Figure 5. The transcriptional expression of CaWRKY40 and the bindings of CaWRKY40 to the promoters of its target genes were enhanced by transient expression of CaCDPK15.

(a) Schematic representation of the typical W-boxes in the promoters of the target genes of CaWRKY40. (b) The transcript level of CaCDPK15 by transient overexpression itself in pepper leaves. GV3101 cells containing the construct of 35S:CaCDPK15 was infiltrated into pepper leaves, which were harvest at 24 hpi to isolate the total RNA for transcriptional expressional assay of CaCDPK15 by real-time RT-PCR. Expression values are normalized by the expression levels of CaACTIN and 18s rRNA. (c) The bindings of CaWRKY40 to the promoters of its target genes were potentiated by transient expression of 35S:CaCDPK15 in pepper plants. The GV3101 cells carrying the construct of 35S:CaWRKY40-HA and that containing 35S:CaCDPK15-Flag were mixed at a ratio of 1:1 and were co-infiltrated into pepper leaves, with GV3101 cells containing 35S:00 as mock. The leaves were harvested at 48 hpi for chromatin preparation, the isolated chromatins were digested with micrococcal nuclease and the acquired DNA collections with 300–500 bp in length were used as templates for real-time RT-PCR to assay the bindings of CaWRKY40 to the promoters of its target genes, for each target gene of CaWRKY40, a specific primer pair flanking each typical W-box was designed and the one (primer pair based on 1 W in CaPR1 promoter, 1 W in CaNPR1 promoter and 2 W in CaDEF1 promoter, respectively) that amplified product was used in the real-time RT-PCR analysis. Relative enrichment levels of samples of the CaWRKY40 transient overexpression were set to 1 after normalization by input. (a,b) Data are the means ± SD from at least three independent experiments. Asterisks indicate statistically significant differences compared with 35S:00 (EV) and 35S:CaWRKY40/35S:00 (EV). (t-test, **P < 0.01).