Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst

Abstract

The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of Plasmodium zygotic development and ookinete-to-oocyst transition. To achieve this we used in vitro and in vivo cross-fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage-specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of Plasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.

Figure 1

Imaging of wt_green_230p and wt_red_230p parasite lines derived from cross‐fertilization assays. A. Purified ookinetes from in vitro cross‐fertilization assays. B. Ookinetes crossing the mosquito midgut epithelium 24 hpi. C. Confocal microscopy sections of 7‐day‐old oocysts on the basal side of A . gambiae midgut epithelium. D. Confocal microscopy sections of sporozoites from homogenized A . gambiae salivary glands 21 dpi. Images were taken at ×63 magnification for the purified in vitro ookinetes and at ×40 magnification for mosquito tissues.

Figure 2

Allelic expression of fluorescent reporters placed under the control of a constitutive gene promoter. A. wt_green_230p x wt_red_230p. B. Δp47green_230p x wt_red_230p. C. Δp48/45green_230p x wt_red_230p. D. wt_green_ssu x wt_red_230p. Each part consists of three panels. The first composite panel is a collection of six representative fluorescence microscopy pictures of A . gambiae midguts fed on mice co‐infected with two transgenic parasite lines as indicated at the bottom of each part, taken at 24 and 48 hpi respectively. The GFP, mCHERRY and a combination of the two channels (merge) are shown. The second panel is a graph showing the distribution and median number of GFP‐positive, mCHERRY‐positive and GFP/mCHERRY double‐positive parasites per midgut at 24 and 48 hpi. The collective results from three biological replicates are shown, where n is the total number of parasites counted. The third panel shows a genogram summarizing the results of these cross‐fertilization experiments. Squares correspond to male gametocytes, circles correspond to female gametocytes, small ellipses correspond to ookinetes and large ellipses correspond to early stage oocysts. The colour of the outline indicates the genotype, while the fill‐in colour indicates the phenotype. Yellow outline indicates gfp/m C herry heterozygotes, while yellow fill‐in colour indicates GFP/mCHERRY double‐positive parasites. Black lines indicate the crosses and the resulting progeny. Horizontal black lines indicate the median parasite number. Stars indicate statistical significance determined with the Mann–Whitney U‐test (***P < 0.001; **P < 0.01; *P < 0.05).

Figure 3

Allelic expression of fluorescent reporters placed under the control of zygote/ookinete specific promoters. A. wt_cht_pgreen_230p x wt_cht_pred_230p. B. wt_ctrp_pgreen_ssu x wt_cht_pred_230p. Each part consists of four panels. The first composite panel is a collection of three representative fluorescence microscopy pictures of in vitro cultured ookinetes initiated with blood from mice co‐infected with two transgenic parasite lines as indicated at the top of each part. The GFP, mCHERRY and a combination of the two channels (merge) are shown. The second panel shows a representative fluorescence microscopy picture of an A . gambiae midgut fed on mice co‐infected with the two transgenic parasite lines taken at 24 hpi. The third panel is a graph showing the distribution and median number of GFP‐positive, mCHERRY‐positive and GFP/mCHERRY double‐positive ookinetes in the in vitro culture. The fourth panel shows a genogram summarizing the results of these crosses. Squares correspond to male gametocytes, circles correspond to female gametocytes and ellipses correspond to ookinetes. The colour of the outline indicates the genotype, and the fill‐in colour indicates the phenotype. Yellow outline indicates gfp/m C herry heterozygotes, while yellow fill‐in indicates to GFP/mCHERRY double‐positive parasites. Black lines show the crosses and the resulting progeny.

Figure 4

Models explaining the maternal phenotype related to the constitutively expressed reporters. A. Maternal mRNA (mat‐mRNA) contribution. Both parental alleles are silenced during zygotic development. Maternal mRNA is supplied by the female gametocyte and translated. B. Paternal allele silencing. The parental allele is silenced during zygotic development. The maternal allele is de novo transcribed and translated. C. Paternal allele silencing and maternal mRNA contribution. A combined model, whereby the paternal allele is silenced, while maternal mRNA is supplied by the female gametocyte and the maternal allele is de novo transcribed and translated. Squares correspond to male gametocytes, circles correspond to female gametocytes, small ellipses correspond to ookinetes and large ellipses correspond to early stage oocysts. The colour of the outline indicates the genotype, and the fill‐in colour indicates the phenotype. Yellow outline indicates gfp/mCherry heterozygotes, while yellow fill‐in indicates GFP/mCHERRY double‐positive parasites.