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.