Fig. S1. A. clustalw alignment of PPLP2 encoded by P. berghei gene PBANKA_143240 (top) and P. falciparum gene PF3D7_1216700 (bottom). The predicted signal peptide is indicated by a yellow line and the membrane attack complex/perforin (MACPF)-like domain by a green dashed line (amino acids 488–712 in P. berghei, 562–786 in P. falciparum). The apicomplexan perforin-like (ApiPLP) motif (W-x(2)-[FL]-[FI]-x(2)-[FY]-G-T-H-x(7)-G-G) is marked with a solid blue line (all below the sequences). The regions chosen for production of the two PPLP2 antisera are indicated by a light red line (antiserum A) and by a dark red line (antiserum B); both lines above the sequences.

B. Specificity of PPLP2 antiserum A. No PPLP2-specific signal was detected in P. berghei male gametocytes lacking pplp2. The SET protein (green) was used as a marker of the nucleus of male gametocytes.

C. No PPLP2 signal was detected in P. berghei WT female gametocytes. The characteristic small female nucleus was stained with the SET antibody and the PVM was stained for the SEP1 protein (both green). Both of these antisera were produced in rabbits. The PPLP2 antiserum A is of mouse origin and the secondary antibody conjugated to Alexa Fluor 555 (red). These experiments were carried out in parallel with labelling of WT male gametocytes, which were positive for PPLP2.

D. Specificity of PPLP2 antiserum B. No labelling was detected on gametocytes immunolabelled with a mouse antiserum against the maltose-binding protein (MBP) tag. An antibody against the Pfs230 antigen (red) identified the gametocyte.


Fig. S2. Immunofluorescence assays of P. falciparum gametocytes at 15 min post activation showed PPLP2 labelling (green) in association with shed membranes (indicated by arrows). The exflagellating microgametocyte is highlighted by labelling of Pfs230 (red). Arrowheads indicate microgametes. The corresponding differential interference contrast (DIC) images are shown. Bar, 5 μm.


Fig. S3. Experimental genetic strategy for disruption of pplp2 in Plasmodium berghei.

A. Maps of the WT pplp2 locus, the disruption vector pΔpplp2, and the pplp2 locus after integration. The WT locus comprises a single exon encoding the PPLP2 protein. A double-cross-over strategy was employed to replace the middle of the coding region with the DHFR/TS cassette. The two targeting fragments are depicted as double arrows in blue. Primers used for diagnostic PCR (black arrows), the expected fragments (purple double arrows) and their size are indicated. Note that the DHFR/TS cassette is not shown to scale.

B. Diagnostic PCR reactions on gDNA from WT (lanes 1, 4 and 7) and the clone pplp2(−) cl 1 (lanes 2, 5 and 8). A non-cloned population from an independent transfection was also included (lanes 3, 6 and 9). The absence of contaminating WT parasites in the transfected parasites is evidenced by the primer pair peraF/peraR. The primer pairs are indicated on the top and molecular weight markers to the left.

C and D. Southern blot analysis of pplp2(−) cl 1.

C. The pplp2 locus of WT (top) and mutant parasite (bottom) gDNA. The DNA was digested with EcoRI and HindIII and the sizes of the fragments generated are shown.

D. Southern blot analysis of gDNA from WT and the pplp2(−) cl 1 digested by EcoRI and HindIII. The blot was hybridized with a mixed probe containing the two fragments used for the targeting construct (red lines in C).


Fig. S4. Measurements of the diameter of the super-flagella of the pplp2(−) mutant.

A. Exflagellating male gametocytes were stained with the TAT mAb recognizing the tubulin of the axonemes. (a–k) Axonemes of the pplp2(−) mutant. (l) A representative example of WT single axoneme. Scale bar 5 μm.

B. Measurement of axonemes of the pplp2(−) mutant and comparison with WT single axonemes. The diameter of the axonemes was measured using ImageJ. Forty WT axonemes were measured and the average size determined was 300 nm ± 62 nm (standard deviation). The average diameters of three measurements per mutant axonemes and the calculated number of axonemes per flagella are shown in the table for each panel a–k.


Fig. S5. Examples of super-flagella of 1,10-phenanthroline-treated P. falciparum gametocytes. Gametocytes were activated in the presence of 1,10-phenanthroline as described (Sologub et al., 2011). At 20 min post activation, the PVM had ruptured, while the EM was still detectable. (A and B) Axonemes had clustered to bundles, forming a super-flagellum. The axonemes reside inside the same flagellar membrane. AX, axoneme; EM, erythrocyte membrane; PDM, parasite-derived membrane; SF, super-flagellum. Bar, 2 μm (A), 0.5 μm (B).


Table S1. Sequence of the oligonucleotide primers used in this work.


Movie S1. Exflagellation of WT male gametocyte. Separate flagella are visible. In the final frames an exflagellation centre is formed. Images captured at one frame per second for 100 s. (avi)


Movie S2. Exflagellation of pplp2(−) mutant. A single beating super-flagellum is visible. Images captured at one frame per second for 54 s. (avi)


Movie S3. Four exflagellating pplp2(−) mutant males, each having one super-flagellum. No exflagellation centres are formed. Images captured at one frame per second for 90 s. (avi)


Movie S4. One exflagellating pplp2(−) cell. In this case two flagella are seen in close proximity to each other. Images captured at one frame per second for 58 s. (avi)


Movie S5.pplp2(−) sample treated with saponin before activation. Eight separate flagella are attached to the residual cell. Images captured at one frame per second for 84 s. (avi)


Movie S6. WT sample treated with saponin before activation. Eight separate flagella are attached to the residual cell. Images captured at one frame per second for 120 s. (avi)


Movie S7.pplp2(−) sample treated with equinatoxin II before activation. Eight separate flagella are attached to the residual cell. Images captured at two frames per second for 88.5 s. (avi)


Movie S8.pplp2(−) sample treated with saponin 14 min after activation. A male with a persisting super-flagellum is visible. Images captured at one frame per second for 82 s. (avi)

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