Fig. 5: OsNPR1 plays a positive role in activating JA signaling.

a, b Co-IP assays showing that OsNPR1 interacts with OsJAZ9, OsMYC2 or OsMYC3. OsNPR1 and OsJAZ9, OsMYC2 and OsMYC3 were transiently co-expressed in tobacco leaves. c Protein competition analyzed by Co-IP assays in planta. OsJAZ9-FLAG and OsMYC2-MYC or OsMYC3-MYC were infiltrated with or without OsNPR1-GFP in leaves of N. benthamiana, HA-GFP serves as negative control. The samples were harvested at 48 hpi for coimmunoprecipitation with FLAG beads. d OsNPR1 elevated the transcriptional activation activity of OsMYC2. (Upper) Schematic diagram of the dual-LUC assays. The promoters of OsMADS1 and OsNOMT with a firefly luciferase (LUC) were used to construct the pOsMADS1::LUC and pOsNOMT::LUC vectors as the reporters. Renilla luciferase (REN) was the internal control. OsNPR1 and OsMYC2 were the effectors. (Lower) The OsMADS1 and OsNOMT promoters were activated by OsMYC2 protein, and this activation were highly enhanced by co-expression with OsNPR1 in N. benthamiana. The LUC/REN ratio represents the relative LUC activity. Error bars represent SD, values are means ± SD (n = 3 biologically independent replicates per genotype). Significant differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * at the columns indicate significant differences (p ≤ 0.05). e Schematic diagram of OsMADS1 and OsNOMT genes with exons indicated as black boxes for ChIP-qPCR analyses. Black triangles denote the CACGTG (G-box) motifs. Arrowheads denote the transcription start sites. P1 and P2 denote the corresponding amplicons for qPCR. f ChIP-qPCR analyses of OsNPR1 binding to the G-box from OsMADS1 and OsNOMT promoters in NIP and OsNPR1-7# plants using OsNPR1-specific polyclonal antibodies. Error bars represent SD, values are means ± SD (n = 3 biologically independent replicates per genotype). Significant differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * at the columns indicate significant differences (p ≤ 0.05). g ChIP-qPCR analyses of OsNPR1 binding to the G-box from OsMADS1 and OsNOMT promoters in NIP and Ri-m2m3-6# plants using OsNPR1-specific polyclonal antibodies. Error bars represent SD, values are means ± SD (n = 3 biologically independent replicates per genotype). Significant differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * at the columns indicate significant differences (p ≤ 0.05). h Viral symptoms in OsNPR1-7# (n = 25), OsNPR1-7#/Ri-m2m3 (n = 22), Ri-m2m3 (n = 25) transgenic plants and NIP (n = 21) in response to RSV infection. The phenotypes were observed and photos taken at 30 dpi. Scale bars = 5 cm, 4 cm or 1 cm. i The relative mRNA levels of RSV CP in RSV-infected OsNPR1-7#, OsNPR1-7#/Ri-m2m3-6#, Ri-m2m3-6# transgenic and NIP rice plants as detected by RT-qPCR at 30 dpi. Error bars represent SD, values are means ± SD (n = 3 biologically independent replicates per genotype). Significant differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * at the columns indicate significant differences (p ≤ 0.05). j The accumulation of RSV CP protein in RSV-infected OsNPR1-7#, OsNPR1-7#/Ri-m2m3-6#, Ri-m2m3-6# transgenic and NIP rice plants. CBB serves as the loading control to monitor input protein amounts. Experiments in (a)–(c) and (j) were repeated three times with the similar results. Source data including uncropped scans of gels (a–c and j) and p values of statistic tests (d, f, g and i) are provided in the Source data file.