A single valine to leucine switch disrupts Plasmodium falciparum AP2-G DNA binding and reveals GDV1’s role in ap2-g activation

Domain: chemistry
Published: 2026-01-17
Editorial outcome: minor_revision

Abstract
Sexual commitment in Plasmodium parasites is essential for malaria transmission, yet the signaling events initiating sexual conversion in only a subpopulation of parasites remain unclear. We discovered a single valine (V) to leucine (L) mutation in a transcription factor required for P. falciparum gametocytogenesis (AP2-G) that abrogates sexual differentiation. AP2-G.L does not bind the ap2-g consensus motif, GnGTAC, or stimulate gene transcription, including autoregulation. The GDV1-dependent expression of AP2-G.L demonstrates GDV1’s critical role in the initial activation of the silent ap2-g locus in the absence of functional AP2-G. While AP2-G.V is required for MSRP1 expression, providing a marker that discriminates early from late sexually committed schizonts. Together this work demonstrates that V in AP2-G plays a critical role in DNA binding, highlighting the functional importance of this specific region for malaria transmission as well as the key role of GDV1 in the initial activation of ap2-g expression and induction of sexual differentiation.

Introduction
Malaria transmission is a global public health concern which accounted for 263 million clinical cases and more than half a million of deaths in 20231. A critical step in human-to-human transmission via mosquito is the production of sexual stage Plasmodium parasites, called gametocytes. Sexual differentiation begins in a subpopulation of blood stage parasites during the asexual replication cycle, but the percent of parasites that commit to sexual development varies in vitro and in vivo2,3. In contrast to relatively low in vitro gametocyte-commitment rates (GCR) (<30%)2 in P. falciparum laboratory adapted strains and clinical isolates, in vivo symptomatic or asymptomatic malaria studies show dramatic variability in GCRs (0-78%)3,4. The factors underlying this quantitative variability in gametocyte production remain undefined and make it difficult to target and eliminate the infectious reservoir. This GCR variation coupled, with the high prevalence of asymptomatic malaria infections, which can be as high as 60-80% in some regions5–7, and the inability of commonly used antimalarials to kill mature gametocytes8 are major challenges to malaria control programs. Defining the molecular mechanisms underlying the regulation of sexual commitment should facilitate the design of new transmission blocking strategies.

Two genes have been found to be required to initiate sexual commitment in P. falciparum, gametocyte development protein 1 (gdv1: PF3D7_0935400)9 and a plant-like apetala2 transcription factor (ap2-g: PF3D7_1222600)10. GDV1 and AP2-G are required for ap2-g expression3,11, while AP2-G also activates downstream genes needed for the progression of sexually committed rings to continue gametocyte development10,12. In a sexually committed blood stage parasites, the ap2-g locus is epigenetically silenced by histone 3 lysine 9 trimethylation (H3K9Me3)13 and this histone modification is stabilized by heterochromatin protein 1 (HP1: PF3D7_1220900)11,14. As the blood stage parasite matures, the start of DNA replication marks entry into the schizont stage of the life cycle. During schizogony, it has been proposed that in the subpopulation of schizonts that express GDV1, the ap2-g locus is de-repressed11, but the induction and regulation of this decision has not been established. There is evidence that once GDV1 is expressed, it interacts with HP-1 and this interaction has been proposed to contribute to ap2-g de-repression by removing HP111, but the detailed mechanism that confers specificity to the release of HP-1 only at the ap2-g locus and only in some parasites remains to be elucidated. Once expressed, AP2-G induces its own expression, termed autoregulation, by binding to the core AP2-G DNA recognition motif GnGTAC that is present in the 5’ flanking region of ap2-g10,12. This motif is also present upstream of a number of other genes, some of which are upregulated in the presence of AP2-G in schizonts and/or the next generation of blood stage parasites, called rings, initiating gametocyte differentiation. The reasons different AP2-G-dependent genes are expressed at different times after AP2-G expression remains unclear as does the regulation of initial expression of GDV1 and AP2-G.

In higher eukaryotes, de-repression of epigenetically silent genes has been described through post-translation modifications15. Phosphorylation of histone 3 (H3) serine 10 has been demonstrated to play a critical role in release of HP1 from lysine 9 trimethylated H316–18. This change allows RNA polymerase access to transcribe the previously silenced genes. In P. falciparum, several studies have consistently shown phosphorylation of H3 serine 29 and 3319–21 in trophozoite and schizont stages, which is when sexual commitment is also thought to be initiated. It is possible that phosphorylation of H3 serine 29 and/or 33 or both could play critical roles in releasing HP1 from ap2-g locus bound H3K9Me3.

We sought to evaluate whether serine/threonine kinase (STK2: PF3D7_0214600), which has a nuclear localization signal, could be one of the kinases playing a role in the phosphorylation of H320,22. We used reverse genetics to define the role of STK2 in gametocytogenesis but, unexpectedly, whole genome sequencing of the stk2 knockout parasites revealed a single valine to leucine mutation in ap2-g. We show that the replacement of valine with leucine in the presence and absence of stk2 abrogates AP2-G binding to the upstream core GnGTAC motif, autoregulation, downstream gene expression and gametocyte production. Here we have identified a highly robust system that can be utilized to study the transcriptional activation of ap2-g by an upstream regulator, GDV1, and monitor the activation of ap2-g specific targets.

Results
Determining the genetic locus underlying a complete loss of gametocytogenesis
In the course of evaluating kinases for a role in gametocytogenesis, we generated a serine/threonine kinase (STK2: PF3D7_0214600) knockout line (3D7.stk2Δ) in a gametocyte-competent P. falciparum 3D7 strain (Supplementary Fig. 1a–c). Asexual growth was normal consistent with the previous piggyBac transposon insertion mutagenesis prediction that stk2 is dispensable during asexual growth23. In marked contrast, 3D7.stk2Δ clones did not produce immature or mature gametocytes (Fig. 1a, b, Supplementary Fig. 1d). To determine whether the block was at sexual commitment or maturation of sexually committed rings, we quantified gametocyte-committed ring stage parasites using the validated early gametocyte biomarker ap2-g4. Ap2-g expression levels were significantly reduced in 3D7.stk2Δ ring stage parasites (Fig. 1c), suggesting a block in sexual commitment similar to that observed in a previously reported gametocyte deficient line, F12 which has a premature stop codon in ap2-g at bp 392310, and a gametocyte inducible line NF54.gdv1.3xha.fkbp in the absence of Shield1 (Shld1-). In the gametocyte inducible line gdv1 is tagged with an in-frame ligand-dependent degradation domain (FKBP). In the absence of Shield 1 (Shld1) ligand the GDV1-FKBP chimeric protein is targeted for proteasomal degradation24 and therefore not available to activate ap2-g expression and sexual commitment. The presence of Shld1 blocks degradation of GDV1-FKBP and sexual commitment proceeds normally.

To confirm the phenotype and define the cellular localization of STK2, we used the original double crossover plasmid to generate an inducible STK2 reporter line (NF54.stk2.gfp.fkbp) as well as a standard knockout (NF54.stk2Δ) in a gametocyte competent Pf strain NF54. Surprisingly, the NF54.stk2Δ and NF54.stk2.gfp.fkbp lines with and without Shld1 all produced gametocytes and had GCRs similar to the parental line NF54 (Fig. 1d, e). These results strongly suggest that the loss of gametocytogenesis in 3D7.stk2Δ clones was independent of stk2.

The gdv1 and ap2-g loci previously associated with gametocyte production9,10, remained intact in the 3D7.stk2Δ line and were free from the known ap2-g nonsense (F12) or frameshift (GNP-4) mutations10, or nonsense mutations (K561*, Q578*) in gdv125,26 (Supplementary Fig. 2). To extend our search, we sequenced the whole genome of the 3D7.stk2Δ line. Whole genome analysis confirmed the lack of deletions or mutations conferring premature termination of gdv1 or ap2-g. However, ap2-g had a missense mutation (g.6487G>T) (Fig. 1f) that resulted in a switch from valine (V) to leucine (L) in the AP2 domain (V2163L). This V2163L mutation was the only missense SNP identified in 3D7.stk2Δ not present in wild type NF54 (Supplementary Table 1). The AP2-G AP2 domain is 50 aa long (AP2-G 2162-2210)22,27 and highly conserved across the Plasmodium species infecting human, rodent and monkeys with all these species having a valine at the position corresponding to V2163 (Fig. 1g). Additionally, in the global P. falciparum isolate repositories analyzed to date, no mutations have been reported in the AP2-G AP2 domain (PlasmoDB22, MalariaGEN28) (Fig. 1h). These results suggest that the AP2-G AP2 domain is under strong selective pressure in the field as expected for a gene required for transmission. The 3D7.stk2Δ line was therefore designated as 3D7.stk2Δ//ap2-g.L and wild type (wt) or mutant ap2-g designated ap2-g.V or ap2-g.L, respectively.

The AP2-G V2163L substitution is sufficient for a loss of gametocytogenesis
To confirm that ap2-g.V2163L prevents gametocytogenesis, we swapped the V allele with an L allele in a gametocyte competent P. falciparum strain, NF54, using a forward mutation editing approach (Fig. 2a), generating transgenic mutant lines (NF54.ap2-g.L and NF54.ap2-g.L.gfp.fkbp). While both lines have a V to L replacement, the NF54.ap2-g.L.gfp.fkbp line also has GFP.FKBP inserted in frame to regulate AP2-G.L. The whole genomes of both lines were sequenced and compared to the parental line, NF54, to rule out the presence of any unintended mutations. None of the lines expressing AP2-G.L produced immature or mature gametocytes (Fig. 2b, c), which is the same phenotype shown by original 3D7.stk2Δ//ap2-g.L clones (Fig. 1a, b). To strengthen our hypothesis, we also tested whether reversing the L2163 mutation back to V can restore gametocytogenesis in the 3D7.stk2Δ//ap2-g.L line, as well as the NF54.ap2-g.L.gfp.fkbp line (Fig. 2d). Correcting the mutation to V restored the rate of gametocytogenesis to that observed in the original parental lines (NF54 or 3D7) (Fig. 2e, f). This forward and reverse mutation editing approach firmly supports the valine to leucine mutation, not the stk2 knockout or another mutation, as the cause of the loss of gametocytogenesis in the 3D7.stk2Δ//ap2-g.L line.

V2163L switch disrupts DNA binding
AP2 domain of AP2-G was described earlier22,27, and V2163 is located at the N-terminus of the domain (Fig. 1h). MOTIF search (https://www.genome.jp/tools/motif/) analysis also predicted the same amino acids as an AP2 domain (aa 2162 to 2210, i-Evalue = 0.00053) in which V2163 is the second aa (Fig. 3a). Changing V2163 to L to mimic the observed mutations shifted the start of the predicted AP2 domain downstream by 11 aa decreasing its length (Fig. 3a) and increasing the independent Expectation Value (i-Evalue = 0.0023) indicating reduced confidence in the motif prediction. Eleven aa shorter AP2 domains were also predicted when V2163 was substituted by any of the other aa (Fig. 3a, Supplementary Fig. 3), except isoleucine which was predicted to cause only a six aa shift (Fig. 3a). Consistent with the MOTIF search predicted AP2 domain, AlphaFold 3D structure modeling29,30 also predicted the structure of the same amino acids (aa 2162-2210) with a high confidence (Fig. 3b–d). MutationExplorer was then used to evaluate changes to the AP2-G structure following the leucine 2163 mutation. The analysis predicted a minimal overall structural destabilization (Total energy difference with parent = −25.5) with localized changes in stability within (Fig. 3e, f) and in close proximity to the AP2 domain (Supplementary Table 2), suggesting the possibility of a structural alteration in the mutated AP2 domain that could alter DNA binding.

These computational predictions are consistent with V2163 being critical for DNA binding and functional activity31. To confirm the in-silico prediction, we generated recombinant proteins encoding the AP2 domain (AP2-G 2150-2220, 60 aa) containing mutated (AP2-G.L) or wild type amino acid (AP2-G.V) as well as P. berghei wild type PbAP2-G. The recombinant proteins were purified from the soluble fraction of E. coli extracts and tested in an electrophoretic mobility shift assay (EMSA) for binding to a biotinylated oligonucleotide (GTGTACAC) that includes the core PfAP2-G DNA recognition motif GnGTAC10,12. In contrast to the wild type recombinant PfAP2-G domain (AP2-G.V) and PbAP2-G domain, the mutant recombinant protein (PfAP2-G.L) did not bind the GTGTACAC oligo (Fig. 3g). Both the in vitro and in silico results suggest that mutated AP2-G.L would not bind to the core GnGTAC motif in genomic DNA and therefore no longer act as a functional transcription factor to activate gene expression. An alteration in DNA binding is also consistent with the inability of AP2-G.L to support ap2-g transcription and gametocyte production in the 3D7.stk2Δ//ap2-g.L line (Fig. 1a–c).

GDV1 activates ap2-g in the absence of AP2-G or its autoregulation
Parasite lines expressing AP2-G.L provide tools to investigate the initiation of ap2-g expression in the absence of AP2-G autoregulation and interrogate the separate roles of GDV1 and AP2-G in this initiation process. Ap2-g expression is highly dynamic during schizogony, therefore first we did an RNA time-course using a gametocyte inducible line (NF54.gdv1.3xha.fkbp//ap2-g.V) expressing wild type AP2-G to identify the best time to compare expression between lines. As described previously, in the gametocyte inducible line GDV1-FKBP is stabilized by Shld1 ligand, and without Shld1 GDV1-FKBP gets degraded, as a result ap2-g is not expressed and there is no sexual commitment. Prior to the experiments, parasites were maintained in the absence of Shld1 to eliminate contamination with gametocyte-committed parasites produced during earlier asexual cycles that also express ap2-g and could interfere in the accurate determination of ap2-g expression initiation. After Shld1 addition to half the parasite cultures, ap2-g transcription was monitored every other hour starting from 28 hours post invasion (hpi) through 42 hpi in both Shld1 (+) and (-) conditions (Fig. 4a). This time course covers the trophozoite to schizont developmental transition. The ap2-g RNA time-course revealed that in the presence of Shld1-stabilized GDV1, ap2-g transcripts first increased over baseline (~two-fold) at 32 hpi and this increase was maintained until 36 hpi. After 36 hpi, ap2-g transcripts rose dramatically, until plateauing at 40 hpi. Six days later stage III-IV gametocytes were detected in the culture (Fig. 4b). In contrast, in the absence of Shld1 there was no increase in ap2-g transcript (Fig. 4a), and gametocytes were not produced (Fig. 4b).

From the time course, 40-44 hpi was selected to compare ap2-g expression in lines with wt gdv1 and wt or mutant ap2-g tagged with fkbp (ap2-g.V.fkbp//wt gdv1, ap2-g.L.fkbp//wt gdv1), allowing Shld1-mediated regulation of AP2-G. In addition, fkbp-tagged gdv1 lines expressing non-tagged wt or mutant ap2-g (gdv1.3xha.fkbp//ap2-g.V or gdv1.3xha.fkbp//ap2-g.L) were included to compare the effect of the presence or absence of GDV1 on wt or mutant AP2-G expression and activity. Ap2-g transcription at schizont stage was completely disrupted by GDV1 knockdown in the absence of Shld1, regardless of whether the line expressed wt or mutated ap2-g (Fig. 4c). In contrast, in the fkbp-tagged ap2-g wt or mutant lines expressing wt GDV1, as well as a gametocyte-deficient line, F12, which expresses wt GDV1 but has a truncated AP2-G lacking the AP2 domain10, ap2-g was still expressed (~16 fold or more) in the presence or absence of Shld1. However, ap2-g expression was higher in ap2-g.V.fkbp line (Shld1+) than the ap2-g.L.fkbp line (Shld1+) or either line in the absence of Shld1. These results are consistent with wt GDV1 initiating ap2-g expression in all these lines, while only Shld1-stabilized wt AP2-G.V stimulated AP2-G mediated autoregulation. Ap2-g transcript levels were also higher in the presence of Shld1 in the gdv1.fkbp//ap2.g.V line than the gdv1.fkbp//ap2-g.L line (Fig. 4c) again indicating that during schizogony wt AP2-G enhances its own transcription.

In the next generation, only parasites expressing both AP2-G.V and GDV1 [3D7.stk2Δ//ap2-g.V, NF54-gdv1.3xha.fkbp//Ap2-g.V (Shld1+)] expressed all of the AP2-G dependent early gametocyte genes4 tested (Fig. 4d, e, Supplementary Fig. 4a–g) that contain at least one copy of the upstream AP2-G DNA recognition motif, GnGTAC. In contrast, none of these early gametocyte genes were expressed by parasite lines expressing AP2-G.L (3D7.stk2Δ//ap2-g.L, NF54.ap2-g.L), truncated AP2-G (F12) or those lacking GDV1 [NF54.gdv1.3xha.fkbp//ap2-g.V (Shld1-)]. These results clearly demonstrate that after GDV1 initiates ap2-g expression AP2-G.V, not AP2-G.L, stimulates ap2-g autoregulation during schizogony and downstream gene expression in the progeny consistent with the need for DNA binding to mediate these effects (Fig. 4f).

Quantification of ap2-g.V & ap2-g.L and gdv1 translation
A nanoluciferase (nLuc) reporter was used to quantify and compare the time course of ap2-g.V, ap2-g.L, and gdv1 translation using a series of transgenic lines (3D7.stk2Δ//ap2-g.V.fkbp.p2a.nLuc, 3D7.stk2Δ//ap2-g.L.fkbp.p2a.nLuc and NF54.gdv1.p2a.nLuc). The P2A peptide that causes ribosome skipping32,33 was included upstream of nLuc to allow the production of two separate proteins from a single RNA. We first confirmed a linear increase in nLuc signal with increasing parasite number (Supplementary Fig. 5). The assay was then used to monitor nLuc activity from 20 to 44 hpi and again 9 and 22 h later (53 hpi & 66 hpi) in the next generation rings. The time course data shows that signal from gdv1-linked nLuc was first detected at 26 hpi, which is prior to ap2-g-linked nLuc activity, as expected for an upstream regulator. Gdv1-linked nLuc activity increased rapidly until 34/35 hpi, then started to plateau (Fig. 5a). This early GDV1 activity is also supported through live imaging of a GDV1-tdTomato reporter line (683.gdv1.3xha.tdTom.fkbp) in which the GDV1-tdTomato fluorescent signal was first detected at 28-30 hpi which corresponds to the late trophozoite to early schizont transition prior to the first nuclear division (Fig. 5b). As schizogony progressed, GDV1 expression continued and a punctate pattern around each nucleus was observed (Supplementary Fig. 6). In contrast to gdv1-linked nLuc, ap2-g.V-linked nLuc activity was first detected at 31 hpi and continued to increase until 41 hpi after which activity plateaued, consistent with its transcription time-course (Fig. 4a). The time course of Ap2-g.L-linked nLuc signal was similar to ap2-g.V-linked nLuc activity during schizogony but levels were 2-3 fold lower in magnitude than ap2-g.V-linked nLuc activity, even throughout the plateau at 39-44 hpi (Fig. 5a). In the next generation ring stage parasites (53 & 66 hpi) ap2-g.V-linked nLuc activity remained high, which is in marked contrast with the ap2-g.L-linked nLuc line that had ~12 fold less nLuc activity and no gametocyte conversion (Fig. 5a, Supplementary Fig. 7). The increased ap2-g.V-linked nLuc activity in comparison to ap2-g.L-linked nLuc activity in schizonts (38-40 hpi) and next generation rings stage parasites (10-15 hpi) was confirmed using additional ap2-g.L (NF54.ap2-g.L.p2a.nLuc, 3D7.stk2Δ//ap2-g.L.fkbp.p2a.nLuc) and ap2-g.V (NF54.ap2-g.V.p2a.nLuc, 3D7.stk2Δ//ap2-g.V.fkbp.p2a.nLuc) parasite lines with NF54 or 3D7 backgrounds. Consistently there was a significant increase in nLuc activity in schizonts (3-4 fold: Fig. 5c) and rings (4-10 fold: Fig. 5d) stages in ap2-g.V over ap2-g.L parasite lines with the same parent as well as a significant increase in gametocyte conversion rate in the ap2-G.V lines (Fig. 5e). This pattern is consistent with an increase in AP2-G.V, not AP2-G.L protein expression due to AP2-G.V-dependent autoregulation. The nLuc activity observed in ap2-g.L lines in schizont stages strongly support wt GDV1 driving the initial ap2-g.V or ap2-g.L transcription, that is then augmented in the ap2-g.V lines due to autoregulation. The markedly lower levels of ap2-g.L-linked nLuc activity in the next generation rings are consistent with the decrease in GDV1-linked nLuc activity in rings (Fig. 5a) as well as the lack of functional AP2-G.V to maintain expression in the absence of GDV1. The findings also highlight the importance of AP2-G.V expression in gametocyte maturation.

To directly assess the timing of the requirement for GDV1 in ap2-g transcription initiation, we generated NF54.gdv1.3xha.fkbp//ap2-g.V.p2a.nLuc transgenic reporter that allows monitoring of ap2-g activity by regulating GDV1 with Shld1. As expected in the absence of Shld1 there was no ap2-g.V-linked nLuc activity (Fig. 5f) and following the addition of Shld1 at ring stages (16 hpi) ap2-g.V-linked nLuc activity began at ~33 hpi and started to plateau at 40 hpi (Supplementary Fig. 8a). To determine when GDV1 was no longer needed for ap2-g.V expression, Shld1 was added to tightly synchronized cultures in a 24 well plate at 16 hpi and then removed from replicate wells at defined time points from 30 hpi to 44 hpi (Supplementary Fig. 8b). Ap2-g.V-linked nLuc activity was assessed in all wells at 44 hpi (schizonts) and 56 hpi (rings). The GCR of each culture was also measured (Supplementary Fig. 8b). Early GDV1 removal (<40 hpi) resulted in lower ap2-g.V-linked nLuc levels in both schizonts (44 hpi: Fig. 5g) and the next generation rings (56 hpi: Supplementary Fig. 8c), as well as lower gametocyte production. Levels of ap2-g.V-linked nLuc activity gradually increased after 32-34 hpi reaching a plateau at ~40 hpi, after which time removing GDV1 had little effect (Fig. 5g). This controlled GDV1 exposure indicates it is required until ~40 hpi, for maximal ap2-g.V expression during the trophozoite-schizont transition.

Dynamics of AP2-G autoregulation and downstream gene induction
Ap2-g.mScarlet (mSc) reporter lines (3D7.stk2Δ//ap2-g.L.mSc or 3D7.stk2Δ//ap2-g.V.mSc) were generated to compare AP2-G.V and AP2-G.L protein expression patterns and identify sexually committed schizonts. Live imaging and flow cytometry show a subpopulation of schizonts (9-11%) positive for AP2-G.V.mSc which is similar to the 8-10% day 4 GCR (Fig. 6a, Supplementary Fig. 9a-b), suggesting the positive schizonts progressed to gametocytes. In contrast, none of the ap2-g.L.mSc lines were positive and there was no GCR (Fig. 6a, Supplementary Fig. 9a-b). It is possible that the fluorescence assays are not sensitive enough to detect the amount of AP2-G.L generated from the low transcript levels induced by GDV1 in the absence of AP2-G autoregulation or, alternatively, that the DNA binding activity of AP2-G.V is required for protein expression.

To extend the expression profile of sexually committed schizonts beyond ap2-g, the RNA time course of msrp1 (ID# PF3D7_1335000) another gene expressed in sexually committed schizonts was tested. Msrp1 transcription increases over baseline only in the AP2-G.V line and the time course was slightly later than ap2-g RNA (Supplementary Fig. 10a), consistent with earlier reports that it is AP2-G regulated3,4,12. Protein expression was tracked by tagging MSRP1 with mNeonGreen (mNG) in 3D7 and NF54 parental lines (3D7.stk2Δ//ap2-g.L.mSc//msrp1.mNG, 3D7.stk2Δ//ap2-g.V.mSc//msrp1.mNG, NF54.msrp1.mNG//ap2-g.V.mSc and NF54.msrp1.mNG//ap2-g.L.mSc). AP2-G.V.mSc and MSRP1.mNG were co-expressed in a subpopulation of schizonts, while no signal from either protein was detected in the AP2-G.L lines using fluorescence microscopy (Fig. 6a, b, Supplementary Fig. 10d (lower panels)). AP2-G.V.mSc expression was punctate and perinuclear (correlation coefficient with DNA=0.56±0.08). MSRP1 was present throughout cytoplasm and tended to be localized to the merozoite periphery in mature schizonts (46-48 hpi) (Fig. 6b (top panel), Supplementary Fig. 10b). Using flow cytometry AP2-G+MSRP1+ double positive schizonts were also only found in the AP2-G.V lines and they were in the same proportion as the D4 GCR (Fig. 6c, Supplementary Fig. 10e) and a similar correlation was found between MSRP1+ schizonts and D4 GCR in the NF54.msrp1.mNG line (Supplementary Fig. 10c) suggesting MSRP1 can be used as another marker of sexual commitment. In some experiments, low background fluorescence (<3×103 fluorescence intensity) was detected in the AP2-G.L lines using flow cytometry, but this variation is likely due to differences in the background fluorescence of different RBC preparations.

Analysis of MACS-purified schizonts from a dual reporter line (NF54.ap2-g.V.mSc//msrp1.mNG) indicated schizonts single positive for AP2-G.mSc, not MSRP1.mNG, had less DNA, suggesting they were younger than schizonts double positive for AP2-G.mSc and MSRP1.mNG (Fig. 6d). In these dual reporter lines there was also no MSRP1.mNG single positive schizonts, only double positive AP2-G.mSc / MSRP1.mNG schizonts, consistent with MSRP1 expression being dependent on AP2-G. The time course of AP2-G.V.mSc and MSRP1.mNG (NF54.ap2-g.V.mSc//msrp1.mNG) expression was further evaluated every hour from 28-46 hpi to understand the dynamics of sexual commitment (Fig. 6e, Supplementary Fig. 11). AP2-G.mSc single positives schizonts were first observed from 39-40 hpi, typically in 4-5 nuclear schizonts, and none of the schizonts with less than three nuclei were positive. Approximately 2 hrs later (41-42 hpi), these AP2-G positive schizonts turned positive for MSRP1.mNG. Again, no single positive MSRP1.mNG schizonts were detected (Supplementary Fig. 11). AP2-G and MSRP1 expression was much later than GDV1.tdTom, which was detected prior to nuclear division at 28-29 hpi (Fig. 5b, Supplementary Fig. 6). These distinct MSRP1.mNG expression patterns allow differentiation of early and late sexually committed schizonts and demonstrate the distinct dynamics of AP2-G.V and downstream gene induction.

Methods
Parasite culture
Plasmodium falciparum parasite strains (NF54, 3D7) or transgenic or mutant lines, were cultured in complete RPMI medium containing RPMI1640, 25 mM HEPES, 100 μg/ml of hypoxanthine, and 0.3 mg/ml of glutamine (KD Biomedical, Columbia, MD) supplemented with 25 mM NaHCO3 (pH 7.3), 5 μg/ml of gentamicin, and 10% human serum (Interstate Blood Bank, Memphis, TN) or 0.5% Albumax II (Gibco, USA) in an atmosphere of N2:O2:CO2 90:5:5. Sorbitol treatment (5%, 10–30 min at 37°C) or MACS® LS columns (Miltenyi Biotec, Auburn, CA) were used to synchronize parasites in ring or schizont stages, respectively. Parasitemia [(parasite-infected RBCs/total RBCs) *100] was evaluated through microscopy of cultures smears stained with 10% Giemsa for 15m (Sigma-Aldrich, St Louis, MO). Light microscope images were captured using QC Capture 2.9.13 software.

P. falciparum gametocyte phenotyping
The gametocyte conversion rate (GCR) of a single cycle was determined by quantifying parasitemia of a synchronized ring stage culture (Day 0) then adding N-acetylglucosamine (50 mM) (NAG) to block further asexual growth. The plate/flask was then cultured for five or seven days (D4 or D6) with daily media changes without RBC supplementation and the D4 or D6 gametocytemia (stage II-III or III-IV) determined (Supplementary Fig. 1d). Parasites were quantified using Giemsa stained culture smears or flow cytometry, which is described in the next section. The GCR of each sample was calculated using the formula (D4 or D6 gametocytemia / D0 ring parasitemia) x 100.

Flow cytometry and data analysis
Parasite cultures and uninfected RBCs (negative control) were resuspended at 0.02% hematocrit (~2,000 cell/μl) in 1x SA buffer (154 mM NaCl, 9.27 mM glucose, 10 mM Tris-HCl, pH 7.4) with 500 nM MitoProbe DiIC(5) (Cat: M34151) and incubated at 37°C in the dark for 30 min prior to analysis on an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ) or Attune Nxt flow cytometer (Invitrogen, USA). The forward and side scatter signals were used to select the total RBC population and then the DiIC(5) (Ex 640nm/Em 675 ± 25nm) was used to identify Pf infected RBC.

A Cytek Aurora Spectral Analyzer flow cytometer (USUHS, Biomedical Instrumentation Center facility) was used to identify parasite infected RBC positive for mScarlet (mSc) or mNeonGreen (mNG) or both fluorescent protein signals. Hoechst 33342 DNA stain (Invitrogen, USA, Cat: H3570), 1:500 dilution, 10 minutes staining) was used to quantitate and identify the stage of infected RBCs (iRBCs). The proportion of mSc and or mNG positive iRBCs observed in total or stage-specific iRBCs was determined using FlowJo v10.10.0. A retrogating strategy was used to visualize and quantify only AP2-G.mSc positive iRBCs or MSRP1.mNG positive iRBCs from MACS-column purified parasites having a range of younger to mature schizonts based on the Hoechst 33342 signal. BD Accuri C6 Software, Attune software v5.3.0, and Spectral Flo v3.03 were used to analyze Accuri, Attune Nxt flow, and Cytek Aurora flow cytometers, respectively.

Plasmid engineering (CRISPR & non-CRISPR) and transgenic line generation
The stk2 knockout lines (3D7.stk2Δ.wr or NF54stk2Δ.wr) were generated using a double crossover plasmid (pCC1) in the absence of Cas9. To generate this plasmid, two homologous fragments were introduced in the plasmid using SacII + SpeI and NcoI + AvrII restriction enzymes. T4-DNA ligase was used to ligate the restriction enzyme-digested PCR products into the plasmid. The inducible stk2 line was generated using a single crossover plasmid (p1605.stk2.gfp.fkbp.wr) (Supplementary Fig. 12a) to tag the C-terminus of stk2 with green fluorescent protein (GFP) followed by the FKBP-destabilization domain (FKBP)3. This single crossover plasmid was also used to generate an ap2-g mutated line (NF54.ap2-g.L.gfp.fkbp.wr). To generate the single crossover plasmid construct, a single homologous region (600–1500 bp) of the target gene was PCR amplified, digested with XhoI + AvrII restriction enzyme and ligated into XhoI + AvrII digested plasmid using T4-DNA ligase. Primer DNA sequences for stk2 and ap2-g genes are given in Supplementary Table 3.

E. coli electrocompetent cells (DH10B) were transformed with the ligation reaction and grown overnight on ampicillin (100 μg/ml) containing LB plates. Ampicillin-resistant insert positive colonies were assessed using restriction enzyme digestion or by PCR and then further verified by DNA sequencing using Geneious prime® 2025.2.2 software. DNA sequence-confirmed colonies were expanded, and plasmid was purified using the NucleoBond Xtra Plasmid Maxi prep kit (Macherey Nagel, USA). Purified plasmid reconstituted in 1x cytomix buffer containing KCl (120 mM), CaCl2 (0.15 mM), K2HPO4/KH2PO4 (10 mM, pH 7.6), HEPES (25 mM, pH 7.6), EGTA (2 mM, pH 7.6), and MgCl2 (5 mM) was stored at -20°C. 100–150 μg plasmid DNA per transformation was used to transform parasites using established transformation protocols3. Approximately 5% rings were transformed (Day 0) and on D2 appropriate drug was added for selection: WR99210 (2.5 nM) (Jacobus Pharmaceuticals, Plainsboro, NJ) or Blasticidin (2.5 nM) (Gibco, USA). Resistant parasites from three independent transformations were selected and grown for 3 weeks without drug before reapplying the appropriate drug. The resistant parasites obtained were screened for the presence of single or double-crossover chromosomal integration (Supplementary Fig. 1b and 12b) using PCR with primers listed in Supplementary Table 3 and selected for cloning by limiting dilution. Transformed lines were maintained in the appropriate drug/s.

CRISPR/Cas9 gene editing was used to generate the majority of the gdv1, ap2-g and msrp1 transgenic lines. Single plasmid (pDC-cam-cas9-gfp.wr)52 or two-plasmid based CRISPR/Cas9 gene editing was employed to transform gametocyte competent laboratory strains (3D7, NF54) or gametocyte deficient precursor transgenic lines (3D7.stk2Δ-WR, NF54.ap2-g-L-gfp-fkbp-WR). pDC-cam-cas9.gfp.wr has a repair template (flanked by EcoRI + AatII), a guide RNA sequence and Cas9 expression cassettes52 and this plasmid is also called a single-guide plasmid (SGP). We used this SGP plasmid to replace ap2-g.V2163 with ap2-g.L2163 (pDC-cam.ap2-g.L2163.wr) or vice versa ap2-g.L2163 with ap2-g.V2163 (pDC-cam.ap2-g.V2163.bsd), and also to tag the C-terminus of gdv1 with 3xha.fkbp (pDC-cam.cas9.gdv1.3xha.fkbp-WR) or p2a.NanoLuc (pDC-cam.cas9.gdv1.p2a.NanoLuc-BSD) or tdTomato.fkbp (pDC-cam.cas9.gdv1.tdTomato.fkbp-BSD), and the msrp1 with mNeonGreen (pDC-cam.cas9.msrp1.mNeonGreen-DSM) (Supplementary Fig. 12c-d). To tag the c-terminus of ap2-g with a number of different reporters, we modified the SGP and generated a two-guide plasmid (TGP) by replacing Cas9 with a 2nd gSeq cassette using infusion cloning (Supplementary Fig. 12d-e). Using this TGP, we tagged the c-terminus of ap2-g with fkbp (pDC-cam.ap2-g(L or V).fkbp), mScarlet (pDC-cam.ap2-g(L or V).mSc), fkbp.p2a.NanoLuc (pDC-cam.ap2-g(L or V).fkbp.p2a.NanoLuc) or p2a.NanoLuc (pDC-cam.ap2-g(L or V).p2a.NanoLuc). The WR drug resistance marker was replaced with either BSD or DSM1 depending on the requirement. When TGP was used for the transformation, if the parasite line used for transformation lacked Cas9 expression a second plasmid pUF-DSM53 was used as the Cas9 source.

Guide RNA sequence (20 nt) with NGG PAM was designed using e-CRISP web-based tools54 that provide only a few but very efficient guide sequences per gene. The gRNA seq was digested with BbsI, a type IIS restriction enzyme, ligated into the plasmid using T4-DNA ligase and Stellar competent cells were used for the transfection. All other modifications (single or multiple donor fragments, drug resistance markers or the second gRNA cassette) were done using infusion cloning. To use this system, PCR fragments generated with infusion cloning compatible primers and then infused with the appropriate restriction enzymes digested plasmid. Insert positive colonies were identified and the NucleoBond Xtra Plasmid Maxi kit was used to purify plasmid for transformation as described above in the non-CRISPR plasmid section. The DNA sequences of the In Fusion PCR primers and gRNAs used are listed in Supplementary Table 3.

Fresh, uninfected RBCs were transformed using 100–150 ug of each plasmid, washed with RPMI, mixed with 0.2–1% MACS purified schizont stage parasites and incubated in triplicates in a 12 well culture plate. On D3 (ring stage) appropriate drug/s were applied for three generations/cycles (~ 6 days) to select transformed parasites and then removed. Parasites recovered after drug/s selection were tested for editing using PCR (Supplementary Fig. 12c) and confirmed by DNA sequencing. Transformed parasites were cloned after genotype verification using a limiting dilution method and clones were further tested by DNA sequencing.

Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR)
RNA was extracted from parasites preserved in 1x RNA Shld1 (Zymo Research, USA) using RNA Micro kit (Zymo Research, USA), as per the manufacturer’s instructions. RNA was eluted in 36 μl RNAse-free water and purified RNA (4.0 μl or 8.0 μl) was additionally treated with ezDNAse™ before cDNA synthesis using SuperScript™ IV VILO™ Master Mix (Invitrogen, USA). A 1:20 dilution of cDNA was made and 2 μl from this dilution was used for each RT-qPCR well. RT-qPCR (QuantStudio 7 Pro) was performed using gene-specific primers designed using the web-based tool Primer3Plus v3.3.055 (Supplementary Table 3) and Fast SYBR Green PCR Master Mix (Applied Biosystems, USA) as described earlier3. Primer efficiency was tested by serial dilution and ranged from 85 to 105%. All samples were run in duplicate and tested for both the gene of interest and the control constitutive gene, arginyl tRNA (schizont stage) or skeleton binding protein 1 (sbp1) (ring stage) on the same plate. Data was analyzed using QuantStudio™ Design & Analysis Software v2.6.0, and the ΔCt values were determined by subtracting the mean cycle threshold (Ct) value for arginyl tRNA or sbp1 from the mean Ct of each test gene. The relative quantity (2-ΔΔCt) was calculated for each gene using RNA from NF54-gdv1-3xha-fkbp parasites grown in the absence of Shld1 or gametocyte deficient lines (3D7.stk2Δ or NF54-ap2-g-L).

Whole genome sequencing
Whole Genome Sequencing was conducted using a short-read workflow. Genomic DNA was used as input for library preparation after mechanical shearing and subsequent end-repair, a-tailing and ligation with unique dual index adapters. Sequencing libraries were assessed for size distribution and absence of adapter dimers before sequencing on an Illumina NovaSeq 6000 using a 151+8+8+151 bp read parameter for run conditions. An average 26,329,252 sequences per samples were generated and subsequently mapped using the Burrows-Wheeler Alignment56 against P. falciparum 3D7 reference genome (PlasmoDB)22. The average percentage of mapped reads per isolate was 78% and an average 74x coverage was achieved across the genome. The Genome Analysis Toolkit57 was then used to identify single nucleotide polymorphism (SNP) variants per isolate (Supplementary Table 1). Whole genome DNA Sequencing data generated in this study have been deposited in the BioProject/SRA under the accession codes PRJNA1260958: SRR33482688, SRR33482687, SRR33482686, SRR33482685, SRR33482684, SRR33482683, SRR33482682.

In-silico prediction of AP2 domain and structural visualization of AP2-G-DNA interaction
MOTIF search (https://www.genome.jp/tools/motif/), a web-based tool was used to predict the AP2 domain in-silico. This web tool has definitions of known protein domains, including AP2 domains derived from plants to higher eukaryotes. The independent Expectation Value (i-Evalue) represents the number of motifs with a similar statistical score that would be expected to occur by chance in a random dataset of the same size. Lower i-Evalue indicates that the motif is highly unlikely to have occurred randomly, suggesting it is a biologically significant pattern. This tool predicts a domain based on amino acid sequence similarity. We provided wild type or mutated amino acid sequences of AP2-G as an input and for each sequence an AP2 domain was predicted with a significance score. To visualize the 3D structure, an AlphaFold29,30 predicted AP2-G structure was used (AF-Q8I5I9-F1-model_v4.pdb), which was generated using full length AP2-G. Domains were identified using the predicted local distance difference test (pLDDT) model confidence score (pLDDT=70 or higher). For modeling the impact of AP2-G.L2163 mutation on the 3D structure, the MutationExplorer58 web tool was used. To do this, the predicted 3D structure (AF-Q8I5I9-F1-model_v4.pdb) was loaded to MutationExplorer and L2163 mutation was introduced. This tool compares the global and individual residue specific energy changes from wild type AP2-G. The structure stabilizing (positive) and de-stabilizing (negative) energies were recorded with a threshold of >0.5.

Electrophoretic mobility shift assay
Plasmodium falciparum (Pf-AP2-G-L2163, PfAP2-G.V2163) and P. berghei (PbAP2-Gwt) recombinant proteins were prepared as described earlier59. These recombinant proteins were expressed as glutathione S-transferase (GST: pGEX-4T1, GE Life Sciences) fusion proteins and purified from the soluble fraction of sonicated bacterial material. Electrophoretic mobility shift assay (EMSA) was performed using Light Shift EMSA kits (Thermo Scientific)10 using 2 μg of protein and 20 fmol of biotinylated probe59. Briefly, probes and recombinant DNA-binding domains were incubated at RT for 20 min before loading on a 0.5x TBE-buffered 6% polyacrylamide gel. Complexes were separated at 100 V until the dye front had migrated 3/4 of the gel length. Probes were then transferred overnight onto Nylon membranes, immobilized by UV (302 nm) cross-linking for 15 min, and visualized using strepdavidin-HRP conjugate and luminol detection solution (BioRad).

Parasite live imaging
MACS-column purified schizont stage parasites were used for visualizing mScarlet or mNeonGreen or tdTomato fluorescent protein. Live parasites re-suspended in 1x SA were stained with Hoechst 33342 DNA stain (1:500 dilution, 10m) and washed once in 1x SA before imaging. Live imaging was performed using Zeiss inverted fluorescence microscope (AxioObserver 7) under 630x magnification and ZEN v3.0 (blue edition) in-built analysis software. For co-localization, Z-Stack images were deconvolved using ZEN v3.0 in built software and the weighted correlation coefficient was used for the DAPI (DNA) and mSc (AP2-G) signals as well as the mSc (AP2-G) and mNG (MSRP1) signals. A total of five schizont’s Z-Stack images were analyzed, and ~15 layers of images through the center of each schizont were included in correlation coefficient analysis. The mean and standard error of the correlation coefficients for each schizont was used to determine colocalization.

Protein quantification using nLuc system
Nanoluciferase (nLuc) bioluminescence signal was measured using a Nano-Glo® Luciferase Assay System (Cat# N1110, Promega, USA) and the Cytation 5 Biotek imaging system as per manufacturer’s instruction. Gen 5 v3.08 software was used to quantitate bioluminescence signals. Tightly synchronized parasite cultures (Sorbitol-MACS-sorbitol) were used to measure nLuc signals at defined time points or developmental stages (schizonts or rings). Nano-substrate was used in 1:50 dilution in the assay buffer (30 μl) with an equal volume of culture (30 μl) making final dilution of 1:100. The number of parasites in each comparison group was quantified using flow cytometry or hemocytometer and used to normalize the bioluminescence signal. In the time-course experiment, a background signal subtraction was applied. Mean nLuc signal obtained from 16-22 hpi was subtracted from each data-point measured later from 24 to-44 hpi. In generation 2, the ring stage nLuc signal was compared between samples containing the same number of parasites without a background subtraction. For the fold change analysis (Fig. 5c, d), nLuc signal was presented relative to nLuc signal observed in ap2-g.L2163 line.

Data analysis
GraphPad Prism 10.6.1 statistical package was used to evaluate differences between parasite lines. All comparisons between wt and stk2Δ clones, Shld1 (+) and Shld11 (-) or ap2-g-L2163 and ap2-g-V2163 groups were analyzed using a Kruskal-Wallis test followed by Dunn’s multiple comparison. ANOVA test followed by Bonferroni’s or Turkey’s multiple comparison was performed after evaluating the normal distribution of data using a Quantile-Quantile plot. Two groups were compared using Mann-Whitney test. All statistical tests were two-sided and the p value of each comparison is indicated. Correlations between groups or conditions were assessed using Microsoft excel (Microsoft Excel 2013) to determine the Pearson correlation coefficient.

Plasmids and parasite lines source, and authentication
P. falciparum wt (Nf54, 3D7) and mutant F12 strains were obtained from B.E.I Resources (USA) and Prof Manuel Llinas’ lab (Pennsylvania State University, USA), respectively. Plasmid with tdTomato (pGEMT-PT2A-iRFP670-Tdtomato-GFP: Addgene plasmid #111817)33 or mScarlet (pmScarlet_C1: Addgene plasmid #85042)60 or mNeonGreen (ER-mNeonGreen: Addgene plasmid #137804)61 or NanoLuciferease (pUAS-NanoLuc: Addgene plasmid #87696)62 were obtained from Addgene Inc (USA).

Genomic DNA from the parasite lines was analyzed for the presence or absence of the appropriate chromosomal insertions/modifications/alleles using targeted and whole genome sequencing. The parasite lines were reassessed periodically throughout the study for the genotype stability using targeted sequencing of PCR amplicons spanning the modified region of the genome. Wild type lines (NF54 and 3D7) were also tested to confirm that they continued to produce gametocytes. Giemsa-stained culture smears of P. falciparum infected and uninfected red blood cells were assessed for normal morphology by light microscopy. The following lines were analyzed by whole genome sequencing - Wild type parasite (NF54), gametocyte deficient line 3D7.stk2Δ, ap2-g mutation introduced (NF54.ap2-g.L2163 and NF54-Ap2g-L2163-gfp.fkbp) or mutation corrected (3D7.stk2Δ.ap2-g.v2163 and NF54.ap2-g.V2163) transgenic lines.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the current manuscript, Prajapati et al have discovered a novel SNP in ap2-g that leads to a single amino acid substitution in AP2-G (V2163L) in the DNA binding domain. The authors have conclusively shown the association of the SNP with the inability of the mutant AP2-G to bind the canonical DNA sequence abrogating the transcription of downstream sexual stage genes and abrogating the gametocyte production. The authors have corrected the mutation in the mutant background and vice versa to conclusively show the association of the SNP with the phenotype. The role of GDV1 as the upstream factor to ap2-g in the sexual commitment is also demonstrated. Requirement of functional AP2-G is clearly demonstrated for sustained ap2-g transcript expression using various transgenic parasites. The results obtained have been analysed to reach justified conclusions. The study is well planned and designed with proper controlled experiments. The study is of relevance to better understand the signalling events during sexual commitment that would be useful for the screening and identification of transmission blocking molecules. I have few queries and comments for authors related to the work:\nIt is not clear what was the rationale for targeting STK2 that is the foundation of the study. There could be many other kinases that might be involved in the phosphorylation event.\nFig. 2 legend: since the p value does not show ns in the figure, it can be omitted from the legend. Same suggestion applies to other figures as well.\nFig. 3: The i-Evalue representing the size and interaction strength of AP2 domain to bind the DNA sequence is more (0.0023) for the V-L mutant than the wt.V (0.00053). As I get from the description of the parameter, the expected value should have been reverse.\nFig. 4a. It is not clear how many times the experiment was performed to determine the expression of ap2-g transcript in the NF54.gdv1.3xha.fkbp parasites with and without Shld1\nFig. 5a: The Gdv-1 linked nLuc activity seems to be highest at 39-40 hpi and plateau after that. The results under ‘Quantification of ap2-g.V….’ however states the peak expression ta 34 hpi. It will be useful to mention GDV-1tdTomato to provide an instant reference that the expression of GDV-1 is being monitored.\nFig 5g. The effect of Shld1 removal to destabilize GDV1 <40 hpi is not very different in the AP2G nanoLuc signal and GCR% in the main figure 5g compared to the supplementary Figure S8c. The GCR% in 40 and above hpi of shld1 removal is comparable to 38 and 39 hpi, then how the boundary of dotted box indicating the developmental phase that requires GDV1 for gametocyte production was determined? Although there is a striking increase in the nanoLuc signal in Fig. S8a however that difference is not reflected in the GCR% in Fig. 5g?\nFig 6a and b. it will be useful to mention the parasites in the IFA panel for ready understanding of the figure.\nThe co-localization coefficient may be shown to further strengthen the statement in line 285-287 “AP2-G.V.mSc expression was punctate and co-localized with the nuclear stain, while in mature schizonts MSRP1 was concentrated around the periphery of the merozoites (Fig 6b (top panel), Fig S10b).\nThere are single positive AP2-G.mSc cells with high enough expression of AP2-G however still there is no expression of MSRP1 evident in those cells. Is it known what percentage of total AP2-G positive cells form gametocytes?\nAlthough the proportion of mNeonGreen single positive cells (0.45) is very less in Fig. 6d however, in the Fig.6c the mutation corrected and the mutant line show 1.71 and 2.05 percent cells that are single mNeonGreen positive, respectively. These percentages are greater than the single positive mScarlet (1.19 and 1.87) AP2-G expressing cells. These panels seem to suggest that MSRP1 expression exist in cells without AP2-G?\nThe possibility of a functional AP2-G driving the expression of an ‘unknown factor’ that leads to the increased induction of ap2-g transcript cannot be ruled out. Therefore, the sentences in the Discussion section Line 312-314 needs to be modified accordingly. The authors have briefly discussed this aspect (Lines 332-333).\nThe SNP in the ap2-g locus (6487G>T) was found while stk2 was knocked out in the 3D7 background and not in the NF54. The authors have made an argument that a sub-population of 3D7 may have this SNP. Was it consistently observed in all the clones of 3D7.STK2ko?\nMinor Comments/Suggestions:\nThe authors can refer to the latest malaria figures as reference 1.\nLine 68: Should be rewritten as ‘Once expressed, AP2-G’\nLine 89: use of ‘generated’ may be avoided for better readability.\nLine 91: stk2 is misspelled.\nThe last sentence in Main (from line 93-96) needs to be rephrased to make it simpler to understand. One suggestion is as follows: Here we have identified a highly robust system that can be utilized to study the transcriptional activation of ap2g by an upstream regulator, GDV1 and monitor the activation of ap2g specific targets.\nLine 159: the isoleucine mutant will cause 5 aa shift not 6.\nLine 182: should be every hour\nLine 219: The sentence ‘We first confirmed that the nLuc signal was linear through the parasitemias used in the experiments (Fig S5).’ needs to be rephrased’.\nLine 400: should be rewritten as ‘In Plasmodium, a conservative…’.\nLine 406: Wolbachia should be italicized.\nI could not locate ‘f’ in Fig. S2.\nFig. S1. It will be useful for the readers and quick understanding of the PCR results to represent the reactions with primer sets i.e., instead of DCO1, P2+P5 and likewise.\nIt is not clear whether the NF54.gdv1.3xha.fkbp is generated in this study as no description of its generation was found in the material and methods. In case it is used earlier please provide a reference.", "reviewer_2": "The authors address the important question of how the most lethal human malaria parasite commits to sexual development, a process that is critical for parsite transmission to mosqutitoes. Prompted by the discovery of a spontaneous missense mutation in ap2g, a gene encoding a known master regulator transcription factor for commitment to sexual development, this work adds new detail to our understanding of how ap2g operates downstream of another known factor, GDV1. In particular, the data demonstrate that GDV1 controls ap2g expression early, before an autoregulatory loop is thought to lock ap2g in the on-state, which is then thought to turn on a larger number of target genes.\nConceptually, this work is not entirely novel but, importantly, it consolidates and corroborates previous data in a way that I find compelling and suitable for publication in Nature Communications. This is technically challenging work in a difficult system which uses many new conditional lines and reporter systems in carefully designed and well-executed experiments. Particular strengths an innovative approaches include (1) the careful comparison of inducible lines for GDV1 and AP2G-L and -V and the inclusion of F12 as another loss-of-function mutant, (2) the independent analysis of both transcript abundance, translation through a luciferase fusion gene and protein level through an mScarlet fusion, (3) the use of different phenotypic readouts ranging from gametocyte conversion rates to early marker gene expression and MSRP1 expression.\nI find the manuscript well written. The analysis is thorough and the work is well executed. The statistical analysis is appropriate and methods are largely described clearly.\nI think Figure 3 and related text passages need some improvement. The biological significance of where an algorithm for motif identification draws a domain boundary is not clear to me. The statement that most amino acid substitutions affect where a particular algorithm draws the boundary is therefore difficult to interprete. They are not saying that the substitution would affect folding of the domain, right? How did they model the structures in 3a? This is not clear from methods or figure legend. Or did they just take an existing structure and drew the different amino acids into it? I.e. is this a cartoon or data? I see how the substitution is in the DNA binding site, but can they model how the substitution would affect folding and binding?\nFigure 3c: Is there evidence that the binding is sequence specific? Have they for instance competed with the same or an unrelated unlabelled oligo? What is the evidence the expressed domain is present in the same quantity and has not precipitated? Do they know it is folded?\nMinor points:\nPage 5 Changing V2163 to 154 L to mimic the observed mutation shifted the start of the predicted AP2 domain downstream by 155 11 aa which decreases size and interaction strength (i-Evalue). To understand the significance of this obervation the reader would need to know precisely which tool was used and how it predicts AP2 domains.\nSomewhere around line 178 the non-expert needs to be told that the inducible line expresses GDV1 fused to a degron stabilised by Shld1 and that Shld1 induces gametocytogenesis by stabilising GDV1-FKBP.\nLine 190-193: Please break up this sentence to make it easier to understand.\nFigure 3 legend: What do they mean by “…at the interface of (the?) AP2-G-DNA binding complex.” This sounds as if the complex was binding something.\nFig. 4 a, c: What are the units on the vertical axes?\nIf I compare in Fig. 4c bars 1 vs. 3 or 5 vs. 6, it is tempting to interprete the difference as an early sign of autoregulation but the effect size appears small. Could autoregulation be more important to boost expression later and to lock ap2g into an active state when there are many additional target genes? The L mutant or F12 line never reach this later state, of course. Perhaps this could be discussed more."}, "author_response": "Over the years we and others have tested numerous kinases for a role in sexual commitment, but either did not get transformants or they had no effect on Pf sexual commitment. Stk2 that can target serine and has a nuclear localization signal, seemed reasonable to add to the list. Honestly, we were a bit surprised when there was a phenotype and that gdv1 and ap2-g were intact, at least when analyzed by length\nFig. 2 legend: since the p value does not show ns in the figure, it can be omitted from the legend. Same suggestion applies to other figures as well.\nResponse: We have removed ns from Fig legends where it is irrelevant.\nFig. 3: The i-Evalue representing the size and interaction strength of AP2 domain to bind the DNA sequence is more (0.0023) for the V-L mutant than the wt.V (0.00053). As I get from the description of the parameter, the expected value should have been reverse.\nResponse: We added that an increase in the independent Expectation Value (i-Evalue) reduces confidence in the motif prediction (Line 159-160) and in the methods section added a more detailed explanation of the value, “The independent Expectation Value (i-Evalue) represents the number of motifs with a similar statistical score that would be expected to occur by chance in a random dataset of the same size. Lower i-Evalue indicates that the motif is highly unlikely to have occurred randomly, suggesting it is a biologically significant pattern” (Line 581-585). We have also modified S3 fig legend.\nFig. 4a. It is not clear how many times the experiment was performed to determine the expression of ap2-g transcript in the NF54.gdv1.3xha.fkbp parasites with and without Shld1\nResponse: Since the ap2-g RNA time course included 8 sequential RNA sample collections for both Shld1 (+) and (-) conditions, demonstrated a consistent pattern over 14 hours that matched with our previous single time point data (Fig 4c), and mirrored data from an independent time course done in duplicate monitoring ap2-g-linked nanoLuc in a different parasite line (Fig 5a), the entire RNA time course of NF54.gdv1.3xha.fkbp was performed once (Fig 4a).\nFig. 5a: The Gdv-1 linked nLuc activity seems to be highest at 39-40 hpi and plateau after that. The results under ‘Quantification of ap2-g.V….’ however states the peak expression ta 34 hpi. It will be useful to mention GDV-1tdTomato to provide an instant reference that the expression of GDV-1 is being monitored.\nResponse: The text was changed to more accurately describe the change in activity by indicating that “Gdv1-linked nLuc activity increased rapidly until 34/35 hpi, then started to plateau” (Line 238-239, 341-342). The reference to GDV-1.TdTomato was mentioned earlier in the sentence for clarity (Line 239-241).\nFig 5g. The effect of Shld1 removal to destabilize GDV1 <40 hpi is not very different in the AP2G nanoLuc signal and GCR% in the main figure 5g compared to the supplementary Figure S8c. The GCR% in 40 and above hpi of shld1 removal is comparable to 38 and 39 hpi, then how the boundary of dotted box indicating the developmental phase that requires GDV1 for gametocyte production was determined? Although there is a striking increase in the nanoLuc signal in Fig. S8a however that difference is not reflected in the GCR% in Fig. 5g?\nResponse: We agree that AP2-G nano expression increases consistently from 32-34 hpi to 40 hpi, so that the change between 38-40 hpi is not dramatic and this was a reason to put a soft boundary (dotted). We added a new sentence indicating that the “Levels of ap2-g.V-linked nLuc activity gradually increased after 32-34 hpi reaching a plateau at ~40 hpi and after which removing GDV1 had little effect.” (line 277-280). And included a ~ sign before the 40 hpi figure.\nFig S8a is the baseline data showing the time course of ap2-g-linked nanoLuc expression from 28-44 hpi in the constant presence or absence of Shld1. Fig 5g is the GCR% when Shld1 is only present for a defined time.\nFig 6a and b. it will be useful to mention the parasites in the IFA panel for ready understanding of the figure.\nResponse: We have included “Schizont stage” in Fig 6A+B live imaging panels.\nThe co-localization coefficient may be shown to further strengthen the statement in line 285-287 “AP2-G.V.mSc expression was punctate and co-localized with the nuclear stain, while in mature schizonts MSRP1 was concentrated around the periphery of the merozoites (Fig 6b (top panel), Fig S10b).\nResponse: We have included the co-localization coefficient for AP2-G and DNA stain (Lines 300-303, 614-620). Based on the DNA and AP2-G signal (co-localization coefficient=0.56±0.08), we modified the text to refer to AP2-G as perinuclear. The co-localization coefficient of the overlap of MSRP1 with AP2-G was 0.21±0.03 indicating its location in the periphery.\nThere are single positive AP2-G.mSc cells with high enough expression of AP2-G however still there is no expression of MSRP1 evident in those cells. Is it known what percentage of total AP2-G positive cells form gametocytes?\nResponse: AP2-G expression begins between 38-39 hpi, while MSRP1 expression does not start until ~2 hr later and in a time course experiment (Fig S11) we find that all the MSRP1 positive cells were AP2-G positive and that over time the AP2-G single positive cell population decreases as the double positive cell population increases. From this data we suggest that the single AP2-G-mSc positive schizonts from ap2-G.V.mSc//msrp1.mNG line are younger and once mature it will be double positive. We worked hard to synchronize parasites to a 2 hour window, but there are still growth difference over the next 40 that leads to some diversity in the population. The close correlations of the % GCR with the % double positive cells in the ap2-G.V.mSc//msrp11.mNG line and the % AP2-G single positive cells in the ap2-G.V.mSc line (Fig S9a) suggest that vast majority of AP2-G positive schizonts are destined to produce gametocytes.\nAlthough the proportion of mNeonGreen single positive cells (0.45) is very less in Fig. 6d however, in the Fig.6c the mutation corrected and the mutant line show 1.71 and 2.05 percent cells that are single mNeonGreen positive, respectively. These percentages are greater than the single positive mScarlet (1.19 and 1.87) AP2-G expressing cells. These panels seem to suggest that MSRP1 expression exist in cells without AP2-G?\nResponse: No single MSRP1.mNG positive schizonts were observed by microscopy in the dual reporter lines. However, by flow cytometry the proportion of the cells that are weakly single positive (<3x103 fluorescence intensity) does vary from experiment to experiment as seen in the flow data shown in Fig 6 c and d. One major factor contributing to the variation is differences in the background fluorescence of the RBC cells in the culture. To control for RBC variation, we did a time course (Fig S11), monitoring development every hour from 37 to 44 hpi and again at 46 hpi, which clearly demonstrated the early appearance of single positive AP2-G.mSc cells followed by the appearance of double positive cells and a later decline in the single positive AP2-G.mSc cells. In this experiment there were only a few cells in the single positive MSRP1.mNG gate. This time course and the lack of single positive MSRP1.mNG cells by fluorescence microscopy increases our confidence that MSRP1 is only expressed in AP2-G positive cells. We have modified the text to better describe the background variation (Lines 307-310).\nThe possibility of a functional AP2-G driving the expression of an ‘unknown factor’ that leads to the increased induction of ap2-g transcript cannot be ruled out. Therefore, the sentences in the Discussion section Line 312-314 needs to be modified accordingly. The authors have briefly discussed this aspect (Lines 332-333).\nResponse: We modified the original Lines 312-314 (now 333-334) to read “…AP2-G.V-dependent regulation of itself or another factor plays a major role in maintaining ap2-g expression and driving downstream gene expression.”\nThe SNP in the ap2-g locus (6487G>T) was found while stk2 was knocked out in the 3D7 background and not in the NF54. The authors have made an argument that a sub-population of 3D7 may have this SNP. Was it consistently observed in all the clones of 3D7.STK2ko?\nResponse: It was only an assumption that the mutant allele might be present in the culture at very low frequency. The reviewer is correct that except for the identification of this mutation in an experiment carried out in a separate laboratory, our group only identified this mutation in clones from a single transformation even though we performed many transformations with the 3D7 parental line. Consequently, we have removed the statement from the discussion.\nMinor Comments/Suggestions:\nThe authors can refer to the latest malaria figures as reference 1.\nResponse: We have updated malaria figures in the manuscript (lines 40-41).\nLine 68: Should be rewritten as ‘Once expressed, AP2-G’\nResponse: We corrected this (Line 69).\nLine 89: use of ‘generated’ may be avoided for better readability.\nResponse: We removed “generated”.\nLine 91: stk2 is misspelled.\nResponse: We corrected this (Line 91).\nThe last sentence in Main (from line 93-96) needs to be rephrased to make it simpler to understand. One suggestion is as follows: Here we have identified a highly robust system that can be utilized to study the transcriptional activation of ap2g by an upstream regulator, GDV1 and monitor the activation of ap2g specific targets.\nResponse: We replaced line 93-96 with your suggestion (now Lines 92-94).\nLine 159: the isoleucine mutant will cause 5 aa shift not 6.\nResponse: Thanks for catching this. The number listed in the table for the start of the AP2 domain and the red text did not match. We have corrected this in Fig 3a.\nLine 182: should be every hour\nResponse: This is a time course that included samples from every two hours (Fig 4a: 28, 30, 32, 34, 36, 38, 40, and 42 hpi), so we expressed as every other hour (now Line 195). In Fig 5a, only GDV1 was monitored for every hour.\nLine 219: The sentence ‘We first confirmed that the nLuc signal was linear through the parasitemias used in the experiments (Fig S5).’ needs to be rephrased’.\nResponse: We rephrased the sentence to read “We first confirmed a linear increase in nLuc signal with increasing parasite number” (Linse 234-235).\nLine 400: should be rewritten as ‘In Plasmodium, a conservative…’.\nResponse: We corrected the sentence (Line 422).\nLine 406: Wolbachia should be italicized.\nResponse: We italicized Wolbachia (Line 428).\nI could not locate ‘f’ in Fig. S2.\nResponse: We relabeled f, it was misplaced while rearranging figure panel.\nFig. S1. It will be useful for the readers and quick understanding of the PCR results to represent the reactions with primer sets i.e., instead of DCO1, P2+P5 and likewise.\nIt is not clear whether the NF54.gdv1.3xha.fkbp is generated in this study as no description of its generation was found in the material and methods. In case it is used earlier please provide a reference.\nResponse: We replaced DCO1 with P2+P5 and similarly others also in this series. The NF54.gdv1.3xha.fkbp line was generated for this study and was included in the methods (Lines 510-511, 517-518, Fig S12d).\nThe original structure in Fig 3a was modeled using Rosetta software with the two different amino acid sequences. We have replaced this with Alphafold prediction AF-Q81519-F1 and used the MutationExplorer web tool to evaluate changes due to the V2163L mutation. The model predicted changes in several amino acids located close to L2163 in the AP2 DNA binding domain. To follow up we also used Alphafold to predict the structure of the recombinant proteins used in the EMSA. Again, the core AP2 domain structure was not disrupted, consistent with the recovery of soluble recombinant protein that was used in the EMSA.\nThe MOTIF search webtool provides the AP2-G motif boundary which is only prediction, but the MutationExplorer also predicts changes in the AP2 domain that could alter DNA binding. The MOTIF search tool doesn’t measure protein characteristics such as folding. It only provides a motif/domain prediction if the query protein sequence shows significant similarity with their annotated database. The definition of AP2 domain and their boundaries in ap2 genes including ap2-g, has been described earlier (PMID: 16040597 and 30959972). We have modified results section to better describe these predictions (Lines 155-169).\nFigure 3c: Is there evidence that the binding is sequence specific? Have they for instance competed with the same or an unrelated unlabelled oligo? What is the evidence the expressed domain is present in the same quantity and has not precipitated? Do they know it is folded?\nResponse: AP2-G binding motif was previously defined using a protein binding microarray (PBM) with wt recombinant AP2-G protein (rAP2-G) that was generated using the same protocol used in this study (PMID: 21060817). Based on that work, we used an oligonucleotide that contains a consensus GnGTAC sequence which binds wt PfAP2-G and also rAP2-g from the distantly related rodent parasite P. berghei. The wt and mutant PfAP2-G recombinant proteins contained the same amino acid region (aa 2150-2220) and were purified as GST fusion proteins from the soluble/supernatant fraction of sonicated E. coli material. There were no signs of misfolding. The protein was present in the soluble fraction and based on the protein quantification we used the same quantity of both wt or mutant AP2-G in two different dosages (3 mg and 6 mg). The results and methods section have been revised to include this additional description (Lines173-174, 598-600).\nMinor points:\nPage 5 Changing V2163 to 154 L to mimic the observed mutation shifted the start of the predicted AP2 domain downstream by 155 11 aa which decreases size and interaction strength (i-Evalue). To understand the significance of this obervation the reader would need to know precisely which tool was used and how it predicts AP2 domains.\nWe included a more detailed explanation of the Motif Search results for the AP2-G sequence with the various mutations at the position corresponding to V2163 (Lines 155-162). This program provides a motif/domain if the query protein sequence shows significant similarity with annotated databases. “The independent Expectation Value (i-Evalue) represents the number of motifs with a similar statistical score that would be expected to occur by chance in a random dataset of the same size. Lower i-Evalue indicates that the motif is highly unlikely to have occurred randomly, suggesting it is a biologically significant pattern”, lines 581-586.\nSomewhere around line 178 the non-expert needs to be told that the inducible line expresses GDV1 fused to a degron stabilised by Shld1 and that Shld1 induces gametocytogenesis by stabilising GDV1-FKBP.\nResponse: We have included a description of the gametocyte inducible parasites line for non-experts at Lines 109-113, 189-191. “In the gametocyte inducible line gdv1 is tagged in-frame with a ligand-dependent degradation domain (FKBP). In the absence of Shield 1 (Shld1) ligand the GDV1-FKBP chimeric protein is targeted for proteasomal degradation and therefore not available to activate ap2-g expression and sexual commitment. The presence of Shld1 blocks degradation of GDV1-FKBP and sexual commitment proceeds normally”. We have also referred to this in lines 189-191.\nLine 190-193: Please break up this sentence to make it easier to understand.\nResponse: Lines 190-193 have been simplified and now presented in two sentences (now lines 204-208).\nFigure 3 legend: What do they mean by “…at the interface of (the?) AP2-G-DNA binding complex.” This sounds as if the complex was binding something.\nResponse: We have removed this explanation as we replaced Fig3a with the AlphaFold and MutationExplorer analysis (Lines 162-169).\nFig. 4 a, c: What are the units on the vertical axes?\nResponse: We corrected the y-axis label (expression fold change).\nIf I compare in Fig. 4c bars 1 vs. 3 or 5 vs. 6, it is tempting to interprete the difference as an early sign of autoregulation but the effect size appears small. Could autoregulation be more important to boost expression later and to lock ap2g into an active state when there are many additional target genes? The L mutant or F12 line never reach this later state, of course. Perhaps this could be discussed more.\nResponse: Correct, the difference between 1 & 3 and 5 & 6 is due to the ability of wt AP2-G, not mutant AP2-G to enhance its own expression and that autoregulation is magnified in the next generation rings. Together the results suggest. GDV1 is required to light the fire so to speak (ie stimulate the initial ap2-g expression), but wt AP2-G is needed for the fire to continue (increase its own expression as well as downstream genes in mature schizonts and the next generation rings). To highlight this, we added the following phrases to the end of the sentences on Line 211 and 357, which are now Lines 217-218 “again indicating that during schizogony wt AP2-G enhances its own transcription.” and Lines 360-361 “consistent with the requirement for wt AP2-G to activate sexual differentiation."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Most of the comments have been satisfactorily addressed by the authors. The following two comments need to be addressed.\nFig. 4a. It is not clear how many times the experiment was performed to determine the expression of ap2-g transcript in the NF54.gdv1.3xha.fkbp parasites with and without Shld1\nSingle experiment can be accepted based on the reflection of the result corroborated by other experimental data. Just wondering whether technical replicates were included as is usually done for the transcript analysis experiments. If yes, it would be nice to plot the data with the error bars.\nFig 6a and b. it will be useful to mention the parasites in the IFA panel for ready understanding of the figure.", "reviewer_2": "The authors have responded adequately to the reviewers' questions. I have no further comments."}, "author_response": "Fig. 4a: Since the ap2-g RNA time course included 8 sequential RNA sample collections for both Shld1 (+) and (-) conditions, demonstrated a consistent pattern over 14 hours that matched with our previous single time point data (Fig 4c), and mirrored data from an independent time course done in duplicate monitoring ap2-g-linked nanoLuc in a different parasite line (Fig 5a), the entire RNA time course of NF54.gdv1.3xha.fkbp was performed once (Fig 4a).\nSingle experiment can be accepted based on the reflection of the result corroborated by other experimental data. Just wondering whether technical replicates were included as is usually done for the transcript analysis experiments. If yes, it would be nice to plot the data with the error bars.\nResponse: We replaced Fig 4a with technical replicates and indicated in the legend that error bar derived from technical replicates.\nFig 6a and b. it will be useful to mention the parasites in the IFA panel for ready understanding of the figure.\nResponse: We have included “Schizont stage” in Fig 6A+B live imaging panels.\nThe stage of the parasite is evident by the punctation pattern. What I meant to say was the identity of the parasite i.e., 3D7.stk2Δ//ap2-g.L.mSc and 3D7.stk2Δ//ap2-g.V.mSc. Similarly, for ‘b’.\nResponse: Thank you for clarification. We have included the parasite line names in all images, including supplementary figures."}]

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