Mutant/mutation
Three different transgenic lines were produced that express GFP under control of different fragments of the sub1 promoter region. These constructs were integrated into a silent locus into the genome.

The first two constructs, named SUB1/prom1 and SUB1/prom2, included the DNA sequence spanning the region between 1279 bp and either 25 bp or 450 bp upstream of the pbsub1 ATG, respectively including or excluding an intron present in the pbsub1 5’ UTR. The resulting transgenic parasite lines were both found to specifically display reporter gene expression in the cytoplasm of gametocytes, with no detectable expression in asexual blood stage parasites. We then generated a third construct, where a larger DNA segment, extending from 2385 bp to 450 bp upstream of the pbsub1 ATG was fused to gfp. In the obtained transgenic line, named SUB1/prom3, the reporter gene was expressed both in gametocytes and schizonts

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.
The subtilisin-like protease SUB1 is expressed both in liver and blood schizonts, then secreted into the PV from vesicular organelles called exonemes just prior to schizont rupture. The proteolytic activity of SUB1 has been shown to be important for maturation and egress of invasive merozoites from both infected erythrocytes and hepatocytes.
During Plasmodium falciparum merozoite egress, PfSUB1 carries out the maturation of a family of papain-like proteins called the serine repeat antigens (SERAs), while for merozoite maturation the role of SUB1 is to undertake the processing of several merozoite surface proteins (MSPs).

Phenotype
Three different transgenic lines were produced that express GFP under control of different fragments of the sub1 promoter region. These constructs were integrated into a silent locus into the genome.

The first two constructs, named SUB1/prom1 and SUB1/prom2, included the DNA sequence spanning the region between 1279 bp and either 25 bp or 450 bp upstream of the pbsub1 ATG, respectively including or excluding an intron present in the pbsub1 5’ UTR. The resulting transgenic parasite lines were both found to specifically display reporter gene expression in the cytoplasm of gametocytes, with no detectable expression in asexual blood stage parasites. We then generated a third construct, where a larger DNA segment, extending from 2385 bp to 450 bp upstream of the pbsub1 ATG was fused to gfp. In the obtained transgenic line, named SUB1/prom3, the reporter gene was expressed both in gametocytes and schizonts

These results suggested that the pbsub1 gene includes two distinct regulatory regions: one, closest to the ATG, that specifically drives gene expression in the sexual stages and another, further upstream, necessary to drive expression also in asexual stages. In all the SUB1/prom lines, the GFP fluorescence signal significantly increased in activated male gametocytes, indicating upregulation of pbsub1 gene expression during male gamete formation.

Additional information
From the paper:
'It was recently shown that PbSUB1 is also expressed in P. berghei sexual stages, but the function of the enzyme in gametocytes/gametes and its potential substrates in these stages are still unknown. Here we show that PbSUB1 is selectively expressed in male P. berghei gametocytes and that it localizes to male osmiophilic bodies (MOBs). Using conditional depletion of PbSUB1 expression or enzyme activity, we show that the protease plays a role in male gamete egress.
We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation.'

'We next investigated the subcellular localization of PbSUB1 in mature P. berghei gametocytes by immunofluorescence (IFA) using an anti-PbSUB1 antibody. Antibody specificity was confirmed in gametocytes by double IFA with an anti-HA monoclonal antibody on a P. berghei line in which the endogenous pbsub1 gene had been modified with a triple HA-tag at the C-terminal end [see mutant RMgm-973). The two signals co-localized in gametocytes, producing a punctate pattern. Double IFA with antibodies to the nuclear protein SE translocation proto-oncogene like (SET), which is highly abundant in male gametocytes and with the female gametocyte marker G377, showed that PbSUB1 expression in gametocytes is male-specific. Of one hundred SUB1-positive gametocytes examined, none showed a fluorescence signal with the G377 antibody. Co-localization experiments with an antibody against the Male Development Protein 1 (MDV1), a gametocyte-specific protein associated with OBs in both P. berghei male and female gametocytes, showed that PbSUB1 specifically localizes to these secretory organelles in males (MOBs)'

Other mutants