Mutant/mutation
The mutant lacks expression of LISP2 and PLASMEI2 and expresses GFP under the control of the constitutive eef1a promoter.

Protein (function)
LISP2: The protein contains a predicted Plasmodium 6-cysteine motif. Several studies analysing expression of this protein in P. berghei provide evidence for specific expression in liver stages. The protein has been named LISP2 and sequestrin.
PLASMEI2: Mei2 is a member of the largest family of RNA binding proteins (RBPs) – those that contain a RNA recognition motif (RRM), a stretch of 70-90 amino acids that contain two consensus RNA-interacting motifs, RNP1 and RNP2. RRM-containing proteins are subdivided into ten separate families (RRM_1 thru RRM_10) based on shared amino acid identities between members of each family and Mei2 contains a C-terminal RRM_2, thought to be unique to fungi and plants.
Plasmodium contains a single Mei2-like gene (from herein referred to as PlasMei2). In the P. yoelii rodent malaria model, Plasmei2 is only expressed during liver stage development and is localized in distinct cytoplasmic structures reminiscent of P-granules

Phenotype
The P. yoelii lisp2− plasmei2− GAP exhibits complete late liver stage developmental arrest.

Additional information
The P. yoelii lisp2− plasmei2− GAP exhibits complete late liver stage developmental arrest.

We  compared late liver stage development of the lisp2− plasmei2− dual gene deletion parasite with lisp2− and plasmei2− single gene deletion parasites as well as wild-type parasites. Groups of Swiss Webster (SW) mice were challenged i.v. with 50,000 sporozoites of each strain and liver stage developmental progression was measured based on luciferase activity at 43 h, a time point late in liver stage development but before the complete maturation and liver stage-to-blood stage transition of wild-type parasites. Parasite development, based on luciferase expression was indistinguishable between single and dual gene knockout parasite strains and wild-type parasites, suggesting that all three GAP progress to late liver stage development. To further assess the phenotype of liver stage development, parasites were visualized by indirect immunofluorescence assay (IFA) at 43 h of liver stage development, using antibodies recognizing the PVM protein Hep17 and the endoplasmic reticulum protein BiP. Liver stages of GAP developed to late schizogony and appeared similar to the wild type based on expression patterns of Hep17. However, the plasmei2− liver stages showed a DNA segregation phenotype and aberrant BiP expression, and this phenotype was also observed in the lisp2− plasmei2− liver stages.

To quantify liver stage growth of the gene knockout parasite lines, liver stage size was determined at 43 h in comparison to that of the wild type, and no significant differences were seen among all analyzed strains. Thus, P. yoelii lisp2− plasmei2− GAP retains the late-liver stage arresting phenotype of the single gene deletion parasites and phenotypically resembles the plasmei2− single gene knockout. To determine whether the lisp2− plasmei2− GAP persisted in the liver, we measured liver stage luciferase activity of the lisp2− plasmei2− GAP over time after sporozoite inoculation in C57BL/6 mice. At 72 h, both lisp2− plasmei2− GAP and fabb/f− GAP luciferase activities had significantly decreased, with lisp2− plasmei2− GAP activity at background levels, whereas fabb/f− GAP luciferase activity was still significantly higher than background. This suggests that the fabb/f− GAP persists for longer than the lisp2− plasmei2− GAP. At 96 h, both GAP had luciferase activities comparable to background.

As we had observed the lowest frequency of breakthrough infections among single gene knockouts in the plasmei2− parasite, we next determined if comparable high doses of lisp2− plasmei2− GAP would lead to breakthrough infection. We thus performed challenges i.v. with 200,000 or 500,000 lisp2− plasmei2− sporozoites in cohorts of highly susceptible BALB/cByJ mice for each dose. We did not observe any breakthrough to blood stage infection (0/29 for 200,000 and 0/26 for 500,000. This finding shows that the P. yoelii lisp2− plasmei2− gene knockout combination constitutes a synthetic lethal phenotype in which two sublethal single gene deletions synergize to cause a completely penetrant lethal phenotype. In consequence, the lisp2− plasmei2− GAP is completely attenuated at late liver stage.

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