Summary

RMgm-1034
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0934000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Name tag: mCherry
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 10 May 2014, 20:50
  *RMgm-1034
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24798694
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-1028
Other information parent lineThis parent line expresses GFP under the control of the HSP70 promoter. The reporter gene is introduced into the silent 230p locus using the GOMO method of transfection.
The mutant parasite was generated by
Name PI/ResearcherRisco-Castillo, V; Silvie, O.
Name Group/DepartmentCentre d’Immunologie et des Maladies Infectieuses
Name InstituteFaculté de Médecine Pierre et Marie Curie
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-1034
Principal nameRON4::mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageRON4-mCherry displayed a typical punctuate distribution in schizonts and apical localization in merozoites, indicating correct targeting to the rhoptries. No expression in gametocytes.
Normal blood stage development.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteRON4-mCherry is expressed in both oocyst-derived and salivary gland sporozoites, with an apical distribution consistent with rhoptry localization.
Normal numbers of sporozoites are formed that have normal infectivity in vitro (in HepG2/CD81 cells) and in vivo
Liver stageRON4-mCherry was not detected in 24 h liver stages but were expressed in late (72 h) liver stage parasites. Punctuate mCherry-positive structures could be distinguished over diffuse fluorescence of the parasite cytoplasm, consistent with localization to the rhoptries of forming hepatic merozoites.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of RON4

Protein (function)
RON4 is a rhoptry protein. While microneme proteins are typically involved in gliding motility, adhesion to substrates and host cell recognition, rhoptry proteins have been implicated in MJ (moving junction) and PV formation as well as host cell modifications.
A recent conditional mutagenesis approach in P. berghei has shown that RON4 plays a critical role during hepatocyte infection (see RMgm-685).

Phenotype
Phenotye analyses indicate expression of RON4 in rhoptries of merozoites and sporozoites.

Additional information
In the paper evidence is presented that RON2 and RON4 are lost following rhoptry discharge during merozoite and sporozoite entry. In contrast, analyses indicate that RAP2/3 is secreted into the parasitophorous vacuole during infection. Evidence is presented that sporozoite rhoptry discharge occurs only in the presence of CD81, providing the first direct evidence for a role  of CD81 during sporozoite productive invasion (the host membrane protein CD81 is required for infection of hepatocytes by P. falciparum and P. yoelii sporozoites. CD81 acts at an  early stage of infection, possibly at the entry step, but the mechanisms involved are still unknown).

RON4-mCherry could be already detected in maturing liver schizonts, whereas RAP2/3-mCherry and RON2-mCherry were only observed in very late liver stages

Other mutants
See here for other RON2 mutants
See here for other RON4 mutants
See here for other RAP2/3 mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0934000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerunknown
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