A single valine to leucine switch disrupts AP2-G DNA binding and reveals GDV1's role in activation.

Sexual commitment in parasites is essential for malaria transmission, yet much remains unknown about the underlying signaling events initiating sexual conversion in a subpopulation of parasites. We discovered a single valine (V) to leucine (L) mutation in an Apetala 2 (AP2) transcription factor required for gametocytogenesis, that abrogates sexual differentiation and confirmed this with forward and reverse mutation editing. Mutated AP2-G.L does not bind the consensus motif, GnGTAC, or stimulate AP2-G-dependent gene transcription including autoregulation. We then used AP2-G.L parasite lines as tools to demonstrate the critical role of GDV1 in the initial activation of the silent locus during the trophozoite to schizont transition in the absence of functional AP2-G and its autoregulation. Additionally, we show that AP2-G.V is required for MSRP1 expression, which can be used to distinguish early and late sexually committed schizonts. Together this work demonstrates that valine in AP2-G plays a critical role in DNA binding, highlighting the functional importance of this specific region for malaria transmission as well as the critical role of GDV1 in the initial activation of expression and induction of sexual differentiation. The reporter lines generated allow further study of signaling pathways or screening of factors regulating sexual commitment.