Two members of a Nodule‐specific Cysteine‐Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules

SUMMARY Legumes have evolved a nitrogen‐fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat‐Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host‐produced nodule‐specific cysteine‐rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen‐fixing symbiosis in M. truncatula. Significance Statement Our results demonstrate the requirement of NCR086/NCR314, which are members of the nodule‐specific cysteine‐rich peptide family, for effective symbiotic interaction between Medicago truncatula and the tested rhizobia strains. The NCR086/NCR314 peptides are encoded by a highly similar gene pair in M. truncatula A17 that was duplicated following the separation of lines M. truncatula A17 and Medicago littoralis R108.