1. MTB1 to MTB3 Are Direct Transcriptional Targets of MYC2.
MTB1 to MTB3 Are Direct Transcriptional Targets of MYC2. (A) Schematic representations of MTB1, MTB2, and MTB3 genes showing the primers and probe used for ChIP-qPCR assay and EMSA. (B) ChIP-qPCR analysis of MYC2 enrichment on the chromatin of MTB1, MTB2, and MTB3. Chromatin of MYC2-GFP-9# plants was immunoprecipitated using anti-GFP antibody, and the immunoprecipitated DNA was quantified by qPCR. The enrichment of target gene promoters is displayed as a percentage of input DNA. Data represent means ± sd (n = 3). ACTIN2 was used as a nonspecific target. (C) EMSA showing that MBP-MYC2 directly binds to the promoters of MTB1, MTB2, and MTB3. The MBP protein was incubated with the labeled probe to serve as a negative control; mutated probes were used as a negative control. Ten- and 20-fold excesses of unlabeled or mutated probes were used for competition. Ten- and 20-fold excesses of MBP protein were used for competition. Mu, mutated probe in which the G-box motif 5ʹ-CACATG-3ʹ was replaced with 5ʹ-AAAAAA-3ʹ. (D) and (E) RT-qPCR assays showing wound-induced expression of MYC2, MTB1, MTB2, and MTB3 in wild-type and jai1 plants (D) and MTB1, MTB2, and MTB3 in wild-type, MYC2-RNAi-3#, and MED25-AS-5# plants (E). Eighteen-day-old seedlings of the indicated tomato genotypes with two fully expanded leaves were mechanically wounded using a hemostat on both leaves for the indicated times before extracting total RNAs for RT-qPCR assays. Data represent means ± sd (n = 3). In (B) and (E), asterisks indicate significant differences from the wild type according to Student’s t test at **, P < 0.01 and ***, P < (Supplemental Data Set 2).
Yuanyuan Liu et al. Plant Cell 2019;31:
©2019 by American Society of Plant Biologists