Figure.5 CaHSFA5 was directly targeted and transcriptionally regulated by CaNAC2c under HTS. A. The CATGTG containing fragment and a CATGTG free fragment within the CaHSFA5 promoter used for primer pairs design for ChIP-PCR to assay the direct targeting ofCaHSFA5 by CaNAC2c. B. ChIP-PCR showed that CaHSFA5 was directly targeted by CaNAC2c. Chromatins were isolated fromCaHSFA5-GFP transiently overexpressed pepper leaves, which was sheared into fragment of 300-500 bps in length. The DNA was immunoprecipited with antibodies of GFP, the acquired DNA was used as template with specific primer pair of CATGTG containing promoter region. C. ChIP-qPCR showed that enhanced ability of CaNAC2c to bind toCaHSFA5 promoter under HTS. D. the binding of promoter fragment of CaHSFA5 by CaNAC2c was confirmed by MST using CaNAC2c-GFP fusion protein transiently overexpressed in pepper leaves and immunoprecipited by antibody of GFP and a CATGTG containing promoter fragment. E. the binding of promoter fragment of CaHSFA5 by CaNAC2c was confirmed by EMSA using prokaryotically expressed CaNAC2c-GST and promoter fragment of CaHSFA5 containing CATGTG or its mutant version (GGGGGG). F. CaHSFA5 was downregulated byCaNAC2c silencing under HTS.G. CaHSFA5 was upregulated byCaNAC2c overexpression in N. benthamiana plants or byCaNAC2c transient overexpression in pepper leaves. In C, F and G, data presented are means ± standard error (SE) of four replicates, different uppercase letters above the bars indicated significant differences among means (P < 0.01), as calculated with Fisher’s protected LSD test.