SEN1 is responsible for molybdate transport into nodule symbiosomes for nitrogen fixation in Lotus japonicus

Symbiotic nitrogen fixation (SNF) in legume root nodules requires a steady supply of molybdenum (Mo) for synthesis of the iron-Mo cofactor for nitrogenase in bacteroids. For this nutrient to be exported by the host plant it must cross the peribacteroid membrane (PBM), however, the molybdate transporter responsible has not yet been identified. A Lotus japonicus symbiotic mutant, sen1, forms nodules that do not fix nitrogen; it has nodule defects and bacteroid degradation. The biochemical function and subcellular localization of SEN1 protein remains to be elucidated. Here, we found a new phenotype in which the SEN1 mutation resulted in increased Mo accumulation in the nodule host fractions but decreased Mo accumulation in the bacteroids at 10 days post inoculation. We identified the molybdate efflux transport activity of SEN1 via heterologous expression in yeast. SEN1 was expressed exclusively in nodules, and its expression was stable in response to varying Mo supply in nutrient solution. In situ immunostaining verified that the SEN1 protein is localized, in part, to the PBM in the rhizobium-infected cells. Taken together, these results confirmed that SEN1 is responsible for mediating molybdate efflux from the cytosol of nodule host cells to the symbiosomes for SNF. Furthermore, SEN1 mutation reduced the expression of nifD and nifK, suggesting that SEN1 may be pertinent to iron-Mo-cofactor assembly. This work fills the knowledge gap regarding how molybdate is allocated from the host plant to the bacteroids; such knowledge is critical for developing new SNF biological systems in non-legume plants.