Data. Mbengueet al. (2010). Plant Cell 10.1105/tpc.110.075861...Supplemental Figure 6. PUB1-RNAi...
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2 0 4 0 6 0 8 0 1 0 0 M t P U B 1 : - - - - - - - - - - - - - M N D P R S K M M I S P G L L P T E S L L D S L I L I S N E V S S M Q K F P - - L V Q I K N V S S M I R R I K L L S S L F E E I Q E S D S P L P P S S I L C F I E I F S V I T : 8 5 L j S G A 0 1 9 0 2 : - - - - - - - - - - - - - M N D A V S A M M I S S G L L P T G T L L D S L I H I S N E V G S M E K L P - - L V H T R N V S S M I R R I K L L S S L F E E I Q E T D S P L P P S S I L C F T E L Y S V I T : 8 5 G m 0 2 g 4 3 1 9 0 : - - - - - - - - - - - - - - - - - - - - - M V S P G L L P S G T L L D S L I H V C N E V G S M E K F P - - L V H T R N V S T M I R R I K L L S S L F E E I Q E T D T P L P P S S I L C L T E L F S V I R : 7 7 L j B 0 2 K 2 0 : - - - - - - - - - - - - - - - - - - - - M M I S S T F L P N K S L L D S L I N I S N E A S S M E K F P - - F L H A R N V T S M I R R I K V L S S L F E E I Q V S D T P L P P S S I L C F T E F F S L I T : 7 8 P t 0 8 s 0 7 1 6 0 : - - - - - - - - - - - - - - - - - - - - - - - - - - - M P T G S L M K S L I H I S N E V A S V E K L P - - F S Q F R N I S T M I R R I K F L S F L F E D I R E T N S P L P P S S L L C L T E L F S V I Q : 7 1 P t 1 0 s 1 9 3 3 0 : - - - - - - - - - - - - - M N V A L P P I M I S S V F L P T G S L V E S L I H I S N E V A S V E K L P - - F V Q F R N I S T M I R R I K L L S S L F E D I R E T N S P L P P S S I L C L T E L F S V I Q : 8 5 V v 2 9 3 3 3 0 0 1 : - - - - - - - - - - - - - M D V A L P T L M V S S G F L P T G S L L E S L I H I S N E V A S M E R L P - - V I Q V R N I S T M I R R I K L L S S L F E E I H E M N T P L P P S S I L C L T E L F S V I R : 8 5 B n A R C 1 : M A T D S A M F A S S R R R Q S P S L E A F L S P V D L S D V P L L Q T L S S I S S E I V S C F S N A R F S F Q R R N T R S L I R K V Q V F A V L L Q H L A P - E S S L D P T A V L C F K E L Y L L L H : 9 9 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 M t P U B 1 : R V K V L I Q E C T D G S S L W S L I Q L D F I S N Q F F V L V K E M G R A L D I L P - L N L L N V A Q D I K E Q V D L L H K Q S K R V E L E L F I D P R E V Q R R E N L F E V M S K N C L Q N K K T N : 1 8 4 L j S G A 0 1 9 0 2 : R V K D L I Q E C K D G S T L W G L I Q L E L I S N Q F Y V L A K E L G R A L D I L P - L K L L N I T A D T K E Q V Q L L Q K Q A K R - - V E L L I D P R E L Q R R E Q L L Q V M A N N S L R N K K - - : 1 8 0 G m 0 2 g 4 3 1 9 0 : R V K V L I Q D C K D G S S L W S L I Q L E F I S N Q F Y V L V K E M G R A L D I L S - L S L L N V T S D I K E Q V E L L H K Q A K R - - A E L L I D P R E L H R R E Q L I Q - - - - - - - - - K K - - : 1 6 3 L j B 0 2 K 2 0 : K V K L L I Q E C K D G S N L W G L I Q L E F I S N Q F Y V L A K E M G R V L D I L P - V S L L N I T T D M R E Q L E I L H K Q T K K - - I E L F I D P R D L Q R R D Q L L Q V M A N K S L P H N K - - : 1 7 3 P t 0 8 s 0 7 1 6 0 : R V K L L I Q R C K G G S S L W G L I Q T E F L S N Q F H A L V K E M G G A L D I L P - L N L L N L S T D T R E Q V E L L H K Q A K R - - V D L L V D P Q E L Q R R E E L L Q I M A S N N Q K N S R - - : 1 6 6 P t 1 0 s 1 9 3 3 0 : R V K L L I Q G C K D G S S L W G L I Q T Q F L S N Q F H V L V K E M G G A L D I L P - L S L L N L S A D T R E Q V E L L H R Q A K R - - F D L L V D P R E L Q R R E E L L Q I M T R N N Q K N S G - - : 1 8 0 V v 2 9 3 3 3 0 0 1 : R V K S F I Q G C K D A S S L W S L V Q T E L I S S Q F H V V V K E M G R A L D I L P - L S L L N I T A D I R E Q V E L L H K Q A K R - - V D L F V D P R E L Q R R E E L L Q V M A T N N E K N A K - - : 1 8 0 B n A R C 1 : H S K F L L R Y C A H S S K L W L L L Q S P S L S S F F H D L S K D Y S T L L D V F L P V D S L C L N D D V R E Q V Q L L H M Q H Y V - - - D D N N D E T L R N R L Y S F L D E F E N G S V P N S E - - : 1 9 4 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 M t P U B 1 : N N K G F I D F V K V E E I M C S I G L R T L S D Y V E E I S K L E V E A Q N Q A G T G G L I V V S N I N N L M S L V S Y T K S M V F R N - - - D G E S E E C K P I S M F L Y N K S K I H D N D S S S S : 2 8 1 L j S G A 0 1 9 0 2 : - N K G F I D F G K V E D I L S S I G L R T P S D Y D G E I S N L E V E A Q N Q A G T G G L I V V S N I N N L I S L V S Y S K S M I F R N G E N D R D E E E C K P L S L I L Y N K N - - H D - - - S S S : 2 7 4 G m 0 2 g 4 3 1 9 0 : - - K G L V D F G K V E E I L S S I G L R T P S D Y E E E I S K L E A E A Q N Q A G T G G L I V V S N I N N L I S L M C Y S K S M I F K E G E S D T K E D - - - - - - - - L Y D S S - - - - - - - S S S : 2 4 6 L j B 0 2 K 2 0 : - S K G F I D L R K L E E I L S S I G F R S P S D Y D R E I S K L E A E A Q N Q A G T G G L M V V S N I N N L L C V V S H A K S M F F E D G E S K K D E R N C K K L S E F L Y V K I H D S S - - - S S S : 2 6 9 P t 0 8 s 0 7 1 6 0 : - N K G F V D F V K V K E V F S C I G L R S P L D Y E E E I S K L E A E A E K Q A G T G G L I V V S N I N N L I S L V T Y S K S V I F I D R H E E E I K E N F K Q L S A S M N R N Q D V S - - - - S S S : 2 6 1 P t 1 0 s 1 9 3 3 0 : - N K G F D D F V K L K E V L S C I G L R S P L E Y E E E I S K L E A E A E K Q A G T G G L I V V S N I N N I I S L V T S S K S V I F V D R D K E E I K E N F K Q R S A F M N R N Q D V S - - - - S S S : 2 7 5 V v 2 9 3 3 3 0 0 1 : - N K A L V D F R E V K E I L S S I G L R S P L D Y D E E I S K L E A E A Q K Q A G T G G L I V V S N I N N L I S L V L Y S K S M I F S E E H N E K T D E A F K K R S A S F R M N N D H S - - - - S S S : 2 7 5 B n A R C 1 : - - - - - - - - E L R F F F F E K L A I K D P T S Y R E E I E F L E E Q I K S H G - C D L D P T R S V I N G F I D I T R Y V M F L L F K I E D G N E I K K Q R K R L I S E E I E N T F T T - - - - - - - : 2 7 8 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 M t P U B 1 : S S F S Q S M M T V N I P D E F R C P I S L D L M R D P V I V S S G H T Y D R I S I A E W I N - S G H H T C P K S G Q R L I H T A L I P N Y A L K S L V H Q W C Y E N N V K M N E A I T - - - - K N N N : 3 7 6 L j S G A 0 1 9 0 2 : S S M S I S V I P - N I P D E F R C P I S L D L M R D P V I V S S G H T Y D R N S I A Q W I N - T G H H T C P K S G Q R L I H T A L I P N Y A L K S L V Q Q W C Y E N N V P V N E P M N S - - - L L N N : 3 6 9 G m 0 2 g 4 3 1 9 0 : Q S M T P - - - - - N V P D E F R C P I S L D L M R D P V I V S S G H S Y D R I S I A Q W I N - S G H H T C P K S G Q R L I H T A L I P N Y A L K S L V Q Q W C H D N N V P V D E P T T E - - - G N K N : 3 3 7 L j B 0 2 K 2 0 : Q S M T V - - - - - D I P D E F R C S I S L Q L M R D P V I L S S G H T Y D R N S I A K W I N - S G H H T C P K S G Q R L I H T A L I P N F A L K S L I Q Q W C Q E N K V P V N D P T S L - - - L S E G : 3 6 0 P t 0 8 s 0 7 1 6 0 : Q S I L P - - - - - N I P D E F R C P I S L D L M K D P V I V A S G H T Y D R N S I A Q W I N - S G H H T C P K S G K R L I H T S L I P N Y A L K S L V H Q W C Q D N N V P L I E N S T S S S S K F E R : 3 5 5 P t 1 0 s 1 9 3 3 0 : Q S I L S - - - - - N I P D E F R C P I S L D L M K D P V I V A S G H T Y D R N S I A Q W I N - S G H Q T C P K S G Q R L I H M A L I P N Y A L K S M V H Q W C Q D N N V P L I D D S S S S F S K S E S : 3 6 9 V v 2 9 3 3 3 0 0 1 : Q S A I L - - - - - N I P D E F R C P I S L D L M R D P V I V A S G H T Y D R N S I A Q W I N - T G H N T C P K S G M K L I H M A L I P N Y A L K S L V H Q W C R E N N I Q L I E S T S S S S S D L G R : 3 6 9 B n A R C 1 : - - - - - - - - - - T L P K D F I C S I S L N L M N D P V I I S T G Q T Y D R T S I A R W I H Q E G R S T C P K T G Q K L V D L S F V S N L A L R H L T T L W C E V T G L S H D S P K E S - - - - - - - : 3 6 1 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 M t P U B 1 : S S S K R H K N E N A I D H I S E N K A S K D A V K M T A E F L V G K L A T G S T D I Q R Q S A Y E I R L L A K T G M D N R R I I A E V G A I P F L V T L L V S K D S R I Q E H V V T A L F N L S I Y D : 4 7 6 L j S G A 0 1 9 0 2 : S R S K K N L K E E A I D H I S A N K A A S D A V K M T A E F L V G K L A T G S V D I Q R Q A A Y E L R L L A K S G T N N R S I I P E A G A I P F L V T L L A S Q D S R T Q E H A V T A L F N L S I L D : 4 6 9 G m 0 2 g 4 3 1 9 0 : S S K K - - L K E D A V D H I S A N K A A A D A V K M T A E F L V G K L A T G S A D I Q R Q A A Y E L R L L T K T G M V N R S V I A E V G A I P F L V T L L G S Q D S R I Q E H A V T A L F N L S I F D : 4 3 5 L j B 0 2 K 2 0 : N N S K K N L N E N A V D Q V S T N Q G S T D S V K M T A E F L V G K L A T G S A D I Q M K A A Y E L R L L A K S G M N N R R I I A E A G A I P F L V T L L G S N D S R I Q E N A V T A L L N L S I F D : 4 6 0 P t 0 8 s 0 7 1 6 0 : S S S K S K L S E K A I E H I S A T K A A M D A V K M T A E F L V G K L A M G S P E I Q R Q A A Y E L R L L A K T G M D N R K I I A E A G A I P F L V T L L S S T D P R I Q E N A V T A L L N L S I F D : 4 5 5 P t 1 0 s 1 9 3 3 0 : S S G K S K L S E K A I D H I S A T K A A M D A V K M T A E F L V G K I A M G S P E I Q R Q A T Y E L R L L A K T G M G N R R I I A E A G A I P F L V T L L S S T D P K T Q E N A V T A M L N L S I L E : 4 6 9 V v 2 9 3 3 3 0 0 1 : S N S M R K S C E K A V D H I S A T K T A M D A V K M T A E F L V G K L A T G S P E I Q R Q A A Y E L R L L A K T G M D N R R I I A E A G A I P F L V T L L S S H D P R I Q E N A V T A L L N L S I F D : 4 6 9 B n A R C 1 : - - - - - - - - - - - L P K V F Q T R A S T E A N K A T L S I L V Q N L A H G S - - - - E L A A G E I R V L T R T V T E T R T L I V E T G A I P Y L R S L L K S Q N A V A Q E N A V A S I F N L S I D E : 4 4 6 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 M t P U B 1 : N N K I L I M A A G - A I D N I V E V L E F G K T M E A R E N A A A A I Y S L S M I D D C K V Q I G A S S R A I P A L V G L L K E G T I I G K R D A A T A L F N L A V Y N P N K L S I V K S G A V T L L : 5 7 5 L j S G A 0 1 9 0 2 : N N K I L I M V A G - A V D N I V E V L E S G K T M E A R E N A A A A I Y S L S M V D D C K V Q I G A C T R A I P A L V A L L K E G T P I G K R D A A S A I F N L A V Y N P N K S S I V K A G A V P L L : 5 6 8 G m 0 2 g 4 3 1 9 0 : N N K I L I M A A G - A V D S I V E V L E S G K T M E A R E N A A A S I Y S L S M V D E C K V Q I G G R P R A I P A L V E L L K E G T P I G K R D A A S A L F N L A V Y N P N K V S V V K A E A V P V L : 5 3 4 L j B 0 2 K 2 0 : N T K I L I M A A G - A V E N I I G V L E S G K S M E A R E N A A A A I F S L S M V D E C K V T I G T H P R A I P A L I G L L K E G T A T G K R D A A T A L F N L A V Y K H N K A S I V H A G A V T L L : 5 5 9 P t 0 8 s 0 7 1 6 0 : N N K I L I M A A G - S I D S I I N V L E S G K T M E A R E N A A A T I F S L S I I S D C K V T I G T R P R A F S A L V G L L R E G T A T G K K D A A S A L F N L S V Y N A N K A S V V V A G A V P L L : 5 5 4 P t 1 0 s 1 9 3 3 0 : N N K T L I M S A G - S I D S I I D V L E S G K T M E A R E N A A A T I F S L S I I N D C K V T I G T R P R A F S A L V G L L R E G T S A G K K D A A S A L F N L S V Y E A N K A S V V V A G A V P L L : 5 6 8 V v 2 9 3 3 3 0 0 1 : N N K I L I M A A G - A I D N I V D V L Q S G K T M E A R E N A A A A I F S L S M I D D C K V T I G A H P R A M P A L V A L L R E G T S A G K R D A A T A L F N L V V Y S A N K G S A V V A G A V P L L : 5 6 8 B n A R C 1 : A N R S L I V E E H D C L E P I M S V L V S G L T M R A K E I A A A T L Y T L S S V H D Y K K A I A N A D G C I E A L A L V L R N G T V R G K K D A V Y A L H S L W L H P D N C S L M V K R G G V S A L : 5 4 6 6 2 0 6 4 0 6 6 0 6 8 0 7 0 0 M t P U B 1 : V E L L M D D K A G I T D D S L A V L A V L L G C S E G L E E I K N S K S L V P L L I D L L R F G S V K G K E N S I T L L L G L C K E E G E L V A M R L L A N P R S I P S L Q S L A A D G S L R A R R K : 6 7 5 L j S G A 0 1 9 0 2 : V E L L M D D K A G I T D D V L A V L A V L Q G C S E G L E E I R - - - - - - - E I I K A F G W P - - - - - - - - - T F L F G F F F K N - - - - - - - - - - - - - - - - - - - - - - L G G Q L K R G K - : 6 2 9 G m 0 2 g 4 3 1 9 0 : V E L L M D D K A G I T D D A L A V L A L L L G C S E G L E E I R N S R A L V P L L I D L L R F G S V K G K E N S I T L L L G L C K Q E G E V V A R R L L A N P R S I P S L Q S L A A D G S L R A R R K : 6 3 4 L j B 0 2 K 2 0 : I E L L M D D K A G I T D D A L A V L A L L L G C S Q G L E E I R N S K E L V P L L V D L L R I G S A R G K E N S I T L L L G L C K E E G E L V A S R L L A N P R S V P S L Q S L A A V G S L R A Q R K : 6 5 9 P t 0 8 s 0 7 1 6 0 : I E L L M D D K A G I T D D A L A L L A L L S G C S E G L E E I R Q S R I L M P M V I D L L R F G S T K G K E N S I T L L L G L C K D G G E E V A R R L L I N P R S I P S L Q S L S T D G S L K A R R K : 6 5 4 P t 1 0 s 1 9 3 3 0 : V E M L M D D K A G I T D D A L A L L A L L L G C S E G L E E I R K S K V L V P L L I D L L R F G S T K G K E N S I T L L L G L C K D G G E E V A R R L L I N P R S I P S L Q S L S A D G S L K A R R K : 6 6 8 V v 2 9 3 3 3 0 0 1 : I E L L M D D K A G I T D D A L A V L A L L L G C P D G L E E I R K S R I L V P L L I D L L R F G S P K G K E N S I T L L L G L C K D G G E E V A R R L L L N P R S I P S L Q S L V A D G S L K A R R K : 6 6 8 B n A R C 1 : V G A L G - - E E A V A E K V A W V L G V M A T E S L G A E S I G R E E T V V T G L M E L M R C G R P R G K E K A I A T L L Q L C T A G G A V V T E K V V K T P A L A V L T R K L L L T G T D R A K R K : 6 4 4 7 2 0 M t P U B 1 : A D A L L R L L N R C C S Q P H H S L - : 6 9 4 L j S G A 0 1 9 0 2 : - - - - - - - - - - - - - - - - - - - - : - G m 0 2 g 4 3 1 9 0 : A D A V L R F L N R C C S Q P H H C V - : 6 5 3 L j B 0 2 K 2 0 : A D A L L R L L N R C C S Q P H H S L - : 6 7 8 P t 0 8 s 0 7 1 6 0 : A D A L L R L L N R E N I Q K R V - - - : 6 7 1 P t 1 0 s 1 9 3 3 0 : A D A L L R L L N R C C S Q S H N P V G : 6 8 8 V v 2 9 3 3 3 0 0 1 : A D A L L R L L N R C C S Q S H S T V G : 6 8 8 B n A R C 1 : A V S L S K V C K G C D Q N T Q R - - - : 6 6 1 U‐box ARM ARM ARM ARM ARM UND Supplemental Figure 1. Annotated alignment of PUB1 with Closely Related Proteins. Putative Mt PUB1 orthologues from Lotus japonicus (Lj), Glycine max (Gm), Populus trichocarpa (Pt) and Vitis vinifera (Vv) and ARC1 from Brassica napus (Bn) were used for the alignment. The position of the UND, U-Box and ARM repeats are shown. Similar alignments (Supplemental Datasets 1 and 2) were used to generate Supplemental Figures 4 and 5 and the alignments were trimmed between the arrows before phylogenetic analyses. Supplemental Data. Mbengue et al. (2010). Plant Cell 10.1105/tpc.110.075861
Transcript of Data. Mbengueet al. (2010). Plant Cell 10.1105/tpc.110.075861...Supplemental Figure 6. PUB1-RNAi...
20 40 60 80 100 MtPUB1 : -------------MNDPRSKMMISPGLLPTESLLDSLILISNEVSSMQKFP--LVQIKNVSSMIRRIKLLSSLFEEIQESDSPLPPSSILCFIEIFSVIT : 85LjSGA01902 : -------------MNDAVSAMMISSGLLPTGTLLDSLIHISNEVGSMEKLP--LVHTRNVSSMIRRIKLLSSLFEEIQETDSPLPPSSILCFTELYSVIT : 85Gm02g43190 : ---------------------MVSPGLLPSGTLLDSLIHVCNEVGSMEKFP--LVHTRNVSTMIRRIKLLSSLFEEIQETDTPLPPSSILCLTELFSVIR : 77LjB02K20 : --------------------MMISSTFLPNKSLLDSLINISNEASSMEKFP--FLHARNVTSMIRRIKVLSSLFEEIQVSDTPLPPSSILCFTEFFSLIT : 78Pt08s07160 : ---------------------------MPTGSLMKSLIHISNEVASVEKLP--FSQFRNISTMIRRIKFLSFLFEDIRETNSPLPPSSLLCLTELFSVIQ : 71Pt10s19330 : -------------MNVALPPIMISSVFLPTGSLVESLIHISNEVASVEKLP--FVQFRNISTMIRRIKLLSSLFEDIRETNSPLPPSSILCLTELFSVIQ : 85Vv29333001 : -------------MDVALPTLMVSSGFLPTGSLLESLIHISNEVASMERLP--VIQVRNISTMIRRIKLLSSLFEEIHEMNTPLPPSSILCLTELFSVIR : 85BnARC1 : MATDSAMFASSRRRQSPSLEAFLSPVDLSDVPLLQTLSSISSEIVSCFSNARFSFQRRNTRSLIRKVQVFAVLLQHLAP-ESSLDPTAVLCFKELYLLLH : 99 120 140 160 180 200 MtPUB1 : RVKVLIQECTDGSSLWSLIQLDFISNQFFVLVKEMGRALDILP-LNLLNVAQDIKEQVDLLHKQSKRVELELFIDPREVQRRENLFEVMSKNCLQNKKTN : 184LjSGA01902 : RVKDLIQECKDGSTLWGLIQLELISNQFYVLAKELGRALDILP-LKLLNITADTKEQVQLLQKQAKR--VELLIDPRELQRREQLLQVMANNSLRNKK-- : 180Gm02g43190 : RVKVLIQDCKDGSSLWSLIQLEFISNQFYVLVKEMGRALDILS-LSLLNVTSDIKEQVELLHKQAKR--AELLIDPRELHRREQLIQ---------KK-- : 163LjB02K20 : KVKLLIQECKDGSNLWGLIQLEFISNQFYVLAKEMGRVLDILP-VSLLNITTDMREQLEILHKQTKK--IELFIDPRDLQRRDQLLQVMANKSLPHNK-- : 173Pt08s07160 : RVKLLIQRCKGGSSLWGLIQTEFLSNQFHALVKEMGGALDILP-LNLLNLSTDTREQVELLHKQAKR--VDLLVDPQELQRREELLQIMASNNQKNSR-- : 166Pt10s19330 : RVKLLIQGCKDGSSLWGLIQTQFLSNQFHVLVKEMGGALDILP-LSLLNLSADTREQVELLHRQAKR--FDLLVDPRELQRREELLQIMTRNNQKNSG-- : 180Vv29333001 : RVKSFIQGCKDASSLWSLVQTELISSQFHVVVKEMGRALDILP-LSLLNITADIREQVELLHKQAKR--VDLFVDPRELQRREELLQVMATNNEKNAK-- : 180BnARC1 : HSKFLLRYCAHSSKLWLLLQSPSLSSFFHDLSKDYSTLLDVFLPVDSLCLNDDVREQVQLLHMQHYV---DDNNDETLRNRLYSFLDEFENGSVPNSE-- : 194 220 240 260 280 300 MtPUB1 : NNKGFIDFVKVEEIMCSIGLRTLSDYVEEISKLEVEAQNQAGTGGLIVVSNINNLMSLVSYTKSMVFRN---DGESEECKPISMFLYNKSKIHDNDSSSS : 281LjSGA01902 : -NKGFIDFGKVEDILSSIGLRTPSDYDGEISNLEVEAQNQAGTGGLIVVSNINNLISLVSYSKSMIFRNGENDRDEEECKPLSLILYNKN--HD---SSS : 274Gm02g43190 : --KGLVDFGKVEEILSSIGLRTPSDYEEEISKLEAEAQNQAGTGGLIVVSNINNLISLMCYSKSMIFKEGESDTKED--------LYDSS-------SSS : 246LjB02K20 : -SKGFIDLRKLEEILSSIGFRSPSDYDREISKLEAEAQNQAGTGGLMVVSNINNLLCVVSHAKSMFFEDGESKKDERNCKKLSEFLYVKIHDSS---SSS : 269Pt08s07160 : -NKGFVDFVKVKEVFSCIGLRSPLDYEEEISKLEAEAEKQAGTGGLIVVSNINNLISLVTYSKSVIFIDRHEEEIKENFKQLSASMNRNQDVS----SSS : 261Pt10s19330 : -NKGFDDFVKLKEVLSCIGLRSPLEYEEEISKLEAEAEKQAGTGGLIVVSNINNIISLVTSSKSVIFVDRDKEEIKENFKQRSAFMNRNQDVS----SSS : 275Vv29333001 : -NKALVDFREVKEILSSIGLRSPLDYDEEISKLEAEAQKQAGTGGLIVVSNINNLISLVLYSKSMIFSEEHNEKTDEAFKKRSASFRMNNDHS----SSS : 275BnARC1 : --------ELRFFFFEKLAIKDPTSYREEIEFLEEQIKSHG-CDLDPTRSVINGFIDITRYVMFLLFKIEDGNEIKKQRKRLISEEIENTFTT------- : 278 320 340 360 380 400 MtPUB1 : SSFSQSMMTVNIPDEFRCPISLDLMRDPVIVSSGHTYDRISIAEWIN-SGHHTCPKSGQRLIHTALIPNYALKSLVHQWCYENNVKMNEAIT----KNNN : 376LjSGA01902 : SSMSISVIP-NIPDEFRCPISLDLMRDPVIVSSGHTYDRNSIAQWIN-TGHHTCPKSGQRLIHTALIPNYALKSLVQQWCYENNVPVNEPMNS---LLNN : 369Gm02g43190 : QSMTP-----NVPDEFRCPISLDLMRDPVIVSSGHSYDRISIAQWIN-SGHHTCPKSGQRLIHTALIPNYALKSLVQQWCHDNNVPVDEPTTE---GNKN : 337LjB02K20 : QSMTV-----DIPDEFRCSISLQLMRDPVILSSGHTYDRNSIAKWIN-SGHHTCPKSGQRLIHTALIPNFALKSLIQQWCQENKVPVNDPTSL---LSEG : 360Pt08s07160 : QSILP-----NIPDEFRCPISLDLMKDPVIVASGHTYDRNSIAQWIN-SGHHTCPKSGKRLIHTSLIPNYALKSLVHQWCQDNNVPLIENSTSSSSKFER : 355Pt10s19330 : QSILS-----NIPDEFRCPISLDLMKDPVIVASGHTYDRNSIAQWIN-SGHQTCPKSGQRLIHMALIPNYALKSMVHQWCQDNNVPLIDDSSSSFSKSES : 369Vv29333001 : QSAIL-----NIPDEFRCPISLDLMRDPVIVASGHTYDRNSIAQWIN-TGHNTCPKSGMKLIHMALIPNYALKSLVHQWCRENNIQLIESTSSSSSDLGR : 369BnARC1 : ----------TLPKDFICSISLNLMNDPVIISTGQTYDRTSIARWIHQEGRSTCPKTGQKLVDLSFVSNLALRHLTTLWCEVTGLSHDSPKES------- : 361 420 440 460 480 500 MtPUB1 : SSSKRHKNENAIDHISENKASKDAVKMTAEFLVGKLATGSTDIQRQSAYEIRLLAKTGMDNRRIIAEVGAIPFLVTLLVSKDSRIQEHVVTALFNLSIYD : 476LjSGA01902 : SRSKKNLKEEAIDHISANKAASDAVKMTAEFLVGKLATGSVDIQRQAAYELRLLAKSGTNNRSIIPEAGAIPFLVTLLASQDSRTQEHAVTALFNLSILD : 469Gm02g43190 : SSKK--LKEDAVDHISANKAAADAVKMTAEFLVGKLATGSADIQRQAAYELRLLTKTGMVNRSVIAEVGAIPFLVTLLGSQDSRIQEHAVTALFNLSIFD : 435LjB02K20 : NNSKKNLNENAVDQVSTNQGSTDSVKMTAEFLVGKLATGSADIQMKAAYELRLLAKSGMNNRRIIAEAGAIPFLVTLLGSNDSRIQENAVTALLNLSIFD : 460Pt08s07160 : SSSKSKLSEKAIEHISATKAAMDAVKMTAEFLVGKLAMGSPEIQRQAAYELRLLAKTGMDNRKIIAEAGAIPFLVTLLSSTDPRIQENAVTALLNLSIFD : 455Pt10s19330 : SSGKSKLSEKAIDHISATKAAMDAVKMTAEFLVGKIAMGSPEIQRQATYELRLLAKTGMGNRRIIAEAGAIPFLVTLLSSTDPKTQENAVTAMLNLSILE : 469Vv29333001 : SNSMRKSCEKAVDHISATKTAMDAVKMTAEFLVGKLATGSPEIQRQAAYELRLLAKTGMDNRRIIAEAGAIPFLVTLLSSHDPRIQENAVTALLNLSIFD : 469BnARC1 : -----------LPKVFQTRASTEANKATLSILVQNLAHGS----ELAAGEIRVLTRTVTETRTLIVETGAIPYLRSLLKSQNAVAQENAVASIFNLSIDE : 446 520 540 560 580 600 MtPUB1 : NNKILIMAAG-AIDNIVEVLEFGKTMEARENAAAAIYSLSMIDDCKVQIGASSRAIPALVGLLKEGTIIGKRDAATALFNLAVYNPNKLSIVKSGAVTLL : 575LjSGA01902 : NNKILIMVAG-AVDNIVEVLESGKTMEARENAAAAIYSLSMVDDCKVQIGACTRAIPALVALLKEGTPIGKRDAASAIFNLAVYNPNKSSIVKAGAVPLL : 568Gm02g43190 : NNKILIMAAG-AVDSIVEVLESGKTMEARENAAASIYSLSMVDECKVQIGGRPRAIPALVELLKEGTPIGKRDAASALFNLAVYNPNKVSVVKAEAVPVL : 534LjB02K20 : NTKILIMAAG-AVENIIGVLESGKSMEARENAAAAIFSLSMVDECKVTIGTHPRAIPALIGLLKEGTATGKRDAATALFNLAVYKHNKASIVHAGAVTLL : 559Pt08s07160 : NNKILIMAAG-SIDSIINVLESGKTMEARENAAATIFSLSIISDCKVTIGTRPRAFSALVGLLREGTATGKKDAASALFNLSVYNANKASVVVAGAVPLL : 554Pt10s19330 : NNKTLIMSAG-SIDSIIDVLESGKTMEARENAAATIFSLSIINDCKVTIGTRPRAFSALVGLLREGTSAGKKDAASALFNLSVYEANKASVVVAGAVPLL : 568Vv29333001 : NNKILIMAAG-AIDNIVDVLQSGKTMEARENAAAAIFSLSMIDDCKVTIGAHPRAMPALVALLREGTSAGKRDAATALFNLVVYSANKGSAVVAGAVPLL : 568BnARC1 : ANRSLIVEEHDCLEPIMSVLVSGLTMRAKEIAAATLYTLSSVHDYKKAIANADGCIEALALVLRNGTVRGKKDAVYALHSLWLHPDNCSLMVKRGGVSAL : 546 620 640 660 680 700 MtPUB1 : VELLMDDKAGITDDSLAVLAVLLGCSEGLEEIKNSKSLVPLLIDLLRFGSVKGKENSITLLLGLCKEEGELVAMRLLANPRSIPSLQSLAADGSLRARRK : 675LjSGA01902 : VELLMDDKAGITDDVLAVLAVLQGCSEGLEEIR-------EIIKAFGWP---------TFLFGFFFKN----------------------LGGQLKRGK- : 629Gm02g43190 : VELLMDDKAGITDDALAVLALLLGCSEGLEEIRNSRALVPLLIDLLRFGSVKGKENSITLLLGLCKQEGEVVARRLLANPRSIPSLQSLAADGSLRARRK : 634LjB02K20 : IELLMDDKAGITDDALAVLALLLGCSQGLEEIRNSKELVPLLVDLLRIGSARGKENSITLLLGLCKEEGELVASRLLANPRSVPSLQSLAAVGSLRAQRK : 659Pt08s07160 : IELLMDDKAGITDDALALLALLSGCSEGLEEIRQSRILMPMVIDLLRFGSTKGKENSITLLLGLCKDGGEEVARRLLINPRSIPSLQSLSTDGSLKARRK : 654Pt10s19330 : VEMLMDDKAGITDDALALLALLLGCSEGLEEIRKSKVLVPLLIDLLRFGSTKGKENSITLLLGLCKDGGEEVARRLLINPRSIPSLQSLSADGSLKARRK : 668Vv29333001 : IELLMDDKAGITDDALAVLALLLGCPDGLEEIRKSRILVPLLIDLLRFGSPKGKENSITLLLGLCKDGGEEVARRLLLNPRSIPSLQSLVADGSLKARRK : 668BnARC1 : VGALG--EEAVAEKVAWVLGVMATESLGAESIGREETVVTGLMELMRCGRPRGKEKAIATLLQLCTAGGAVVTEKVVKTPALAVLTRKLLLTGTDRAKRK : 644 720 MtPUB1 : ADALLRLLNRCCSQPHHSL- : 694LjSGA01902 : -------------------- : -Gm02g43190 : ADAVLRFLNRCCSQPHHCV- : 653LjB02K20 : ADALLRLLNRCCSQPHHSL- : 678Pt08s07160 : ADALLRLLNRENIQKRV--- : 671Pt10s19330 : ADALLRLLNRCCSQSHNPVG : 688Vv29333001 : ADALLRLLNRCCSQSHSTVG : 688BnARC1 : AVSLSKVCKGCDQNTQR--- : 661
U‐box
ARM
ARM ARM
ARM ARM
UND
Supplemental Figure 1. Annotated alignment of PUB1 with Closely Related Proteins.Putative Mt PUB1 orthologues from Lotus japonicus (Lj), Glycine max (Gm), Populus trichocarpa(Pt) and Vitis vinifera (Vv) and ARC1 from Brassica napus (Bn) were used for the alignment. The position of the UND, U-Box and ARM repeats are shown. Similar alignments (Supplemental Datasets 1 and 2) were used to generate Supplemental Figures 4 and 5 and the alignments were trimmed between the arrows before phylogenetic analyses.
Supplemental Data. Mbengue et al. (2010). Plant Cell 10.1105/tpc.110.075861
Supplemental Figure 2. Interaction of PUB1 with LYK3 in N. benthamiana.PUB1 interacts preferentially with LYK3 rather than BRI1 or LRRII.1 in N. benthamiana.PUB1ARM interacts less than PUB1 with LYK3. The expression level of each receptor was tested by fusion to full length YFP (left panels). BiFC experiments were performed by co-expression of the indicated split-YFP pair combinations: Yn = N-terminal domain of YFP; Yc = C-terminal domain of YFP (right panels). Leaves were imaged by epi-fluorescence using identical exposure settings. Identical post-treatment parameters were applied to all images. Bars represent 200 μm.
LYK3‐Yc / Yn‐PUB1ARM
BRI1‐Yc / Yn‐PUB1
LYK3‐Yc / Yn‐PUB1
LRRII.1‐YFP
Non transformed
LYK3‐YFP
BRI1‐Yc / Yn‐PUB1ARMBRI1‐YFP
LRRII.1‐Yc / Yn‐PUB1 LRRII.1‐Yc / Yn‐PUB1ARM
Supplemental Figure 3. Detection of GST LYK3-IR in the in vitro Trans-phosphorylation of PUB1 experiment.The same proteins as used in Figure 3E were loaded as indicated. On the left panel, protein staining shows the size and quantity of the purified proteins. On the right panel, LYK3-IR detected by anti-GST antibodies. The ∼70 kDa recombinant LYK3 kinase indicated by < is LYK3 while the lower band is a contaminant of LYK3 purification.
‐GSTProtein staining
<72100130
5540
MBP
MBP‐PUB1
MBP‐PUB1UNDGST‐LYK3‐IR
kDa
Supplemental Figure 4. Phylogenetic Relationships between the UND-PUB-ARM Proteins of M. truncatula and Selected UND-PUB-ARM Proteins from other Higher Plants.The analysis includes all known UND-PUB-ARM proteins of M. truncatula (Mt) and A. thaliana (At) and related, functionally characterized proteins from B. napus (Bn), Nicotiana tabacum (Nt), and O. sativa (Os). Analyses were made on whole protein alignments (Supplemental Dataset 1) using a neighbor-joining method with 1000 bootstrap replicates (shown as percentages). Mt PUB1 is circled.
Lj6CM0118Vv35825001NtACRE276Pt04s06040Pt11s06840Mt2g021720Gm15g09260Gm08g12610Gm05g29450BnAF024625At1g29340Os05g39930At5g01830LjT28O03Vv37474001Pt16s14150Pt06s10810At1g60190At1g10560Mt5g035790Vv28839001Vv16120001Mt8g125550LjT19C19Pt15s08550Pt12s08030LjB02K20LjSGA01902Gm02g43190MtPUB1Vv29333001Pt10s19330Pt08s07160Os06g51130Os08g37570Vv36257001Pt09s16740Pt04s21490At5g42340Pt02s01090Vv14430001Gm18g47120Lj1T10B06Mt7g088030Os02g49950At1g23030At1g71020Vv30470001Gm20g01640Mt4g130390Os06g01304At3g54850Vv18150001Pt10s23330Pt08s03560Gm20g32340Gm10g35220Lj1CM0147OsSPL11Os03g16824At2g28830At3g46510Vv24348001Pt01s24550Pt09s03500Gm11g14910Gm12g06860At1g27910Pt08s17710Mt3g103550At1g67530At1g24330At4g36550Mt5g020450At3g54790NtPUB4At2g23140At5g67340
100
48
100
93
100
92
100
100
100
98
100
94
100
54
100
100
100
Supplemental Figure 5. Phylogenetic Analysis of Higher-Plant UND-PUB-ARM Proteins.Phylogenetic relationships between PUB1 and other plant UND-PUB-ARM proteins illustrating clustering of related proteins. Proteins from the following species were used: A. thaliana (At), B. napus (Bn), G. max (Gm), L. japonicus (Lj), M. truncatula (Mt), Nicotiana tabacum (Nt), O. sativa(Os), P. trichocarpa (Pt) and V. vinifera (Vv). Analyses were made on whole protein alignments (Supplemental Dataset 2) using a neighbor-joining method with 1000 bootstrap replicates (shown as percentages on the major clusters). Mt PUB1 is arrowed.
Supplemental Figure 6. PUB1-RNAi Construct Efficiency.The PUB1-RNAi construct efficiently down-regulates PUB1 gene expression. PUB1 mRNA quantification was determined after 8 h of NF treatment of lyk3-4 roots transformed with the PUB1-RNAi construct or the vector control. Roots from 17 control and 18 PUB1-RNAi plants were tested. PUB1 mRNA quantification was normalized against two reference genes in each sample tested (relative units). The standard deviation between samples is represented.
Constructs Destination vectors RestrictionsForward Reverse (5' and 3')
proPUB1 (Restriction)
GCAGAGGATCCGGGTCCA TCCCCCGGGTTTGTGATTAGTGTCAGAAATTAGATTACTCT
pBI101 BamHI - SmaI
RNAi PUB1 (Gateway) GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGAATGATCCTAGATCAAAGATGATGATATCTCC
GGGGACCACTTTGTACAAGAAAGCTGGGTCTTGGATTGTTTATGCAAAAGATCAACTTGCT
pK7GWiWG(II) + ubq10::DsRED1
LYK3-IR for Y2H screen (Restriction, Stop codon)
GAATTCCGTTATAGACACAAGTCAATT AAACTGCAGTCATCTAGTTGACAACAGATTTATGAG pGB (Y2H bait) EcoRI - PstI
LYK3-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAAATACTTCCAAAAGAAGGAAGAAGAGAAAACT
GGGGACCACTTTGTACAAGAAAGCTGGGTTTCTAGTTGACAACAGATTTATGAGAGATTGATTTTC
PGB GW (Y2H bait)
BRI1-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGAGAGATGAGGAAGAGACGGAGAAAGAAAG
GGGGACCACTTTGTACAAGAAAGCTGGGTTTAATTTTCCTTCAGGAACTTCTTTTATACTCATATCA
PGB GW (Y2H bait)
LRRII.1-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGACACCATCAAAAGATCAGACACAAAAGT
GGGGACCACTTTGTACAAGAAAGCTGGGTATCTTGCTCTGGAAAGTTGTATAGCTTC
PGB GW (Y2H bait)
LYK2-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGATACTTCAAAAAGAAGGAAGAAGAGAAAACT
GGGGACCACTTTGTACAAGAAAGCTGGGTTTCTCACTGACAAGAGATTTATGATAGTTTGATTTTCA
PGB GW (Y2H bait)
LYK4-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGATACTTTCGCAAGAAGAATGGAGAAGAGT
GGGGACCACTTTGTACAAGAAAGCTGGGTTATTTGAATCATGCTCCACATCAAGACTCA
PGB GW (Y2H bait)
NFP-IR (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAGATACTTTCGCAAGAAGAATGGAGAAGAGT
GGGGACCACTTTGTACAAGAAAGCTGGGTCACGAGCTATTACAGAAGTAACAACATGAGTAGCT
PGB GW (Y2H bait)
PUB1 (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGAATGATCCTAGATCAAAGATGATGATATCTCC
GGGGACCACTTTGTACAAGAAAGCTGGGTTCAAAGAATGATGAGGTTGAGAACAACACC
pAMpAT35S-Yn-GW, pAMpAT35S-3HA-GW
GCTTACCATGGGGTACCCA TCCCCGCGGTCACAAAGAATGATGAGGTTGAGAACA
BRI1 (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAAGACTTTTTCAAGCTTCTTTCTCTCTGTAAC
GGGGACCACTTTGTACAAGAAAGCTGGGTTTAATTTTCCTTCAGGAACTTCTTTTATACTCATATCA
pAMpAT35S-GW-YFP, pAMpAT35S-GW-Yc
PUB1 (Gateway, Stop codon)
PGAD GW (Y2H prey), pGEX GW (GST fusion)
PUB1UND (Gateway, Stop codon)
PGAD GW (Y2H prey), pGEX GW (GST fusion)
PUB1UNDU-Box (Gateway, Stop codon)
PGAD GW (Y2H prey)
PUB1ARM (Gateway, No Stop codon)
GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGAATGATCCTAGATCAAAGATGATGATATCTCC
GGGGACCACTTTGTACAAGAAAGCTGGGTTAGCATTCTCATTCTTGTGCCTTTTGCT
PGAD GW (Y2H prey), pAMpAT35S-Yn-GW
PUB1 (Restriction, Stop codon)
ATTCGAGCTCAATGAATGATCCTAGATCAAAGATGA GCAGGTCGACTCACAAAGAATGATGAGGTTGAGA pMAL (MBP fusion) SstI - SalI
PUB1UND (Restriction, Stop codon)
ATTCGAGCTCAAGTAAAATACATGATAATGATAGTTCTTCT
GCAGGTCGACTCACAAAGAATGATGAGGTTGAGA pMAL (MBP fusion) SstI - SalI
Recovered from the Y2H screen
Primers
(PCR on pAMpAT35S-3HA-PUB1)3HA-PUB1 (Restriction, Stop codon)
Supplemental Table 1. Primers used in this study
NcoI - SstI
Recovered from the Y2H screen
Recovered from the Y2H screen
pGREEN35S
Supplemental Methods
Phylogenetic and Protein Sequence Analyses
The Medicago truncatula EST data base (Journet et al., 2002) and the Medicago truncatula Gene Index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=medicago) were used to access cDNAs. The A. thaliana UND-PUB-ARM protein sequences were obtained from the TAIR database (http://www.arabidopsis.org/) using the accessions given in Mudgil et al. (2004). The M. truncatula proteins were obtained from the M. truncatula genome sequence database release 3.0 using either BLASTP with default values or by using the REMORA serveur (Carrere and Gouzy, 2006; Courcelle et al., 2008) and the following workflow: BLASTP using the U-Box of PUB1 (E-value = 10), followed by BLASTP on the protein subset using the PUB1 ARM repeat 3 (E-value = 0.001), see Supplemental Figure 1 for the sequences used. The following sequences were obtained from the NCBI database using the following accessions: ARC1 (AAB97738), ACRE276 (AAP03882), PUB4 (AAO61490), SPL11 (AAT94161). Other plant sequences were obtained by BLASTP using the protein sequence of PUB1 and the L. japonicus genome database (http://www.kazusa.or.jp/lotus/index.html) or Phytozome V5.0 (http://www.phytozome.net/). Protein alignments used CLUSTALX (Thompson et al., 1997) and minor adjustments were made either to improve the alignments or to delete the N-terminal and C-terminal extensions using GeneDoc (Nicholas et al., 1997). Phylogenetic analyses were then performed on the alignments using the PHYLIP package of programs (Felsenstein, 1989) on the Pasteur Institute Server (http://bioweb.pasteur.fr/seqanal/phylogeny/), using default parameters unless otherwise stated. PROTDIST with 1000 bootstrap analyses was used to calculate the distance between the sequences before analysis with the neighbor-joining program NEIGHBOR and calculation of the consensus tree using CONSENSE. Trees were viewed and edited using TREEVIEW (Page, 1996).
Protein domains were identified with the PFAM (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/) databases. Protein topology prediction used TMHMM at the Expasy server (http://www.expasy.ch/).
Supplemental References Carrere, S., and Gouzy, J. (2006). REMORA: a pilot in the ocean of BioMoby
web-services. Bioinformatics 22: 900-901. Courcelle, E., Beausse, Y., Letort, S., Stahl, O., Fremez, R., Ngom-Bru, C.,
Gouzy, J., and Faraut, T. (2008). Narcisse: a mirror view of conserved syntenies. Nucleic Acids Res. 36: 485-490.
Felsenstein, J. (1989). PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5: 164-166.
Journet, E.P., van Tuinen, D., Gouzy, J., Crespeau, H., Carreau, V., Farmer, M.J., Niebel, A., Schiex, T., Jaillon, O., Chatagnier, O., Godiard, L., Micheli, F., Kahn, D., Gianinazzi-Pearson, V., and Gamas, P. (2002). Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis. Nucleic Acids Res. 30: 5579-5592.
Nicholas, K.B., Nicholas, H.B.Jr., and Deerfield, D.W. (1997). GeneDoc: Analysis and Visualization of Genetic Variation. EMBNEW.NEWS 4: 14.
Page, R.D.M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Comput. Appli. Biosci. 12: 357-358.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882.
