Abstract

Nitrogen is vital for all living organisms. Even though it is the most abundant gas in the atmosphere, organisms cannot use free nitrogen from the air for their metabolism. Legumes can overcome this nitrogen limitation through a symbiotic relationship with soil bacteria (Rhizobium spp.). Sinorhizobium meliloti can convert inert nitrogen to ammonium and nitrate with nitrogenase enzyme activity in a specialized root organ named ‘nodule.’ In the presence of low nitrogen concentration, some signal exchange triggers infection thread formation, where bacteria invade plant cells. When the bacteria enter the plant cell through infection thread, it surrounds by plant cell membrane. Then, this bacteroid structure differentiates to a symbiosome, which can fix nitrogen. Different factors and peptides highly regulate the differentiation processes. More than 600 NCR peptides were characterized in the Medicago truncatula plant, and moved NCR peptides and other factors from ER to symbiosome by the DNF1-dependent nodule-specific protein secretory pathway.

DNF1 protein is located on the ER membrane, a subunit of a signal peptidase complex responsible for cleaving N-terminal signal sequences of differentiation-related cargo proteins to direct them to the symbiosome. In dnf1 mutants, it is shown that the transportation of those proteins stops, and those proteins are trapped and accumulate in the ER. Also, microscopic analysis indicates that bacteroids cannot develop as symbiosomes and are arrested at early stages of differentiation. So, it is crucial to elucidate other proteins that have roles in the dependent nodule-specific protein secretory pathway. However, using classical protein-protein interaction (PPI) methods have several limitations to performing highly sensitive analysis: inefficient extraction, purification steps, reaction condition differences between in vivo and in vitro, and difficulties of capturing weak and transient interactions. Enzyme-catalyzed proximity labeling (PL) approaches overcome the limitations of those PPI methods. In the presence of a biotin substrate, an engineered biotin-ligase enzyme (miniTurbo, mT) can biotinylate proximal proteins close to the protein of interest. The stabilization of covalently attached biotins makes in-depth proteomic assays, such as immunoprecipitation and mass spectrometry, possible.

Here, we focused on identifying interaction partners of DNF1 in the symbiotic nitrogen fixation pathways using the mini turbo proximity labeling approach for the first time. Therefore, Medicago truncatula seedlings are infected by the Agrobacterium rhizogenes ARqua1 strain for hairy root transformation. Transgenic plants inoculated by Sinorhizobium meliloti for nodule initiation. Nodules were collected at 21 days post-inoculation and then treated with biotin. Biotinylated proteins are immunoprecipitated for in-depth proteomic analysis to identify interacted partner candidates of DNF1.