An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis

Abstract

In the nitrogen-fixing symbiosis between legume hosts and rhizobia, the bacteria are engulfed by a plant cell membrane to become intracellular organelles. In the model legume Medicago truncatula, internalization and differentiation of Sinorhizobium (also known as Ensifer) meliloti is a prerequisite for nitrogen fixation. The host mechanisms that ensure the long-term survival of differentiating intracellular bacteria (bacteroids) in this unusual association are unclear. The M. truncatula defective nitrogen fixation4 (dnf4) mutant is unable to form a productive symbiosis, even though late symbiotic marker genes are expressed in mutant nodules. We discovered that in the dnf4 mutant, bacteroids can apparently differentiate, but they fail to persist within host cells in the process. We found that the DNF4 gene encodes NCR211, a member of the family of nodule-specific cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that NCR211 acts to promote the intracellular survival of differentiating bacteroids. The greatest expression of DNF4 was observed in the nodule interzone II-III, where bacteroids undergo differentiation. A translational fusion of DNF4 with GFP localizes to the peribacteroid space, and synthetic NCR211 prevents free-living S. meliloti from forming colonies, in contrast to mock controls, suggesting that DNF4 may interact with bacteroids directly or indirectly for its function. Our findings indicate that a successful symbiosis requires host effectors that not only induce bacterial differentiation, but also that maintain intracellular bacteroids during the host-symbiont interaction. The discovery of NCR211 peptides that maintain bacterial survival inside host cells has important implications for improving legume crops.nitrogen-fixing symbiosis | Sinorhizobium meliloti | Medicago truncatula | legume | NCR antimicrobial peptides

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula

“…This similarity is in line with the observations that some synthetic cationic peptides (pI > 9.0) showed strong bactericidal activities when applied to the free-living bacteria (19,22,23). Synthetic NFS2 − peptide of A17, but not NFS2 + peptide of DZA315, also possessed a mild bactericidal activity causing bacterial cell death of Rm41 in vitro, as evidenced by a reduction of colony-forming units in plating assays (Fig.…”

“…5B). This observation suggests that the in vitro antimicrobial activity of the peptide is not necessarily correlated with its in planta function (22,23). The differential in vitro activity of the two peptides was also reflected by their effects on induction of membrane permeability (Fig.…”

“…This region encompasses, among other genes, Medtr8g465280, which encodes an NCR peptide (17). Because NCRs have been shown to be host effectors that mediate terminal bacteroid differentiation required for nitrogen fixation in the Medicago-Sinorhizobium symbiosis (18,19,(22)(23)(24), we postulated that Medtr8g465280 was a candidate gene of NFS2.…”

Ensifer glycinis sp. nov., a rhizobial species associated with species of the genus Glycine

“…Controversial debates on the priority and combination of Sinorhizobium and Ensifer persisted, and Sinorhizobium is still being used widely in unofficial journals of bacterial taxonomy (Blanquet et al, 2015;Kim et al, 2015;Li et al, 2016), especially in studies of the symbiotic relationship between rhizobia and legumes. To end the debate, the Judicial Commission of the International Committee on Systematics of Prokaryotes made a final decision that Ensifer takes priority over Sinorhizobium and should be used at least in official journals, such as the International Journal of Systematic and Evolutionary Microbiology (Young, 2010).…”

The evolution of determinate and indeterminate nodules within the Papilionoideae subfamily

“…Although Medicago has numerous NCR genes, individual NCRs (e.g. MtNCR169, MtNCR211) are essential for full bacteroid development, as mutations in these NCR genes results in incomplete bacteroid development (Horvath et al, 2015;Kim et al, 2015). Further, the introduction of the MtNCR035 gene into Lotus is sufficient to induce the formation of enlarged bacteroids (Van de Velde et al, 2010).…”