Mutant/mutation
The mutant lacks expression of B9 (PBANKA_080810). The mutant does not contain a drug selectable marker (-sm) which has been removed from mutant RMgm-932 by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC)(Braks et al., 2006, Nucleic Acids Res 34, e39).

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 S6
Generation of a P. berghei Δb9 mutant which is selectable marker-free (PbΔb9-sm)
A. Schematic representation of the generation of PbΔb9-sm using the marker-recycling method by negative selection. The b9 disruption construct pL1439 (see Figure S5) containing the hdhfr::yfcu selectable marker (black arrows) flanked by the recombination sequences (3’pbdhfr, shaded boxes) targets the b9 locus by double cross-over homologous recombination at specific target regions (gray boxes). Δb9-a mutant is obtained after positive selection with pyrimethamine (see Figure S5). Subsequently, marker-free Δb9-sm mutant is selected by negative selection using 5-FC. Only parasites that have excised the hdhfr::yfcu marker from their genome (PbΔb9-sm), achieved through a recombination/excision can then survive (and be selected) the negative selection procedure. B. Southern blot analysis confirms the correct genotype of PbΔb9-sm. DNA digested with NdeI was hybridized with a probe recognizing the 5’UTR of b9 probe. The localization of NdeI restrictions sites (N) and the expected size of the fragments are shown in A: WT (wild type).

Other mutants
RMgm-932: mutant lacking expression of B9 in the WT background. B9 was disrupted by double cross-over homologous recombination using a plasmid DNA construct.
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-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.