Fig. 3.

SbWRKY1 and SbZNF1 bind to and transactivate the SbMATE promoter. For both (A and B) ChIP-qPCR and (C and D) transactivation assays, protoplasts from Arabidopsis stably transformed with promoter constructs were transfected with pEarleyGate104 containing the indicated TFs or with the empty vector (minus sign). Schematics of the SbMATE promoter constructs are shown: the general structure MITE region is depicted in B: the 243-bp MITE element (unit “b”) is flanked by 100-bp (unit “a”, 100-bp repeat) and 20-bp (unit “c”) sequences, and the number of identical a-b-c triplets varies in different promoter alleles. The MITE-containing a-b-c triplets (MITE repeats) is terminated by a single 100-bp “a” unit with either an 8-bp deletion (depicted by an inverted black triangle, present in SC283 and Tx430) or a 12-bp deletion (inverted white triangle, present in BR012), which results in the 88- or 92-bp terminal fragments. ChIP-qPCR with (A) a truncated SbMATE promoter from the SC283 line extending to position −2,102 bp [−2102promoter(p)] and (B) from BR012 extending to position −5,299 bp [−5299promoter(p)] and containing four copies of the MITE repeats (4×), followed by the 88-bp terminal. Arrows represent primer (SI Appendix, Table S2) positions. Data were normalized to the input (control) for each sample and are expressed as the fold-enrichment vs. preimmune IgG serum controls. Error bars indicate SEM (n = 3), and asterisks indicate significant differences (P < 0.05). (C and D) Transactivation in Arabidopsis protoplasts. Protoplasts stably transformed with (C) −2102pSC283::GUS and transfected with pEarleyGate104 with or without [pEarley(−)] the sorghum TFs. Results are the mean ± SD of three independent experiments. *P < 0.05; Scott–Knott test. (D) Transactivation with the promoter region of Tx430 (−4214pTx430::GUS) and BR012 (−5299pBR012::GUS), with one and four MITE repeats (n), respectively. The error bars represent the limits of the nonparametric bootstrap confidence interval of 95% with n = 4.