Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes.The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane.Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling.Whereas RopGEFs have been found to be phosphorylated proteins,the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear.We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth.Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes.AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain.Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes,similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5.Collectively,our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.