Mutant/mutation
The mutant lacks expression of Py235EBP-1 (PY01365), one member of the py235 gene family.
(The gene model of P. yoelii X (XNL) is shown; however, the gene has been deleted in P. yoelii YM; sequence differences exists between members of the py235 (reticulocyte binding protein) gene family between these two laboratory strains)

Protein (function)
Of the Plasmodium erythrocyte adhesion ligand families identified to date, one of the most studied is the erythrocyte binding ligand family (EBL), which includes P. falciparum erythrocyte binding antigen (EBA)-175 and the Duffy binding protein (DBP) of P. vivax and P. knowlesi, located in the apical organelles of the merozoite.

A second group of high molecular mass adhesion proteins, which was first described in the rodent malaria parasite P. yoelii as Py235 is the reticulocyte binding-like (RBL) super family, so named because of sequence homology with the reticulocyte binding protein RBP-1 and RBP-2 of P. vivax. In P. vivax, these proteins are thought to be involved in erythrocyte selection as they bind to reticulocytes but not mature erythrocytes thereby restricting P. vivax to the invasion of reticulocytes. P. falciparum contains a small group of genes coding for proteins with similarities to Py235 and PvRBP, the PfRH family. In contrast to the PvRBP and PfRH gene families, which are small, the Py235 multigene family contains at least 11 members. Analysis of the sequences on fifteen contigs identified in the P. yoelii genome database, which represent members of the Py235 gene family (and some of which are incomplete), show they have overall conserved structural elements.
The Py235 proteins have been implicated in the selection, recognition and invasion of erythrocytes. Populations of P. yoelii asexual blood stage parasites express multiple members of the py235 gene family. The member Py235EBP-1 (encoded by the single copy gene PY01365) is recognized by mAb 25.77.

Phenotype
Host cell preference (reticulocytes, normocytes), growth/multiplication rate of asexual blood stages was not different from wild type parasites.
Evidence is presented that there was no compensatory significant upregulation of expression of any of the other Py235 genes in the mutant parasite.
It is suggested that that the protein PY01185 (designated PY235EBP-2) is able to fulfill the role of Py235EBP-1 by serving as an invasion ligand (see additional information).

The pehenotype of an independent mutant lacking expression of  PY01365 (RMgm-615) has been reported. The phenotype of this mutant is different from the phenotype reported in this study (see 'Additional Information' below).

Additional information
The mAb 25.77 had previously been used to identify Py235EBP-1, the product of the PY01365 gene. By IFA, this mAb gives a punctuate pattern of fluorescence in the WT parasite line. A similar pattern was also observed for the PY01365-KO parasite line, even though Py235ebp-1 is no longer being expressed. Evidence is presented that mAb 25.77 also recognizes two other members of the Py235 family: gene PY01185 and gene PY05995/PY03534. The PY01365, PY01185, and PY05995/PY03534 sequences form a discrete subset of the family with a degree of similarity in pairwise alignment of greater than 80% at the amino acid sequence level. Evidence is presented that the protein encoded by  PY01185 binds to erythrocytes and has been designated Py235 erythrocyte binding protein-2 (Py235EBP-2), as the second known erythrocyte binding protein from this family.
The similar growth and invasion phenotype of mutant and wild type asexual blood stages may suggest that that the PY01185 gene product (PY235EBP-2) is able to fulfill the role of Py235EBP-1.

The Py235 family of proteins belongs to the reticulocyte binding-like (RBL) super family, with homology with the reticulocyte binding protein RBP-1 (PVX_098585) and RBP-2 (PVX_121920) of P. vivax. P. falciparum contains a small group of genes coding for proteins with similarities to Py235 and PvRBP, the PfRH family (for example see the reticulocyte binding protein 2 homologue a (PF13_0198) and the reticulocyte binding protein 2 homologue b (MAL13P1.176)).

In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. Based on these results it is proposed that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

In an independent study the phenotype has been analysed from an independent P. yoelii mutant lacking expression of PY01365 (see RMgm-615). This mutant showed a different (delayed) course of parasitemia in BALB/c mice and BALB/c mice showed a delayed death from infection. Moreover data is presented indicating that disruption of PY01365 leads to a change in the overall transcription pattern of different members of Py235.

Other mutants
RMgm-615: An independent mutant lacking expression of PY01365