Mutant/mutation
In the 'promoter swap' mutant the promoter of the gama gene has been replaced by the promoter of ctrp gene (PBANKA_0412900). In addition, the mutant expresses the reporter GFP-Luciferase under the constitutive eef1a promoter.

In the manuscript two additional promoter swap mutants are described. In these mutants the promoter of the gama gene has been replaced by the promoter of cap380 gene (PBANKA_1218100) and the trap gene (PBANKA_1349800), respectively.

Selected promoters were CTRP, expressed only in ookinetes; CAP380, which is expressed throughout ookinete-oocyst development; and TRAP, which is expressed primarily in mature midgut sporozoites and salivary gland sporozoites

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.02.24.481744

Protein (function)
GAMA is a micronemal protein with a C-terminal GPI-anchor. P. berghei GAMA consists of 625 aa with a predicted molecular mass of ~71 kDa, (signal peptide cleavage results in a 69 kDa mass). In P. falciparum, two additional asparagine rich repeat regions result in a larger 738 aa protein (predicted molecular weight ~85 kDa, 83 kDa after cleavage of the signal peptide). For P. berghei, SignalP-5.0 predicts a signal peptide with a cleavage site at S21/L22. For P.falciparum, cleavage is predicted at the same locus, A21/L22.
P. berghei and P. falciparum share 44.8% identity across the full-length sequence, with areas of higher conservation found towards the N-terminus (residues 64-347 = 60% identity) and C-terminus (residues 501-700 = 60% identity) split by a central repeat region of low shared identity (residues 348-500, with predicted disorderly structure. The C-terminal transmembrane domain is predicted to be cleaved and appended with a GPI anchor. Downstream of signal peptide cleavage, P. falciparum contains seven cysteine residues prior to the central repeat region and one downstream which are conserved across all Plasmodium species investigated. P. berghei and other rodent infective species of Plasmodium bear an additional cysteine at position 152 in the alignment. GAMA is present in all Plasmodium species and indeed all other intracellular apicomplexans.

Phenotype

A mutant lacking GAMA (∆GAMA; RMgm-5188) showed the following phenotype:
Wild type-like blood stage development. Wild type numbers of ookinetes are produced. (Strongly) reduced oocyst production and strongly reduced numbers of midgut- and salivary gland sporozoites. Evidence is provided for a defect in the egress of sporozoites from oocysts. Sporozoites show strongly reduced motility. (Midgut) sporozoites are unable to establish an infection in mice (after intravenous injection). 

The promoter swap mutants, (ctrp)gama, (cap380)gama and (trap)gama showed the following phenotype:
1. (ctrp)gama
Wild type numbers of oocyst. Reduced numbers of salivary gland sporozoites. (Midgut) sporozoites showed a strongly reduced capacity to establish an infection in mice (after intravenous injection).

2. (cap380)gama
Oocysts exhibited a striking developmental defect, whereby oocysts exhibited lower level GFP expression that wild type and did not grow beyond ~day-five size. No sporozoite formation

3.(trap)gama
Reduced oocyst production; no (strongly reduced) egress of sporozoites from oocysts; however, motility of midgut sporozoites was not affected. Reduced capacity to establish an infection in mice by midgut sporozoites (after intravenous injection). However, salivary gland sporozoites showed wild type like infectivity

In conclusion: in none of the promoter-swap mutants, the promoter swap could rescue (completely) the phenotype(s) observed in the mutant lacking expression of GAMA (∆GAMA; RMgm-5188)

Additional information

Other mutants