Mutant/mutation
The mutant lacks expression of CRK5 and expresses GFP under the constitutive eef1a promoter

Protein (function)
From the Abstract: 'Cell cycle transitions are generally triggered by variation in the activity of cyclindependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.'

Phenotype
Normal gametocyte production. Only a few microgametocytes formed active exflagellation centres after activation. CRK5-KO parasites showed a significant reduction in ookinete conversion compared to the parental control line. A > 30 fold decrease in the number of CRK5-KO oocysts present in A. stephensi mosquitoes allowed to feed on infected mice. Oocysts showed aberrant morphology. No formation of viable sporozoites. No blood stage infection developed in mice after infection by bite of infected mosquitoes.

Additional information
- Previous attempts to disrupt P. berghei crk5 had suggested the gene is essential for asexual blood-stage proliferation (Tewari et al., 2010). However, using long sequence homology regions to replace crk5 with a T. gondii DHFR/TS resistance marker resistant KO-parasites were obtained.
- We tagged the endogenous crk5 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin in a strain expressing the Tir1 protein (Philip and Waters, 2015). Addition of the AID/HA tag to the CRK5 C-terminus imposed a significant fitness cost, with a 2-fold decrease in exflagellation in the absence of auxin, but importantly, depletion of CRK5-AID/HA by one hour of auxin treatment of mature gametocytes prior to activation resulted in a dramatic reduction in exflagellation.

Evidence in this study is presented that:
- CRK5 is a key regulator of gametogony and sporogony in the mosquito
- Phosphoproteome kinetics point to direct phosphorylation of the pre-replicative complex by CRK5
- CRK5 is required for both S- and M- phases during P. berghei gametogony
- We observed an average 2-fold fewer polyploid (>2N) gametocytes 1 min post-activation , demonstrating a dependency on CRK5 for DNA replication during male gametogony.
- Cells depleted of CRK5 are unable to correctly assemble or maintain the spindle, with a greater than two-fold decrease of visible spindles at 1 min pa in absence of CRK5
- CRK5 is part of an atypical nuclear cyclin/CDK complex
- CRK5, SOC2 ((PBANKA_1442200)and CDKrs (PBANKA_0824400; (CDK regulatory subunit, CDKrs) have a similar location and complementary functions during gametogony. CDKrs is a protein related to Cks1 and CksHs2, two CDK-associated proteins in Saccharomyces cerevisiae and and human, respectively.
- CRK5, SOC2 and CDKrs have a similar location and complementary functions during gametogony
- SOC2 expression does not follow a temporal cyclin pattern during gametogony and the CSC complex (CRK5/SOC2/CDKrs complex) is stable during the first round of mitosis
- CSC is dynamically phosphorylated during the first round of replication


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