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. 2020 Oct 20;11(5):e02092-20. doi: 10.1128/mBio.02092-20

FIG 1.

FIG 1

Toxoplasma gondii SLY mediates ER to Golgi vesicle transport. (A) Indirect immunofluorescence assay (IFA) showing the endogenously C-terminally tagged SLY localized to the Golgi. GRASP-GFP, parasite Golgi marker. (B) Western blot showing auxin (IAA)-induced SLY-mAID-HA degradation in intracellular parasites within 2 hours. Catalase, loading control. (C) Plaque assay of the TgSLY-mAID-HA and Tir1 parental strain in the presence or absence of IAA. TgSLY-mAID-HA displays a severe defect in the lytic cycle in the presence of IAA. (D) Parasites lacking TgSLY are impaired in intracellular replication. Error bars represent the ± standard deviation (SD) from three independent experiments. (E) IFA of TgSLY-mAID-HA parasites ± IAA (24 hours). GRASP-GFP failed to localize to the Golgi upon TgSLY degradation. GAP45, marker of the parasite pellicle. Parasites were allowed to grow for 24 hours in the absence of IAA and incubated with IAA for another 24 hours. (F) Electron micrographs reveal severe morphological defects of the Golgi apparatus and endoplasmic reticulum in SLY1-iKD parasites (marked with asterisk) upon auxin treatment (+IAA) compared to the typical Golgi and ER marked with arrowheads in the control (–IAA). Some organelles are highlighted as a, apicoplast; c, conoid; n, nucleus; m, mitochondrion; and r, rhoptry. Scale bars = 1 μm. (G) IFA of TgSLY-mAID-HA parasites ± IAA (24 hours). Trafficking of the GPI-anchored SAG1 protein to the parasite plasma membrane is affected in the absence of TgSLY. GAP45, marker of the parasite pellicle. Parasites were allowed to grow for 24 hours in the absence of IAA and incubated with IAA for another 24 hours. (H) IFA of TgSLY-mAID-HA parasites ± IAA (24 hours). Trafficking of the microneme localized MIC2 protein is affected in the absence of SLY. GAP45, marker of the parasite pellicle. Parasites were allowed to grow for 24 hours in the absence of IAA and incubated with IAA for another 24 hours. (I) IFA of TgSLY-mAID-HA parasites ± IAA (24 hours). Trafficking of GRA1 protein to the dense granules is affected in the absence of TgSLY. GAP45, marker of the parasite pellicle. Parasites were allowed to grow for 24 hours in the absence of IAA and incubated with IAA for another 24 hours. (J) IFA of TgSLY-mAID-HA parasites ± IAA (24 hours). Trafficking of Cpn60 protein to the apicoplast is not affected in the absence of TgSLY. Actin, parasite cytosol. Parasites were allowed to grow for 24 hours in the absence of IAA and incubated with IAA for another 24 hours. Scale bars for all IFA = 7 μm.