Fig. 2. RON13 is a luminal rhoptry protein that is not secreted during invasion.

a IFAs of RH-, ARO-YFP- (green), RON11-YFP- (green), or RON13-YFP- (green) expressing parasites transiently transfected with cytosolic nanobodies targeting YFP fused to a myc-tag (YFPnb-myc). ARO is a protein associated with the cytosolic face of the rhoptry membrane and RON11 is a type III transmembrane protein with its C-terminal domain exposed in the parasite cytoplasm. The myc signal (magenta) observed at the basal part of the parasite is unspecific. Left panels show the schematic topology of the proteins and their ability to bind the cytosolic YFPnb-myc. Scale bar = 2 μm. Image representative of three biologically independent experiments. b Principle of the experimental design for the FRET-based rhoptry secretion assay used to determine if RON13 (orange) is secreted into the host cell during the invasion. Toxofilin (blue) is a soluble rhoptry protein secreted into the host cell. c Gating strategy for quantification of fluorescein⁺ cell (green gate; λ = 550 nm) and coumarin⁺ cell (violet gate; λ = 450 nm) frequency for RON13-BLA- and Toxofilin-BLA-infected cell monolayer (yellow gate) analyzed by flow cytometry. d, e Frequency of fluorescein⁺ cells (λ = 550 nm, d) or coumarin⁺ cells (λ = 450 nm, e) in each condition (mean ± SD; n = 3 biologically independent experiments). Statistical significance was assessed by a one-way ANOVA significance with Tukey’s multiple comparison. Source data are provided as a Source data file.