Fig. 6

Interaction of NRSYM1 with CLE-RS2 and LjNIR1 promoters. a A schematic diagram of the location of DNA fragments used for ChIP-qPCR analyses and electrophoretic mobility shift assay (EMSA) in the 5 kb promoter regions of CLE-RS2 and LjNIR1. The numbering of fragments within each promoter region corresponds to those referenced in (b–e). (b, c) qPCR analysis to examine NRSYM1 binding with the (b) CLE-RS2 and (c) LjNIR1 promoter regions after ChIP. DNA fragments were co-immunoprecipitated with polyclonal anti-myc antibody from chromatin suspensions prepared from pLjUBQ:NRSYM1-myc roots that were incubated with 0 or 10 mM KNO₃ for 24 h without rhizobia. The PCR products were quantified by comparison with products amplified using primers specific to LjUBQ. The fold enrichment of nitrate-induced NRSYM1 binding was calculated as the ratio between +KNO₃ and –KNO₃-immunoprecipitated amplification signals. Error bars indicate SEM (n = 3 independent pools of roots). (d, e) EMSA showing NRSYM1-binding with the NRE/NBS of the CLE-RS2 and LjNIR1 promoter (Supplementary Fig. 8b). Biotin-labeled probes were incubated with NRSYM1(531–976)-myc (+) or in vitro translation products without template (−). The CLE-RS2 promoter 1m and LjNIR1 promoter 3m1 contain mutations in the NRE/NBS of CLE-RS2 promoter 1 and LjNIR1 promoter 3, respectively (Supplementary Fig. 8b; Supplementary Table 2). e NRSYM1 (531–976)-myc and a labeled CLE-RS2 promoter 1 probe were incubated with their respective competitors. Non-labeled probes were used as competitor DNA at an excess molar ratio (–, 1:0; +, 1:20; ++, 1:100). Arrowheads indicate locations of the band shift. f Real-time RT-PCR analysis of GUS expression in WT and nrsym1-1 transgenic hairy roots expressing each GUS construct (Supplementary Fig. 8d). Each cDNA sample was prepared from total RNA derived from an uninoculated whorl of roots grown in the presence of 10 mM KNO₃ for 24 h. Transgenic roots were identified by GFP fluorescence. The expression of GFP was used as the reference. Error bars indicate SEM (n = 5 independent pools of roots). *P < 0.05 by Student’s t-test. ns, not significant. Degrees of freedom are shown above the t-values