Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene tagging
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27982038 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone |
8417HP
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Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | Currà C, Siden-Kiamos I |
Name Group/Department | Foundation for Research and Technology-Hellas |
Name Institute | Institute of Molecular Biology and Biotechnology |
City | Heraklion |
Country | Greece |
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Name of the mutant parasite |
RMgm number | RMgm-4063 |
Principal name | ORP2::mCherry |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | In oocysts ORP2::mCherry was readily detected in the cytoplasm in oocysts at day 7. Cytoplasmic localization was maintained in oocysts until day 12 and before sporozoite formation. When sporozoites had formed and were ready to egress from the oocyst, ORP2 was relocalized to the oocyst periphery where it partly co-localized with Cap380 (PBANKA_1218100), a protein of the oocyst capsule. |
Sporozoite | In oocysts ORP2::mCherry was readily detected in the cytoplasm in oocysts at day 7. Cytoplasmic localization was maintained in oocysts until day 12 and before sporozoite formation. When sporozoites had formed and were ready to egress from the oocyst, ORP2 was relocalized to the oocyst periphery where it partly co-localized with Cap380 (PBANKA_1218100), a protein of the oocyst capsule. No mCherry signal was detected in sporozoites after examining the cells in immunofluorescence assays (IFA) |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation
The mutant expresses of C-terminal mCherry-tagged version of ORP2.
Protein (function)
In the paper it is shown that each of two HFD (histone-fold domain)-containing proteins of P. berghei gene (IDs PBANKA_0902500 and PBANKA_1303400), which are named oocyst rupture protein 1 (ORP1) and ORP2, have essential and similar functions in the rupture of the oocyst capsule. Evidence is presented that protein interaction via the HFD is critical for function. ORP1 is located in the oocyst capsule, whereas ORP2 is re-localized from the oocyst cytoplasm to the capsule at the time when mature sporozoites have formed.
The histone-fold domain (HFD) is found in histones and in proteins with a role in transcriptional regulation such as the TATA-box-binding protein-associated factor TAFII and in the CCAAT-binding transcription factor subunits NF-YB and NF-YC. The only example of a protein with a HFD acting outside the nucleus is son-of-sevenless, a protein with multiple domains containing two HFDs, which are involved in binding to lipids. The HFD is B70 amino acids in length and forms three helices separated by small linker sequences. In a heterodimer the proteins are organized in head-to-tail orientation, resulting in a compact ‘handshake’ interaction. In the well-studied NF-Y complex, the heterodimer NF-YB and NF-YC interacts with a third subunit, NF-YA. Although the heterodimer binds DNA in a nonspecific manner, the NF-YA subunit confers binding specificity to the CCAAT motif.
ORP1 (PBANKA_0902500, 950 amino acids) contains a carboxyterminal HFD. ORP2 has an amino-terminal HFD, which is most similar to NF-YC of plants and animals, and HAP5 of yeast. Both HFDs comprises the three a-helices characteristic of the HFD and the short aC helix found in NF-YB/NF-YC proteins. Neither of the two proteins contains any other recognizable motifs and outside the HFD there is only a low degree of similarity comparing the two. The two proteins are considerably longer than NF-YB and NF-YC of animals. BLAST searches of the Plasmodium genome failed to reveal any protein with similarity to NF-YA, which suggested that these two proteins may not be part of a classical NF-Y DNA-binding complex.
Phenotype
No mCherry signal was detected in asexual stages, gametocytes, ookinetes or sporozoites after examining the cells in immunofluorescence assays (IFA)
In oocysts ORP2::mCherry was readily detected in the cytoplasm in oocysts at day 7. Cytoplasmic localization was maintained in oocysts until day 12 and before sporozoite formation. When sporozoites had formed and were ready to egress from the oocyst, ORP2 was relocalized to the oocyst periphery where it partly co-localized with Cap380 (PBANKA_1218100), a protein of the oocyst capsule.
Additional information
See RMgm-4060 for a mutant lacking ORP2 (PBANKA_1303400).
See RMgm-4059 for a mutant lacking ORP1 (PBANKA_0902500).
See RMgm-4061 for a mutant with a mutated histone-fold domain (HFD) of ORP1. Phenotype analyses of this mutant indicates that HFD is essential for ORF function
See RMgm-4062 and RMgm-4063 for mutants expresing GFP-tagged ORP1 version and a mCherry-tagged version of ORP2. Analyses of these mutants show that ORP1 is located in the oocyst capsule, whereas ORP2 is re-localized from the oocyst cytoplasm to the capsule at the time when mature sporozoites have formed.
Other mutants
See above |