The Single-Cell Transcriptome Program of Nodule Development Cellular Lineages in Medicago truncatula

Legumes can establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules after root exposure to lipo-chito-oligosaccharides secreted by the bacteria. Nodule development initiates with anticlinal mitotic divisions in the pericycle and endodermal and inner cortical cells, establishing cell lineages that ultimately form each nodule compartment. We characterized these lineages by isolating and sequencing the transcriptome of Medicago truncatula single nuclei derived from uninoculated roots and roots undergoing early nodule development at 24, 48, and 96 hours after inoculation. To enrich samples for cells responding to the rhizobia, we complemented the analysis of the Medicago wild-type genotype A17 with a mutant for the autoregulation of nodulation, sunn-4. Analysis of cell lineage trajectories derived from the cortex indicates that their transcriptome is initially enriched for cytokinin perception and signaling while repressing auxin accumulation. As these cells differentiate to form nodules, expression of genes related to auxin biosynthesis, transport, and signaling was enhanced, while genes involved in cytokinin degradation were activated as lineages bifurcated to form the nodule meristem and infection zones. While the contribution of auxin and cytokinin in nodule development has been recognized, this single-cell resource quantifies the expression of each of their regulators, receptors and targets as cells divide and differentiate to form each nodule compartment.