Supplemental Data. P rez-Nadales and Di Pietro (2011 ......Mar 22, 2011 · ca nv n tk s s s qnqs...

Sc MQ F P F A C L L S T L V I S GS L A R A S P F D F I F GN G T QQA QS QS E S QGQV S F T N E A S QD S S T T S L V T A Y S QGV H S H QS A T I V S A T I S S L P S TWY D A S S T S Q T S V S Y A S Q E S D Y A V 110Ca - - - - - - - - - - - - - M L A N V K L N L V T A L Y V L S Y V S V V N A Y QQ E N E I T P A D N I D K R A GA I GN F F R D F T N S I F GN D N S E V N Q P S T N GA T S T GH F F G P S I P S T S T H QQ T P G E T S N 97Ag - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MR V V T L A G L S V L L S A S P A L A D A Y A A A 26Af - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MR P A T L V A A L L S L GG L E L V A A 21Fg - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MH V K S F V L A L A L A S S S V V E 19Fo - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MH A K N F I V A L A L A S S S V V E 19Mg - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1Nc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MGW L V P G P L A K I L GR E K C G F V E 22

Sc N QN S WS A S T N Q L P S T S T T S Y Y A P T F S T S A D F A A S S V N A A S D V S T A S V P I D T S A N S I P F T T T S N I E T T T S A P L T S D T P L I S T S T MS A A D N V F S S A N P I S A S L T T T D S S E S F 220Ca N V N T K S S S QN QS P S T S P T S T V A A A A A T - S S S P V A S T R P A S T S E QK QQ E E T T A R QS T S P A T T A T T S N T P P S P S T S K E T P T S N T A Q T S S A N N N QQS S N T A A P S T S V I Q P - S T 205Ag A P P A D I V D Y GR H Q P MV R R S D N E T V QA S P T A L GV L P S L L A S I A N E I E S R L D P I N K K T T S S T T L S V A T N T K S S S S R S S A T G K L H GS S K S S E T A A S - - - - - - - - - - - - - - - - - 119Af E N A Q E L K P R F I P K Q F K R H V V H G P D S S - A T T S S N - - - - T N I H L D K R A D V N P F I S Y F N S L L QN G P T A T S S A P L V S L P I I V S I D A S GV T H T I T P T - - - - - - - - - - - - - - - - - - 108Fg A N R P R I Y Y P R QV K R E V V N N A A A A P A V P - E N QV - - - - - - V P D L E K R D P QN S F L D R L F S N N N D D K A S T GD K E S G I N F D P T I P L G L K R P G P S A S A P P A L V L Q P T T T S MA - - - - 118Fo A N R P R MY I P R QV K R E V E N N - - - A P V A P - E N Q P - - - - - - A P V I E R R E P A D S F I K S L I D K N S R K QA S Q - - - E D GV N F D P T L P L G F K K S G P S A S A P - A L I I Q P T T S - - - - - - - 108Mg - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MH N F S K L A V A F V A A A S F A S A E P E T K A K V E R P I I Y F P R H I K R Q F A N T T T - - - - - - - - - - - - - - - - - - - - - - 48Nc R I GA GA R GR MK R K R L GH R D L A GA GD V D - GD L A G E L R S F G L F V K F T R Y D Y L L T T A L L A A S Y V A G E H V G P T K P K Y Y F P K H V K R QY A N A T I T S N D A P I S T I D T S S D A L S Q T T K 131

Sc D Q T S T A GA I P V QS S A D F S S S S E I L V QS S A D F S S P S S P T T T D I S L S A A P L Q T S E S S S F T T A S A A L P V S S T D V D GS S A S P V V S MS A A GQ I A S S S S T D N P T MS E T F S L T S T E V 330Ca S E V H V QS QQ T S T T P N T P T S S P N T P T T S E A A P T T S A A P T T S E A P V T P S T S E V V P N T P T T S E A P N T P T T S E A P V T P S T S E V V P N T P T T S K A P N T P T T S - - - - - - - - - E A P A T 306Ag - - - - - - - - - - - - - - - - - S D T A D S A S T S A P L T S S N T R S T E S T E S S A V T S S T A K K G T K T P T K G L S E S S S T K T I D S E T I K P L S E P T L S S GH S H S R K T L S - - - - - - - - I P T S Y L 204Af - - - - - - - - - - - - - - - - - - - - - - - - - P T P S P S A E T S S D A T S GS T L E P S S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A T D QS A P N N V P A T A - - - - - - - - - L S T A Q T 151Fg I E E P E T T E P A E A T D A P I E T D T G I L L A P S G I V T S K T A V K S T Q P A K T S A A A P K E E T S A A P V E T E T - - - - V V E T E E P A V T T E A P K E E T K L A A T T A P A V K - - - - - - - - - E P E S V 215Fo E A L P E T T E P A V S T D A P T E A D T G I L L A P S G I V T S K T A A Q P A E P A K T T A A A V K E P S T E A P K K E E T K P A A T S D V E A K P A T T E K P K E E T K P A A T T A P A A E - - - - - - - - - E P S S V 209Mg - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P A S E A S S S T S R P P P I P V P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E T S S F S S S A S S S - - - - - - - - - S A Q E L 83Nc R E T T S N D QS E S L S D I P F V K S T S S R F L T S S E P Q S E S E S D T S R V T T V V I A S T V Y V S P S Q P T T A D L S T A G T A A P S A P A V D T D F V S S T D S T A GS S D S T S Q - - - - - - - - - G T T G L 232

Sc D GS D V S S T V S A L L S A P F L Q T S T S N S F S I V S P S V S F V P S QS S S D V A S S S T A N - - - - - - - - - - - - - - - - - - - - - V V S S S F S D I P P Q T S T S GS V V S V A QS A S A L A F QS S T E V Y 419Ca P T T S E A P N T P T T S E A P V T P T T S E V V P T T S T QGD A V S T S S T S V T E Q T T L T S - - - - - - - - - - - - - - - - - - - - - S T Q L P P T T A S T T Q T S T P E A S D S P K P S S T S I E T P S T S T F E 395Ag T N T E A K P T P S A E T S E E MS S T T D K - - - - - - - - - - - - - S K H K T K K E K H K S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T T T S G T D A I S S S E T D 254Af S S P A K T D T S D N S Q - - - - - - - - - - - - - - - - - - - - - - T A P A S S P A T S S G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N T V S D T L S A V V S S V L GD 193Fg N T P K E K P - V D S K T E E P A P V K T T - - - - - - - - - - - - - - E P K V E Q P A S T P - - - - - - - - - - - - - - - - - - - - - - - S K K T V I E T K T E S - - - - - - - - - - - - P V E T K P - - A V V S T T K K 273Fo K T P E K K P V V E T K T E A P V P V E T V T E P A V V E T K T E K P V E S K T E K P S A T K G L L D P V E S I L T S V L P V A S D P K K E T K P P A I E T K T E E P A V E T T E K P K T E P E E T K P - - A A V P T T K E 317Mg T A S R Q P T S I D E F F - - - - - - - - - - - - - - - - - - - - - - - - - - S T L S D A L T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T D S T P F S - - - - - - - - 111Nc S S S A T D N S S T D L L G F P T G I S S GS - - - - - - - - - - - - - T T E S S L S D S T T G - - - - - - - - - - - - - - - - - - - - - - - - - - G T S S GA T S S P E S T P G T S L GS T T D S S T S S P V T A T D R V 303

Sc GA S A S S T MS S L L S T T S L QS T T L D S S S L A S S S A S S S D L T D Y GV S S T A S I P L L S A S E QA S T S S S F S V V S P S V S F V P S QS S S D V A S T S A P S V V S S S F S Y T S L QA GGS S M T N P S 529Ca QD P T T T S S V G T P S S E Q P Q P T T T S E S A V T S N S P T Q E - - S T S L V E P T T S S L E S S N T P T P N P S T S E A Q P S T S A S QA P P D T T S S A P A P E L S S S N A D F S N S V L H S S E T T S L V N P T 503Ag L S S T I T D G L S T L T T P T S D S MS Q L S A T S S I D S S L S GS Q I P S A T S S L S S T GK A T N S A I S A S L S S G T S S T S S GS H L P S GS P T D S S V S G I P T S G L T S S S A QS S GS G I P T S G L T S 364Af P S S S T S T S P P V S S S A T D GS K A P E S - - - - - - - - - - - - - - - - S T T V S A A Q T S QA K T D P S D N S Q T T P A S S T P A T S S GN V V S D A L GG - - - - - - - - - - - - - V GG L V S S V L GN P S S 274Fg G L L D P V E T L L S S L L P V P A E S K T T Q P K A V N T E T T K P K E - - E T Q P A P A E T Q P A P A E T Q P A P V E T E T V Q P A D T E T K S P S V E K P A P T T K E G L L D P V E T L L S S I L P V P A E T E T K D 381Fo G L L D P V E S I V S S V V A D P A D S K T T E P K P V N T E T K K - - - - - - A E P A K T D S K - - - K E T Q P A P V E T E T A K P A E P E T T S P A A E K P A P T T K G - L L D P V E T L L S S V L A D P A D N K T K D 417Mg QR P A T S GA GR S S A T GD V T P I I V P S S - - - - - - - - - - - - - - - - A S P P S T A V K P GS V S A L T T S QN S T S A A T S E S V T S P GS T S - - - - - - - - - - - - - - - - - - - - - - - - G P A G T P E 181Nc D S S A S A S A S A S A S T S A S D N T I L P S S GA N V T S S F A S - - T T D A V S S S S S V T K T A S S S S P T T D S A S S S A S V T GS V T S P GS I A N T T S S A I S A S S S S S V V D I L S F L N P G F GGA S S 411

Sc S S T I V Y S S - - - - S T GS S E E S A A S T A S A T L S GS S S T Y MA GN L Q S Q P P S T S S L L S E S QA T S T S A V L A S S S V S T T S P Y T T A GGA S T E A S S L I S S T S A E T S QV S Y S QS T T A L Q T 635Ca D S Q I D S S S T T D A V S QA T T E P T S E N T P T A A S S V T A N D I N S A QS S A P T S N A D A E T A S S P V S E QS L A T GS Q T S L D T T A GA S S T A S E A T A E N L S T F G T D GS S D A S Q T I A E T T S N 613Ag S S A QS S A S G I P T S G L T S S S A QS S GS G I P T S G L T S S S A QS S GS G I P T S G L T S S S A E S S S S G I P T S G L T S S S T QS S GS G I P T S S L S S S S T QS S GS G I P T S S L S S S S E S A V S G 474Af S T I A V P - - - - - - - - T A T E A K S T GS S A T E S N D P S T N S T T P T GS S S P A A P V E T T P T P A A S S S G L L D T V A S D L S G I L P T A S S S L A S GA A T T D S S N A D G T V T H S T V V I P S S T GN 376Fg P Q P D Q T QS E - - - - - P L P E E T G T P T K E P QR Q E S K P Q T D D Q T L P T V S P P S D E P T A A P G T A Q T S E GG L L D P V E T L L S S L L P V D P S D V K T T S P E E T A A P G I E P T S A A G E E T GS E 486Fo P T P D Q T E S I - - - - - S Q P E E T G T P T K E P QR D N S K P Q T D GQ T L P T F S P P A D K T T A A P N A G E T S K D G I L D P V E T L L S S V L P N Q P - D V K T T S P E E S S A P - V D S Q P T S A A P N GS E 520Mg S S S A S D - - - - - - - - - F T S A V A T S R A S T A T S N T G L I P E T T I L P T T A T S N T G L I P E T T I L P T T A S L S T A E S A V T P S I T S S - - - A S S S G I L I A P T GV V T P T S S S - - - - - - - - - 270Nc S A S A T D S T T V L GS V A N T T S A A I S A T D V A S S S A S A T D S T T A L G S V A N T T S A A I S A T D V A R S S A S A T D V A S S S A A S T D A S GS L T S A S A L P T V S S GV I S N T T S P D S S S S L QV L 521

Sc S S F A S S S T T E GS E T S S QG F S T S S V L V QM P S S I S S E F S P S Q T T T QMN S A S S S S QY T I S S T G I L S QV S D T S V S Y T T S S S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 712Ca S P D QS V V T P S A S A S P D V S T L P T GS E S G T S L V S GS E T S I D T N T V A S GS T V I P E S S N I P T QS P S QS V V S S D A A A S N V S T GS A T T D S L A GS E T GV Q P I S S S A T G T S E P V F S S E 723Ag S L I S S A Y S S L S S I S S D N I S I S S T D S A GS S H L S H A L P T S S S V I I P I S S T P I P Q E L T M T GN S S L S T V R S G T E I S T I S T S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 551Af N S S P E A S P S P T P S P S GV L D P L T S A V T D L L P G - - - - - - - - - - - S A S S S A V T N GS S GA E T S - - - - - - - - - - A S T D L GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 431Fg Q T V S GA P V T N P E T T G L P E L I P - T S I L P E P T L P L D E T D L L P I P T D I T D V T S I L P E L S S P V T V S P A I P T T L A P - E E S Q T G L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 563Fo Q T V S A A P S T E P E T T G P P E L I P - T S I L P D P - - - - - - T D I L P V P - - - T D V T S A V P E P S S P V T V S P V I P T T V S P P E QS E S GS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 589Mg S T E D P V F D G I G T L I S S I V S S V S T V L Q P N G T A P - - - - - - - - - - - - V T T T P N T S V D V A T - - - - - - - - - - - - - T P V D I A S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 322Nc T P P T T T V E P I S T A N A T T V E T A S T V T A T D GS A S T GW L P P I I V L P S I R T S S T S A A D V A N S T A P A L V S G T GR A T N S T I G T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 598

Sc - - - - - - - - - - - - - - - - - - - - - - - - - - - S V S QV S D T P V S Y T T S S S S V S QV S D T P V S Y T T S S S S V S QV S D T P V S Y T T S S S S V S QV S D T P V S Y T T S S S S V S QV S D T S V P S T S S 795Ca Y N S S E G T T S L V V P T N S E L S S T V T GS S E T A A T A I N S E S V L T GS S D T A A T V T GS E S I L T GN T E T S A T A I A S E S T L T GS T T GA T D S A A T T I A S E S V L T G T S D A S A T V I P S E S A 833Ag - - - - - - - - - - - - - - - - - - - - - - - - - - - A V T S V S S T K S E T T T S V P L S T S R S A N H S A V I T P S S S P L T I L P S S S S T T V V S T S E GH K I S S T F N GS I S S H T L A S H S T F S L T S S S V 634Af - - - - - - - - - - - - - - - - - - - - - - - - - S T V P V S V P A V T P S A T S S S G L GG I L G P I L S S A GS T GV I P - - - - - - - - - - - - - - - - - - - - T A S T H T S S F V GV P T G L T S I L I P GGS S G 496Fg - - - - - - - - - - - - - - - - - - - - - - - - - - - - P D L T S F V D P N T K L - - - - - I D P T E S P N V T L P D A T S GV G T E P A E T E L P V P T T E P V N G T E T Q P A N G T E T Q P A P T G E P E T T GA P E P 640Fo - - - - - - - - - - - - - - - - - - - - - - - - - - - - P D L T S F L D P N T K F P Q T GS V G P T D A P N T T V P E V T S G I D V K - - - - - - P E P T S E P I N - - - - - - - - G T E T I P N V T G E P Q T T GA P E P 657Mg - - - - - - - - - - - - - - - - - - - - - - - - - - T T A S D T L S P T T A V V S T T G - - - - - - - P V T S V Q T L P P V S T - - - - - - - - - - - - - - - - - - - P T A N G T V T S - - - P P V D S Q T T V L P T T T P 377Nc - - - - - - - - - - - - - - - - - - - - - - - - - - A T A S E I I A T E D P L I T S S S S S G I L L A P T GV V N E T T S A S T N L I E S L A S N V A S L V GS I L I P T GN G T A T E P T A P V T T S D S V T I P S A T E 682

Sc R S S V S QV S D T P V P S T S S R S S V S Q T S S S L Q P T T T S S Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 831Ca L T GS T T T P I A S E S V L T G T T S A D V S GA T T I GS E S I F T G T T E S T G T P L P T A S G T E S L D T T V A T G T S V S E QS GV E T A L S T Q P T T G T E A T V T S GV S QS E Q T G T S A V T GV T E S S E 943Ag S MS S F V T S S S QV P E E V T P T P QS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 656Af S E S P T S Y T Q I S P S V P T T A P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 515Fg T E D V T E A P T - - - V A P T A T GA A S E E P T GN - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 665Fo T D V N T E E P T N I T Q T A V P T GD A T N E P S GN T T A P A T E P A T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 695Mg - - - - G L S S D T I V T S P GV T A N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 393Nc P Y A S GS GS GV I T P S A S L T T S Q P K P P MMS S G I V S I P I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 718

Sc - - - - - - - R F T I S T H GA L S E S S S V S QQA S E I T S S I N A T A S E Y H S I Q T T A A T QS T T L S F T D A N S S S A S A P L E V A T S T P T P S S K A S S L L L T P S T S S L S QV A T N T N V Q T S L T T E 934Ca Q I QS GA T T P A T T T A S D A T A V T N A S E A S A E S QA T T T A A S E A T T GS QV T T A A S E A A T E S QA T T A A S E A A T E S QA T T A A S E A A T E S QA T T P A S E P A T GS QV T S E V T P A T V P S S 1053Ag - - - - - - - - - - - - - - - - - T E T E Y P QS T R P S N T S T L Y S S S R E T Y P Y T T T L S Y I P S T T S A D K T S E E S Y S A P E V T T L A P L S D S T QS S QA S T T L S S QD S T T L T T S S T S T R P T TWG 749Af - - - - - - - - - - - - - - - - - - - - - - - S V T S T A V I P GS S D T A I GS S I A V T GS T GV S S T L P T P T A D F S S S A I Q T L S T T E Q - - - T T P S P S T T P E T T T A A P T T T T T S T T Y P P E T T T - 598Fg - - - - - - - - - - - - - - A T E P A T E P I D T A T D S V P T E L P A T Q T GD E T N G T A T E E P T S P A T GV E T E S A P V A T D I QQ T S A A V P T - - V S E V Q P T I Q P T S Q P P V E T E T Q P Q T Q P E P V P 759Fo - - - - - - - - - - - - E P A T E P A T K P V D T A S GS V P T GS P A T E T D A E T N G T A T A E P T K P A T GV E T QS A P - A S E G E Q T S A V A P - - - - T D E Q P S V A P T S Q P P V E T E T K - - - - P E P E P 784Mg - - - - - - - - - - - - - - - - - - - - - - - - - - S T QV P - T T V P T T I P T T Q P P V T E P T I T P T V L P P S - - - P N N T V P S N T T T Q L P - - P T QA P T L T Q L P T T T T S P A L T T P A T T P S V A P T S 471Nc - - - - - - - - - - GN S T V S S T K A V E S G P T T T G I P GS S V P V I P GN S S I P V T S MS D T T V V L P P V T L I N S T T V A A N S S T I I P V I V T S A S A E S S V P V S S L E P V T T T QV L I P P P V P T T 818

Sc S T T V L E P S T T N S S S - - - - - - - - - - - - - - T F S L V T S S D N NWW I P T E L I T QA P E A A S T A S S T V G - - - - - - - G T Q T M T L P H A I A A - - A T QV P E P E GY T L - - I T I G F K K A L N Y E 1019Ca V T A A D T A A T S I I T S P P A S A E P S S E V T A V A P S A A T T S S T K NW L P S S L V I A E T P S S N A S K S T E S I V QA S A T GA S T S G L P R A I T P - - E T T T T P G F D Y QV - - I T V G F K S A L N Y P 1159Ag - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K N A P S GNW L P T V I I T A T E Q E V V S T E S P QN T QS K P K P T A T P N I Q T L P QV I A A P D R V T P K K D Y T L - - I T I G F K K Q L N Y P 824Af - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T E N T D F V P S S I L V E P - - - T T T T Q L S T - - A E T A T G T N R P A Q L P GS I S P A N G L T S P - P P D S T L - - ~ I Q L G F N GK L R Y S 665Fg T T L V P T A A E P V T S I R - - - - - - - - - - - - - - - - - P T A T L T N T N NW L P T T I M F E P - T S I P A A P T QA - - T E T S T S T G L P A N I P R V I L P - N D P N K P I P E GS R S I Q I G F L F P F N Y K 848Fo T T L V P T A A V P V T S I R - - - - - - - - - - - - - - - - - P T A T L T N T N NW L P T T I V F E P - T S I P A A P S Q P - - T E T S T S T G L P A N I P R V I L P - N D P N K P I P E GS V P I Q L G F L F P F N Y K 873Mg - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A T S S A N S N D DW L P T T I I V QA P L P S T T GS S T N - - A P S S A P T V L P S D L P K I I N P S D D I T E P L G P D MM E - - I QV A F K F A L N Y R 547Nc T I N Y P P V T QY T P T - - - - - - - - - - - - - - - - - A T V S N M P P GY N S P T T I L V D Q P P P A Q T H T V S F - - V QQ T T A T A L P T D L P K A I A P - D D T T A P K P E D N V F - - I Q I G F N Y G F N Y P 906

Sc F V V S E P - K S S A Q I F GY L P E A L N T P F K N V F T - - - N I T V L Q I V P L QD D S L N - Y L V S V A E V Y F P T A E I E E L S N L I T N S S S A F Y T - - D GMG T A K S MA A MV D S S I P L T G L L H D S N 1122Ca F V V E N S - I S S A Q I F QY L P R V L K Y P F N GD K S - L QN V S V R R L I P Y - T A S N I D Y T I T V A E V Y F P K D S V K A L GS F I T T P GS A I Y R - - N P D S V L QA L A S L I D S R I P L T G L V T D D Q 1264Ag F I A MN P - F A N A Q I F D Y L P G I L N Y P F N F E Y A - - - D I S V I Q L V P L K A N S K D - Y I A T I A QV Y F P S E K V D E L A K L V V D T N S E L Y S - - S Y D A N MK V F A S L I D P Q L P L T G L A GK G F 927Af F V A T T P - L S S S Q I F L Y I P QG L I Y A L E I L G - - - K D I A M F A I Q P Y D N S A S T GY I A T V A L A Y I P T D K V D T L R K L L R S P L S K L Y Q - - Q P N E S V K T L F S M I D P S I P L V V G E S GS S 769Fg F L A R N T - V A A A Q L F K Y L P K G L A D A GG F S K - - - D R I L I E K I I P MD T QS QWGY I T A L A K I H Y P E N M L D S L QA D L M T P N S L L Y N - - N D L E I V R N L T S V I N N K I D I F G - - - - - N 947Fo F L A R N T - V A A A Q L F K Y L P K G L A D A GG F S K - - - D R I L V S K I V P L D T QS QWGY I T A L A K I H Y P E N M I D S L QA D L I T P N S P L Y N - - N D L E I V R N L T S V I N T K I D I F G - - - - - D 972Mg F I T N E N P N A GA Q I F E Y L P K S L K Y M E G L T E E QK K R L QV L R V V P L N T E QQ L GY V T S V A I A TW P K A F F P Q L R L D V K T P F S Q F Y QN T S N GM L A H N L T M L V N P A I D I L P GA T L D G 657Nc F V A K N N - N A A A Q I F R L L P E A L A F A S S I E S - - - H R V R V T K L M P MN T V N T L GY W T T L A V V S Y P QA Y V E S L R L D V K I A S S P L Y N N P T P - - L V Y N L T MQ I N P A I D I I L GS T L I G 1010

Sc S N S GGS S D GS S S S N S N S GS S GS GS N S N S GV S S S S GN S Y QD A G T L E Y S S K S N S N V S T S S K S K K K I I G L V I GV V V GGC L Y I L F M I F A F K Y I I R R R I QS Q E I I K N P E I S - S I S 1231Ca QV S GS S S D S N P S T N - S Y GS MD I V S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N T K V A D K GR I A G I T I GA A A GC G L Y M T L MV L L F R K F R K S N K A L - E L P I T D S E S N L G F 1342Ag S S S GS T T QN K P H P D A D N S I GR S GA L GG P Y N P - - - - - - - - - - - - - - - - - - T N S K S A A V P MS K A K L T G I V V GS I C GV T L Y I A L M I V GV R Y F L L R S R GGV E L QD N H S F Y S S N S 1019Af S GS G L Y S D S G I S GS GS S S D GN GS GS N S GN D A - - - - - - - - - - - - - - - - - - - D A GA S R S A S A R A S S V G I GV GV V C GA A A Y GA GM FWV A R R Y R K K R Q L - H R R S N - - - - - - S T M 853Fg T D N GA S S D T GD S D D S G L GGN D G F GN S G E - - - - - - - - - - - - - - - - - - - - - - - - - GD K T A K Q K A T T A G I A V GA V G L S V MY GA A M F L V A R R Y K R K K Q L GH R R A S S I GS S QR S S 1032Fo S D T D N N T D N GN S D D GN S GN R D V F GN S G E - - - - - - - - - - - - - - - - - - - - - - - - - GD K S A K Q K A T T A G I A V GA V G L S V MY GA A M F L V A R R Y K R K K Q L GH R R A S S I GS S QR S S 1057Mg K P A G - - A GS G T GGN GS N G P N D V F N N D N N S - - - - - - - - - - - - - - - - - - - - - - - - T N QS A T Q R G T V A G I A F GA V S L A A A Y GA A M F I V A R R Y K K K R QA - H R R S S S V A - - - T P S 737Nc D GS GGD S GS N P S N A GN N GN A D P F T N N N N - - - - - - - - - - - - - - - - - - - - - - - - - GN Q T S QQ R G T T A G I V GGA V A V A A A Y GA A M F V V A R R Y K R K K QA - H R R A S S L GG - - S P S 1092

Sc S S E F GG E K N Y N N E K R MS V Q E S I T QS MR I QN - - - - WMD D S Y Y GH G L T N N D S T P T - - - - - - - - R H N T S S S I P K I S R P I A S QN S L GWN E V - 1306Ca S D E D S S M L E S S S G F S A I F S R I N H GGV L T GD P - - - - - - - - N GGGD D MMMMN N N N - - - - - - - - - - - N N L R P N N I S E P V QA S N S L GWY H - 1409Ag GGS S D M I T S S E I P P T L Y D D K S L F V T A N N A R MMR A S V T P S MK V D NWMD Y N N S V N G L GD N N A T H S F V N GK I T K I S G P I A S E N S L GWD P - - 1105Af E Q T S E GR GA GS L F A - - - - - - - - - - - - - - - - - - - - - - - - - A G GR L S R N S QN S R - - - - - - - - - - - - GS GR GQM I S A P V MA E N S L GWN - - - 901Fg E MQY N GN GS P A L MGGA L MS R D F S N Y GA QG P - QG E QA P A R P G GR D S H GS GR S G - - - - - - - - - - MGN S A R T A F I S A P V A A E N S L GWN - - - 1106Fo E MQY T GN GS P A L MGGA L MS R D F S A Y GA T - - - H A E QGQV R P G GR D S H GS GR S G - - - - - - - - - - MGN S A R T A Y I S A P V A A E N S L GWN - - - 1129Mg E MR QS G - - S P A L MGGA L L S R D F T H Y GGV MG - - - - - - - - P A G GR E S H GS N GS GR - - - - - - - - S A GN S A R T A G I S A P V A Q E N S L GWN - - - 804Nc D MQQMGGGS P A L MGGA L L S R D F T GY GGV A GGA A A A GA V A P GGR D S H GS GR S G - - - - - - - - - - MG L S S R T A Y I S A P V A A E N S L GWN - - - 1167




























Sc S S E F GG E K N Y N N E K R MS V Q E S I T QS MR I QN - - - - WMD D S Y Y GH G L T N N D S T P T - - - - - - - - R H N T S S S I P K I S R P I A S QN S L GWN E V - 1306Ca S D E D S S M L E S S S G F S A I F S R I N H GGV L T GD P - - - - - - - - N G GGD D MMMMN N N N - - - - - - - - - - - N N L R P N N I S E P V QA S N S L GWY H - 1409Ag GGS S D M I T S S E I P P T L Y D D K S L F V T A N N A R MMR A S V T P S MK V D NWMD Y N N S V N G L GD N N A T H S F V N GK I T K I S G P I A S E N S L GWD P - - 1105Af E Q T S E GR GA GS L F A - - - - - - - - - - - - - - - - - - - - - - - - - A G GR L S R N S QN S R - - - - - - - - - - - - GS GR GQM I S A P V MA E N S L GWN - - - 901Fg E MQY N GN GS P A L MGGA L MS R D F S N Y GA QG P - QG E QA P A R P G GR D S H GS GR S G - - - - - - - - - - MGN S A R T A F I S A P V A A E N S L GWN - - - 1106Fo E MQY T GN GS P A L MGGA L MS R D F S A Y GA T - - - H A E QGQV R P G GR D S H GS GR S G - - - - - - - - - - MGN S A R T A Y I S A P V A A E N S L GWN - - - 1129Mg E MR QS G - - S P A L MGGA L L S R D F T H Y GGV MG - - - - - - - - P A G GR E S H GS N GS GR - - - - - - - - S A GN S A R T A G I S A P V A Q E N S L GWN - - - 804Nc D MQQMGGGS P A L MGGA L L S R D F T GY GGV A GGA A A A GA V A P G GR D S H GS GR S G - - - - - - - - - - MG L S S R T A Y I S A P V A A E N S L GWN - - - 1167





















































































Positively charged cluster

C-terminus

PRD

TM

Sc mucin repeats





























Supplemental Figure 1. Sequence alignment of fungal Msb2 proteins. The alignment shows the predicted amino acid sequences of Msb2 orthologues from S. cerevisiae (Sc), C. albicans (Ca), A. gossypii (Ag), A. fumigatus (Af), F. graminearum (Fg), F. oxysporum (Fo), M. oryzae (Mg) and N. crassa (Nc). Highly conserved residues are shaded in black, moderately conserved residues are shaded in grey.

Supplemental Data. Pérez-Nadales and Di Pietro (2011). Plant Cell 10.1105/tpc.110.075093

1

Supplemental Figure 2. F. oxysporum Msb2 does not complement signalling function in S. cerevisiae.(A) Physical maps of the gene replacement construct containing the F. oxysporum Msb2 ORF and terminator with the kanamycin resistance cassette, and of the S. cerevisiae MSB2 gene locus. The relative positions of the primers used for PCR analysis of transformants are indicated. (B) Amplification of genomic DNA of S. cerevisiae strain PC538 and three derived transformants using the indicated combination of primers. An amplification product indicative of homologous insertion of the construct at the MSB2 locus is detected in transformants T1 and T2, but not in the ectopic transformant E1. (C) RT-PCR of cDNA obtained from S. cerevisiaestrain PC538 and three derived transformants using the indicated combination of primers. An amplification product indicative of the presence of the F. oxysporum Msb2 transcript is detected in transformants T1, T2 and E1. (D) Cells of the indicated S. cerevisiae strains were spotted onto SC medium lacking histidine to visualize activation of the FG pathway-dependentFUS1:HIS3 reporter gene (left panel), or onto YPD medium (middle panels) and washed after 48 h (right panels) to determine agar invasion.

YGR015C

F.oxysporum Msb2 ORF (3390 bp)

kanMX4

Fo Msb2 terminatorYGR016W

Scmsb2-C3

Msb2-ORF-as

Msb2-ORF-s

S. cerevisiae MSB2 ORF (3921 bp)

YGR015CYGR016W YGR014W

Scmsb2-as

Scmsb2-s

500 bpA

SC-his

YPD

Before After

PC538

T1

E1

T2

PC948msb2

DB

C

Scmsb2-C3/Msb2-ORF-as

PC538

PC538+FoMsb2::kanMX4

T1 E1 T2

PC538

PC538+FoMsb2::kanMX4

T1 E1 T2

0,5 Kb

Msb2-ORF-s/Msb2-ORF-as

2,8 Kb


2

B

C

Dwt msb2

msb2 +Msb2-HA

#13 #53

1.8 Kb

M

4.5 Kb3.5 Kb

wt msb2

msb2 +Msb2

#C1 #C2M

fmk1msb2

9.7 Kb

5.5 Kb

wtmsb2

#16 #21 #23 ect.#57

1 kb

msb2-knock1

M13for

M13rev

msb2-knock2msb2-nest3

msb2-nest4

msb2-ORF-s

msb2-comp2

msb2-for2EcoRI

msb2-ORF-as

probe

EcoRI

EcoRI

msb2-3´HA-as

A

Msb2 ORF

hph cassette

TtrpC HygR PgpdA

HAHA-s

Supplemental Figure 3. Targeted disruption of F. oxysporum Msb2.(A) Physical maps of the Msb2 locus and the gene replacement construct obtained by PCR fusion ( msb2 allele). Relative positions of the primers used for generation of the gene disruption construct and PCR analysis of transformants and complemented strains, the probe used for Southern analysis, and the HA epitope in the msb2+Msb2HA strains are indicated.(B) Southern blot hybridization analysis of the wild-type strain 4287, different msb2 mutants and an ectopic transformant. Genomic DNA treated with EcoRI was hybridized with the probe indicated in (A). Molecular sizes of the hybridizing fragments are indicated on the left. (C) Amplification of genomic DNA of the indicated strains using forward primer nest3 and msb2-ORF-as indicated in (A), to differentiate the wild-type allele from that corresponding to the

msb2 allele. (D) PCR amplification of genomic DNA of the indicated strains using forward primer HA-s, specific for the HA epitope nucleotide sequence, and reverse primer msb2-3´HA-as depicted in (A).


3

Supplemental Figure 4. Msb2 contributes to growth of aerial mycelium and colony hydrophobicity under conditions of nutrient limitation.The indicated strains were grown for five days on yeast peptone glucose (YPD) or Minimal Medium (MM). A 100- l drop of water stained with Bromophenol blue was deposited at a distance of 5 mm from the edge of the fungal colony. Plates were incubated for 2 h at room temperature and photographed.

YPD

msb2 fmk1msb2+Msb2

fmk1 msb2wt

MM


4

Supplemental Figure 5. Growth of different strains in submerged culture.Growth of the wild-type strain (blue diamonds), msb2#16 (pink squares), msb2#57 (yellow triangles), msb2+Msb2 (blue triangles), fmk1 (purple diamonds) and fmk1 msb2 (brown squares) was measured in microtiter wells containing liquid yeast peptone glucose (YPD) (A) or Minimal Medium (MM) (B), by determining the optical density at 595 nm at different time points after inoculation. Error bars represent standard errors calculated from 3 wells.

0

0.2

0.4

0.6

0.8

1.0

0 10 14 17 20 24 36 40 42 45

0

0.2

0.4

0.6

0.8

0 10 14 17 20 24 36 40 42 45

Hours after inoculation

Opt

ical

dens

ity(5

95 n

m)

A

B

Opt

ical

dens

ity(5

95 n

m)


5

YPD

wt msb2 fmk1msb2+Msb2

fmk1msb2

Ethanol

Menadione

NaCl 0.4 M

NaCl 0.8 M

NaCl 1 M

Supplemental Figure 6. Msb2 is not required for the oxidative and osmotic (salt) stress response.Colony phenotype of the indicated strains grown on yeast peptone glucose (YPD) or YPD supplemented with 10 µg/ml menadione or with the indicated concentrations of NaCl. Plates were spot-inoculated with 105 microconidia, incubated three days at 28°C and scanned.


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Supplemental Figure 7. Phosphorylation of the Fmk1 and Mpk1 MAPKs in different mutants.(A) Msb2 is required for full levels of Fmk1 phosphorylation. Total protein extracts from the indicated strains germinated for 14 h in PDB and subsequently transferred for 30 min onto plates containing MM were submitted to immunoblot analysis as described in Fig. 5. A monoclonal -actin antibody was used as an additional loading control. (B) Phosphorylationof the Fmk1 and Mpk1 MAPKs in different mutants. Total protein extracts from the indicated strains germinated for 14 h in PDB (0´) and subsequently transferred for 30 min onto plates containing MM (30´) were submitted to immunoblot analysis with anti-phospho-p44/42 MAPK antibody ( -P-erk), using an extended exposure time that allows simultaneous detection of P-Fmk1 (lower band) and P-Mpk1 (upper band). A monoclonal -actin antibody was used as loading control. Relative positions of P-Fmk1 and P-Mpk1 are indicated to the right.

-actin

-P-erk

0´ 30´ 0´ 30´ 0´ 30´ 0´ 30´

wt msb2 fmk1 fmk1msb2 mpk1 fmk1

mpk1

0´ 0´

P-Mpk1P-Fmk1

B

fmk1msb2

msb2+Msb2wt msb2#

16 fmk1

-P-erk

-erk

-actin

msb2#57

A


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Supplemental Figure 8. Expression of MAPK genes in different fungal strains.mRNA abundance of the indicated genes was measured 6 h after transfer of the strains toMM plates using quantitative real time PCR. Relative expression levels represent mean Ctvalues normalized to actin gene expression levels and relative to expression values in thewild type strain. Bars represent standard errors calculated from four biological replicates. Values with the same letter are not significantly different according to the Mann-Whitney test (p 0.05).

Mpk1

0,5

1

1,5

2

2,5

wt msb2 fmk1msb2+Msb2

fmk1msb2

Rel

ativ

eex

pres

sion

aab

b

aa

Rel

ativ

eex

pres

sion Hog1

a

0

0,2

0,4

0,6

0,8

1

1,2

1,4

b

a

b

b

Fmk1

0,2

0,4

0,6

0,8

1

1,2

1,4

Rel

ativ

eex

pres

sion a

b

a

c c


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Supplemental Figure 9. Msb2 regulates a subset of Fmk1-dependent functions related to invasive growth.(A) Msb2 is not required for vegetative hyphal fusion, mycelial aggregation and root adhesion. Strains were grown overnight in PDB diluted 1:50 with water and supplemented with 20 mMglutamic acid. Upper: Fungal cultures were observed with a Leica DMR microscope using the Nomarsky technique. Arrows point to fusion events between hyphae and conidial germ tubes of the wild-type and msb2 strains. Bar, 10 m. Middle: Fungal cultures were transferred to a Petri dish and photographed. Note the presence of mycelial aggregates in the wild type and

msb2 strains. Bar, 1 cm. Lower: Roots of tomato seedlings were immersed for 36 h in microconidial suspensions, washed by vigorous shaking in water and observed with a binocular microscope. Adhering mycelium is visible as a white mass covering the roots. Bar, 1 cm. (B) Msb2 contributes to pectinolytic activity and penetration of cellophane membranes. Extracellular pectinolytic activity on plates containing polygalacturonic acid (PGA) was visualized as a contrasting halo underneath the fungal colony. Penetration of cellophane membranes was determined by growing colonies for four days on a MM plate covered by a cellophane membrane (before). The cellophane with the fungal colony was removed and plates were incubated for an additional day (after). Bar, 1 cm.

Before

After

B

PGA

A wt fmk1msb2+msb2

fmk1 msb2msb2#57msb2#16

Aggreg.

Fusion

Rootadhesion


9

WT

msb2#57

fmk1

msb2 +msb2

fmk1 msb2

B

WT

msb2#16

fmk1msb2+msb2

msb2#57

A

Supplemental Figure 10. Msb2 contributes to invasive growth on living fruit tissue.Tomato (A) and apple (B) fruits were inoculated with 5 × 104 microconidia of the indicated strains, incubated at 28ºC for four days and photographed.


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Supplemental Figure 11. F. oxysporum penetrates roots preferentially through openings at the junctions of epidermal cells. Scanning electron microscopy analysis of tomato roots, 24 h after inoculation with microconidia of the F. oxysporum f. sp. lycopersici wild-type strain (A,B), fmk1 mutant (C), or of uninoculated roots (D). (A,B)Fungal hyphae scanning the root surface and entering roots through openings at intercellularjunctions. Insets show magnifications of penetration sites. Arrows point to penetration events (A,B) oropenings visible in uninoculated tomato roots. c, conidium.

c

A

50 m

B

20 m

D

20 m

cC

50 m


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Supplemental Table 1. Primers used in this study. Name Sequence 5'-3'

pGemT M13for CGCCAGGGTTTTCCCAGTCACGAC M13rev AGCGGATAACAATTTCACACAGGA

Fomsb2 msb2-5'-HA-s GCAGATGGAGGCGCTGAAGCAT

msb2-5'-HA(722)-as GGCATAGTCAGGAACGTCATATGGATA- AGTCTCGGTGGCAGGGCTGCC

msb2-3'-HA-s TATCCATATGACGTTCCTGACTATGCC- ACTGATGCGGAGACCAACGGCAC

msb2-3'-HA-as CTGAACAACACCACCGCTTCCC msb2-for2 GAGATTCCAACAATAGCAGATG msb2-comp2 GCAATCCGCGCCCAATAGAC msb2-rev CAGGTCGCCAGGATGGAGCC msb2-HA-s CCATATGACGTTCCTGACTATG msb2-nest3 CAAGCATCAAAGGCGTCGTC msb2-nest4 CTCACGCCTAACGCCTCCAA

msb2-knock1 GTGACTGGGAAAACCCTGGCG- AGTTGGATACTGTTTGGTGATTG

msb2-knock2 TCCTGTGTGAAATTGTTATCCGCT- ACTCGTTAGCAAGATTGTTCCTC

msb2-ORF-s TGCCCCCACAGATGAGCAAC msb2-ORF-as GGATCTTGGCGAGAGCAGTG

qPCR 09795-for (fpr1) CCCAAGAAGAACCCTGCTCC 09795-rev (fpr1) GAGTAGGGGTTGGAGCCGC Chs3-12 GTGTCATGGGGAACAAAGGG Chs3-18 CCTGTAACCCCAAAAGTATGT ChsV-20 GCACAATTTGGCTGAGCTTAT ChsV-36B GGATCCCTACAATTGGCCAGAAAGCA act-2 GAGGGACCGCTCTCGTCGT act-q6 GGAGATCCAGACTGCCGCTCAG Fmk1-F1 CCGGTTTCATGACTGAATACGT

Fmk1-R1 ATCGTGGTAAGGCTCAAGGTAT

FoHog1-F1 GTCACTTATGGGATCGCGATC

FoHog1-R1 TAGTGGCGGTTGTATCAGGCT

FoMpk1-F1 CCATCAAGAAGGTCACCAATGT

FoMpk1-R1 AGCAACGTACTCGGTCATGTAT

Yeast compl. Msb2-PstI-F1 CTGCAGATCCCTCGCGAGACCCGTTT

Msb2-Sac2-R1 CCGCGGAACACCACCGCTTCCCATCG

Fomsb2-S1 ACCTGGCCAACCTTTTTTAATGCTCTTGACCTTTCTTTTCTCTTGCGTACGCTGCAGGTCGAC

Fomsb2-S2 TACAAGTTGACATGTATCCTATCATCCAGAACCTGCTCTCATTGTATCGATGAATTCGAGCTCG

Fomsb2-C1 CTATTTTTATTGACTTTTCATTAGGCTTCCTAATTATACCCATCTATGCACGCAAAGAACTTCAT

Fomsb2-C2 ATGAAATAAGTAGGTATCGTTATAAGTTTATAAGGTTATGCAAGCTGGACCAAACACACCTAAAA


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Supplemental Data. P rez-Nadales and Di Pietro (2011 ......Mar 22, 2011 · ca nv n tk s s s qnqs...

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Transcript of Supplemental Data. P rez-Nadales and Di Pietro (2011 ......Mar 22, 2011 · ca nv n tk s s s qnqs...