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).