CaHSFA5 is directly targeted by CaNAC2c upon HTS but not upon RSI
By co-expression assay in pepperhub, it was found CaHSFA5 was co-expressed with CaNAC2c under HTS (Table S5). To test ifCaHSFA5 is one of target genes of CaNAC2c, we study the binding of CaNAC2c to promoter of CaHSFA5 by ChIP-PCR. The result showed that the specific primer pair of CaHSFA5 promoter produced clear DNA band, while the control primer pair did not produce any DNA band, indicating that CaNAC2c directly targetsCaHSFA5 (Figure 5A). Moreover, the binding of CaNAC2c to the promoter fragment ofCaHSFA5 was significantly enhanced by HTS (Figure 5B and C). MST confirmed the binding of CaNAC2c to its putative binding site in theCaHSFA5 promoter in vitro (Figure 5D). By EMSA, prokaryotically expressed CaNAC2c-GST bound promoter of CaHSFA5,importantly, cis -element CATGTG was found to be responsible for the binding of CaNAC2c to the promoter of CaHSFA5 , since its mutation version GGGGGG blocked the binding (Figure 5E). In addition, CaHSFA5 was found to be repressed by CaNAC2csilencing in pepper plants challenged with HTS, while upregulated byCaNAC2c -TO in pepper plants. Similarly, NbHSFA5 was upregulated by CaNAC2c -OE in NB plants (Figure 5F and G). All these results indicate that CaHSFA5 is directly and positively regulated by CaNAC2c.
To assay the role of CaHSFA5 in pepper thermotolerance and immunity against RSI, the effect of CaHSFA5 silencing was studied. We successfully and specifically silenced CaHSFA5 by VIGS in pepper plants (Figure 6H). Similar to CaNAC2c, CaHSF5A acts negatively in pepper growth, since theCaHSF5A silenced pepper plants exhibited an enlarged size in leaves, stems and roots (Figure S7). In addition, the CaHSFA5 silenced plants exhibited thermo sensitiveness compared to the wide type plants(TRV:00 ) with or without pretreated with nonlethal HTS, displayed with higher mortality rate as well as decreased Fv/Fm and △F/Fm’ (Figure 6B-E). Consistently,CaHSFA5 silencing significantly impaired upregulation ofCaHSP24 and CaHSP70 upon HTS while did not affect transcript level of CaDEF1 , indicating that CaHSFA5 acts as positive regulator in thermotolerance (Figure 6I-K). By contrast, the silencing of CaHSFA5 did not affect the resistance of pepper plants to RSI (Figure 6F and G).
To further assay the role of CaHSFA5 in the function of CaNAC2c, NbHSFA5, the ortholog of CaHSFA5 in N. benthamiana genome, was specifically and successfully silenced in CaNAC2c overexpressingN. benthamiana plants by VIGS, its effect on growth and response to RSI or HTS were checked. The result showed that the silencing ofNbHSFA5 significantly restored the growth inhibited (Figure S8) in CaNAC2c overexpressing N. benthamiana plants, but did decreased its thermotolerance (Figure S9B-D) and not affected itsR. solanacearum resistance (Figure S9E and F).