GmTNRP1, associated with rhizobial type-III effector NopT, regulates nitrogenase activity in the nodules of soybean (Glycine max)

Soybean is a rich source of protein and oil for humans. It forms a symbiotic association with rhizobia to fix atmospheric nitrogen for its growth and development. Rhizobial type-III effectors act as important signaling molecules in symbiosis. However, they are poorly studied. In this study, mutation of TtsI in Sinorhizobium fredii HH103 reduced nodule numbers and dry weights in SN14 (cultivated soybean) and ZYD00006 (wild soybean). Moreover, it reduced nitrogenase activity in SN14 but not in ZYD00006. Based on the differences in nitrogenase activity between SN14 and ZYD00006 inoculated with HH103 and the TtsI mutant, two conditional quantitative trait loci associated with nitrogenase activity were identified in chromosome segment substitution lines derived from SN14 and ZYD00006. Using transcriptome analysis, single-nucleotide polymorphism (SNP) analysis, and qRT-PCR, Glyma.13G150000 (GmTNRP1) was identified as a candidate gene in soybean that was associated with nitrogenase activity, along with NopT (a type-III effector). In addition, GmTNRP1 (an LRR-RK family protein located on the cell membrane) was observed to be induced by NopT to negatively regulate nitrogenase activity. In transgenic hairy roots with GmTNRP1 silencing, no significant difference was observed in terms of nitrogenase activity between nodules inoculated with the NopT mutant and those inoculated with HH103 (wild type). However, the nitrogenase activity in the nodules decreased after overexpression of GmTNRP1. SNP located in the GmTNRP1 promoter affected its expression in response to NopT. Haplotype analysis revealed that SN14 belonged to Hap1 and ZYD00006 belonged to Hap2, which explained the different results in the two varieties. To the best of our knowledge, this is the first study to identify a soybean gene associated with a rhizobial type-III effector in the nodules. GmTNRP1 can negatively regulate nitrogenase activity in the nodules. This study enhanced the understanding of the mechanism underlying symbiotic association between soybean and rhizobia.