Scientific issues
The symbiosis of rhizobia and leguminous plants to form nodules is the key to achieve symbiotic nitrogen fixation, but the cell types and molecular regulatory mechanisms of definitive nodule development and biological nitrogen fixation in leguminous plants are still unclear.
Design of Experiments
Single-cell nuclear transcriptome (snRNA-seq): Soybean seedlings were inoculated with rhizobia, and samples were taken after 14 days, and the samples were divided into N groups (containing nodules and roots) and R groups (containing roots only);
Conventional transcriptomes: N and R groups;Knockout of the roots and nodules of mutant plants GMCRE1S and wild-type (WT) plants that knock out the cytokine receptor gene.
Key results of the study
1. Construction of snRNA-seq transcription map of soybean root nodules
Data were obtained from 23,063 cells in group N and group R and 19,712 cells, respectively, and gene expression levels were highly correlated with the common transcriptome, demonstrating the reliability and accuracy of snRNA-seq data. A total of 21 cell clusters were identified by snRNA-seq, among which 20 were nodule-specific cells, and most of the known nodule genes were specifically expressed in these three clusters, which further indicated that the three clusters contained nodule-specific cell types, indicating that soybean nodule-specific cells were successfully captured by snRNA-seq. 
Figure 1 Identification of 21 cell clusters in soybean roots and nodules by snRNA-seq
2. Identification of nodule-specific cell typesThe cell types were identified by GUS staining, and 21 cell clusters were completely annotated into 17 different cell types, among which 6 types were unique to soybean nodules, including mesocolumn-related procambium cells, phloem-related meso-columnar cells, endocortex, outer cortex, non-infected cells, and infected cells. 
Fig.2 Cell type annotation of 21 clusters in roots and nodules
3. Metabolic process of soybean stereotyped nodulesSoybean (stereotyped) and alfalfa (amorphous) nodules differ in structure, nitrogen-fixing products, and bacteroides. Through the enrichment of high-expression genes in soybean and alfalfa root nodules, the differences in metabolism and development between the two root nodule types were further improved, and it was found that the metabolic processes of acylurea dependence in the two nodules were different. 
Fig.3 Comparison of the two root transcriptomes
4. Reconstruction of soybean nodule-specific cell differentiation trajectoriesRNA Velocity was used to reconstruct the differentiation process of soybean nodules, and two new genes were identified, namely GmbHLH93 and GMSCL1, which were used to verify their functions in the process of nodule formation by using the hairy root overexpression system. 
Figure 4 Utilization of RNAvelocityReconstruction of soybean nodulation trajectories and identification of driver genes
5. The cytokine receptor gene gmcre1 plays an important role in the process of nitrogen fixation in soybean nodulationBecause the genes that are highly expressed in soybean nodules are mainly enriched in "cytophrein signaling", by focusing on this pathway, it was found that the cytol** receptor gmcre1a b c d was highly expressed in the endothelial layer of root nodules and vascular cells. The role of GMCRE1 in the formation of soybean nodules was verified by constructing the variant plant GMCRE1s.
Fig.5 Study of genes related to cytosin signaling pathway during nitrogen fixation in soybean nodulation
Conclusion
In this study, the intrinsic mechanism of nitrogen fixation in soybean was analyzed by using single-cell nuclear transcriptome, six nodule-specific cell types were identified, and the important role of cytosin receptor gmcre1 in root nodule development and nitrogen fixation was determined, which determined a new direction for the study of the nitrogen fixation mechanism of rhizobia in leguminous plants.