Table 1

Primers used in this study"

Fig. 1

Amino acid sequence alignment (A) and phylogenetic tree (B) analysis of GmCYS2 with its homologs in some other plants The main cystatin conserved motifs are in yellow, red, green, and blue boxes in panel A. The scale in panel B represents genetic similarity, indicating the proximity relationships among species."

Fig. 2

Sequence analysis of His-GST-GmCYS2 fusion protein 5-10: His tag; 14-233: GST tag; 244-250: TEV cleavage site; 251-355: GmCYS2 protein sequence (Removing signal peptide of 26 amino acids at N end)."

Fig. 3

Expression, antibody detection and purification of GmCYS2 protein in vitro A: SDS-PAGE analysis of protein expression in BL21 (DE3) strain; B: Western blot analysis of protein with anti-His antibody; C: SDS-PAGE analysis of purified protein. M1: protein marker; M2: Western marker; M3: protein pre staining marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Non induced whole cell lysate. 1: Whole cell lysate induced under 15°C for 16 h; 2: Whole cell lysate induced under 37°C for 4 h; NC1: Supernatant of non-induced cell lysate; NC2: The precipitation of non-induced cell lysate; 3: Supernatant of cell lysate induced under 15°C for 16 h; 4: The precipitation of cell lysate induced under 15°C for 16 h; 5: Supernatant of cell lysate induced under 37°C for 4 h; 6: The precipitation of cell lysate induced under 37°C for 4 h; 7: Purified protein from supernatant of cell lysate induced under 37°C for 4 h."

Fig. 4

Inhibitory activity of the recombinant protein GmCYS2 in extracts from nodules in different periods in vitro Samples were extracts of 30-day- and 60-day-old nodules. Reaction buffer added with purified GmCYS2 protein and the specific substrates cathepsin L-like, B-like, and H-like, respectively. Values are triplicate measurements. * P < 0.05."

Fig. 5

Effect of GmCYS2 overexpression on symbiosis in L. japonicus A: Transgenic plants at 30 days after inoculation with MAFF303099 showing hairy roots expressing pMUb: GmCYS2 (GmCYS2-OX) or the empty vector pU1301 (CK). Bar = 10 mm; B: Mean numbers of nodules per plant of L. japonicus; C: The relative ratios of various groups divided by nodule number per plant; D: GUS identification of positive hairy roots; E: Detection of GmCYS2 gene expression in positive hairy roots by RT-PCR; M: DL2000 DNA marker; 1: plasmid as positive control; 2: empty vector control; 3-5: composite plants."

Table 2

Statistics of nodule number of composite L. japonicus overexpressing GmCYS2"

Fig. 6

Detection of transcription levels of GmCYS2 and symbiotic marker genes in transgenic hairy roots qPCR analysis of the transcript levels of GmCYS2 and symbiotic marker genes NIN, ENOD40-1, and ENOD40-2 in the GmCYS2-OX and control hairy roots. * P<0.05, ** P<0.01."