The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants’ needs.

In Rhizobium-legume symbiosis, the bacteria are converted into nitrogen-fixing bacteroids. In many legume species, differentiation of the endosymbiotic bacteria is irreversible, culminating in definitive loss of their cell division ability. This terminal differentiation is mediated by plant peptides produced in the symbiotic cells. In Medicago truncatula more than ∼700 nodule-specific cysteine-rich (NCR) peptides are involved in this process. We have shown previously that NCR247 and NCR335 have strong antimicrobial activity on various pathogenic bacteria and identified interaction of NCR247 with many bacterial proteins, including FtsZ and several ribosomal proteins, which prevent bacterial cell division and protein synthesis. In this study we designed and synthetized various derivatives of NCR247, including shorter fragments and various chimeric derivatives. The antimicrobial activity of these peptides was tested on the ESKAPE bacteria; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli as a member of Enterobacteriaceae and in addition Listeria monocytogenes and Salmonella enterica. The 12 amino acid long C-terminal half of NCR247, NCR247C partially retained the antimicrobial activity and preserved the multitarget interactions with partners of NCR247. Nevertheless NCR247C became ineffective on S. aureus, P. aeruginosa, and L. monocytogenes. The chimeric derivatives obtained by fusion of NCR247C with other peptide fragments and particularly with a truncated mastoparan sequence significantly increased bactericidal activity and altered the antimicrobial spectrum. The minimal bactericidal concentration of the most potent derivatives was 1.6 µM, which is remarkably lower than that of most classical antibiotics. The killing activity of the NCR247-based chimeric peptides was practically instant. Importantly, these peptides had no hemolytic activity or cytotoxicity on human cells. The properties of these NCR derivatives make them promising antimicrobials for clinical use.

Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as ief (Infection-related Epidermal Factor) in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene, is activated by the ERN1-branch of the Nod Factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development suggesting that combined action of these proteins plays a role in nodule infection.