Mutant/mutation
In the 'promoter swap' mutant: the promoter of RON11 is replaced by the promoter of msp9 (PBANKA_1443300). In addition the mutant expresses GFP under the constitutive eef1a promoter.

Protein (function)
Rhoptry neck protein 11 (RON11) was first described as localized to the rhoptry neck in Toxoplasma tachyzoites (Beck et al., 2013). It has an architecture of seven transmembrane domains plus two putative calcium binding EF-hand domains, and is highly conserved in the Apicomplexa, suggesting an important role in rhoptry-mediated functions.

Phenotype
RON11 is essential for blood stage development/multiplication (see RMgm-103, RMgm-190 for unsuccessful attempts to disrupt the apc3 gene). In the 'promoter-swap' mutant the promoter of ron11 is replaced by an 'asexual blood stage’ promoter that is silent (or has a strongly reduced activity) in sporozoites (the promoter of msp9, PBANKA_1443300).

The mutant shows the following phenotype: Normal blood stage development/multiplication. Normal sporozoite formation in oocysts. Normal release of sporozoites in hemocoel. Normal numbers of hemocoel sporozoites. However strongly reduced (120-200 fold) numbers of sporozoites in salivary glands. Hemocoel derived mutant sporozoites show strongly reduced infectivity in mice compared to wild type hemocoel derived sporozoites (10.000 fold reduction in liver load after intravenous injection of sporozoites). Evidence is presented for strongly reduced attachment of sporozoites to hepatocytes in vitro and reduced motility and adhesion of sporozoites. In contrast to PbTRAP, no PbRON11 signal was detected on the surface or the apical tip of activated control sporozoites, as visualized using anti-PbRON11N antibodies, indicating that PbRON11 remains inside the rhoptries during gliding.

Additional information
PbRON11 expression during sporozoite maturation in mosquitoes was also examined using sporozoites collected from midguts, haemolymph, and salivary glands of infected Anopheles stephensi at day 23 post-feeding. Western blot analysis using anti-RON11 antibodies revealed that PbRON11 was detected migrating at approximately 100 kDa in all sporozoite samples, indicating that PbRON11 is synthesized early in sporozoite formation and the protein is present until after sporozoites invade salivary glands. This finding also suggests that PbRON11 is not cleaved during sporozoite maturation in mosquito bodies.
To examine PbRON11 localization, IFA was performed on P. berghei schizonts and sporozoites. PbRON11 was detected as a punctate pattern in mature schizonts. PbRON11 signal was distributed somewhat widely in the anterior part of sporozoites collected from midguts, and was concentrated in the apical region of sporozoites collected from haemolymph or salivary glands.. IEM was performed to refine localization of PbRON11. PbRON11 was demonstrated to localize to the neck portion of rhoptry of merozoites.
However, the signal for PbRON11 in sporozoites was not abundant by IEM using specific antibodies. Therefore, we generated transgenic parasites expressing PbRON11 fused with a c-Myc-tag at the C-terminus (RMgm-4645). PbRON11 tagged with c-Myc was confirmed to localize to the rhoptry neck region in merozoites. PbRON11 tagged with c-Myc in sporozoites collected from salivary glands was localized to rhoptries, but not restricted to the neck portion.

To examine whether pbron11 transcription was decreased in PbRON11-cKD sporozoites, quantitative RT-PCR was performed using sporozoites collected from midgut oocysts of PbWT-GFP, PbRON11-cont, and PbRON11-cKD. It was confirmed that transcription of pbron11 in sporozoites of PbRON11-cKD cl1 and cl2 was 80 to 90 times less than that of PbWT-GFP or PbRON11-cont. PbRON11-cont, PbRON11-cKD cl1 and cl2) in schizonts and sporozoites was compared by western blot analysis. The intensity of the 100 kDa band corresponding to PbRON11 was equivalent in schizonts among four parasite lines, indicating that PbRON11 production is normal in schizonts of mutant lines. In contrast, PbRON11 signal intensities in PbRON11-cKD cl1 and cl2 sporozoites collected from midgut oocysts were less than 1/20 of that in PbRON11-cont

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