Mutant/mutation
The mutant lacks expression of B9 (PBANKA_080810).

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. B9 transcripts are found in sporozoites. Evidence is presented for translational repression of b9 transcripts in P. berghei sporozoites. The B9 protein is found in (young) liver stages. For P. falciparum B9 evidence is presented for a location at the plasmalemma membrane of liver stages. P. falciparum and P. berghei mutants lacking expression of B9 abort development during development of young liver stages

Phenotype
Normal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 PbΔb9 sporozoites none of the mice developed  blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 PbΔb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that PbΔb9 parasites arrest early after invasion of hepatocytes. PbΔb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of PbΔb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.
Combined, these analyses demonstrate that PbΔb9 has a critical role during early liver stage development, although a few liver stages can complete liver-stage development in the absence of B9. This phenotype is comparable to the phenotype of mutants lacking expression of the 6-Cys proteins P52 and P36.

Additional information
See also mutant RMgm-929 which expresses mCherry under the control of b9 regulatory sequences. No mCherry fluorescence signals were detected in blood-stages, oocysts and sporozoites, despite the presence of b9 transcripts in sporozoites. Strong fluorescence signals were detected in hepatocyte-culture 5 hours after the addition of mCherryb9 sporozoites. The non-fluorescent sporozoites indicate that b9 transcripts are translationally repressed and that the B9 protein is generated after a developmental switch to intrahepatic development. mCherry expression was observed both in intra-hepatic stages and in extracellular sporozoites that had been activated and  started to ‘round up’. Five hours post infection (hpi) fluorescence signals decreased; at 15hpi weak signals were detected in all parasites and no fluorescence was detected at 24hpi and 32hpi.

Supporting Figure S5
Generation of P. berghei mutants lacking expression of B9 (PbΔb9)
A. Schematic representation of the construct pL1439 targeting b9 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the hdhfr::yfcu selection marker (SM). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). B. Schematic representation of the construct pL1499 targeting b9 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the hdhfr selection marker (SM). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). The pL1499 construct was generated by an adapted ‘Anchor-tagging’ PCR-based method employing a 2-step PCR reaction. In the first PCR step two-flanking fragments of b9 were amplified from genomic DNA with the primers 4667/4557 (5’) and 4558/4668 (3’). Both primer 4557 and 4558 have 5’-terminal extensions homologues to the hdhfr selectable marker cassette (SM) obtained from plasmid pL0040 by digestion with restriction enzymes XhoI and NotI. Primers 4667 and 4668 have 5’-terminal overhang with an anchor-tag suitable for the second PCR step. In this step the fragments were annealed to either side of the SM with anchor-tag primers 4661/4662, resulting in the second PCR fragment. To remove the ‘anchor’, the second PCR fragment was digested with Asp718 and ScaI as primer 4667 contained an Asp718 restriction enzyme site and 4668 contained a ScaI site. C. Diagnostic PCRs (left) and Southern analyses of separated chromosomes (right) confirm the correct integration of the constructs in Δb9-a and Δb9-b. Primer pairs and amplicon sizes are shown in A. Δb9-a: selectable marker (M, primers 4698/4699); 5’-integration event (5’; primers 4288/L1858); 3’-interation event (3’; primers 4239/4289); ORF (O; primers 4737/4738). Δb9-b: selectable marker (M, primers L307C/3187); 5’-integration event (5’; primers 4288/4470); 3’-interation event (3’; primers 4471/4289); ORF (O; primers 4737/44738). For Southern analyses, pulsed field gel-separated chromosomes were hybridized with a 3’UTR pbdhfr probe. Mutant Δb9-a has been generated in the reference P. berghei ANKA line cl15cy1. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8 and the endogenous dhfr-ts gene located on chromosome 7. Mutant Δb9-b (right hand side) mutant has been generated in the reference P. berghei ANKA PbGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8, the reporter GFP-Luccon construct on chromosome 3, and the endogenous dhfr-ts gene located on chromosome 7.

Other mutants
RMgm-933: mutant lacking expression of B9 in the GFP-Luciferase expressing background. B9 was disrupted by double cross-over homologous recombination using a linear construct generated by the 'Anchor-tagging' PCR-based method.
RMgm-934: mutant lacking expression of B9. The mutant does not contain a selectable marker.
RMgm-935: Triple knock-out mutant lacking expression of B9, P52 and P36
RMgm-936: P.yoelii 17XNL mutant lacking expression of B9 in the GFP-Luciferase expressing background.