Fig 5. Knockdown/depletion of NEK1 impairs male gamete formation and blocks parasite transmission.

(A) Auxin (IAA) treatment of NEK1-AID/HA parasites abolished exflagellation (error bars show standard deviation from the mean; technical replicates from 3 independent infections. (B) Exflagellation centres per field at 15-min post activation. n = 3 independent experiments (>20 fields per experiment). Error bar, ± SEM. (C) Percentage ookinete conversion from zygote. n = 3 independent experiments (>100 cells). Error bar, ± SEM. (D) Total number of GFP-positive oocysts per infected mosquito in NEK1Clag compared to WT-GFP parasites at 14-day post infection. Mean ± SEM. n = 3 independent experiments. (E) Mid guts at 10× and 63× magnification showing oocysts of NEK1Clag and WT-GFP lines at 14 days post infection (dpi). Scale bar = 50 μm in 10× and 20 μm in 63×. (F) Rescue experiment showing male-derived allele of NEK1clag confers defect in ookinete formation and is complemented by “female” mutant parasite lines (Δdozi and Δnek4). Male mutant line (Δhap2) did not complement the phenotype. Shown is mean ± SD; n = 3 independent experiments. The data underlying the figures A, B, C, D, and F can be found in S1 Data. (G, H). Volcano plots showing significantly down-regulated (red, Log₂ fold ≤ −1, q value < 0.05) and up-regulated genes (green, Log₂ fold ≥1, q value < 0.05) in NEK1clag compared to wild-type lines in non (0 min)-activated gametocytes and 30-min activated gametocytes. (I). Expression heat map showing affected kinases. The data underlying the figures G, H, and I can be found in S2 Table.