Fig. 5: bHLH57 accounts for the highly induced expression of SBRR1-R by specifically binding to its promoter.
From: Natural variation in SBRR1 shows high potential for sheath blight resistance breeding in rice
a, Y1H assay to identify potential bHLH57 (basic helix-loop helix 57) binding site. M1 and M2 represent two fragments of the 256-bp insertion. The CACCGG motif is found only in the M2 fragment. b, EMSA to confirm the direct binding of bHLH57 to CACCGG motif. GST-EV, glutathione S-transferase-empty vector. c, ChIP-qPCR assay for the enrichment of the P2 region of the SBRR1-R promoter by transcription factor bHLH57 (n = 3). Enriched values are normalized to the input. An unrelated OsActin1 intron region (ACTIN) was used as a negative control. IgG-immunoprecipitated DNA was used as a control. d, Dual-luciferase reporter assay to study the effects of bHLH57 on SBRR1-R gene expression (n = 6). 35S, cauliflower mosaic virus 35S promoter. GFP, green fluorescent protein. REN, Renilla luciferase. LUC, firefly luciferase. e, RNA expression levels of the SBRR1 gene in TG394, T394-SBRR1R and their bhlh57-ko plants (n = 3). HPI, hours post inoculation. f, ShB lesions of bHLH57 knockout lines (T1 generation) in TG394 and T394-SBRR1R backgrounds scored 14 days after R. solani inoculation in greenhouse (n = 24). g, ShB lesions of bHLH57 overexpression lines (T1 generation) in TG394 and T394-SBRR1R backgrounds scored 14 days after R. solani inoculation in greenhouse (n = 21). Data in c–g are presented as means ± s.d. Statistical significances in d and c were determined by a two-sided Student’s t-test. Different lowercase letters in e–g indicate significant differences (P < 0.05) based on one-way ANOVA with Duncan’s multiple range test. Scale bar, 5 cm in f and g. The Y1H, dual-luciferase reporter and EMSAs were done independently three times.
