Plasmodium parasites have two actin isoforms. Actin I is ubiquitously expressed, while the second actin isoform is expressed in the sexual stages and ookinetes. Reverse genetic analysis revealed two phenotypes in parasites lacking the protein: a block in male gametogenesis (exflagellation) and a second phenotype in oocyst development, dependent upon the expression of the gene in female gametocytes. Here, we report that the genetic complementation of two independent mutants lacking actin II does not fully restore wild-type function. Constructs were integrated in the c-rrna locus, previously used for expression of transgenes, in order to determine the dependence of expression on actin II flanking genomic regions. Partial restoration of male gametogenesis was achieved when the transgene contained, in addition to the coding region, 1.2 kb upstream of the actin II open reading frame. Another transgene, which comprised 2.7 kb of actin II 5′ flanking regions and the cognate 3′ downstream sequence, fully restored exflagellation. However, in both complemented strains, oocyst development was severely impaired compared to the WT. These data suggest that male gametocyte expression of actin II is dependent upon extensive flanking regions, while female expression requires even longer genomic sequences for correct expression of the gene.
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MA was supported by a grant from the program Bilateral Research and Technology Cooperation Greece-France, The General Secretariat for Research and Technology, Ministry of Education, Greece (grant number 2013SE01380004). This work was performed in the framework of the BIOSYS research project, Action KRIPIS, project no. MIS-448301 (2013SE01380036) funded by the General Secretariat for Research and Technology, Ministry of Education, Greece, and the European Regional Development Fund (Sectoral Operational Programme: Competitiveness and Entrepreneurship, NSRF 2007–2013)/European Commission).
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed.
Maria Andreadaki and Elena Deligianni contributed equally to this work.
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Online Resource Figure. S1. Genotyping of complementation lines. A. Diagnostic PCR using primer pairs L739 and L635 indicative of left integration and L665 and L740 indicative of right integration in the c- or d-rrna locus (Franke-Fayard et al. 2004). Primers recognizing the GAPDH locus were used for quality control of the gDNA (Andreadaki et al. 2014). Templates were derived from: gDNA of WT (lanes 1, 3, 5) and actIIC2 (lanes 2, 4, 6). The genotyping by PCR of actIIC has been reported previously (Deligianni et al. 2011). B. Southern blot analysis of gDNA originating from WT and the two cloned complementation lines digested by HindIII and EcoRI. The blot was hybridized with a 675 bp fragment corresponding to the ets probe specific for the c- and d-rrna loci (Franke-Fayard et al. 2004). In WT the probe recognizes an 8.7 kb (arrow) and 17 kb and fragment corresponding to the c- and d-type, respectively, as well as two weaker, non-specific bands of 10 kb and 3 kb (asterisks). These were detected in two independent experiments in WT gDNA. After integration of the construct in the c-rrna locus a 3 kb fragment is detected, while a 10.4 kb fragment is detected after integration in the d-rrna locus. Both complementation lines were integrated in the c-rrna locus as the 17 kb fragment is still present, while the 8.7 kb is no longer detected. (PDF 717 kb)
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Andreadaki, M., Deligianni, E., Nika, F. et al. Expression of the Plasmodium berghei actin II gene is controlled by elements in a long genomic region. Parasitol Res 115, 3261–3265 (2016). https://doi.org/10.1007/s00436-016-5133-z
- Gene expression