Supporting Online Material for

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1 Supporting Online Material for Plant Peptides Govern Terminal Differentiation of Bacteria in Symbiosis Willem Van de Velde, Grigor Zehirov, Agnes Szatmari, Monika Debreczeny, Hironobu Ishihara, Zoltan Kevei, Attila Farkas, Kata Mikulass, Andrea Nagy, Hilda Tiricz, Beatrice Satiat-Jeunemaître, Benoit Alunni, Mickael Bourge, Ken-ichi Kucho, Mikiko Abe, Attila Kereszt, Gergely Maroti, Toshiki Uchiumi, Eva Kondorosi,* Peter Mergaert *To whom correspondence should be addressed. This PDF file includes: Materials and Methods Figs. S1 to S4 Table S1 References Published 26 February 2010, Science 327, 1122 (2010) DOI: /science

2 Supporting Online Material Material and Methods Plant and bacterial growth WT and dnf1-1 M. truncatula Jemalong and L. japonicus Myakojima MG20 were grown and inoculated for nodulation with S. meliloti strains 1021 and Mesorhizobium loti MAFF303099, respectively, as described (S1). Constructs were introduced into Agrobacterium rhizogenes strains ARqua1 and A4TC24 for M. truncatula and L. japonicus transformation (S2), respectively. Molecular methods The promoter of NCR035 (a 1kb fragment upstream of the ATG) was obtained by an Amplified Fragment-Length Polymorphism (AFLP) based PCR protocol as described (S3) and recombined in the Gateway vector pdonrp4-p1r according to the manufacturer s instructions (Invitrogen). Open reading frames (ORF) of NCR001, NCR007, NCR035, NCR053, NCR084, NCR121, NCR122, and NCR274 (S4) were amplified from nodule cdna and recombined in the Gateway vector pdonr221 according to (S5). Entry clones for the MtLb1 promoter (2.1 kb upstream of ATG) (S6), the GUS and mcherry ORFs and the 35S terminator were obtained in the Gateway vectors pdonrp4-p1r, pdonr221 and pdonrp2r-p3, respectively. For NCR expression and pmtlb1::gus promoter analysis in L. japonicus, entry clones were recombined in the binary vector pkm43gw (S7). For mcherry fusions, the binary vector pk7m34gw (S7) was modified to facilitate the transformation procedure by inserting a rold promoter driven egfp-er reporter cassette. 1

3 Entry clones were recombined in the resulting vector. Sequences of used primers will be provided upon request. Gene expression analysis by RT-PCR was done as described (S4). Primer sequences can be obtained upon request. Antibodies The mature regions of NCR001 and NCR084 were amplified from nodule cdna and cloned into the expression vector pbadgiii/a (Invitrogen). Recombinant proteins were purified according to the manufacurer s instructions and used for immunization of rabbits by a commercial service (Agro-bio) resulting in antibody ab1. A second rabbit anti-ncr001 antibody (ab2) was generated against the synthetic peptide AFERTETRMLTIPC (Genscript). Antibodies were purified on activated thiol Sepharose 4B (Pharmacia) covalently bound with the corresponding peptide. The ER-specific KDEL antibody (MAC 256) was obtained from Abcam. Protein analysis Crude protein extracts of roots, nodules, bacteroids (S5) and cultered S. meliloti were prepared by extraction with 8M urea, 100 mm NaH 2 PO 4, 10 mm Tris Cl ph 7.0 in a 3:1 (v/v) buffer:sample ratio. Protein samples were separated by SDS-PAGE in a 20% acrylamide gel as described (S8). For Western blot analysis, proteins were blotted on Optitran BA-S 85 reinforced nitrocellulose 0.45 µm (Gentaur). Pre-immune and polyclonal antibodies were used with 1:2,000 dilutions and secondary anti-rabbit IgG coupled to horseradish peroxidase (Calbiochem) in 1:10,000 dilution for detection. Proteins in the range of ~3-5 kda (indicated with brackets in Fig. 1A) from a bacteroid extract were cut out from SDS-PAGE gels, destained, trypsin-digested (S9, S10) and analyzed 2

4 by liquid chromatography-mass spectrometry with a Q-TOF machine (Waters). Data were processed with Mascot Distiller (v ) software (Matrix Science). For protein identification, the NCBI database was searched using the ProteinProspector (v5.3.1) package (UCSF). Histological analysis and light microscopy Sections (7 µm) of Technovit (Heraeus Kulzer) embedded WT and dnf1-1 nodules were stained with toluidene blue as described (S11) and observed with a Leica DMI 6000B inverted microscope. Bacteroids were isolated and observed as described (S1). Histochemical GUS staining in transgenic L. japonicus nodules was done as described (S12) with the following modifications: no pre-fixation was done and the buffer contained 1 mm final concentration of potassium ferrocyanide and potassium ferricyanide. After staining, nodules were immediately embedded in 6% agarose, sectioned (70 µm) with a Leica VT1200S vibratome and mounted in deionised water for imaging. Imaging was done with a Leica DMI 6000B inverted microscope. (Immuno)fluorescence localisation and confocal microscopy For immunolocalisations, harvested nodules were fixed in 3% paraformaldehyde in MTSB buffer (S13) for 40 min under vacuum. After washing in MTSB buffer, nodules were embedded in 6% agarose in water and cut in longitudinal 70 µm sections using a Leica VT1200S vibratome. These sections were then incubated for 15 min in 0.1 M glycine, 0.1 M ammonium chloride in MTSB and washed 5 times with MTSB. For the subsequent steps the InsituPro robot (Intavis Ag) was used according to the following protocol (all steps at room temperature): washing with MTSB/0.1% triton (6 x 10 min); pre-incubation in 3% BSA in MTSB/0.1% triton (1h); incubation with affinity purified primary antibody (1:200 dilution) in 3

5 3% BSA in MTSB/0.1% triton (6h); washing with MTSB/0.1% triton (8 x 10 min); incubation with secondary antibody anti-rabbit IgG-Alexa fluor 633 or anti-rat IgG-Alexa 488 (Invitrogen) (1:500) in 3% BSA in MTSB/0.1% triton (3h); washing with MTSB/0.1% triton (8 x 12 min); washing with MTSB (5 x 10 min). For the SYTO13 nucleic acid staining, nodules sections were incubated for 5 minutes with 1 µm SYTO13 in H 2 O. Sections were mounted in deionised water for confocal imaging. For localisation of the mcherry translational fusions, 70 µm longitudinal sections of transgenic nodules embedded in 6% agarose in water were cut with a Leica VT1200S vibratome and mounted in deionised water for confocal imaging. Fluorescence images were acquired at 1024x1024 pixels resolution with the confocal laser scanning microscope TCS SP2 from Leica, using the 10X water-immersion and 63X oilimmersion objectives and Leica software. Images were processed with Adobe Photoshop for adjustment of contrast and brightness. Electron microscopy Ultrastructural observations and immunogold labelling were done as described (S14). Handcut nodules were fixed in 3% paraformaldehyde/0.5% glutaraldehyde, embedded in LR white medium grade resin (Agar Scientific). Immuno-staining was done with NCR001 primary antibody (1:200) and anti-rabbit IgG 15 nm gold secondary antibody (1:50) (Agar Scientific). Embedding was done with the Leica EM AFS2, ultrathin sections were cut with a Leica UC6 ultramicrotome, and observed with a JEOL 1400 transmission electron microscope at 120kV. For scanning electron microscopy, hand-cut nodules were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer ph 7.2 for 4 hours. After rinsing with 0.05M cacodylate buffer (2 x 15 min) and distilled water (2 x 5 min) the samples were dehydrated as followed: 30% ethanol (30 min), 50% ethanol (30 min), 70% ethanol (30 min), 80% ethanol (30 min), 90% 4

6 ethanol (20 min), 95% ethanol (20 min) and 100% ethanol (2 x 30 min). The ethanol was replaced with t-butanol in four steps (EtOH: t-buoh, ratio 1:1 (1h); EtOH: t-buoh, 1:2 (1h); t-buoh (2 x 1h). Samples were then freeze-dried in a t.bufreeze Dryer VFD-21 (Vacuum Device Inc.), fixed on an aluminum stub and coated with gold\palladium on an ion Coater IB- 2 (Eiko Engineering Co.) before imaging on a Hitatchi S-4100H scanning electron microscope. In vitro NCR peptide activities Synthetic NCR peptides were purchased from Innovagen and Proteogenix. Treatments were done on S. meliloti 1021 cultures grown to OD 600 of 0.3, washed in 10 mm sodium phosphate buffer ph 7.0 and diluted to OD in the same buffer. 200 µl bacteria were treated with peptides at the indicated concentrations and incubated for 1-3h at 30 C. Membrane integrity was tested by addition of 10 µg/ml PI while the metabolic activity by CTC staining at 4 mm concentration. Fluorescence was measured in a Tecan InfiniteM200 plate reader. Data handling (normalization and statistics) was performed using Excel. Each measurement was done in triplicate (biological repeats). In the cell kill assays, bacterium suspensions with or without peptide treatment were diluted and plated out in triplicate on selective medium. Colonies were counted after 2 days of incubation at 30 C. DNA measurements on PI stained, peptide treated and control suspensions were performed with a PARTEC cyflow cytometer. For microscopy observation of NCR treated bacteria, S. meliloti cells were diluted until OD in minimal medium (10 mm Na phosphate ph 7.0; 6.25 mm NH 4 NO 3 ; 12 mm Na succinate; 1X Gamborg s vitamin solution (Sigma); trace elements: 50 µm H 3 BO 3, 0.5 µm CuSO 4, 0.5 µm Na 2 MoO 4, 0.7 µm CoCl 2, 1 µm ZnSO 4, 13 µm MnSO 4 ) supplemented with 1.25 µm NCR035, NCR055, NCR057, NCR084, NCR224, NCR247 and incubated for 24 hours at 30 C. After incubation, PI was added to a final concentration of 10 µg/ml, cells were 5

7 appropriately diluted and visualised with a Leica DMI 6000B inverted fluorescence microscope. For FITC-NCR035 localisation, log phase S. meliloti cultures supplemented with 46 nm FITC-NCR035 were fixed on a polylysine-coated slide and FITC fluorescence images were taken with a confocal laser scanning microscope TCS SP2 from Leica or a Zeiss AxioObserver Z1 epifluorescence microscope. 6

8 Fig. S1. Controls for immunogold localisation of NCR001. (A-D) Immunogold localization of NCR001 on ultrathin M. truncatula nodule sections was carried out with two independent antibodies, ab1 (A and C) and ab2 (B and D) (see Materials and Methods). Images are taken from the nitrogen fixing (A and B) and senescencing (C and D) nodule cells. (E, F) Control sections treated with pre-immune serum (E) or without primary antibody (F). Gold particles are seen as black dots in (A-D) but are absent in the controls (E) and (F). b, bacteroid. Scale bars: 1 µm. 7

9 Fig. S2. Nodule and bacteroid differentiation and NCR expression in the dnf1-1 M. truncatula signal peptidase complex mutant. (A and B) Toluidine blue-stained longitudinal WT (A) and dnf1-1 (B) nodule sections at 15dpi and 25dpi, respectively show the normal nodule structure with the typical nodule zones in both nodules (I, meristem; II, infection zone; II-III, interzone; III, fixation zone). (C and D) Bacteroid containing symbiotic cells from WT (C) or dnf1-1 (D) nodules. (E and F) Magnification of (C) and (D), respectively. Bacteroids (arrows) are elongated in the WT symbiotic cells (E) but small in the mutant (F). (G and H) Heat-killed and PI stained bacteroids, isolated from WT (A) and dnf1-1 (B) nodules show that bacteroids are not elongated and thus not terminally differentiated in the dnf1-1 nodules. (I) RT-PCR expression analysis of NCR001, NCR084, NCR035 and the constitutive histone H3-like gene in WT roots and 25 dpi WT and dnf1-1 nodules. (J) SDS-PAGE and coomassie blue staining of nodule and bacteroid protein extracts from WT and dnf1-1 plants. (K) Western blot analysis of the same extracts with anti-ncr001 antibody. * indicates migration of NCR001, ** indicates migration of non-processed NCR001. Scale bars: 100 µm (A and B); 10 µm (G- H). 8

10 Fig. S3. Heterologous expression of NCR035 from the M. truncatula leghemoglobin 1 promoter in transgenic L. japonicus nodules. (A and B) Promoter activity of the MtLb1 promoter in L. japonicus nodules. (A) MtLb1 promoter driven GUS activity is confined to the nitrogen fixing symbiotic cells in a longitudinal section of a L. japonicus nodule transformed with pmtlb1::gus. (B) Magnification of a symbiotic cell from (A). (C-E) Targetting of NCRs to symbiosomes in transgenic L. japonicus nodules. (C) A longitudinal section of a L. japonicus nodule transformed with pmtlb1::ncr035-mcherry shows fluorescent signal in the symbiotic cells. (D) Magnification of a symbiotic cell from (C) and (E) a region of a symbiotic cell from (D) localises the fluorescent signal to the symbiosomes (asterisks), in a similar way as the localization of the same fusion protein in M. truncatula nodules. Scale bars: 100 µm (A and C); 10 µm (B and D); 2 µm (E). 9

11 Fig. S4. In vitro activities of NCR peptides. (A) Respiration activity of S. meliloti cells measured by CTC fluorescence after treatment with NCR035 and the peptide antibiotic polymyxin B, used as a control. (B) Ca 2+ inhibition of NCR035 activity: left panel, membrane permeabilisation activity measured by PI uptake and right panel, inhibition of bacterial reproduction detected by a plating assay. (C) Membrane permeabilisation activity of FITC- NCR035 and NCR035 measured by PI uptake. Data are means ±SD (N=3). 10

12 1: NCR169 (TC41305) MGEMFKFIYTFILFVHLFLVVIFEDIGHIKYCGIVDDCYKSKKPLFKIWKCVENVCVLWYK 2: NCR138 (BG583579) MAKFSMFVYALINFLSLFLVETAITNIRCVSDDDCPKVIKPLVMKCIGNYCYFFMIYEGP 3: NCR053 (TC31903) MTHISKFVFALIIFLSIYVGVNDCKRIPCKDNNDCNNNWQLLACRFEREVPRCINSICKCMPM 4: NCR068 (TC36358) MAQILMFVYFLIIFLSLFLVESIKIFTEHRCRTDADCPAREL>PPEYLKCQ>YGGMCRLLIKKD 5: NCR136 (TC41544) MAHKFVYAIILFIFLFLVAKNVKGYVVCRTV>EDDCPPDTRDLRYRCLNGKCKSYRLSYG 6: SMc00870 (CAC ) AtpE Probable ATP synthase subunit C transmembrane protein MEAEAAKYIGAGLACLGMAGTALGLGNIFGSYLSGALRNPSAADGQFGRLVFGFAVTEALGIFSLLIALLLLFAV Table S1. Identification of small proteins from purified bacteroids by mass spectrometry. Eight peptides determined by mass spectrometry were identified by ProteinProspector. They corresponded to five different plant-encoded NCR peptides and one S. meliloti small protein. The accession number and sequence of the identified small proteins are shown and the peptides identified by mass spectrometry are indicated in red. Two independent peptides were identified for NCR169 as well as for NCR068 (underlined with a dashed and full line, respectively). The amino acids indicated in blue in NCR068 and NCR136 represent changes between the identified peptides and the sequence of the database accessions that likely result from variability among the Medicago genotypes. The presence of a S. meliloti protein is in agreement with the bacteroid origin of the sample. 11

13 References to Supplementary Materials S1. P. Mergaert et al., Proc. Natl. Acad. Sci. U. S. A. 103, 5230 (2006). S2. A. l. Boisson-Dernier et al., Mol. Plant-Microbe Interact. 14, 695 (2001). S3. P. Ratet, A. Porcedu, M. Tadege, K. S. Mysore, in The Medicago truncatula Handbook, U. Mathesius, E. P. Journet, L. W. Sumner, Eds. (2006). S4. P. Mergaert et al., Plant Physiol. 132, 161 (2003). S5. B. A. Underwood, R. Vanderhaeghen, R. Whitford, C. D. Town, P. Hilson, Plant Biotechnol. J. 4, 317 (2006). S6. P. Gallusci, A. Dedieu, E. P. Journet, T. Huguet, D. G. Barker, Plant Mol. Biol. 17, 335 (1991). S7. M. Karimi, B. De Meyer, P. Hilson, Trends Plant Sci. 10, 103 (2005). S8. H. Schagger, Nat. Protocols 1, 16 (2006). S9. J. Rosenfeld, J. Capdevielle, J. C. Guillemot, P. Ferrara, Anal. Biochem. 203, 173 (1992). S10. U. Hellman, C. Wernstedt, J. Gonez, C. H. Heldin, Anal. Biochem. 224, 451 (1995). S11. W. Van de Velde et al., Plant Physiol. 141, 711 (2006). S12. A. Sessions, D. Weigel, M. F. Yanofsky, Plant J. 20, 259 (1999). S13. J. Friml, E. Benkova, U. Mayer, K. Palme, G. Muster, Plant J. 34, 115 (2003). S14. C. Hawes, B. Satiat-Jeunemaitre, in Plant Cell Biology, C. Hawes, B. Satiat- Jeunemaitre, Eds. (Oxford University Press, 2001), pp