The protozoan parasite Toxoplasma gondii encodes dozens of phosphatases, among which a plant-like phosphatase absent from mammalian genomes named PPKL, which is involved in regulating brassinosteroid signaling in Arabidopsis, was identified in the genome. Among the Apicomplexa parasites, T. gondii is an important and representative pathogen in humans and animals. PPKL was previously identified to modulate the apical integrity and morphology of the ookinetes and parasite motility and transmission in another important parasite, Plasmodium falciparum. However, the exact function of PPKL in the asexual stages of T. gondii remains unknown. The plant auxin-inducible degron (AID) system was applied to dissect the phenotypes of PPKL in T. gondii. We first analyzed the phenotypes of the AID parasites at an induction time of 24 h, by staining of different organelles using their corresponding markers. These analyses were further conducted for the parasites grown in auxin for 6 and 12 h using a quantitative approach and for the type II strain ME49 of AID parasites. To further understand the phenotypes, the potential protein interactions were analyzed using a proximity biotin labeling approach. The essential role of PPKL in parasite replication was revealed. PPKL is localized in the apical region and nucleus and partially distributed in the cytoplasm of the parasite. The phenotyping of PPKL showed its essentiality for parasite replication and morphology. Further dissections demonstrate that PPKL is required for the maturation of daughter parasites in the mother cells, resulting in multiple nuclei in a single parasite. The phenotype of the daughter parasites and parasite morphology were observed in another type of T. gondii strain ME49. The substantial defect in parasite replication and morphology could be rescued by genetic complementation, thus supporting its essential function for PPKL in the formation of parasites. The protein interaction analysis showed the potential interaction of PPKL with diverse proteins, thus explaining the importance of PPKL in the parasite. PPKL plays an important role in the formation of daughter parasites, revealing its subtle involvement in the proper maturation of the daughter parasites during division. Our detailed analysis also demonstrated that depletion of PPKL resulted in elongated tubulin fibers in the parasites. The important roles in the parasites are potentially attributed to the protein interaction mediated by kelch domains on the protein. Taken together, these findings contribute to our understanding of a key phosphatase involved in parasite replication, suggesting the potential of this phosphatase as a pharmaceutic target.

中文翻译：

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类植物蛋白磷酸酶 PPKL 调节弓形虫寄生虫的复制和形态

原生动物寄生虫弓形虫编码数十种磷酸酶，其中在基因组中发现了一种哺乳动物基因组中不存在的植物样磷酸酶，名为 PPKL，它参与调节拟南芥中的油菜素类固醇信号传导。在顶复门寄生虫中，弓形虫是人类和动物中重要且具有代表性的病原体。PPKL 先前已被确定可调节另一种重要寄生虫恶性疟原虫中动动子的顶端完整性和形态以及寄生虫的运动和传播。然而，PPKL 在弓形虫无性阶段的确切功能仍不清楚。应用植物生长素诱导降解决定子（AID）系统来剖析弓形虫中 PPKL 的表型。我们首先通过使用相应的标记对不同的细胞器进行染色，分析了 AID 寄生虫在 24 小时诱导时间的表型。使用定量方法对在生长素中生长 6 和 12 小时的寄生虫以及 AID 寄生虫的 II 型菌株 ME49 进一步进行了这些分析。为了进一步了解表型，使用邻近生物素标记方法分析了潜在的蛋白质相互作用。PPKL 在寄生虫复制中的重要作用被揭示。PPKL 位于寄生虫的顶端区域和细胞核，部分分布在细胞质中。PPKL 的表型分析表明其对于寄生虫复制和形态的重要性。进一步的解剖表明，PPKL 是母细胞中子代寄生虫成熟所必需的，从而导致单个寄生虫中产生多个细胞核。在另一种弓形虫ME49菌株中观察了子代寄生虫的表型和寄生虫形态。寄生虫复制和形态方面的实质性缺陷可以通过基因互补来弥补，从而支持了 PPKL 在寄生虫形成中的重要功能。蛋白质相互作用分析显示了 PPKL 与多种蛋白质的潜在相互作用，从而解释了 PPKL 在寄生虫中的重要性。PPKL 在子代寄生虫的形成中发挥着重要作用，揭示了其在分裂过程中对子代寄生虫的正常成熟的微妙参与。我们的详细分析还表明，PPKL 的消耗导致寄生虫中微管蛋白纤维伸长。寄生虫中的重要作用可能归因于蛋白质上的 kelch 结构域介导的蛋白质相互作用。总而言之，这些发现有助于我们了解参与寄生虫复制的关键磷酸酶，表明这种磷酸酶作为药物靶点的潜力。