Overexpression of CaNAC2cenhanced basal and acquired
thermotolerance, resistance to RSI but decreased growth ofN.benthamiana plants
To further confirm the results from CaNAC2c silencing and
transient overexpression, we assayed the effect of ectopic
overexpression of CaNAC2c (CaNAC2c -OE) inN.benthamiana plants on thermotolerance and RSI resistance. We
generated transgenic N.benthamiana plants of 35S:CaNAC2c ,
a total 9 T0 plants were acquired, by strict
self-pollination, their corresponding T1 and
T2 lines were acquired. Two T2 lines,CaNAC2c-OE1# and CaNAC2c-OE2# , with high levels ofCaNAC2c transcripts were selected for further assay (Figure
4A). It was found that compared to
the wild type plants, the growth and development of two lines were
significantly inhibited, manifested by their smaller size in leaves,
roots and stems, fewer numbers of leaves and flowers (Figure S5),
confirming the result from CaNAC2c silencing in pepper plants
that CaNAC2c acts as negative regulator in pepper growth and
development.
In parallel, it was also found that CaNAC2c -OE N.
benthamiana plants exhibited significantly enhanced tolerance to HTS
(Figure 4B), displayed by their significantly lower mortality rate
calculated after 1–42 h of HTS with or without pretreatment of
nonlethal HTS (Figure 4C). In addition, Fv/Fm and △F/Fm’ were found to
be enhanced, ion leakage and H2O2accumulation was decreased by CaNAC2c -OE (Figure
S6).Consistently, the transcript levels of thermotolerance relatedNbAPX , NbHSP18 and NbsHSP were upregulated
byCaNAC2c -OE in N. benthamiana plants compared to the
control plants, and this upregulation was amplified by HTS (Figure 4D).
These results also confirmed that CaNAC2c acts as positive regulator in
thermotolerance. The transcript levels of immunity related marker genes
such as NtDEF1 were assayed, the results showed that transcript
abundances of these genes were not altered in by CaNAC2c -OE upon
HTS (Figure S9I).
To assay the effect of CaNAC2c -OE on immunity against RSI, theCaNAC2c -OE N. benthamiana plants were challenged with RSI,
an obvious wilt symptoms in the wild type plants were observed at 7 dpi,
while the transgenic plants exhibited only slight wilt symptoms, and
lower levels of disease index and population of R.solanacearum in
transgenic plants were found (Figure 4E, F and G). Consistently,
enhanced transcript levels of immunityrelated genes such asNtDEF1 were found in the CaNAC2c -OE N. benthamianaplants upon RSI, while thermotolerance related genes did not alter in
their transcript levels in the transgenic plants upon RSI (Figure S9I).