Fig. 4: Natural variations in OsNLP4^indica enhance binding affinity for NRE.

a Electrophoretic mobility shift assay (EMSA) to detect OsNLP4^japonica and OsNLP4 ^indica binding to the NRE. EMSAs were performed using a biotin-labeled NRE cis-fragment from the OsNIA1 gene as a probe and an unlabeled fragment as a competitor. Data are representative of three independent experiments, with similar results. b Dual-luciferase assays. The diagram depicts the effectors and reporter. The effector genes OsNLP4^indica and OsNLP4^japonica were under the control of the CaMV 35S promoter. The fragment containing the NRE cis-element from the promoter of OsNIA1 was fused to the LUC gene as a reporter. Firefly LUC and REN luciferase activities were detected by transient dual-luciferase reporter assays, and the LUC:REN ratio was calculated. c NRE binding to OsNLP4^japonica and OsNLP4^indica, as measured by microscale thermophoresis (MST). K_d: dissociation constant. d EMSA to detect OsNLP4^japonica and OsNLP4^indica binding to the OsD3 promoter. e Dual-luciferase reporting transient assays showed that OsNLP4^indica had a greater ability to inhibit OsD3 expression than OsNLP4^japonica. Data are representative of three independent experiments, with similar results. f The expression levels of OsD3 in the basal tissues of XS134 and NILs (NIL-1, NIL-2) plants cultured in soil for 30 days. g EMSA to detect OsNLP4^japonica and OsNLP4^indica binding to the OsRFL promoter. Data are representative of three independent experiments, with similar results. h Dual-luciferase reporting transient assays showed that OsNLP4^indica had a greater ability to induce OsRFL expression than OsNLP4^japonica. i The expression levels of the OsRFL in the XS134, NIL-1, and NIL-2 plants at the branch meristem formation stage (young panicles, 0.5 cm). The values are the means ± SDs (n = 3 replicates for b, c, e, f, h, and i). The different letters above the bars denote significant differences (P < 0.05) according to a two-sided Duncan’s multiple range test. Source data are provided as a Source Data file.