Osa* 1 MKLVQREAEKL.ALHNAGFLAQKRLARGLRLNYTEAVALIAAQILEFVRD......GD...RTVTDLMDLGKQLLGRRQVLPAVPHLLETVQVEGTFMDGTKLITVHDPISSDDGNLELA Bdi* 1 MKLVPREAEKL.ALHGAGFLAQKRLARGLRLNYTEAVALIASQILEFVRD......GD...KSVTDLMDLGKQMLGRRQVLPAVPHLLDTVQVEGTFMDGTKLITVHDPISSDDGNLELA Sbi* 1 MKLLPREADKL.ALHNAGFLAQKRLARGLRLNYTEAVALIAAQILELIRD......GD...KTVTDLMDLGKQLLGRRQVLPAVPYLLHTVQVEGTFVDGTKLVTVHDPISLDDGNLELA Zma* 1 MRLLPREADKL.ALHNAGFLAQKRLARGLRLNYTEAVALIAAQILELIRD......GD...KTVTDLMDLGKQLLGRRQVLPAVPYLLHTVQVEGTFVDGTKLVTVHDPISLDDGNLELA Ath* 1 MKLLPREIEKL.ELHQAGFLAQKRLARGIRLNYTEAVALIATQILEFIRD......GD...KSVAELMDIGRQLLGRRQVLPAVLHLLYTVQVEGTFRDGTKLVTVHEPISLENGNLELA Aly* 1 MKLLPREIEKL.ELHQAGFLAQKRLARGIRLNYTESVALIATQILEFIRD......GE...KSVAELMDIGRQLLGRRQVLPAVVHLLYTVQVEGTFRDGTKLVTVHEPISLENGNLELA Olu 1 MRLTPCEIDKLNILFAAGRVAQRRLARGMRLNHPEAVALIAMQCVELVRDPKFDKDGERRALTSVEVQDLGKRVLGRRHVLDGVPELVGDVQIETTFDDGTKLITIHDAICADDVDLELA Ota 1 MRLTPCEVDKLGALVAAGRLAQRRLARGIRLNHPEAVALIAMQCVEFARDPNFVIRGDARALTAAEVQDLGKRVLGRRHVLEGVPELVGDVQIETTFDDGTKLVTIHDAVCADEVDLELA Ppa* 1 MKLSPREVNKL.TLHGAGVLAQKRLARGLRLNHPEAIALIATQVLEFIRE......G....KSVAELMDLGKRMLGRRQVMPGVPYLVESVQVEGTFRDGTKLVTIHEPFSSEDGDLALA Smo* 1 MRLGPKEIDKL.LLHEAGFLAQKRLARGLPLNYPEAVALIAAQVLEFIRE......N....KSVAELMDLGRQLLGKRQVLPEVGHLLEMVQVEGTFADGTKLVTVHHPIVKENGDLALA Cen 1 MKLSPREVEKL.GLHNAGYLAQKRLARGVRLNYTEAVALIASQIMEYARD......GE...KTVAQLMCLGQHLLGRRQVLPAVPHLLNAVQVEATFPDGTKLVTVHDPISRENGELQEA Gma2* 1 MKLSPREVEKL.GLHNAGYLAQKRLARGLRLNYTEAVALIATQIMEFARD......GE...KTVAQLMCIGKHLLGRRQVLPEVQHLLNAVQVEATFPDGTKLVTVHDPISCEHGDLGQA Gma1* 1 MKLSPREIEKL.DLHNAGYLAQKRLARGLRLNYVETVALIATQILEFVRD......GE...KTVAQLMCIGRELLGRKQVLPAVPHLVESVQVEATFRDGTKLVTIHDLFACENGNLELA Mtr* 1 MKLCQREIEKL.QLHNAGFLAQKRLARGLKLNYPEAVALIATQIVEFVRN......GD...KTVSELMSIGRELLGRRQVLSAVPHLLETVQVEATFHDGTKLITVHDPIARENGNLVLA Csa* 1 MKLSPKELDKL.GLHNAGFLAQKRLARGLRLNYTEAVALIATQILEFARN......GD...KSVAELMELGPKLLGRRQVLPAVPHLVDSVQVEGTFPDGTKLVTVHNPFEEENGNLELA Cpa* 1 MKLTPREVEKL.GLHNAGYLAQKRLARGLRLNYAEAVALIATQILEFVRD......GE...KSVAELMDIGRQLLGRRQVLPAVPNLLESVQVEGTFLDGTKLITIHDPIASENGNLELA Mes* 1 MKLTPRELEKL.GLHNAGYLAQKRLARGLRLNYSEAVALIATQILEFIRD......GD...KTVAELMDVGKCLLGRRQVLPAVPHLLDSVQIEGTFPDGTKLVTVHNPIASENGNLELA Ptr* 1 MKLTPREVDKL.GLHNAGFLAQKRLARGRKLNYTEAVALIASQILEFVRD......GD...KSVAELMDIGKQILGRRQVLPAVPHLLDTVQVEGTFPDGTKLITVHNAIASENGNLELA Mal 1 MKLTPREIEKL.DLHNAGFLAQKRLARGLRLNYTEAVALIATQILEFVRD......GD...KTVAELMDIGRQLLGRRQVLPAVPHLLDTVQVEGTFPDGTKLITIHDAISSEEGNLELA Mgu* 1 MKLAPREIEKL.MLHNAGALAQKRLARGVRLNHVEAVALIATQILEFVRN......GD...KSVVELMDIGRQLLGRRQVLPSVPYLLETVQVEGTFPDGTKLITIHDPISCENGNLELA Stu* 1 MKLAPREIEKL.MLHNAGYLAQKRLARAQLLNYTEAVALIATQVLEFVRD......GD...KSVAELMDIGRQLLGRRQVLPTVPHLLDCVQVEGTFPDGTKLITIHDPIACENGNLDLA Vvi 1 MKLSPREVDKL.LLHNAGFLAQKRLASGLRLNYTEAVALIASQILAFVRE......GE...KTVAELMDIGKQLLGRRQVLPAVPHLLHTVQVEGTFPDGTKLVTVHDAIASENGNLDLA Spo 1 ..MQPRELHKL.TLHQLGSLAQKRLCRGVKLNKLEATSLIASQIQEYVRDG.........NHSVADLMSLGKDMLGKRHVQPNVVHLLHEIMIEATFPDGTYLITIHDPICTTDGNLEHA Kae A1 MELTPREKDKL.LLFTAALVAERRLARGLKLNYPESVALISAFIMEGARDG..........KSVASLMEEGRHVLTREQVMEGVPEMIPDIQVEATFPDGSKLVTVHNPII ♦1 ♦2 ♦3 Osa 111 LHGSFLPVPSLEKFS.....SVGVDDFPGEVRFCSGHIVLNLHRRALTLKVVNKADRPIQIGSHYHFIEANPYLVFDRQRAYGMRLNIPAGTAVRFEPGDAKTVTLVSIGGRKVIRGGNG Bdi 111 LHGSYLPVPSHEIFS.....GSDADDSPGEVHFCSGRIILNLHRRALTLKVVNKADRPIQIGSHYHFIEANPYLIFDRKRAYGMRLNIPAGTAVRFEPGDSKRVTLVSIGGQKVIRGGNG Sbi 111 LHGSFLPVPSPEKFS.....SDDVEEYPGEIHYSSTRIVLNLHRRALTLKVVNKADRPIQIGSHYHFIETNPYLVFDRKRAYGMRLNILAGTAVRFEPGDAKSVTLVSIGGHKVIRGGNG Zma 111 LHGSFLPVPSPEKFS.....SDDVEEYPGEIHYSSSRIVLNLHRRALSLKVVNKADRPIQIGSHYHFIETNPYLVFDRKRAYGMRLNILAGTAVRFEPGDAKSVILVSIGGHKVIKGGNG Ath 111 LHGSFLPVPSLDKFPE....VHEGVIIPGDMKYGDGSIIINHGRKAVVLKVVNTGDRPVQVGSHYHFIEVNPLLVFDRRKALGMRLNIPAGTAVRFEPGERKSVVLVNIGGNKVIRGGNG Aly 111 LHGSFLPVPSLDKFPE....AHE.DVIPGDMKYGDGSIIINHGRKALVLKVVNTGDRPVQVGSHYHFIEVNPLLVFDRRKALGMRLNIAAGTAVRFEPGERKSVKLVNIGGNKVIRGGNG Olu 121 LLGSFLPKPSEKAFPPA...TKEKTPAPGEVRTSKDSLELLAGRPKRKLKVTNTCDRPIQVGSHFHLIEANRFLTFDRRRAYGARLAIPSGTAVRFEPGESKTVNMVNIAGNRVVKGGNN Ota 121 LLGSFLPKPNAEAFPPA...AKEKTPAPGEICTSKDSLELLAGRLKRKLKITNTCDRPIQVGSHYHLIETNRFLKFDRRRAYGARLAIPSGTAVRFEPGESKTVSVVDIAGKRVVKGGNN Ppa 110 LHGSFLPVPPLGAFHF....HME.AMYPGKLITEKGEIVINQGRRAVMLTVSNRADRPIQIGSHYHFIETNPYLCFDREKAYGMRLNISAGSAVRFEPGDTKRVTLVSIGGKKVIKGGNG Smo 110 LHGSFLPVPSVDVFPE....FQE.QPAPGMLVLAPGHIFINAGRKGLKLTVSNTADRPIQVGSHYHFVETNPYLVFDREKSYGMRLNILAGTAVRFEPGETKTVALVSIGGNSVIRGGNG Cen 111 LFGSLLPVPSLDKFAE....TKEDNRIPGEILCEDECLTLNIGRKAVILKVTSKGDRPIQVGSHYHFIEVNPYLTFDRRKAYGMRLNIAAGTAVRFEPGDCKSVTLVSIEGNKVIRGGNA Gma2 111 LFGSFLPVPSLDKFAE....NKEDNRIPGEIIYGDGSLVLNPGKNAVILKVVSNGDRPIQVGSHYHFIEVNPYLTFDRRKAYGMRLNIAAGTAVRFEPGDSKSVKLVRIGGNKVIRGGNG Gma1 111 LFGSFLPVPSLDKFTE....NEEDHRTPGEIICRSENLILNPRRNAIILRVVNKGDRPIQVGSHYHFIEVNPYLTFDRRKAYGMRLNIAAGNATRFEPGECKSVVLVSIGGNKVIRGGNN Mtr 111 LFGSFLPVPSLDIFTE....NNEDNVIPGEIKTEDRMVILNAGREAVSLKVVNNGDRPVQVGSHYHFIEVNPYLTFDRRKAFGKRLNIASGTTTRFEPGESKSVILVSIGGNKVIQGGHN Csa 111 LEGSFLPVPSPEKFP.....LMESSVVPGEIICPNDKISINVGRKAVRLSVVNKGDRPIQVGSHYHFIEVNPSLVFDRSKAYGMRLNISAGSATRFEPGDPKSVTLVAIGGNQVIRGGNG Cpa 111 LYGSFIPVPSLDKFPA....IED.SKIPGEIIFGYGSISLNHGRKAVILKVVNTGDRPVQVGSHYHFIEVNPYLVFDRSKAYGMRLNIPAGTATRFEPGETKSVILVSIGGRKVIRGGNG Mes 111 LHGSFLPVPLLDKFPP....IED.NEIPGGFVFGYGNITINPGRKAVMLKVVNYGDRPIQVGSHYHFIETNPSLYFDRMKAYGMRLNILAGTAIRFEPGDCKSVLLVSIGGKKVIRGGNG Ptr 111 LQGSFLPVPSLDKFPA....IED.NEIPGAIIFGDGNVIINSGRKAVTLKVINTGDRPIQVGSHYHFIETNRSLLFDRRKAHGMRLNIPAGTAIRFEPGESKSVVLVSIGGKQVIKGGNG Mal 111 LRCSFLPVPSSEKFT.....RTEDDVHPGEIIFRSGDITLNPYRRAVVLKVINTGDRPVQIGSHYHFIEVNPSLVFDRKKAYGMRLNIPAGTATRFEPGENKSVKLVSIGGKRVIRGGNA Mgu 111 LHGSFFPVPSLDKFP.....PVEICNIPGELFFGPGRITLNLGRKAIVLKVTNTGDRPIQVGSHYHFIEVNPYLVFDRRKAYGLRLNIPAGTATRFEPGDTKSVTLVRIGGEQVIRGGNN Stu 111 LHGSFLPVPPQEKFP.....VIEDSKIPGQMCFGGGLIVLNPQRKAVILKVTNTGDRPIQVGSHYHFIEVNPSLIFDRMKALGMRLNIPAGAATRFEPGETRSVVLIGISGKKVIRGGNA Vvi 111 LHGSFLPVPSVDKFP.....DMEDDRIPGEIRYGGGTIMLNSCRKAIVLRVTNTGDRPIQVGSHYHFIEVNPALVFDRRKAHGMRLNIPAGTATRFEPGETKRVSLVRIGGKQVIRGGNC Spo 109 LYGSFLPTPSQELFPLEEEKLYAPENSPGFVEVLEGEIELLPNLPRTPIEVRNMGDRPIQVGSHYHFIETNEKLCFDRSKAYGKRLDIPSGTAIRFEPGVMKIVNLIPIGGAKLIQGGNS Kae B1 MPGEYHVKPGQIALNTGRATCRVVVENHGDRPIQVGSHYHFAEVNPALKFDRQQAAGYRLNIPAGTAVRFEPGQKREVELVAFAGHRAVFGFRG ♦4 ▲ ♦5 ♦6 K I Osa 226 IADGAVNRSQLNEVMEKVIANGFGHEDYPDSSEGIIG...DGTHDYSVDHEKYASMYGPTTGDKIRLGDTDLFAEIEKDYAIYGDECIFGGGKVLRDGMGQSAGYPASDCLDTVVTNAVV Bdi 226 IADGAVNSSQLNEVIKKVTENGFGHEDYPDASEGLIG...DGTLDCSIDHEKYCSMYGPTTGDKIRLGDTDLFAEIEKDFAVYGDECLFGGGKVLRDGMGQSAGYPASACLDTVITNAVV Sbi 226 IADGPIDSSRLNEVMQKVNANSFGHEDYPDAREGLIG...DGPFDCTVDREKYASIYGPTTGDKIRLGDTNLYAEIENDFAIYGDECVFGGGKVLRDGMGQATGYPESSCLDTVITNAVV Zma 226 IADGPIDSSSLNVVMQKVNANSFGHEDYPDAREGIIG...DGSFDCTVDHEKYASIYGPTTGDKIRLGDTNLYAEIEKDFAFYGDECIFGGGKVLRDGMGQASGYPESFCLDTVITNAVV Ath 227 IVDGLVDDVNWTVLMETMERRGFKHLEDIDASEGIAG..EDPRFTTMISREKYANMYGPTTGDKLRLGDTNLYARIEKDYTVYGDECVFGGGKVLREGMGQGIEQAEALSLDTVITNSVI Aly 226 IVDGLVDDVNWTVVMEIMERRGFRHLEDADASEGIVG..EDPRFTTTISREKYANMYGPTTGDKLRLGDTNLYARIEKDYTVYGDECVFGGGKVLREGMGQGIEQSEALSLDTVITNSVI Olu 238 LVNGPATADRLDEVMKRVIDGGFGHVDADDLGEG......EP...LMIPRHKYAHMYGPTIGDRVRLGDTNLYITPERDLTMKGEESKFGGGKTLREGMSQQAGVGDADSLDTIITNALI Ota 238 LVNGPATADRLEEVMKRVIDGGFGHAEADDLGEG......EA...LMIPRQKYAHMYGPTVGDRVRLGDTNLYITPERDLTMKGEESKFGGGKTLREGMSQQAGVGDADSLDTIITNALI Ppa 225 LASGPVDHSRLPTIMESILAKNFLHAHESNALTGVSG..VDSTLTFKVDKEHYAHIYGPTTGDKVQLGDTNLCAKVEKDYTFYGDECQFGGGKVLRDGMGQASGCGENETLDTVITNALV Smo 225 IAEGPVDSSRLPKIMEELSLRNFGHKQQNDALP.......VEDASCPISREVYTNIFGPTTGDKIKLGDTELYAQIEKDFTFYGDECKFGGGKVIRDGMGQGTGYKADETLDTVITNAVV Cen 227 IADGPVNETNLEAAMHAVRSRGFGHEEEKDASEGFTKEDPNCPFNTFIHRKEYANKYGPTTGDKIRLGDTNLLAEIEKDYALYGDECVFGGGKVIRDGMGQSCGHPPAISLDTVITNAVI Gma2 227 IADGQVNETNLREAMEAVCKRGFGHKEEEDASEGIT.GDPDSPFTTIIPREEYANKYGPTTGDKIRLGDTDLFAKIEKDFALYGDECVFGGGKVLRDGMGQSCGDPPAISLDTVITNAVI Gma1 227 IADGPVNDSNCRAAMKAVVTRGFGHVEEENAREGVT..GEDYSLTTVISREEYAHKYGPTTGDKIRLGDTDLFAEIEKDFAVYGDECVFGGGKVIRDGMGQSSGHPPEGSLDTVITNAVI Mtr 227 IVCGPVNDSKCIAAMEAVRTRGFKHKEDENAREGIT..GEDYSLTKLIPREEYANKYGPTIGDKIRLGDTNLFAEIEKDFAAYGDECVFGGGKVIRDGMGQSCGHSPDGSFDTVITNAVV Csa 226 IADGPVDSSKLKDVMEAVHARGFKHVEENNAREGIA..GIDDEFTTRLSREDYANRYGPTTGDKVRLGDTDLYAEIEHDFSVYGDECVFGGGKVIREGMGQSCGHPPTLSLDTVITNAVI Cpa 226 IVDGPVDDANLQAVMNSIKIRGFGNLEEANSSEGVTG..RDSAFTTVVSREAYGNMYGPTVGDKIRLGDTDLFAEIESDFTIYGDECVFGGGKVIREGMGQACGHAPSDSLDTVITNALI Mes 226 IVDGPVDDANYTAVSETIKSKGFGNKEEENAREGVTG..EDYDFTTVVSREAYANMYGPTTGDKIRLGDTNLYAEIERDFAVYGDECVFGGGKVIRDGMGQSCGVHPFDSLDTVITNAVI Ptr 226 IVDGPVDHENWTNIMGNIRRREFGNREEENASEGVIG..EGSAFNNTISREAYANMYGPTAGDKIRLGDTNLYAEIERDFAFYGDECVFGGGKVIRDGMGQSCGHQPADSLDTVITNAVV Mal 226 IVDGPVDDAKWEEVLEALSARGFGNKEEENASEGITG..ENLDFTAVISREAYANIYGPTTGDKIRLGDTNLYTEIERDFAVYGDECVFGGGKVLRDGMGQACGYPPDGALDTVITNAVI Mgu 226 IADGQVNDTNITSVMKAVHEGRYGFSEEANAMEGVIE..QGSSFSYSMSHETYANMYGPTTGDKIRLGDTDLLAEIERDYAVYGDECVFGGGKVLRDGMGQASGYQLSDCLETVITNAVI Stu 226 IADCPVDDAKVMTLMGALSEGGFGHLEEPNPREGVVG..EESCFSFSMTHEEYANMFGPTTGDRIRLGDTDLFAEIEKDFGIFGDECVFGGGKVLRDGMGQACGYPPADCLDTVITNAVV Vvi 226 IIDGPVDDTNITAVMESESMVRFGHSEEAHVSEGVIG..EDPDLAIRMSHEAYANMYGPTTGDKIRLGDTELYAEIESDFAVYGDECVFGGGKVIRDGMGQACMYAAAECVDTVITNAVV Spo 229 LSKGVFDDSRTREIVDNLMKQGFMHQPESPLNMPLQS.....ARPFVVPRKLYAVMYGPTTNDKIRLGDTNLIVRVEKDFTEYGNESVFGGGKVIRDGTGQSSSKSMDECLDTVITNAVI Kae B95 EVMGPLEVNDE MSNISRQAYADMFGPTVGDKVRLADTELWIEVEDDLTTYGEEVKFGGGKVIRDGMGQG.QMLAADCVDLVLTNALI ♦7

Osa 343 IDYTGIYKADIGINGGLIVAIGKAGNPDVMDMDGVNEEMIVGVNTEVIAAEGMIVTAGGIDCHVHFICPQLAEEAIASGITTLVGGGTGPAHGTCATTCTPSPSHMKLMLQSTDELPINM Bdi 343 IDYTGIYKADIGIKDGLIIAIGKAGNPDVMD..GVHSNMIVGVNTEVIAAQGMIVTAGGIDCHVHFICPQLAEEAIASGITTLVGGGTGPAHGTCATTCTPAPSQMKLMLQSTDEIPINM Sbi 343 IDYTGIYKADIGIKDGLIVAIGKAGNPDVMD..GVHSNMIVGVNTEVIASEGMIVTAGGIDCHVHFICPQLAEEAIASGITTLVGGGTGPAHGTCATTCTPAPSQMKLMLQSTDQLPINM Zma 343 IDYTGIYKADIGIKGGLIVAIGKAGNPDVMD..GVHNNMIVGVNTEVIASEGMIVTAGGIDCHVHFICPQLAEEAIASGITTLVGGGTGPAHGTCATTCTPAPSQLKLMLQSTDQLPINM Ath 345 IDYSGIYKADIGIKNGHIVGIGKAGNPDTMHG..VQNNMLIGNKTEVIAGEGMIVTAGAIDCHVHFICPQLVYEAVSSGITTMVGGGTGPAYGTRATTCTPSPFDMKLMLQSTDSLPLNF Aly 344 IDYSGIYKADIGIKNGHIVGLGKAGNPDTMHG..VQSNMLIGNKTEVIAGEGMIVTAGAIDCHVHFICPQLVYEAVSSGITTMVGGGTGPAYGTRATTCTPSPFDMKLMLQSTDSLPLNF Olu 349 VDHSGIYKADIGIKDGHIVGIGHGGNPDVAD...VTPGMVVGVNTEAIAAEGCIVTAGALDTHIHYICPQLCTEAVASGITTLLGGGTGPASGTCATTCTPSAAHMQFMLETTDALPLNF Ota 349 VDHSGIYKADIGIKDGHIVGIGHGGNPDIAD...VTPGMVVGVNTEAIAAEGCIVTAGALDTHIHYICPQLCVEAVASGITTLLGGGTGPASGTCATTCTPSAAHMRFMLETTDTLPLNF Ppa 343 IDHTGIYKADIGIKGGIIVGIGKAGNPDVMDG..VTEGMIVGVNTEAIAGEGMILTAGGIDSHVHYICPQLADEAIAAGLTTLIGGGTGPAHGTCATTCTPSSEHMRLMLQATDEIPLNI Smo 338 IDYTGIYKADVGIKHGYICGIGKAGNPDVMDA..VSSNMIVGVNTEVVAGEGLILTAGGIDSHVHFICPQLSTEAIASGLTTLIGGGTGPAHGTCATTCTPAVDQMRLMLRSTDDFPINF Cen 347 IDYTGIIKADIGIKDGLIASIGKAGNPDIMNG..VFSNMIIGANTEVIAGEGLIVTAGAIDCHVHYICPQLVYEAISSGITTLVGGGTGPAAGTRATTCTPSPTQMRLMLQSTDDLPLNF Gma2 346 IDYSGIIKADIGIKDGLIVSIGKAGNPDIMDD..VFFNMIIGANTEVIAGEGLIVTAGAIDCHVHYICPQLVDEAISSGITTLVGGGTGPTAGTRATTCTPAPSQMKLMLQSTDDLPLNF Gma1 345 IDYTGIIKADIGIKDGLIISTGKAGNPDIMND..VFPNMIIGANTEVIAGEGLIVTAGAIDCHVHFICPQLVYDAVTSGITTLVGGGTGPADGTRATTCTPAPNQMKLMLQSTDDMPLNF Mtr 345 VDYTGIFKADIGIKDGLIASIGKAGNPDVMHG..VN..MIFGANTEVIAGEGLIVTAGAIDCHVHFICPQLVYEAVSSGITTLVGGGTGPADGTRATTCTPAPNQMQMMLQSTDDLPLNF Csa 344 IDHSGIFKTDIGIKDGFIMTLGKAGNPDVMDG..VFSDLIIGANTEVIAGEGLLVTAGAIDCHVHFICPQLAYEAISSGITTLVGGGTGPAAGTCATTCTPSPVQMRMMLQSTDDLPLNF Cpa 344 IDYSGIFKADIGIKGGFIVALGKSGNPDIMDG..VFPNLIIGVNTEVIAGEGMIVTAGAIDCHVHFICPQLAYEAISSGITTMIGGGTGPADGTRATTCTPSPLQMKLMLQSTDGLPLNF Mes 344 IDYSGIYKADIGIRCGLIAAIGKAGNPDIMND..VHPEMIIGVNTEVIAGEGMIVTAGAIDCHVHFICPQLAYEAISSGITTLVGGGTGPADGTRATTCTPAPTQMKLMLQSTDDLPLNF Ptr 344 IDYSGIYKADIGIKDYLIHAIGKSGNPDVMN...VPSDMTIGVNTEVIAGEGMIVTAGGIDCHVHFICPQLAFESISSGITTLVGGGTGPADGTRATTCTPAPSHMKLMLQSTDDLPLNF Mal 344 IDYSGIFKADIGIRDGLIVSLGKAGNPDIMDG..VFSNMIIGVNTEVIAGEGKIITAGAIDCHVHFICPQLAYEAIASGITTLVGGGTGPAEGTRATTCTPAPSHMKLMLQSTDDLPLNF Mgu 344 IDYTGIYKADIGIKDGYITSIGKAGNPDIMND..VSPDMIIGVNTEVIAGEGMIVTAGAIDCHVHFICPQLAYEAITSGITTLVGGGTGPAHGTRATTCTPAPFHMKLMLQSTDELPLNF Stu 344 IDYTGIFKCDIGIKDGHIVSLCKAGNPDIMD.....SDAIIGVNTEVIAGEGMIVTAGAIDCHVHFICPQLAYEAISSGITTMVGGGTGPAHGTRATTCTPGHVHMELMLQSTDEIPLNF Vvi 344 IDYTGIFKADIGIKDGLIVSLGKAGNPDIMHG....AHMIIGVSTEVIAGEGMIVTAGAIDCHVHFICPQLAYEAISSGITTLVGGGTGPADGTRATTCTPAASHMKFMLQSTDDLPLNF Spo 344 IDHTGIYKADIGIKNGYIVGIGKAGNPDTMDN..IGENMVIGSSTDVISAENKIVTYGGMDSHVHFICPQQIEEALASGITTMYGGGTGPSTGTNATTCTPNKDLIRSMLRSTDSYPMNI Kae C76 VDHWGIVKADIGVKDGRIFAIGKAGNPDIQPN....VTIPIGAATEVIAAEGKIVTAGGIDTHIHWICPQQAEEALVSGVTTMVGGGTGPAAGTHATTCTPGPWYISRMLQAADSLPVNI ♦8 N N ♦9 not in Smo Osa 463 GFTGKGNTTKPDGLAEIIKAGAMGLKLHEDWGSTPAAIDNCLSVAEAFDIQVNIHTDTLNESGCVEHTIAAFKDRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTLNTVDE Bdi 461 GFTGKGNTAKPDGLPEIIKAGAMGLKLHEDWGSTPAAINNCLCVAEAFDIQVNIHTDTLNESGCVEHTIAAFKDRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTSNTVDE Sbi 461 GFTGKGNTSKPEGLAEIIKAGAMGLKLHEDWGTTPSAIDNCLSVAEDFDIQVNIHTDTLNESGCVEHTIAAFKDRAIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTSNTVDE Zma 461 GFTGKGNTSKPEGLAEIIKAGAMGLKLHEDWGTTPSAIDNCLSVAEDFDIQVNIHTDTLNESGCVEHTIAAFKGRAIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTSNTVDE Ath 463 GFTGKGNTAKPLELRHIVEAGAMGLKLHEDWGTTPAAIDNCLAVAEEYDIQVNIHTDTLNESGFVEHTINAFRGRTIHTYHSEGAGGGHAPDIIRVCGVKNVLPSSTNPTRPYTKNTVDE Aly 462 GFTGKGNTAKPLELQHIVEAGAMGLKLHEDWGTTPAAIDNCLAVAEEYDIQVNIHTDTLNESGFVEHTINAFRGRTIHTYHSEGAGGGHAPDIIRVCGVKNVLPSSTNPTRPYTKNTVDE Olu 466 AFTGKGNTASPEGLHEIIKAGAVGMKLHEDWGTTPAAIDNCLTIAEEYDVAVTIHTDTLNESCCVEKSIEAFKGRTIHTYHSEGAGGGHAPDIIKVCGEKMVLPSSTNPTRPYTKNTVDE Ota 466 AFTGKGNTASPEGLHEIIKAGAVGMKLHEDWGTTPAAIDNCLTIAEEYDVAVTIHTDTLNESCCVEKSIEAFKGRTIHTYHSEGAGGGHAPDIIKVCGEKMVLPSSTNPTRPYTKNTVDE Ppa 461 GFTGKGNTSDVEGLPEIIRAGAIGLKLHEDWGTTPAAIRNCLNVADEYDIQVTIHTDTLNESGCVEQSIEAFGGRTIHTYHSEGAGGGHAPDIIKVCGLPNILPSSTNPTRPYTVNTIDE Smo 456 GFTGKGNSSKVEGLDDIIRAGAIGLKLHEDWGTTPAAIDRCLDVADKFDIQVTIHTDTLNESGCVEHSIAAFKNRTIHTYHSEGAGGGHAPDIIKVCGLPNVLPSSTNPTRPFTKNTIDE Cen 465 GFTGKGSSSKPDELHEIIKAGAMGLKLHEDWGSTPAAIDNCLTIAEHHDIQINIHTDTLNEAGFVEHSIAAFKGRTIHTYHSEGAGGGHAPDIIKVCGIKNVLPSSTNPTRPLTSNTIDE Gma2 464 GFTGKGSSSKPDELHDIIKAGAMGLKLHEDWGSTPAAIDSCLTVADQYDIQINIHTDTLNEAGFVEHSIAAFKGRTIHTYHSEGAGGGHAPDIIKVCGMKNVLPSSTNPTRPLTLNTIDE Gma1 463 GFTGKGNSAKPDELHEIIRAGAMGLKLHEDWGTTPAAIDSCLTVADQYDIQVNIHTDTLNESGFVEHTIAAFKGRTIHTYHSEGAGGGHAPDIIKVCGEKNVLPSSTNPTRPYTHNTIDE Mtr 461 GFNGKGNCAKPDELHEIVKAGAMGLKLHEDWGTTPATIHNCLTVAEQYDIQVNIHTDTLNESGFVEHTIAAFEGRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTSPFTLNTIDE Csa 462 GFTGKGNSSKPDELYGIVRAGAMGLKLHEDWGTTPAAIDNCLTVAEKYDIQVNIHTDTLNESGFVEHTIAAFKERTIHTYHSEGAGGGHAPDIIRVCGVKNVLPSSTNPTRPFTMNTVDE Cpa 462 GFTGKGNSAKPDELHEIIRAGAMGLKLHEDWGSTPAAIDNCLTVAEQYDIQVNIHTDTLNESGFVEHTIAAFKKRTIHAYHSEGAGGGHAPDIIKVCGVENVLPSSTNPTRPFTSNTIDE Mes 462 GFTGKGNGAKPNELHNIVKAGAMGLKLHEDWGSTPAAIDTCLTVAGEYDIQVNIHTDTLNESGFVEHTIAAFNGRTIHTYHSEGAGGGHAPDIIKVCGVENVLPSSTNPTRPYTSNTIDE Ptr 461 GFTGKGNAAKPEELHKIIRAGAMGLKLHEDWGTTPAAIDNCLTVADEYDVQANIHTDTLNESGFVEDTIAAFKGRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPYTSNTIDE Mal 462 GFTGKGNSSTPDELHEIIKAGAMGLKLHEDWGTTPAAIDNCLAVAELHDVQVNIHTDTVNESGFVENTIAAFKGRTIHAYHSEGAGGGHAPDIIRVCGVKNVLPSSTNPTRPFTSNTIDE Mgu 462 GFTGKGNSSKEEGLHEIIKAGAMGLKLHEDWGTTPAAIDKCLSVAELYDIQVNIHTDTLNESGFVEHTIAAFKDRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTTNTVDE Stu 459 GFTGKGNSSKADGLHEIIKAGAMGLKLHEDWGTTPAAIDMCLTVADQYDIQVNIHTDTLNESGFVEHTIAAFKGRTIHTYHSEGAGGGHAPDIIKVCGVKNVIPSSTNPTRPFTLNTVDE Vvi 460 GFTGKGNSAKPDGLHEIIRAGAMGLKLHEDWGTTPAAIDNCLTVAEQYDIQVNIHTDTLNESGFVEHTIAAFKDRTIHTYHSEGAGGGHAPDIIKVCGVKNVLPSSTNPTRPFTSNTIDE Spo 462 GLTGKGNDSGSSSLKEQIEAGCSGLKLHEDWGSTPAAIDSCLSVCDEYDVQCLIHTDTLNESSFVEGTFKAFKNRTIHTYHVEGAGGGHAPDIISLVQNPNILPSSTNPTRPFTTNTLDE Kae C192 GLLGKGNVSQPDALREQVAAGVIGLKIHEDWGATPAAIDCALTVADEMDIQVALHSDTLNESGFVEDTLAAIGGRTIHTFHTEGAGGGHAPDIITACAHPNILPSSTNPTLPYTLNTIDE ♦10 not in Smo N S ▲ ♦11 N N♦12 Osa 583 HLDMLMVCHHLDRNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVITRTWQTANKMKRQRGRLPISSSPDAAEDNDNFRIRRYIAKYTINPAIVNGFSDFVGSVEV Bdi 581 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVIIRTWQTANKMKVQRGRLPGSGDSDPSKDNDNFRIRRYIAKYTINPAIVNGFSDFVGSVEV Sbi 581 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVITRTWQTANKMKVQRGSLPGSADSNAAQNNDNLRIRRYIAKYTINPAIVNGFSDFVGSVEV Zma 581 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRVGEVITRTWQTANKMKVQRGSLPGSGDANAAPDSDNLRIRRYIAKYTINPAIVNGFSDFVGSVEV Ath 583 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVISRTWQTADKMKAQRGAIDPNMA.....DDDNSRIKRYIAKYTINPAIANGFADLIGSVEV Aly 582 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVISRTWQTADKMKAQRGAIDPSMA.....DDDNSRIKRYIAKYTINPAIANGFADLIGSVEE Olu 586 HLDMLMVCHHLDPEIPEDVAFAESRIRAETIAAEDVLHDMGALSIMASDSQAMGRVGEVIRRTWQTAHSNKEQRGFLEEDAN....SGADNVRVKRYVAKYTINPAIAHGMSHKVGSLEV Ota 586 HLDMLMVCHHLDPEIPEDVAFAESRIRAETIAAEDVLHDMGALSIMASDSQAMGRVGEVIRRTWQTAHSNKEQRGFLEEDAN....SGADNFRVKRYVAKYTINPAIAHGMSHKVGSLEV Ppa 581 HLDMLMVCHHLNKNISEDVSFAESRIRGETIAAEDILHDMGAISMMSSDSQAMGRIGEVITRTWQTAHKMKSQRKQLPETRN....DDNDNLRIRRYIAKYTINPAIAHGVSHLIGSVEV Smo 576 HLDMLIVCHHLNRNIPEDVAFAESRIRNETIAAEDILHDMGAISMMSSDSQAMGRIGEVITRSWQTAHKMKLQRGPLPEDRE....NNNDNFRVRRFIAKYTINPAVAHGVSHIVGSIEV Cen 585 HLDMLMVCHHLDREIPEDLAFAHSRIRKKTIAAEDVLNDIGAISIISSDSQAMGRVGEVISRTWQTADKMKAQTGPLKCDSS.....DNDNFRIRRYIAKYTINPAIANGFSQYVGSVEV Gma2 584 HLDMLMVCHHLNREIPEDLAFACSRIREGTIAAEDILHDIGAISIISSDSQAMGRVGEVISRTWQTANKMKVQRGPLQPGES.....DNDNFRIKRYIAKYTINPAIANGFSQYVGSVEV Gma1 583 HLDMLMVCHHLNKNIPEDVAFAESRIRAETIAAEDILHDKGAISIISSDSQAMGRIGEVISRTWQTADKMKSQRGPLQPGE......DNDNFRIKRYVAKYTINPAIANGLSQYVGSVEA Mtr 581 HLDMLMVCHHLDKNCPEDVAFAESRIRAETIAAEDILHDMGAISIIASDSQAMGRIGEVISRTWQTANKMKSQRGPLQPDDS.....DNDNFRIKRYVAKYTINPAIANGLSRYIGSVEV Csa 582 HLDMLMVCHHLDRNIKEDVAFAESRIRKETIAAEDILHDMGAISIISSDSQAMGRIGEVISRTWQTAHKMKLAR....PSSS.....DNDNLRIKRYVSKYTINPAIANGFSQYVGSVEV Cpa 582 HFDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVICRTWQTAHKMKTQRGLLGPNGS.....DNDNFRIKRYIAKYTINPAIVNGISDFIGSVEV Mes 582 HLDMLMVCHHLDKNIPEDVAFAESRIRSETIAAEDILHDMGAISIISSDSQAMGRIGEVISRTWQTADKMKLQRGSIGPDGS.....DNDNFRIKRYIAKYTINPAVANGFAELIGSIEV Ptr 581 HLDMLMVCHHLDKNIPEDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVISRTWQTAHKMKSQRGLIGPGGS.....DNDNFRIRRYIAKYTINPAIANGLAKFVGSVEV Mal 582 HLDMLMVCHHLDKNIPEDVKFADSRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVIARTWQTAHKMKSQRGSIDPNGS.....NNDNLRIKRYVAKYTINPAIANGISQYVGSVEV Mgu 582 HLDMLMVCHHLDKNIREDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVICRTWQTANKMKSVRGPLESSAP.....QNDNLRIKRYIAKYTINPAIACGFSKYVGSVEV Stu 579 HLDMLMVCHHLCKNSREDVAFAESRIRAETIAAEDILHDMGAISIISSDSQAMGRIGEVICRTWQTAHKMKLFRGPLDIDGS.....DNDNFRIKRYIAKYTINPAIANGISQFVGSVEV Vvi 580 HLDMLMVCHHLDKDIPEDVAFAESRIRAETIAAEDILHDMGAISIIASDSQAMGRIGEVIIRTWQTAHKMKLQRGSLDASGV.....DNDNLRIKRYIAKYTINPAIANGFSRFVGSIEV Spo 582 ELDMLMVCHHLSRNVPEDVAFAESRIRAETIAAEDILQDLGAISMISSDSQAMGRCGEVISRTWKTAHKNKLQRGALPEDEG....SGVDNFRVKRYVSKYTINPAITHGISHIVGSVEI Kae C312 HLDMLMVCHHLDPDIAEDVAFAESRIRRETIAAEDVLHDLGAFSLTSSDSQAMGRVGEVILRTWQVAHRMKVQRGALAEETG.....DNDNFRVKRYIAKYTINPALTHGIAHEVGSIEV ♦13 ▲A ▲ N ♦14 ♦ 15

Osa 703 GKLADLVIWKPSFFGAKPEMVIKGGAIACANMGDPNASIPTPEPVMMRPMFGAFGGAGSANSIAFVSKAAKEAGVAVQYKLGKRVEAVGRVRGLTKLNMKLNDALPKIDVDPETYTVTAD Bdi 701 GKLADLVLWKSSFFGAKPELIIKGGAIAWANMGDPNASIPTPEPVMMRPMFGAFGKAGSSNSIAFVSKAAKEAGVASEYKLSKRVEAVGGVRGLTKLDMKLNDALPKIEVDPETYTVSAD Sbi 701 GKLADLVLWKPSFFGAKPELVVKGGAIAWANMGDPNASIPTPEPVVMRPMFGAFGKAGSSNSIAFVSKAAKEAGVAMEYKLEKRVEAVGGVRGLTKLDMKLNDALPRIEVDPETYTVTAD Zma 701 GKLADLVLWKPSFFGAKPELVVKGGAIAWANMGDPNASIPTPEPVVMRPMFGAFGKAGSSNSIAFVSKAAKEAGVATEYRLEKRVEAVGRVRGLTKLDMKLNDALPKIEVDPETYTVTAD Ath 698 KKLADLVIWQPAFFGAKPEMIIKGGNIAWANMGDANASIPTPEPVISRPMFGAFGKAGSENSVAFVSKAALRKGVKELYGLKKRVVAVSNVRQLTKLDMKLNDALPEITVDPETYVVTAN Aly 697 KKLADLVIWQPAFFGAKPEMIIKGGNIAWANMGDANASIPTPEPVISRPMFGAFGKAGSENSVAFVSKAALRNGVKELYGLKKRVVAVSNVRQLTKLDMKLNDALPDITVDPETYIVTAN Olu 702 GKFADVVIWKPAFFGAKPEIVVKGGQIAWAQMGDPNASIPTPEPVIMRGMFGALKPG..KTCIAFVSAAAAAADVGAEYGLNKRVEAVVKCRGLSKDDMVLNNACPRIEVDPETYEVRAD Ota 702 GKFADIVIWKPAFFGAKPEIVVKGGQIAWAQMGDPNASIPTPEPVIMRGMFGSLKPG..KTCIAFVSAAAAAADVGTEYGLYKRVEAVVKCRGLSKDDMILNNACPKIEVDPETYEVQAD Ppa 697 GKLADLVLWQPGHFGAKPEMVLKCGFIAWAQMGDANASIPTPEPKLMRPMFGAHGKACSSQSIAFVSKAALEAGVKAAYGLQKRVEAVQNVRNIGKADMKLNNATPVIEVDPETYHVTAD Smo 692 GKLADLVLWKPGFFGAKPELVIKGGDVAWAQMGDANASIPTPEPVIMRPMFAACGKAASSSSIAFVSKAAKDLNVGQAYGLTKRIEAVKNVRNLSKADMKLNSETPCIEVDPESYEVTAD Cen 700 GKLADLVMWKPSFFGTKPEMVIKGGMVAWADIGDPNASIPTPEPVKMRPMYGTLGKAGGALSIAFVSKAALDQRVNVLYGLNKRVEAVSNVRKLTKLDMKLNDALPEITVDPESYTVKAD Gma2 699 GKLADLVMWKPSFFGAKPEMVIKGGVVAWADMGDPNASIPTPEPVKMRPMFGTLGKAGGALSIAFVSKAAVDQRVHALYGLNKRVEAVGNVRKLTKLDMKLNDSLPQITVDPDNYTVTAD Gma1 697 GKLADLVLWKPSFFGAKPEMVIKGGEVAYANMGDPNASIPTPEPVIMRPMFGAFGKAGSSHSIAFVSKAALDEGVKASYGLNKRVEAVKNVRKLTKRDMKLNDTLPQITVDPETYTVTAD Mtr 696 GKLADLVLWKPSFFGAKPEMVIKGGDIAWANMGDANASIPTPEPVIMRPMFGAFGKAGRANSIAFVSKAALDYGVKALYGLDKRVEAVDNVRKLSKLDMKLNDALPEITVDPETYTVTAD Csa 693 GKFADLVLWKPAFFGAKPEMVIKGGIIAWANMGDPNASIPTPEPVLMRPMFGAFGKAGSANSIAFVSKEAVNIGIKAMYGLEKRVEAVGNVRKLTKLDMRWNDALPLIEVDPETYTVKAD Cpa 697 GKLADLVLWKPSFFGAKPEMVIKGGAIAWANMGDPNASIPTPEPVILRPMFGAFGKAGSANSIAFVSKAALVSGVKELYGLEKRVEAVGNVRGLTKHHMKLNDALPNITVDPETYRVTVD Mes 697 GKLADLVLWKPSFFGAKPEMVLKGGVIAWAEMGDPNASIPTPEPVISRPMFGAFGKAASANSIAFVSKIAADNGIKDSYGLSKRVEAVGNTRKLTKLDMKLNDALPDITVDPETYTVTAN Ptr 696 GKLADLVLWKPSFFGAKPEMVIKGGAIAWANMGDANASIPTPEPVISRPMFGAFGKAGSTHSIAFVSKEAADNGIKAEYELDKRVEAVGGVRKLTKLDMKLNDALPDITVDPETYTVTAD Mal 697 GKLADLVLWKPSFFGAKPEMIIKGGVIAWANMGDPNASIPTPEPVLMRPMFGAFGKAGSANSIAFVSKVAADNGIKNLYGLQKSVRAVNNVRKLTKLDMKLNDALPNITVDPETYTVTAD Mgu 697 GKLADLIIWKPAFFGTKPEMVVKGGTIAWSDMGDPNASIPTPEPVTMRPMFGAFGKAASSNSIAFVSKVALDLGIKEHYGLNKRLEAVSNVRKLTKLDMKLNDALPQITVDPEAYTVTAD Stu 694 GKLADLVVWKPSFFGAKPEMVIKGGVIAWSNMGDPNASIPTPEPVTMRPMFGAFSKAASSNSIAFVSKAALDAGIKDSYRLNKRVEAVTNVRNISKLDMKLNDALPDIKVDPETYTVTAD Vvi 695 GKVADLVLWNPSFFGAKPEMVIKGGVIAWANMGDPNASIPTPEPVMMRPMFGAFGKAGSANSIAFVSKVAAECGIKTHYGLTKRVEAVGNVRRLTKLDMKLNDALPVITVDPETYTVTAD Spo 698 GKFADLVLWDFADFGARPSMVLKGGMIALASMGDPNGSIPTVSPLMSWQMFGAHDPE...RSIAFVSKASITSGVIESYGLHKRVEAVKSTRNIGKKDMVYNSYMPKMTVDPEAYTVTAD Kae C427 GKLADLVVWSPAFFGVKPATVIKGGMIAIAPMGDINASIPTPQPVHYRPMFGALGSARHHCRLTFLSQAAAANGVAERLNLRSAIAVVKGCRTVQKADMVHNSLQPNITVDAQTYEVRVD ♦16 ♦17 Osa 821 GEVLRCQPTPTVPLSRNYFLF Bdi 819 GEVLTCQPATTVPLSRNYFLF Sbi 819 GEVLTCQPAPTVPLSRNYFLF Zma 819 GEVLTCQPAPTLPLSRNYFLF Ath 816 GEVLTCAPADSVPLSRNYFLF Aly 815 GEVLTCAPADSVPLSRNYFLF Olu 818 GVVLKSQPAQELPLARRYFIV Ota 818 GVVLKSQPAQELPLARRYFIV Ppa 815 KVPLVCEPAESLPLSQTYFLF Smo 810 NIPLVCSPAEKLPLATNYFLF Cen 818 GKLLCVSEATTVPLSRNYFLF Gma2 817 GEVLTSFATTFVPLSRNYFLF Gma1 815 GEVLTCTAAKTVPLSRNYFLF Mtr 814 GEVLTCAAATTVPLSRNYFLF Csa 811 GEVLTCQPATSVPLSRNYFLF Cpa 815 GEVLTCNAATSVPLSQNYFMF Mes 815 GEVLSCPASTTVPLSRNYFIF Ptr 815 GEVLTCPAATTVPLSRNYFLF Mal 815 GEVPTCDAATTVPLSKNYFLF Mgu 815 GEVLTCTAAATVPLSRNYFLF Stu 812 GTALTCPPATTVPLSRNYFLF Vvi 813 GVTLSCPAATTVPLSRNYFLF Spo 813 GKVMECEPVDKLPLSQSYFIF Kae C545 GELITSEPADVLPMAQRYFLF

Supplemental Figure S1. Multiple alignment of plant ureases with ureases from Schizosaccharomyces pombe and Klebsiella aerogenes. Osa, Oryza sativa, ssp. Nipponbare; Bdi, Brachypodium distachyon; Sbi, Sorghum bicolor; Zma, Zea mays; Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Olu, Ostreococcus lucimarinus, gi 145343758; Ota, Ostreococcus tauri, gi 151357863; Ppa, Physcomitrella patens; Smo, Selaginella moellendorfii; Cen, Canavalia ensiformis, gi 225714; Gma2, Glycine max 05g27840 embryo specific urease; Gma1, Glycine max 11g37250 ubiquitous urease; Mtr, Medicago trunculata; Csa, Cucumis sativus; Cpa, Carica papaya; Mes, Manihot esculenta; Ptr, Populus trichocarpa; Mal, Morus alba, gi 222143560; Mgu, Mimulus guttatus; Stu, Solanum tuberosum, gi 14599413; Vvi, Vitis vinifera, gi 225425840; Spo, Schizosaccharomyces pombe, gi 5731944; Kae, Klebsiella aerogenes urease subunits (A, gi 137084), (B, gi 137077) and (C, gi 137070). Sequences were either obtained from Genebank (gi numbers given) or predicted from completed plant genome projects (using Phytozome v5.0; www.phytozome.net). For Osa, Zma, Smo, Mtr and Ptr the coding sequence prediction in Phytozome was manually modified to better match the plant urease consensus. Two sequence differences for the Oryza sativa sequence from cultivar Hunan late indica No. 2 are annotated above the alignment. Alignments were generated with ClustalW and shading was performed with boxshade (www.ch.embnet.org). Amino acids of the active site in urease from K. aerogenes are highlighted by red background. N, nickel binding; S, substrate binding; A, catalytic acid. Black triangles mark amino acids that are not directly in the active site but have been experimentally confirmed to be important for enzyme activity in urease from K. aerogenes (Uniprot, www.uniprot.org). A red triangle marks the glycine residue that was found to be important for flexible hinge movement of the K. aerogenes -subunit to allow Ka-UreD and Ka-UreF binding for urease activation. Rhombs and numbers label canonical intron positions (exceptions are commented) generally found in ureases of all plant species for which complete genome sequences are available (organism names marked with * at position 1). The labeled amino acid is at least partially encoded by the exon preceding the intron.

MEAEAAM..AAA.AAATG Osa* 1 ..............................MEAEAAMAPAAAPAAATGAVRVEKVRGRSAVTRCFAKYPLKLIAP.SKAGRASSGAAWLYAITYGGGIVSGDIISCTVAVGDGCAAAMTT Bdi* 1 ...................................MELDAEPAAATTGVVRVEKVRGRSAVTRCFAKYPLKIIVP.SKVGHASSGAAWLYALTYGGGIVSGDRISCTVSVGDGCTAAMTT Sbi* 1 .........................................MAEAATGAVRVERVRGRSALTRCFARYPLKLIAP.SKVGPSSCDAVWLYALTYGGGIVSGDTVSCTVSVGDGCTVAITT Ath* 1 ............................................MATGKVVVEKVGGRSTATSCFSKYPLKFLLP.SKAAPAGTDVVWIYSITYGGGIVSGDSISCEFTIGDGCTAVITT Aly* 1 ............................................MATGKVVVEKVGGRSTATSCFSKYPLKFLLP.SKAAPAGTDVVWIYSITYGGGIVSGDSILCEFTIGDGCTAVITT Ppa* 1 .....................MAFMDSGRRSGWEKLLGPSFKDRALTGVIKVDNVAGKSAVTRTFAKYPLKFLLP.NKIVPSGIDAVWIYAISYGGGIVSNDSISQRVEVGPSCTAVITT Gma1* 1 MSYGAVVTHQSGSCTSLIQSNQRVALPSVDGRGRIMRSGEAVREMEMGSVVVEKVGGRSSVTSCFSRYPLKFIIP.KKVGSSKTDAVWVYALNYGGGIVSGDNISCKFSVGDTCTMVLTT Csa* 1 ...........................................MEVDGAVVVEKVAGKSAVTRCFSKYPLKFIIP.RKVGPSKTDCIWIYTLNYGGGIVSGDSISCELTVKDGSNAVLTT Cpa* 1 ............................................MERAKIAVEKVGSKSTVTRCFSKYPLKFIVP.LKAAPSNIDAVWIYSLTYGGGIVSGDLISCEFDIGDSCTLVLTT Mes* 1 ............................................METGKITVEKVNGKSTVTRCFSKYPLKFIIP.MKVVPSKTDAVWIYTLTYGGGIVSGDSISCEFNIGDGCTTVLTT Ptr* 1 ...........................................MERTGKVVVEKVGGKSTVTRCFSKYPLKFIVP.SKAGPCKTDSVWIYSLTYGGGIVSGDSISCEFEIGDGCTTVFTT Mal 1 ..................................MRESELRKEMEQTGTAVVEKVGDKSTVTRCYSKYPLKFIVP.KKLGNSKTDAVWIYTLTYGGGIVSGDSISCDFTISDGCTTVLTT Mgu* 1 ............................................MERGKVVVERVGGKSKVTRCFCKYPLKFIVPNKVGAAAAADAVWIYTITYGGGIVSGDSIAFDVTVGDGCTAVFTT Vvi* 1 ............................................METGFVAVEKIGGRSTVTRCFSKYPLKFIIP.RKVGSSITDAVWIYSLTYGGGIVSGDSISCGFTIGDGCTTVLTT Sly 1 ............................................METGKVIVEKVRGKSTLTKCFSKYPLKFINP.KNVAPSQTDVVWIYAITYGGGIVSGDSIVCDYTIGDGCTTVLTT Rco* 1 ............................................METGKVTVDKVGGKSTVTRCFSKYPLKFIIP.TKVGPSKTDAVWIYTLTYGGGIVSGDSISCEFIIGHGCTAVLTT Spo 1 ..........................................MEDKEGRFRVECIENVHYVTDMFCKYPLKLIAP...KT..KLDFSILYIMSYGGGLVSGDRVALDIIVGKNATLCIQS Kae 1 ...................................MLPPLKKGWQATLDLRFHQAGGKTVLASAQHVGPLTVQRP....FYPEEETCHLYLLHPPGGIVGGDELTISAHLAPGCHTLITM ♦ Gma1 only ♦1 not in Sbi ♦2 not in Csa jpred P 997004678745666404278888458885245676447057743 77777777458899985488558634588999864888178873 jpred B 8777777774588999985588658999861688457526 77777745888854787745524588999865888468853 Osa 90 QASTKVYKAV.DSKCSEQVLEARVGEDALFALIPDPVTCFSMARYHQKQVFHVFPN.SNLVVVDWFTSGRYESGEKWNFSFYKSINHILLED........QPLFIDSVLLEQSS...NFS Bdi 85 QASTKVYKAV.GLKCSEQVLEATVGKDALLAAIPDPVTCFSTARYYQKQVFHVSGD.SNLVLVDWFTSGRYESGEKWDFNSYKSVNHILSEEH.......QPLFIDSVLLEQGS...NCS Sbi 79 QASTKVYKAV.GSKCSEQLLEARVGEDALLAVIPDPVTCFSTARYHQKQVFHVSAN.SNLVVVDWFTSGRYESGEKWDFSFYKSVNHIFLGD........QPLFIDSTLLEQGS...TYS Ath 76 QSSTKVYKAI.GSKCSEQILEARIGSEALLVVIPDPVTCFSTARYYQKQIFRLLSD.SNLVLVDWITSGRHANGEKWDFEFYKSINNVYLEDD.......HPLFLDTVLLEKRS...IQS Aly 76 QSSTKVYKAI.GSKCSEQTLEARIGSEALLVVIPDPVTCFSTARYFQKQIFRLLSD.SNLVLVDWITSGRHANGEKWDFEFYKSINNVYLEDD.......HPLFLDTVLLEKRS...IQT Ppa 99 QSSTKIYKSV.EGKFCEQILQAYVGREGFFAVLPDPITCFKNSKYTQVQEFYLAAD.ANLVLIDWMTSGRVDNGESWEFELYKSINHIYLEDVKADCSKSTPLFLDCLCLEQGV...GTS Gma1 120 QGSTKVYKSV.GSKCSQQILEARVGSNALLAIIPDPVTCFSTARYCQKQVFCVLPD.SNLVMVDWITSGRHESGEKWDFDLYRSTNNIFLEDG.......QPLFLDTMLLDKEK...IGC Csa 77 QASTKVYKSR.GEELSEQLLEARIGSDALLAVLPDPVTCFATARYAQKQVFRVGSG.SSLVLVDWFTSGRHGSGEIWEFDLFKSTNQIFLEDG.......HPLFFDTVLLERGG...INT Cpa 76 QASTKVYKSI.DSRCSEQVLEARVGSGALFVVIPDPVTCFSTARYSQKQIFRVVSD.SNLVIVDWITSGRHESGEKWDFELYKSTNHIFLDQD.......QPLFLDTVLLEQGS...IVS Mes 76 QASTKVYKSL.GSKCSEQFLEARIGSDSLLAVIPDPVTCFSTARYSQKQVFRVLSD.SSLVIVDWITSGRHESGEKWDFEFYKSTNNIFLDHD.......QPLFLDTVFLEQGK...IAT Ptr 77 QASTKVYKSV.GSRCSAQFLEVTVGSDALLAILPDPVTCFSTARYSQKQVFRVLLD.SNLVIVDWFTSGRHESGEKWDFDLYKSTNNIFLDDN.......QPLFLDTVLLEQQS...ISP Mal 86 QASTKVYKSL.GSKCSVQVLEARVGNNALLAVIPDPVTCFSTARYTQKQVFRMLSD.ASLIVVDWVTSGRHESGEKWDFDLYKSTNHIFLEDN.......EPLLLDTVLLEKGS...MSS Mgu 77 QSSTKVYKSL.GSKSSQQTLEASVGQDALLVVIPDPVTCFSTAKYSQIQNFKLASG.SSLLLVDWITSGRHESGEKWAFSHFKTTNRILLDDN.......EPVFLDTMLLEQES...NSS Vvi 76 QASTKVYKSV.GSKCSEQVLEARIGSNALLAIIPDPVTCFSTARYSQKQVFRVFSD.SCLVIVDWITSGRHATGEKWDFELYKSSNHIFLD.D.......QPLFLDTVLLEQGS...VSS Sly 76 QASTKVYKAV.GTKISEQVLEARIGSNAFLAVIPDPVTCFSTAKYSQKQVFKVMSD.SSLLLVDWITSGRHETGEKWNFDLYRSMNNIFHNDD.......EPLFLDTALLEQGT...CSD Rco 76 QASTKVYKSL.GSKCSEQILEARIGSDSLLAVIPDPVTCFSTARYSQKQIFRVLSD.SSLVVVDWITSGRHESGEKWDFELYKSSNNIFLDDD.......QPLFLDTVLLEQGS...AGI Spo 74 QGNTKLYKQIPGKPATQQKLDVEVGTNALCLLLQDPVQPFGDSNYIQTQNFVLEDETSSLALLDWTLHGRSHINEQWSMRSYVSKNCIQMKIPAS..NQRKTLLRDVLKIFDEPNLHIGL Kae 82 PGASKFYRSS..GAQALVRQQLTLAPQATLEWLPQDAIFFPGANARLFTTFHLCAS.SRLLAWDLLCLGRPVIGETFSHGTLSNRLEVWVDN........EPLLVERLHLQEGE....LS ♦3 ♦4 ♦ Ppa only ♦5 Jpred P 5855788886 236899999876268855887537646677773588778885488 86688851013677777777642221322121578 88512356664488 746 Jpred B 7876265378 85689989898547741014677776777745888898875587 24788999874268877777764341312020578 88846888744777 77 Osa 197 IADRMQEYNVVAMVILLGPKLKHIQDQMQDEVKKMMSVQLRPPTSAGGRYSTRS....Q.PLHPQRPPIIASCSPFGRMGTGMVARITAVSTESVYSFLRHHLAALEPFLGACPYPAS.. Bdi 193 IAERMQEYNVVAMVVLLGPKLKQIQDKMQDEVKNMMSVQLRPPTSGGGRYATRP....Q.PLHPQRPPLIASCSPFGRTGTGMVAQVVAVSTESVYSFLRHHLAALEPFLGAAPYSAS.. Sbi 186 IVERMQEYNVIAMVVLLGPKLRHIQDKMQDEVKKLMSGQLRPPTSGGSLYTMRS....QLPQHPQRPQLVASCSPFGRTGTGMVARVAAVNTELAYSFLRHRLAELEPFLGAPPYAAS.. Ath 184 IAERMQDYQAIAMVILFGAKLKEIQKQVQENVKNMMSEQLQLSYSSRRHKSES.....SSRNRFMKPEFIASCSTFGPEGKGVVVRIASDSTESVYNFLRQQLAELEPVLGQAPYA.... Aly 184 IAERMQDYHAIAMVILFGAKLKEIQKQVQENVKNMMSEQLQILCS.RRHKSES.....SSSNRFMKPEFIASCSTFGPEGKGVVIRIASDSTESVYNFLKQQLAELEPLLGQAPYA.... Ppa 214 VAERMRGFHVVANMVIYGPKLAAFRVKVQKKVQELTQKAFTRRKSCDLTARLRDSSLSAESPSTSDPLLFVSCSSIGPANEGLVVRAVASTTALMYDFFKEQLAHIDSLIGACPYAGR.. Gma1 228 VQEHMHNYQVIAMIVLLGPKMQYIQNLVQDHVKKVMSEQLQHPSAAWSHQRDK......ADHFITKPSFIASCSAFGPKKIGLLVRVAAETTESVYKFLRHQLAPLEPMIGVPPY..... Csa 185 IIERMHGYQVIAMVVILGPKVKNIRDQVRENVKMIMGEQLHSPFTSARGPQMKM....NSNRLLTKPEIIASSSVFGPMGIGTVVRIAAMETETVYRFLQQQLASMETLLGVPPYK.... Cpa 184 IAERMQEYQVIAMVILLGPSLKHIQNQVQENVKRMMSEQLHMPSTSAGHHYRSN....SDPKRLAKPTIIASCSNFGPKGVGVVVRVAAITTESVYRFLQRQLVGLEPMIGISPFL.... Mes 184 ITERMHGYQVVAMVIILGPKLKHIQTQVQENVKRIMSEQLHMPFTGLGGHTKS.....NSSICFTKPPFIASCSLFGPKGVGVVVRIGALTTESVYKFLQQQLAGLEPLIGVLPYR.... Ptr 185 ITERMRGYQVIAMIILLGPKLKHIQSEVQENVKRMMSEQLHIPFTGLSGCAQS.....NS.RHFTKPSFIASCSVFGPKGIGVIVRVAAMTTESVYKFLQHQLVGMEPLIGVLPYH.... Mal 194 ITERMQEYQVVAMVVFWGPKMKHIQNQVQEEVRRMMTEQLQFYSPASAQRMKR.....SSTYCTNKPNFIVSSSVFGPQGKGVVVRIVATTTESVYTFLQHQLAGLEPLLGLSPYH.... Mgu 185 IADRLQDYQAIAMIVLWGPKLKPIQNQIQEDVKKLMSRQFRMPTIGSG..........FADSKLPKPSFLASCTTFGPQGTGVVIRIASTTTESVYKFLQHQLASMEAFLGASPYS.... Vvi 183 IAERMKDYQVIAMLVLLGPTLKHIQNQVQEDVKRMMSEQLRFPSTATGRHR.......SSDHRLLKPTFIASCSPFGPKGIGVVVRIAATTTESVYSFLRHQLAGMEPLLGVPPYC.... Sly 184 IAERMQDYQVIAMVILLGPKLKHVQNQIQEDVKKIMSQSLHMPTIGSRQSTSR.....HNDHHLTKPSFLASCSIFGPKGIGVVTRIAAMTTESVYNFLQHQLSSMEPLLGVKPYSYAS. Rco 184 IRERMRDYQVIAMVILLGPKLKHIQSQVQENVKRIMSEQLHMPFSGLSGNVKS.....NSSTFFTKPPFIASCSLFGPKGIGIVVRIAAMTTESVYRFLQHQLADMEPLIGVLPYR.... Spo 192 KAERMHHFECIGNLYLIGPKFLKTKEAVLNQYRNKEKRISKTTDS.................SQMKKIIWTACEIRS....VTIIKFAAYNTETARNFLLKLFSDYASFLDHETLRAFWY Kae 187 SIAERP..WVG..TLLCYPATDALLDGVRDALAP.....LG.......................LYAGASLTDRLLTVRFLSDDNLICQRVMRDVWQFLRPHLTGKSPVLPRIWLT.... ♦6 ♦7 Jpred P 777753603222111207748899999999999999875378888763677764 2 6787777458885378887458888875065588999999875367740025778899 Jpred B 634311 231 255258855789999999875 26 8877414774588887458647899999999999998752688777788899

Supplemental Figure S2. Multiple alignment of plant UreD proteins with UreD from Schizosaccharomyces pombe and Klebsiella aerogenes. Osa, Oryza sativa, ssp. Nipponbare; Bdi, Brachypodium distachyon; Sbi, Sorghum bicolor; Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Ppa, Physcomitrella patens; Gma1, Glycine max 02g20690; Csa, Cucumis sativus; Cpa, Carica papaya; Mes, Manihot esculenta; Ptr, Populus trichocarpa; Mal, Morus alba, gi 222143566; Mgu, Mimulus guttatus; Vvi, Vitis vinifera; Sly, Solanum lycopersicum, gi 31096385; Rco, Ricinus communis; Spo, Schizosaccharomyces pombe, gi 2104425; Kae, Klebsiella aerogenes, gi 731078. Sequences were either obtained from Genebank (gi numbers given) or predicted from completed plant genome projects (using Phytozome v5.0; www.phytozome.net). For Ptr, Gma1 and Mgu the coding sequence prediction in Phytozome database was manually modified to better match the plant UreD consensus or the available EST data. The Osa (Nipponbare) sequence was deduced from cDNA cloned in this work. Differences in the N-terminal sequence between ssp. Nipponbare and Indica Hunan late no. 2 are annotated above the alignment. These differences result from a microsatellite-like sequence in the 5’ end of rice ureD coding sequences. Alignments were generated with ClustalW and shading was performed with boxshade (www.ch.embnet.org). Rhombs and numbers label canonical intron positions (exceptions are commented)

generally found in ureDs of all plants for which complete genome sequences are available (organism names marked with * at position 1). The labeled amino acid is at least partially encoded by the exon preceding the intron. The gene from P. patens only contains one intron in a non-canonical position. Jpred P: a secondary structure prediction for UreD from O. sativa using the plant UreD alignment without the microbial sequences (Spo and Kae). Jpred B: a secondary structure prediction for UreD of K. aerogenes using an alignment of 100 bacterial UreD sequences obtained with NCBI Concise Microbial Protein BLAST (www.ncbi.nlm.nih.gov/genomes/prokhits.cgi) using the Kae sequence as query. Secondary structure predictions were performed using the Jpred 3 server (www.compbio.dundee.ac.uk/www-jpred/index.html). Prediction confidence is given as numerical value (highest confidence = 9) and predicted -helical regions are marked in blue while -folds are marked in green.

Osa 1 MERVMECDYPASKKNKVVHPMDCEMKEEPTN.....AASMNQHSLWSQWQLLDSILPTGGFAHSYGLEAAMQSRMVNNPEELRSFVVQVLEN.TGSLLLPFVCCANK..SP.DAATWVKL Bdi 1 ....MERDHTALKKMRLDDAADSPMTEVLAT.....ASSMNQQLFWSQWQLLDSILPTGGFAHSCGLEAAMQSRIVNNPEDLRLFLLQALEN.IGSLLLPFVYCASK..SP.DATTWVKL Sbi 1 ..MLMESDSPVSKKSRLFHTEDCEMEEVPSN.....AVGVNPSLQWTQWQILDSILPTGGFAHSYGLEAAMQSRMVNNQDDLKSFVIQVLDN.TGSLLLPFVYCASK..SP.DAAAWIKL Zma 1 ....MEPGSPAPKKSRLVHSADCEMEEAQAPSSSNAAGGVNQSLHWTQWQILDSILPTGGFAHSYGLEAAMQSRVVNDQEDLRSFVVQVLDS.AGSLLLPFVHCACAGKSPGDAAAWAKL Ath 1 ......................MEEDERRDIV....MSRASSCMQWSQWQLLDSILPTGGFAHSFGLEAAIQTRLVSSPEDLETHIIHVLDN.TASLLLPFVYSALK..SP.DIETWHKL Aly 1 ......................MEVDERSDIV....MSRTASCMQWSQWQLLDSILPTGGFAHSFGLEAAVQTRLVSSPEDLETHIIHLLDN.TASLLLPFVYSALK..SP.DIETWHKL Ppa 1 .....................MASEGEEGEVP....SQRDAGAVAWTIWQLIDSVLPTGGFAHSFGLESAAQAGLVFDAHTLEKFAVTAMEN.TGSLLLPFVFAAVEG.RVMSLEEWIGL Smo 1 ......................MDLPAKKSQG...........SDWVVWQLVDSFFPTGGFAHSYGLEAAVQAGLVSDSKSLDTFIKSTLEN.SGSLLLPFVSASFK..LP.DVAGWIEL Gma2 1 ......................MQVNEGHNRP....C.SDP.FLQWSKWQLLDSLLPTGGFAHSFGLEATVQSHLVSNSNDLKTFVIHILEN.TGSLLLPFVYSASM..LP.NLETWHKL Gma1 1 ......................MQVNEEHNKP....C.SDP.FLQWSQWQLLDSLLPTGGFAHSFGLEAAVQCHLVSDSNDLKTFVIHILEN.TGSLLLPFVYSASM..LP.NLETWHKL Mtr 1 ......................MQTNEEREKP....V.FENSFLQWSQWQLLDSILPTGGFAHSFGLEAAVQSRLVSDSNELKTFLIHVLEN.TGSLFLPFVYLSCM..SP.NMETWHKL Csa 1 ......................................MDDKHCHWSQWQLLDSILPTGGFAHSFGLEAAIQAQIVSSPDDLKTFVIHLLDN.TGSLFLPFVHSATQ..SP.DFETWKKN Cpa 1 ......................MEMNGGS...............DWSQWQLLDSILPTGGFAHSFGLEAAVHARVVSCPQGLQTYVTHVLDN.TGSLLLPFVYSAAL..SP.SLETWHKL Mes 1 ..................MEDDMIIDKKRRKI....A.STDFLLQWSQWQLLDSILPTGGFAHSFGLEAAMQARIILGHEDFKNQVIHILEN.AGSLLLPFVYSATL..SP.DLDTWQRL Ptr 1 ..................MEGKKEIDGAKEKP....A.STSFSLHWSQWQLIDSILPTGGFAHSFGIEAAIQARVILNPEDFQTYVIHVLEN.TGSLLLPYVYSAAM..CP.DLDNWRKL Mal 1 ....................MKMETDNEREKS....SKVEPSVLQWSQWQLLDSILPTGGFAHSLGLVINPIP.PISSHEDLQTFTIHLLHN.TGSLLLPYVYAAAT..AP.DTATWRRL Mgu 1 .....................CEGEPNEDNAE....SSTAGPLQLWSQWQLLDSILPTGGFAHSFGLEAAMQSRLVVSSEDLKTYIIHTLEN.TGSLLLPFVYSATI..SP.NTQSWHNL Sly 1 ...................MENMEEKIEGSLQ....F.APKELLQWSQWQLLDSVLPTGGFAHSFGVEAAIQARLVSGPEDLRTFVIHILEN.TGSLLLPFVYTLNT..SP.NVETWYKL Vvi 1 ..................MEVPMEIDKGSHGP....A.STNALLQWSQWQLLDSILPTGGFAHSCGLEAAFQAHMISGPEDLQTYVLHVLEN.TGSLLLPFVYSANM..SP.NLETWHKL Spo 1 .....................................MTDSQTETHLSLILSDTAFPLSSFSYSYGLESYLSHQQVRDVNAFFNFLPLSLNS.VLHTNLPTVKAAWE..SP...QQYSEI Kae 1 ......................................MSTAEQRLRLMQLASSNLPVGGYSWSQGLEWAVEAGWVLDVAAFERWQRRQMTEGFFTVDLPLFARLYRACEQGDIAAAQRW Hpy 1 .............MDKGKSVKSTEKSVGMPPKTPKTDNNAHVDNEFLILQVNDAVFPIGSYTHSFGLETYIQQKKVTNKESALEYLKANLSSQFLYTEMLSLKLTYESALQQDLKKILGV Jpred 99888877777640101145653567776645 6776468999999875378888746899999987447887568999999998852 52665689999985 47 66899999 2WGL < HHHHHHHHHHHH HHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHH HHHHHHHH Model HHHHHHHHHHH HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH Osa 112 DQLLEAMLTNEVSRKASMSQGSALLRVAASVFTEI.QSLQDLRQTFLGSKI.....VSFHHAPIFGLICGLVGFDSETTQRAYMFVTMRDVISAATRLNLIG..PLAASVLQHQVAEDAE Bdi 108 DQLLEATLTNEVGRKASTSQGSALLRVAASVFTEI.HSLQDLRKNFLGSTS.....VSFHHAPLFGLICGLVGFDCETTQRAYMFVTMRDVISAATRLNLIG..PLAASVLQHQVAPDAE Sbi 110 DQLLEATLTNEVARKASTSQGSALLRVAASVFTES.QALQDLRRTFLGSKS.....VSFHHASIFGLICGLVGFDSETAQRAYMFVTMRDVFSAATRLNLIG..PLAASVLQHQVAPDAE Zma 116 DRLLDATLTNEVARKASAAQGSALLRVAASVFAEV.PALQELRRTLLGSKS.....VSFHHAPVFGLVCGLVGFDGETAQRAYMFVTMRDVLSAATRLNLIG..PLAASVLQHQLAPDAE Ath 91 DGILNATLTNQVSSKASMSQGSALFRIAASVFTEV.PNLKMIRDASLGSKN.....VCFHHAPIFGLVCGLLGMDSETSQRAYLFVTLRDVLSAATRLNIVG..PMGASVMQHRIAIVTE Aly 91 DGILNATLTNQVSSKASVSQGSALFRIAASVFTEI.PNLKMIRDASLGSKN.....VYFHHAPIFGLVCGLLGMDPETSQRAYLFVTLRDVLSAATRLNIVG..PMGASVMQHRIAIVAE Ppa 94 DCALHAVMSNGVARAASVGQGKALLRLAGGVFKEIAGEVAAMRVSVMGKPPR....AHGHHALVVGRLLGLLGVDASTAQRAYLYLTLRDVLSAATRLNLVG..PMEAALLQHRHAQLAE Smo 84 DRLLNATLSNHVARRASSSQGSALLRTAATVYPDL.AELGELRGVVRAGR......ASGHHAGVFGIVCGLLKLDALTCQRAYLYLTLRDVLSAATRLNLVG..PLQAAAMQQSLRGFGE Gma2 89 DKILDATLTNEVGRKASISQGSALMRVASAVFSEV.PSLKTMRDASLGLET.....VSFHHAPVFGLICGALGFDRTSSQRAYMFITMRDVISAATRLNLIG..PLGAALLQHRVAPIAE Gma1 89 DKILDATLTNEVGRKASISQGSALMRVASAVFSEV.PSLKTMRDTSLGLGT.....VSFHHAPVFGLICGALGFDKTSSQRAYMFITMRDVISAATRLNLIG..PLGAALLQHQVAPNAE Mtr 90 DRILDATLTNEVGRKASISQGSALMRVASAVFAEI.PSLKTMRDSSMKLGT.....VSFHHAPIFGLTCGALGFDSTSSQRAFMFITMRDVVSAATRLNLIG..PLGAALLQHQVAPIAE Csa 79 DMLLDAMLTNEVSRKASVTQGSALMRVSAIVFSEI.PSLKAMRENLYGTGA.....VSFHHAPIFGLICGLLGWDGTMSQRAYLFITLRDVISAATRLNLVG..PLGAAVLQHQLAFVAE Cpa 80 DRMLDATLTNEVSRKASVSQGSALMRVAATVFPEV.PSFKTMRDVSLTSRA.....VSFHHAPIFGLICGLLGIDSGTSQRAYMFVTMRDVISAATRLNLVG..PMGAAVMQHQISLVAE Mes 94 DKILDATLTNEVSRKASIAQGSALMRVAAAVFTEL.PYLKAMRDACIGSGA.....VSFHHAPIFGMICGLLGMDSETSQKAYIFITMRDAISAATRLNLVG..PLGAAVLQHQLCVVAE Ptr 94 DRMLDATLTNEVSRKASVSQGSALMRVAAAVFTEI.PSLKIMREMSLGSGI.....VAFHHAPVFGIVCGLLGMDSETSQRAYMFITLRDAFSAATRLNLVG..PLGAAVLQHQVSIAAE Mal 92 DKALDATLTNEVARKASIAQGSALMRVAAAVFADTGPSIKAMRDSCLGGSR.....MNFHHAPVFGLVCGLLGMDSATAQRGYMFITMRDVISAATRLNLVG..PLGAAVMQHRIALLAE Mgu 92 DKTLNATLTNEIARKASIAQGSALMRVAASVFTEI.PCLKTMRAAALSGG......VYFHHAPVFGLVCGLLGFDAETTQRAYMFITMRDVISAATRLNLVG..PLGAAVLQHNVGPIAE Sly 93 DKILDATLTNEVSRKASISQGSALLRVAAAVFQEV.PYFKTMREVSLASGA.....VRFHHAPIFGLVCGLLGLNAETSQKAYLFITMRDVVSAATRLNLVG..PLGAAVLQHQLAANAE Vvi 94 DRMLDATLTNEVGRKASIAQGSALMRVAATVFSEV.PSLKMMRSNSLGSGT.....VAFHHAPIFGLVCGLLGLDVGISQRAYMFITMRDVISAATRLNLIG..PLGAAKLQHDIAIAAE Spo 78 EDFFESTQTCTIAQKVSTMQGKSLLNIWTKSLSFFVTSTDVFKYLDEYERRVRSKKALGHFPVVWGVVCRALGLSLERTCYLFLLGHAKSICSAAVRLDVLTSFQYVSTLAHPQTESLLR Kae 83 TAYLLACRETRELREEERNRGAAFARLLSDWQPDCPPPWRSLCQQSQLAG..............MAWLGVRWRIALPEMALSLGYSWIESAVMAGVKLVPFG..QQAAQQLILRLCDHYA Hpy 108 EEVIMLSTSPMELRLANQKLGNRFIKTLQAMNELDMGEFFNAYAQKTKDPT.........HATSYGVFAASLGIELKKALRHYLYAQTSNMVINCVKSVPLS..QNDGQKILLSLQSPFN Jpred 99999998727899998727999999875378888 746889887438863 5358558899999988534689999999999999886104378710 7899999999999999 2WGL HHHHH HHHHHHHHHHHHHHHHHHH HHHHHHHHH H.........HHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH H..HHHHHHHHHHH Model HHHHHH HHHHHHHHHHHHHHHHHHHHH HHHHHH HHH..............HHHHHHH HHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH Osa 224 RMVQKWKDRGVEEATQTSPLLDALQGCHAYMFSRLFCT Bdi 220 KMVQKWRDRDVSEASQTAPLLDALQGCHAYMFSRLFCS Sbi 222 GMMQKWRDRDVSEASQTAPLLDVLQGCHAYMFSRLFSS Zma 228 RMVRKWRDRDVSEASQTAPLLDAVQGCHAYMFSRLFCS Ath 203 TVLEKWMNREAGEACQTSPLLDVVQGCHGYLFSRLFCS Aly 203 TVLEKWMDREASEACQTSPLLDVVQGCHGYLFSRLFCS Ppa 208 RVMGKYANRSVHEAHQIAPLLDTLQGSQTLLFSRLFCS Smo 195 AVVRKCADRGVEDACQVSPLLDTAQACHDHLFSRLFCS Gma2 201 VILEKWMNRVVEEACQTMPLLDTVQGCHGYLFSRLFSS Gma1 201 VILEKWMNRAVDEACQTMPLLDTVQGCHGYLFSRLFSS Mtr 202 VILEKWMNRDVEEACQTMPLLDTVQGCHGYLFSRLFSS Csa 191 DILKRWMNRPVEEACQTVPLLETVQGCHSCLFSKMFCS Cpa 192 VLTKKWMDRVAEDACQTAPLLDTVQGCHGYLFSRLFCS Mes 206 SVLEKWMDHTVEEACQTAPLLDTLQGCHSYLFSRLFCS Ptr 206 TMLKRWMNREVEDACQTAPLLDTLQGCHGYLFSRLFCS Mal 205 EIVIKWMDRSVEDACQVAPLLDTVQGCHAYLFSRLFCS Mgu 203 SLSIKWMNRGVEEACQTCPLLDTIQGCHGYLFSRLFCS Sly 205 DLSKKWMNRPVEEACQTSPLLDTIQGCHGYLFSRLFCS Vvi 206 EMSKKWMNRKFEEACQTAPLLDTVQGCHAYLFSRLFCS Spo 198 DSSQLALNMQLEDTAQSWYTLDLWQGRHSLLYSRIFNS Kae 187 AEMPRALAAPDGDIGSATPLAAIASARHETQYSRLFRS Hpy 217 QLIEKTLELDESHLCTASVQNDIKAMQHESLYSRLYMS Jpred 99999874478856899999999999999999987289 2WGL HHHH > Model HHHHHHH HHHHHHHHHHHH HHH

Supplemental Figure S3. Multiple alignment of plant UreF with UreF from Schizosaccharomyces pombe, Klebsiella aerogenes and Helicobacter pylori. Osa, Oryza sativa, ssp. Nipponbare; Bdi, Brachypodium distachyon; Sbi, Sorghum bicolor; Zma, Zea mays; Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Ppa,

Physcomitrella patens; Smo, Selaginella moellendorfii; Gma2, Glycine max 14g04380; Gma1, Glycine max 02g44440; Mtr, Medicago trunculata; Csa, Cucumis sativus; Cpa, Carica papaya; Mes, Manihot esculenta; Ptr, Populus trichocarpa; Mal, Morus alba, gi 222143564; Mgu, Mimulus guttatus; Sly, Solanum lycopersicum, gi 31096389; Vvi, Vitis vinifera; Spo, Schizosaccharomyces pombe, gi 2239223; Kae, Klebsiella aerogenes, gi 137097. Sequences were either obtained from Genebank (gi numbers given) or predicted from completed plant genome projects (using Phytozome v5.0; www.phytozome.net). For Vvi and Mtr the start site was manually corrected after comparing with sequences from expressed sequence tags. Alignments were generated with ClustalW and shading was performed with boxshade (www.ch.embnet.org). Jpred: a secondary structure prediction for UreF from O. sativa using the alignment without Hpy and the Jpred 3 server (www.compbio.dundee.ac.uk/www-jpred/index.html). Prediction confidence is given as numerical value (highest confidence = 9) and predicted -helical regions are marked in blue while -folds are marked in green. 2WGL: experimentally determined secondary structure of UreF from H. pylori (pdb accession number: 2WGL; the structure of the C- and N-terminal ends was not determined). Model: secondary structure elements (for UreF from K. aerogenes) of the bacterial UreF model from Salomone-Stagni et al., 2007.

Vvi 1 MASQDQMALRH H Osa* 1 ..............................................................................MASHDHDHDHHHHHSHDDGDHHHSHHQ...DGSHGGG...GG Bdi* 1 ..............................................................................MASQDHHHDGGHSHSHDEGGHHHHSH...GDAAAAGGRGTGA Sbi* 1 ......................................................................................MASHDHHHSHEHEHEHGHE.DGGGHGSGG..... Zma* 1 ....................................................................................MASHDHHHSHSHDDHHHHSHG.DGGGHAAGG..... Ath* 1 .......................................................................................MASHDHHHHHHDHEHDHDRKSDGGEGKA..... Aly* 1 .....................................................................................MASHDHHHHHHHHDHEHGHDRKSDGGEGKS..... Olu 1 ..............................................................................................MSGDAHAHAHSHGSDEVEFG...... Ota 1 ............................................................................................MSGDAHAHAHAHADGNDDVEFG...... Msp 1 .....................................................................MGCGEKDCDGTDGHGHGHSHGGDAQAHGHSHGDAGGDATPLIQDGEGKDGI Ppa* 1 .....................................................MEGHSHSHDHSHDHSHDLSHGHSHDHSHDHSHDDHTHDHDHQHGLDHSHAHP.............AQ Smo* 1 ..........................................................................................MAEHHHHHDHGEAHG..............A Gma* 1 ..........................................................................MASEGDHHHHHHHHQDHDHHHHDHDHHHHHD..........GEGET Mtr* 1 ................................................................................MASGGDHTHVHDHDHHHHDHDHKHG............... Csa* 1 ........................................................................MASQDTHHHHHHHDGHDHHHHHHDGHDHHHTHE..........KPKGD Mes* 1 ...............................................................................MASHDHHAHDHHHHHHDQDHHHDTHN............NQT Ptr* 1 ............................................................................MASQDHHTHDHHHHDHDHHHHQHDHTHDE.............KT Mal 1 ...............................................................................MASDDHHHHHDHHHHHDHDHDHDHHG...........DSRT Mgu* 1 ...........................................................................MASHHHHEHVDHDHDHGHGNEHHHHHHTHE............... Stu 1 ..............................................................................MASSGHHMHDHDHHDHDNHHHHHHTHE...........DTKA Vvi* 12 DHKHHHDHHHDHHHDHHHGHKHGHHGHHGHHHHRHHHHRRHPDNKEPSNDQHHPSSDDQKQKESDSHDDDHDCDGNDSHGEDYEPGNDHHHESSDDHKHHHHGHP...........DMKA Rco* 1 .................................................................................MDSSHDHHHTHDHEHDHQHHRHPN.............EK Spo 1 ...................................................................MAIPFLHKGGSDDSTHHHTHDYDHHNHDHHGHDHHSHD............... Kae 1 ........................................................................................................................ ♦ 1 not Gma + Mtr Osa 37 .SWVGEDGRVWHSHDGLAPHSHEPIYSPGDFSKRA..PPLISRRFAERAFTVGIGGPVGTGKTALMLALCRSLR.EKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Bdi 40 GSWVGEDGRLWHSHDGLAPHSHEPIYSAGDFSKRA..PPLDSRRFADRAFTVGIGGPVGTGKTALMLALCTCLR.DKYSLAAVTNDIFTKEDGEFLVKHGALPEE.RIRAVETGGCPHAA Sbi 29 .SWVGEDGRVWHSHDGLAPHSHEPIYSPGDFTKRA..PPLASRNFADRAFTVGIGGPVGTGKTALMLALCRFLR.DKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Zma 31 .SWVGEDGRVWHSHDGLAPHSHEPIYSPGDFTKRA..PPLASRDFADRAFTVGIGGPVGTGKTALMLALCRFLR.DKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Ath 29 .SWVGKDGKVYHSHDGLAPHSHEPIYSPGYFSRRA..PPLHDRNFSERAFTVGIGGPVGTGKTALMLALCRFLR.DKYSLAAVTNDIFTKEDGEFLVKNGALPEE.RIRAVETGGCPHAA Aly 31 .SWVGKDGKVYHSHDGLAPHSHEPIYSPGYFSRRA..PPLNDRNFSERAFTVGIGGPVGTGKTALMLALCRFLR.DKYSLAAVTNDIFTKEDGEFLVKNGALPEE.RIRAVETGGCPHAA Olu 21 .........ELHAHDGLKPHRHERLYGPGSFAKRRKGAKVRAQTFADRAYTVGIGGPVGTGKTALTLALCRALR.DAYDVTAVTNDIFTREDGEFLIANDALGDANRIRAVETGGCPHAA Ota 23 .........ASHAHDGLKPHRHERLYGPGSFAKRRKGSTVRGQTFADRAYTVGIGGPVGTGKTALALALCRALR.DAYDVTCVTNDIFTKEDGEFLIANDALGDANRIRAVETGGCPHAA Msp 52 VYYRAPDGRVLHSHDGLKPHSHDPIPSPGHFERRR..PRKERGDYDERAFTVGIGGPVGTGKTALMLQLCRHFTKESRDICAVTNDIFTREDGEFLTRHEALEAG.RIRAVETGGCPHAA Ppa 55 GEFVGADGKIYHSHDGLAPHTHEPLESPGFFSRRA..APLTTRDFKERGFTVGIGGPVGTGKTALMLALCETLR.DKYSIAAVTNDIFTEEDGEFLIKHGALAPE.RIRAVQTGGCPHAA Smo 17 NEWKGPDGKLYHSHDGLAPHTHEQLDSPGYFNRRP..LALQSRNFAERAFTVGIGGPVGTGKTALMLALCQALR.DKYSIAAVTNDIFTREDAEFLVKNGALAPE.RIRAVETGGCPHAA Gma 37 NSWVGKDGKVYHSHDGLAPHSHEPIYSPGYFTRRA..PPLLNRNFNERAFTVGIGGPVGTGKTALMLALCELLR.ENYSLAAVTNDIFTKEDGEFLVKHKALPEE.RIRAVETGGCPHAA Mtr 26 DSFIGADGKVYHSHDGLAPHSHEPIYSPGFFSRRA..QPLINRDFNERAFTVGIGGPVGTGKTALMLALCQNLR.DRYSLAAVTNDIFTKEDGEFLVKHKALPEE.RIRAVETGGCPHAA Csa 39 SSFVGADGRVYHSHDGLAPHSHEPIYSPGFFTRRA..PPLLTRNFNERAFTVGIGGPVGTGKTALMLALCTFLR.DKYSLAAVTNDIFTKEDGEFLVKHGALPEE.RIRAVETGGCPHAA Mes 29 TSWVGPDGRVYHSHDGLAPHSHEPIYSPGYFSRRA..PPIVTRDFNERAFTVGIGGPVGTGKTALMLAICQFLR.DKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Ptr 32 SSRVGPDGRVYHSHDGLAPHSHEPIYSPGFFSRRA..QPILTRDFNERAFTVGIGGPVGTGKTALMLSLCKLLR.DKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Mal 31 GAWVGPDGRVYHSHDGLAPHSHEPIYSPGSFSKRA..PPLLTRDFNERAFTIGIGGPVGTGKTALMLALCKFLR.DKYSLAAVTNDIFTKEDGEFLVKNGALPEE.RIRAVETGGCPHAA Mgu 31 ESWVGPDGKVYHSHDGLAPHSHEPIESPGYFSRRA..PPLVNRDFNERAFTIGIGGPVGTGKTALMLALCEYLR.DRYSLAAVTNDIFTKEDGEFLVKHGALPEE.RIRAVETGGCPHAA Stu 32 TSWVGADGKVYHSHDGLAPHSHEPIYSPGYFSRRA..PPLNDRNFNERAFTIGIGGPVGTGKTALMLALCKLLR.EKYSLAAVTNDIFTKEDGEFLIKHGALPEE.RIRAVETGGCPHAA Vvi 121 ESWVGPDGKLYHSHDGLAPHTHEPIYSPGYFSRRA..PPLLTRNFHERAFTVGIGGPVGTGKTALMLALCQCLR.EKYSLAAVTNDIFTKEDGEFLVKHGALPEE.RIRAVETGGCPHAA Rco 27 TSWLGADGRVYHSHDGLAPHSHEPIYSPGFFSRRA..PPILTRDFSERAFTVGIGGPVGTGKTALMLAICKFLR.DKYSLAAVTNDIFTKEDGEFLIKNGALPEE.RIRAVETGGCPHAA Spo 39 SSSNSSSEAARLQFIQEHGHSHDAMETPGSYLKREL.PQFNHRDFSRRAFTIGVGGPVGSGKTALLLQLCRLLG.EKYSIGVVTNDIFTREDQEFLIRNKALPEE.RIRAIETGGCPHAA Kae 1 ..........................................MNSYKHPLRVGVGGPVGSGKTALLEALCKAMR.DTWQLAVVTNDIYTKEDQRILTEAGALAPE.RIVGVETGGCPHTA <------> P-loop motif ♦ 2 not Mtr ♦ 3 not Mtr Y Osa 152 IREDISINLGPLEELSNLCKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLIINKTDLAPAVGADLAVMERDALRMREGGPFVFAQVKHGVGVEEIVNH Bdi 156 IREDISINLGPLEELSNLYKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLSVMERDALRMREGGPFVFAQVKHGVGVEEIVDH Sbi 144 IREDISINLGPLEELSNLYKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLAVMERDALRMREGGPFVFAQVKHGVGVEEIVNQ Zma 146 IREDISINLGPLEELSNLYKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLSVMERDALRMREGGPFVFAQVKHGVGVEGIVNH Ath 144 IREDISINLGPLEELSNLFKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSAGDKIPRKGGPGITQADLLVINKTDLAAAVGADLSVMERDSLRMRDGGPFVFAQVKHGLGVEEIVNH Aly 146 IREDISINLGPLEELSNLFKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSAGDKIPRKGGPGITQADLLVINKTDLAAAVGADLSVMERDALRMRDGGPFVFAQVKHGLGVEEIVNH Olu 131 IREDISSNLNAVEDLTAAYPDCDLCLMESGGDNLAANYSRELADYIVYVIDVCGGDKIPRKGGPGVTQADLLVVNKCELADAVGASLEVMERDAKIQREDGPVVMAQVKKGVGVREIAQH Ota 133 IREDISSNLNAVEELTAAYPNCDLCLMESGGDNLAANYSRELADYIVYVIDVCGGDKIPRKGGPGVTQADLLVVNKCELADAVGASLDVMERDAKIQREDGPVVMAQVKKGVGVREIARH Msp 169 IREDISCNLTEVENLTREYSPEFVLLE.SGGDNLAANFSRELADYIIYVIDVCGGDKIPRKGGPGVTQADLLVINKTELADAVGASLEVMARDSKKQRDTGPFVMAQVKRGVGVGEIAEH Ppa 171 IREDISINLGPLEDLSQTFKADILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLADAIGADLKVMERDSLRMRDGGPFVFAQVKHKIGVSDIVNH Smo 133 IREDISINLGPLEELSTKFSADILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAQAVGADLKVMERDALRMRDGGPFVFAQVKHGVGLQDIIDY Gma 153 IREDISINLGPLEELSNLFKADILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAIGADLAVMQRDALRMRDGGPFVFAQVKHKIGVEEIGNL Mtr 142 IREDISINLGPLEELSNLYKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAQAIGADLGVMERDALRMRDGGPFVFAQVKHNVGVEEIVNH Csa 155 IREDISINLGPLEELSNLYKTDILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLATAVGADLAVMERDALKMRDGGPFVFAQVKHGVGVGEIVNH Mes 145 IREDISINLGPLEELSKLFKTDILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLAVMERDALRMRDGGPFVFAQVKHGLGVEEIVNH Ptr 148 IREDISINLGPLEELSNLFKADLLLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLVVMERDALRMRDGGPFVFAQVKHGLGIEEIVNH Mal 147 IREDISINLGPLEELSNLFKADILLCE.SGGDNLAANFSRELADYIIYIIDVSAGDKIPRKGGPGITQADLLVINKTDLAPAVGADLAVMERDALRMRDGGPFVFAQVKHGVGIEEIVNH Mgu 147 IREDISINLGPLEELSNLYKADILLIE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDIAAAVGADLSVMERDSLRMRDGGPFVFAQVKHGVGVKQIVDH Stu 148 IREDISINLGPLEELSNLYKADILLCE.SGGDNLAANFSRELADYIIYIIDVSAGDKIPRKGGPGITQADLLVINKTDLAAAVGADLSVMERDALRMRDGGPFVFAQVKHGVGVEDIVNH Vvi 237 IREDISINLGPLEELSNLFKVDILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLAPAVGADLAVMERDSLRMRDGGPFIFAQVRHGVGIEDIVDH Rco 143 IREDISINLGPLEELSKLFKTDILLCE.SGGDNLAANFSRELADYIIYIIDVSGGDKIPRKGGPGITQADLLVINKTDLASAVGADLTVMERDAVRMRDGGPFVFAQVKHGVGVEEIVNH Spo 156 IREDVSGNLVALEELQSEFNTELLLVE.SGGDNLAANYSRDLADFIIYVIDVSGGDKIPRKGGPGITESDLLIINKTDLAKLVGADLSVMDRDAKKIRENGPIVFAQVKNQVGMDEITEL Kae 77 IREDASMNLAAVEALSEKFGNLDLIFVESGGDNLSATFSPELADLTIYVIDVAEGEKIPRKGGPGITKSDFLVINKTDLAPYVGASLEVMASDTQRMRGDRPWTFTNLKQGDGLSTIIAF ♦ 4 ♦ 5 ♦ 6

Osa 271 ILQAWEIATGNKRR..... Bdi 275 VLRAWEMATGASRR..... Sbi 263 ILQAWEIVTGNKRR..... Zma 265 ILQAWEIATGNKRR..... Ath 263 VMHSWEHATGKKRQ..... Aly 265 VMHSWEHATGKKRQ..... Olu 251 ILDDWAAQTGREKKPF... Ota 253 ILADWSERTGRAEKPFNP--UreD Msp 288 ILAAWTEATGGERNKRAKT Ppa 290 VLKAYEESKKPHHHAHED. Smo 252 VLHAWEDATKRKRSRR... Gma 272 VLQAWEAATGNKRH..... Mtr 261 VLQAWEAATGKKRH..... Csa 274 IIQAWEAATGKKRH..... Mes 264 ILQAWEGATGKKQH..... Ptr 267 ILQGWEVATGKKRH..... Mal 266 VLQAWEAATGKKRH..... Mgu 266 VLGAWEAATGKKPH..... Stu 267 ILQAWEVATGNKKR..... Vvi 356 VLHAWEVATGEKRH..... Rco 262 VLQAWEVATGKKQH..... Spo 275 ILGAAKSAGALK....... Kae 197 LEDKGMLGK..........

Supplemental Figure S4. Multiple alignment of plant UreG with UreG from Schizosaccharomyces pombe and Klebsiella aerogenes. Osa, Oryza sativa, ssp. Nipponbare; Bdi, Brachypodium distachyon; Sbi, Sorghum bicolor; Zma, Zea mays; Ath, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Olu, Ostreococcus lucimarinus, gi 145342523; Ota, Ostreococcus tauri, gi 116055197 (C-terminal fused UreD sequence removed); Msp, Micromonas species RCC299, gi 255084295; Ppa, Physcomitrella patens; Smo, Selaginella moellendorfii; Gma, Glycine max; Mtr, Medicago trunculata; Csa, Cucumis sativus; Mes, Manihot esculenta; Ptr, Populus trichocarpa; Mal, Morus alba, gi 222143562; Mgu, Mimulus guttatus; Stu, Solanum tuberosum, gi 13161908; Vvi, Vitis vinifera; Rco, Ricinus communis; Spo, Schizosaccharomyces pombe, gi 14268501; Kae, Klebsiella aerogenes, gi 149341. Sequences were either obtained from Genebank (gi numbers given) or predicted from completed plant genome projects (using Phytozome v5.0; www.phytozome.net). For Zma, Ppa and Vvi the coding sequence prediction in Phytozome was manually corrected using expressed sequence tag data. Differences in the sequence between ssp. Nipponbare and Indica Hunan late no. 2 are annotated above the alignment. Alignments were generated with ClustalW and shading was performed with boxshade (www.ch.embnet.org). Rhombs and numbers label canonical intron positions (exceptions are commented) found in ureGs of all plant species for which complete genome sequences are available (organism names marked with * at position 1). The labeled amino acid is at least partially encoded by the exon preceding the intron.

00 1 2

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AN AN U AN+U ANdark

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Supplemental Figure S5. Relative urease and arginase activity changes of rice plants in response to different N sources and darkness. A, 8-day-old plants were transfered from sterile agar media containing 5 mM ammonium nitrate (AN) to media either containing 5 mM urea (U) or 5 mM urea and 5 mM ammonium nitrate (AN + U) or no nitrogen (no N). Controls were placed on 5 mM ammonium nitrate (AN) and an additional set of plants was placed on AN and kept in darkness (carbon starva-tion). For three days shoot urease and arginase activities relative to total protein were monitored in three biological replicates (error bars are sd). Activities before transfer (day 0) were set 100%. B, As in A but activities were determined in roots. Three biological replicates were taken at day 0 and day 3.

A

B

SHOOT

ROOT

no N U + AN U+

Supplemental Figure S6. Phenotype of rice plants grown under different N regimes. Plants grown for 10 days without nitrogen (no N), with 5 mM urea (U), with limiting 0.25 mM ammonium nitrate (+), with 5 mM amonium nitrate (AN), and with 5 mM urea and 0.25 mM amonium nitrate (U+) as N source. Two representative plants are shown for each treatment.

Supplemental Table S1. AUG initiation codons in the 5’ leader sequence of ureF transcripts from different plants species

number of AUG upstream of intron

number of AUG in the intron

number of AUG downstream of intron

Arabidopsis thaliana 0 2 0 Brachypodium distachyon

1a 3 0

Cucumis sativus 5 15 1 Glycine max 02g44440

0 42 0

Glycine max 14g04380

1a 18 1

Manihot esculenta 0 32 0 Medicago trunculata 0 42 0 Oryza sativa 0 25 0 Populus trichocarpa 0 5 0 Sorghum bicolor 2 9 0 Vitis vinifera 0 49 0 a AUG located within the first nine 5’ bases of the transcript. At a distance smaller than 12 nucleotides from the 5’CAP the AUG is not efficiently recognised as initiation codon. Jackson RJ, Hellen CUT, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Reviews Molecular Cell Biology 11: 113-127

Supplemental Table S2. Amino acid quantification in shoot and root of rice plants grown under different N regimes. shoot noa ANa Ua U+a +a mean sd mean sd mean sd mean sd mean sd alanine 1.24 0.05 2.81 0.12 2.47 0.14 2.09 0.20 1.90 0.05

arginine 0.31 0.01 6.24 0.18 2.23 0.16 3.12 0.29 0.28 0.01

asparagine 0.91 0.09 29.10 1.01 37.73 1.59 45.37 2.88 2.74 0.14

aspartate 1.44 0.04 1.67 0.14 2.52 0.12 2.61 0.22 1.23 0.02

citrulline 0.14 0.01 0.13 0.00 0.12 0.00 0.09 0.01 0.05 -

cysteine 0.90 0.18 5.34 0.78 1.47 0.30 1.87 0.09 0.58 0.03

glutamate 1.33 0.09 2.30 0.08 3.76 0.25 3.78 0.34 1.52 0.06

glutamine 3.37 0.28 32.30 0.62 26.68 1.40 28.59 1.92 4.64 0.08

glycine 0.53 0.05 3.61 2.71 1.28 0.16 1.91 0.11 0.41 0.02

histidine 1.18 0.04 0.64 0.04 0.44 0.03 0.56 0.04 0.80 -

isoleucine 0.59 0.02 0.76 0.04 0.38 0.02 0.54 0.05 0.60 0.01

leucine 0.36 - 0.81 0.03 0.38 0.02 0.47 0.04 0.33 0.01

lysine 0.49 0.01 1.10 0.05 0.50 0.01 0.64 0.04 0.40 0.02

methionine 0.21 0.01 1.68 0.11 1.34 0.09 0.96 0.09 1.23 0.05

ornithine 0.32 0.01 0.61 0.02 0.29 0.00 0.30 0.01 - -

phenylalanine 0.26 0.01 0.29 0.01 0.21 0.01 0.32 0.03 0.19 -

proline 0.29 0.08 0.67 0.05 0.37 0.06 0.33 0.04 0.19 -

serine 1.31 0.07 7.98 0.35 3.92 0.22 4.45 0.35 1.52 0.04

threonine 0.51 0.01 2.35 0.12 1.17 0.07 1.73 0.11 0.57 0.01

tryptophan 0.90 0.04 0.30 0.01 0.16 0.01 0.23 0.02 0.66 0.02

root noa ANa Ua U+a +a mean sd mean sd mean sd mean sd mean sd alanine 0.41 0.39 0.43 0.02 0.47 - 0.43 0.01 0.29 - arginine 0.13 0.22 0.15 0.08 0.46 0.04 0.62 0.02 0.05 0.07 asparagine 0.17 0.02 3.28 0.12 16.77 0.11 17.35 0.58 0.35 0.07 aspartate 0.78 - 0.92 0.01 1.78 0.03 2.01 - 0.76 0.02 citrulline 0.09 0.04 0.05 0.01 0.22 0.26 0.04 - 0.03 - cysteine 0.40 0.18 0.23 0.32 0.24 0.34 0.53 - 0.37 0.39 glutamate 0.68 0.01 0.61 0.03 0.82 0.02 0.91 - 0.52 0.01 glutamine 1.16 0.05 2.85 0.17 18.90 0.66 20.04 0.91 1.31 0.12 glycine 0.24 0.02 0.62 0.03 0.35 0.01 0.37 0.01 0.47 0.01 histidine 0.36 - 0.36 0.03 0.47 0.04 0.49 0.04 0.20 - isoleucine 0.19 0.15 0.16 0.04 0.20 0.01 0.24 0.01 0.06 0.02 leucine 0.21 0.23 0.22 0.12 0.16 0.01 0.18 0.01 0.11 - lysine 0.34 0.30 0.40 0.12 0.40 0.01 0.48 0.07 0.23 - methionine 0.87 0.81 0.79 0.02 0.72 - 0.78 0.08 0.55 - ornithine 0.23 - 0.27 0.07 - - - - - - phenylalanine 0.13 0.12 0.14 0.02 0.09 0.01 0.11 0.01 0.06 - proline 0.39 0.38 0.22 0.06 0.08 0.01 0.07 0.02 0.17 0.01 serine 0.64 0.01 1.35 0.06 1.07 0.03 1.17 0.01 0.57 0.01 threonine 0.22 0.21 0.40 0.02 0.32 0.01 0.36 0.02 0.18 - tryptophan 0.31 0.27 0.28 0.02 0.29 0.01 0.28 0.02 0.13 - a no, without nitrogen; AN, with 5 mM ammonium nitrate; U, with 5 mM urea; U+ with 5 mM urea and 0.25 mM ammonium nitrate; +, with limiting 0.25 mM ammonium nitrate as N source in the growth media.

Supplemental Table S3. Primers used in this study No. sequence comments 1021 TGTCTCTATATCAGGAGATTTG AtureF, differential

splicing, reverse 1033 GTGAACGATTCCTGGACCTGCCTC Atactin 2, forward 1034 GAGAGGTTACATGTTCACCACAAC Atactin 2, reverse 1683 TTCATGATGCAGGATATGGGCAAG OsureD, first PCR,

reverse 1694 ACATATGAAGCTGGTACAAAGGGAG Osurease, second

PCR, forward 1695 ACTCGAGCTAAAAGAGGAAGTAATTCCGAGA Osurease, second

PCR, reverse 1697 ACTCGAGTCAGGTGCAAAACAGCCTA OsureF, second

PCR, reverse 1698 ACCATGGCGTCTCACGACCAC OsureG, second

PCR, forward 1699 AGGATCCCTATCGACGCTTGTTGCC OsureG, second

PCR, reverse 1701 AGGATCCTCATGATGCAGGATATGGGC OsureD, second

PCR, reverse 1705 AAAGAAAGAAAGAGCATGGAGGC OsureD, first PCR,

forward 1723 AAAGAAAGAAAGACCATGGAGGC OsureD, second

PCR, forward 1775 TCGAGAAAAGCGATATAAAACA pXS1-pat, forward 1776 TATGTTTTATATCGCTTTTC pXS1-pat, reverse 1777 TCGAGAAAAGCGATATAAAAC pXS2-pat, forward 1778 CATGGTTTTATATCGCTTTTC pXS2-pat, reverse 1860 TCGAGCTCTTCAAACAAATAACACCAAAAATGGCATC

TTGGTCTCATCCTCAATTTGAAAAAGGCGCCCATATGG pXNS1pat-Strep, forward

1861 AATTCCATATGGGCGCCTTTTTCAAATTGAGGATGAG ACCAAGATGCCATTTTTGGTGTTATTTGTTTGAAGAGC

pXNS1pat-Strep, reverse

1862 TCGAGCTCTTCAAACAAATAACACCAAAAATGGCATCT TGGTCTCATCCTCAATTTGAAAAAGGCGCCTCCATGG

pXNS2pat-Strep, forward

1863 AATTCCATGGAGGCGCCTTTTTCAAATTGAGGATGAG ACCAAGATGCCATTTTTGGTGTTATTTGTTTGAAGAGC

pXNS2pat-Strep, reverse

1970 TTCATATGGAACGGGTAATGGAATG OsureF, second PCR, forward

2265 GACGTCGAGCGGAGCGAAG Osarginase, first PCR, forward

2266 GCCCTGAATCTGAATGGATGCTC Osarginase, first PCR, reverse

2267 TGAATTCAAAATGGGCGGCGTGGCGGC Osarginase, second PCR,forward

2268 TCCCGGGCTTGGAGATCTTGGCTGTGAGCTC Osarginase, second PCR,reverse

2438 ATCACTCATCTTCTCCGACG AtureF, differential splicing, forward

2439 AACAAATGACCTCAACTCCTCTG OsureF differential splicing, reverse

2440 CCCTCAATCCAACATCTATTCC OsureF differential splicing, forward 1

2441 AGTTCAGTGTGAGAACATGGTAATG OsureF differential splicing, forward 2

2537 GCGGATCCATGAAGCTGGTACAAAGGGAGGCGGA Osurease first PCR, forward

2538 AAGGATCCCTAAAAGAGGAAGTAATTCCGAGATAGTG Osurease first PCR, reverse

2539 C GAGGATCCATGTACATTTTCATGACTCGAA OsureF, first PCR, forward

2540 CCGGATCCTCAGGTGCAAAACAGCCTAGAGAAC OsureF, first PCR, reverse

2541 CGGGATCCATGGCGTCTCACGACCACG OsureG, first PCR, forward

2542 CCGGATCCCTATCGACGCTTGTTGCC OsureG, first PCR, reverse