pa - plant cell · pa pa ho pa ho pa pa ... the respective double-stained cross sections of...

Host: maize

Host: cowpea

Host: Lotus japonicus

*

*

*

*

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A

B

C

ho ho

pa

pa

ho

pa

ho

pa

pa

pahoho

pa

Supplemental Figure 1. Host specificity of the parasitic plant P. japonicum. (A - C) Interaction of P. japonicum with maize (A), cowpea (B) and Lotus japonicus (C) roots. On the right are shown the respective double-stained cross sections of haustoria embedded with Tecnovit 7100 solu-tion stained with Safranin O and Fast Green dyes. Note that L. japonicus is a nonhost for P. japonicum. Black arrows point to the interface between the host and parasite. Asterisks mark the haustoria, pa: parasite root and ho: putative host root. Bars on the left photographs corres-pond to 200 µm and bars on the right cross sections correspond to 50 µm.

Supplemental Data. Ishida et al. Plant Cell (2016) 10.1105/tpc.16.00310

Supplemental Figure 2. P. japonicum grown in rhizotron with or without rice hosts. P. japonicum plants were grown in rhizotron chamber without (left) or with (right) rice hosts. Parasites are labeled with pa and hosts are labeled with white arrowheads in the right panel. Bar: 1 cm.

Parasitic stageAutotrophic stage

papa papa

pa


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DMBQtreatment

Host root exudatetreatment

Homolog to Arabidopsis

Lipase(CUST_17498_PI426107926)

GlutathioneS-transferase

(CUST_20810_PI426107926)

Quinone Reductase 2

(CUST_18852_PI426107926)

Aux-IAATF

(CUST_13707_PI426107926)

Gibberellin 2-beta-dioxygenase

(CUST_25712_PI426107926)

Time (h)Time (h)

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Host root exudatetreatment

CytokininOxidase 3

(CUST_30762_PI426107926)

Glc7-InteractingProtein 1

(CUST_33571_PI426107926)

Pirin(CUST_40645_PI426107926)

Glucan1,3-beta-

glucosidase(CUST_45134_PI426107926)

Legend: DMBQ (RT-qPCR)

Mock (RT-qPCR)

DMBQ (Microarray)

Host exudate (RT-qPCR)

Mock (RT-qPCR)

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Supplemental Figure 3. RT-qPCR validation of the expression profiles of selected genes. Fold--induction of gene expression after DMBQ (solid orange lines) or mock (solid blue lines on the left column) treatment. For comparison, microarray data are shown in the graphs of DMBQ-trea-ted roots (solid gray lines). Host rice root exudate (dashed orange lines) or mock (dashed blue lines) treatments. Data are shown as log2 fold-induction of treated roots compared to non-trea-ted roots at 30 min, 1 h, 3 h, 6 h, 24 h and 48 h of treatment period. Two-week-old P. japonicum plants were grown vertically to prevent the roots from entering the agar media, thereby forcing the roots to be in contact with 10 µM DMBQ solution (on the left) or with rice root exudate (on the right). The values are the average of at least two biological replicates with at least two tech-nical replicates. Error bars indicate ± standard errors (SE). Each experiment used pooled RNA extracted from the roots of total 20-30 plants. Asterisks indicate significant differences (p value<0.05) compared to mock-treatment in RT-qPCR experiments. Primers specific for the reference Pj-PTB gene were used for normalization.


A B C D

Supplemental Figure 4. P. japonicum roots at 48 h after rice root exudate treatment. (A) Non-transformed roots in normal growth conditions and (B) non-transformed roots treated 48 h with rice root exudate. (C-D) Magnification of host-induced haustoria in non-transfor-med roots. Root tissues were cleared with chloral hydrate and observed under bright-field microscope (D). The black bar corresponds to 500 µm and white bars correspond 250 µm. White arrows indicate haustoria.


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noisserpxE eneG(L

ogFC

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Supplemental Figure 5. Expression profiles of early-responsive genes. Clusters of transcripts where the peak of expression occurred before 3 h of 10µM DMBQ treatment. Lines corres-pond to average of transcriptional expression of all the members of each cluster. The line colors represent the first (blue), second (red) and third (black) biological experiments. The gray bars are ± standard errors (SE). Complete table is shown in Supplemental Data Set 2.


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Supplemental Figure 6. Expression profiles of late-res-ponsive genes. Clusters of transcripts where the peak of expression occurred after 3 h of 10 µM DMBQ treat-ment. Lines correspond to average of transcriptional expression of all the members of each cluster. The line colors represent the first (blue), second (red), and third (black) biological experiments. The gray bars are ± standard errors (SE). Complete table is shown in Supplemental Data Set 2.

Supplemental Data. Ishida et al. Plant Cell (2016) 10.1Supplemental

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Supplemental Figure 7. Expression profiles of down-regulated genes. Lines correspond to average of transcriptional expres-sion of all the members of each cluster. The line colors represent the first (blue), second (red), and third (black) biological experiments. The gray bars are ± standard errors (SE). Complete table is shown in Supplemental Data Set 2.


TvQR2

PjQR2

TvQR1

PjQR1

0.2

154 bp 93 bp 837 bp

PjQR1: 331 aa

PjQR2: 205 aa

93 bp 93 bp 266 bp 407 bp100 bp

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Supplemental Figure 8. QR2 rather than QR1 is differentially regulated during parasitism. (A) The UPGMA phylogenetic tree based on full-length protein alignment of Pj-QR1, Pj-QR2 and their homologs in T. vesicolor, Tv-QR1 and Tv-QR2. Bootstrap values are shown in percentage at the internal nodes. (B) Schematic view of the Pj-QR1 and Pj-QR2 genes, black boxes repre-sent exons and lines are introns. The bar corresponds to 100 bp. (C) Relative expression levels of QR1 and QR2 measured by RT-qPCR. Values correspond to the ratio between the expres-sion of 10 µM DMBQ-treated roots compared to mock-treated roots (0.1%(v/v) DMSO) sampled at 0.5 h, 1 h, 3 h, 6 h, 24 h and 48 h after treatment. Data represent the average ± standard errors (SE) of three independent biological replicates .


10 20 30 40 50 60 70 80 90 100. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |PjYUC1 1 M - - - G N Y L K E I E - - - - G - - K T AH - D - P YC A K I - G - - - S - KR - C VW V PG P V - I VG AG P SG L A A A AC L R E - KG V P S L V V ER SN - C I A S L WQ L K T YD R L R L H L 80AtYUC6 1 M - - - - - - - - - - E - - - - G - - K L AH - D - - H - - R - - G - M T S PR R I C V - V T G P V - I VG AG P SG L A T A AC L K E - R G I T S V L L ER SN - C I A S L WQ L K T YD R L H L H L 72AtYUC2 1 M - - - - E F V - - T E T L - - G - - KR I H - D - P Y - V E - - - - - - E - T R - C L M I PG P I - I VG SG P SG L A T A AC L K S - R D I P S L I L ER S T - C I A S L WQ H K T YD R L R L H L 76AtYUC1 1 M - - - - - - - - - - E - - - - S - - - - - H - - - PH - N K T - D - - - Q - T Q H I I L VH G P I - I I G AG P SG L A T S AC L S S - R G V P S L I L ER SD - S I A S L W K S K T YD R L R L H L 69AtYUC4 1 M - - - - G T C - - R E - - - - S - - - - - E - - - P - - - - - - - - - - - - T Q - - I F V PG P I - I VG AG P SG L A V A AC L SN - R G V P S V I L ER T D - C L A S L WQ KR T YD R L K L H L 65PjYUC2 1 M - - - - - - - - - - F - - - - N - - - - F S - - - D - - - D G - D T I - P - R R - C VW VN G P V - I VG AG P SG L A V A AG L KQ - Q G V P F V I L ER AD - C I A S L WQ N R T YD R L K L H L 69PjYUC4 1 M - - - - - - - - - - F - - - - S - - - - F T - - - D - - - T - - D - I F S - R R - C VW VN G P V - I VG AG P SG L A V A AG L K E - H G V P F V V L ER AD - C I A S L WQ KR T YD R L K L H L 68AtYUC9 1 M - - - - - - - - - - E - - - - N - - - M F R - L M A S - - E - - E - Y F S ER R - C VW VN G P V - I VG AG P SG L A T A AC L H D - Q G V P F V V V ER SD - C I A S L WQ KR T YD R L K L H L 73AtYUC5 1 M - - - - - - - - - - E - - - - N - - - M F R - L M G S - - E - - D - S - SD R R R C I W VN G P V - I VG AG P SG L A T A AC L R E - EG V P F V V L ER AD - C I A S L WQ KR T YD R I K L H L 73AtYUC8 1 M - - - - - - - - - - E - - - - N - - - M F R - L M D Q - - D Q - D - L - T N N R - C I W VN G P V - I VG AG P SG L A T A AC L H E - Q N V P F V V L ER AD - C I A S L WQ KR T YD R L K L H L 73AtYUC7 1 M C - N N N N - - - T SC V - - N I S SM L Q - - - P - - - E - - D - I F S - R R - C I W VN G P V - I VG AG P SG L A V A AD L KR Q E - V P F V I L ER AN - C I A S L WQ N R T YD R L K L H L 80AtYUC3 1 M YG N N N K - - - K S - I - - N I T SM F Q N L I P - - - EG SD - I F S - R R - C I W VN G P V - I VG AG P SG L A V A AG L KR - EG V P F I I L ER AN - C I A S L WQ N R T YD R L K L H L 85PjYUC3 1 M - - - - N - - - - - H C V P PN - - - M - T - L - D - - - Q - - E A F F S - G R - C I I VN G P V - I VG AG P SG L A V A AC L R R - Q G V P F V I V ER A S - C I A S L WQ N R T YD R L S L H L 75AtYUC10 1 M - - - - - - - - - - E - - - - - - - - - - - - - - - - - - - - - - - - - - - T - - - V - V - - - V - I VG AG P AG L A T S VC L N Q - H S I PN V I L E K ED I - Y A S L W K KR A YD R L K L H L 53AtYUC11 1 M - - - - - - - - - - E - - - - - - - - - - - - - - - - - - - - - - - - - - - K E - - I K I L - - V L I I G AG P AG L A T S AC L N R - L N I PN I V V ER - D VC S A S L W KR R S YD R L K L H L 57Clustal Consensus 1 * : : : * : * : * * : * * * . : . * : . : : : * : * * * * : : : * * * : * * * 36

110 120 130 140 150 160 170 180 190 200. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PjYUC1 81 P KQ F C E L P L M P F P K T - F P T Y P S KQ Q - F VR Y L ED Y AR R F G I R P V F G R R V VR A E F D - G L L G F W - - R V - - - A - T - - - A - G - - E - - G - - - - E T S V E - Y VC - R W L 157AtYUC6 73 P KQ F C E L P I I P F PG D - F P T Y P T KQ Q - F I E Y L ED Y AR R F D I K P E F N Q T V E S A A F D EN L - G M W - - R V - - - - - T - - - S VG - - E - EG - - - - - T T - E - Y VC - R W L 148AtYUC2 77 P KD F C E L P L M P F P S S - Y P T Y P T KQ Q - F VQ Y L E S Y A EH F D L K P V F N Q T V E E A K F D R R C - G L W - - R V - - - R - T - - - T - G - - - - - G K KD - E T M - E - Y V S - R W L 154AtYUC1 70 P KH F C R L P L L D F P E Y - Y P K Y P S KN E - F L A Y L E S Y A SH F R I A PR F N KN VQ N A A YD S S S - G F W - - R V - - - K - T - - - H - D - - - - - N - - - - - T - - E - Y L S - KW L 142AtYUC4 66 P KH F C E L P L M P F P KN - F P K Y P S K - Q L F I S Y V E S Y A AR F N I K P V F N Q T V E K A E F D D A S - G L W - - N V - - - K - T - - - Q - D - - - - - G - - - - - V - - - - Y T S - T W L 137PjYUC2 70 P K K F C Q L P S F P F P - D H Y PD Y P S KR Q - F I E Y L EC Y AR N F D I R PQ F N EC VQ S A K YD E PC - R L W - - R V - - - R - T T - R S - E - - N - - A - - A - E V - - E - Y I C - Q Y L 147PjYUC4 69 P KQ F C Q L P K F P F P - D H Y P E Y P S KR Q - F I E Y L EC Y AQ H F D I N PH F N EC VQ S A K YD E AC - C L W - - R V - - - K - T V - - G - P - - D - - G - - G - E V - - E - Y I C - Q W L 145AtYUC9 74 P K K F C Q L P KM P F P - D H Y P E Y P T KR Q - F I D Y L E S Y AN R F D I K P E F N K S V E S AR F D E T S - G L W - - R V - - - R - T T - - S - D - - - - - G - - E - EM - - E - Y I C - R W L 149AtYUC5 74 P K K VC Q L P KM P F P ED - Y P E Y P T KR Q - F I E Y L E S Y AN K F E I T PQ F N EC VQ S AR YD E T S - G L W - - R I - - - K - T T - - S - S - - S S SG - - S - EM - - E - Y I C - R W L 152AtYUC8 74 P KQ F C Q L P KM P F P ED - F P E Y P T KR Q - F I D Y L E S Y A T R F E I N P K F N EC VQ T AR F D E T S - G L W - - R V - - - K - T V - - S K S - - E S T Q - - T - E V - - E - Y I C - R W L 153AtYUC7 81 P KQ F C Q L PN L P F P ED - I P E Y P T K YQ - F I E Y L E S Y A T H F D L R P K F N E T VQ S A K YD KR F - G L W - - R V - - - Q - T V L R S - E L - - - L G - - YC E F - - E - Y I C - R W L 161AtYUC3 86 P KQ F C Q L PN Y P F PD E - F P E Y P T K F Q - F I Q Y L E S Y A AN F D I N P K F N E T VQ S A K YD E T F - G L W - - R V - - - K - T I - - S - N M G Q - L G - - SC E F - - E - Y I C - R W I 166PjYUC3 76 P KQ F C Q L PH A P F P - E S Y P E Y P SR SQ - F VD Y L E S Y AR R F Q I Q PR F G E T VR H A VH D Q AC - G L W - - R V - - - R - T - - - S - A - - D - - G - - G - E T - - E - Y I C - R W L 151AtYUC10 54 A K E F C Q L P F M PH G R E - V P T F M S K E L - F VN Y L D A Y V AR F D I N PR YN R T V K S S T F D E SN - N KW - - R V V A EN - T V - - T - - - - - - - G - - - - E T - - E V YW S - E F L 131AtYUC11 58 A KQ F C Q L PH M P F P SN - T P T F V S K - L G F I N Y L D E Y A T R F N VN PR YN R N V K S A Y F - K - D - G Q W I V K V V - - N K T - - - T - - - - - - - A - - - - L I - - E V Y - S A K F M 133Clustal Consensus 36 . * . . * . * * . * : : : * : * : : * . . * : * : . . * : . * . : * * . : : 62

210 220 230 240 250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PjYUC1 158 I V A T G EN A E A V V P A I EG - M - A E F G - - G - D V V - H T SG Y KN G G - F Y EG K - R V L V VG C G N SG M E VC L D L - - C N H H A T P S L V VR D A VH I L PR EM L G K S T F G L SM 247AtYUC6 149 V A A T G EN A E P V V PR F EG - M - D K F A A AG - - V V KH T C H Y K T G G D F - AG K - R V L V VG C G N SG M E VC L D L - - C N F G AQ P S L V VR D A VH V L PR EM L G T S T F G L SM 240AtYUC2 155 V V A T G EN A E E VM P E I D G - I - PD F G - - G - P I L - H T S S Y K SG E I F - S E K - K I L V VG C G N SG M E VC L D L - - C N F N A L P S L V VR D S VH V L PQ EM L G I S T F G I S T 244AtYUC1 143 I V A T G EN AD P Y F P E I PG - R - K K F S - - G G K I V - H A S E Y K SG E E F - - R R Q K V L V VG C G N SG M E I S L D L - - VR H N A S PH L V VR N T VH V L PR E I L G V S T F G VG M 233AtYUC4 138 V V A T G EN A E P V F PN I PG - L - K K F T - - G - P V V - H T S A Y K SG S A F - AN R - K V L V VG C G N SG M E V S L D L - - C R YN A L PH M V VR N S VH V L PR D F F G L S T F G I AM 227PjYUC2 148 I V A T G EN A ER V A P E I D G - L - ED F G - - G - E V I - H AC D Y K SG E S Y - R G K - R V L V VG C G N SG M E V S L D L - - C N H G A K P AM V VR S S VH V L PR E V F G K S T F E V A V 237PjYUC4 146 V V A T G EN A ER V V P E I D G - L - K E F G - - G - E V I - H AC D Y K SG EG Y - KG K - K V L V VG C G N SG M E V S L D L - - C N H D A K P AM V VR S S VH V L PR E I F G R S T F E L AM 235AtYUC9 150 V V A T G EN A ER V V P E I N G - L M T E F D - - G - E V I - H AC E Y K SG E K F - R G K - R V L V VG C G N SG M E V S L D L - - AN H N A I T SM V VR S S VH V L PR E I M G K S T F G I S V 240AtYUC5 153 V V A T G EN A E K V V P E I D G - L T T E F E - - G - E V I - H SC E Y K SG E K Y - R G K - S V L V VG C G N SG M E V S L D L - - AN H N AN A SM V VR S S VH V L PR E I L G K S S F E I SM 243AtYUC8 154 V V A T G EN A ER VM P E I D G - L - S E F S - - G - E V I - H AC D Y K SG E K F - AG K - K V L V VG C G N SG M E V S L D L - - AN H F A K P SM V VR S S L H VM PR E VM G K S T F E L AM 243AtYUC7 162 V V A T G EN A E K V V P E F EG - L - ED F G - - G - D V L - H AG D Y K SG ER Y - R G K - R V L V VG C G N SG M E V S L D L - - C N H D A S P SM V VR S S VH V L PR E V L G K S T F E L S V 251AtYUC3 167 V V A T G EN A E K V V PD F EG - L - ED F G - - G - D V L - H AG D Y K SG G R Y - Q G K - K V L V VG C G N SG M E V S L D L - - YN H G AN P SM V VR S A VH V L PR E I F G K S T F E L G V 256PjYUC3 152 V V A SG EN A E K V V P E F EG - A - AD F G - - G - R M M - H AC E Y K SG K E F - G G K - R V L V VG C G N SG M E V A L D L - - T N H H A F P S L V VR S S VH V L PR E I L G K S T Y E I AG 241AtYUC10 132 V V A T G EN G D G N I PM V EG - I - D T F G - - G - E I M - H S S E Y K SG R D F - KD K - N V L V VG G G N SG M E I S F D L - - C N F G AN T T I L I R T PR H V V T K E V I - H - - - - L G M 216AtYUC11 134 V A A T G EN G EG V I P E I PG L V - E S F Q - - G - K Y L - H S S E Y KN G E K F - AG K - D V L V VG C G N SG M E I A YD L S KC N - - AN V S I V VR SQ VH V L T R C I V - R - - - - I G M 219Clustal Consensus 63 : . * : * * * . : * . * * * : * : * * . * : : : * * * * * * * * * * : . * * . * : : : * * : : . : . . : . 105

310 320 330 340 350 360 370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PjYUC1 248 W L L KW F PM R L VD R F L L V A SW L L - L G D T G R F G L D R PR VG P L E L KN L S - G K T P V L D VG T L - D K I K SG D I K V Y PG - I - Q R L - S - H Q S - V E F VD - G - R - K E - S F 335AtYUC6 241 F L L KW L P I R L VD R F L L V V SR F I - L G D T T L L G L N R PR L G P L E L KN I S - G K T P V L D VG T L - A K I K T G D I K VC SG - I - R R L - KR H E - - V E F - D N G - K - T E - R F 328AtYUC2 245 S L L KW F P VH V VD R F L L R M SR L V - L G D T D R L G L VR P K L G P L ER K - I KC G K T P V L D VG T L - A K I R SG H I K V Y P E - L - KR V - M - H Y S - A E F VD - G - R - VD - N F 332AtYUC1 234 T L L KC L P L R L VD K F L L L M AN L S - F G N T D R L G L R R P K T G P L E L KN V T - G K S P V L D VG AM - S L I R SG M I Q I M EG - V - K E I - T - K KG - A K F M D - G - Q - - E KD F 321AtYUC4 228 T L L KW F P L K L VD K F L L L L AN - S T L G N T D L L G L R R P K T G P I E L KN V T - G K T P V L D VG A I - S L I R SG Q I K V T Q A - V - K E I - T - R N G - A K F L N - G - K - - E I E F 315PjYUC2 238 F M L KW L P L W L VD K VM L A L AW V V - L G S T D K YG L KR P V VG P L E L KN AH - G K T P V L D I G A L - ER I R SG D I Q V V PG - V - KR F - T - R - G M V E L V T - G E K - L E - - I 325PjYUC4 236 L M L KW L P L W L VD K V L L F L SWM I - L G N M E K F G L KR P S L G P L E L KN - H Q G K T P V L D I G A L - ER I R SR D I Q V V PG - I - K K F - S - K - G S V E L VN - G E K - L E - - I 323AtYUC9 241 M M M KW L P L W L VD K L L L I L SW L V - L G S L SN YG L KR PD I G PM E L K SM T - G K T P V L D I G A L - E K I K SG D V E I V P A - I - KQ F - S - R - H H V E L VD - G Q K - L D - - I 328AtYUC5 244 M L M KW F P L W L VD K I L L I L AW L I - L G N L T K YG L KR P T M G PM E L K I V S - G K T P V L D I G AM - E K I K SG E V E I V PG - I - KR F - S - R - SH V E L VD - G Q R - L D - - L 331AtYUC8 244 KM L R W F P L W L VD K I L L V L SWM V - L G N I E K YG L KR P EM G PM E L K S V K - G K T P V L D I G A I - E K I R L G K I N V V PG - I - KR F - N - G - N K V E L VN - G EQ - L D - - V 331AtYUC7 252 T M M KWM P VW L VD K T L L V L T R L L - L G N T D K YG L KR P E I G P L E L KN T A - G K T P V L D I G A I - SM I K SG K I K I V AG - I A K - F - G - P - G K V E L VD - G - R V L Q - - I 339AtYUC3 257 T M M K YM P VW L AD K T I L F L AR I I - L G N T D K YG L KR P K I G P L E L KN K E - G K T P V L D I G A L - P K I R SG K I K I V PG - I I K - F - G - K - G K V E L I D - G - R V L E - - I 344PjYUC3 242 T M M KW V P VW V VD R V L L A A AR L I - L G S T E EH G L KR P S I G P L Q L KD T Q - G R T P V L D I G A V - S K I R SG D I K I V PG - I - K K F - V - R AG - V E L VN - G D L - L Q - - V 329AtYUC10 217 T L L K Y A P V AM VD T L V T T M A K I L - YG D L S K YG L F R P KQ G P F A T K L F T - G K A P V I D VG T V - E K I R D G E I Q V I N G G I - G S I N G - K - T - L T F EN - G H K - Q D - - F 305AtYUC11 220 S L L R F F P V K L VD R L C L L L A E L R - F R N T SR YG L VR PN N G P F L N K L I T - G R S A T I D VG C VG E - I K SG K I Q V V T S - I - KR I EG - K - T - V E F I D - G N T - KN - - V 307Clustal Consensus : : : * : : . * : . * * * * * * : * * : : . . : * : * : * : : : : : . : * : .

410 420 430 440 450 460 470 480 490 500. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PjYUC1 336 D A I I L A T G Y K SN V P F W L K - E - S - E - M F S E KD G L P KR P F PN - G W KG - EC G L Y A VG F T - KR G L L G A SM D A KN I A - AD I - E SC WR A E A K - - - H - - - - L - S A I S 418AtYUC6 329 D A I I L A T G Y K SN V P SW L K - E - N - K - M F S K KD G F P I Q E F P E - G WR G - EC G L Y A VG F T - KR G I SG A SM D A KR I A - ED I - H KC W KQ D - E - - - Q - - - - L - Q - - - 407AtYUC2 333 D A I I L A T G Y K SN V PM W L K - G VN - - - M F S E KD G F PH K P F PN - G W KG - E SG L Y A VG F T - K L G L L G A A I D A K K I A - ED I - E - V - - Q - - R - - - H - - F - L - P - - - 408AtYUC1 322 D S I I F A T G Y K SN V P T W L Q - G - G - D - F F T - D D G M P K T P F PN - G WR G - G KG L Y T VG F T - R R G L L G T A SD A V K I AG E - I G D Q - W - R D - E I - - K - - G - - - S - - - 399AtYUC4 316 D S I I L A T G Y K SN V PD W L K - E - N - S - F F T - K EG M P K T P F PN - G W KG - E KG L Y T VG F T - R R G L SG T A YD A V K I A - ED I T D Q - W - M - - K F - - N - - G P L - S - - - 394PjYUC2 326 D S V V L A T G YR SN V P YW L Q - E - S - E - F F G - KN G M P K A P F PH - G W KG - KG G L Y A VG F T - R R G L SG A S AD A V K I S - R D I A K - VW - R D - D M K - R - - K - M - Q - - - 405PjYUC4 324 D S V V L A T G YC SN V P YW L Q - E - N - D - F F S - KN G F P K A P F PN - G W KG - K AG L Y A VG F T - K KG L SG A S AD AM K I S - Q D I G K - VW - KD - D L K - Q - - K - K - Q - - - 403AtYUC9 329 D A V V L A T G YR SN V P SW L Q - E - S - E - F F S - KN G F P K S P F PN - AW KG - K SG L Y A AG F T - R KG L AG A S VD A VN I A - Q D I G N - VW - R E - E T KR Q - - K - M - R - - - 409AtYUC5 332 D A V V L A T G YR SN V P SW L Q - E - N - D - L F S - KN G F P K S P F PN - AW KG - K SG L Y A AG F T - R KG L AG A S AD A VN I A - Q D I G N - VW - R E - E T KR Q - - K - M - R - - - 412AtYUC8 332 D S V V L A T G YR SN V P YW L Q - E - N - E - F F A - KN G F P K T V AD N N G W KG - R T G L Y A VG F T - R KG L SG A SM D A V K I A - Q D I G S - VW - Q L - E T K - Q P T K - - - R - - - 413AtYUC7 340 D S V I L A T G YR SN V P SW L K - E - N - D - L - G - E I G I E KN P F P K - G W KG - K AG L Y A VG F T G R - G L SG A S F D AM S V A - H D I AN - SW - K E - E T K - Q - - Q - I - K - - - 418AtYUC3 345 D S V I L A T G YR SN V P SW L K - D - N - D - F F S - D D G I P KN P F PN - G W KG - E AG L Y A VG F T - R KG L F G A S L D AM S V A - H D I AN - R W - K E - E S K - Q - - Q - K - K - - - 424PjYUC3 330 D S V I F A T G Y S SN V P L W L K - E - S - E - F F T - KD G F PR S P F PN - G W KG - KD G L Y A VG F A - R KG L AG A SM D A V K V A - Q D I G K - L W - N Q - E T - - M - - - - M - Q - - - 407AtYUC10 306 D A I V F A T G Y K S S VC N W L E - D - Y - E Y VM K - KD G F P K A PM P K - H W KG E KN - L YC AG F S - R KG I AG G A ED AM S V A - D D I - - - - - - R - - S - - - I - - - - L - A - - - 378AtYUC11 308 D S I V F A T G Y K S S V S KW L E VD - D G D - L F N - EN G M P KR E F PD - H W KG - KN G L Y S AG F G - KQ G L AG I SR D AR N I A - R D I - - - - - - D - - S - - - L - - - - VC G - - - 382Clustal Consensus 139 * : : : : * * * * * . * * * : . . * : . * : * * * . * * : * : * : * * : : * 171

510 520. . . . | . . . . | . . . . | . . . . | . . . . | .

PjYUC1 419 S PC S - M - Q KH I L SD - Q L I N E L - I - - N 438AtYUC6 407 - - C K - L - G KR - M KR - K F - S E SD C G G N 426AtYUC2 408 - - - - - L - A - R - - P - - Q - - - H - - C - - - 415AtYUC1 399 - - T R N M C S SR - F V - - - F T S K - - - - - S 414AtYUC4 394 - - C R N I C S SH - I I H L H F - N K - - - - - S 411PjYUC2 405 - - - K - V PN - H - - R R - C I - S T - - - - - F 417PjYUC4 403 - - - K - V P T - H - - R R - C I - S T - - - - - F 415AtYUC9 409 - - - R N V - G - H - - R R - C I - S V A - - - - - 421AtYUC5 412 - - T R - V - G - H - - R R - C I - S V A - - - - - 424AtYUC8 413 - - SR - - - G - S - L R R - C I - SQ Q - - - - F 426AtYUC7 418 - - - T - V A T R H - - R R - C I - SH - - - - - F 431AtYUC3 424 - - - T - A A AR H - - R R - C I - SH - - - - - F 437PjYUC3 407 - - - K - - - N - H - - - - - S V - G L - - - - - A 415AtYUC10 378 - - - - - - - T - L - - KN - - - - N - - - - - - - 383AtYUC11 382 - - - R - - - S - S - - KN - K L - S K - - - - - - 391Clustal Consensus

Supplemental Figure 9. Alignment of Pj-YUCs and At-YUCs amino acid sequences. The sequen-ces were aligned using CLC Genomics Workbench (v4.8) and the figure was generated by BioE-dit. Residues are background-shaded according to their identity or similarity scores calculated from protein similarity matrix BLOSUM62 with the default cut-off value in BioEdit software. Amino acids with similar property are shaded with the same color.


A Bright field MergeVenus

B

Supplemental Figure 10. Expression pattern of DR5 promoter in P. japonicum roots. Confocal images of transgenic hairy roots carrying pDR5rev:3xVenus-N7. (A) at root apical meristem. (B) mature zone. Bars correspond to 50 µm.


No fluorescence

pHG8YUC3-2

pHG8YUC3-1

Pj-YUC1

4

3

2

1

No fluorescence

pHG8YUC3-2

pHG8YUC3-1

No fluorescence

pHG8YUC3-2

pHG8YUC3-1

Pj-YUC2 Pj-YUC4

Rel

ativ

e ex

pres

sion

leve

l

Supplemental Figure 11. Transcript levels of YUC homologs in transgenic roots. Trans-cript levels were measured by RT-qPCR in transgenic roots transformed with non-fluo-rescent hairy roots, pHG8-YUC3-1 and pHG8YUC3-2 after 48 h in contact with host Ara-bidopsis roots using primer pairs specific for the Pj-YUC1, Pj-YUC2 and Pj-YUC4 genes. Data correspond to the average ± standard error of three to five biological replicates with 5-15 independent transgenic roots per experiment.


6

4

2root

leng

th (m

m)

No fluorescence

pHG8YUC3-2

pHG8YUC3-1

0.4

0.3

0.2

0.1Num

ber

of la

tera

l roo

ts

No fluorescence

pHG8YUC3-2

pHG8YUC3-1

0.5

A B C

Non-fluorescent roots pHG8 YUC3-1 pHG8 YUC3-2

bright field bright field bright fieldYFP YFP YFP

D E

Supplemental Figure 12. Morphology of transformed hairy roots. (A - C) Photographs of transgenic roots transformed with the control (A) or silencing constructs (B, C) after 48 h of DMBQ treatment, under bright field (left) and fluorescence (right). Arrows point to haustoria. Bars correspond to 1 cm. (D) Root length of transgenic roots. (E) Number of lateral roots per transgenic root. Data correspond to the average ± standard errorof three to five biological replicates with 5-15 independent transgenic roots per experiment.


Per

cent

age

of tr

ansg

enic

root

s w

ith a

t lea

st o

ne h

aust

oriu

m (%

)

60

20

40

80

pHG8YFP pHG8YUC3-1Non-fluorescent roots

*

Supplemental Figure 13. RNAi lines of P. japonicum roots targeted to YUC3 show reduced haustorium formation induced by rice root exudates. Graph shows the percentage of transgenetic roots with at least one haustorium. Roots transformed with empty vector pHG8-YFP and no-fluorescent hairy roots were used as control. Non-fluorescent roots represent those transgenetic hairy roots without the T-DNA containing the fluorescent protein gene. Root silenced for Pj-YUC3 gene carries the construct pHG8YUC3-1. Aste-risk (*) represent α = 0.01 by t-Test assuming unequal variances. Values represent avera-ge ± SD of three to six independent experiments with 10 to 40 transformed roots per expe-riment. Total n = 99, 90 and 89 in no fluorescent roots, pHG8-YFP and pHG8YUC3-1, respectively.


A Venus

GFP

Bright field

Bright field

Merge

Merge

B

CProAtPGP4

Pro

AtP

GP

4:3x

Ven

us-N

7P

roA

tRP

S5a

:H2B

-GFP

Cre-GR AtHSPter

ProAtRPS5a PjYUC34xTer AtHSPter

LoxP

LoxP

Supplemental Figure 14. Expression patterns of At-PGP4 promoter and At-RPS5a promoter in P. japonicum. (A) At-PGP4 promoter expression in P. japonicum root transformed with AtPGP4:3x-Venus-N7. (B) At-RPS5a promoter expression in P. japonicum root transformed with AtRPS5a::H2B-GFP. Bars correspond to 50 µm. (C) Schematic view of AtPGP4>>PjYUC3 construct.


Supplemental Table 1 - Total number of reads obtained in each RNA-seq library

Library 1 Illumina Hi-seq2000

Library 2

Library 3

Illumina Hi-seq2000

Roche 454 FLX

AutotrophicStage

Haustoria

Mixed

24,537,964 reads(90 bp reads)

25,841,404 reads(90 bp reads)

888,638 reads(average 333.8 bp)

2,208,416,760 bp

2,325,726,360 bp

296,626,260 bp

Sequencer Material Read Total sequence


Supplemental Table 2. Investigation on the rice contamination in P. japonicum unigenes

e-value threshold Number of contigs matchedwith rice sequences*

Minimum alignment length

37 bp

54 bp

92 bp

73 bp

238

198

176

160

1e-10

1e-20

1e-30

1e-40

*Number of P. japonicum unigenes that show local alignment in the indicated e-value and 100% identity with rice cDNAs (ver 6.1)


Supplemental Table 3: Summary of de novo assembly of P. japonicum transcriptome

Values

Number of contigs

Total residues (bp)

Minimum legth (bp)

Maximum length (bp)

Average length (bp)

N50 length (bp)

Putative protein codinga

Putative proteins with start codon

Blast hits to Arabidopsisb

Blast hits RefSeq-protb

Blast hits to nrb

57,939

47,011,167

300

12,710

812

1,090

47,817 (82.5%)

17,044 (35.6 % of predicted proteins)

34,897 (60.23%)

38,372 (66.23%)

37,259 (64,3%)a Number of ESTScan-predicted protein sequences more than 50 aab Blastx with threshold e-value less than 1e-10.


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