Summary

RMgm-4657
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0921600; Gene model (P.falciparum): PF3D7_1126800; Gene product: RNA-binding protein, putative (PbSR-MG)
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 7 August 2019, 18:11
  *RMgm-4657
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) : 31370870
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherYeoh LM, McFadden GI, Ralph SA
Name Group/DepartmentDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institut
Name InstituteThe University of Melbourne
CityParkville
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4657
Principal namePbSR-MG KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production. Activation of female gametocytes normal. Male exflagellation, however, strongly reduced. Flagella stained with an anti-tubulin antibody remain curled within the gametocyte body. Motility of male free gametes strongly impaired.
Fertilization and ookineteNot tested
OocystAblation of PbSR-MG also resulted in fewer oocysts per mosquito host. To test if this difference solely reflected the defect in exflagellation, we repeated the mosquito infection using equal numbers of in vitro generated ookinetes. Despite exposing mosquitoes to equal infective load, we still observed a significant difference in oocyst numbers in PbSR-MG KO compared to wild-type parental parasites.
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbSR-MG (similar mutants were made in the following background parent lines: a reporter P. berghei ANKA line expressing mCherry in female gametocytes and GFP in male gametocytes (RMgm-164) and in a reporter line expressing tdTomato in all blood stages (RMgm-4178).

Protein (function)
Serine-arginine–rich (SR) proteins are major regulators of alternative splicing, with regular expression levels necessary for maintaining normal alternative splicing and viability. One SR protein, PBANKA_0921600, is specifically upregulated in male gametocytes of P. berghei and downregulated in female gametocytes, and phylogenetic analysis identifies this gene as an orthologue of a known splicing factor in T. gondii. We named PBANKA_0921600 PbSR-MG (P. berghei SR protein male gametocytes).

Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization (see mutant RMgm-4658)

Phenotype
Normal gametocyte production. Activation of female gametocytes normal. Male exflagellation, however, strongly reduced. Flagella stained with an anti-tubulin antibody remain curled within the gametocyte body. Motility of male free gametes strongly impaired.
Ablation of PbSR-MG also resulted in fewer oocysts per mosquito host. To test if this difference solely reflected the defect in exflagellation, we repeated the mosquito infection using equal numbers of in vitro generated ookinetes. Despite exposing mosquitoes to equal infective load, we still observed a significant difference in oocyst numbers in PbSR-MG KO compared to wild-type parental parasites.

Additional information
Localizing an integrated, hemagglutinin-tagged (HA-tagged) PbSR-MG confirmed its expected nuclear localization (see mutant RMgm-4658).

Evidence is presented for the following: 
- RNA-seq analysis revealed differences in alternative splicing to be heavily skewed by sex in the mutant lacking PbSR-MG. Ten percent of genes had modified alternative splicing in male gametocytes, while only 3.9% of female-specific and 0.90% of asexual-stage genes were affected. Loss of PbSR-MG had minimal impact on transcription, altering the overall expression of only 0.5% of genes. 
- There were 247 genes that had both male-specific changes in alternative splicing and were male genes regulated by PbSR-MG. Hence, 79% (247/314) of the genes regulated by PbSR-MG were originally identified as having alternative splicing changes specific tomale gametocytes. Fromthe opposing perspective, only 20% (247/1238) of all male-specific alternative splicing changes are regulated by PbSR-MG. This contrasts with female gametocytes, where only 5.4% (73/1340) of the female-specific alternative splicing changes are affected by the deletion of PbSR-MG. Presumably, there are other unidentified factors regulating female-specific alternative splicing. 
- When PbSRMGwas ablated, the number of alternatively spliced genes detected was relatively unchanged for asexual parasites  modestly increased for female gametocytes, and most increased for male gametocytes. Many genes were alternatively spliced in both wild-type and mutant parasites, suggesting that much alternative splicing is regulated by factors additional to PbSR-MG in all three stages. 

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0921600
Gene Model P. falciparum ortholog PF3D7_1126800
Gene productRNA-binding protein, putative
Gene product: Alternative namePbSR-MG
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-240478
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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