1 Supporting experimental procedures 1 2 Methods S1 3 Plasmid construction 4 For promoter-GUSanalysis and complementation tests,Gateway compatibledestination 5 vectors,pEpi168::GW-p35S::GFP and pEpi308::GW-p35S::GFP, were constructed as 6 follows: Genomicsequences of theLjEXPA7promoter regions (168 and 308 bp) were 7 amplified by PCR.Each fragment was digested with DraI and SpeI and then inserted8 into theHindIII (blunt)-SpeI siteof p35S::GW-p35S::GFP(P35S:GFP-gw; Banba et al., 9 2008). For the construction of the pEpi400::GW-p35S::GFP vector, the genomic 10 sequence of the LjEXPA8 promoter region (400 bp) was amplified by PCR. The11 fragment was digested with HindIII and SpeI and then ligated with12 p35S::GW-p35S::GFP. For co-transformation with CCaMK under the control of 13 pEpi308 and an inactive truncated CCaMK under the control of p35S, a Gateway 14 compatible destination vector, pEpi308::CCaMK-p35S::GW, was constructed by 15 introducing the reading frame cassetteC.1 (RfC.1) of theGateway vector conversion 16 system (Invitrogen, Carlsbad, CA, USA) into the XhoI (blunt)-XhoI (blunt) site of17 pEpi308::CCaMK-p35S::GFP.All primer sets used for construction arelisted in Table18 S2.19 Information of entry clones of symbioticgenes and GUSplus(GUS+) and conversion 20 of these entry vectors into pEpi destination vectors are described below. cDNA21 sequences of NFR5 and NUP85 were amplified by PCR and cloned into 22 pENTR/D-TOPO (Invitrogen,Carlsbad,CA,USA). cDNA sequence of NUP133was 23 amplified by PCR and digested with SacII and AscI and then inserted into the24 2 SacII-AscI site of pENTR/D-TOPO. The entry clones constructed in this study, and1 entry clones of GUS+ (Yano et al., 2008),NFR1 (Nakagawa et al., 2011),CASTOR,2 POLLUX,CCaMK,CCaMKT265D (Banba et al., 2008), an inactive truncated CCaMK 3 (CCaMK1-340) (Shimodaet al.,2012),CYCLOPS(Yano et al.,2008),NSP1(Yokotaet 4 al.,2010) and NSP2(Yokotaet al.,2010),wereconverted with thedestination vectorof5 pEpi308::GW-p35S::GFP by the LR reaction (Gateway LR clonase II EnzymeMix;6 Invitrogen,Carlsbad,CA,USA).7 8 Methods S2 9 RNA isolation from root hairs 10 Surface-sterilized seeds were germinated on sterilized filter paper on 1.5% agar 11 containing half-strength B&Dmedium(Broughton and Dilworth 1971) in squareplastic12 plates (Eiken Chemical Co.,Tokyo,Japan,http://www.eiken.co.jp/).Theplasticplates 13 were incubated vertically for 2 days in agrowth cabinet with a 16 h-day/8h-nightcycle14 at 24°C,and then theseedlings wereinoculated withasuspension ofDsRed-labeled M.15 loti.Theplants weregrown for an additional 2 days with theroots shielded fromlight.16 The roots were then collected and dropped immediately into a 100-ml stainless steel17 beaker filled with liquid nitrogen. The samples were stirred gently with a magnetic 18 stirrer for 20 min. In this step, the root hairs were cut off from the roots.The liquid 19 nitrogen containingroot hairs was then filtered through astainless testing sievewith a20 mesh apertureof 500 µm.Liquid nitrogen was evaporated in a50-mL sampletubeand 21 root hairs werecollected.Theroots trapped by thetesting sievewerealso collected,as 22 “stripped roots”.Total RNAs wereisolated by theCTAB method (Chang et al.,1993)23 with somemodifications.Briefly,thecollected roothairs or stripped rootswereground 24 3 to afine powder in liquid nitrogen,immediately dissolved in theextraction solution (2%1 CTAB,100 mMTris pH9.5,20 mMEDTA,1.4MNaCl,1%2-mercaptoethanol),and 2 then incubated at 65°C for 10 min.After two successive extractions with chloroform,3 RNAs wereprecipitated using LiCl (final concentration 2.5 M) at -20°C overnight.The 4 precipitated RNAs were collected by centrifugation, and were dissolved in distilled 5 water. As necessary, the samples were further purified using an RNeasy Mini Kit6 (Qiagen,http://www.qiagen.com/) according to the manufacturer’s instructions.7 8 References 9 Banba,M.,Gutjahr,C.,Miyao,A.,Hirochika,H.,Paszkowski,U.,Kouchi,H.and10 Imaizumi-Anraku,H. (2008) Divergence of evolutionary ways among common sym11 genes: CASTOR and CCaMK show functional conservation between two symbiosis12 systems and constitutetheroot of acommon signaling pathway.Plant Cell Physiol.,49,13 1659-1671.14 Broughton,W.J.and Dilworth,M.J. (1971) Control of leghaemoglobin synthesis in 15 snakebeans.Biochem.J.,125,1075-1080.16 Chang, S., Puryear, J. and Cairney, J. (1993) A simple and efficient method for17 isolating RNA frompinetrees.Plant Mol.Biol.Rep.,11,113-116. 18 Nakagawa,T.,Kaku,H.,Shimoda,Y.,Sugiyama,A.,Shimamura,M.,Takanashi,19 K., Yazaki, K., Aoki, T., Shibuya, N. and Kouchi, H. (2011) From defense to 20 symbiosis: limited alterations in the kinase domain of LysM receptor-like kinases are 21 crucial for evolution of legume-Rhizobiumsymbiosis.Plant J.,65,169-180.22 4 Yokota, K., Soyano, T., Kouchi, H. and Hayashi, M. (2010) Function of GRAS 1 proteins in root nodulesymbiosis is retained in homologs of anon-legume,rice.Plant 2 Cell Physiol.,51,1436-1442.3