|Additional remarks phenotype|
The mutant lacks expression of the RNA binding protein Puf2 and expresses GFP under the constitutive eef1α promoter.
The roles of Puf (Pumilio and fem-3 mRNA binding factor) proteins are diverse yet intimately involved in the translational regulation of developmental and differentiation factors in organisms as diverse as yeast, C. elegans, Drosophila and humans. Two such proteins, Puf1 (PFE0935c) and Puf2 (PFD0825c), are known in the human malaria parasite, P. falciparum, with orthologs in all Plasmodium spp. characterized. The Plasmodium Puf proteins have the typical highly conserved organization that includes the eight tandem copies of the PUM RNA binding domain (or Pumilio Homology Domain, PHD) at the carboxyterminus of the protein and expectedly PfPuf2 was shown to bind RNA in vitro. In P. falciparum evidence has been reported for a role for Puf2 in gametocyte development although Pfpuf2 is most highly transcribed in sporozoites (see also 'Additional Information').
The phenotype analyses indicate that Puf2 is not essential for blood stage development, production of gametocytes, ookinetes, oocysts and sporozoites. However, sporozoite infectivity is affected. Evidence is presented that sporozoites in the absence of Puf2 'prematurely' initiate development into the early livers stage (EEF's) inside salivary glands (see below). The phenotype analyses indicate that the RNA binding protein Puf2 regulates the transition of sporozoites into liver stage forms.
Evidence is presented that sporozoites in the absence of Puf2 'prematurely' initiate development into the early livers stage (EEF's) inside salivary glands. This is based on morphological changes of sporozoites in salivary glands, at day 18-27after mosquito infection, resembling transformation of sporozoites into the early liver stage after hepatocyte invasion.
Evidence is presented that mutant sporozoites have reduced levels of mRNA transcripts of the genes puf1 (PBANKA_123350) and uis1/ik2 (PBANKA_020580; PFA0380w).
Independent mutants lacking expression of Puf2 have been published (RMgm-515, RMgm-516, RMgm-591). A few aspects of the phenotype of these parasites are different from the mutants described here: i) no effect on gametocyte production was reported; ii) sporozoites showed reduced capacity to invade hepatocytes in vitro.
In P. falciparum Puf2 expression has been demonstrated in gametocytes and disruption of Pfpuf2 (PFD0825c) resulted in increased gametocyte production and increased differentiation of male gametocytes (Miao J. et al., 2010, 123-1039-1049). The phenotype of the P. falciparum mutants lacking expression of Puf2 has not been analysed during mosquito development.
See mutants RMgm-513, RMgm-514 and RMgm-616 for P. berghei mutants lacking expression of Puf1 ((PBANKA_123350) . Phenotype analyses of these mutants indicate that Puf1 is not essential for blood stage development, production of gametocytes, ookinetes, oocysts and infective sporozoites and for mosquito transmission and liver stage development .
Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565). After transfection with a KO vector a strong PCR signal diagnostic for gene disruption was observed in transfected populations indicating that this gene is not essential for asexual proliferation. Cloning will however be required to validate this interpretation for this
RMgm-616: mutant lacking expression of Puf 1((PBANKA_123350) which expresses GFP under the constitutive eef1α promoter
RMgm-513: A mutant lacking expression of Puf1 (PBANKA_123350)
RMgm-514: A mutant lacking expression of Puf1 (PBANKA_123350) which expresses GFP under the constitutive eef1α promoter
RMgm-515: An independent mutant lacking expression of Puf2
RMgm-516: An independent mutant lacking expression of Puf2 which expresses GFP under the constitutive eef1α promoter
RMgm-591: A mutant lacking expression of both Puf1 and Puf2 which expresses GFP under the constitutive eef1α promoter