Fig. 1

Construction and auto-activation test of the bait plasmid pGBKT7-GmNFR1α-pk (A) M: trans 2K Plus II DNA marker; 1: isolation of GmNFR1α-pk cDNA; 2: pGBKT7-GmNFR1α-pk digested by EcoR I and Sal I; 3: pGBKT7 digested by EcoR I and Sal I. (B) positive control (pGBKT7-53 and pGADT7-T). (C) negative control (pGBKT7-lam and pGADT7-T). (D) pGBKT7-GmNFR1α-pk and pGADT7."

Fig. 2

Screening of yeast two-hybrid cDNA library (A) positive clones on QDO/X/A plate; (B) PCR detection of inserts from yeast two-hybrid cDNA library."

Fig. 3

Interaction between GmNFR1α and GmLbc2 (A) yeast two-hybrid analysis of interaction between GmNFR1α-pk and GmLbc2. 1: positive control (pGBKT7-53 and pGADT7-T); 2: negative control (pGBKT7-lam and pGADT7-T); 3: pGBKT7-GmNFR1α-pk and pGADT7-GmLbc2. (B) BiFC analysis of interaction between GmNFR1α and GmLbc2."

Fig. 4

Amino acid sequence alignment analysis of GmLbc2 and its homologous proteins in some other plants Gm: Glycine max; Gs: Glycine soja; Cc: Cajanus cajan; Va: Vigna angularis; Vr: Vigna radiata; Mt: Medicago truncatula; Lj: Lotus japonicus; As: Astragalus sinicus."

Fig. 5

Phylogenetic tree of GmLbc2 and its homologs The scale represents genetic similarity, indicating the proximity relationships among species. Abbreviations are the same as those given in Fig. 4."

Fig. 6

Effect of GmLbc2 overexpression on nodulation in L. japonicus (A) phenotype of hairy roots expressing p1301U (CK). (B) phenotype of hairy roots overexpressing GmLbc2 (GmLbc2-OX); photographs were taken at 30 d after inoculation, Bars = 5 mm. (C) mean number of nodules per plant with standard deviation (SD) of L. japonicus expressing empty vector pU1301 (CK) of GmLbc2-OX at 30 days after inoculation with M. loti, **P<0.01. (D) transcript levels of GmLbc2, NIN, Enod40-1, and Enod40-2 in CK and GmLbc2-OX hairy roots detected by qRT-PCR."