Generation of <i>ipis2-</i>[GFP-Luc;mCherry] and <i>ipis3</i>-[GFP-Luc;mCherry] was achieved through double homologous recombination and confirmed by PCR.

(A) Homology regions (labeled as the 5’ and 3’ genomic regions) flanking the gene of interest were cloned into the GOMO GFP-Luciferase vector. The resulting parasites lack either IPIS2 or IPIS3, express both mCherry and GFP-luciferase, and contain a DHFR-γFCU drug resistance cassette. (B) Successful integration of the construct was verified by PCR amplification of the products indicated in grey in panel A. Produ cts were sequenced to confirm the genomic editing. (C) Excision of the mCherry and drug resistance expression cassette from the recycled lines was confirmed by the presence of product using the mCherry excision (mCherry exc) and absence of the 3’ integration (3’) product. (TIF)</p

Phenotypes of <i>ipis2</i>- and <i>ipis3</i>- in mosquito and blood infection stages.

(A) Midguts of mosquitoes were harvested 14 days after an infectious blood meal and microscopy was used to determine the presence or absence of oocysts. (B) The number of sporozoites per mosquito were calculated at least 17 days following infected blood meal. (C) Liver stage ipis2- and ipis3- parasites grow similarly to wild-type parasites in vitro. HepG2 cells infected with either wild-type, ibis2-[GFP-Luc;mCherry], or ibis3-[GFP-Luc;mCherry] sporozoites were fixed 48 hours after infection, and the parasites were stained with antibodies against PbHSP70. Exo-erythrocytic forms were imaged with the fluorescence microscope, and their size was determined using FIJI. ns, not significant, unpaired t-test. (D) Parasitemia in the blood of mice following infection with 1000 sporozoites was quantified by microscopy from Giemsa-stained blood smears. *, P ipis2- and ipis3- are less pathogenic in mice than wild-type P. berghei. Survival of mice after infection with 1000 sporozoite. n = 5 (ipis2-), n = 10 (ipis3-). ns, not significant **, P (TIF)</p

PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3), PBANKA_1400700 and putative <i>P</i>. <i>vivax</i> orthologs localize to discrete structures in the cytoplasm of infected erythrocytes.

A) PBANKA_0524300-mCherry, PBANKA_0623100-mCherry, and PBANKA_1400700-mCherry colocalize with the IBIS marker IBIS1-GFP in the erythrocyte cytoplasm. Infected cells were fixed and labeled with anti-GFP and anti-mCherry antibodies to amplify the fluorescent signal. Scale bars, 5 μm. B) Erythrocytes infected with P. berghei expressing the HA-tagged P. vivax proteins PvTRAg8 or PvTRAg2, which are putative orthologs of IPIS2 and IPIS3, respectively, were fixed and labeled with anti-HA antibodies. Scale bars, 5 μm.</p

<i>ipis2-</i> and <i>ipis3-</i> contribute to blood-stage growth efficiency during <i>P</i>. <i>berghe</i>i infection.

A) The appearance of ipis3- parasites in the blood following sporozoite injection is delayed in comparison to wild-type. C57BL/6 mice were infected with 1,000 sporozoites of either ipis2-, ipis3- or wild-type parasites, and blood-stage infections were monitored starting from day 3 post-infection with Giemsa-stained blood smears. Statistics: Log-rank (Mantel-cox) test; n = 5 (ipis2-), n = 10 (ipis3-). B) The growth of ipis2- and ipis3- parasites in the liver does not differ significantly from wild-type P. berghei. The relative parasite burden in the liver was measured by qPCR of RNA extracted from livers forty-two hours after intravenous injection of either ipis2-, ipis3-, or wild-type sporozoites. Levels of Pb18s rRNA were normalized to levels of mouse GAPDH RNA in each infected liver. Statistics: unpaired two-tailed t-test; n = 5; ns: not significant. C) The ipis2- and ipis3- lines do not grow as efficiently as wild type in the blood of co-infected animals. Mice were infected with equal amounts of wild-type P. berghei-infected erythrocytes and ipis2-[mCherry; PyrS] or ipis3-[mCherry; PyrS] infected erythrocytes. Parasitemia was measured by flow cytometry between days 4–7 after infection. * p ipis2- line, a statistically significant increase in mCherry signal was detected; however the extent was negligible. * p <0.05, unpaired two-tailed t-test.</p

Integration of IPIS3-mCherry and IPIS2-mCherry expression into the <i>ipis2</i>- and <i>ipis3</i>- lines, respectively.

(A) ibis3-[GFP-Luc; PyrS] or ibis2-[GFP-Luc; PyrS] were used as recipient lines for transfection of IPIS2 and IPIS3 B3D+mCherry vectors, respectively. (B) Successful integration of the plasmid was confirmed by PCR. The absence of product for the 5’ and 3’ integration PCRs in the wild type, ipis2-[GFP-Luc;PyrS], and ipis3-[GFPLuc;PyrS] were negative controls. The PCR reactions labeled “IPIS2” or “IPIS3” amplified a region in the IPIS2 or IPIS3 genes, which are absent from the recipient knockout lines. (TIF)</p

Mass-spec results of immunoprecipitation experiments.

(XLSX)</p

mCherry tagged PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3) colocalize with IBIS1-GFP at the liver-stage parasitophorous vacuole.

A) Protein features of candidate proteins that coprecipitated with IBIS1. B) Hepatoma cells were infected with sporozoites after the cross-fertilization of IBIS1-GFP and either PBANKA_0524300-mCherry, PBANKA_0623100-mCherry or PBANKA_1400700-mCherry transgenic parasite lines. Cells were fixed at indicated time points and the mCherry and GFP signals were amplified by using anti-RFP and anti-GFP antibodies. Scale bars, 10 μm.</p

Primers used in this study.

(PDF)</p

Localization of mCherry tagged PBANKA_0524300 (<i>IPIS2</i>), PBANKA_0623100 (<i>IPIS3</i>), and PBANKA_1400700 in the liver stage of infection.

(A) Hepatoma cells were infected with transgenic parasites expressing the indicated proteins fused to the fluorescent protein mCherry. After fixation at the time points indicated, the infected cells were labelled with an anti-RFP antibody to amplify the mCherry signal. Scale bars, 10 μm. (B) P. berghei expressing both GFP- and mCherry-tagged proteins following cross fertilization were imaged by confocal microscopy 48 hours after infection of hepatoma cells. Scale bars, 10 μm (TIF)</p

Immunoprecipitation of <i>P</i>. <i>berghei</i> proteins from infected red blood cells identifies proteins that co-precipitate specifically with IBIS1-mCherry and EXP2-mCherry.

A) The indicated mCherry-tagged proteins were immunoprecipitated from lysates of late stage P. berghei using RFP-specific nanobodies and co-precipitating proteins were identified by LC-MS/MS. B) P. berghei proteins that were detected in the IBIS1-mCherry sample in at least two of the experimental replicates are depicted in the heatmap, which indicate the mean normalized LFQ intensities of detected proteins of three independent experiments. Each row represents a protein detected in the IBIS1-mCherry eluates. The columns represent the immunoprecipitates prepared from the mCherry-expressing parasite lines indicated in (A) (1: IBIS1-mCherry, 2: mCherry, 3: PEXEL-mCherry, 4:EXP2-mCherry). Proteins that were enriched in the IBIS1-mCherry pulldown relative to at least two of the three controls (top 10% of the normal distribution) are annotated. Those in bold were enriched relative to all three controls. Proteins marked with a pink dot were followed up further in this study. C) P. berghei proteins that were detected in the EXP2-mCherry sample in at least two of the experimental replicates are depicted in the heatmap, which indicate the mean normalized LFQ intensities of detected proteins of three independent experiments. Each row represents a protein detected in the EXP2-mCherry eluates. The columns represent the immunoprecipitates prepared from the different mCherry-expressing parasite lines indicated in (A). Proteins marked with dark blue dots are confirmed to play roles in PTEX-mediated export, and those marked with light blue dots were previously found biochemically associated with components of the PTEX complex [33,34].</p