Regulation of MILDEW RESISTANCE LOCUS-O trafficking by calmodulin-binding domains

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.