Post-Doctoral Associate University of Florida Gainesville, Florida
Single-cell RNA sequencing has revolutionized the investigation of transcriptomes by allowing the definition of gene expression in each cell type that constitutes a tissue or organ. In this study, we applied single-nuclei RNA sequencing (snRNA-seq) to investigate the transcriptional response of Medicago truncatula roots to Sinorhizobium meliloti infection. Roots were harvested at four-time points, control (0h), 24h, 48h, and 96h after infection, using two genotypes: Jemalong A17 and the hypernodulation mutant sunn-4. The datasets were integrated, aggregating 36,868 nuclei in 29 clusters, which were annotated based on cell type markers. Significant changes were observed in the population of cells in response to the infection, including the appearance of clusters over time. These changes were more pronounced in sunn-4, which showed the enrichment of cells responding to the infection. We characterized the transcriptional changes related to the root hair infection and the pericycle and cortex response to S. meliloti, including cortical cells originating the nodule meristem and the infected cells of the developing nodule. Pseudotime analysis reconstructed the developmental trajectories of those cell types by ordering them according to their transcriptional states and identifying genes that potentially govern their development. The high resolution obtained by snRNA-seq allowed the dissection of the expression of genes involved in root nodule symbiosis (RNS), pinpointing their transcription to specific cell types at each time point. Our dataset revealed that members of well-established gene families have distinct expressions, and their expression in specific cell types may reveal the genes in each family that were co-opted for RNS. For example, the knockdown of a member of the STYLISH family, specifically expressed in cells responding to the infection, confirmed its involvement in RNS. Experimental validation of other genes is in progress and may reveal new players governing the transcriptional changes necessary for the establishment of root nodule symbiosis.