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).