Transient expression of CaNAC2c in pepper leaves
For transient expression analysis, GV3101 cells harboring35S:CaNAC2c-GFP (using 35S:GFP as control) was grown overnight and then resuspended in induction medium (10 mM MES, 10 mM MgCl2, 200 µM acetosyringone, pH 5.6) to OD600 = 0.8, approximately 1 ml was infiltrated into the leaves of pepper plants at the eight-leaf stage using a syringe without a needle, and then the infiltrated leaves were collected at the indicated time points for further use.
Generation of transgenicCaNAC2c-overexpressingN. benthaminana plants
Leaf discs of N. benthamiana were transformed with GV3101 harboring 35S:CaNAC2c-GFP vector followed the method of Regneret al (F et al., 1992). and Bardonnet al (N, F, MA, & L, 1994). 19 independent T0 transgenic N. benthamiana lines were selected by hygromycin (5 mg l-1) selection and further confirmed by PCR and quantitative real-time RT-PCR (qRT-PCR). The T0 plants were then self-pollinated and their seeds individually harvested. Similarly, corresponding seeds of T2 and T3 were acquired. Two T3 transgenic lines that exhibited moderate levels of CaNAC2c transcripts without phenotypic abnormality were selected for further analysis.