Single‐cell Transcriptome Profiling Reveals Gene Regulatory Networks and Key Genes in the Root Epidermis and Cortical Cells Associated with Early Nodulation in Glycine Max

The legume crop soybean forms a symbiosis with rhizobia to fix atmospheric nitrogen (N) in specialized organs called root nodules. However, the mechanisms regulating early infection of the root epidermis and nodule-primordium formation in the cortex for proper nodule formation remain unclear in soybean. Here, we report a single-cell transcriptome analysis of mock- and rhizobia-inoculated soybean roots at 4 days after inoculation, an important control point for autoregulation of nodulation and nodule-primordium formation. We profiled 21,500 cells and detected 12 major cell clusters, and identified 193 infected-cell-specific, 205 epidermis-specific and 180 cortex-specific DEGs. Gene-ontology enrichment and gene-regulatory network analyses uncovered key pathways such as reactive oxygen species-mediated hormone signaling involved in coordinating defense signaling and symbiotic pathways. We also identified and functionally validated an ethylene-activated circuit comprising GmWRKY6.3/6.4 transcription factors and select downstream GmNod19 targets, in which genes act as positive regulators by promoting infection-thread formation during early nodulation, thereby shaping nodule formation. This study showcases how single-cell transcriptomics and gene-regulatory networks provide hypotheses for identification and characterization of previously unappreciated regulatory circuits, broadens our understanding of precise genetic control underlying symbiosis establishment, and underscores how functional diversification of nodulation genes has occurred across legumes.