Silencing of LRIM1 and APL1 together has the same effect on midgut infections of GFP-expressing P. berghei as silencing either gene alone. Experiments were performed 7 days post infection with injection of dsGFP serving as the control. Horizontal lines indicate the median. (A) Oocyst numbers in susceptible mosquitoes. Whether injected together or separately dsLRIM1 and dsAPL1 treated mosquitoes show a significant (P<0.001) increase of oocysts numbers compared with the control but no difference between them. (B) Live oocysts (green dots) and melanized ookinetes (grey squares) in refractory mosquitoes. Both the increase in live and the decrease in melanized parasites in dsLRIM1 and dsAPL1 injected mosquitoes are significant (P<0.001) compared with the control. (C) Phase contrast (top row) and fluorescent (bottom row) photomicrographs of parasites in the midguts of refractory mosquitoes following injection of dsRNA. Inset in the control shows a melanized ookinete at higher magnification.

The LRIM1/APL1C complex interacts with TEP1-C. (A) A. gambiae cultured cells were co-transfected with transgenes for LRIM1^HIS and APL1C^HIS (+) or GFP (−) as a control. His-tagged proteins were affinity purified from conditioned medium (CM). Starting CM and captured (Bound) samples were analyzed by western blot with anti-His (left panel) or TEP1 antibodies (right panel), or silver stained (middle panel). Indicated are: black arrows, LRIM1^HIS and APL1C^HIS monomers; black arrowhead, LRIM1^HIS/APL1C^HIS complex (black); white arrowheads alternative LRIM1^HIS and APL1C^HIS complexes; red arrow, putative TEP1-C bands; black double, TEP1-F and TEP1-C. (B) TEP1 western blot of hemolymph extracted from dsGFP, dsLRIM1, dsAPL1 and dsTEP1 injected mosquitoes. Probing with SRPN3 served as a loading control. (C) Confocal analysis of TEP1 immunolocalization (red) in midgut epithelia from susceptible mosquitoes 30 hours post-infection with GFP-expressing P. berghei (green). A mixture of live (GFP⁺, open triangles), dead (TEP1⁺, white arrows) and dying (GFP⁺ and TEP1⁺, box and inset) parasites are detected in control injected (dsGFP) mosquitoes. In contrast, no dead or dying parasites were observed in mosquitoes injected with dsLRIM1, dsAPL1, or dsTEP1 in three independent experiments. Scale bars in dsGFP panel and inset are 10μm.

LRIM1 and APL1C circulate in the hemolymph as a disulfide-bonded multimeric complex. (A) Schematic representation and predicted molecular weights of the LRIM1 and APL1C proteins. Features indicated: asterisk, predicted N-linked glycosylation site; red line, cysteine residue; black bracket, conserved cysteine-rich region. Colored boxes denote: black, signal peptide; white, PANGGL repeat; blue, LRR repeats; green, coiled-coil domain. Orange horizontal lines show the location of peptides used to generate antibodies. (B) LRIM1 and APL1C western blots of hemolymph before (−) and after (+) treatment with PNGase-F. Arrows indicate the position of the endogenous proteins and open arrowheads indicate deglycosylated forms. (C) LRIM1 and APL1C western blots of hemolymph under non-reducing (NR) and reducing (R) conditions (D) LRIM1 co-immunoprecipitates with the APL1C complex. Hemolymph (H) was used for immunoprecipitation with an antibody against APL1C (+) or mock treated (−). Left and middle panels show western blot analysis of samples run under NR conditions and probed with APL1C and LRIM1 antibodies, respectively. Right panel shows western blot under R conditions, probed with LRIM1 antibody. Immunoprecipitated complex detected with APL1C antibody (white arrowhead), LRIM1 antibody (white arrowhead) and LRIM1 monomer (arrow) are indicated. (E) Silencing by injection of either dsLRIM1 or dsAPL1 results in specific loss of the LRIM1/APL1C complex from the hemolymph compared to control (dsGFP). Probing with SRPN3 served as a loading control. The LRIM1 and APL1C blots were cropped at the position of the complex since no other bands were detected.