indicated in cells. These numbers are shown for both the dataset used for the tree on Figure 3B (out of 997 bootstrap trees) and for a wider dataset including all curated systems (out of 974 trees), after the mark ‘‘&’’ (Text S1). (PDF) Figure S3 Individual phylogenies of the NF-T3SS core proteins with flagellar homologs. These trees were obtained with NF-T3SS proteins, and did not include flagellar proteins. (A) The eight collapsed individual trees. Branches with low support value (bootstrap,80%) were collapsed. Supports for some critical relationships in the rooted trees can be found in Table S4, black arrows indicate position of the flagellum sequences in the ‘‘rooted’’ versions of these trees (not shown). (B) These trees are the same as the trees presented in panel A, except that branches with bootstrap values below 80% were not collapsed. (PDF) Figure S4 Maximum likelihood phylogeny of the NF-T3SS using the concatenation of the eight core genes with homologs in the flagellum. The eight individual trees are displayed in Figure S3. For more clarity, strain names were replaced by the corresponding species names when the systems were monophylet- ic. Names in red correspond to the model NF-T3SSs used to build the protein profiles of the NF-T3SS core genes. Bootstrap supports are all shown for relationships between families, but within families bootstrap supports are indicated only when below 80%. The ‘‘Gene content’’ panel indicates the presence (black for clear homologs, or brown if found in a distant or unrelated clade- specific family with weak or no sequence similarity) and absence (grey) of other conserved NF-T3SS genes (see Materials and Methods). When not found in the protein dataset with our Hmmer search or with our clustering method, traces of genes were sought by performing tBlastn searches on complete genomes. These hits could correspond either to pseudogenes, fast evolving sequences, or sequencing errors. If traces were retrieved, the gene appears with a salmon box. The ‘‘Ecology’’ panel indicates bacterium/ eukaryote associations. Large monophyletic clades correspond to the NF-T3SS families (colored boxes). ‘‘Chlamy’’ stands for Chlamydiales, ‘‘Desulfo’’ for Desulfovibrionales, ‘‘Myxo’’ for Myxococcales, ‘‘Rhizo’’ for Rhizobiales, and ‘‘Bups’’ for Burk- holderia pseudomallei group. The black arrow indicates the position of the flagellum sequences in the ‘‘rooted’’ version of this tree (Figure S5), and the scale of the tree is given in substitutions per site. (PDF) Figure S5 Concatenate phylogeny of flagellar and non-flagellar T3SSs based on SctNJQRSTUV and their respective flagellar homologs (see Tables S1 or S4 for gene names). Branch supports correspond to the occurence of the splits in 100 rapid bootstrap trees. Color boxes surround the different NF-T3SS families. References 1. Lee VT, Schneewind O (2001) Protein secretion and the pathogenesis of bacterial infections. Genes Dev 15: 1725–1752. 2. Nogueira T, Rankin DJ, Touchon M, Taddei F, Brown SP, et al. (2009) Horizontal gene transfer of the secretome drives the evolution of bacterial cooperation and virulence. Curr Biol 19: 1683–1691. 3. Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62: 379–433. 4. Ghosh P (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68: 771–795. 5. Galan JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444: 567–573. 6. Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4: 811–825. 7. McCann HC, Guttman DS (2008) Evolution of the type III secretion system and its effectors in plant-microbe interactions. New Phytologist 177: 33–47. Origins and Diversification of T3SS Protocol S1 (DOC) Table S2 (DOC) Yellow for Myxo, brown for Chlamy, red for Hrp2, orange for Ysc, green for Rhizo, cyan for Hrp1, violet for SPI2, and dark purple for SPI1. Two levels of taxonomy are indicated on the right. See Dataset S2 for equivalence between system codes and replicon names. (PDF) Analysis of the F-/V- ATPase family. Table S1 Nomenclature of NF-T3SS and flagellar components in various systems. (DOC) List and justification of excluded NF-T3SS systems. Table S3 List of model genomes used to build flagellar protein families and profiles. (DOC) Table S4 Support values (percentage of rapid bootstraps) for critical relationships in rooted phylogenies of T3SS genes. (DOC) Table S5 List of model genomes used to build NF-T3SS protein families and profiles (in red on Figure S4). (DOC) Analysis of the F-/V- ATPase trees. The phylogeny of NF-T3SS. Checking phylogenetic signal consistency with Prunier. Tracing the origins of Rhizo secretins. Text S1 (DOC) Text S2 (DOC) Text S3 (DOC) Text S4 (DOC) Acknowledgments We thank Bertrand Ne´ron and the team of the MOBYLE platform for the design and implementation of the query system and of the visualization tools. We thank Rob Knight for discussions, and Vincent Daubin and Claude Parsot for comments and suggestions on earlier versions of this manuscript. We thank the two editors and three anonymous reviewers for their constructive criticisms and suggestions. Author Contributions Conceived and designed the experiments: SSA EPCR. Performed the experiments: SSA. Analyzed the data: SSA EPCR. Contributed reagents/ materials/analysis tools: SSA EPCR. Wrote the paper: SSA EPCR. 8. Kosarewicz A, Konigsmaier L, Marlovits TC (2012) The blueprint of the type- 3 injectisome. Philos Trans R Soc Lond B Biol Sci 367: 1140–1154. 9. Attree O, Attree I (2001) A second type III secretion system in Burkholderia pseudomallei: who is the real culprit? Microbiology 147: 3197–3199. 10. Rainbow L, Hart CA, Winstanley C (2002) Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei. J Med Microbiol 51: 374–384. 11. Sun GW, Gan YH (2010) Unraveling type III secretion systems in the highly versatile Burkholderia pseudomallei. Trends Microbiol 18: 561–568. 12. Hansen-Wester I, Hensel M (2001) Salmonella pathogenicity islands encoding type III secretion systems. Microbes Infect 3: 549–559. 13. Martinez-Becerra FJ, Kissmann JM, Diaz-McNair J, Choudhari SP, Quick AM, et al. (2011) A broadly protective Shigella vaccine based on Type III secretion apparatus proteins. Infect Immun 80: 1222–1231. 14. Kline T, Felise HB, Sanowar S, Miller SI (2011) The Type III Secretion System as a Source of Novel Antibacterial Drug Targets. Curr Drug Targets 13: 338–351. PLOS Genetics | www.plosgenetics.org 12 September 2012 | Volume 8 | Issue 9 | e1002983