TheMedicago truncatulavacuolar iron Transporter-Like proteins VTL4 and VTL8 deliver iron to symbiotic bacteria at different stages of the infection process

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule-specific Vacuolar iron Transporter-Like (VTL) proteins inMedicago truncatula. Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA-seq data showing differential expression ofVTL4andVTL8during early and late infection, respectively. Thevtl4insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly-arrangedVTL4-VTL8genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression ofVTL8alone. Using a newly developedluxreporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed invtl4mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. These data indicate that VTL8, the closest homologue of SEN1 inLotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids.