Identification And Application Of Key Genes Of Symbiotic Interaction Of Soybean Rhizobia By Single-Cell RNA Sequencing

Soybean is the most economically important legume worldwide,and is also an important foundational and strategic material related to the national economy and people’s livelihood.China is a major consumer and importer of soybeans,with a high degree of external dependence.Therefore,high soybean yield has become a major national demand.Low yield per unit area is a "bottleneck" problem in soybean production in China.Symbiotic nitrogen fixation can provide more than 65% of nitrogen for soybeans and is also one of the determining factors for soybean high yield.Research showed that nodule number and mass,as well as seed yield,increased curvilinearly upward with increasing microbial population size of Bradyrhizobium japonicum.However,more nodules are not always correlated with yield increases,since the rhizobia fix nitrogen for their host plant in exchange for carbohydrates,had certain antagonism.Thus,understanding the molecular mechanism underlying this specialized symbiotic relationship would suggest strategies to improve soybean production,while reducing synthetic nitrogen(N)fertilizer inputs in agriculture systems.Symbiotic nitrogen fixation is a cost-effective and environmentally friendly means to acquire substantial N in legumes,however,the identification of key genes responsible for symbiosis establishment and SNF in soybean lags that in the model plants Medicago truncatula and Lotus japonicas due to its genome complexity.Here,we conducted transcriptome profiling of soybean root during the early stage of soybean-rhizobium interaction.Through single-cell RNA sequencing(sc RNA-seq)and fsiunctional verification and other molecular biological methods,the molecular mechanism of symbiosis between soybean and rhizobia was explored.The main research findings are as follows:(1)We performed deep sequencing analysis of mock-and rhizobia-inoculated soybean roots 4 days after inoculation,using single-cell RNA sequencing.We obtained data from5,083 cells,with 2,437 cells from control and 2,646 cells from treated group.(2)We profiled 5,083 cells and detected 12 major cell clusters,including three cortexand root hairy cell-specific clusters.We identified 1,898 rhizobia inoculation-responsive genes,of which 969 were specific to cortex and root hairy cell clusters.Subsequent high dimensional weighted gene co-expression network(hd WGCNA)and Gene Ontology(GO)enrichment analysis detected 69 decisive differentially expressed hub genes(DEHGs)and pathways such as carbohydrate metabolism,cell death,stress responses,and organismal development during SNF.(3)We overexpressed each gene individually in the soybean root hair system.Compared to plants transformed with empty vectors,overexpression of each target gene resulted in their higher expression level.Of the three genes tested,overexpressing GmNOD19 showed the most significant effect on nodule formation.We also detected a significant effect on nodule growth for two out of three repeats when overexpressing GmGER2.By contrast,we observed no significant difference when GmTIP1 was overexpressed compared to empty vector(EV)plants across all three repeats,although we confirmed the overexpression of the gene.Overall,our data suggest that both GmNOD19 and GmGER2 are functional genes that participate in SNF.That genes discovered in the present work will serve as candidates for further genetic functional analyses and the construction of regulatory networks to advance our knowledge of SNF in soybean.The screening of efficient nitrogen fixation genes is of great significance for improving the symbiotic nitrogen fixation efficiency of soybeans,laying a theoretical foundation for the breeding of efficient nitrogen fixation varieties in soybeans.