Effect of BFD2 (TGME49_311100) knockout on T. gondii gene expression during BFD1 induction

The acute-to-chronic stage transition in Toxoplasma gondii involes a global restructuring of the parasite transcriptome. Prior work identified a single transcription factor (BFD1) that is both necessary and sufficient to establish the chronic-stage program, making it the master regulator of this process. Recently, we found that parasites lacking a CCCH-type zinc finger RNA-binding protein (BFD2) are also unable to produce chronic forms. To understand how BFD2 aids in establishing the BFD1 transcriptional program, we knocked out BFD2 in a BFD1-conditional expression strain, wherein BFD1 is induced by treatment with the small molecule Shield-1. Comparative RNA-seq analysis was performed on conditional BFD1 expression strains in the presence (DD-BFD1) and absence of BFD2 (DD-BFD1/∆bfd2), after 48h of treatment with either 3 µM Shield-1 to induce BFD1 expression or an equivalent volume of ethanol (i.e. vehicle). Experiments were performed in biological triplicate (separate infections) and technical duplicate (i.e. samples split in two at the time of harvest and processed in parallel for all subsequent steps, from RNA isolation through library prepartion and sequencing).

刚地弓形虫(Toxoplasma gondii)的急性向慢性阶段转换，涉及寄生虫转录组的全局重塑。既往研究已鉴定出单一转录因子（transcription factor）BFD1，其对于建立慢性阶段发育程序既是必需的也是充分的，因此是该过程的主调控因子。近期本研究发现，缺失CCCH型锌指RNA结合蛋白(CCCH-type zinc finger RNA-binding protein) BFD2的弓形虫，同样无法产生慢性阶段虫体。为解析BFD2如何助力BFD1转录程序的建立，我们在BFD1条件性表达菌株中敲除了BFD2，该菌株的BFD1可通过小分子Shield-1处理诱导表达。我们分别使用3 μM Shield-1诱导BFD1表达，或以等体积乙醇（即溶剂对照）处理48小时后，对存在BFD2的DD-BFD1菌株与缺失BFD2的DD-BFD1/Δbfd2菌株开展了比较RNA测序(RNA-seq)分析。实验设置了三次生物学重复（独立感染实验）与两次技术重复：即收获样本时将每份样品均分为两份，后续从RNA提取、文库制备到测序的全部步骤均平行处理。