana Encephalartos.middelburgensis Penstemon.cobaea Dacrydium.xanthandrum Lamium.purpureum sorghum Esterhazya.campestris domesticated.barley thale.cress Encephalartos.inopinus Retrophyllum.minus Chelone.obliqua Hirschfeldia.incana Afrocarpus.gracilior Camelina.sativa Aureolaria.pedicularia (349samples) Encephalartos.friderici.guilielmi Orobanche.pinorum cacao rape Podocarpus.coriaceus Aegilops.tauschii e and white boxes indicating presumed absence in the genome or transcriptome data from the taxon. Encephalartos.ngoyanus Sitka.spruce Orthocarpus.bracteosus Encephalartos.cerinus Macrozamia.plurinervia Cycas.thouarsii Dacrydium.balansae Polygonatum.pubescens Genlisea.aurea Encephalartos.ghellinckii Zea.mays Phtheirospermum.japonicum Populus.tremula black.cottonwood Castilleja.fissifolia Encephalartos.arenarius Pedicularis.julica Brachypodium.distachyon Castilleja.crista.galli date.palm Podocarpus.acutifolius cucumber Schizopetalon.walkeri tomato peach potato Ceratozamia.huastecorum Dacrydium.elatum Encephalartos.turneri Coffea.canephora Amborella.trichopoda Afrocarpus.usambarensis Nageia.formosensis Cucumis.melo.subsp..melo Rhodomonas.sp.CCMP768 Guillardia.theta.CCMP2712 Goniomonas.pacifica.CCMP1869 Gtheta Prymnesium.parvum.Texoma1 Emiliania.huxleyi.CCMP1516 Ehuxleyi Pleurochrysis.carterae.CCMP645 Emiliania.huxleyi.374 Isochrysis.galbana.CCMP1323 Emiliania.huxleyi.CCMP370 Isochrysis.sp.CCMP1244 Chrysochromulina.polylepis.CCMP1757 Emiliania.huxleyi.379 Pavlova.sp.CCMP459 Isochrysis.sp.CCMP1324 Emiliania.huxleyi.PLYM219 Gephyrocapsa.oceanica.RCC1303 Bnatans Lotharella.globosa.CCCM811 Aureoumbra.lagunensis.CCMP1510 Aanophagefferens Ectocarpus.siliculosus Chattonella.subsalsa.CCMP2191 Ochromonas.sp.CCMP1393 Pseudopedinella.elastica.CCMP716 Heterosigma.akashiwo.CCMP2393 Pteridomonas.danica.PT Aureococcus.anophagefferens Nannochloropsis.gaditana Heterosigma.akashiwo.CCMP3107 Dinobryon.sp.UTEXLB2267 Ngaditana Heterosigma.akashiwo.CCMP452 Vaucheria.litorea.CCMP2940