Effect of depleting BFD1-regulated gene-expression factors (TGME49_253790, TGME49_208020, TGME49_224630, TGME49_306620, TGME49_311100) on the Toxoplasma transcriptome during alkaline stress

The transcription factor BFD1 is the master regulator of the Toxoplasma chronic-stage differentiation program, which is induced under alkaline stress. Prior work indicated that BFD1 transcriptionally activates five other putative RNA- and DNA-binding proteins during differentiation, suggesting potential secondary roles in shaping the Toxoplasma chronic-stage transcriptome. To describe the distinct genetic cohorts controlled by these factors, we generated individual knockdown strains for each candidate using the auxin inducible degron system (TIR1/AID) and examined the transcriptomic effects of their depletion under alkaline stress. Overall design: Comparative gene expression RNA-seq analysis of conditional knockdown strains of ME49 Toxoplasma, cultured for 96h under alkaline stress in either the presence or absence of TGME49_200385 (BFD1), TGME49_306620, TGME49_311100, TGME49_224630, TGME49_253790, or TGME49_208020. Experiments were performed in biological duplicate (separate infections).

转录因子BFD1是弓形虫（Toxoplasma）慢性阶段分化程序的主调控因子，该分化程序可由碱性胁迫诱导激活。既往研究显示，BFD1在分化过程中能够转录激活另外5种推定的RNA及DNA结合蛋白，提示其在调控弓形虫慢性阶段转录组的过程中存在潜在的次级调控功能。为阐明上述因子所调控的独特遗传群，我们借助生长素诱导降解系统（auxin inducible degron system，TIR1/AID）为每个候选靶标构建了独立的敲降菌株，并检测了碱性胁迫条件下这些靶标被耗尽后的转录组变化。总体实验设计：对ME49株弓形虫的条件性敲降菌株开展比较基因表达RNA测序（RNA-seq）分析，受试菌株在碱性胁迫环境中培养96小时，分别设置添加与不添加TGME49_200385（BFD1）、TGME49_306620、TGME49_311100、TGME49_224630、TGME49_253790及TGME49_208020的实验组与对照组。本实验设置生物学重复（独立感染实验）共2次。