Mutant/mutation
The mutant lacks expression of IPIS2. In addition it expresses GFP-Luciferase under control of the constitutive eef1a promoter. It does not contain a drug-selectable marker (removed by negative selection; GOMO-constructs were used to delete the gene; see mutant RMgm-1027).

Protein (function)
PBANKA_052430 is tryptophan/threonine-rich antigen, putative. Different Plasmodium species express multiple tryptophan-rich proteins which are encoded by genes located near/in subtelomeric regions that often do not have a syntenic location between different species. The orthology between genes of different Plasmodium species is therefore less clear.
In this study the protein was identified by co-precipitation with exported Plasmodium proteins present in membrane structures (Maurer's cleft-like) in the cytoplasm of the infected red blood cell. The protein does not have predicted secretion or export signals, but do have a tryptophan-threonine rich domain (PF12319) that is conserved in several proteins across Plasmodium species. 
Unlike IBIS1, which appears to be exclusive to the rodent Plasmodium species, tryptophan-rich domain-containing proteins are found across the Plasmodium genus. Syntenic orthologs of IPIS2 are conserved across the Plasmodium clade and are expressed in P. vivax, P. knowlesi, P. malariae and P. cynomolgi. Notably, no syntenic IPIS2 ortholog exists in the Laverania Plasmodium species P. reichenowi or P. falciparum. IPIS3 has syntenic orthologs only in the rodent Plasmodium species.

Phenotype
(Slightly) reduced growth/multiplication rate of asexual blood stages. Schizonts show reduced sequestration in inner capillaries of organs and are detected in the peripheral blood circulation (fifteen-fold more schizonts in the peripheral blood compared to wild type). Evidence for reduced binding to host CD36 in in-vitro assays).

Normal/wild-type-like sporozoite formation and liver stage development.

Additional information
PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3) and PBANKA_1400700, were found to be localized to IBIS1-positive membranes in the cytoplasm of infected red blood cells. The first two proteins both in red blood cells and (in the PVM of) liver stages. These proteins were termed intra-erythrocytic Plasmodium-induced structure proteins 2 and 3 (IPIS2 and IPIS3, respectively). PBANKA_1400700 was only exported into red blood cells but remained in the cytoplasm of liver stages.

Analyses of mCherry-tagged proteins (see RMgm-5227, RMgm-5228, RMgm-5229) showed the following:

- IPIS2 and IPIS3 localizes to punctate structures in the cytoplasm of infected red blood cells
- A previous study reported exported GFP-tagged PBANKA_1400700 evenly distributed throughout the erythrocyte cytoplasm. In contrast, we detected the exported fusion protein in a punctate pattern. In both this study and the previous study, much of the fluorescently tagged PBANKA_1400700 remained associated with the parasites.
- All three proteins were also detected in the liver infection stage. PBANKA_0524300-mCherry and PBANKA_0623100-mCherry were first detected 24 hours post-infection in the developing liver-stage forms and surround the parasite in a pattern indicative of the liver-stage parasitophorous vacuole and associated tubovesicular network. PBANKA_1400700-mCherry was detected throughout the liver stage, but it was not secreted from the parasite despite the presence of a signal peptide.
- To determine if the localization of these three proteins overlaps with IBIS1, the parasite lines in which these proteins are tagged with mCherry were crossed with a  line in which IBIS1 is tagged with GFP via co-infections of mosquitoes. Signals from PBANKA_0524300-mCherry and PBANKA_0623100-mCherry overlapped considerably with the IBIS1-GFP signal surrounding the liver-stage parasites and in infected red blood cells, colocalize with IBIS1-GFP. 
Although the majority of PBANKA_1400700-mCherry remains parasite-associated, the exported mCherry signal colocalizes with IBIS1-GFP.

In order to determine if the putative orthologs from P. vivax also targeted the membrane structures in the cytoplasm of Plasmodium-infected red blood cells, we generated P. berghei lines that expressed HA-tagged PvTRAg8, the syntenic P. vivax ortholog of IPIS2 (PVP01_0532600) or HA-tagged PvTRAg2, which shares similarity to IPIS3 (PVP01_0202200). These genes were integrated into the P. berghei genome such that they are expressed under the control of the IPIS2 or IPIS3 promoters, respectively. Both PvTRAg8-HA and PvTRAg2-HA are exported efficiently by P. berghei and localize to discrete structures in the cytoplasm of infected erythrocytes.

The localization of IPIS2 and IPIS3 to the intraerythrocytic membrane structures and their requirement for efficient blood-stage growth prompted us to investigate whether the membrane structures are still formed in the cytoplasm of erythrocytes infected with the ipis2- and ipis3- knockout lines. In order to visualize these structures in the knockout lines, we tagged IPIS2 with the fluorescent protein mCherry in the IPIS3-deficient line, and likewise, tagged IPIS3 with mCherry in the line lacking IPIS2. The IPIS proteins still appear in punctate structures distributed across the erythrocyte cytoplasm in the absence of either IPIS2 or IPIS3

Other mutants