Mutant/mutation
The mutant lacks expression of the small subunit (ssu) of S1(c-type) ribosomal rna gene unit.
In addition, it contains a DiCre expression cassette. The Dicre gene (the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively) is under control of the constitutive hsp70 promoter. The DiCre expression cassette is introduced into the silent p230p locus. In addition it expresses mCherry and does not contain a drug-selectable marker (see below).

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.11.14.623520

Protein (function)
P. yoelii contains four distinct copies of the ribosomal RNA gene units (A-D), each encoding the following rRNA molecules: large subunit (LSU; 28 S) rRNA, small subunit (SSU; 18 S) rRNA and 5.8 S rRNA .
Based on differences in sequence and expression the four gene units are divided into the blood stage A-type (a- and b-unit) and S-type (S1, S2 or c- and d-units).  The A-type ribosomes are present in the liver and blood stages of the parasite, and the S-type ribosomes are the predominant types produced during development in the mosquito.
P. berghei and P. yoelii have two A-type rRNAs (historically called the A and B Units) located on chromosome (Chr.) 7 and Chr. 12 that maintain near 100% sequence identity to one another. These A-type rRNAs have sequence variation when compared to the S1 rRNA (historically called the C Unit, found on Chr. 5) and S2 rRNA (D Unit, found on Chr. 6). The essentiality of the S-type ribosomes has been investigated in two related rodent malaria species, although discrepancies exist in their results. In P. berghei, neither S-type ribosome type was essential for sporozoite development, leading to the hypothesis that only one S-type ribosome was required in a dose-dependent manner to allow for proper mosquito-stage development. In contrast, deletions within the S2 rDNA in P. yoelii led to oocysts that did not mature, indicating that the S2 rRNA was essential for full development of mosquito-stage parasites.

The rapamycin-inducible Cre recombinase (DiCre) uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycine-?binding protein (either FKBP12 or FRB, the rapamycine-binding domain of the FKBP12 rapamycine-associated protein mTOR). Rapamycine-induced heterodimerization of the two components restores recombinase activity (rapamycine-?mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn-60, called Cre60) each fused to a different rapamycine-binding protein (FKBP12 and FRB respectively) results in the reconstitution of Cre recombinase activity).
This site–specific DiCre recombinase recognizes short, 34 bp sequences called loxP sites and catalyses the excision or inversion of the floxed (flanked by loxP) DNA segment. 
 
Phenotype
From the paper: 
'Plasmodium yoelii S-type rDNA loci are dispensable in blood-stage parasites Prior investigations into S-type ribosome essentiality in P. berghei and P. yoelii demonstrated that sequences within S-type rDNA loci could be individually deleted in blood-stage parasites. The discrepancy between these studies exists in the phenotypes observed in mosquito-stage parasites with deletions of the S1 or S2 rDNA (also called the C- and D-loci, respectively), which could have arisen due to the different gene targeting strategies used. To address this apparent discrepancy and overcome past technical limitations, we leveraged a dimerizable Cre recombinase (DiCre) system in Plasmodium yoelii to first create clean deletions of the entire rDNA of either S-type ribosome. This system allows for the recycling of the selectable marker for pyrimethamine resistance, allowing us to then target the second S-type locus for subsequent deletion. To delete either the S1 (PY17X_0522400) or S2 (PY17X_0625700) rDNAs, we used conventional reverse genetics targeting upstream and downstream of the entirety of either S-type rDNA locus. The template provided for recombination contained a deletion cassette that conferred pyrimethamine resistance and constitutive GFP expression flanked by LoxP sites, further flanked by homology arms specific to either S1 or S2. Linearized plasmids were transfected into a P. yoelii line that had constitutive DiCre and mCherry expression. Similar to previous studies, we were able to readily delete the S-type rDNAs individually in blood-stage parasites. Transgenic parasites were selected for using pyrimethamine and confirmed by genotyping PCR, and enriched for using FACS for GFP+ and mCherry+ parasites. These enriched populations of transgenic parasites were then treated with rapamycin to excise the DNA encoding pyrimethamine resistance and GFP expression to generate parasites with clean deletions of the entire S1 or S2 rDNA locus, hereby called PyΔS1 (RMgm-5631) or PyΔS2 (RMgm-5632). Excision of the deletion cassette in our PyΔS1 and PyΔS2 parasite lines allowed for the reuse of the selectable marker for pyrimethamine for a subsequent genetic modification. PyΔS1 clone 1.1 was transfected with the linearized plasmid containing the deletion cassette with homology arms for S2. An identical workflow as described above was followed and we generated a clean deletion of the S2 rDNA locus in our ΔS1 clone 1.1, thereby generating the first Plasmodium parasite line null of either S-type rDNA, hereby called PyΔS1ΔS2 (RMgm-5633)'.

Blood stage infection:
Reduced first wave of blood stage parasitemia, as well as significantly lower parasitemia throughout infection. Despite the the reduced first wave of parasitemia, PyΔS1 parasites maintained the first wave of male gametogenesis. Contrastingly, deletion of the S2 rDNA did not affect blood-stage parasitemia but decreased the first wave of male gametogenesis. The PyΔS1ΔS2 parasites phenocopied the PyΔS2 parasites, with no effect on blood-stage parasitemia and a decreased first wave of male gametogenesis.

Mosquito infection:
The prevalences of infection for all S-type deletion lines were comparable to the DiCre control, indicating that an S-type ribosome is not required to transmit to and establish mosquito infection. Both individual S-type deletion lines had decreased intensity of mosquito infection, although the most pronounced decrease was observed in the PyΔS2 parasites. PyΔS1ΔS2 parasites did not have this pronounced decrease, but rather had a similar intensity of infection to the PyΔS1 parasites. These data indicate that the presence of the S1 ribosome in the absence of the S2 ribosome leads to a larger decrease in either parasite transmission or early mosquito-stage development.
The prevalence of infection for all S-type deletion lines were comparable to the DiCre control, indicating that an S-type ribosome is not required to transmit to and establish mosquito infection. Both individual S-type deletion lines had decreased intensity of mosquito infection, although the most pronounced decrease was observed in the PyΔS2 parasites. PyΔS1ΔS2 parasites did not have this pronounced decrease, but rather had a similar intensity of infection to the PyΔS1 parasites. These data indicate that the presence of the S1 ribosome in the absence of the S2 ribosome leads to a larger decrease in either parasite transmission or early mosquito-stage development.
The diameters of 7-day old oocysts of parasite lines lacking an S2 ribosome (PyΔS2 and PyΔS1ΔS2) were, on average, 3-4μm smaller than control oocysts. Healthy, matured oocysts that had released sporozoites or were undergoing sporogony were observed in the control line and PyΔS1. In both parasite lines lacking the S2 ribosome, PyΔS2 and PyΔS1ΔS2, we observed healthy, mature oocysts but also observed an increased frequency of oocysts that failed to mature.
Despite the presence of these failed oocysts, all S-type deletion lines produced sporozoites that migrated to the salivary glands, demonstrating that neither S-type ribosome is essential for sporozoite development.The S-type deletion lines had no statistically significant decrease in salivary gland sporozoite loads

Additional information
Plasmodium yoelii A-type rDNA loci can also be deleted in blood-stage parasites. It is generally assumed that the highly abundant A-type rRNAs of asexual blood stage parasites are essential, which aligns with a previous unsuccessful attempt to delete either A-type rDNA locus in P. berghei. To test this assumption, we targeted the two A-type loci in P. yoelii independently by using homology arms upstream and downstream specific to the rDNA locus located on either Chr7 or Chr12. Surprisingly, the individual A-type rDNA loci could be deleted. Genotyping revealed that one out of four transfection populations included transgenic parasites with the respective A-type locus deleted; however, these transgenic parasites were rapidly outcompeted by remaining wild type parasites in the population with continued passaging. This demonstrated that while either A-type locus could be individually deleted, the resulting decreased abundance of the A-type ribosome in blood-stage parasites substantially decreased parasite fitness.

DiCre recombinase mediated excision of targeted DNA sequences in vivo was achieved by injecting mice intraperitoneally with 4 mg/ml rapamycin (Fisher Scientific, Cat #AAJ62473MF) in 5% v/v PEG-400 (VWR, Cat # 200002-062), 5% v/v Tween-80 (VWR, Cat # VWRVM126-100ML), and 4% v/v DMSO (Fisher Scientific, Cat #PI85190) at a dosage of 4 mg rapamycin/kg mouse weight. Mice were injected with rapamycin when blood-stage parasitemia hit 1-2% and 48 hours later were euthanized by CO2 and exsanguinated by cardiac puncture for blood-stage parasite collection. Finally, transgenic parasites were cloned by limiting dilution approaches and clonality was validated by genotyping PCR.

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