Additional remarks phenotype | Mutant/mutation
The mutant expresses a FLAG tagged version of Argonaute 2 (Argo2) under control of the constitutive hsp70 promoter. In addition it expresses GFP under control of the constitutive eef1a promoter.
The Argonaute 2 construct is integrated by double cross-over integration in a locus on P. berghei chromosome 6 (Sil6 locus) between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220).
The mutant is drug-selectable marker free. Introduction of the hsp70-Argonaute 2 expression cassette is performed using positive selection. After cloning of the transgenic parasites the drug-selectable marker cassette is removed by applying negative selection, resulting in selection of parasites that have removed the positive/negative selectable marker hdhft/yfcu by homologous recombination.
This mutant has been used to express specific AgoshRNA's and evidence is provided for (partly) gene-silencing.
Protein (function)
In most eukaryotes, a tool for gene-siliencing is available in the form of RNA interference (RNAi), an endogenous pathway for gene regulation on the mRNA level that can be usurped by using small interfering or short hairpin RNAs (siRNAs or shRNAs, respectively) as exogenous RNAi triggers. However, Plasmodium species lack the canonical RNAi machinery, including the key enzymes Dicer that processes transcribed shRNAs into siRNAs, as well as Argonaute 2 (Ago2) that, when loaded with the siRNA, binds and cleaves target mRNA. Interestingly, though, a noncanonical RNAi pathway has recently been described in mammalian cells that requires only Ago2 to process a special type of shRNAs. These so-called AgoshRNAs have a shorter stem and loop than conventional shRNAs, which prevents their recognition by Dicer and instead facilitates direct loading into, and processing by, Ago2. The resulting protein-RNA complex then binds to a complementary target mRNA and causes its cleavage and degradation. The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called.
AgoshRNA.
Phenotype
Expression of Ago2 in blood and liver stages is shown. The mutant had (slightly) reduced growth of blood stages and produced decreased numbers of oocysts and sporozoites.
This mutant has been used to express specific AgoshRNA's and evidence is provided for (partly) gene-silencing.
Additional information
We found remarkable differences between the transcriptome of blood stages of the parental PbGFPcon and PbAgo2 (with or without AgoshRNA). Specifically, in PbAgo2, we observed a significantly lower abundance of female gametocyte-specific transcripts that prior work had shown to be down-regulated upon deletion of DOZI.
Other mutants |