Symbiotic nitrogen fixation provides most of the nitrogen required for soybean growth. Rhizobial nodulation outer proteins (Nops) have been reported to influence host specificity during symbiosis establishment. However, the host proteins that interact with Nops remain unknown. In this study, we generated Sinorhizobium fredii HH103 mutants (HH103ΩNopL, HH103ΩNopT, and HH103ΩNopLΩNopT) and analysed the nodule number (NN) and nodule dry weight (NDW) of 12 soybean germplasms after inoculation with wild-type S. fredii HH103 or the mutant strains. The analysis of chromosome segment substitution lines revealed quantitative trait loci (QTLs) associated with NopL and NopT interactions. A total of 22 QTLs for the 2 nodule traits were detected and mapped to 12 different chromosomes in the soybean genome. Eight and fifteen QTLs were found to be associated with NN and NDW, respectively. Furthermore, 17 candidate genes were selected for further analyses. Considering the results of reverse-transcription quantitative PCR, we propose that the protein products of these 17 candidate genes interact with NopL and NopT.

Soybean is a cereal crop with high protein and oil content which serves as the main source of plant-based protein and oil for human consumption. The symbiotic relationship between legumes and rhizobia contributes significantly to soybean yield and quality, but the underlying molecular mechanisms remain poorly understood, hindering efforts to improve soybean productivity. In this study, we conducted a transcriptome analysis and identified 22 differentially expressed genes (DEGs) from nodule-related quantitative trait loci (QTL) located in chromosomes 12 and 19. Subsequently, we performed functional characterisation and haplotype analysis to identify key candidate genes among the 22 DEGs that are responsive to nitrate. Our findings identified GmTCP (TEOSINTE-BRANCHED1/CYCLOIDEA/PCF) and GmNLP (NIN-LIKE PROTEIN) as the key candidate genes that regulate the soybean nodule phenotype in response to nitrogen concentration. We conducted homologous gene mutant analysis in Arabidopsis thaliana, which revealed that the homologous genes of GmTCP and GmNLP play a vital role in regulating root development in response to nitrogen concentration. We further performed overexpression and gene knockout of GmTCP and GmNLP through hairy root transformation in soybeans and analysed the effects of GmTCP and GmNLP on nodulation under different nitrogen concentrations using transgenic lines. Overexpressing GmTCP and GmNLP resulted in significant differences in soybean hairy root nodulation phenotypes, such as nodule number (NN) and nodule dry weight (NDW), under varying nitrate conditions. Our results demonstrate that GmTCP and GmNLP are involved in regulating soybean nodulation in response to nitrogen concentration, providing new insights into the mechanism of soybean symbiosis establishment underlying different nitrogen concentrations.

Large amounts of nitrogen fertilizer are annually applied to improve soybean yield. However, the overuse of nitrogen fertilizers has resulted in significant environmental pollution. Biological nitrogen fixation is an ecological and environmentally friendly method to increase soybean yield. However, the signaling pathway and function of genes in the plant host regulated by rhizobia under the symbiotic relationship remain unknown. In this study, the chromosome region in soybean responsive to Sinorhizobium fredii HH103 was identified using chromosome-segment-substituted lines produced from a cross between cultivated soybean SN14 and wild soybean Zyd00006. In addition, candidate genes associated with nodulation traits and regulated by S. fredii HH103 were identified. In total, three quantitative trait loci (QTLs) containing 68 genes were identified on chromosomes 02, 05, and 20. The differentially expressed genes among the QTL regions were determined using RNA-seq and qRT-PCR. Glyma. 05G240500, a potential gene responding to S. fredii HH103 and positively regulating soybean nodulation, was identified. To explore the relationships between haplotypes and soybean nodulation phenotypes, SNPs found in the regulatory areas of Glyma.05G240500 haplotype were investigated. Our study revealed the role of Glyma.05G240500 in symbiosis and provided a reference for facilitating symbiotic nitrogen fixation in the field and for marker-assisted selection.