Fig. 7: GmRR11d acts as a downstream regulator of GmNARK in soybean.

a, b The expression of GmRR11d in roots a and nodules b of wide-type cv Bragg and GmNARK mutant nts1007. Seven-day-old-plants were inoculated with USDA110. The roots were harvested at 0, 1, 3, 5, and 10 DAI and the nodules were harvested at 6, 8, 10, 12, 14, 16, 18, and 25 DAI. Data are presented as means ± SD from three biological repeats. More than 9 samples were analyzed in three independent biological repeats. Asterisks indicate significant differences relative to the wild type control. Two-sided Student’s t-test, *p < 0.05; p** < 0.01; ***p < 0.001. c The expression of GmRR11d in shoots and roots of Bragg/Bragg, Bragg/nts1007, nts1007/nts1007 and nts1007/Bragg grafting plants. Data are presented as means ± SD from three biological repeats and 6 samples were collected for expression analysis. Different letters indicate significant differences at P < 0.05 (One-way ANOVA). d The expression of GmRR11d in split-roots of Bragg and nts1007. -R/-R and +R/ + R treatment as negative and positive control. Data are presented as means ± SD from three biological repeats and 12 roots were collected for expression analysis. Different letters indicate significant differences at p < 0.05 (Two-way ANOVA) e qRT-PCR analysis of GmRR11d in empty vector (EV1) and 35 S:GmRR11d transgenic hairy roots of Bragg and nts1007 plants. Data are presented as means ± SD from three biological repeats and more than 10 roots were collected for expression analysis Asterisks indicate significant differences relative to the EV1 control. Two-sided Student’s t-test, **p < 0.01. f Nodule number of Bragg and nts1007 plants expressing EV1 and 35 S:GmRR11d at 21 DAI. Data are presented as means ± SD from three biological repeats and more than 10hairy roots were collected for analysis. Different letters indicate significant differences at p < 0.05 (Two-way ANOVA). g Phenotypes of nodules from individual hairy roots of Bragg and nts1007 plants expressing EV1 and 35 S:GmRR11d at 21 DAI. h BAP treatment increases the enrichment of GmRR11d in the promoter of GmNIN1a. DNA fragments were co-immunoprecipitated with anti-FLAG antibodies from chromatin suspensions prepared from 35 S:GmRR11d-FLAG or control (EV1) samples treated with or without 10⁻⁷ M BAP. The DNA fragments were normalized to the input data. Data are presented as means ± SD from three biological repeats. Different letters indicate significant differences at p < 0.05 (one-way ANOVA). i A proposed working model for GmRR11d-mediated nodulation inhibition. At low nitrogen conditions, rhizobia infection induces GmNSP1a to activate GmNIN1a that promotes nodulation and autoregulation of nodulation (AON). At the early infection stage, the level of GmRR11d is low and CK level is suitable for nodulation, GmNSP1s complexes with GmNSP2 to activate GmNIN expression; activation of AON induces expression of B-type regulator GmRR11d which interacts with GmNSP1a and suppresses its transcriptional activation of GmNIN1a. Meanwhile, AON-induced CK accumulation enhances binding of GmRR11d to the GmNIN1a promoter to repress its expression induced by CK and to activate CK signaling, thereby inhibiting nodulation of soybean.