The establishment of symbiotic interactions between leguminous plants and rhizobia requires complex cellular programming activated by Rhizobium Nod factors (NFs) as well as type III effector (T3E)-mediated symbiotic signaling. However, the mechanisms by which different signals jointly affect symbiosis are still unclear. Here we describe the mechanisms mediating the cross-talk between the broad host range rhizobia Sinorhizobium fredii HH103 T3E Nodulation Outer Protein L (NopL) effector and NF signaling in soybean. NopL physically interacts with the Glycine max Remorin 1a (GmREM1a) and the NFs receptor NFR5 (GmNFR5) and promotes GmNFR5 recruitment by GmREM1a. Furthermore, NopL and NF influence the expression of GmRINRK1, a receptor-like kinase (LRR-RLK) ortholog of the Lotus RINRK1, that mediates NF signaling. Taken together, our work indicates that S. fredii NopL can interact with the NF signaling cascade components to promote the symbiotic interaction in soybean.

In some legume–rhizobium symbioses, host specificity is influenced by rhizobial nodulation outer proteins (Nops). However, the genes encoding host proteins that interact with Nops remain unknown. We generated an Ensifer fredii HH103 NopP mutant (HH103ΩNopP), and analyzed the nodule number (NN) and nodule dry weight (NDW) of 10 soybean germplasms inoculated with the wild-type E. fredii HH103 or the mutant strain. An analysis of recombinant inbred lines (RILs) revealed the quantitative trait loci (QTLs) associated with NopP interactions. A soybean genomic region containing two overlapping QTLs was analyzed in greater detail. A transcriptome analysis and qRT-PCR assay were used to identify candidate genes encoding proteins that interact with NopP. In some germplasms, NopP positively and negatively affected the NN and NDW, while NopP had different effects on NN and NDW in other germplasms. The QTL region in chromosome 12 was further analyzed. The expression patterns of candidate genes Glyma.12g031200 and Glyma.12g073000 were determined by qRT-PCR, and were confirmed to be influenced by NopP.

Soybean is a legume crop rich in protein and oil. Symbiotic nitrogen fixation plays an important role in the growth of soybean. The type-III effectors such as NopT and NopP are the important signaling factors for the establishment of symbiosis in soybean. In this study, the analysis of nodulation in soybean after inoculation with HH103ΩNopT, HH103ΩNopP, and HH103ΩNopT&NopP indicated crosstalking between NopT and NopP. Further, we aimed to identify the genes of soybean involved in the pathway underlying the crosstalk between NopT and NopP using RNA-seq analysis. Five of the identified candidate genes were confirmed to be induced by NopT and NopP. The expression of GmPBS1 significantly increased to a much larger extent than that of the other four genes after soybean was inoculated with HH103ΩNopT, HH103ΩNopP, or HH103ΩNopT&NopP. The interaction between NopT and GmPBS1 was confirmed via bimolecular fluorescence complementation. Finally, nodulation analysis after GmPBS1 overexpression in the hairy roots indicate that GmPBS1 can regulate the negative effect of NopP on the nodulation, and this regulation is related to NopT. Collectively, our results suggested that during the nodulation in soybean, NopT and NopP have a crosstalking network and GmPBS1 is the hub gene.

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.

In soybean (Glycine max)-rhizobium interactions, the type III secretion system (T3SS) of rhizobium plays a key role in regulating host specificity. However, the lack of information on the role of T3SS in signaling networks limits our understanding of symbiosis. Here, we conducted an RNA sequencing analysis of three soybean chromosome segment substituted lines, one female parent and two derived lines with different chromosome-substituted segments of wild soybean and opposite nodulation patterns. By analyzing chromosome-linked differentially expressed genes in the substituted segments and quantitative trait loci (QTL)-assisted selection in the substituted-segment region, genes that may respond to type III effectors to mediate plant immunity–related signaling were identified. To narrow down the number of candidate genes, QTL assistant was used to identify the candidate region consistent with the substituted segments. Furthermore, one candidate gene, GmDRR1, was identified in the substituted segment. To investigate the role of GmDRR1 in symbiosis establishment, GmDRR1-overexpression and RNA interference soybean lines were constructed. The nodule number increased in the former compared with wild-type soybean. Additionally, the T3SS-regulated effectors appeared to interact with the GmDDR1 signaling pathway. This finding will allow the detection of T3SS-regulated effectors involved in legume-rhizobium interactions.