Mutant/mutation
The mutant expresses the plant auxin receptor transport inhibitor response 1 (Oryza sativa TIR1) under control of the ookinete specific soap promoter. The TIR1 is (C-terminal) tagged with FLAG. In addition, it expresses CDC50c C-terminal tagged with AID  
 
Protein (function)
TIR1 and AID: The plant hormone auxin induces rapid proteasomal degradation of certain proteins by a specific E3 ubiquitin ligase. To implement this system in Plasmodium, only two transgenic components are needed: a plant auxin receptor called transport inhibitor response 1 (TIR1) and a proteins of interest (POI) tagged with an AID 9degron) motif. In these engineered parasite lines stably expressing the plant TIR1, the auxin functions as a molecular glue promoting specific interaction between the ubiquitin ligase complex and AID-tagged POI, which triggers proteasomal degradation of the latter.

CDC50c: P4-ATPase interacts with the co-factor protein, CDC50, which is required for trafficking of the complex from ER to plasma membrane and for flippase activity. A search of the Plasmodium genomes identified three paralogs of cdc50 genes: cdc50a (PY17X_0619700); cdc50b (PY17X_0916600); and cdc50c (PY17X_0514500) (see RMgm-4490)

Phenotype
The Tir1::Flag fusion protein is highly expressed in ookinetes (see RMgm-4946). 
From the paper:
'To investigate the potential function(s) of CDC50C in mosquito transmission, we attempted to disrupt the cdc50c gene but failed to obtain mutants, suggesting an essential role of CDC50C in parasite asexual blood stages. This observation is in agreement with the recent global knockout screening results in Plasmodium falciparum and Plasmodium berghei. Therefore, we applied an auxin-inducible degron (AID)–based protein degradation system we recently adapted in P. yoelii, which allows rapid depletion of a target protein fused to an AID motif with the aid of auxin/indole-3-acetic acid (IAA). The C terminus of the endogenous cdc50c locus was tagged with the sequence encoding the AID::6HA in the soap-Tir1 strain. The resulting cdc50c::aid parasite exhibited the expected cell periphery localization of CDC50C in ookinetes, indicating no marked effect of AID tagging on CDC50C localization. IAA treatment (1 mM for 1 hour) markedly reduced the CDC50C protein abundance but had little impact on P28 levels. Of note, in vitro gliding motility of the IAA-treated ookinetes is comparable to controls. Furthermore, we used a membrane feeding experiment using ookinetes treated with either IAA or EtOH to test the effect of CDC50C depletion on parasite transmission. Notably, IAA-treated ookinetes were lysed during midgut epithelium traversal. Consistent with these observations, IAA-treated ookinetes developed a much lower number of oocysts in mosquito midguts compared to the controls at 7 days pf. Together, these results strongly suggest a critical role of CDC50C in safeguarding ookinetes during midgut traversal, similar to that of its binding partner ATP7' (see mutant RMgm-5046) 

Additional information

From the paper:
'CDC50C showed peripheral localization in ookinete. CDC50C is predicted to contain two transmembrane helices and a large exocytosolic loop. Similar to ATP7, CDC50C is sensitive to ookinete trypsin treatment and permeabilization, indicating the PPM localization of CDC50C in ookinetes of the tagged parasite cdc50c::6HA. To determine whether CDC50C interacts with ATP7, we generated a double-tagged strain atp7::6HA/50c::3V5, wherein ATP7 and CDC50C were tagged with 6HA and 3V5, respectively. ATP7 and CDC50C showed marked colocalization at the cell periphery in the mature ookinetes. This colocalization was also observed in another independent double-tagged strain atp7::6HA/50c::4Myc. Furthermore, immunoprecipitation using an HA antibody indicated that ATP7 is bound to CDC50C in the atp7::6HA/50c::4Myc ookinete lysates. Last, we performed a proximity ligation assay (PLA), an immunohistochemical method to detect protein interaction in situ. A robust PLA signal was detected at the ookinete periphery in the atp7::6HA/50c::4Myc parasites when both anti-HA and anti-Myc primary antibodies were present, indicative of ATP7 and CDC50C interaction. 

From mutant RMgm-4946:
We used the CRISPR/Cas9 method to tag two genes, cdc50c (PY17X_0514500) and fbxo1 (PY17X_1120000) with the AID motif and interrogate the expression of these two proteins with auxin. The eef1a-Tir1 line allows efficient degradation of the AID-tagged endogenous protein in the asexual schizont and sexual gametocyte stages, while the soap-Tir1 line allows protein degradation in the ookinetes.
To generate the plasmid for tagging the cdc50c (PY17X_0514500) with an AID degron and sextuple HA fusing epitope (AID::6HA), the C-terminal 518 bp of coding region and 529 bp of the 3’UTR of the cdc50c gene were amplified as the homologous left and right arm, respectively. One sgRNA was designed to target the Cterminal coding region of cdc50c genes. To generate the plasmid for tagging the fbxo1 (PY17X_ 1120000) with the AID::6HA, the C-terminal 498 bp of coding region and 521 bp of the 3’UTR of the fbxo1 gene were amplified as the homologous left and right arm, respectively. One sgRNA was designed to target the C-terminal coding region of fbxo1 genes.
To assess the degradation kinetics of targeting proteins, the asexual blood stage parasites, schizonts, gametocytes, and ookinete culture were incubated with 1 or 4 mM IAA (auxin; Indole 3-acetic acid, Sigma Aldrich, I2886) at 37℃. 

We noticed that in the ookinetes of eef1a-Tir1/cdc50c::aid parasites, we failed to detect a  clear depletion of 50C-AID even after 3 hours of auxin treatment, which is likely due to the relatively lower Tir1 expression in the ookinetes compared to that in the asexual blood stage and gametocytes. Therefore, we attempted to engineer a transgenic line to achieve higher Tir1 expression in the ookinetes. To that end, we used the promoter of soap (PY17X_1040200), a gene specifically and highly expressed in the ookinetes and early oocysts, to drive the Tir1 expression. Using the CRISPR/Cas9 method, we replaced the eef1a promoter with the soap promoter (1999bp) at the upstream of Tir1 in the eef1a-Tir1 parasites (RMgm-4945), and generated a new line designated as soap-Tir1 (RMgm-4946). As expected, the Tir1 protein is highly expressed in the ookinetes, but not in the asexual blood stage of the soap-Tir1 parasites 

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