Characterizing the molecular interactions of Nodule-specific Cysteine Rich peptides with bacterial proteins and DNA in legume-rhizobia symbiosis

Abstract

The symbiosis between the legume Medicago sativa and the soil bacterium Sinorhizobium meliloti is regulated by Nodule-specific Cysteine Rich (NCR) peptides, a family of plant-derived disulfide-containing peptides. Following bacterial infection of root nodule cells, these peptides drive terminal bacterial differentiation into polyploid, elongated, branched ‘bacteroid’ form specialized for atmospheric nitrogen fixation. Although a few NCRs are known to be essential for symbiosis, the mechanisms through which they modulate bacterial physiology and metabolism remain largely elusive. This project aimed to characterize NCR peptide interactions with bacterial proteins, and their potential to directly bind DNA to uncover functional consequences on bacterial and plant phenotypes. AlphaFold Multimer predicted peptide-protein interactions were validated using biochemical, genetic, and biophysical approaches, including yeast display assay, yeast two-hybrid assay, and biolayer interferometry (BLI). Cationic NCRs and bacterial protein interactions were tested using BLI, isothermal titration calorimetry (ITC), and mass photometry. Crosslinking mass spectrometry was used to generate an unbiased proteome-wide map of in vivo interactions between NCR peptides and bacteroid proteins. NCR-DNA interactions were also examined via gel-based retardation assays, BLI, circular dichroism and mass photometry. Finally, to assess the biological significance of these interactions, the putative symbiotic protein FixU was targeted for genetic modification. S. meliloti fixU knockout and overexpression strains were analyzed using growth curve assay in the presence and absence of NCR peptides. The fixU knockout strain was further evaluated in planta by inoculation into M. sativa, and plant phenotypes were compared with those inoculated with wild-type bacteria. NCR peptides effect on S. meliloti thioredoxin (TrxA) activity was assessed using a fluorometric assay monitoring the reduction of eosin-labeled insulin disulfides. These findings collectively provide insights into NCR-mediated host manipulation of bacteria, with implications for optimizing biological nitrogen fixation, improving legume crop productivity, and exploring NCR peptides as novel antimicrobial therapeutics.