Mutant/mutation
The mutant lacks expression of histone H3.3 and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.

Protein (function)
Two conserved histone H3 proteins (canonical H3 and variant H3.3; PBANKA_0108800 and PBANKA_1117100) were identified as gene annotations in the Plasmodium genomic database (http://www.plasmodb.org). Both histone H3 proteins consist of 136 amino acids with highly conserved sequence homology. The amino acid sequence alignment indicated only eight amino acid differences between these proteins. In addition, the secondary structure prediction revealed four conserved α-helices in both histone H3 proteins. Moreover, the predicted models, built against canonical H3 and H3.3, indicated a high degree of structural similarity between these proteins. The three-dimensional structural prediction based on AlphaFold2 revealed a highly conserved structure between the two histone H3 proteins.
H3.3, a histone H3 variant, is highly conserved in all eukaryotes from yeast to humans, but its function varies among species. H3.3 is deposited into transcribed genes, promoters, and enhancer regions, and it is considered as a mark of transcriptionally active genes in flies, plants, mice and human.However, H3.3 is also incorporated into heterochromatin structures at telomeres, thereby contributing to the maintenance of genomic integrity. In mammals, H3.3 plays an essential role in embryonic development. H3.3 is required for the establishment of heterochromatin in the mouse embryo, and it contributes to chromosome segregation and the maintenance of genome integrity during embryonic development. H3.3 depletion leads to the dysfunction of heterochromatin structures and chromosome distribution, resulting in developmental arrest.
Histone post-translational modifications regulate transcriptional activation in asexual blood stage malaria parasites, consequently controlling the repression of multiple virulence genes and determining sexual stage commitment. Histone H3 modifications are associated with the expression of the var gene family of virulence factors. Histone H3 lysine 9 (H3K9) trimethylation in the 5′ flanking region of var gene contributes epigenetically to repress var gene expression. The replacement of H3K9 acetylation is a key step toward unlocking the silenced var genes. In the genus Plasmodium, H3.3 is highly conserved, and it has diverse localization patterns including both conserved and parasite-specific features. It has been reported that canonical H3 is 'likely essential' for asexual blood stage growth/multiplication, but H3.3 is not essential for asexual blood stage growth/multiplication in malaria parasites. And also it has been reported that Plasmodium H3.3 specifically marks the promoter region of the virulence gene families in its active and poised state, but not the inactive state, and it could contribute to the antigenic variation process, which is critical for pathogenicity.

Phenotype
See also RMgm-5528 for a mutant (Δh3.3_a) lacking expression of H3.3 (that expresses GFP under control of the constitutive eef1a promoter). Another h3.3 gene deletion mutant (Δh3.3_b) was generated (RMgm-5529) using the P. berghei reporter line 820cl1m1cl1, in which male gametocytes express GFP and female gametocytes express RFP.

Reduced growth/multiplication rate of asexual blood stages. Male gamete production (exflagellation) comparable to wild type.
Fertilized zygotes exhibited an early-stage growth arrest with strongly reduced mature ookinetes (mainly round, retort and degenerated zygotes).
Reduced numbers of oocysts with smaller size compared to wild type. No sporozoites in salivary glands.

Additional information
Another h3.3 gene deletion mutant (Δh3.3_b) was generated (RMgm-5529) using the P. berghei reporter line 820cl1m1cl1, in which male gametocytes express GFP and female gametocytes express RFP. This parasite line allowed to perform male and female gametocyte selection/purification and detailed analysis. Δh3.3_b strain exhibited similar phenotypes as Δh3.3_a. This line produced numbers of male and female gametocytes comparable to wild type parasites, indicating that H3.3 is not required for production of female gametocytes. Western blot analysis revealed histone H3 (PBANKA_0108800) protein expression was comparable between the mutant and WT.

To examine the expression of H3.3 in male and female gametocytes, a HA-tagged h3.3-complemented mutant (h3.3::ha (c)_b) was  generated in Δh3.3_b based on the 820cl1m1cl1 line (see RMgm-5530). h3.3::ha (c)_b displayed similar asexual growth rate and production numbers of salivary gland sporozoites as the WT. Male or female gametocytes were sorted into GFP-expressing male gametocytes and RFP-expressing female gametocytes by cytometry. Western blot analysis revealed that the majority of H3.3-HA was detected in female gametocytes.

RNA-seq analysis of purified male and female gametocytes of Δh3.3_b (RMgm-5529) revealed that in both male and female Δh3.3-b gametocytes, the expression of stage-specific genes essential for sexual development was not decreased upon the loss of H3.3, indicating that H3.3 might not play a crucial role in the transcriptional regulation of stage-specific gene expression in male and female gametocytes.

Other mutants