The putative ion channel DMI1 localizes to the nuclear envelope and regulates nuclear calcium spiking during early symbiotic signaling

Two major plant-microbes symbiotic interactions are legume nodulation and arbuscular mycorrhization. Several genes required for the development of these symbioses have been cloned in model legumes. Among them, the Medicago truncatula DMI1 protein shares strong homologies with prokaryotic ion channels such as MthK. Two DMI1 homologs in Lotus japonicus, named CASTOR and POLLUX, have been localized in plastids. However, our confocal and electron microscopy by high pressure freezing and cryo-substitution method places DMI1 on both inner as well as outer nuclear membranes. The N-terminal soluble domain of DMI1 is sufficient but not necessary to target the protein to nuclear envelope. This domain is also required for the functionality of DMI1. The soluble C-terminus of DMI1 contains an RCK (regulator of the conductance of K super(+)) domain which in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. A dmil mutant lacking the entire C-terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast mutant towards those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants.