Differentiation trajectories and biofunctions of symbiotic and un-symbiotic fate cells in root nodules of Medicago truncatula

The root nodule is a complex symbiotic nitrogen fixation factory,in which cells are highly heterogeneous.However,the differentiation trajectories and interconnection of nodule cells remain largely unknown.In this study,we set up a modified protocol for nodule protoplast preparation and performed a single-cell RNA sequencing profiling of the indeterminate Medicago truncatula nodule.We designated 13 cell clusters with specific expression patterns in 14-day post inoculation nodules and constructed a spatial and functional cellular map based on experimental data and bioinformatic analyses.Pseudotime analysis further revealed that two groups of apical meristematic cells develop into symbiotic and un-symbiotic fate cells along their particular trajectories.Biofunction analysis of each cell cluster revealed their particularity and interrelation,especially that the un-infected cells in nitrogen fixation zone are also involved in nitrogen assimilation by undertaking the asparagine synthesis.Collectively,our data offer an important resource for investigating the mechanism of nodule organogenesis and symbiotic nitrogen fixation.