Fig. 5: GmRR11d directly targets the promoter of GmNIN1a and represses GmNIN1a expression.

a qRT-PCR analysis of GmNIN1a and GmENOD40-1 expression in rhizobia-infected hairy roots expressing 35 S:GmRR11d or RNAi-GmRR11d at 2 DAI. The expression levels were normalized against the geometric mean of soybean GmELF1b. Data are presented as means ± SD from three biological replicates. More than 12 roots were analyzed in three independent biological repeats. Asterisks indicate significant differences relative to the EV1 or EV2 control. Two-sided Student’s t-test, *p < 0.05; **p < 0.005. b–d GUS assays showing the specific binding of GmRR11d to the GmNIN1a promoter and the inhibitory effect of GmRR11d on GmNIN1a in N. benthamiana leaf (b, c; Scale bars = 1 cm) and in transgenic soybean hairy root (d; Scale bars = 100 μm). The proGmNIN1a and proGmNIN1a-DM contain normal (GGATT) and mutated (AAAAA) GmRR11d binding sites in GmNIN1a promoter, respectively. The construct harboring proGmNIN1a:GUS or proGmNIN1a-DM was co-transformed with empty vector (EV1) or 35 S:GmRR11d into epidermal cells of N. benthamiana leaves and transgenic hairy roots of soybean, and GUS staining were performed 48 h after transformation in N. benthamiana leaves and 2 DAI in transgenic hairy roots. Data are presented as means ± SD from three biological replicates. Different letters indicate significant differences at p < 0.05 (one-way ANOVA). e Diagram of the GmNIN1a promoters. GmRR11d binding site (GGATT) shown as purple boxes. The fragments marked by the letters A to G indicate the regions examined in ChIP-qPCR assays. Region A contains two GmRR11d binding sites. f ChIP-qPCR assay showing binding of GmRR11d to the GmNIN1a promoter. DNA fragments were co-immunoprecipitated with anti-FLAG antibody from chromatin suspensions prepared from 35 S:GmRR11d-FLAG or empty vector (EV1) samples. DNA fragments corresponding to the regions indicated in e were analyzed by qRT-PCR. The DNA fragments were normalized to the input data. Data are presented as mean ± SD of three biological repeats. Asterisks indicate significant differences relative to the EV1 control. Two-sided Student’s t-test, **p < 0.01.