Mutant/mutation
The mutant lacks expression of CDPK3.
The mutant does not contain the hdhfr/yfcu drug-selectable marker cassette. This cassette has been removed by negative selection.

Protein (function)
CDPK3 belongs to an expanded family of Ca²⁺ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. In plants, CDPKs translate Ca²⁺ signals generated by external stimuli into cellular responses, thereby regulating cell division and differentiation, the development of tolerance to stress stimuli and the specific defense responses to pathogens.
CDPK3 has a role in the transformation of the mature ookinete into the oocyst stage (see RMgm-154).

Phenotype
CDPK3 has a role in the transformation of the mature ookinete into the oocyst stage (see RMgm-154).

The mutant in this study was generated to  screen for genetic interactions among protein kinases. In this study a role of CDPK4 during erythrocytic (asexual blood stage) proliferation has been found.

From the paper:
'In P. falciparum schizonts and P. berghei sporozoites, PKG controls microneme secretion, a process that is also critical to sustain gliding in ookinetes. The PKG-inhibitor C2 blocks the secretion of the ookinete microneme protein CelTOS-3xHA into the culture supernatant, specifically in the inhibitor-sensitive line, indicating that signalling through PKG is required for microneme secretion also in ookinetes. However, depletion of CDPK1 and chemical inhibition of CDPK4 does not affect secretion of CelTOS-3xHA either individually, or in combination. In marked contrast, deletion of CDPK3, an ookinete-specific CDPK needed for optimal gliding, does reduce secretion of CelTOS-3xHA. Complementation of cdpk3 deletion ascertained that this effect was due to the absence of CDPK3 expression. Furthermore, CDPK3 does not appear to interact functionally with CDPK4, since addition of inhibitor 1294 does not decrease motility further in the CDPK3-KO. Altogether, this suggests that the main function of CDPK3 is to control microneme secretion downstream of PKG but independently of CDPK4, while CDPK1 and CDPK4 perform complementary functions in supporting efficient gliding.'

'The role of CDPK3 in ookinete gliding was unknown and we show here that it controls microneme secretion.'

From the Abstract:
Most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito.

Additional information
To search for genetic interactions between P. berghei protein kinase genes, parasites from a panel of mutant clones lacking a specific kinase were negatively selected for loss of the selection marker and then transfected with a pool of barcoded gene knockout (KO) vectors to inactivate another kinase in the same background. The competitive growth rate of each mutant within the pool was measured during days 4–8 post infection by barcode sequencing. For the background lines, we focussed on the CDPK family and on the two atypical MAP kinases.
In preliminary experiments, we found that a double mutant of map1 and map2 showed normal asexual growth, and the double KO mutant was included in the screen as a single recipient background to identify interactors of either gene. Due to the essential role for PKG in calcium mobilisation upstream of CDPKs, we also included the inhibitor-resistant PKGT(619Q)-3xHA line and its inhibitor-sensitive control, PKG-3xHA. The library of KO vectors was comprised of 37 targeting vectors for protein kinases and 6 characterised vectors targeting unrelated genes for use as references.

Other mutants