Investigating the interactions between Nodule-specific Cysteine Rich peptides and bacterial proteins in legume-rhizobia symbiosis

Abstract

The symbiotic relationship between the legume Medicago truncatula and the soil bacterium Sinorhizobium meliloti is mediated by a class of disulfide-containing peptides called NCR (Nodule-specific Cysteine-Rich) peptides. Once the bacteria enter the nodule cells of the root, the plant releases these peptides, which then bind to its bacterial targets and cause terminal differentiation of the bacteria into a nitrogen-fixing ‘bacteroid’ form. While some information is known about what peptides are essential for symbiosis, little is known about how these peptides control the metabolism and growth of their target symbiont. This project aimed to elucidate the interactions between the NCR peptides and their target bacterial proteins. An AlphaFold Multimer screen was the first step to shortlist candidate NCR peptide-bacterial protein interactor pairs. The yeast surface display technique, which involves making a fusion of the peptide to an outer-cell wall protein of yeast, is used in conjunction with flow cytometry, to look for overlapping signals from peptide and potential target protein and hence verify these computational findings in the wet lab. A yeast two-hybrid assay was also developed to screen more interactions in an unbiased proteome-wide manner. Lastly, expression systems ranging from bacterial, fungal, and cell-free expression were tested and optimized for expression and purification of NCR peptides for more direct protein-protein interaction studies. Once elucidated, the knowledge of interactions involving antimicrobial NCR peptides can be harnessed to develop novel therapeutic strategies to aid the antimicrobial resistance crisis which is directly responsible for an estimated 4.95 million documented deaths in total.