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Fig. S1. Generation of Py-ICP-myc parasite line and its colocalization with Pf- and Py-ICP antibodies.

A. ICP-myc was generated by integrating a second copy of the Py-ICP gene fused with a quadruple myc tag at the C-terminus into the Py genome. The ICP gene (without the stop codon) with 1.5 kb of sequence upstream from the start codon was amplified from WT Py genomic DNA and inserted into the b3D-myc vector. The construct was linearized with BsmI and transfected into Py blood-stage schizonts with subsequent injection into SW mice. Successful transgenics were selected by pyrimethamine treatment. The integration of the ICP-myc vector into Py was confirmed by PCR using a b3D-myc-specific forward primer and a locus-specific reverse primer (int). This band is only present in the ‘Py-ICP-myc’ sample, but not in WT. The ICP-specific open reading frame (ORF) test showed positive bands in both WT and ICP-myc as, in addition to the myc-tagged copy, there is another intact copy of ICP in the ICP-myc parasites.

B. A rabbit polyclonal antibody (ICP AB) was generated against purified recombinant Py-ICP protein and the specificity of the antibody was analysed in an IFA. The expression of ICP in Py-ICP-myc parasites was detected using anti-myc antibodies and ICP antibody. Colocalization of myc with ICP antibody was detected in blood-stage parasites (BS), salivary gland sporozoites (Spz) and in vivo 43 h pi liver-stage parasites (LS), showing that Py-ICP antibody can detect ICP specifically. Nuclei were stained with DAPI. Scale bar, 5 μm.

C. In Py-ICP-myc salivary gland sporozoites, the anti-myc staining pattern colocalizes with that of the P. falciparum ICP antibody.

D. Western blot analysis of lysates from Py, Py-ICP-myc and Py-ICP-RFA BS parasites. ICP expression was detected using Py-ICP polyclonal antibodies (Py-ICP AB). Myc-tagged and RFA-tagged ICP run higher than untagged protein antibody. Processing events are detected in wild type and recombinant Py-ICP proteins. As Py-ICP-myc contains both the tagged and untagged copies of Py-ICP, the Py-ICP antibody recognizes both the myc-tagged (open arrows) and untagged (closed arrows) protein. Py-ICP-RFA was generated by replacement of the entire Py-ICP locus, thus only the tagged protein is identified via western blot (grey arrows).

Fig. S2. Generation of Py-ICP-RFA.

A. Py-ICP-RFA was generated using double-crossover recombination strategy to replace the endogenous ICP gene with a copy whose C-terminus was fused with a regulatable, fluorescent affinity (RFA) tag (Muralidharan et al., 2011) that consists of GFPmut2, an E. coli DHFR degradation domain (DDD) and a single HA epitope. Successful transgenics were selected by pyrimethamine treatment. The successful double-crossover replacement into the Py-ICP locus was confirmed by PCR. Primers specific to the ICP ORF showed a positive band only in the WT. Furthermore, ‘T1’ and ‘T2’ primers only amplified bands from the Py-ICP-RFA parasite.

B and C. The expression of ICP in Py-ICP-RFA blood stages was visualized using antisera combinations of either Py-ICP antibody with anti-HA (B) or anti-GFP with anti-HA (C). Colocalization of all three antisera was observed in IFAs.

D. Western blot analysis of lysates from blood-stage parasites was also performed on Py-ICP-RFA. Similar ICP bands were detected by polyclonal rabbit Py-ICP antibody and mouse monoclonal anti-HA antibody.

Fig. S3. Mass spectrometry of recombinant Py-ICP fragments. High and low-molecular-weight fragments of recombinant Py-ICP were analysed by mass spectrometry. The signal peptide of Py-ICP is underlined and boxed regions designate peptides identified during mass spectrometry.

A. Peptides from the high-molecular-weight band spanned the full length of the protein sequence of Py-ICP, confirming that this band corresponds to full-length Py-ICP.

B. Peptide hits from the lower-molecular-weight band correspond to the C-terminus of Py-ICP only.

Fig. S4. Py-ICP is expressed in oocyst and midgut sporozoites. Oocysts (A) and midgut sporozoites (B) were dissected from Anopheles stephensi mosquitoes 10 days after infected with Py-ICP-myc parasites and expression of ICP was monitored by an immunofluorescence assay (IFA) using a rabbit anti-myc antibody. Oocyst and midgut sporozoites were detected with a mouse anti-circumsporozoite protein (CSP) antibody. Nuclear staining was achieved with DAPI. Differential interference contrast (DIC) and fluorescent images were captured and processed using deconvolution. Scale bar is 5 μm.

Fig. S5. ICP localizes to the interior of early liver stage parasites but is released during late liver stage in vitro. Salivary gland sporozoites were isolated from Anopheles stephensi mosquitoes infected with Py-ICP-myc parasites. Isolated sporozoites were incubated with HepG2:CD81 cells seeded on the previous day. Parasites were allowed to invade and develop in vitro for 4 (A), 24 (B) or 43 (C) h before being fixed for IFA. Cells were stained with DAPI to stain the nuclei of both parasites and host hepatocytes. ICP was observed to be confined inside the PVM during early liver stage (A and B) but was released during late liver stage (C). Scale bar, 5 μm.

Fig. S6. Py-ICP may be essential in blood stage. (A) A double-crossover homologous recombination strategy was used to attempt to delete the Py-ICP gene. Two ∼ 800 bp DNA fragments were amplified from both the 5′UTR and 3′UTR regions of the ICP gene and were inserted into a b3D.DT∧H.∧D vector, which contains a pyrimethamine selectable marker (TgDHFR). The construct was linearized and transfected into Py blood-stage schizonts followed by injection into SW mice. Double recombinant parasites were not detected after pyrimethamine treatment by genotyping after several attempts. Only WT or 3′ single-crossover recombinant parasites were detected after several drug selection cycles (B). A single-crossover recombination strategy was used to attempt to knockout the ICP gene. A ∼ 1000 bp fragment was amplified from the middle portion of the ICP gene (distal to both the start and stop codons) and was inserted into a b3D.DTH.D vector as described above. The construct was linearized via digestion with EcoRI and was transfected into Py blood-stage schizonts followed by injection into SW mice. No successful recombination was detected after pyrimethamine treatment.

Fig. S7. Western blot analysis of ICP immunoprecipitations. The presence of epitope tagged Py-ICP following immunoprecipitation (IP) was confirmed by immunoblot (IB).

A. ICP was precipitated from Py-ICP-myc and Py-GFP (control) blood-stage parasite lysates using antibodies against the myc epitope (αmyc). Probing of blots with the Py-ICP antisera revealed the presence of Py-ICP in the precipitate of Py-ICP-myc, but not from Py-GFP lysates.

B. Py-ICP-RFA was precipitated from lysate using GFP antibodies (αGFP). Recombinant Py-ICP concentrated in the precipitate and was depleted from lysate flow-through. In contrast, Py-ICP remained in the flow-through lysate when immunoprecipitated with control mouse IgG.

Table S1. Co-immunoprecipitation experiments.

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Movie S1. Py-ICP containing vesicles are highly dynamic. Py-ICP-RFA-infected RBCs were immobilized on poly-lysine slides coated with antibody against the RBC protein Ter-119. Py-ICP-RFA parasites express recombinant ICP fused to GFP and Py-ICP localization was monitored via GFP expression. ICP localizes to the parasite cytoplasm and to vesicles. These vesicles are highly dynamic and move around in the RBC. Movie consists of 100 images taken over 86 s. Movie is sped up 8.6X.

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Movie S2. Infected RBCs are non-motile after adherence to a slide. Py-ICP-RFA-infected RBCs were immobilized on poly-lysine slides as described in Movie S1. DIC pictures were taken to ensure that the RBC is non-motile and all movement in Movie S1 comes from the parasite. Movie consists of 100 DIC images taken over 86 s. Movie is sped up 8.6X.

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