MTB1 Antagonizes MYC2 for DNA Binding.

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MTB1 Antagonizes MYC2 for DNA Binding. MTB1 Antagonizes MYC2 for DNA Binding. (A) Schematic representations of TomLoxD and JA2L showing the amplicons and probe used for ChIP-qPCR assay and EMSA. Positions of the transcription start site (TSS) and transcription termination site (TTS) are indicated. (B) and (C) ChIP-qPCR analysis of the enrichment of MYC2 (B) and MTB1 (C) on the chromatin of TomLoxD and JA2L in MYC2-GFP-9# and MTB1-GFP-5# plants, respectively. (D) and (E) ChIP-qPCR analysis of the enrichment of MYC2 (D) and MTB1 (E) on the G-box regions of TomLoxD and JA2L in MYC2-GFP-9# and MTB1-GFP-5# plants, respectively, upon wounding. Plants were treated with mechanical wounding for the indicated times before cross-linking. (F) and (G) ChIP-qPCR assays of the impaired enrichment of MYC2 on the G-boxes of TomLoxD and JA2L due to MTB-RNAi in MYC2-GFP-9# and MYC2-GFP-9#/MTB-RNAi-8# plants (F) and MTB1-OE in MYC2-GFP-9# and MYC2-GFP-9#/MTB1-OE-11# plants (G) upon wounding. Plants were treated with mechanical wounding for 1 h before cross-linking. For (B) to (G), chromatin of each sample was immunoprecipitated using anti-GFP antibody and quantified by qPCR. The enrichment of target gene promoters is displayed as a percentage of input DNA. Data represent means ± sd (n = 3). For (D) to (G), ACTIN2 was used as a nonspecific control. For (F) and (G), asterisks indicate significant differences detected using Student’s t test (*, P < 0.05 and **, P < 0.01; Supplemental Data Set 2) when compared with MYC2-GFP. (H) EMSA showing that MBP-MTB1 interferes with MBP-MYC2 to bind to DNA probes from the TomLoxD promoter in vitro. Biotin-labeled probes were incubated with a fixed amount of MBP-MYC2 and an increasing amount of MBP-MTB1 or MBP, and the free and bound DNAs were separated on an acrylamide gel. The MBP protein was incubated with the labeled probe to serve as a negative control; mutated probes were used as a negative control. Mu, mutated probe in which the G-box motif 5ʹ-ACCATGTG-3ʹ was deleted. Yuanyuan Liu et al. Plant Cell 2019;31:106-127 ©2019 by American Society of Plant Biologists