Actin–membrane interactions mediated by NETWORKED 2 in Arabidopsis pollen tubes through associations with Pollen Receptor‐Like Kinase 4 and 5

During fertilization, Pollen Receptor‐Like Kinases ( PRK s) control pollen tube growth through the pistil in response to extracellular signals, and regulate the actin cytoskeleton at the tube apex to drive tip growth. We investigated a novel link between membrane‐integral PRK s and the actin cytoskeleton, mediated through interactions between PRK s and NET 2A; a pollen‐specific member of the NETWORKED superfamily of actin‐binding proteins. We characterize NET 2A as a novel actin‐associated protein that localizes to punctae at the plasma membrane of the pollen tube shank, which are stably associated with cortical longitudinal actin cables. NET 2A was demonstrated to interact specifically with PRK 4 and PRK 5 in Nicotiana benthamiana transient expression assays, and associated at discreet foci at the shank membrane of Arabidopsis pollen tubes. Our data indicate that NET 2A is recruited to the plasma membrane by PRK 4 and PRK 5, and that PRK kinase activity is important in facilitating its interaction with NET 2A. We conclude that NET 2A– PRK interactions mediate discreet sites of stable interactions between the cortical longitudinal actin cables and plasma membrane in the shank region of growing pollen tubes, which we have termed Actin‐Membrane Contact Sites ( AMCS s). Interactions between PRK s and NET 2A implicate a role for NET 2A in signal transduction to the actin cytoskeleton during fertilization.