Fig. 9: APR2-APRp2-APRp4 module regulates apical anchorage of APR-SPMT.

a Yeast two-hybrid (Y2H) assay for screening the APR2 directly interacting protein. Using two fragments F1 (1–600, aa) and F2 (601–1394, aa) of APR2 as the bait. Yeast expressing paired bait-pray constructs were grown under the restrictive conditions to detect their interactions. Representative results from two independent experiments. b PLA detecting protein interaction between APR2 and ARA1, APRp2 and APRp4 in ara1::6HA;apr2::gfp, aprp2::6HA;apr2::gfp and aprp4::6HA;apr2::gfp ookinetes. Scale bars: 5 μm. Two independently performed experiments with similar results. c Representative images from negative staining TEM (NS-TEM) of 17XNL, ∆ara1, ∆aprp2, and ∆aprp4 ookinetes. More images are shown in Supplementary Fig. 8A. Scale bars: 200 nm. Quantification of distance between SPMT and the apical membrane-like layer was shown in lower right panel. n is the number of ookinetes in each group from three independent experiments. Values are means ± SD. From left to right: ns P = 0.43, ****P = 2e-29, and ****P = 6e-33, by two-sided Mann–Whitney test. d Mature ookinete conversion rate. Values are means ± SEM (n = 3 biological replicates), From left to right: *P = 0.02 and *P = 0.02, by two-sided t test. e Ookinete cell shape. θ is the angle between the two lines of nucleus-apical and nucleus-basal as illustrated in Fig. 1k. Quantitation of θ in right panel. n is the number of ookinetes tested. Boxes show medians with interquartile ranges, whiskers: min to max show all points. From left to right: ****P = 2e−05 and ****P = 3e−10, by two-sided Mann–Whitney test. Scale bars: 5 μm. Two independently performed experiments with similar results. f Ookinete gliding motility measured using the in vitro Matrigel-based assay. n is the number of ookinetes tested. Boxes show medians with interquartile ranges, whiskers: min to max show all points. From left to right: ****P = 2e−09 and ****P = 7e−08, by two-sided Mann–Whitney test. g Midgut oocysts count in mosquito 7 dpi. n is the number of mosquitoes dissected. Red horizontal lines show mean value of oocyst numbers. From left to right: ****P = 2e−11 and ****P = 4e−20, by two-sided Mann–Whitney test. h Proposed model showing the module of APR2-APRp2-APRp4 in regulating the stabilization of APR and apical anchorage of APR-SPMT.