Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of SUB1 (subtilisin-like protease 1). The mutant expresses the yeast Flp recombinase under the control of the uis4 promoter  and contains a FRTed ORF (open reading frame) of the sub1 locus. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome. This mutant has been generated by replacement of the endogenous sub1 gene by an sub1 gene with a FRTed ORF in the mutant RMgm-269 that expresses Flp and GFP.
The sub1 ORF is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269). Removal of the FRTed ORF of sub1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the ORF of sub1 which was flanked by FRT sequences.

Protein (function)
The subtilisin-like protease SUB1, a bacterial-like serine protease, is a Plasmodium merozoite product essential for the parasite erythocytic cycle. In P. falciparum, SUB1 was shown to have a dual function. It is crucial for merozoite egress from erythrocytes and renders released merozoites competent for a new round of erythrocyte invasion. In the process of P.falciparum merozoite egress from erythrocytes, SUB1 carries out the maturation of the family of papain-like proteases called serine repeat antigens (SERAs). It remains unknown whether the SERAs have a direct role in membrane disruption, or whether they in turn maturate other effectors, but it is established that their SUB1-dependent maturation is essential for the egress of merozoite. The reported role of SUB1 in P.falciparum merozoite invasion is to undertake the processing of merozoite surface proteins (MSPs), including MSP1. After SUB1 maturation, the membrane-anchored Cterminal 42-kDa fragment of MSP1, remains associated with the other MSP1 fragments and additional partners, including MSP6 and MSP7.

Phenotype
The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence SUB1 expression specifically in liver stages. Removal of the FRTed ORF of sub1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the FRTed ORF of sub1.

PCR analysis of genomic DNA of the mutant sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1.
The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced liver stages at 24 and 48 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number and size to that of wild type sporozoites.
Evidence is presented for normal development of liver merozoites but a reduced egress of liver merozoites from the mature liver schizonts. Evidence is presented that the reduced egress is due to lack of rupture of the PVM indicating a role of SUB1 in PVM rupture. in addition, evidence is presented for a role of SUB1 in processing of SERA3 and MSP1. SUB1-deficient liver stage merozoites are unable to establish a blood stage infection.

Additional information
See also RMgm-972 for an independent mutant containing a FRTed ORF of the sub1 locus. Analyses of liver stages of this mutant provided evidence for the following: 48 hour mutant liver stages were significantly smaller than wild type liver stages. The majority of the PbSUB1-deficient parasites completely lacked a detectable MSP1 signal. Multiple nuclei were observed in the early PbSUB1-deficient schizonts, indicating normal nuclear replication, but in later stages many of the schizont nuclei appeared condensed and abnormal. Mutant liver stages were deficient in merosome formation. SUB1-deficient liver stages are unable to establish a blood stage infection.

Other mutants
RMgm-972: An independent mutant containing a FRTed ORF of the sub1 locus
RMgm-973: A mutant expressing a HA-tagged version of SUB1