Quantification of Organellar Genome Copy Number

Tachyzoites (iPYK1 or iPYK1-Δpyk2 strain) with or without 3 or 5 days’ ATc treatment were forced to egress by needle passage and purified for DNA extraction using the EasyPure genomic DNA kit (Transgen Biotech, Beijing, China). Thirty ng DNA from each sample was subject to qPCR analysis, using primers (listed in Table S1) designed to amplify fragments from the apicoplast genome (EF-Tu), mitochondrial genome (CytB), and nuclear genome (UPRT), respectively, as previously described (43 (link)– (link)45 (link)). The qPCR was performed using the Power SYBR green PCR master mix (Toyobo Co., Ltd., Osaka, Japan), and all reactions were performed on the ABI ViiA 7 detection system (Life Technologies, Inc., Rockville, MD, USA). The threshold cycle (C_T) values for the nuclear genome amplification were used as a normalization reference to compare apicoplast and mitochondrial genome abundance across samples. The 2^ΔΔCT method was used to estimate the changes of apicoplast and mitochondrial genome abundance after ATc treatment. Every sample was independently tested three times, each with two technical replicates.