The Plasmodium falciparum 3D7 strain was maintained in continuous culture using fresh human O+ erythrocytes at 2% hematocrit in complete RPMI 1640 medium supplemented with 0.5% Albumax II, 0.2% sodium bicarbonate, 25 mM HEPES, and 50 μg/mL hypoxanthine. Cultures were incubated at 37°C under controlled atmospheric conditions (5% O₂, 5% CO₂, and 90% N₂), with medium replacement every 48 hours (Lu et al., 2021). For parasite synchronization, early ring-stage cultures were treated with 5% sorbitol at 37°C for 15 minutes to lyse mature-stage parasites, followed by washing with complete medium. Late-stage schizonts were isolated using a Percoll-sorbitol gradient (40% and 70% layers prepared in RPMI medium) by centrifugation for 20 minutes at 37°C without brake application. The schizont-enriched interface was carefully collected and washed with complete medium before reinvasion.

The plasmid construction was performed as previously described. Based on the pL6CS plasmid, we designed specific sgRNA sequences targeting the tcf25 locus, and homology arms (approximately 1 kb each) were amplified by PCR from genomic DNA. Site-directed mutagenesis or HA-tag insertion was performed through overlap extension PCR, with the modified sequence flanked by the homologous arms. The final constructs were cloned into the pL6CS vector and Sanger sequencing prior to maxiprep purification. For transfection, 100 μg of the constructed plasmid and 100 μg of pUF1-Cas9 plasmid (dissolved in 150 μL of water) were co-transfected into fresh erythrocytes via electroporation. Transfected erythrocytes were immediately infected with Percoll-enriched schizont-stage parasites. The cultures were maintained under standard conditions until parasitemia reached 8-12%, typically after 1–2 replication cycles. Positive selection was initiated using WR99210 and DSM1, with drug pressure maintained for 3–4 weeks. Following morphological confirmation of normally developing parasites by Giemsa-stained thin blood smear microscopy, genomic DNA was isolated using the TIANamp Genomic DNA Kit (DP304; TIANGEN) according to the manufacturer’s protocol. Successful modification was initially screened by PCR, followed by agarose gel electrophoresis. PCR products of expected size were gel-purified and confirmed by Sanger sequencing, ensuring the absence of unintended mutations. This process successfully generated endogenous mutant parasite strains in the tcf25 gene. The primers used in this study are listed in Supplementary Table S1 .

Wild-type (WT) and tcf25 gene knockout parasite strains were cultured in vitro. Following strict synchronization, the parasite growth window was restricted to 6 hours. At the trophozoite stage, initial parasitemia was adjusted to 0.1%, with parasites divided into WT and tcf25 gene knockout groups. Cultures were maintained for four life cycles, and parasitemia was determined by microscopic examination during each trophozoite stage. A growth curve was generated and analyzed using GraphPad Prism 9.0.0.

Synchronous cultures of WT and tcf25_ko parasites were established at 2% initial parasitemia at 8–16 hours. After re-invasion, ring-stage parasitemia was determined by Giemsa-stained thin blood smears (parasitemia 1). Then, parasites were treated with 50 mM N-acetyl-D-glucosamine (GlcNAc; A3286, Sigma-Aldrich) in complete RPMI 1640 medium for 4 consecutive days at the next cycle to selectively eliminate asexual stage parasites while allowing gametocyte development. Gametocytemia was assessed by morphological identification in Giemsa-stained smears (parasitemia 2). The sexual conversion rate was calculated using the formula: Sexual conversion rate (%) = parasitemia 2/parasitemia 1. Each experiment included three biological replicates.

The tcf25-HA-tagged strain was maintained in vitro. Synchronization was achieved through sorbitol treatment at the ring stage. For immunofluorescence assays, parasites were harvested at different stages and immediately fixed in ice-cold 4% paraformaldehyde at 4°C. Fixed parasites were adhered to glass slides, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked with 1% BSA for 1 hour at room temperature. Immunostaining was performed using rabbit anti-HA multiclonal antibody (1:500 dilution; Cat#HA721750, HUABIO) followed by iFluor™ 488 Conjugated Goat anti-rabbit secondary antibody (1:500 dilution; Cat# HA1121, HUABIO), with each incubation for 1 hour at room temperature. Between incubations, slides were washed three times with PBS. For nuclear visualization, samples were stained with DAPI, then fluorescence images were acquired using a laser scanning confocal microscope (STELLARIS 5, Leica Microsystems). Image processing and analysis were performed using ImageJ software.

Western blotting was performed as previously described with modifications (Shang et al., 2022). Briefly, synchronized schizont-stage parasites were collected, and total protein was extracted by boiling at 100°C for 5 min. Proteins were separated on 8% SDS-polyacrylamide gels and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in PBST for 1 hour at room temperature, followed by incubation with antibodies overnight at 4°C. After three washes with PBST, the membrane was incubated with HRP-conjugated secondary antibody (1:5000 dilution; Cat#HA1001, HUABIO) for 1 hour at room temperature. Protein bands were detected using enhanced chemiluminescence (ECL) reagent and visualized using a ChemiDoc™ XRS+ imaging system with Image Lab™ Software (Bio-Rad).

Parasite cultures of WT and tcf25_ko strains were synchronized using our established growth assay protocol. Briefly, schizont-stage parasites were enriched by Percoll gradient centrifugation followed by ring-stage synchronization to achieve a narrow 6-hour growth window. After re-invasion, parasites were harvested at two developmental stages: ring (10–15 hpi) and schizont (40–45 hpi). Two biological replicates were used in this experiment. Then, parasite pellets were homogenized in TRIzol reagent (Transgen) and stored at -80°C. Total RNA was isolated using the TransZol Up Plus RNA kit (ER501) according to the manufacturer’s protocol. For RNA-seq library construction, 500 ng of high-quality total RNA per sample was used for the VAHTS Universal V10 RNA-seq Library Prep Kit (Vazyme, NR616). Briefly, poly(A)+ RNA was enriched using magnetic beads, followed by fragmentation and first-strand cDNA synthesis. After second-strand synthesis and adapter ligation, libraries were amplified with 14 cycles of PCR. Final library quality was assessed by Qubit 2.0 fluorometer and Agilent 2100 system. Paired-end sequencing was performed on an Illumina NovaSeq 6000 platform at BMK Biotechnology Co., Ltd (Beijing, China).

RNA-seq raw data were quality-trimmed using Trimgalore (v0.6.6) with default parameters, removing adapter sequences and low-quality bases. The cleaned reads were then aligned to the Plasmodium falciparum reference genome (PlasmoDB-v64) using HISAT2 (v2.2.1). Resulting SAM files were converted to BAM format and sorted using Samtools (v1.12). PCR duplicates were marked and removed using Picard tools (v2.26.0), followed by the generation of read count matrices with featureCounts (v2.0.1) against gene annotations. Read counts were analyzed using DESeq2 (v1.46.0) in R (v4.4.2). Approximately 20 million reads per sample were retained for downstream analysis. Genes with low counts were filtered prior to analysis. Differential expression was determined by applying thresholds of |log₂ fold change| > 1 and p-value < 0.05.

Parasite cultures of WT and tcf25_ko strains were strictly synchronized as previously described. Ring-stage samples at approximately 10–15 hpi were collected from two independent clones. Total RNA was extracted using the manufacturer’s protocol. 500 ng of total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme, R323). RT-qPCR was performed with the following thermal cycling program: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 54°C for 20 s, 56°C for 7 s, 59°C for 7 s, and 62°C for 27 s. The seryl-tRNA synthetase (PF3D7_0717700) gene was used as an internal reference for normalization. Relative gene expression levels were calculated using the 2^-ΔΔCt method. All samples were analyzed in triplicate using three biological replicates. The primers used are listed in Supplementary Table S1 .

Synchronous schizont-stage parasites (approximately 1×10^9 parasites) were cross-linked with formaldehyde and quenched with 0.125 M glycine. Erythrocytes were lysed using 0.15% saponin in PBS, followed by three washes with PBS. The parasite pellet was resuspended in lysis buffer and mechanically disrupted using a Dounce homogenizer. Chromatin was sheared to an average size of 100–500 bp using a sonicator. The sheared chromatin was centrifuged to remove debris. Then the supernatant was incubated with Protein A/G magnetic beads for 2 hours at 4°C with rotation. After magnetic separation, a small aliquot of the supernatant was reserved as an input control and stored at -80°C. The remaining chromatin was incubated overnight at 4°C with rotation in the presence of anti-HA antibody (2 μg) and Protein A/G magnetic beads (25 μL/mL). Then the immune complexes were washed sequentially with: low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE buffer. Finally, chromatin was eluted with the elution buffer by rotation for 30 min at room temperature. Cross-links were reversed by adding NaCl to 200 mM final concentration and incubating overnight at 45 °C. Samples were then treated with RNase A and Proteinase K. DNA was purified using the PCR Purification Kit. Libraries were prepared with the VAHTS Universal DNA Library Prep Kit for Illumina V4 (Vazyme) following the manufacturer’s instructions and sequenced on the Illumina NovaSeq 6000 platform (150 bp paired-end) at BMK Biotechnology. We used technical replicates in this experiment.

Raw sequencing reads were quality trimmed using TrimGalore as described. The cleaned paired-end reads were aligned to the genome using Bowtie2 (v2.4.4). Resulting SAM files were converted to sorted BAM format using SAMtools (v1.15). Significant binding sites were identified by performing peak calling on two biological replicates using MACS2 (v2.2.7.1) with a threshold of q-value < 0.05. Peaks present in both replicates (overlap ≥ 60%) and with a fold enrichment greater than 1.8 were selected using Bedtools intersect (v2.31.1). These high-confidence peaks were subsequently annotated as target genes. Read coverage was normalized and visualized using bamCoverage to generate bigWig files with RPKM normalization. And Integrative Genomics Viewer was used for manual inspection of specific loci. All statistical analyses and visualizations were performed in RStudio (v4.4.2) using ggplot2 (v3.5.2) and ChIPseeker (v1.30.3) packages.

Significantly differentially expressed genes were functionally annotated, and Gene Ontology (GO) enrichment analysis was performed in the PlasmoDB website (https://plasmodb.org/plasmo/app), with significance determined by the hypergeometric test (p < 0.05). All statistical analyses and visualizations were generated using ggplot2 (v3.5.2) in RStudio.

The P. falciparum orthologue of TCF25, PF3D7_0506800, contains a coding region of 2,790 bp, which is composed of three exons and encodes a protein of approximately 110 kDa. In this study, we conducted a comprehensive multiple sequence alignment of the TCF25 functional domain sequences (304-868aa) from P. falciparum and two other diverse species: Saccharomyces cerevisiae and Homo sapiens. Our analysis revealed significant sequence conservation within the N-terminal region of the functional domain, suggesting strong evolutionary pressure to preserve this segment. However, compared to yeast Rqc1 and human Nulp1, PfTCF25 appears to have retained distinct evolutionary features, indicating that this protein may perform specialized functions in Plasmodium spp. ( Figure 1A ).

Previous studies have indicated that tcf25 is a dispensable gene for asexual blood-stage development (Zhang et al., 2018). Therefore, we successfully deleted tcf25 using a CRISPR/Cas9-based strategy. Given the considerable length of the tcf25 gene, we selected a 112 bp region near the start codon as the target for deletion. The upstream and downstream DNA regions flanking the target sequence were PCR-amplified and cloned into the pL6CS vector, which has been previously employed for gene editing in P. falciparum (Shang et al., 2021). A 20 bp guide RNA sequence was also inserted into the vector. The resulting construct was co-transfected with the pUF1-Cas9 plasmid into WT parasites ( Figure 1B ). The deletion of the tcf25 locus (tcf25_ko) parasite lines was confirmed by PCR and Sanger sequencing ( Figures 1C, D ). To assess the potential effect of tcf25 deletion on asexual parasite replication, we conducted in vitro growth assays comparing WT and tcf25_ko strains over three replication cycles. Parasitemia was monitored by microscopic examination of Giemsa-stained thin blood smears. We observed a slight upregulation in the tcf25_ko strain during the third cycle. However, no significant difference was observed between WT and tcf25_ko strains in the fourth cycle ( Figure 1E ). These findings suggest that tcf25 is dispensable for asexual blood-stage replication.

Since the absence of tcf25 does not affect the asexual replication in the blood stage, we further investigated its role in transcriptional regulation. RNA-seq was performed between WT and tcf25_ko parasite strains at the ring and schizont stages ( Figure 2A ). Comparative RNA-seq analysis of WT and tcf25_ko parasites identified 192 differentially expressed genes (DEGs) in the ring stage, with 24 upregulated and 168 downregulated in the knockout strain ( Figure 2B ; Supplementary Tables S2 , S3 ). Upregulated genes were predominantly associated with antigenic variation, while Gene Ontology (GO) enrichment analysis of downregulated transcripts revealed significant enrichment for lipid metabolic process, carbohydrate derivative biosynthetic process, cell cycle process, microtubule cytoskeleton organization and meiotic cell cycle ( Figure 2C ). At the schizont stage, 57 DEGs were detected (4 upregulated, 53 downregulated) ( Figure 2D ; Supplementary Tables S2 , S3 ). GO analysis linked these downregulated genes to biological processes involved in interaction with host, evasion of host immune response, obsolete pathogenesis, and actin filament bundle assembly ( Figure 2E ). These findings demonstrate that tcf25 deletion exerts genome-wide transcriptional effects in P. falciparum.

To explore consistently altered genes, we examined the intersection of downregulated DEGs after tcf25 deletion at both the ring and schizont stages. The data revealed 17 consistently downregulated genes in the tcf25_ko strain, including gexp04, pfg14-748, and g27/25, which were associated with gametocyte development ( Figures 3A, B ) (Healer et al., 1997; Eksi et al., 2005). To evaluate the impact of tcf25 deletion on sexual commitment and development, we induced gametocytogenesis in WT and tcf25_ko parasite lines and monitored developmental progression via morphological analysis of Giemsa-stained thin blood smears. To ensure reproducibility of the tcf25 deletion phenotype, two independently generated tcf25_ko clones were used in this experiment. The tcf25_ko clone 1 line exhibited a marked reduction in gametocyte, with conversion efficiency decreased by approximately 69% compared to WT strain, while clone 2 displayed a reduction of approximately 74% in gametocyte production ( Figure 3C ). Nevertheless, gametocytes from the tcf25_ko strain displayed normal morphology and retained the ability to reach maturation, indicating that TCF25 is critical for efficient sexual commitment, while it is dispensable for gametocyte development ( Figure 3D ). To elucidate the molecular basis of this phenotype, we examined the transcriptional changes in gametocytogenesis-associated genes in tcf25_ko strain at the ring and schizont stages. Comparative expression analysis revealed a global downregulation of genes in sexual commitment and development ( Figure 3E ). Notably, ap2-g, the master transcriptional regulator of gametocytogenesis, was downregulated in the knockout line, although this reduction did not reach statistical significance ( Figures 3F, G ) (Kafsack et al., 2014). These findings demonstrate that tcf25 deficiency disrupts the transcriptional activation of gametocyte conversion and development genes.

To characterize the expression profile of tcf25 across the intraerythrocytic development cycle, we analyzed recent transcriptomic data assessing mRNA abundance during asexual proliferation (R, T, and S) and gametocyte development (day2-10) (Shang et al., 2021). The data indicated that tcf25 exhibits relatively stable expression throughout these stages, with only a modest elevation during the trophozoite stage ( Figure 4A ). This suggests a potential functional role for TCF25 in both asexual and sexual stages. To determine the subcellular localization of TCF25, we generated a tcf25::ha-tagged parasite line ( Figure 4B ). HA-tagged TCF25 protein was successfully detected by Western blot analysis ( Figure 4C ). Immunofluorescence assay (IFA) confirmed that TCF25 is expressed throughout asexual blood stages, with nuclear localization and cytoplasmic distribution ( Figure 4D ). This nuclear enrichment suggests TCF25 may function as a nuclear regulatory factor. To systematically identify TCF25’s molecular targets, we performed ChIP-seq using synchronized schizont-stage parasites. Peak calling analysis revealed 44 high-confidence TCF25 binding genes distributed across the parasite genome ( Supplementary Tables S4 , S5 ). GO analysis revealed that these target genes are associated with biological process involved in interaction with host, cytoadherence to microvasculature, mediated by symbiont protein, evasion of host immune response, etc. ( Figure 4E ). Genomic mapping identified significant TCF25 enrichment in gene bodies, with additional occupancy detected at promoter regions and 3’ UTRs ( Figures 4F, G ). We next categorized TCF25-bound peaks by genomic distribution, revealing occupancy across diverse regulatory elements: promoter (14%), exon (82%), and 3’ UTR (4%) ( Figure 5A ). Integrated analysis of ChIP-seq and transcriptomic data demonstrated that during the ring stage, TCF25-bound genes showed significantly upregulated expression relative to non-bound genes (p < 0.05). This regulatory pattern was absent during the schizont stage ( Figure 5B ). These results imply that TCF25 likely acts as a transcriptional repressor.

To investigate the role of TCF25 in gene expression, we integrated ChIP-seq data with transcriptomic profiles from WT and tcf25 knockout parasites. Intersection analysis between TCF25-bound target genes and DEGs from transcriptomic data identified 8 overlapping genes ( Figure 5C ). This subset was predominantly composed of rRNA genes. Considering the well-established regulatory functions of ApiAP2 transcription factors in orchestrating parasite stage transitions, we performed comprehensive transcriptional profiling to characterize expression alterations in ApiAP2 genes upon tcf25 knockout. The analysis of all 30 ApiAP2 transcription factors revealed stage-specific dysregulation: 93% (28/30) demonstrated reduced expression during the ring stage, while 63% (19/30) were downregulated in schizont stages (Singhal et al., 2024). The most pronounced effects occurred within the ap2-g subfamily, with ap2-g, ap2-g3, and ap2-g4 showing the greatest suppression, consistent with their known functions in sexual commitment ( Figure 5D ). Together, these results demonstrate that TCF25 mediates gametocytogenesis through the ap2-g pathway.

As a member of the RQC complex, TCF25 is critically involved in ribosomal rescue pathways by facilitating the disassembly and release of stalled 60S subunits from arrested ribosomes, thereby maintaining translational fidelity. In P. falciparum, four distinct rRNA species (28S, 5.8S, 5S, and 18S rRNA) are encoded as gene clusters distributed across chromosomes 1, 5, 7, 8, 11, 13, and 14. These rRNAs can be classified into two major types: A-type and S-type. The A-type rRNAs (A1 and A2) are expressed during the asexual blood stage, whereas the S-type rRNAs (S1 and S2) are active in the gametocyte and sporozoite stages (Fang et al., 2004). Genome-wide binding profiling revealed significant enrichment of TCF25 specifically at rDNA clusters on chromosomes 5, 7, and 14, which primarily encode A-type rRNAs. IGV visualization demonstrated predominant localization within coding regions, along with peaks at promoter elements. Chromosome 7 exhibited the strongest TCF25 binding signal among all rDNA loci ( Figures 6A–C ). Given the limitations of RNA-seq in accurately quantifying rRNA levels, we performed RT-qPCR to measure rRNA abundance in two independent tcf25_ko clones. Notably, both clones exhibited a significant upregulation of 28S rRNA transcripts (p < 0.01), a core structural component of the 60S ribosomal subunit ( Figures 6D, E ). This finding suggests that TCF25 deficiency may impair the recycling of 60S subunits, leading to their compensatory overproduction. Collectively, these results suggest the critical role of TCF25 in ribosome biogenesis and ribosomal subunit homeostasis.