Expression level, polymorphism analysis, and localization of ROP18. (A) Expression level of ROP18 in the parental lines (type II, ME49, and type III, CEP) and 18 F₁ progeny. Data are displayed as fold-difference relative to the type III parent. The genotype for each of the F₁ progeny at the ROP18 locus is indicated by the bar color (white or black). (B) Variation in the percentage of type I, II, and III SNP polymorphisms across chromosome VIIa. The entire chromosome was divided into 10-kb windows, and the number of SNPs of each type in each window [based on EST assemblies; see (5)] was divided by the number of sites with data from all three strains to compute a polymorphism percentage. The number of each polymorphism type in the ROP18 coding region is also shown. *Accession number AM075204 (28). **Downloaded from (16) (Gene model 20m.03896). (C) Immunofluorescence assay of type III:ROP18_II showing HA-specific staining (green) of HA-tagged ROP18 colocalizing with the rhoptry marker ROP2/3/4 (red) in human foreskin fibroblasts 40 hours post inoculation. (D) Immunofluorescence assay of type III:ROP18_II showing HA-specific staining of tagged ROP18 in human foreskin fibroblasts 2 hours post inoculation. Punctate staining indicates the location of ROP18-HA that has been secreted into the infected host cell (22). Color scheme same as in (C).

Effect of expressing the type I and III strain allele of ROP16 in the type II Prugniaud strain (PRUΔhxgprt) on virulence in mice. Mice were infected intraperitoneally with 5000 tachyzoites of type II (PRUΔhxgprt) or an engineered version expressing an HA-tagged copy of the ROP16 allele from the type III CEP strain (type II:ROP16_III) or the type I RH strain (type II:ROP16_I). For PRUΔhxgprt, 24 mice were used, 10 for type II:ROP16_III, and 14 for type II:ROP16_I. Infections were verified in all the survivors on the basis of the presence of Toxoplasma-specific antibodies. Results from three independent experiments were pooled.

Genetic mapping of virulence phenotypes of F₁ progeny from II × III crosses. BALB/c and CBA/J mice were infected with 100,000 or 100 tachyzoites from 40 different F₁ recombinant progeny from II × III crosses, and mortality was recorded daily for 40 days. As there were no significant differences between the two different mouse strains, results were pooled. Three phenotypes are represented: (i) “high-dose survivability,” log₁₀ survival time (in days) after injection of 100,000 parasites (black line); (ii) “avirulence,” a binary trait defined as no mortality at any dose (red line); (iii) “low-dose survivability,” log₁₀ survival time (in days) after injection of 100 parasites (blue line). Plots indicate the log-likelihood association of phenotypes with markers aligned across the genome. Marker positions (in cM) are given by tick marks. Significance levels determined by 1000 permutations are indicated by horizontal lines [upper lines are significant; lower line is suggestive (P = 0.1)]. Because the significance levels for all three of the phenotypes differed by less than 0.1 LOD unit, only one significance line is drawn for all three. (A) Primary genome scan (see text). (B) Secondary genome scan after the effect of the major virulence peak on chromosome XII, evident in (A) and cosegregating with the SAG3 marker, is neutralized by making it a covariate.

Effect of expressing the type II allele for ROP18 in a type III strain background (CEP) on virulence in mice. Mice were infected intra-peritoneally with either CEPΔhxgprt complemented with HXGPRT alone (CEP*) or CEP parasites complemented with the type II allele for ROP18 along with 588 bp of upstream sequence (type III:ROP18_II). Infections were verified in all the survivors based on the presence of Toxoplasma-specific antibodies, and only seropositive survivors are represented on the graph. Ten mice were used for all strain and dose combinations except for type III:ROP18_II-100 (eight mice) and type III:ROP18_II-10 (seven mice).