Additional file 1 of The plant-like protein phosphatase PPKL regulates parasite replication and morphology in Toxoplasma gondii
Creators: name: Wu, Xi-Ting
name: Gao, Xu-Wen
name: Wang, Qiang-Qiang
name: He, Kai
name: Bilal, Muhammad Saqib
name: Dong, Hui
name: Tang, Yi-Dan
name: Ding, Hui-Yong
name: Li, Yue-Bao
name: Tang, Xiao-Yan
name: Long, Shaojun
Publisher: name: figshare
Year: 2024
Subjects: subject: Physical Sciences not elsewhere classified
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Evolutionary Biology
subject: Environmental Sciences not elsewhere classified
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. Diagnostic PCR of the PPKL-AID line. The integration of the AID fragment at the endogenous locus was assayed by PCR, which showed the absence of the fragment in the parental line (1) but the presence of the fragment in the AID fusion line (2). Figure S2. Organelle stability assay in the PPKL-AID parasites grown in auxin. (A) Parasites were grown in the presence or absence of auxin for 24 h, followed by IFA using organelle protein markers ACP for the apicoplast, ROP5 for the rhoptries, IPT for the endoplasmic reticulum, Cen1 for the centriole, MIC2 for the micronemes, and GRA7 for the dense granule. The protein markers GAP45 and IMC1 were used to label the parasite outline for observing the morphology. Three independent experiments were performed, with similar outcomes. (B) Extracted cytoskeleton in the parasites. Parasites were grown in the absence or presence of auxin, followed by extraction of the cytoskeleton by deoxycholate treatment and tubulin detection. Scale bars = 2 μm. Figure S3. Potential interacting proteins identified by TurboID. (A–C) IFA and western blot detection of the TurboID-PPKL fusion line. (A) The parasites were grown for 24 h and processed for IFA using antibodies against Ty and GAP45 for detection of the fusion protein. (B–C) Detection of biotinylated proteins in the TurboID-PPKL line grown in the absence (−) or presence (+) of biotin (500 μM) for 1 h. Fluorescent streptavidin reagents were used to detect biotinylated proteins in the parasite line. Actin served as the control for western blot. (D) Analysis of differential candidates identified in the proximity proteome of PPKL. The volcano plot depicts fold changes in the proteome by comparing the hits in the TurboID with those in the parental line. The P-values and fold changes were analyzed and plotted by log10 and log2, respectively. The heat map is shown for the log10 of P-values. The hits with P&lt;0.05 were labeled with numbers listed by sequential orders in the table (Table S4). (E–F) The interaction of differential protein candidates retrieved from (D) was predicted using the STRING database, and the sequential order numbers were listed in the interacting network (E). The candidates in the core interactome of PPKL were listed with information on CRISPR fitness scores(34), the sequential order, and known information published. (G) Gene Ontology analysis of the 28 proteins with P&lt;0.05. The hits were identified by statistical analysis in (D) and uploaded for GO analysis in ToxoDB. Table S1. Lines used in this study. Table S2. Plasmids used in this study. Table S3. Primers used in this study.