Soybean is an important source of plant protein and a key crop in cereal-legume intercropping systems. Unlocking its biological nitrogen fixation potential holds profound significance for promoting sustainable ecological agriculture. Our previous research identified a soybean apoptosis inhibitor, GmBI-1, which forms a heteroprotein complex with the soybean nodulation factor receptor GmNFR1α and plays a positive regulatory role during early rhizobial infection. This study aims to screen for proteins interacting with GmBI-1 using a yeast two-hybrid library and investigate their functions in the nodulation and nitrogen fixation process, thereby providing a theoretical basis for further elucidating the molecular regulatory network of symbiotic nitrogen fixation in soybean.

The yeast two-hybrid system was employed to screen an AD-cDNA library from soybean roots and root nodules, aiming to isolate and identify potential interacting proteins of GmBI-1. The identified interacting proteins were annotated and functionally predicted, followed by analysis of their tissue-specific expression. Taking one of the library proteins, GmNod44, as the research target, bioinformatics analysis was conducted on it. Further validation of the interaction between GmBI-1 and GmNod44 was performed using yeast two-hybrid retesting (Y2H) and tobacco in vivo luciferase complementation imaging (LCI) assays. Additionally, co-localization of GmBI-1 and GmNod44 proteins was observed in Arabidopsis protoplasts. Moreover, GmNod44 was overexpressed using Agrobacterium rhizogenes-mediated hairy root transformation in soybean to investigate its biological function in nodulation and nitrogen fixation.

Screening of the yeast two-hybrid library identified 18 potential interacting proteins of GmBI-1, including late nodulins, leghemoglobins, serine/threonine protein kinases, cysteine oxidases, cytidine triphosphate synthases, ubiquitin-conjugating enzymes, and other proteins related to immunity and post-translational modifications. Tissue expression analysis revealed that six genes exhibited relatively high expression levels in roots, while four genes showed higher expression in root nodules. Among them, GmNod44 was specifically and highly expressed in root nodules. Phylogenetic analysis indicated that soybean GmNod44 shares the highest homology with wild soybean GsNod22. GmNod44 and GmBI-1 were confirmed to interact in both yeast and tobacco in vivo assays and were co-localized to the endoplasmic reticulum in Arabidopsis protoplasts. Following overexpression of GmNod44, the number of nodules on composite soybean hairy roots significantly increased, and the expression levels of the interacting gene GmBI-1 and three nodulation marker genes-Apyrase GS52, Calmodulin, and Lb1-were significantly upregulated. Nitrogenase and reactive oxygen species (ROS)-scavenging enzyme activities in the nodules markedly rose, while cysteine protease activity significantly decreased. The above results indicate that overexpression of GmNod44 can promote soybean nodulation, enhance the nitrogen fixation capacity of nodules, and delay nodule senescence.

The late nodulin GmNod44 interacts with the apoptosis inhibitor GmBI-1 and positively regulates the process of nodulation and nitrogen fixation in soybean.