FIGURE 5.

CaHSL1 was directly regulated by CaWRKY40 at the transcriptional level. (A) ChIP assay showed that the W-boxes in the promoter of CaHSL1 are bound by CaWRKY40. CaWRKY40-HA was transiently overexpressed in pepper leaves, the leaves were collected at 24 hpi and the DNA-protein complexes were immunoprecipitated using anti-HA antibodies. The immunoprecipitated DNA was employed as template for PCR using primer pairs specific to the fragment containing the W-boxes or without the W-box within the CaHSL1 promoter. PW, specific primer pair for the fragment containing W-boxes within the promoter of CaHSL1; PCK, specific primer pair of the W-box free fragment within the promoter of CaHSL1. (B) Effect of transient overexpression of CaWRKY40 on transcript levels of CaHSL1 in pepper plants with or without HTHH challenge by qRT-PCR; (C) Effect of CaWRKY40 silencing by VIGS in pepper leaves with or without HTHH challenge on transcription of CaHSL1 by qRT-PCR. In (B,C), CaHSL1 transcript levels in CaWRKY40 transient overexpressing or CaWRKY40-silenced pepper plants were compared with those in mock-treated control plants, which were set to a relative expression level of “1.” Data show the mean ± SD from four biological replicates; error bars indicate standard error. Different upper-case letters on the bars indicate significant differences between means (p < 0.01), as determined by Fisher’s protected LSD test.