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Abstract Flowering plant sexual reproduction relies on the communication between the pollen tube and synergid cells to induce pollen tube bursting. During this process, the MILDEW RESISTANCE LOCUS-O (MLO) protein NORTIA (NTA) is polarly trafficked from the Golgi, where it is inactive, to the filiform apparatus, where it is functional in synergids. MLOs were recently described as calcium channels and have been proposed to be negatively regulated through calmodulin (CaM) binding at a conserved C-terminal calmodulin-binding domain (CaMBD). To determine whether CaM binding is necessary for MLO function during pollen tube reception, C-terminal truncations and CaMBD point mutations were made in NTA. Point mutations were also generated in a constitutively filiform apparatus-localized chimeric NTA containing the MLO1 C-terminus. In this study, we demonstrate that mutating the MLO1 and NTA CaMBD reduces the ability for MLOs to function during pollen tube reception. This is in part due to altered subcellular localization of the CaMBD mutants in synergids. We showed that the CaMBD is not necessary for Golgi localization of MLOs, but is necessary for efficient trafficking and total protein accumulation at the filiform apparatus. Our results suggest an additional role for CaM binding as a regulator of MLO trafficking in addition to its previously proposed role as a negative regulator of MLO Ca2+ channel activity. 

Summary Root hair tip‐growth involves coordinated Ca²⁺and ROS signaling to promote growth while maintaining tip integrity. MILDEW RESISTANCE LOCUS‐O (MLO) proteins act downstream of FERONIA (FER) receptor‐like kinases in pollen tubes and synergids to regulate calcium dynamics. This study uses a constitutively active MLO (faNTA) to identify a new role for the FER/MLO signaling module in regulating [Ca²⁺]_cytoscillations in growing root hairs.faNTA was used as a tool to manipulate Ca²⁺influx infermutants. Light sheet fluorescence imaging was used to image the reporter R‐GECO1 to observe [Ca²⁺]_cytdynamics during root hair elongation in various genotypes.We show that faNTA is sufficient to restore normal root hair development, [Ca²⁺]_cytoscillations, and ROS levels tofer‐4. MLO15 was identified as a regulator of root hair tip growth based on disrupted root hair growth and [Ca²⁺]_cytsignatures inmlo15‐4. We also link the FER/MLO module to ROS accumulation by showing that faNTA is sufficient to restore ROS levels infer‐4root hairs, but is unable to complement the burst root hairs ofrbohc.We propose that MLOs act downstream of FER to mediate Ca²⁺influx and promote ROS production to regulate root hair tip growth.