Deciphering the structure, localization and interaction partners of DNF2 in symbiotic nitrogen fixation

The nitrogen cycle relies on symbiotic nitrogen fixation. Although they are unable to use nitrogen directly, plants need nitrogen in the soil in forms that can be used by their metabolism. By establishing a symbiotic association with rhizobia, legumes get over this limitation. Communication between Medicago truncatula and Sinorhizobium meliloti triggers root hair curling and the formation of special organ called nodule, where bacteria produce ammonia and trade it with the host plant for carbon and reducing power. The nitrogen-fixing bacteria in legumes convert atmospheric nitrogen into usable forms of nitrogen, enabling high-yield agriculture. The molecular mechanism of symbiotic nitrogen fixation is being studied by identifying the interacting proteins of the DNF2 protein in Medicago truncatula. DNF2 protein, a phosphatidylinositol-specific phospholipase C enzyme, allows intracellular bacteria to survive within the plant by inhibiting the plant's immune system. However, limited knowledge exists regarding the proteins associated with DNF2 and its structure. Aim of the project is identifying the interacting proteins of DNF2 in Medicago truncatula, demonstrating their localization and purify it using the Tobacco protein expression system to elucidate 3D structure of the DNF2. For structural analysis, Nicotiana benthamiana transient expression system is optimized, nickel-NTA affinity chromatography is performed to purify DNF2 and X-ray diffraction method used to elucidate the 3D structure of DNF2 protein. To observe subcellular localization of DNF2, confocal laser scanning microscopy used and DNF2 protein was found to be localized in the apoplast. Finally, a mass spectrometry approach was used to identify the protein's interaction partners, and some protein candidates were identified. Elucidating the function and structure of DNF2 is an important step towards eliminating synthetic nitrogen fertilizers and sustainability in the future.