Fig. 2

PyDMT1 hypomorphs are associated with reduced growth and suppressed pathogenicity. a A schematic showing positions of nine guide RNAs and homologous recombination arms designed for Pydmt1 knockout. Each guide RNA and flanking homologous recombination arms were cloned in the vector expressing Cas9, and parasites were selected with pyrimethamine after transfection. All failed in repeated trials. Different homologous recombination arms were also tried in vain. b Single crossover strategy for PyDMT1 knockdown in P. yoelii. The strategy was designed in such a way that after a successful crossing-over, the 4.1-kb region upstream from the translation start of the neighboring gene would remain intact, leaving only the Pydmt1 regulatory region truncated at the insertion point. Arrows indicate various truncations upstream of Pydmt1 CDS had been tried to make the hypomorphic mutant. c qPCR detection of Pydmt1 transcript levels for PyDMT1 hypomorphic clones A1 and C2. The control parasites of PyDMT1-HA and hypomorphic clones A1and C2 were purified from mouse blood and subjected to qPCR using primers targeting the 3’ segment of PyDMT1. PyDMT1 transcript abundance was normalized to tubulin. Shown are mean values + / − SD. **P < 0.01; N = 3. d Protein levels of Pydmt1-A1 and Pydmt1-C2 as detected by western blots. HA antibody was used for detection. The specificity of the HA antibody was demonstrated with none-HA carrying parasite strain (Figure S2d), suggesting all the three bands from the western blot were derived from PyDMT1. e Reduced growth of the Pydmt1 hypomorphic mutants. Parasitemia of BALB/c mice following infection (i.v.) with 10⁶ PyDMT1 or PyDMT1 hypomorphic iRBC was determined by counting iRBC in Giemsa-stained blood smears(N = 5, one-way ANOVA and Tukey’s multiple comparison test, **P < 0.01, ***P < 0.001). f Loss of pathogenicity for the Pydmt1 hypomorphic mutants. Shown here is the survival of BALB/c mice inoculated with 10.⁶ control P. yoelii or hypomorphic PyDMT1 parasite-infected iRBC (N = 5, log-rank test, ***P < 0.001)