Supplemental Data. Polymorphic Members of the lag Gene. Family Mediate Kin Discrimination. in Dictyostelium. Current Biology, Volume 19

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1 Supplemental Data Polymorphic Members of the lag Gene Family Mediate Kin Discrimination in Dictyostelium Rocio Benabentos, Shigenori Hirose, Richard Sucgang, Tomaz Curk, Mariko Katoh, Elizabeth A. Ostrowski, Joan E. Strassmann, David C. Queller, Blaz Zupan, Gad Shaulsky, and Adam Kuspa Supplemental Experimental Procedures Strains, cell growth and development Cells were grown in shaking suspension in HL-5 nutrient broth and harvested for experiments at the logarithmic growth phase [1]. Antibiotics were added as necessary for selection of transformants and for strain maintenance (10 µg/ml G418, 5 µg/ml Blasticidin S, or 100 µg/ml Hygromycin B). Cells were developed on nitrocellulose filters [1] or on non-nutrient agar [2] as indicated. Gene disruptions and GFP and RFP transformations were performed in the laboratory wild-type strain AX4 [3]. Wild isolates (Supplement Table S1) were grown on SM-agar plates in association with Klebsiella aerogenes bacteria, cells were collected, washed away from bacteria and genomic DNA was extracted as described [1]. The cada strain TL97 [4] and the csaa strain T10 [5] were obtained from the Dictyostelium stock center. The lagd1 strain was described before [6]. Gene Disruption by homologous recombination To disrupt lagb1 we amplified DNA segments of approximately 900 bp upstream and downstream of the gene by PCR with the following primers: 5 CCA TCT GCA GTT CAT TTT TGT AAA ACT TGT AGC ATT 3 and 5 CCA GGA TCC TGA GAA TGG TTC ATC TGA ATG G 3 for the region upstream of the gene and 5 AAC ACT AGT CCA GCA GTA ATA AAT AAT TTC ATC CA 3 and 5 CCA GGA TCC TCA AAC AGT GTG ATG GGA AAA 3 for the downstream region. The PCR fragments were cloned into the BSR-based plpblp plasmid [7] using standard cloning methods. To disrupt lagc1 we 1

2 amplified the 5 region by PCR from the plagc-gal plasmid [6] using the following primers: 5 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTG TAC TCA AAA TCT GAA TCC CAC TAC TAC TAA 3 and 5 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CAT CTA GAT TTT ATC AAT GAT TTT TTT TAT TTT GAA TTT GGC 3 which include the Gateway system attb1 and attb2 sequences, respectively (underlined). The PCR product was cloned into pdonr221 with the BR clonase II kit (Invitrogen) according to the manufacturer s recommended protocol. The lagc1 3 region was amplified by PCR from AX4 genomic DNA with the following primers: 5 TTT CTA GAT TAG ATC TAA AAA AAA ATG TAA ATT TAA AAT CAC AAA TCA CAT ATG TGT AAA ATT AAA ACT TAA C 3, and 5 ACG TTT TAG GTT GTA GTT TGC TTA TAT GCA A 3. That PCR product was cloned into the plasmid containing the 5 region using standard cloning methods. A Hygromycin B resistance cassette was excised from peztn::tet R - A15hyg R, a plasmid derived from peztn:tet R -bs R by replacing the Blasticidin S resistance gene with a hygromycin resistance gene [8], and cloned in between the 5 and 3 fragments. The knockout plasmids were confirmed by sequencing, linearized and transformed into AX4 cells and selection for drug resistance was used to isolate putative transformants. PCR screening was used to screen for the lagb1 gene deletion among the Blasticidin-resistant cells for the lagc1 deletion among the Hygromycin-resistant cells. Positive clones were confirmed by a modified Southern blot analysis method as described [1]. Fluorescence labeling of cells A Red Fluorescence Protein (RFP) vector with G418 resistance: pa15/dsred was constructed by PCR amplification of the fast-maturating DsRed1 variant, DsRed-Express, from pdsred-express (Clontech) with the following primers: 5' AAA GGT ACC GAG CTC TCC CAT ATG GT 3' and 5' TTT CTC GAG ACG CGT TAC AGG AAC AGG TGG TGG 3' and cloned into pdxa- HC [9]. The tdtomato coding sequence was amplified by PCR from a plasmid containing RFP-tdTomato [10] with the following primers: 5 TTG GTA CCA TGG 2

3 TGA GCA AGG GCG AGG AG 3 and 5 TTA CGC GTT ACT TGT ACA GCT CGT CCA TGC C 3 and cloned into pa15/dsred instead of the DsRed sequence using standard techniques to generate pa15/tdtomato. A Gateway-based actin15 overexpression vector with hygromycin B resistance: We cloned the Gateway reading frame cassette A from the Gateway Vector Conversion Systems (Invitrogen) into pdxa-3h-hygro [11] with the attr1 site immediately downstream of the actin15 promoter. The resulting vectors are named pa15gw-hygr. GFP and RFP vectors with hygromycin B resistance: The GFP and RFP-tdTomato sequences were cloned into pdonr221 (Invitrogen). GFP with 6xHis at the N-terminal end was amplified by PCR with the following primers 5 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA AAA ATG CAT CAT CAT CAT CAT CAT C 3 and 5 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TAT TTG TAT AGT TCA TCC ATG CCA T 3 (underlined: attb1 and attb2, respectively). RFP-tdTomato with 6xHis at the N-terminal end was amplified with the following primers: 5 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA AAA ATG CAT CAT CAT CAT CAT CAT C 3 and 5 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TAC TTG TAC AGC TCG TCC AT 3 (underlined: attb1 and attb2, respectively). The PCR products were cloned into pdonr221 using the BP clonase II enzyme mix (Invitrogen) according to the manufacturer s recommended protocol. The plasmids were named pentr-gfp and pentrtdtomato. Subsequent transfers of the GFP and RFP genes to pa15gw-hygr were done with the LR clonase II enzyme mix (Invitrogen) according to the manufacturer s recommended protocol. The final constructs were named pa15gw/gfp-hygr and pa15gw/tdtomato-hygr, respectively. Strain construction GFP- and RFP- labeled strains were generated in various genetic backgrounds. AX4 was transformed with pdxa-gfp2 [12] to obtain AX4-GFP or with pa15/tdtomato to generate AX4-RFP. pdxa-gfp2 and pa15/tdtomato were also transfected separately into the cada strain TL97 [4] and into the 3

4 lagd1 strain [6]. lagc1 cells labeled with GFP or RFP were generated from AX4-GFP and AX4-RFP by homologous recombination of the lagc1-deletion vector as described above. The csaa strain T10 [5] was labeled with GFP and RFP, separately, by transformation with the pa15gw/gfp-hygr and A15GW/tdTomato-hygR plasmids, respectively, using the hygromycin B resistance, because of T10 is G418 resistant. TL97 and T10 were obtained from the Dictyostelium stock center. Segregation assays on a solid substrate For co-development on a solid substrate, strains were grown in suspension in pure populations as above, mixed at 1:1 ratios and deposited on non-nutrient agar at a final cell density of 2.5x10 5 cells/cm 2. The plates were incubated at 22 C in a humid chamber and photographs were taken either with transmitted light microscopy or with epi-fluorescence microscopy at the appropriate wavelengths. In double-labeling experiments, images were overlaid without further manipulation and the results are shown in color photographs. Adhesion and segregation assays in liquid suspension AX4, AX4-RFP, lagc1 and lagc1 -GFP cells were grown in pure populations as above. The cells were developed in pure populations on nitrocellulose filters. Aggregates were harvested at various times during development and dissociated into single cells by trituration in 20 mm potassium phosphate ph 6.5, 40 mm EDTA [13]. Single cells were collected by filtration through a cell strainer (40 µ mesh), counted and adjusted to a density of 1 x 10 6 cells / ml in 20 mm potassium phosphate ph 6.5. The cells were mixed at a proportion of 4:1:4:1 (AX4 : AX4-RFP : lagc1 : lagc1 -GFP) and shaken at 100 RPM for 1 hour in room temperature. Cell suspensions were photographed with epi-fluorescence microscopy and the images were processed as above. Sporulation Efficiency 4

5 Cells were grown and developed as described above for 30 hours. Sporulation efficiency was measured as described [14] with a minor modification the detergent used was 0.1% NP40. Each strain was tested in two independent biological replications. Each biological sample was developed on 3 filters and samples from each filter were counted thrice. Sporulation efficiency was calculated as the fraction (%) of spores recovered relative to the number of cells that were developed. RNA Extraction and Quantitative RT-PCR Vegetative cells (5 x 10 7 total) were collected by centrifugation from the growth medium and resuspended in 500 µl Trizol reagent (Invitrogen). Samples were incubated at room temperature for 5 minutes and stored at 80 C. Developing cells were collected by scraping from nitrocellulose filters with a sterile spatula and resuspended in 500 µl Trizol as above. RNA was extracted according to the manufacturer s recommended protocol. RNA concentration was calculated by absorbance at 260 nm. To calculate mrna expression levels in the different strains, cdna was produced by reverse transcription. Briefly, 1 µg of RNA was treated with 1 unit of DNase I at room temperature for 15 minutes. The reaction was stopped by addition of EDTA, and the DNase I was inactivated by a 10-minute incubation at 65 C. Half of the reaction was used to produce cdna and the other half as a negative control (without Reverse Transcriptase) for potential genomic DNA contamination. cdna synthesis and quantitative PCR were done as described [2] with the exception that the SuperScript II reverse transcriptase (Invitrogen) was used for reverse transcription. Quantitative real-time PCR reactions were performed with the following primer pairs; Ig7 was used as the normalizing RNA: Ig7 5 TTA CAT TTA TTA GAC CCG AAA CCA AGC 3 and 5 TTC CCT TTA GAC CTA TGG ACC TTA GCG 3 ; lagb1 5 TAA GTT TGC ACC ACC AAC CA 3 and 5 CCG AGA TAC ATC TCA TTT GGA A 3 ; lagc1 5 AAC ATT ACC TCC TGT CAT TTA TCG 3 and 5 CTT GGT CCT GAA CGA ACT CC 3 ; lagd1 5 GTT TAT TCG GCA CCA AAT CG 3 and 5 TGG TGA AGC ATA TCG TAA TTG 5

6 AA 3 ; lage1 5 CAT TTG CTC CTC CAA CCA TT 3 and 5 TTT GGA GCG CTT TCA TAA CC 3 ; lagh1 5 TCA TTG TGC CAG GTT GTG G 3 and 5 TTG GAG AAA GTG ATT GGT TCG 3 ; lagi1 5 GGT CCA AAT GAT AAA ACT TG 3 and 5 CAC TAC CAT TAC CAT TAC CA 3 ; lagu1 5 AAC CAT GCA CAC AAC ATG AAA T 3 and 5 CAT GCT TTG CAC GAA CTT TTA G 3 ; lagv1 5 CCA CCT ATA GCA TGG GAG GT 3 and 5 GAT GTT GGT ACA GGC CCA CT 3. Each strain was developed in two independent replicates and each RNA sample was analyzed thrice by quantitative RT-PCR. Data and standard deviations were calculated across the 6 replicates. Sequencing genes from wild strains Genomic DNA was extracted as described [1]. To sequence the lagb1, lagc1, lagd1 and lage1 genes, we used one set of primers to amplify the entire structural gene (ORF plus introns) from genomic DNA and another set of primers to amplify smaller fragments from the initial PCR product. The gene-specific primer pairs used to amplify the structural gene were: lagb1 5 TCA TTT ATT TGA AAA GGG GTA TTT TT 3 and 5 TGT TTT CAA CAA TGC AAC ACA A 3 ; lagc1 5 CAC ACT CAA GCT CAT TCA CCA 3 and 5 TGC CAT TAA ATC ACC ACA CG 3 ; lagd1 5 GGT TGT GTG TTT TGT GTG TGA A 3 and 5 GGA ACC AGA TTC ACC GAG TTT A 3 ; lage1 5 CAT TTG CTC CTC CAA CCA TT 3 and 5 TTT GGA GCG CTT TCA TAA CC 3. Each nested PCR primer pair contained one of two universal primer tags that were later used for sequencing. The universal primer tags were: 5 GGT CCG GCG AGT GG 3 and 5 CCG ACG CCA GCT CG 3. The gene-specific portions of the nested PCR primer sequences were as follows. For lagb1 5 TCA TTT ATT TGA AAA GGG GTA 3 ; 5 GCA CCA CTA GGG TTG GTG TC 3 ; 5 CTT GAA TCC AGG GTG TGG TC 3 ; 5 ACA CCG GTC ACA TCG AAA AT 3 ; 5 TGT CAC ATT CTC CGC TGA AG 3 ; 5 TTC ACT ACC ACC GTC ACC AA 3 ; 5 TGC AAC ATT GGA AGA ACC TG 3 ; 5 GCC ACA TGG TAC AGC AGG A 3 ; 5 TGG GTC TGG TGG TCT TGT TT 3 ; 5 ATG GTT GGT GGT GCA AAC TT 3 ; 5 6

7 GCG GTG GAG TGG TTA CTA TCA 3 ; 5 GCG GCA AAT GAA ATT AAT GC 3 ; 5 GTT GAT GGT CAG TAC TTT ATT G 3 ; 5 TGT TTT CAA CAA TGC AAC ACA A 3 ; 5 TGG TAG GGG CAA ATC ATT TTT 3 ; 5 TTG GGT ATG GTA CCA GTT TC 3 ; 5 TGC TAC AAG TTT TAC AAA AAT GAA AGT 3 ; 5 TAC CAT ATC CAA TGT ACC AA 3 ; 5 TGT ATT TGA TTT ATA TTC ACC GTC A 3 ; 5 AAC ATG TAC CAT TCC AAG TCG 3 ; 5 CAA GGT TCA TAA AAC CAG GTT GA 3 ; 5 TCA GTA CTT TAT TGC TGA TAT TTT CCA 3 ; 5 TTG AAA CAA CGT CTG ATG AGT G 3. For lagc1 5 AAC CCG AAG CAT CAA ATG 3 ; 5 ACT CAA GCT CAT TCA CCA 3 ; 5 CAT TTG ATG CTT CGG GTT 3 ; 5 GTA CCA AGA TTT GTA CCA 3 ; 5 TCC CGG TCC AAT GTC TGT 3 ; 5 TGA ACA TGG TGA GGC TGA 3 ; 5 CCT CCA CAA GCA AGT GTT 3 ; 5 TGC AAA TGA ATT GGG TGG 3 ; 5 TGC ACC ACC AGA AGT TGA 3 ; 5 CAC TTG AAC CGG AAT GGC 3 ; 5 GGA GTT CGT TCA GGA CCA 3 ; 5 CCA TTA AAT CAC CAC ACG 3 ; 5 AAT TTG GTG CCA TTT CAG TTC 3 ; 5 CAT CAA ATG TAC AAT CCT CAA CAG 3 ; 5 TGA TTT GAT ACT GAT GAT ATT GAT GTT 3 ; 5 GAA ATT AAA TTA TCT CCA TCA CC 3 ; 5 TAA CAA AGC CAA ATT CAA AAT 3 ; 5 TCA AAA GAT GGA TCA GAG AAA 3 ; 5 TTT TAT TAG ATG TAA TAT ATG ATG A 3 ; 5 TTT TCT CAG ATG TAA TAT ATG A 3 ; 5 TTT TAT TAG GTG TAA TAT ATG A 3 ; 5 TGA TTA ATA CCA TCA AAG GA 3 ; 5 TGG TCA ATA CCA TCA AAA GA 3 ; 5 TGA GTA ATA CCA TCA AAA GA 3 ; 5 TTA GTT CAT CAG CAC CAA GT 3 ; 5 TTA ATT GGG CCA TTT TCA TA 3 ; 5 AAT TTT GGT AAT TCA ACA TCA TTA 3 ; 5 TGT TAA GCT TGC ATT GAG AA 3 ; 5 TCT CAA TGC AAG CTT AAC AG 3 ; 5 ATG AAG GAT TTG ATT GGG TA 3. For lagd1 5 GGT TGT GTG TTT TGT GTG 3 ; 5 TGA CAA TCC TCC GTT GGG 3 ; 5 TTC GGC ACC AAA TCG TAA 3 ; 5 ATC ACT GCT GGG AAA CAA 3 ; 5 TGC AAC ATT TCC ACC AAG 3 ; 5 TGG TGC TGA AAT TGA AGG 3 ; 5 TTG TTT CCC AGC AGT GAT 3 ; 5 TTA ATT GGG CCA TTT TCA 3 ; 5 CGT TCA TTC TCC AAA GGT 3 ; 5 ACC AGC ACC AGC AAC ACA 3 ; 5 TTG GAG ATG ATT TGG GTC 3 ; 5 GGA ACC AGA TTC ACC GAG. For lage1 5 TTT TAC ATT GAC CCG CAA TTA G 3 ; 5 CTT CCA TTA CAG TGA TTA TTG G 3 ; 5 GAT CCC GTT GAA AAC CTC AA 3 ; 5 TTT GGC CAG CAG TAA TGG TT 3 ; 5 CAC AAA GGG 7

8 GTA CTT CAC AG 3 ; 5 GTA ACC ATT CCA CCA CCA CA 3 ; 5 GGC CAA AGC TTC AAA TCA AA 3 ; 5 AAT GGT TGG AGG AGC AAA TG 3 ; 5 TAC CAT GTG GTG GTG GAA TG 3 ; 5 TGC TTT TCT GGC TCT TCT CTC 3 ; 5 GTT GAT GGT CAA TAC TTT ATT GC 3 ; 5 AAG CAT GAG CTT CAT TGT ATT A 3. The PCR products were purified using the QIAquick multiwall PCR purification system (Qiagen) according to the manufacturer s recommended protocol and sequenced by a commercial vendor using an Applied Biosystems 3730xl automatic sequencer. GenBank accession numbers of all the sequences we used are provided in Supplement Table S1. Analysis of polymorphic sequences The regions of the genes used for analysis are provided in Supplemental Table S1. Multiple sequence alignments were performed with the Clustal W algorithm in MacVector Intronic sequences were deleted manually and the alignments were adjusted accordingly. We used the Nei and Gojobori method to calculate the number of synonymous (ds) and non-synonymous (dn) nucleotide substitutions per site for a given pair of homologous sequences [15]. The gene coding sequences of a given pair of strains were scanned with a sliding window spanning 101, 81 and 31 aa with essentially identical results (31 aa windows shown), and the dn/ds ratio was calculated at each position. The dn/ds ratios reported are an average across all pair-wise comparisons. Correlation between sequence polymorphism and segregation Motivation: When D. discoideum cells are mixed with genetically similar cells, they readily form chimeric fruiting bodies, but mixing with genetically dissimilar cells results in partial strain segregation [Ostrowski, 2008 #66]. This phenomenon implies the existence of a kin-discrimination system and the properties of lagb1 and lagc1 suggest they might participate in that system. To test that possibility, we examined the correlation between the lagb1 and lagc1 sequence dissimilarity and the published segregation data [Ostrowski, 2008 #66]. We first computed the dissimilarities between the AX4 LagB1 and LagC1 protein 8

9 sequences and the respective sequences of each of 11 wild isolates. We then examined the correlation between the sequence dissimilarities and the published segregation data. We observed a positive correlation between lagb1-lagc1 sequence dissimilarity and segregation, although it was weaker than the correlation with genetic distances inferred from microsatellite length (Supplement Table S2). We then searched the sequences and found 140 overlapping intervals in LagB1 and 24 overlapping intervals in LagC1 protein sequences that exhibited the property of high positive correlation between sequence dissimilarity and strain segregation. We summarized the correlation between these sequences and the observed segregation and found high positive correlation that was statistically significant in both proteins (Supplement Table S2). These results suggest that the protein domains may have a function in segregation. Plotting the highly correlated sequences along the length of the two proteins revealed that in LagB, the correlation was highest between aa (Supplement Fig. S1A) and in LagC the correlation was highest between aa (Supplement Fig. S1B). In both cases, the domains reside near the first predicted Ig-fold of the extracellular domain, consistent with the proposed role in cell-cell interaction. Method: We calculated the Spearman s rank correlation between the calculated protein sequence dissimilarity and the segregation variance values reported by Ostrowski et al. [16] for 12 strains (AX4, NC4, QS32, QS33, QS34, QS36, QS37, QS38, QS40, QS41, QS45, QS113). Both the sequence similarity and the segregation were measured against a common reference strain (AX4). Sequence dissimilarity was calculated in all subintervals between 5 and 13 aa over each protein position. We used three protein similarity matrixes (PAM250, BLOSSUM, GONNET) to calculate similarity and then negated it to obtain dissimilarity. For each subinterval, we then fitted a linear model relating subinterval sequence dissimilarity and segregation. The final model is a composite (ensemble) of all linear models with high Spearman s rank correlation (r -0.65) and high significance (p < 0.025): there were 140 such models for lagb1, and 24 for lagc1, each one represented by the coordinates of the 9

10 sequence subinterval, the sequence similarity matrix used, and the coefficients from the linear regression. When the composite model was applied to a new sequence, each linear model predicted the level of segregation based on the sequence similarity in its specific subinterval. The final prediction of the composite model is an average of the predictions of all the linear models. We built two composite models, one for each gene (lagb1, lagc1) and presented the final prediction as the relative contribution of each model among all models (yaxis) that span each position (x-axis). in Supplement Fig. S1. Finally, we adapted the models built on the AX4 reference data and applied them to predict the segregation of strains QS32, QS33 and QS38 from strain QS32. In that case, the models were adjusted (shifted by a constant value) to predict zero segregation when the reference strain was mixed with self. Figure S1. Correlation between sequence polymorphism and strain segregation. We considered the correlation between the strain segregation data reported by Ostrowski et al. [16] and the predicted amino acid sequence differences between the laboratory strain AX4 and each of 12 different wild strains. For each amino acid position in the sequence (x-axis) we plotted a weight (y-axis, arbirary units), which is proportional to the number of models from the composite that span that particular position (140 models for lagb1 and 24 models for lagc1). Plots for the two genes are shown: LagB1 (A) and LagC1 (B). The predicted protein structures of the respective proteins are shown below each plot. Pink boxes represent hydrophobic domains and blue boxes represent immunoglobulin folds (IPT, TIG or E-set domains). All of the Ig-folds are predicted to be extracellular. 10

11 Table S1. Wild strains and the sequenced genes Strain AX4 a NC4 QS1 QS4 QS8 QS9 QS11 QS14 QS15 QS17 QS18 QS21 QS22 QS23 QS30 QS31 QS32 QS33 QS34 QS36 QS37 QS38 QS39 QS40 QS41 QS42 QS43 QS44 Collection location Little Butts Gap, NC Little Butts Gap, NC Carthage, TX Houston Arboretum Pasadena, TX Nucleotides sequenced and accession numbers Coordinates lagb (2709 bp) Acc. number lagc (2670 bp) Acc. number lagd (2688 bp) Acc. number lage (2739 bp) Acc. number N W FJ FJ FJ FJ N W FJ FJ ; W FJ FJ W FJ FJ FJ ; W FJ FJ FJ W FJ FJ FJ W FJ FJ FJ FJ W FJ FJ W FJ FJ FJ W FJ FJ FJ W FJ FJ FJ FJ ; W FJ FJ FJ ; W FJ FJ W FJ FJ FJ ' N 94 18' W FJ FJ N W FJ FJ FJ FJ N 1-540; 95 4 W FJ FJ FJ N 95 4 W 1-580; FJ FJ Webster, TX Bloomingto n (Lobelia), ' N FJ FJ FJ IN ' W Land Between N FJ FJ FJ Lakes, KY ' W ' N Linden, TX W FJ FJ W FJ FJ FJ ; ' N FJ ; FJ ' W b Indian Gap, TN Mt. Greylock, N FJ FJ MA W Little Butts N Gap, NC W FJ FJ Monteverde, 10 18' N Costa Rica 84 26' W FJ La Malintzi National 1-360; Park, N FJ ; Mexico W Mt. Fuji, N Japan E FJ FJ FJ

12 QS45 QS46 QS47 QS W FJ FJ Forrest City, 34 50' N ; AR 91 28' W FJ FJ St. Louis, N MO W FJ FJ Effingham, N IL W FJ FJ FJ a strains highlighted in gray were used for the analysis of correlation between strain segregation and protein sequence variation b - sequences highlighted in gray were not included in the dn/ds analysis Table S2. Correlations between strain segregation and sequence dissimilarity Microsatellites a Full length protein sequence Model weighted protein sequence lagb1 lagc1 lagb1 lagc1 Spearman s correlation p-value a - recalculated from [16] 12

13 Supplemental References 1. Shaulsky, G., and Loomis, W.F. (1993). Cell type regulation in response to expression of ricin-a in Dictyostelium. Dev. Biol. 160, Huang, E., Blagg, S.L., Keller, T., Katoh, M., Shaulsky, G., and Thompson, C.R. (2006). bzip transcription factor interactions regulate DIF responses in Dictyostelium. Development 133, Knecht, D.A., Cohen, S.M., Loomis, W.F., and Lodish, H.F. (1986). Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Mol Cell Biol 6, Wong, E., Yang, C., Wang, J., Fuller, D., Loomis, W.F., and Siu, C.H. (2002). Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development. Development 129, Harloff, C., Gerisch, G., and Noegel, A.A. (1989). Selective elimination of the contact site A protein of Dictyostelium discoideum by gene disruption. Genes Dev 3, Kibler, K., Svetz, J., Nguyen, T.L., Shaw, C., and Shaulsky, G. (2003). A cell-adhesion pathway regulates intercellular communication during Dictyostelium development. Dev Biol 264, Faix, J., Kreppel, L., Shaulsky, G., Schleicher, M., and Kimmel, A.R. (2004). A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the CreloxP system. Nucleic Acids Res 32, e Abe, T., Langenick, J., and Williams, J.G. (2003). Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development. Nucleic Acids Res 31, e

14 9. Manstein, D.J., Schuster, H.P., Morandini, P., and Hunt, D.M. (1995). Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162, Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, Knetsch, M.L., Tsiavaliaris, G., Zimmermann, S., Ruhl, U., and Manstein, D.J. (2002). Expression vectors for studying cytoskeletal proteins in Dictyostelium discoideum. J Muscle Res Cell Motil 23, Levi, S., Polyakov, M., and Egelhoff, T.T. (2000). Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum. Plasmid 44, Bowers-Morrow, V.M., Ali, S.O., and Williams, K.L. (2002). Cell adhesion during the migratory slug stage of Dictyostelium discoideum. Cell Biol Int 26, Shaulsky, G., Escalante, R., and Loomis, W.F. (1996). Developmental signal transduction pathways uncovered by genetic suppressors. Proc Natl Acad Sci U S A 93, Nei, M., and Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol 3, Ostrowski, E.A., Katoh, M., Shaulsky, G., Queller, D.C., and Strassmann, J.E. (2008). Kin discrimination increases with genetic distance in a social amoeba. PLoS Biol 6, e

15 Figure S1 15