VCF (Variant Call Format) files were downloaded from the MalariaGEN database (https://www.malariagen.net/resource/26), which includes 7113 Plasmodium falciparum samples from 29 countries. To control the quality of samples, the substandard samples were excluded by the following criteria: (1) duplicate samples, (2) samples with low sequencing coverage, (3) samples with unclear geographic origin information, (4) laboratory strains, (5) samples with genotype missing rates >30%, and (6) samples with Fws values (within-host diversity) <0.95. PfAP2-06B gene variants having single nucleotide polymorphisms (SNPs) were screened using bcftools after quality control of the samples, and high-quality SNPs were selected according to the following criteria: (1) exclusion of centromeric, core high-variation, subtelomere repeat, subtelomere high-variation regions, and VQSLOD <0; (2) QUAL>30; and (3) SNP genotype missing rate <20% and (4) biallelic SNPs in coding regions.

Population-level genetic diversity was characterized by expected heterozygosity (H_E ). H_E was calculated using the formula, H_E = [n/(n − 1)][1 − Σp²], where n is the number of genotyped samples and p is the frequency of each allele at a locus. Neutral evolutionary history of the pfap2-06b gene was determined using Tajima’s D test. The pair-wise genetic differentiation between different regions was estimated using the fixation index (F_ST ). All calculations were undertaken using DnaSP (v6.12.03) software.

The Plasmodium falciparum 3D7 strain was cultured in O type fresh human erythrocytes as described; in complete RPMI 1640 medium (Gibco) with 0.5% Albumax I (Invitrogen) and a gas phase maintained under 5% CO2, 5% O2 and 90% N2 at 37°C (Fan et al., 2020). Parasites were regularly synchronized with repeated 5% sorbitol treatments at the ring stage. For growth curve assays, parasites were tightly synchronized to a 5 h window by purification of schizont stages using Percoll-sorbitol gradients (70% Percoll and 40% Percoll) followed by 5% sorbitol treatment 5 h later (Shang et al., 2022).

The pL6cs-ap2-06b-ty1-gfp, pL6cs-pfap2-06b^K3124R , and pL6cs-ap2-06b-2xfkbp-gfp-2xfkbp plasmids were constructed as described (Ghorbal et al., 2014; Zhao et al., 2020). First, the guide RNA was annealed by complementary oligonucleotides and cloned into the pL6cs construct between XhoI and AvrII restriction enzyme sites. Then the 3’UTR region of pfap2-06b with ty1-GFP, the region of pfap2-06b with mutation, or the 3’UTR region of pfap2-06b with 2×fkbp-gfp-2×fkbp were cloned into AflII and AscI restriction sites. The constructed plasmids were verified by sequencing and transformed into Escherichia coli XL10 for amplification and purification. All primers used for construction are listed in Supplementary Table S1 .

Transfections were performed in uninfected red blood cells using 100 μg of purified pL6cs-pfap2-06b-ty1-gfp or pL6cs-pfap2-06b^K3124R plasmid together with 100 μg of pUF1-Cas9-BSD, for the pL6cs-pfap2-06b-2xfkbp-gfp-2xfkbp plasmid, together with 100 μg of pUF1-Cas9-DSM1 and pLyn-FRB-mCherry plasmids followed by the addition of purified schizont stage parasites (Zhao et al., 2020). Subsequently, parasites were cultured in the presence of 2.5 nM WR99210 and 2 μg/ml BSD or 1.5 μM DMS1 (Invitrogen) until live parasites were identified in Giemsa’s solution-stained thin blood smears roughly 3 weeks later. The sequences at the designed integration sites were examined by PCR using genomic DNA template followed by DNA sequencing to confirm the presence of genetic editing. Clones of the transgenic parasite lines PfAP2-06B-GFP, PfAP2-06B^K3124R, and PfAP2-06B-KS were derived by limiting dilution cloning (Fan et al., 2020). Primers used for verification are provided in Supplementary Table S2 .

Parasites were tightly synchronized to a 5 h window. Ring-stage parasites were plated at 0.1% parasitemia in a 6-well plate with 2% hematocrit fresh erythrocytes. For PfAP2-06B^K3124R parasites, the wild-type strain 3D7 was used as a control group. PfAP2-06B-KS parasites were cultured in the presence or absence of rapalog, a ligand that causes FRB and FKBP to form dimers, and thereby remove the target protein from its site of action to achieve knockdown effect (Birnbaum et al., 2017). Parasitemias were monitored periodically by counting trophozoite-stage parasites within Giemsa-stained thin blood smears for three intraerythrocytic growth cycles (Shang et al., 2021).

RSAs were performed as described (Witkowski et al., 2013; Zhang et al., 2017; Siddiqui et al., 2018). Briefly, highly synchronous parasite cultures at the young ring stage (0 to 3 h) were exposed for 6 h to 700 nM DHA or 0.1% dimethyl sulfoxide (DMSO) as the control. The drug was then washed out with RPMI 1640, and parasites were further cultivated further for 66 h under standard in vitro culture conditions. At 72 h after the assay initiation, survival rates were calculated in Giemsa-stained thin smears by counting the viable parasites surviving in DHA-treated versus DMSO-treated cultures. Parasite isolates demonstrating >1% survival were considered to display reduced susceptibility, or partial resistance, to artemisinin (Amaratunga et al., 2014).

Immunofluorescence assays were performed as described to characterize PfAP2-06B localization (Shang et al., 2021). Parasites were harvested at the schizont stage and fixed by 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 10 min, then washed with PBS. Prepared samples were incubated with primary antibody against GFP (Proteintech, 66002-1-Ig) at 1:500 to 1:1,000, followed by a secondary antibody AlexaFluor 488 goat anti-mouse IgG (ThermoFisher Scientific, A11029) at 1:500.The samples were then incubated with rabbit anti-mCherry (Proteintech, 26765-1-AP) at 1:300, followed by secondary antibody AlexaFluor 568 goat anti-rabbit IgG (ThermoFisher Scientific, A11036) at 1:500. For nuclear localization, the samples were washed with PBS and incubated with DAPI for 30 min. Preparations were visualized with a Nikon A1R microscope at 60-100× magnification, and images were acquired with NIS Elements software and processed using Adobe Photoshop.

To determine the sensitivity of parasites to quinolines, IC50 assays of 3-day inhibition tests were performed using chloroquine or amodiaquine. Briefly, 200 μL of a tightly synchronized parasite culture at 1% parasitemia and 2% hematocrit was added into each well of a 96-well plate, followed by chloroquine or amodiaquine drug pressure at a series of concentrations (500 nmol/L, 250 nmol/L, 125 nmol/L, 62.5 nmol/L, 31.25 nmol/L, 15.63 nmol/L, 7.81 nmol/L, and 3.91 nmol/L). After 72 hours of incubation, the parasiticidal effects of the drugs were estimated by the addition of 100 μL of SYBR Green I lysis buffer to each well. After thorough mixing, the plates were incubated for 1 h and then SYBR Green fluorescence values were detected using a microplate reader (490 nm excitation and 530 nm emission wavelengths). IC50 values were calculated by GraphPad Prism. The formulation of SYBR Green I lysis buffer solution is listed in Supplementary Table S3 .

Highly synchronous ring (10–16 hpi), trophozoite (24–30 hpi), and schizont (40–46 hpi) stage parasites were collected in TRIzol. Total RNA purification was achieved using a Direct-zol RNA Kit (Zymo Research). To assay the levels of specific transcripts, 500–800 ng of total RNAs were reverse transcribed (Takara), and quantitative PCR assays were performed in triplicate using the primers shown in Supplementary Table S4 and seryl-tRNA synthetase (PF3D7 0717700) as the endogenous control. PCR thermal cycling was initiated with 30 sec denaturation at 95°C, followed by 40 cycles of 5 sec at 95°C, 20 sec at 54°C, 7 sec at 56°C, 7 sec at 59°C, and 27 sec at 62°C. Gene expression changes were quantified using the ^△△Ct method.

ChIP-Seq assays were carried out in two biological replicates as described, with minor modifications (Santos et al., 2017; Josling et al., 2020). Synchronized parasites were harvested at the schizont stage and cross-linked immediately with 1% paraformaldehyde (Sigma) by rotating for 10 min at 37°C, then quenched with 0.125 M glycine for 5 min on ice. The parasites were resuspended in 50 ml of PBS and lysed with 0.15% saponin for 5 min on ice. The released nuclei were washed several times with PBS, then sonicated using an M220 sonicator (Covaris) at 5% duty factor, 200 cycles per burst, and 75W of peak incident power to generate 100-500 bp fragments. The samples were diluted tenfold with dilution buffer and precleared with Protein A/G magnetic beads (Thermo) for 2 hours at 4°C. The precleared chromatin supernatants, a small part of which were aliquoted as input controls, were incubated overnight at 4°C with 0.5 μg of antibodies against GFP (Abcam, ab290) and 20 μL of Protein A/G magnetic beads. The immunoprecipitates were washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE buffer, then eluted with Elution Buffer. To reverse the cross-link, the eluted samples were incubated overnight at 45°C and treated with RNase A at 37°C for 30 min and Proteinase K at 45°C for 2 hours. Finally, DNA was extracted using a MinElute PCR purification kit (Qiagen, 28006). For library preparation, 1.5 ng of ChIP DNAs were end-repaired (Epicentre No. ER81050), processed with a protruding 3’ A base (NEB No. M0212L), and ligated with adapters (NEB No. M2200L). Agencourt AMPure XP beads (Beckman Coulter) were then used for size selection and purification. Libraries were amplified using a KAPA HiFi PCR Kit (KAPA Biosystems, KB2500) with the following program: 1 min at 98°C; 12 cycles of 10 s at 98°C and 1 min at 65°C; and finally, extension for 5 min at 65°C. Library sequencing was conducted on an Illumina HiSeq Xten platform and generated 150 bp pair-end reads.

To remove residual adapters and low-quality bases, read trimming was conducted with Trimmomatic (Bolger et al., 2014) using a 4 bp window and average window quality above 15. Clipped reads with a minimum length of 50 bp and average read quality above 20 were mapped to the P. falciparum 3D7 genome build 47 using Bowtie2 (Langmead and Salzberg, 2012; Bolger et al., 2014) and default parameters. Peaks were identified using the call-summits option of the MACS2 callpeak function (Zhang et al., 2008) and a q-value cutoff of 0.05. Log2-transformed ChIP/input fold enrichment signals were calculated with the MACS2 bdgcmp function and visualized with Gviz (Hahne and Ivanek, 2016). Genomic Ranges (Lawrence et al., 2013) assigned peaks to nearby target genes if they overlapped 5′ UTRs (<3 kb upstream of the translation start sites), gene body, and 3′ UTRs (<0.5 kb downstream of the translation stop sites). Functions enriched in the target genes were analyzed using malaria parasite metabolic pathways (MPMP) (Ginsburg, 2006), functional gene families, and cluster Profiler (Yu et al., 2012) [Benjamini-Hochberg (BH) adjusted p-value of <0.01]. Peaks were extended +/− 250 bp around summits. Those detected in both biological replicates were reserved for discovery of PfAP2-06B DNA binding motifs between 6 bp and 10 bp using DREME (Bailey, 2011) as compared with random genomic regions of 500 bp.

All data are reported as mean ± the standard deviation (SD) of three duplications. Statistical differences between multiple groups were analyzed using Student’s T test. Statistical analysis was completed using GraphPad Prism software (version 8.0.1, San Diego, CA, USA). Result with P < 0.05 was defined as statistically significant.

To explore the genetic diversity and population genetics of P. falciparum AP2-06B in different geographical strains, the AP2 gene sequences of P. falciparum in the MalariaGEN database were analyzed. After sample quality control, 3683 samples were included in this study; specifically, 1,156 West African, 151 Central African, 382 East African, 808 western Southeast Asian, 955 eastern Southeast Asian, 36 South Asian, 37 South American, and 158 from Oceania. In the pfap2-06b gene (PF3D7_0613800), 733 SNPs were identified, including 516 non-synonymous mutations and 217 synonymous mutations. The number of non-synonymous mutations observed in African samples was higher than that in other regions, and the highest number of non-synonymous mutations was in West Africa (n=345) ( Figure 1A ). A higher genetic differentiation for pfap2-06b was found between Africa and Southeast Asia (F_ST > 0.25) than the respective differentiations within these two regions ( Figure 1B ). There were 22 SNPs with F_ST >0.25 in different regions, among which S256P, L2716I, F2768C, K3124R, N3511D, C3644Y, and M3847F had higher genetic differentiation between Africa and Southeast Asia. The allele frequencies of 6 non-synonymous mutations (R1034C, L2716I, K3124R, N3511D, C3644Y, and M3847V) in PfAP2-06B were greater than 0.05 ( Figure 1C ). Of these, only K3124R was in one of the three putative AP2 DNA-binding domains within PfAP2-06B, which spans approximately aa 3087 to 3136 ( Figure 1D ). K3124R is the mutation located in an AP2-06B domain, and has a significantly higher mutation frequency in Southeast Asia than in other regions.

As mentioned above, AP2-06B is mutated at high frequency in Southeast Asia where drug resistance originated, which suggested that AP2-06B might be related to the development of drug resistance. AP2-06B is a transcriptional regulatory factor, and understanding the identity and function of its target genes may inform its mechanism of action. Therefore, ChIP-seq assays were performed to explore the role of AP2-06B in transcriptional regulation and whether it might impact expression related to the development of resistance, such as the genes pfcrt, pfk13, and pfmdr1. To achieve this, we first constructed a C-terminal GFP tagged PfAP2-06B transgenic parasite line using the CRISPR/Cas9 gene editing system ( Figure 2A ). Correct editing of the 3D7/PfAP2-06B-GFP strain was verified by collecting genomic DNA from the transgenic parasite line and conventional PCR detection with two sets of primers and sequencing ( Figure 2B ). A perinuclear localization of PfAP2-06B was confirmed by immunofluorescence assay ( Figure 2C ). To investigate transcriptional regulation by PfAP2-06B, ChIP-seq assays were performed on the 3D7/PfAP2-06B-GFP schizonts using antibodies against GFP. The results of ChIP-Seq analysis showed that AP2-06B had a significant binding peak in the 5′ UTR of pfk13 and pfcrt, but not on pfmdr1 ( Figure 2D ). Therefore, AP2-06B may induce resistance to artemisinin or quinolines by regulating pfk13 or pfcrt; and since K3214R is a mutation located in one of the three predicted DNA-binding AP2 domains of AP2-06B and may be a key mutation in AP2-06B induced resistance.

The mutation frequency of AP2-06B in Southeast Asia, a region with high drug resistance, is significantly higher than found in other areas. In addition, AP2-06B can bind to the loci of pfk13 and pfcrt, genes which are involved in resistance to artemisinin and quinolines, respectively. As the mutation located in the AP2-06B AP2 domain with a significantly higher mutation frequency in Southeast Asia, we investigated whether K3124R mutation induces resistance to artemisinin and quinolines by regulating pfk13 and pfcrt expression. DNA sequence of the artemisinin-resistant CWX we previously sequenced, and sequences of the quinoline-resistant strains PfDd2 and Pf7G8 were downloaded from PlasmoDB, were analyzed for the genetic diversity of pfap2-06b, compared with the Pf3D7 strain as a reference sequence. In contrast to Pf3D7, the K3124R mutation was found within the AP2-06B AP2 domain of the resistant strains CWX, PfDd2, and Pf7G8 ( Figure 3A ).

To determine whether this mutation can alter the expression of pfk13 and pfcrt, as well as cause artemisinin and quinoline resistance in Plasmodium, the K3124R mutation was introduced into the endogenous pfap2-06b locus of the Pf3D7 strain to generate the edited clone PfAP2-06B ^K3124R using CRISPR/Cas9-mediated genome editing ( Figure 3B ). The AP2-06B region containing the mutation of Pf3D7 and PfAP2-06B^K3124R was verified by PCR ( Figure 3C ) and sequenced to confirm the successful mutation ( Figure 3D ). To investigate the effect of the K3124R mutation on parasite viability, growth curve assays of PfAP2-06B^K3124R were performed, in comparison with the parental 3D7 strain as a wild type control. The result showed that the K3124R mutation had no effect on parasite viability ( Figure 3E ). The sensitivity to artemisinin and quinolines of the Pf3D7 ^K3124R and expression of pfk13 and pfcrt were also assayed. The results showed that the K3124R mutation did not change the expression of pfk13 ( Figure 3F ) or pfcrt ( Figure 3G ) and Pf3D7^K3124R did not alter susceptibility to DHA ( Figure 3H ) or the quinoline antimalarials chloroquine and amodiaquine ( Figure 3I ).

PfAP2-06B^K3124R did not change the expression of pfk13 or pfcrt nor alters susceptibility to DHA or quinoline antimalarials. However, the ChIP-Seq result indicated that AP2-06B had a significant binding peak in the loci of pfk13 and pfcrt, as a transcriptional regulator factor, suggesting that AP2-06B might function as a transcription factor regulating the expression of pfk13 and pfcrt. Therefore, a conditional knockdown strategy was adopted by knock sideways (KS) ( Figure 4A ). KS is based on the ligand-induced dimerization of the proteins FRB and FKBP. The native target is fused with FKBP, whereas FRB is separately expressed with the trafficking information for a different cellular compartment (the ‘mislocalizer’). After addition of the ligand (rapalog) to dimerize FRB and FKBP, the target protein is removed from its site of action by the mislocalizer. This method acts rapidly and avoids the problems of targeting essential genes at the genetic level (Birnbaum et al., 2017). Accordingly, we successfully generated a 3D7/PfAP2-06B-KS transgenic knockdown parasite line and isolated clones by limited dilution cloning. Genomic DNA of the transgenic parasite line was collected, and two sets of primers were used for conventional PCR detection to verify the correct editing of the pfap2-06b locus ( Figure 4B ). Immunofluorescence detection confirmed that the localization of PfAP2-06B is transferred from the nucleus to the cell membrane after the addition of rapalog, indicating that AP2-06B knockdown was successful ( Figure 4C ). To investigate the effect of PfAP2-06B on parasite viability, the PfAP2-06B-KS parasite was maintained with rapalog or ethanol (vehicle) to analyze growth, using the parent strain 3D7 as a control. The growth curve analysis showed that the parasitemia of PfAP2-06B-KS strain decreased by 48% treatment with rapalog for a duration of three life cycles compared with the vehicle group, but had no significant effect on the parent strain 3D7 ( Figure 4D ), suggesting that PfAP2-06B is necessary for the growth of P. falciparum. The sensitivity of the PfAP2-06B-KS parasite to DHA and quinolines was then assayed in the presence of rapalog versus ethanol; and no effect was observed on susceptibility to DHA ( Figure 4G ) or the quinoline antimalarials chloroquine and amodiaquine ( Figure 4H ). Finally, the expression of pfk13 and pfcrt were quantified, and it was shown that the conditional knockdown of PfAP2-06B did not change the expression of pfk13 ( Figure 4E ) or pfcrt ( Figure 4F ).