Mutant/mutation
Two mutants are described that express mutated versions of upstream regions of the rsph9 gene. The TGTCT or TGCACA motifs mutated in upstream regions of rsph9.

Protein (function)
PBANKA_1431500 (RSPH9) encodes a putative radial spoke head protein 9.

The garid gene (PBANKA_0102400) is a target gene of AP2-G, and its transcription is upregulated via conditional induction of AP2-G in P. falciparum and P. berghei. Garid encodes a protein that contains an ARID at its N-terminus and a monopartite nuclear localization signal near its C-terminus. The ARID is a helix–turn–helix motif-based DNA binding domain conserved in a wide variety of eukaryotic species from fungi to vertebrates. In mammals, proteins with this domain are classified into 7 subfamilies, namely ARID1 through ARID5, JARID1, and JARID2. Of these, only ARID3 and ARID5 preferentially interact with AT-rich sequences, and the others have no clear sequence preference.

For information on the gSNF2 protein: see mutant RMgm-5363

Phenotype
Evidence presented for RSPH9 expression in male gametocytes and not in females (see RMgm-5377). For RSHP9, mutations in the TGTCT motifs resulted in a significant reduction (approximately five-fold) of the GFP signal intensity in males. In contrast, mutations in the TGCACA motifs resulted in only a slight reduction of the signal

Additional information
Analysis of a mutant lacking expression of gARID (RMgm-5372) showed the following:
Normal growth/multiplication of asexual blood stages. Female gametocytes were observed, but no male gametocytes were observed in garid(−). No exflagellation was detected in garid(−) after the activation of gametogenesis. No male gametocytes were observed in garid(−). No exflagellation was detected in garid(−) after the activation of gametogenesis. No zygote/ookinete formation. No ookinete and oocyst formation.

To further confirm the absence of male gametocytes in garid(−), garid was knocked out in the gametocyte reporter line 820cl1 (garid[−]820), which have GFP and RFP expression cassettes under the control of male- and female-specific promoters, respectively. FACS analysis detected no GFP-positive parasites in the garid(−)820 parasites compared with the parental 820cl1 wherein approximately 3% of the parasites showed a GFP signal. This confirmed that disruption of garid results in the complete loss of male production.

When garid(−) was crossed with parasites that produce infertile males (p48/45[−]), no zygotes were observed, which was consistent with the absence of male gametocytes in garid(−). In contrast, when garid(−) was crossed with parasites that produce infertile females (p47[−]), parasites could undergo fertilization at a ratio similar to that when p48/45(−) and p47(−) were crossed. However, the ratio of conversion from female to banana-shaped ookinetes was only 19%, which was significantly smaller than the ratio for crossing p48/45(−) and p47(−), which was 40%. This result suggested that although female gametocytes of garid(−) showed normal morphology, they exhibit an abnormality that causes delay or arrest of ookinete development after fertilization.

ChIP-seq analysis of gARID showed that it forms a complex with gSNF2 (PBANKA_0312700, PF3D7_0216000), a core subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element. Moreover, ChIP-seq of gARID in gsnf2-knockout parasites revealed an association of gARID with another cis-regulatory element, which is indicated to play a role in both male and female development.

Notably, the genome-wide peak pattern of the ChIP-seq of gARID was almost identical to that of gSNF2, previously reported. gSNF2 is a core subunit of SWI/SNF2 chromatin remodeling complex expressed in gametocytes, and gsnf2 knockout parasite (gsnf2[−]) showed impaired male gametocyte development. However, the knockout phenotypes are of not the same; gsnf2(−) produces immature male gametocytes, whereas garid(−) completely loses the ability to differentiate into male gametocytes. In addition, female gametocyte development was affected in garid(−) but not in gsnf2(−). Consistent with the differences in their phenotype, the results in the differential expression analyses for garid(−) vs. wild type and gsnf2(−) vs. wild type were also different.

A transgenic line expressing GFP-tagged gARID (gARID::GFP) was generated with the CRISPR/Cas9 system using Pbcas9, and then, gsnf2 was disrupted in the gARID::GFPC (gARID::GFP^C_gsnf2(−)). Analysis is of this mutant provided evidence that gARID is recruited by gSNF2 as a subunit of the SWI/SNF complex on the TGTCT motifs. In a previous study, it was shown that the female-specific transcriptional activator, PFG, is responsible for recognizing the TGTAYRTACA motifs. Therefore, gARID is recruited to the TGTAYRTACA motifs by PFG independent of gSNF2. In addition to the ten-base motif, the TGCACA motif was also enriched around the peak summits identified in the ChIP-seq using gARID::GFP^C_gsnf2(−) suggesting that it could be another important gARID-associated sequence motif.

To thoroughly investigate the property of gARID binding regions other than those on the known motifs, we knocked out both gsnf2 and pfg in gARID::GFPC using CRISPR/Cas9 (gARID::GFP^{C_gsnf2(−)_pfg(−)})

To investigate the function of the TGCACA motif as a cis-regulatory element and the relationship between the TGTCT and TGCACA motifs in male gametocyte development,  reporter assays were performed using endogenous loci. As a target for the reporter assays, the following two genes were selected: calm (PBANKA_1421000, a putative calmodulin gene) as a gene significantly down-regulated in garid(−) but only slightly in gsnf2(−) and rsph9 (PBANKA_1431500, a gene encoding a putative radial spoke head protein 9 homolog, a component of radial spokes which control axonemal dynein activity) as a gene downregulated in both garid(−) and gsnf2(−) . First, each of these genes was tagged with gfp to assess their expression using FACS (CALM::GFP and RSPH9::GFP. Then,  the TGTCT and TGCACA motifs were mutated, either or both, within the peak regions located upstream of calm and rsph9 to assess the roles of these two cis-regulatory elements in the activation of male genes.

The expression of the gfp-tagged genes was analyzed using FACS. Before introducing mutations, both CALM and RSPH9 were specifically expressed in the male gametocytes, which was consistent with the sex-specific transcriptome in which the genes are male-enriched. For calm, the disruption of the TGTCT motif did not show a distinct change in the expression of CALM. However, mutations in the TGCACA motifs resulted in a significant reduction (approximately two-fold) of the GFP signal intensity, and mutations in both motifs showed a similar result. Therefore, this indicates that the TGCACA motif functions as a major cis-regulatory element for the activation of calm. For rsph9, mutations in the TGTCT motifs resulted in a significant reduction (approximately five-fold) of the GFP signal intensity. In contrast, mutations in the TGCACA motifs resulted in only a slight reduction of the signal, indicating a higher contribution of the TGTCT motifs on the activation of rsph9 than that of the TGCACA motifs.

Evidence is presented that that the TGCACA motif functions as an independent cis-regulatory element for the activation of downstream genes. Furthermore, the effect of disrupting the TGTCT motifs and both of the two motifs in the reporter assays was similar to the level of down-regulation in gsnf2(−) and garid(−), respectively. This result is consistent with the implication that the differences between the phenotypes of garid(−) and gsnf2(−) could be due to the function of gARID on the TGCACA motif .

Other mutants