Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

A bacterial membrane protein, BacA, protects Sinorhizobium meliloti against the antimicrobial activity of host peptides, enabling the peptides to induce bacterial persistence rather than bacterial death.

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In , this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.