Deciphering How Plant Peptides Control the Metabolism of Symbiotic Bacteria through Protein Centric Approach.

Biological Nitrogen Fixation (BNF) is a product of a symbiotic relationship between nitrogen-fixing soil bacteria and leguminous plants, where the bacteria provide the plant with fixed nitrogen, and the plant provides carbon sources for the bacterium. Intriguingly, in the legumes belonging to the inverted-repeat lacking clade (IRLC), such as alfalfa, the host turns the bacteria into terminally differentiated bacteroids. These differentiated bacteroids display features like polyploidy, cell elongation, and cell division arrest. This terminal bacteroid differentiation (TBD) transforms rhizobia into highly efficient nitrogen-fixing machinery, yet it also renders them incapable of surviving outside the host. The plant achieves this extreme control over the bacteria by secreting an arsenal of small peptides known as Nodule-specific Cysteine Rich (NCR) peptides that target the bacteria within the nodules. However, despite the significance, the mechanism by which these peptides drive TBD is still unknown and leaves an intriguing gap in the field. As a first step to understanding how NCR peptides modulate the metabolism of bacteria, we performed a large-scale in silico AlphaFold Multimer analysis to identify the interacting protein partners of the peptides. Then, we utilized a yeast display screen to validate these interactions and identified many interesting targets in bacterial proteins, including toxins, proteases, and important symbiotic proteins. We are now performing biochemical and genetic characterization of these interactions. Ultimately, this research will enhance our understanding of how legume peptides control symbiotic bacteria and how this can be exploited for improving nitrogen fixation and, ultimately, plant growth.