Summary

RMgm-4708
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1220300; Gene model (P.falciparum): PF3D7_0321400; Gene product: protein kinase, putative (pPK1, pseudokinase)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Oocyst;
Last modified: 22 January 2020, 16:34
  *RMgm-4708
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31953169
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone P. y. yoelii 17XL
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherIshizaki T, Kaneko O
Name Group/DepartmentGraduate School of Biomedical Sciences
Name InstituteNagasaki University
CityNagasaki
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4708
Principal nameΔpPK1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageReduced growth/multiplication of asexual blood stages. Evidence is provided for reduced invasion of merozoites.
Gametocyte/GameteNormal production of male and female gametocytes. Normal egress of both male and female gametocytes from their host red blood cells after activation. Normal exflagellation rate (i.e. male gamete formation); however, exflagellating males showed (strongly) reduced formation of the characteristic exflagellation centers, i.e. exflagellating males surrounded/attached to uninfected cells.
Fertilization and ookineteNot tested
OocystStrongly reduces formation of oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PypPK1

Protein (function)
PY17X_1220300 encodes a 319 amino acid protein with an expected molecular weight of 38 kDa. The CD-Search webware predicted a protein kinase catalytic domain from amino acid positions (aa) 36 to 278 (E-value = 6.63e-22), and a signal peptide sequence or transmembrane region was not predicted. An active protein kinase has several critical features for phosphorylation activity such as a catalytic aspartate and a Mg binding site. The corresponding regions of PY17X_1220300 were not conserved with the known P. yoelii protein kinases, PyCDPK4, PyCDPK3, PyPKAc, PyGSK3, and PyMAPK2, suggesting that PY17X_1220300 does not possess phosphorylation activity. The relevant regions are widely conserved within orthologs in other Plasmodium spp., suggesting that PY17X_1220300 is a pseudokinase with a conserved role across Plasmodium. Taken together, we designated this protein as P. yoelii pseudo Protein Kinase 1 (PypPK1)

Phenotype
Reduced growth/multiplication of asexual blood stages. Evidence is provided for reduced invasion of merozoites.
Normal production of male and female gametocytes. Normal egress of both male and female gametocytes from their host red blood cells after activation. Normal exflagellation rate (i.e. male gamete formation); however, exflagellating males showed (strongly) reduced formation of the characteristic exflagellation centers, i.e. exflagellating males surrounded/attached to uninfected cells.
Strongly reduces formation of oocysts

Additional information
By analyzing a mutant expressing a C-terminal cmyc-tagged version of PypPK1 (RMgm-4709) evidence is presented that PypPK1 is localized at the apical side of daughter merozoites. evidence is presented for presence of protein in nuclei of both male and female gametocytes. and male gametes. Evidence is presented for plasma location of the protein in ookinetes.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1220300
Gene Model P. falciparum ortholog PF3D7_0321400
Gene productprotein kinase, putative
Gene product: Alternative namepPK1, pseudokinase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA CRISPR/Cas9 plasmid (pDC2-Cas9-gRNA-hdhfr, a kind gift from the Wellcome Genome Campus Advanced Course), was digested with BamHI, and then a DNA fragment containing the P. yoelii U6 promoter and its terminator PCR-amplified from P. yoelii gDNA was ligated using an In-Fusion HD cloning kit (Takara Bio Inc., Shiga, Japan), yielding pDC2-cam-Cas9-PyU6-hDHFR. To generate plasmids to knockout the PY17X_1220300 gene locus or to tag the C-terminal end with Myc epitopes, pDC2-cam-Cas9-PyU6-hDHFR was digested with BbsI and ligated with the gRNA component targeting PY17X_1220300. The plasmid was then digested with HpaI and AatII and DNA fragments for the 5’- and 3’-homologous recombination regions of Py17X_1220300 PCR-amplified from P. yoelii gDNA and hDHFR or hDHFR/yFCU expression cassettes PCR-amplified from pDC2-cam-Cas9-PyU6-hDHFR plasmid or p230p-TRAD4-Rand-mCherry plasmid (kind gift from Dr. Soldati), respectively, were ligated using an In-Fusion HD cloning kit, yielding pDC2-Cas9-PyU6-ΔPypPK1 and pDC2-Cas9-PyU6-PypPK1-myc, respectively. The plasmids for transfection were prepared using a HiSpeed Plasmid Midi Kit (Qiagen, Hilden, Germany)
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6