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1 SI Fig. PrpS is single copy gene k EcoRV EcoRV k 5 BmH Pst c well k HindIII HindIII HindIII Southern lots of Ppver genomic DNA from plnts with SS8 hplotypes, hyridized with PrpS proe.. Genomic DNA digested with EcoRV. The presence of single nd (t.5 k) demonstrtes tht PrpS is single copy gene.. Genomic DNA digested with BmHI (lne ) nd Pst (lne ). This demonstrtes tht PrpS is single copy gene, s only one nd is detected in ech lne. c. Genomic DNA digested with BglII nd hyridized with PrpS proe. Lne contins DNA from plnt with SS8 hplotypes which is to give two nds of pproximtely. nd.8 k, s there is BglII restriction site in PrpS (position +9). Lnes nd 3 contin DNA from plnt with hplotypes S S nd S 3 S respectively. Note tht the PrpS proe does not hyridize with this DNA. Together, these dt demonstrte tht PrpS is single copy gene nd provide evidence tht the relted sequences identified s PrpS, PrpS3 nd PrpS8 re llelic to ech other, rther thn relted/prlogous genes.

2 SI Fig. Crtoon of two possile structurl predictions for PrpS TMHMM MPRSGSVVTL FQFVGGLCTL LGVSVAIKAI FAQDYTLKDL IILIVLVALS 50 PP MPRSGSVVTL FQFVGGLCTL LGVSVAIKAI FAQDYTLKDL IILIVLVALS 50 TMHMM IILGGPITLT CVKLLGLVLH RLSFSEDQKW VVAFGTAAIC DVLLVPKNML 00 PP IILGGPITLT CVKLLGLVLH RLSFSEDQKW VVAFGTAAIC DVLLVPKNML 00 TMHMM PMTIFSFLSS IMICVVAVGW DCDRSGMTEG FLVGFGKLLL VYLIKQDFTF 50 PP PMTIFSFLSS IMICVVAVGW DCDRSGMTEG FLVGFGKLLL VYLIKQDFTF 50 TMHMM SLLCGSVLCL AVVAKFTEGK AEATPNPNLA GKADSPHLIT QA 9 PP SLLCGSVLCL AVVAKFTEGK AEATPNPNLA GKADSPHLIT QA 9 extrcellulr 3 35 mino cid loop 97 c extrcellulr 33 mino cid loop 9 9 trnsmemrne trnsmemrne 8 39 intrcellulr intrcellulr () Alignment of PrpS mino cid sequences with structurl predictions ssigned y TMHMM nd PredictProtein. Amino cids re colourcoded (pink, extrcellulr; red, trnsmemrne; lue, intrcellulr). () nd (c) show crtoons indicting two possile structurl topologies, sed on predictions for PrpS using TMHMM () nd PredictProtein (c). Numers indicte the mino cid residue for PrpS. The overll prediction for the three lleles of PrpS is sed on severl structurl prediction progrmmes (see Full Methods). There is no gold stndrd for prediction, so we cnnot fvour one over nother. The predictions suggest etween 35 trnsmemrne segments. Although TMHMM predicts PrpS nd PrpS 8 with the sme topology, it predicts PrpS 3 with different topology; however, our lignment (Fig d) is strong enough to convince one tht ll of the proteins would shre similr topology, irrespective of differing prediction results. There is strong TMHMM prediction for the region of mino cids 00 in PrpS3, which is pplicle to the other lleles, indicting tht PrpS is likely to hve four trnsmemrne domins. Moreover, this is good numer to mke helix undle in the memrne; while 3 nd 5 TM do exist, they re much rrer. The lignment of the TM helices from these predictions is such tht our loop region is extrcellulr. Although the sequences do not show ny prticulr is in following the "positive inside rule" our experimentl dt using the TMHMM ~35 mino cid extrcellulr loop (indicted here in pink; mino cids 397 in PrpS), indicte tht this region of PrpS intercts with the pistil S protein (see Fig e) nd lso re involved in pollen recognition nd rejection (see Figure 3 nd SI Fig 3). It should e noted tht this is tenttive ssignment of possile structure, nd it is premture to

3 SI Fig 3. The extrcellulr loop region of PrpS is involved in mediting pollen inhiition c A 5mer peptide ws designed for regions of the PrpS 35 mino cid extrcellulr loop region (sequence: DQKWVVAFGTAAICD) nd corresponding rndomized peptide (GVVCAWIFDTAAQKD) ws used in n in vitro SI iossy. These were dded to SIinduced pollen from plnts with hplotype S S 3 (i.e. pollen crrying S or S 3 lleles) to test whether the peptide might ffect the SI phenotype. We compred pollen inhiition in the presence of recominnt PrsS lone (SIinduced) with SI in the presence of PrpS peptide (SI + peptide) or with SI in the presence of rndomized PrpS peptide (SI + control peptide). Ech peptide ws mixed with recominnt PrsS for 0 min t room temperture prior to dding to pollen, which ws grown for hr in vitro. Pollen grins nd tues were scored ccording to two ctegories: inhiition or growth ; minimum of 00 pollen grins/tues ws scored for ech smple. These were selected from five rndomly chosen fields of view using x0 ojective. Dt were nlysed using Fisher s Exct Test for x contingency tles (see Supplementl Tle ). () SIinduced pollen from plnts with hplotype S S 3 in the presence of recominnt PrsS nd PrsS 3, showing strong inhiition of pollen tue growth. () The peptide DQKWVVAFGTAAICD rescued pollen from inhiition, s popultion of longer pollen tues re detectle. Note tht ecuse the pollen ws from heterozygous plnt only mximum of 50% would e expected to e rescued y the PrpS peptide. (c) The rndomized peptide GVVCAWIFDTAAQKD hd no effect on pollen SI phenotype, which showed strongly inhiited pollen nd no evidence of rescue. 3

4 SI Figure. A PrpS ntisense oligonucleotide rescues pollen in n Sspecific mnner Pollen tue length (µm) SI inhiited SI + s rescue SI + s no rescue SI SI + s SI + s Pollen tue length (µm) SI inhiited SI + s no rescue SI + s no rescue SI SI + s SI + s PrpS ntisense (s) or sense (s) oligonucleotides (ODNs) were dded s pretretment to pollen () from plnts with hplotypes SS3 or () with hplotypes S3S8 nd in vitro SI induced y ddition of recominnt PrsS nd PrsS3 to () or recominnt PrsS3 nd PrsS8 to () to generte SI+s or SI+s. Controls comprised SI tretment without ODN dditions (SI). The plots show mesurements of individul pollen tue lengths from typicl experiment (50 in ech ctegory) to show the distriution of pollen tue lengths otined with the SI iossy oligonucleotide rescue experiment. The red line indictes the men pollen tue length otined with n SI tretment (inhiition). () SI + s (middle lock) shows the rescued popultion using the PrpS ntisense oligonucleotide with pollen from SS3 plnts. Note tht not ll pollen tues re rescued; rescue from SIinduced inhiition y the PrpS sodns is expected only to ffect 50% of the pollen tues (crrying the S llele), s the pollen comes from SS3 heterozygote. Therefore the resulting popultion of pollen tues is imodl, comprising of ~50% inhiited tues nd ~50% longer llevited tues (ove the SI men, red line), where the sodns locked the SI rection. Thus, the men vlues shown in Fig 3 re expected to e theoreticl mximum of 50%, s ~50% re inhiited nd 50% re expected to exhiit some recovery of growth. () SI + s (middle lock) shows the equivlent tretment, using the sme PrpS sodns, ut with pollen from S3S8 plnts, where SI is not rescued. This is expected if the PrpS sodns ct in n S llele specific mnner. Sense oligonucleotides (SI+s) hd no effect nd pollen remined inhiited.

5 Supplementry Tle. Nonsynonymous sustitution nd synonymous sustitution rtios (K/Ks) for pollen PrpS nd for pistil PrsS sequences Ks K K/Ks Pollen PrpS /PrpS PrpS /PrpS PrpS 3 /PrpS Men SD= SD= SD=0.099 Pistil PrsS /PrsS PrsS /PrsS PrsS 3 /PrsS N/A Men 0.79 SD= SD= SD=0.089 Ks ttest t (d.f) = (NS) 5

6 Supplementry Tle. PrpS peptides llevite SIinduced pollen inhiition. A 5 peptide (DQKWVVAFGTAAICD), corresponding to prt of the 3 externl domin of prediction of PrpS, nd rndomized versions of this peptide (: GVVCAWIFDTAAQKD nd : FTVDVKDCAAAWGQI) were tested for their effect on SI phenotype (scoring pollen either s grown or inhiited ). Dt were nlysed using Fisher s Exct Test for x contingency tles (some of expected clsses < 5), s shown elow: Tretment Inhiition Growth Totl SI + SI + peptide c d c + d Totl + c + d n Proilities for SI ssys using peptide sequence DQKWVVAFGTAAICD nd rndomized versions GVVCAWIFDTAAQKD nd FTVDVKDCAAAWGQI Replicte Peptide DQKWVVAFGTAAICD Rndomized peptide GVVCAWIFDTAAQKD Rndomized peptide FTVDVKDCAAAWGQI 5.07E09 *** 0.73 NS NS *** 0.35 NS NS 3.E07 *** NS NS 5.799E5 *** E5 ***.893E3 ***