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www.sciencemag.org/cgi/content/full/322/5909/1835/DC1 Supporting Online Material for A Conserved Molecular Framework for Compound Leaf Development Thomas Blein, Amada Pulido, Aurélie Vialette-Guiraud, Krisztina Nikovics, Halima Morin, Angela Hay, Ida Elisabeth Johansen, Miltos Tsiantis, Patrick Laufs* *To whom correspondence should be addressed. E-mail: [email protected] Published 19 December 2008, Science 322, 1835 (2008) DOI: 10.1126/science.1166168 This PDF file includes: Materials and Methods Figs. S1 to S10 Tables S1 to S7 References Supporting Online Materiel to A conserved molecular framework for compound leaf development Thomas Blein1, Amada Pulido1, Aurélie Vialette-Guiraud1*, Krisztina Nikovics1†, Halima Morin1,2, Angela Hay3, Ida Elisabeth Johansen4, Miltos Tsiantis3, Patrick Laufs1§ Material and Methods, 10 Supplementary Figures and 7 Supplementary Tables Material and Methods Plant material A. caerulea seeds were bought from a gardener shop (Jardiland, Maurepas, France). S. lycorpersicum M82 wild type were received from S. Biemelt, the la mutant (accession LA0335) from the Tomato Genetics Ressource Center and the gob mutants from D. Zamir. P. sativum (cv Térèse and line NGB5839) were obtained from C. Rameau and the uni mutant (line JI2171) from J. Hofer. S. tuberosum cv Pompadour were a gift from B. Letarnec. RNA extraction and RT-PCR RNA was extract from partially dissected apices or young leaves of the different species using TRIsol reagent (Invitrogen) according to the manufacturer recommendations. Contaminating DNA was removed by DNaseI treatment (Invitrogen) and the RNA was reverse transcribed (SuperScriptTM II reverse transcriptase, Invitrogen). Gene specific primers used for semiquantitative RT-PCR are listed in Supplementary Table 4. PCR products were observed on ethidium bromide-stained gels or following southern blotting (PsNAM1/2). Cloning of NAM/CUC genes. Aquilegia caerulea Blast analysis identified several Aquilegia formosa X pubescens ESTs with high similarities with the AtCUC2 and AtCUC3 proteins (TC10566 and TC15387, respectively). Partial AcNAM and AcCUC3 mRNAs were amplified from reversed transcribed shoot apex RNA from Aquilegia caerulea using respectively AcNAM1Fw and AcNAM1Rv, and AcCUC3Fw and AcCUC3Rv primers (Primer sequence available in Supplementary Table 5). Pisum sativum Fragments of the NAC domain were amplified from reversed transcribed RNA from small axillary shoots of pea (cv Térèse) using degenerated primers (NACdeg1Fw1 and NACdegRv1; NACdegFw2 and NACdegRv2; degCUC3Fw1 and degCUC3Rv1; degCUC3Fw2 and degCUC3Rv2). The PCR products were cloned into pGEM-T Easy (Promega) and the insert of several independent clones was sequenced. The resulting sequences were positioned on a phylogenetic tree containing several NAC proteins including AtCUC1, AtCUC2 and AtCUC3. The sequences of the NAM/CUC orthologs were extended by RACE-PCR using the The GeneRacerTM kit (Invitrogen) and nested gene specific primers. Solanum lycopersicum A fragment of the SlNAM was amplified from genomic DNA of tomato using nam2 and nam5 primers, cloned into pGEM-T Easy (Promega) and sequenced. The full length SlNAM was isolated from RNA extracted from vegetative apices by RACE-PCR using the GeneRacerTM kit (Invitrogen) and nested gene specific primers. Solanum tubersosum RNA from S. tuberosum (cv solara) was obtained from C. Navarro. A StNAM fragment was obtained by RT-PCR using the tomato SlNAM-based primers SlNAM5’ and SlNAM3’. Blast analysis identified a Solanum chacoense EST (DN983806) showing high similarity to AtCUC3 and used to define two specific primers cuc3uj2 and revcuc3uj3. These primers were used to amplify a fragment of StCUC3 which was then extended by RACE PCR using the GeneRacerTM kit (Invitrogen) and nested gene specific primers. Cardamine hirsuta Fragments of ChCUC2 and ChCUC3 were amplified from reversed transcribed shoot apical meristem Cardamine hirsuta RNA using the Arabidopsis-based primers RLT-FwdAT5G53950 and CUC2 3' and insitu CUC3 L Fwd insitu CUC3 L Rv+T7, respectively. A fragment of ChCUC1 was amplified using the degenerated primers NACdegFw2 and miR164bsdegRv1. These fragments were cloned into pGEM-T and sequenced. The sequences of ChCUC1, 2 and 3 were extended by RACE-PCR. mRNA in situ hybridisations They we performed as described (1) using the probes listed in the Supplementary Table 6 Cardamine transgenic lines and dexamethasone induction. A hairpin cassette was created against the specific domain of ChCUC3 (see Supplementary Table 7) in pKANNIBAL(2) and transferred into the binary vector pGreen0029(3) to create the ChCUC3 RNAi vector. The ChCUC3 RNAi plasmid and the 2x35S:MIR164b plasmid(4) were transferred into Agrobacterium tumefaciens GV3101 and transformed into C. hirsuta as described(5). Ten-day-old KNOTTED1-GR C. hirsuta transgenic lines were induced with 106 M Dexamethasone (SIGMA) as previously described(6) for 2 weeks for the phenotypic analysis and for 2 days for the molecular analysis. The STM-GUS line was described in(7). Virus Induced Gene Silencing Gene specific fragments of the NAM/CUC genes (see Supplementary Table 7) and of the PDS genes (8-10) were introduced into TRV (for A. caerulea and S. lycopersicum) or PEBV (for P. sativum) RNA2. The TRV vectors were kindly provided by E. Kramer. VIGS was done as described for A. caerulea(9) and P. sativum (line NGB5839)(8). For S. lycopersicum, we vacuum infiltrated M82 10 days old plants as described for A. caerulea(9). Silencing of PDS is a good marker for VIGS because it gives a clear phenotype and is routinely used in VIGS experiments including when developmental processes are analysed (eg.8, 10-14). PDS is active in green tissues. NAM and CUC3 on the other hand are expressed in regions of the growing apex. The peak of PDS silencing seen as completely white leaves and of NAM/CUC seen as altered developmental patterns will therefore appear in different parts of the plant. So when silencing peaks, the bleaching phenotype will appear in the leaves whereas the peak in phenotype of NAM/CUC silencing will become visible only later when the young organs that were affected by the silencing expand to a mature organ. Therefore, the developmental effect of NAM/CUC3 silencing and the bleaching of the leaf are not always strictly associated. 1. K. Nikovics et al., Plant Cell 18, 2929 (2006). 2. C. Helliwell, P. Waterhouse, Methods 30, 289 (2003). 3. R. P. Hellens, E. A. Edwards, N. R. Leyland, S. Bean, P. M. Mullineaux, Plant Mol Biol 42, 819 (2000). 4. P. Laufs, A. Peaucelle, H. Morin, J. Traas, Development 131, 4311 (2004). 5. S. J. Clough, A. F. Bent, Plant J 16, 735 (1998). 6. A. Hay, M. Tsiantis, Nat Genet 38, 942 (2006). 7. M. Barkoulas, A. Hay, E. Kougioumoutzi, M. Tsiantis, Nat Genet (2008). 8. G. D. Constantin et al., Plant J 40, 622 (2004). 9. E. M. Kramer et al., Plant Cell 19, 750 (2007). 10. Y. Liu, M. Schiff, S. P. Dinesh-Kumar, Plant J 31, 777 (2002). 11. T. M. Burch-Smith, M. Schiff, Y. Liu, S. P. Dinesh-Kumar, Plant Physiol 142, 21 (2006). 12. Y. Dong, T. M. Burch-Smith, Y. Liu, P. Mamillapalli, S. P. Dinesh-Kumar, Plant Physiol 145, 1161 (2007). 13. B. Gould, E. M. Kramer, Plant Methods 3, 6 (2007). 14. F. Ratcliff, A. M. Martin-Hernandez, D. C. Baulcombe, Plant J 25, 237 (2001). SOM Blein et al., Figure S1 Aquilegia caerulea Pisum sativum Cardamine hirsute Solanum lycopersicum Solanum tuberosum Figure S1. Characteristics of species analysed in this study. We have selected 5 broadly distributed and representative Eudicot species that form compound leaves : a stem Eudicot (Aquilegia caerulea), two Rosids (the Eurosid I, Pisum sativum and the Eurosid II, Cardamine hirsuta) and two close members of the Asterids (the Euasterids Solanum lycopersicum and Solanum tuberosum) .This selection of species covers the two main genetic pathways involved in compound leaf formation, the KNOXI pathway in C. hirsuta and S. lycopersicum (1, 2), and the LEAFY pathway (UNIFOLIATA,UNI) in P. sativum(3, 4), whereas no information regarding compound leaf formation is available for A. caerulea and S. tuberosum. Leaflet primordia arise either basipetally (S. lycopersicum, S. tuberosum and C. hirsuta) or acropetally (P. sativum). These species also show diverse levels of margin dissection as A. caerulea, S. lycopersicum and C. hirsuta have lobes and/or serrations. P. sativum shows leaflet specialisation with tendrils forming at the distal part of the leaf. The Phylogenetic tree is taken from (5). 1. 2. 3. 4. 5. D. Hareven, T. Gutfinger, A. Parnis, Y. Eshed, E. Lifschitz, Cell 84, 735 (1996). A. Hay, M. Tsiantis, Nat Genet 38, 942 (2006). C. E. Champagne et al., Plant Cell 19, 3369 (2007). J. Hofer et al., Curr Biol 7, 581 (1997). THE_ANGIOSPERM_PHYLOGENY_GROUP, Bot. J. Lin. Soc. 141, 399 (2003). SOM Blein et al., Figure S2 A AtNAC1 StCUC3 PsCUC3 AcCUC3 ChCUC3 94 AtCUC3 91 100 ChCUC1 AtCUC1 AcNAM 90 PsNAM2 100 57 100 PsNAM1 StNAM SlNAM ChCUC2 0.1 100 AtCUC2 B ChCUC2 AtCUC2 StNAM SlNAM PsNAM1 PsNAM2 AcNAM ChCUC1 AtCUC1 YLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEA YLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEA HLPPGFRFHPTDEELITYYLLKKVLDCNFTARAIAEVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA HLPPGFRFHPTDEELITYYLLKKVLDCNFTARAIAEVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA HLPPGFRFHPTDEELITFYLLKKVLDNTFTARAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA HLPPGFRFHPTDEELITFYLLKKVLDNTFTARAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA HLPPGFRFHPTDEELITFYLIKKVLDSTFTCRAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA LMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVNLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEA LMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVDLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEA ChCUC2 AtCUC2 StNAM SlNAM PsNAM1 PsNAM2 AcNAM ChCUC1 AtCUC1 GYWKATGKDREIYSSKTCALIGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKT GYWKATGKDREIFSSKTCALVGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKT GYWKATGKDREIFSSKTCALVGMKKTLVFYRGRAPKGEKSNWVMHEYRLDDKFAYHYISRSSKDEWVISRVFQKS GYWKATGKDREIFSSKTCALVGMKKTLVFYRGRAPKGEKSNWVMHEYRLDGKFAYHYISRSSKDEWVISRVFQKS GYWKATGKDREIYSSKTYSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKFAYHFLSRNSKDEWVISRVFQKS GYWKATGKDREIYSSKTYSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKFAYHFLSRNSKDEWVISRVFQKS GYWKATGKDREIYSSRTSSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKLSYHYLSRSSKDEWVISRVFQKS GYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSSWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKS GYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSCWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKS 80 80 80 80 80 80 80 80 80 154 154 154 154 154 154 154 154 154 C StCUC3 PsCUC3 AcCUC3 ChCUC3 AtCUC3 GLPPGFRFHPTDEELITFYLASKVFNATFSAIQIPQVDLNRCEPWELPEVAKMGEREWYFFSLRDRKYPTGLRTNRATGA GLPPGFRFHPTDEELITFYLASKVFKNTFFNVKFAEVDLNRCEPWELPDMAKMGEREWYLFSLRDRKYPTGLRTNRATGA GLPPGFRFHPTDEELITFYLASKVFNGRFCGVEIAEIDLNRCEPWELPDIAKMGERVWYFFSQRDRKYPTGLRTNRATEA GLPPGFRFHPTDEELISFYLASKVFDGGLCGIHITEVDLNRCEPWELPEMAKMGEKEWYFYSLRDRKYPTGLRTNRATTA GLPPGFRFHPTDEELITFYLASKIFHGGLSGIHISEVDLNRCEPWELPEMAKMGEREWYFYSLRDRKYPTGLRTNRATTA StCUC3 PsCUC3 AcCUC3 ChCUC3 AtCUC3 GYWKATGKDREVYSATNGALLGMKKTLVFYKGRAPRGEKTKWVMHEYRLDGDFSY-R-Y-SCKEEWVICRILHKV GYWKATGKDKEVYSNSTRALLGMKKTLVFYKGRAPRGEKTKWVMHEYRLHTHLSP---S-TCKEEWVICRIFHKS GYWKATGKDKEVYSASDDSLLGMKKTLVFYKGRAPRGVKTKWVMHEYRLEGDFSS-F-SHTFKEEWVLCRILQKT GYWKATGKDKEVFGSGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLETDLS-HR--HSCKEEWVICRVFNKT GYWKATGKDKEVFSGGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLENDHS-HR--HTCKEEWVICRVFNKT 151 150 152 151 151 80 80 80 80 80 SOM Blein et al., Figure S2 D ChCUC2 AtCUC2 StNAM SlNAM PsNAM1 PsNAM2 AcNAM ChCUC1 AtCUC1 miR164 5’-AGCACGUGUCCUGUUUCUCCA-3’ 5’-AGCACGUGUCCUGUUUCUCCA-3’ 5’-AGCACGUGCCCUGUUUCUCCA-3’ 5’-AGCACGUGCCCUGUUUCUCCA-3’ 5’-AGCACGUGUCCUGUUUCUCCA-3’ 5’-AGCACGUGUCCUGUUUCUCCA-3’ 5’-AGCACGUGCCCUGUUUCUCCA-3’ 5’-AGCACGUGUCCUGUUUCUCCA-3’ 5’-AGCACGUGUCCUGUUUCUCCA-3’ x.. 3’-ACGUGCACGGGACGAAGAGGU-5’ Figure S2. Phylogenetic analysis of the NAM/CUC genes. A. The amino acids of the NAC domains of the NAM/CUC proteins were aligned using the multiple sequence alignment software ClustalX (v1.83)(1). Maximum likelihood (heuristic search tree) from this multiple sequence alignment were generated with PhyML(2) using optimal amino acide substitution model JTT + G select using ModelGenerator (v0.84)(3). The NAC1 protein from A. thaliana was used as outgroup. Bootstrap confidence values were obtained by 100 replicates and only bootstrap values above 50% are indicated. B, C. Alignments of the NAC domain of the NAM and CUC3 proteins are represented in B and C, respectively. D. Alignment of the miR164 binding site in the NAM genes showing the complementarity with miR164 The phylogenetic analysis on the conserved DNA-binding NAC domain showed that the NAM/CUC genes group either into the NAM clade (AcNAM, SlNAM, StNAM, PsNAM1, PsNAM2, ChCUC1, ChCUC2) or the CUC3 clade (AcCUC3, StCUC3, PsCUC3 and ChCUC3). Distinction between members of the two clades extends outside the NAC domain as all members of the NAM clade contained a putative binding site for miR164, which was absent in the members of the CUC3 clade. The predicted PsNAM1 and PsNAM2 proteins showed 95.2% similarity suggesting that they result from a recent duplication. In contrast, the predicted ChCUC1 and ChCUC2 proteins showed only 52.3% similarity and each showed a higher sequence similarity to the corresponding A. thaliana protein than to the other C. hirsuta gene (86.2% and 84.2% for the CUC1 and pairs, respectively). This suggested that they result from a gene duplication event that took place before the separation between C. hirsuta and A. thaliana 13-19 MYA(4). 1. 2. 3. 4. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, Nucleic Acids Res 25, 4876 (1997). S. Guindon, O. Gascuel, Syst Biol 52, 696 (2003). T. M. Keane, C. J. Creevey, M. M. Pentony, T. J. Naughton, J. O. McLnerney, BMC Evol Biol 6, 29 (2006). M. Koch, B. Haubold, T. Mitchell-Olds, Am J Bot 88, 534 (2001). SOM Blein et al., AcNAM lp m lp S. tuberosum AcNAM lp D B G SlNAM I SlNAM s H fm AcH4 F AcH4 stp J SlNAM st st fm st o s StNAM m L R fm s s fm SlNAM M fm PsNAM1/2 m StCUC3 s S Q s P ChCUC3 m ChCUC2 m fm PsCUC3 m lp ChCUC1 m lp N s O StCUC3 m lp StNAM s E lp m m s stp s AcCUC3 lp AcNAM m lp K P. sativum C C. hirsuta S. lycopersicum A. caerulea A FigureS3 T PsNAM1/2 sp cmp VPsNAM1/2 X sp PsNAM1/2 o cw lp U PsCUC3 W PsH4 Y PsH4 sp cmp sp o cw SOM Blein et al., FigureS3 Figure S3. NAM/CUC and Histone H4 expression patterns in meristem and reproductive structures of selected Eudicots: A. caerulea (A-F), S. lycopersicum (G-J), S. tuberosum (KN), C. hirsuta (O-Q) and P. sativum (R-Y). In A. caerula, AcNAM and AcCUC3 are expressed at the boundaries between leaf primordia (lp) and the meristem (m) during the formation of the rosette leaves (A, B) and after bolting (C). In the floral meristem (fm), AcNAM marks the boundary of the numerous stamen primordia (stp, D). In the vegetative and floral meristem, Histone H4 expression, a marker of dividing cells is downregulated in the domain where AcNAM is expressed (arrows in E and F, compare AcNAM and Histone H4 expression in C-E and D-F respectively) SlNAM marks the boundary of the organ primordia in the apical (G) and floral meristem (H, I) of S. lycopersicum. SlNAM is also expressed at the boundaries between outgrowing ovules (o) and at the boundary between inner and outer locules of the stamens (st in J). StNAM and StCUC3 expression in apical and floral meristems of S. tuberosum (K-N). ChCUC1, ChCUC2 and ChCUC3 show similar expression patterns at the boundary of the primordia and the meristem in C. hirsuta (O-Q) PsNAM1/2 and PsCUC3 are expressed at the boundary between organ primordia and meristem of P. sativum (R, S). Their expression precedes the outgrowth of the primordium in the meristem and remains detectable in the axillary region of older leaves. In the flower, PsNAM1/2 and PsCUC3 are expressed in the boundary region of the floral organs (T, U). Pea flowers contain particular structures called common primordia (cmp) that will be divided into two to generate petal and a stamen primordia. PsNAM1/2 and PsCUC3 expression can be observed in a fine domain corresponding to the boundary between the petal and stamen primordia (arrows in T and U). Within the apex, histone H4 expression is reduced in the region where PsNAM1/2 is expressed (compare V and W, arrow in W). PsNAM1/2 is also expressed at the inner boundary of outgrowing teguments within ovules (X), a domain from which Histone H4 expression is excluded (arrows in Y, compare two successive sections X and Y). cmp: common primordium; cw: carpel wall; fm: floral meristem; m: meristem; lp: leaf primordium; o: ovule; s: sepal; sp: sepal primordium; stp: stamen primordium Bars: 0.1mm S. tuberosum S. lycopersicum SOM Blein et al., FigureS4 A SlNAM ltp1 * ltp2 1 B * 2 StNAM 3 4 ltp1 ltp2 * 1 2 SlNAM C 3 4 5 S. lycopersicum ltp1 1 m 2 m 3 4 7 8 3 4 ltp1’ 5 6 P. sativum D PsNAM1/2 ltp4 ltp3 ltp2 ltp1 1 2 Figure S4. Detailed analysis of NAM genes expression in serial sections of S. lycopersicum (A,C), S. tuberosum (B) and P. sativum (D). A. SlNAM expression in an older S. lycopersicum leaf. Note that SlNAM is not expressed in the centre of the region between leaflet primordia 1 and 2 (ltp1, ltp2) but is asymmetrically associated with the distal part of the leaflet primordium (arrow in 2). No SlNAM expression is observed on the proximal side of the leaflet primordia 1 and 2 (region marked by an asterisk). B. StNAM expression in an intermediary S. tuberosum leaf. No expression of StNAM is observed on the proximal side of the young leaflet primordium (ltp2, region marked by an asterisk). C. SlNAM expression in an young S. lycopersicum leaf. Two leaflet primordia are present (ltp1 and ltp1’). Expression of SlNAM is observed below these two leaflet primordia (arrows) but no sign of further leaflet primordium is observed, indicating that SlNAM expression precedes the outgrowth of the leaflet primordia. D. PsNAM1/2 expression in a young P. sativum leaf. Polarised PsNAM1/2 expression is observed in the distal part of primordia 1, 2 and 3 (arrows). Note that strong PsNAM1/2 expression is observed in distal part of leaflet primordium 4 that has just started to grow out. Leaflet primordia are numbered from the oldest to the youngest. Note that in S. lycopersicum and S. tuberosum leaflet primordia form basipetally whereas they form acropetally in P. sativum. Bars: 0.1mm SOM Blein et al., FigureS5 B H2O NS TRV2 AcPDS NS S TRV2 AcPDS AcCUC3 NS S TRV2 AcPDS AcNAM AcCUC3 NS S 2x35S:GFP _ TRV2 AcPDS AcNAM NS S 2x35S:MIR164B TRV1 + cDNA ChCUC3 RNAi A DNA H2O AcNAM ChCUC1 AcCUC3 ChCUC2 TRV1 Ch CUC3 TRV2 ChEF1 AcActine C PEBV1 + D PEBV2 PEBV2 PEBV2 PsPDS PEBV2 PsPDS PsPDS PsNAM PsPDS PsNAM PsCUC3 PsCUC3 H2O _ PsNAM TRV1 + TRV2 TRV2 SlPDS SlPDS SlNAM H2O DNA SlNAM PsCUC3 TRV1 PEBV1 TRV2 PEBV2 PsEF1 SlGAPDH Figure S5. Semi-quantitive RT-PCR experiments revealing the reduction of the NAM/CUC genes expression following transient or stable gene silencing. A. Transient TRV-based VIGS in A. caerulea. TRV1 refers to the RNA1 of the TRV virus and TRV2 refers to the RNA2 in which fragments of the AcPDS, AcNAM and/or AcCUC3 genes were inserted. B. Stable silencing in stable C. hirsuta. C. Transient PEBV-based VIGS in P. sativum. PEBV1 refers to the RNA1 of the PEBV virus and PEBV2 refers to the RNA2 in which fragments of the PsPDS, PsNAM and/or PsCUC3 genes were inserted. D. Transient TRV-based VIGS in S. lycopersicum. TRV1 refers to the RNA1 of the TRV virus and TRV2 refers to the RNA2 in which fragments of the SlPDS and SlNAM genes were inserted. H20: control PCR with no template, DNA: control PCR with genomic DNA as template, NS: cDNA extracted from non silenced tissues, S: cDNA extracted from silenced tissues. SOM Blein et al., Figure S6 No VIGS A C AcPDS B AcPDS AcCUC3 * * % of plants A. caerulea * A E D 60% AcPDS 50% AcPDS AcNAM 40% AcPDS AcCUC3 30% AcPDS AcNAM AcCUC3 20% 10% AcPDS AcNAM 0% smoothed simple simple entire leaflets dissected leaf leaf 1 F 2 No VIGS G PsPDS D E H 4 PsPDS PsNAM I J F G 6 5 4 3 20% *** PsPDS PsNAM PsPDS PSCUC3 PsPDS PsNAM PsCUC3 PsPDS PsCUC3 10% PsPDS PsNAM PsCUC3 * * 3.5 3 * * * 2.5 2 1.5 1 0.5 Q SlPDS SlNAM R SlPDS SlNAM S number of leaflets 9 II 8 7 50% 6 5 4 3 2 1 0 I T SlPDS SlPDS SlNAM primary secondary intercalary leaflets 40% 30% 20% 10% 0% *** SlPDS *** P 0 % of leaflet fusion No VIGS 1 *** 0% ** 0 Int PsPDS PsNAM 4 1 O PsPDS N PsPDS PsNAM distance leaflet-tendril 7 *** 8 * 9 30% *** % of leaflet and tendril fusion 10 M PsPDS Distance leaflet-tendril L *** K 2 S. lycopersicum PsPDS PsNAM PsPDS PsNAM PsCUC3 number of leaflets+tendrils P. sativum C 3 SOM Blein et al., Figure S6 Figure S6. Leaf phenotype induced by NAM/CUC gene silencing in compound leaf species. A-E: Silencing of AcNAM and AcCUC3 in Aquilegia caerulea A. Control leaf that was not infiltrated. Note the three leaflets (*) which are highly dissected into three structures that are themselves lobed. The two lateral leaflets have been cut. B, C. Leaves silenced for the AcPDS gene and for both the AcPDS and AcCUC3 genes. Note that the architecture is unchanged compared to a leaf that was not infiltrated. D. Successive leaves formed on a plant silenced for the AcPDS and AcNAM genes. Note the progressive smoothening of the leaflet margins from leaf 1 to 3. At the final stage (leaf 4), simple leaves with highly dissected margins are formed. E. Scoring of the leaf phenotypes in the different conditions. “smoothed margins” refers to leaves such as leaf 3 in panel D, “simple dissected leaves” refers to leaves like leaf 4 in panel D and “simple entire leaf” refers to leaves similar to leaf 4 in panel D but with smoothed margins. F-N: Silencing of PsNAM/PsCUC genes in Pisum sativum F. Control leaf that was not infiltrated. G. Leaf silenced for the PsPDS gene. The overall architecture of the leaf is unchanged although the leaf is smaller compared to a leaf that was not infiltrated. H. Leaf silenced for the PsPDS PsNAM genes. The pairs of leaflets are not properly spaced along the rachis and leaflets are fused to the rachis (arrows). I. Leaf silenced for the PsPDS, PsNAM and PsCUC3 genes. Note the fusion of the tendrils (arrows) detailed in the inset (Bar=1mm, for the detail). J. Leaves silenced for the PsPDS PsNAM genes with no leaflet that terminates with a pair of tendrils and a terminal tendril. K. Quantification of the leaflet and tendril number. L. Quantification of leaflet and tendril fusion. M. Leaf silenced for the PsPDS PsNAM genes. The reduction of the leaflet and tendril number is reflected in a longer distance between the last pair of leaflets and the first couple of tendrils. N. Quantification of the distance between the last pair of leaflets and the first couple of tendrils. n=64 for PsPDS, n=14 for PsPDS PsNAM, n=3 for PsPDS PsCUC3, n=20 for PsPDS PsNAM PsCUC3 leaves (rank 17-19) of the main stem were analysed in K, L and N O-T: Silencing of SlNAM in Solanum lycopersicum O. Control leaf that was not infiltrated. “I” and “II” point respectively to a primary and secondary leaflets, “int” points to an intercalary leaflet. P. Leaf silenced for the SlPDS gene. Note that although the size of the leaf is reduced, the overall architecture is unchanged compared to a leaf that was not infiltrated. Q,R. Leaves silenced for the SlPDS SlNAM. Note the absence of secondary leaflets and the reduction of the intercalary leaflet number. The leaflet margins are smoothed (arrowheads) and fusion between leaflets or with the rachis occur (arrow). S. Quantification of leaflet number. T. Quantification of leaflet fusion. n=33 for SlPDS, n=63 for SlPDS SlNAM (rank 5-7) of the main stem were analysed in K, L and N Error bars=SE. Significance of the differences with the control values (PDS control) were assessed by a Student’s t test (*=P<0.05;**=P<0.01; ***=P<0.001) Bars=1cm when another value is not specified. SOM Blein et al., FigureS7 gob2 e3883-m1 G378A G95D A 95 272 273 gob1 e1976-m1 C593T Q167Stop gob3 n5126-m1 28 nt deleted (1001-1030) 556 557 1153 Intron 1, 144nt 100nt Intron 2, 782nt B D C gob3 WT gob3 E WT gob3 G F gob3 H gob3 WT I J WT WT gob3 gob3 gob3 gob3 WT gob3 Figure S7. Molecular and Phenotypic characterisation of the tomato goblet mutants A. LeNAM structure and mutations. The cDNA, the ORF (thick lines) and the introns are shown. The effects of the 3 goblet mutations and the corresponding lines are represented (the modification of the nucleotide and of the amino acid are indicated) B, C. Wild-type and gob3 seedlings showing the fused cotyledons and the absence of leaf development in the gob3 line. D, E. Longitudinal sections through a wild-type and a gob3 seedling revealing the absence of meristem in the gob3 line. F. Regenerated gob3 plant G. Leaves from regenerated wild-type and gob3 plants showing the simplified leaf structure and the smoothed margins of the gob3 line H. Inflorescences from regenerated wild-type and gob3 showing the modified structure of the gob3 plant I. Flowers from regenerated wild-type and gob3 showing the increased sepal fusion of the gob3 plant J. Fruits of a wild-type and a regenerated gob3 plant. Note the outer floral organs fused to the fruit (arrowheads) and the absence of seeds in the gob3 plant Bars=1cm SOM Blein et al., FigureS8 A Wild type 2x35S:MIR164B R4 R8 R4 Wild type C1 2x35S:MIR164B C2 C1 R8 ChCUC3 RNAi C2 C1 C2 2x35S:MIR164B 6 ChCUC3 RNAi 2x35S:MIR164B ChCUC3 RNAi 4 3 2 *** 50% 40% 30% 20% 0% R1 R2 R3 R4 R5 R6 leaf rank R7 R8 R9 R10 10 Rosette leaves 1-10 60% 7 WT 6 2x35S:GFP 5 2x35S:MIR164B 4 3 ChCUC3 RNAi 2 2x35S:MIR164B ChCUC3 RNAi 1 0 C1 C2 leaf rank C3 50% 40% *** 8 *** 9 % of leaflet fusion number of leaflets 60% 10% 1 D C2 70% 7 5 C1 80% 2x35S:GFP 8 R8 2x35S:MIR164B ChCUC3 RNAi WT 9 0 R4 *** 10 R4 30% 20% *** number of leaflets C R8 2x35S:MIR164B ChCUC3 RNAi % of leaflet fusion B ChCUC3 RNAi 10% 0% Cauline leaves 1-3 Figure S8. Leaf phenotype induced by ChCUC1, ChCUC2 and ChCUC3 silencing in Cardamine hirsuta A. Rosette leaf 4 (R4) and 8 (R8). Arrows point to examples of fusion between leaflets or between a leaflet and the rachis. B. First (C1) and second (C2) cauline leaf. Arrowheads indicate smoothed leaflet margins. Arrows point to examples of fusion between leaflets or between a leaflet and the rachis. C. Quantification of the rosette leaf parameters. D. Quantification of the cauline leaf parameters 10 plants analysed. Bars=SE. Student’s t test ***= P<0.001 Bars=1cm SOM Blein et al., A la B FigureS9 SlNAM C uni D PsNAM1/2 E PsCUC3 Figure S9. The NAM/CUC genes act downstream of genes required for leaflet formation in S. lycopersicum and P. sativum. The la mutant forms a simple leaf, which shows some lobes but no leaflets (A). No expression of SlNAM is detected in a young developing leaf of the la mutant (B) in contrast to wild type. SlNAM is still expressed in the meristem (arrow). The uni pea mutant forms a simple leaf (C). The expression of the PsNAM1/2 and PsCUC3 genes is not detected in the developing uni leaf (D,E) in contrast to wild type. PsNAM1/2 and PsCUC3 expression is still observed in the meristem and in the leaf axils (arrows). SOM Blein et al., FigureS10 A B Distal Cell proliferation Leaf axis NAM CUC ltp ltp ltp Leaflet formation Proximal KNOX1 LFY-like Leaf Margin Figure S10. Role for the NAM/CUC genes in leaf dissection and promotion of leaflet formation A. View of a tomato apex showing the developing leaflet primordia (ltp). B. Schematic representation of the role of the NAM/CUC genes during the formation of the leaflet highlighted in red in A. The NAM/CUC genes are expressed in discrete strips along the margin of the leaf primordium (shown here in yellow). This leads in local repression of cell proliferation, and thus to the formation of a groove. At the same time, the NAM/CUC genes promote cell proliferation in the cells proximal to their expression domain, thus promoting leaflet formation. This long distance effect of the NAM/CUC genes may rely on movement of the NAM/CUC proteins themselves, or on the production of a mobile signal. NAM/CUC genes are part of a feed-forward regulatory loop with KNOX1 and LFY-like genes, activating their expression and being activated by them. Supplementary Table 1. Quantification of VIGS effects in Aquilegia caerulea survivorsa VIGS positiveb leaf phenotyped nb of infiltrated plants nb of plants % nb of plants %c nb of plants %e TRV1 + TRV2 AcPDS 96 88 92% 46 52% 0 0% TRV1 + TRV2 AcPDS AcNAM 107 97 91% 58 60% 33 57% TRV1 + TRV2 AcPDS AcCUC3 108 106 98% 65 61% 0 0% TRV1 + TRV2 AcPDS AcNAM AcCUC3 131 121 92% 49 40% 27 55% Constructs a determined 4 weeks after inoculation determined 4 weeks after inoculation as plants with white sectors c of survivors d determined 8 weeks after inoculation as plants that show reduced leaf complexity e of plants showing sectors b Supplementary Table 2. Quantification of VIGS effects in Solanum lycopersicum survivorsa VIGS positiveb leaf phenotyped nb of infiltrated plants nb of plants % nb of plants %c nb of plants %e TRV1 + TRV2 SlPDS 38 37 97% 37 100% 0 0% TRV1 + TRV2 SlPDS SlNAM 50 49 98% 49 100% 38 78% Constructs a determined 4 weeks after inoculation determined 4 weeks after inoculation as plants with white sectors c of survivors d determined 8 weeks after inoculation as plants that show reduced leaf complexity e of plants showing sectors b Supplementary Table 3. Quantification of VIGS effects in Pisum sativum survivorsa VIGS positiveb leaf phenotyped nb of infiltrated plants nb of plants % nb of plants %c nb of plants %e PEBV1 + PEBV2 PsPDS 93 93 100% 79 85% 0 0% PEBV1 + PEBV2 PsPDS PsNAM 95 94 99% 81 86% 45 56% PEBV1 + PEBV2 PsPDS PsCUC3 95 95 100% 80 84% 4 5% PEBV1 + PEBV2 PsPDS PsNAM PsCUC3 92 92 100% 77 84% 17 22% constructs a determined 6 weeks after inoculation determined 6 weeks after inoculation as plants with white sectors c of survivors d determined 6 weeks after inoculation as plants that show reduced leaf complexity e of plants showing sectors b The experiments were repeated twice and the numbers indicated in the tables are the sum of the independant experiments. Supplementary Table 4. Primers used for semi quantitive RT-PCR primer 1 name primer 1 sequence (5'->3') primer 2 name primer 2 sequence (5'->3') Reference Aquilegia caerulea species AcActine gene AcActine-1 GAT GGA TCC TCC AAT CCA GAC ACT GTA AcActine-2 GTA TTG TGT TGG ACT CTG GTG ATG GTG T Kramer et al. (2007) Plant Cell, 19, 750-766 Aquilegia caerulea AcCUC3 Ac CUC3 Fw TCC ATC ACA TAG CTT CTT CTT Ac CUC3 Rv AGC ATC CTG AGG TCC ATA TTG This work Aquilegia caerulea AcNAM1 Ac NAM1 Fw TCA ATG GAG ATC ATT AGT TTT Ac NAM1 Rv TAC CCT TCA CAT AGC TAG CAA This work Cardamine hirsuta ChCUC1 Ch CUC1 Fw3 CAA CAG AAG CTG GTT ACT GGA AA Ch CUC1 Stop TCA GAG AGC AAA CGG CCA GTA ACT This work Cardamine hirsuta ChCUC2 Ch CUC2 Start ATG GAC ATT CCA TAC TAC CAC TA Ch CUC2 Rv1 TAG AGA TCA CCC ATT CAT CCT T This work Cardamine hirsuta ChCUC3 Ch CUC3 Start ATG ATG CTT GCG GTA GAA GAT GT Ch CUC3 Rv2 CCA AAG ACC TCC TTA TCT TTT This work Cardamine hirsuta EF1 EF1af ATG CCC CAG GAC ATC GTG ATT TCA T EF1ar TTG GCG GCA CCC TTA GCT GGA TCA This work Pea Early Browning Virus PEBV1 PEBV1-Rv GAA AGA ACC CAA TCA AT PEBV1-Fw TGG CAT AAA TAT AAT ACT TGT This work Pea Early Browning Virus PEBV2 PEBV2-Rv ATT TTG GGC ACA TAT CAA AC PEBV2-Fw GCT GCG GTT AGA AAT AAG AA This work PsCUC3-Start ATG ATG TTA GCA ATG GAA GA PsCUC3-Stop TTA AAC AAG CTG AGC CTG T This work AGG GAG ACA ACA TGA TTG AG RTPsEF1aRv TAA CCA TTT CCA ATC TGT CC This work Pisum sativum PsCUC3 Pisum sativum PsEF1 Pisum sativum PsNAM1/2 PsNAM-Start ATG GAG GGC TTC TAC CA PsNAM2-Stop TTA ATA ATT CCA CAT GCA AT This work Pisum sativum UNI RTPsUNIFwd GGT ATG CAA AGA AAG CTG RTPsUNIRv ATT GGT GGA AGG GAG GTT TT Bai and DeMason (2006) Plant Cell Physiol, 47, 935-48 Lae/e R TTC AAG TAG AGC ATT TCC CTG TCA LaCS L GGG ACC CCT TCA GTC CAG TA Ori et al. (2007) Nat. Genet. 39, 787-91 RTPsEF1aFwd Solanum lycopersicum LA Solanum lycopersicum SlGAPBH SlGAPDH-F TGG AAT CAG GAA CCC TGA AG SlGAPDH-R GAT CGA CAA CGG AGA CAT CA Jasinski et al. (2007) Planta, 226, 1255-63 Solanum lycopersicum SlLFY SlLFY-Fw1 TGG AGG AAT AAG CGA GAG A SlLFY-Rv1 GGA CAT ACC AAA TGG CTA GTC This work Solanum lycopersicum SlNAM Sl NAM5' ATG GAG ATT TAT CAT CAG ATG CA SlNAM3' TCA GTA GCT CCA CAT ACA GTC AA This work Solanum lycopersicum SlPHAN SlPHAN-F GCT GAA GAG GAT GCT TTG SlPHAN-R TGC TCG TCT TCA GTG AGT GAT C Jasinski et al. (2007) Planta, 226, 1255-63 Solanum lycopersicum SlT6/TKn2 SlT6/Tkn2-F TAA GCA GGA GTT CAT GAA GAA G SlT6/Tkn2-R TAC ATG CAC ACA AGT AAT ATG C Jasinski et al. (2007) Planta, 226, 1255-63 Solanum lycopersicum TKn1 Tkn1-F AGA GAT CTC GTT GAG TAG TTG Tkn1-R GTT AGA ACA TTG AGG ATG AGC Jasinski et al. (2007) Planta, 226, 1255-63 Tobacco Rattle Virus TRV1 TRV1-1 CTT GAA GAA GAA GAC TTT CGA AGT CTC TRV1-2 GTA AAA TCA TTG ATA ACA ACA CAG ACA AAC Hileman et al. (2005) Plant J, 44, 334-341 Tobacco Rattle Virus TRV2 TRV2-1 GGT CAA GGT ACG TAG TAG AG TRV2-2 CGA GAA TGT CAA TCT CGT AGG Hileman et al. (2005) Plant J, 44, 334-341 Supplementary Table 5. Primers used for the isolation of the NAM/CUC genes primer name primer sequence (5'->3') AcCUC3Fw1 TCC ATC ACA TAG CTT CTT CTT AcCUC3Rv1 AGC ATC CTG AGG TCC ATA TTG AcNAMFw1 TCA ATG GAG ATC ATT AGT TTT AcNAMRv1 TAC CCT TCA CAT AGC TAG CAA CUC2 3' TCA GTA GTT CCA AAT ACA GTC cuc3uj2 AGC TTA TCA CTT TTT ACT TAG CCT C degCUC3Fw1 GGT TYC AYC CNA CTG AYG ARG AGC T degCUC3Fw2 GAT CTY AAY MGN TGT GAR CCN TGG GA degCUC3Rv1 CTG CAN ATN ACC CAT TCY TCC TT degCUC3Rv2 TCR AGN CGR TAY TCR TGC AKR ACC CA insitu CUC3 L Fwd ATG ATG CTT GCG GTG GAA GA insitu CUC3 L Rv+T7 TGT AAT ACG ACT CAC TAT AGG GCC TGA TAC TAT TTC GCA AGA C miR164bsdegRv1 TGG AGA ARC AGG RCA CGW GCT C NACdegFw1 TYG AYC TSA ACA AGT GYG AGC CWT GG NACdegFw2 ATG GGR GAG AAR GAG TGG TAC TTY T NACdegRv1 TCR AGN CGR TAY TCR TCC ATR ACC CA NACdegRv2 TAR AAR ACD AGN GTY TTC TTC AT nam2 TAA CTC CAC ATG CAA TCA AGT TCA G nam5 GAG CAC GTG TCC TGT TTC TCC A revcuc3uj3 CGG TAA GAG AAA TCA CCG TCA AGA CG RLT-Fwd-AT5G53950 CAG CCG TAG CAC CAA CAC AA SlNAM3' TCA GTA GCT CCA CAT ACA GTC AA SlNAM5' ATG GAG ATT TAT CAT CAG ATG CA Supplementary Table 6. Probes used for in situ hybridisation gene length Aquilegia caerulea species AcNAM 1141 position primer 1 sequence (5'->3') 1-1140 AcNAMFw1 TCA ATG GAG ATC ATT AGT TTT primer 2 sequence (5'->3') in situ Ac NAM1 T7 TGT AAT ACG ACT CAC TAT AGG GCT ACC CTT CAC ATA GCT AGC AA Aquilegia caerulea AcCUC3 1131 1-1130 AcCUC3Fw1 TCC ATC ACA TAG CTT CTT CTT insitu CUC3 L Rv+T7 TGT AAT ACG ACT CAC TAT AGG GCC TGA TAC TAT TTC GCA AGA C Cardamine hirsuta ChCUC1 844 343-1186 ChCUC1fd2 CCC AAC GGG ACT GAG AAC GAA CA insitu ChCUC1 Rv T7 TGT AAT ACG ACT CAC TAT AGG GCG AAG GAT AGA AGT CAA TAT TTT AAA A Cardamine hirsuta ChCUC2 1156 104-1259 insituCUC2LFwd ATG GAC ATT CCG TAT TAC CA insitu ChCUC2 Rv T7 TGT AAT ACG ACT CAC TAT AGG GCA ACA AAC CCT AAC GGG ACA CAC Cardamine hirsuta ChCUC3 1008 75-1082 insituCUC3LFwd ATG ATG CTT GCG GTG GAA GA insitu CUC3 L Rv T7 TGT AAT ACG ACT CAC TAT AGG GCC TAC AGC TGG AAT CCT AAA Pisum sativum PsCUC3 1242 1-1241 PsCUC3Fd3 CAT TTT CAT TCA TCA TCA TCT A T7-PsCUC3Rv3 TGT AAT ACG ACT CAC TAT AGG GCT ATT CTA CAC ATG TAG AGT TA Pisum sativum PsNAM1/2 1180 15-1194 PsNAM5'UTR1 TAA CGG CTC TAC TCT CTC TCT CAT TT T7PsNAM1Rv TGT AAT ACG ACT CAC TAT AGG GCT TCC ACA TGC AAT CAA GCT Solanum lycopersicum SlNAM 1208 95-1302 SlNAM5' ATG GAG ATT TAT CAT CAG ATG CA insitucucrev4 TGT AAT ACG ACT CAC TAT AGG GCA GCA TGC ATT AAA TTA CGC TGA ACG Solanum tuberosum StCUC3 1166 8-1173 StCUC35' CAT ATC TTG AAA ATC TCT CAC TTT T in situ St CUC3 T7 TGT AAT ACG ACT CAC TAT AGG GCG GCT AAA GGG TAG CTC TTT ATA TT Solanum tuberosum StNAM 1063 1-1062 SlNAM5' ATG GAG ATT TAT CAT CAG ATG CA insitucucrev4 TGT AAT ACG ACT CAC TAT AGG GCA GCA TGC ATT AAA TTA CGC TGA ACG For each probe, its length and position are inidicated as well as the primers used to amplify the DNA template used for the in vitro transcription. The sequence of the T7 promoter is underlined Supplementary Table 7. Sequence used for the transient and stable NAM/CUC gene silencing gene length position primer 1 sequence (5'->3') primer 2 sequence (5'->3') Aquilegia caerulea species AcCUC3 587 579-1165 AcCUC3BamHI GGA TCC ACT ATT CCA TGC CCA CAA C AcCUC3SacI GAG CTC AGC ATC CTG AGG TCC ATA TTG Aquilegia caerulea AcNAM1 598 543-1140 AcNAM1SacI GAG CTC TAG TGG TAC AGG GAG TAA AcNAM1XhoI CTC GAG TAC CCT TCA CAT AGC TAG CAA Cardamine hirsuta ChCUC3 469 616-1084 ChCUC3XbaI TCT AGA TAG CCA AAT CAG CTG CCT C ChCUC3HindIII AAG CTT GCC TAC AGC TGG AAT CCT AAA Cardamine hirsuta ChCUC3 469 616-1084 ChCUC3EcoRI GAA TTC ATA GCC AAA TCA GCT GCC TC ChCUC3KpnI GGT ACC TAC AGC TGG AAT CCT AAA Pisum sativum PsCUC3 508 688-1195 PsCUC3BamHI GGA TCC CCC CAA AAA GCT GTC TAC PsCUC3SacI GAG CTC CAA GAA TCA ATT CCC ATA GGA GC Pisum sativum PsNAM1/2 525 613-1137 PsNAM1SacI GAG CTC ACC GCA AGT GGT TCA AAA AAG PsNAM1XhoI CTC GAG TCA ACC ACC CCT CTT TGC TC SlNAM 511 752-1262 SlNAMSacI GAG CTC CGC CGC AGC TAT CGT AAT C SlNAMXhoI CTC GAG CAA TAC CAC TAA AGC TGC CAC ACG Solanum lycopersicum For each gene, the length and position of the fragment used to induce the silencing are indicated as well as the primers used to amplify it. The sequence of the restrictions sites are underlined