1 ATGGCGCGCCGCGCCGCTTCCCGCGCTGTTGGCGCCCTTCGCTCGGACGGCTCGATCCAA M A R R A A S R A V G A L R S D G S I Q 61 GGGCGAGGAGGCCGCGCGGGGGGCAGTGGCGCCGAGGACGCACGCCACGTGTTCGACGAA G R G G R A G G S G A E D A R H V F D E 121 TTGCTCCGCCGTGGCAGGGGCGCCTCGATCTACGGCTTGAACCGCGCCCTCGCCGACGTC L L R R G R G A S I Y G L N R A L A D V 181 GCGCGTGACAGCCCCGCGGCCGCCGTGTCCCGCTACAACCGCATGGCCCGAGCCGGCGCC A R D S P A A A V S R Y N R M A R A G A 241 GACGAGGTAACTCCCGACTTGTGCACCTACGGCATTCTCATCGGTTGCTGCTGCCGCGCG D E V T P D L C T Y G I L I G C C C R A 301 GGCCGCTTGGACCTCGGTTTCGCGGCCTTGGGCAATGTCATTAAGAAGGGATTTAGAGTG G R L D L G F A A L G N V I K K G F R V 361 GACGCCATCGCCTTCACTCCTCTGCTCAAGGGCCTCTGTGCCGACAAGAGGACGAGCGAC D A I A F T P L L K G L C A D K R T S D 421 GCAATGGACATAGTGCTCCGCAGAATGACCGAGCTCGGCTGCATACCAAATGTCTTCTCC A M D I V L R R M T E L G C I P N V F S 481 TACAATATTCTTCTCAAGGGGCTGTGTGATGAGAACAGAAGCCAAGAAGCTCTCGAGCTG Y N I L L K G L C D E N R S Q E A L E L 541 CTGCACATGATGGCTGATGATCGAGGAGGAGGTAGCCCACCTGATGTGGTGTCGTATGGG L H M M A D D R G G G S P P D V V S Y G 601 ACCACTGTCATCAATGGCTTCTTCAAAGAGGATTCAGACAAAGCTTACAGTACATACCAT T T V I N G F F K E D S D K A Y S T Y H 661 GAAATGCTGGACCGGGGGATTTTACCTGATGTTGTGACCTACAACTCTATTGACAAAGCC E M L D R G I L P D V V T Y N S I D K A 721 ATTGCTGCGTTATGCAAGGCTCAAGCTATGATGGAGGTACTTAACACCATGGTTAAGAAT I A A L C K A Q A M M E V L N T M V K N 781 GGTGTCATGCCTGATTGCATGACATATAATAGTATTCTGCATGGATATTGCTCTTCAGGG G V M P D C M T Y N S I L H G Y C S S G 841 CAGCCGAAAGAGGCTATTGGATTTCTCAAAAAGATGCGCAGTGATGGTGTCGAACCAGAT Q P K E A I G F L K K M R S D G V E P D 901 GTTGTTACTTAA V V T & Supplemental Figure 1. rf5 in HL-CMS line YTA. The 912nt mutant of T to A, marked in red, results in the premature stop condon TAA. 1
1 ATGAATTTCGATCACAATCATGTGGTAATAATGGGTTTGAATCAGAGAGACTCGATCTGG M N F D H N H V V I M G L N Q R D S I W 61 AAACTCCTCAATGATTATAACGTGAACTCGTTGAAGAGAAGGAGACAAGCAGAAATAGAC K L L N D Y N V N S L K R R R Q A E I D 121 GCTTTTTTTGAACCATTTGAGAGGGCGCAGCGTATCCGTTTCAATAACTGGCAGAACGGA A F F E P F E R A Q R I R F N N W Q N G 181 ATAGAGTTGTTAGATGGGGCTGAATGGAGGAACGGCGATATAGTTATCCCTGGAGGCGGC I E L L D G A E W R N G D I V I P G G G 241 GGACCAGTAATTTCAAGCCCCTTGGATCAATTTTTCATTGATCCATTATTTGGTCTTGAT G P V I S S P L D Q F F I D P L F G L D 301 ATGGGTAACTTTTATTTATCATTCACAAATGAATCCTTGTCTATGGCGGTAACTGTCGTT M G N F Y L S F T N E S L S M A V T V V 361 TTGGTGCCATCTTTATTTGGAGTTGTTACGAAAAAGGGCGGGGGAAAGTCAGTGCCAAAT L V P S L F G V V T K K G G G K S V P N 421 GCATGGCAATCCTTGGTAGAGCTTATTTATGATTTCGTGCTGAACCTGGTAAACGAACAA A W Q S L V E L I Y D F V L N L V N E Q 481 ATAGGTGGAAATGTTAAACAAAAGTTTTTCCCTCGCATCTCGGTCACTTTTACTTTTTCG I G G N V K Q K F F P R I S V T F T F S 541 TTATTTCGTAATCCCCAGGGTATGATACCCTTTAGCTTCACAGTGACAAGTCATTTTCTC L F R N P Q G M I P F S F T V T S H F L 601 ATTACTTTGGCTCTTTCATTTTCCATTTTTATAGGCATTACGATCGTTGGATTTCAAAGA I T L A L S F S I F I G I T I V G F Q R 661 CATGGGCTTCATTTTTTTAGCTTCTTATTACCAGCGGGAGTCCCACTGCCATTAGCACCT H G L H F F S F L L P A G V P L P L A P 721 TTTTTAGTACTCCTTGAGCTAATCTCTCATTGTTTTCGTGCATTAAGCTCAGGAATACGT F L V L L E L I S H C F R A L S S G I R 781 TTATTTGCTAATATGATGGCCGGTCATAGTTCAGTAAAGATTTTAAGTGGGTTCGCTTGG L F A N M M A G H S S V K I L S G F A W 841 ACTATGCTATTTCTGAATAATATTTTCTATTTCATAGGAGATCTTGGTCCCTTATTTATA T M L F L N N I F Y F I G D L G P L F I 901 GTTCTAGCATTAACCGGTCTGGAATTAGGTGTAGCTATATTACAAGCTCATGTTTCTACG V L A L T G L E L G V A I L Q A H V S T 961 ATCTCAATTTGTATTTACTTGAATGATGCTATAAATCTCCATCAAAATGAGTAATTTCAT I S I C I Y L N D A I N L H Q N E # AATTGAATAAAAACGAGGAGCCGAAGATTTTAGGGGGCGGGACAAACGCGGAAGTGTATTG CGTTACAAAAAATGACAACTAGCATTTGTTTTTTCATTTCATGTTCGAATTCGTTTTTCGT TGGAAAAACCGACGCCAACGTTAAGATCAGTCTCCTTTCTCTTTTGTTTTGGGAGCAGAGC TTAAAAATCTTATTTTCTTGTGCT 1228 ATGACAAATCTGCTCCGATGGCTCTTCTCCACTACCCGAGGGACTAACGGTCTTCCATAT M T N L L R W L F S T T R G T N G L P Y 1288 TTCATCTTCGGTGTCGTTGTAGGAGGCGCCCTGTTGTTTGCTTTGCTAAAGTATCAGGCC F I F G V V V G G A L L F A L L K Y Q A 1348 CCTCTGTACGACCCGGCTTTATTGGACAAAATCATAGATCATAATATAAAAGCCGGGTAC P L Y D P A L L D K I I D H N I K A G Y 2
1408 CCTATAGAGGTTGACTATTCGTGGTGGGGCACCTCTATTCGTGTAGTCTTTCCTAAGTAA P I E V D Y S W W G T S I R V V F P K # Supplemental Figure 2. The sequences of atp6-orfh79. The first open reading frame (from 1 to 1014nt) encodes the atp6 gene and second open reading frame (from 1228nt to 1467nt) encodes the orfh79 gene. The intragenic fragment of atp6-orfh79 is underlined. 3
Supplemental Figure 3. Expression and verification of MBP-RF5. (A) The expression and purification of MBP-RF5 and MBP-tag with pmal-c2x (NEB). (B) The purified recombinant MBP-RF5 was sequenced by Q-TOF. (C) The measurement of MBP-RF5 with Circular Dichroism. 4
Supplemental Figure 4. Yeast three-hybrid system for detection the interaction between RF5 and intragenic fragments of CMS associated transcripts. (A) Transformants were selected for the presence of plasmids, colonies were assayed for HIS3 reporter activity and 3-AT competition assays. The numbering (1 to 8) is consistent with panel B. (B) The measurements of β-galactosidase activity for the three-hybrid assay and details of hybrid RNA lines 1 to 8. MS2-A +, MS2-B + and MS2-C +, corresponding to A, B and C (diagrammed in Figure 2B), respectively, were ligated into piiims2-2 to produce sense strand RNA probes; MS2-A -, MS2-B - and MS2-C - denote reverse ligation to produce anti-sense strand RNA probes. 5
. Supplemental Figure 5. Investigation of the length of BacterioMatchII two hybrid library. fragments. PCR with ptrgf and ptrgr primers to investigate the length of insert M:DL2000 (TakaRa), 1-18: random single clony. 6
Supplemental Figure 6. The expression of GRP162. Quantitative RT-PCR was carried out with anthers to detect the expression of GRP162. Actin was used as a control and GRP162 was expressed over 2.25 fold in the presence of Rf5 contrast to YTA. Error bars represent SD (tests=3). The average CT value of each triplicate from the RT-PCR experiments was calculated, and the number of positive wells per triplicate was noted. 7
Supplemental Figure 7. The expression and purification of His-GRP and His-RRM. The expression and purification of His-GRP162, His-RRM and His-tag. 8
GRP162 cdna sequence: 1 ATGGCGGCGCCGGATGTTGAGTACCGCTGCTTCGTCGGCGGCCTCGCCTGGGCCACCGAC M A A P D V E Y R C F V G G L A W A T D 61 GACCGCTCCCTCGAAGCCGCCTTCTCCACCTTCGGCGAGATCCTCGAGTCCAAGATCATC D R S L E A A F S T F G E I L E S K I I 121 AACGATCGGGAGACGGGGAGGTCGCGCGGGTTCGGGTTCGTGACGTTCTCGAGCGAGCAG N D R E T G R S R G F G F V T F S S E Q 181 GCCATGCGCGACGCCATCGAGGGGATGAACGGCAAGGAGCTCGACGGCCGCAACATCACC A M R D A I E G M N G K E L D G R N I T 241 GTCAACGAGGCCCAGTCGCGCCGCTCCGGAGGCGGAGGCGGAGGCGGCTACGGCCAGCGC V N E A Q S R R S G G G G G G G Y G Q R 301 GGCGGAGGCGGAGGCTACGGTGGCGGCGGCGGCTACGGTGGTGGCGGCGGCGGTGGCTAC G G G G G Y G G G G G Y G G G G G G G Y 361 GGCCAGCGCCGTGAAGGCGGCTACGGCGGTGGCGGCGGCTACGGTGGCGGCCGTGGCGGC G Q R R E G G Y G G G G G Y G G G R G G 421 GGCGGCGGCTACGGCGGTGGCTACGGCAGCCGCGGCGGCGGCAACTCCGACGGGAACTGG G G G Y G G G Y G S R G G G N S D G N W 481 AGGAACTGA R N & Supplemental Figure 8. Sequences of Glycine Rich protein. cdna sequence and protein sequence of GRP162 were shown above, the peptide marked in blue is synthesized for preparing antibody. RNA Recognition Motif of GRP162 is underlined. 9
Supplemental Table 1. Pollen fertility of T0 transgenics (stained with 1% I2-KI -KI). No. pubi-ppr683 pubi-ppr791 pubi-ppr794 pubi YTA 1 2.38% 44.31% 3.91% 0.23% 0.00% 2 2.21% 45.83% 4.88% 0.32% 0.00% 3 1.34% 49.81% 9.78% 0.51% 0.00% 4 0.35% 44.99% 4.22% 0.02% 0.00% 5 5.41% 51.21% 5.13% 0.00% 0.00% 6 1.62% 53.12% 0.00% 0.16% 0.00% 7 1.77% 45.79% 1.81% 0.21% 0.00% 8 0.00% 46.88% 0.79% 0.34% 0.00% 9 1.59% 8.31% 0.12% 0.00% 10 1.78% 6.43% 0.00% 11 4.16% 0.00% 12 5.53% 0.00% 13 5.43% 0.00% 14 7.33% 0.00% 15 3.31% 0.00% 16 4.32% 0.00% 17 0.00% average 2.85% 47.74% 4.53% 0.21% 0.00% ercentage of fertile pollen 100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% pubi- PPR683 pubi- PPR791 pubi- PPR794 pubi YA 10
Supplemental Table 2. Pollen fertility of T1 of PPR791 transgenics (stained with 1% I2-KI -KI). Percentage of fertile pollen of T1 individuals 42.78% 90.78% 48.74% 95.12% 44.93% 89.76% 91.43% 91.31% 59.42% 51.50% 49.56% 88.69% 98.17% 50.18% 97.61% 49.79% 49.21% 97.62% 98.44% 94.34% 95.12% 99.82% 48.75% 83.14% 49.54% 99.23% 94.32% 50.74% 93.70% 51.84% 53.97% 49.89% 44.85% 84.83% 94.14% 97.65% 49.81% 48.95% umber of T1 18 16 14 12 10 8 6 4 2 0 43% 52% 61% 71% 80% 89% 98% 100% Percentage of Fertile pollen 11
Supplemental Table 3. Pollen fertility of F1 progeny with the PPR791 T0 plant backcross to YTA (stained with 1% I2-KI -KI). percentage of fertile pollen of F1 individuals 50.11% 45.11% 44.89% 45.89% 43.34% 49.82% 49.23% 47.90% 45.80% 46.17% 50.12% 49.74% 49.82% 49.88% 51.34% 48.16% 48.83% 50.38% 49.11% 49.71% 54.29% 51.09% 49.23% 41.93% 50.41% 49.78% 49.81% 51.41% 44.53% 50.93% 52.19% 51.80% 50.81% 51.13% 51.83% 49.02% 52.82% 49.25% 59.21% 53.22% 48.78% 49.78% 45.25% 51.29% 51.31% 51.39% 49.81% 49.37% 49.73% 46.91% 49.89% 51.82% 49.23% average 49.52% 12
Supplemental Table 4. The candidate partner proteins of RF5. No Proteins Accessions 1 Glycan structure- biosynthesis 1 NM_001059777 2 V-atpase subunit NM_001051253 3 Gigantea homologous protein NM_001048755 4 Glycine Rich protein AK289192 5 Hypothetical protein AK241834 6 NAM protein domain containing NM_001062103 7 Copper chaperone homologue CCH contains CT833908 8 WD40-domain containing protein NM_001059777 13
Supplemental Table 5. Primers used in this study. Primer Name Primer Sequences Purpose Rf5-atg-BamHI-F cgggatccatggcgcgccgcgccgc Clone Rf5-tga-BamHI-R cgggatcctcagcagctcaaagattct Clone RF5-MBP-F cgcgaattcgaggtaactcccgacttgtgc Expression RF5-MBP-R ggaattctcagcagctcaaagattctataaag Expression RRM-R GCCGTAGCCGCCTCCGCCTCCGCCT Expression RNApF RNApR ggatcctaatacgactcactataggcttgaatgatgctata AATCTCCATC cccgtcgacatggaagaccgttagtccctcgggt AGTG EMSA EMSA ProbeA-F taatacgactcactataggctcatgtttctacgatc EMSA ProbeB-F taatacgactcactataggcgggacaaacgcggaag EMSA ProbeC-F taatacgactcactatagggaaaaaccgacgccaac EMSA Rf5-Y3F cgcggatccgagaggtaactcccgacttgtgc Yeast 3-hybrid Rf5-Y3R ggaattctcagcagctcaaagattctataaagg Yeast 3-hybrid GRP162-Y3F catgccattgatggcggcgccggatgttg Yeast 3-hybrid GRP162-Y3R ccgctcgagggccccaccgctcagttcctcc Yeast 3-hybrid AF CTCATGTTTCTACGATCTCA Yeast 3-hybrid AR TCTTCGGCTCCTCGTTT Yeast 3-hybrid BF CGGGACAAACGCGGAAGT Yeast 3-hybrid BR GATCTTAACGTTGGCGTCG Yeast 3-hybrid CF GAAAAACCGACGCCAACG Yeast 3-hybrid CR GCAGATTTGTCATAGCACAA Yeast 3-hybrid HptII-F CTGCTCCATACAAGCCAACC PCR HptII-R GACCTGCCTGAAACCGAACT PCR orfh79-f ATGACAAATCTGCTCCGATG PCR orfh79-r TTACTTAGGAAAGACTACAC PCR 14
Supplemental Table 5. Primers used in this study (continued) Primer Name Purpose Name Primer Sequences Purpose atp6-orfh79-f TCCTTGTCTATGGCGGTAA PCR atp6-orfh79-r GAGCAAACCACCACTGTCC PCR ptrgf TGGCTGAACAACTGGAAGCT PCR ptrgr ATTCGTCGCCCGCCATAA PCR Rf5-SalI-BiFC cccgtcgacgcagctcaaagattc BiFC GRP162-BamHI-atg -BiFC ccggatccatggcggcgccggatgt BiFC and Expression GRP162-SalI-BiFC cccgtcgacgttcctccagttccc BiFC and Expression 15