Fig 2. IMC43 is essential for parasite replication and survival.

A) Diagram showing the mAID-3xHA degron tag fused to the C-terminus of IMC43 in a TIR1-expressing strain to facilitate proteasomal degradation upon treatment with IAA. B) IFA showing that the IMC43^AID protein localizes normally to the daughter IMC and is depleted when the parasites are treated with IAA. Magenta = anti-HA detecting IMC43^AID, Green = anti-IMC6. C) IFA showing the broad range of morphological and replication defects observed after treating IMC43^AID parasites with IAA for 24 hours. White arrows point to large gaps in the IMC marked by IMC6. Yellow arrows point to daughter buds present outside of the maternal parasite. Asterisks mark parasites producing more than two daughter buds. All three +IAA vacuoles also display desynchronized endodyogeny, where parasites in the same vacuole are at different stages of replication. Green = anti-IMC6. D) Quantification of vacuoles displaying morphological and/or replication defects after 24 hours of IMC43 depletion. Statistical significance was determined using a two-tailed t test (****, P < 0.0001). E) IFA of IMC43^AID parasites treated with IAA for 40 hours. White arrows point to large gaps in the IMC marked by IMC6. Asterisks indicate swollen parasites. Green = anti-IMC6 F) Diagram of the full-length smOLLAS-tagged IMC43 complementation construct driven by its endogenous promoter (IMC43^WT) and integrated at the UPRT locus in IMC43^AID parasites. G) IFA showing that IMC43^WT localizes normally to the daughter IMC and rescues the morphological and replication defects observed upon treatment with IAA. Magenta = anti-OLLAS detecting IMC43^WT, Green = anti-IMC6. H) Plaque assays for IMC43^AID and IMC43^AID + IMC43^WT parasites grown for seven days -/+ IAA. Depletion of IMC43 results in a severe defect in overall lytic ability, which is fully rescued by complementation with the wild-type protein. Scale bars = 0.5 mm. I) Quantification of plaque number for plaque assays shown in panel H. IMC43-depleted parasites form fewer than 10% as many plaques compared to control. Statistical significance was determined using multiple two-tailed t tests (****, P < 0.0001; ns = not significant). J) Quantification of plaque size for plaque assays shown in panel H. Plaques formed by IMC43-depleted parasites are <1% the usual size. Statistical significance was determined using multiple two-tailed t tests (****, P < 0.0001; ns = not significant). Scale bars for all IFAs = 2 μm.