Fig. S1. Generation of PbTN-XXL transgenic parasite.

A. Schematic of genomic integration of PbTN-XXL. Chr12a and Chr12b (Chromosome 12, 820 043–820 541 bp and 820 841–821 272 bp respectively) are the homology regions used to integrate the linearized vector via double homologous recombination. The sequence between these homology regions is replaced with the vector, leading to expression of TN-XXL under the control of the PbHSP70 promotor region. Primers used in B are indicated.

B. PCR confirming 5′ (P1 P2, 830 bp) and 3′ (P3 P4, 736 bp) integration of the construct and the absence of the unmodified locus (P1 P4, 1448 bp) are shown.

Fig. S2. Effect of ionomycin on sporozoite gliding. Non-activated sporozoites were treated with ionomycin according to the Experimental procedures at concentrations of 0.1, 0.5 and 1.0 µm in PBS/glucose or M199 medium (two independent experiments for each medium, blue and red bars). As positive control, parasites were activated with 5% FBS or 2% bovine serum albumin. PBS/glucose and M199 alone or in the presence of 0.1% DMSO were used as negative controls.

Fig. S3. Generation of TRAPMyc transgenic parasite.

A. The TRAP-Myc parasite was generated by cloning the TRAP open reading frame and inserting a triple Myc tag into the disorganized region of the TRAP ectodomain, after base pair 1482. This was flanked upstream by 1.3kb of the TRAP 5′ region, and downstream by an F3 site (modified FRT) and 1.2 kb of the TRAP 3′ region, and cloned into a vector which contains the hDHFR resistance cassette (grey) flanked by FRT sites. This construct was integrated by double crossover replacing the endogenous TRAP (left PCR using primers P5/P6), using as homology regions the TRAP 3′ and the pUC18 plasmid backbone, into the FlpLtrap receiver strain, a P. berghei NK65 GFP fluorescent strain in which the Flip recombinase was integrated at the TRAP locus (Lacroix et al., 2011). The hDHFR resistance cassette was then excised by cycling the recombinant parasite through the mosquito (right PCR using primers P7/P8). This strain was originally constructed to serve as a receiver for integration of other constructs.

B. Gliding of GFP-control and TRAPMyc sporozoites. Sporozoites were activated with 5% FBS and gliding motility was accessed as described in the Experimental procedures. The bars represent the average ± SEM of parasites gliding in three independent experiments. P-values determined using Student's t-test.

Fig. S4. Immunofluorescence controls. Controls for immunofluorescence labelling of TRAP-Myc sporozoites. Sporozoites were fixed, permeabilized (if indicated), and labelled as described in the Experimental procedures.

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