Fig 1.
Comparative transcriptomics of P. yoelii female gametocytes and zygotes.
(A) A heatmap of sample-to-sample distance hierarchical clustering of biological replicates of RNA-seq data is shown. (B) A Venn diagram illustrates the overlapping detection of transcripts in female gametocytes and zygotes. (C) Volcano plot of differentially abundant transcripts enriched in female gametocytes (red) or zygotes (green). (D) Dot plot of transcript abundance in female gametocytes and zygotes. Differentially abundant transcripts in female gametocytes or zygotes are highlighted as red or green points, respectively. (E) Venn diagram of differentially abundant transcripts in zygotes and the P. yoelii orthologous transcripts that are targets of transcription factors PbApiAP2-O and PbApiAP2-Z is shown [11,27].
Fig 2.
Differential protein expression between female gametocytes and zygotes.
(A) A Venn diagram illustrates the overlapping detection of proteins from the library and biological replicate samples from DIA-MS for female gametocytes and zygotes. (B) A heatmap of sample-to-sample distance hierarchical clustering of biological replicates of DIA proteomics is shown. (C) The total signal for each protein (measured as the sum of all quantified peptide areas for the protein) from the biological replicates of samples (not the DIA-MS libraries) compared between female gametocytes and zygotes is shown. The 25th abundance quartile is demarcated with dashed grey lines; only proteins detected above this threshold were considered. Red circles represent proteins that are significantly differently expressed or found in only one stage. Representative proteins-of-interest are labeled. More abundant/only detected in zygotes: gamete surface 6-cys protein P47, ookinete surface 6-cys proteins P25 and P28, and LCCL domain-containing LAP and CCp proteins. More abundant/only detected in gametocytes: gametocyte surface 6-cys proteins P230 and P48/45, perforin-like protein PPLP2, and gamete egress and sporozoite traversal protein GEST.
Fig 3.
Identification of translationally repressed transcripts in P. yoelii female gametocytes that are translated following fertilization.
(A) The protein abundance ratio and RNA abundance ratio for transcripts and proteins detected in female gametocytes and zygotes were compared to identify transcripts detected in female gametocytes with five-fold enriched protein expression in zygotes (highlighted in black). Transcripts-of-note that are relieved from translational repression are indicated in the inset box. (B) Syntenic P. yoelii orthologs of transcripts identified in P. falciparum female gametocytes as translationally repressed are highlighted in pink (Lasonder et al. [6]). (C) Syntenic P. yoelii orthologs of transcripts identified to interact with PbDOZI in P. berghei gametocytes are highlighted in blue (Guerreiro et al. [21]).
Fig 4.
TurboID proximity proteomics reveals changes to the major translationally repressive protein complex across life stages.
(A and B) The abundance ratios of proteins detected over control biotinylation in gametocytes and zygotes for (A) PyDOZI::TurboID::GFP or (B) PyALBA4::TurboID::GFP are plotted. Abundance ratios were calculated for each protein as . Proteins with an abundance ratio > 4 were considered enriched as interactions with PyDOZI or PyALBA4 over background labeling. The color of lines denote comparisons across sample types: teal lines indicate those not detected in gametocytes but detected in zygotes, red lines indicate those detected in gametocytes but not in zygotes, black lines indicate no difference, orange lines indicate those that are more abundant in gametocytes than zygotes (and detected in both samples), and purple lines indicate those more abundant in zygotes than gametocytes (and detected in both samples). (C) Network of protein interactions-of-note detected in DOZI-TurboID and ALBA4-TurboID experiments in gametocytes (left) and zygotes (right). Proteins in grey circles are detected as proximal interactors of both DOZI and ALBA4, proteins in green circles are only detected as DOZI interactors, and proteins in red circles are only detected as ALBA4 interactors. Colored lines represent interactions previously detected in other immunoprecipitation-mass spectrometry experiments: green lines are interactions detected in PbDOZI IP-MS in gametocytes from Mair et al. [10], red lines are interactions detected in PyALBA4 IP-MS in gametocytes from Munoz et al. [20], and blue lines are interactions detected in PyCCR4-1 IP-MS in blood stages from Hart et al. [87].
Fig 5.
Ultrastructure expansion microscopy of female gametocytes and zygotes.
PyDOZI::GFP-expressing female gametocytes (A) or zygotes (B) were visualized via ultrastructure expansion microscopy (U-ExM) as previously described [75,105]. Parasites were stained with anti-GFP to mark DOZI (yellow) and were counterstained with custom rabbit polyclonal antibodies raised against PyCITH, PyPABP1, and PyALBA4 (magenta). Nucleic acids and proteins were stained with Sytox Far Red (blue) and NHS Ester (gray), respectively. Colocalization is denoted with pink coloration in the merged image of a single z plane. Evidence of apical polarization is seen in NHS Ester staining of zygotes. Scale bars = 2.4 microns.
Fig 6.
A model of the regulation of translational repression across the host-to-vector transmission event.
(Top) Proteins that can be made ahead of transmission in female gametocytes (left) or that are made shortly after fertilization and formation of a zygote (right) are illustrated. (Bottom) Proximity proteomics of DOZI and ALBA4 that associate with the 5’ and 3’ end of mRNAs, respectively, revealed conformational changes in bound mRNAs between female gametocytes and zygotes, consistent with recent reports that demonstrated this by smFISH in other eukaryotes. Widespread compositional changes in the DOZI/CITH/ALBA complex are also evident, especially the handoff of DOZI interactions from NOT1-G to NOT1 across the transmission event.