Sub-cellular localization and functional analyses of Medicago truncatula DMI1 and its homologs involved in legume nodulation and arbuscular mycorrhization

Medicago truncatula Does not Make Infections 1 ( DMI1) and its homologs in Lotus japonicus, CASTOR and POLLUX, are required for the early symbiotic signaling leading to the establishment of legume nodulation and arbuscular mycorrhization. Sub-cellular localization studies using confocal and transmission electron microscopy revealed that this novel group of ion channels resides on the nuclear envelope. In L. japonicus, both CASTOR and POLLUX are required for symbiotic signaling. In contrast, DMI1 and SYM8, two close homologs of POLLUX, are the only reported symbiotic ion channels in M. truncatula and Pisum sativum respectively. In M. truncatula, the MtCASTOR gene does not play a significant role in nodulation and mycorrhization. In cross-species transformation of M. truncatula and L. japonicus mutants, DMI1 alone---but not CASTOR or POLLUX---could rescue a Lotus castor/pollux double mutant indicating a superior symbiotic functionality of DMI1. A single serine to alanine polymorphism between POLLUX and DMI1 in the selectivity filter region was pinpointed by mutational analysis to be solely responsible for this improvement. POLLUX(S329A) acted like DMI1 in M. truncatula and L. japonicus and thus carried improved functionality. A reciprocal modification in the filter of DMI1 conferred loss-of-improvement to the dmi1(A294S) allele. This serine to alanine amino-acid substitution present in DMI1 and SYM8 occurred relatively recently in legume evolution. Protein models and FRET analyses suggest that DMI1 has improved functionality in modulating Nod factor-induced calcium spiking over its homologs CASTOR and POLLUX. Our results indicate that subtle differences in the filter region of these ion channels confer distinct abilities in mediating plant-microbe symbioses.