Whole Genome Sequencing Identifies a Medicago truncatula Tnt1 Insertion Mutant in the VTL8 Gene that is Essential for Symbiotic Nitrogen Fixation

Abstract

Legumes carry out symbiotic nitrogen (N) fixation by forming N-fixing nodules with rhizobia during N deficiency. In an effort to discover new legume genes essential to nodule function using the Medicago truncatula–Sinorhizobium meliloti model legume–rhizobia model system, a forward genetics approach was employed using lines of M. truncatula mutagenized with the tobacco-retrotransposon Tnt1, from which N-fixation defective (Fix−) plants were recovered. The Tnt1 mutagenized lines are in the R108 genetic background. One of these lines, NF11322, has a Tnt1 insertion interrupting a novel nodule-specific gene annotated as an iron transporter. Phylogeny analysis of the encoded protein, now known as M. truncatula Vacuolar iron Transporter-Like 8, MtVTL8, shows that it is a homolog of the Saccharomyces cerevisiae vacuolar iron/manganese transporter CCC1 protein, Arabidopsis thaliana vacuolar iron transporter VIT1 protein, and Lotus japonicus SEN1 protein, required during nodule formation in L. japonicus. The mutation in MtVTL8 co-segregates with the Fix− phenotype and mutants show distinctive similarities with a deletion mutant in the A17 genetic background that includes the MtVTL8 gene. This evidence demonstrates that the defect in NF11322 in MtVTL8 is the causative mutation in this mutant line. MtVTL8 encodes an iron transporter, implicated directly in transporting iron to the internalized rhizobia within nodule infected cells and essential for symbiotic nitrogen fixation.