gain-of-function by transgenic expression [27]. Expression of the type I allele of ROP18 in the type III background reconstitutes the acutely virulent phenotype characteristic of the type I clonal lineage [27]. While additional ROP18I* alleles share similarities with the virulent allele from the clonal type I tested previously (corresponding to group I*a) [27], they also differ significantly from one another, , presumably due to genetic drift (Figure 4A). Hence, we undertook a similar reverse genetic analysis with the newly identified subtypes of ROP18I* in order to determine if they were also capable of conferring acute virulence or if they had lost this trait due to mutation. We chose three representative isolates for this analysis from different branches of the ROP18I* tree that have not been previously analyzed; including RUB (ROP18I*b), MAS (ROP18I*c) and CAST. These strains are all acutely virulent in mice (Table S2). We established three separate transgenic lines from each of the parasite isolates, using standard methods for epitope tagging and isolation of stable lines, as described previously [27]. Expression and localization to the rhoptries was confirmed by immunofluoresence staining of the Ty-epitope tag in reference to the endogenous rhoptry protein ROP1 (Figure 4B). Expression levels comparable to the previously derived transgenic line expressing ROP18Ia* (V1 clone, described previously [27]) were confirmed by western blotting (Figure 4C). Challenging adult outbred mice with these transgenic parasites confirmed that the new alleles of ROP18I* conferred acute virulence. Mice died Population Strucure and Virulence of T. gondii within 15–20 days, even when inoculated with few parasites (i.e. 10 tachyzoites) (Figure 4D). Collectively, these data indicate that the majority of haplogroups of T. gondii express ROP18I* alleles, and that these different variants all confer acute virulence in the mouse model. This finding is significant because it indicates that amino acid substitutions conferring increased virulence have become fixed in a lineage of ROP18I*, which is expressed by a wide range of isolates. The widespread distribution of ROP18I* alleles indicates that this gene and the traits it controls, confers a selective advantages in certain settings. Differences in the Expression of ROP18 Largely Explain Differences in Murine Virulence Previous genetic crosses between laboratory strains of clonal types I and III, attributed most of the variance in virulence to expression differences in ROP18 [26,27]. This difference in expression has been ascribed to an additional DNA segment upstream of the ROP18 gene in the type III lineage that alters transcription [32]. To examine the contribution of ROP18 expression to the variation in virulence among natural isolates, we examined the surrounding genomic regions and tested expression by real-time quantitative PCR (qPCR). Substantial differences in the upstream regions of ROP18 were evident when the three clonal lineages were compared (Figure 5A). Direct DNA Figure 4. Newly discovered ROP18I* alleles confer acute virulence. A) Phylogenic analysis of the ROP18I* alleles reveals three major groups (denoted I*a, I*b, and I*c) plus a single node for strain CAST. Tree depicts the same data as in Figure 1B, plotted with an expanded scale. B) Expression of RPO18I* alleles in transgenic lines and colocalization with ROP1 in the rhoptries was confirmed by immunofluorescence staining. ROP18I* alleles were tagged with Ty (green) and visualized using mAb BB2. Endogenous control detected with rabbit anti-ROP1 (red). Representative clones are shown for each allele subtype of ROP18I*. Scale bar = 5 microns. C) Three separate transgenic lines from representative strains as shown, expressed ROP18I* alleles at comparable levels to the previously described ROP18Ia V1 clone (+control) [27]; vector only control (2control). ROP18 was detected with mAb to the Ty-tag, rabbit anti-actin provided as loading control. D) Mouse survival assays demonstrating that expression of ROP18I* alleles in the type III background resulted in increased mortality. Cumulative mortality following challenge with 10, 100, and 1,000 parasites for each of three clones is plotted as a single line per clone (CL). Control is transgenic type III strain containing only the Ble vector. doi:10.1371/journal.pgen.1000404.g004 PLoS Genetics | www.plosgenetics.org 6 March 2009 | Volume 5 | Issue 3 | e1000404