Tcs sp8 se

The CDS of CmNAC34 missing stop codon was amplified and subcloned into the pCAMBIA1300 vector with a GFP to generate a 35S::CmNAC34-GFP fusion construct, and an empty pCAMBIA1300 vector was used as the positive control. These plasmids were introduced into tobacco leaves through Agrobacterium tumefaciens infiltration. Three days after the injection, tobacco leaves were collected, and the GFP fluorescence was observed using a laser confocal fluorescence microscope (TCS SP8-SE; 158 Leica, Wetzlar, Germany) after being stained by the nucleus marker DAPI.
For transcriptional activation assay, the CDS of CmNAC34 was subcloned into the pGBKT7 vector (Clontech, Mountain View, CA, USA) to produce pGBKT7-CmNAC34 fusion plasmid. The empty pGBKT7 vector and pGBKT7-53 + pGADT7-RecT were used as the negative and positive control, respectively. These plasmids were separately transformed into Y2H Gold yeast cells according to the manufacturer’s instructions (Clontech, Mountain View, CA, USA). The transformed yeast cell was cultured on amino acid-deficient medium SD/-Trp for 3–5 days. Then, several positive colonies were dotted on SD/-Trp-His-Ade with X-α-gal and incubated for 3–5 days at 30 °C to evaluate the transcriptional activity.

To investigate the subcellular localization of PuMYB21 and PuMYB54, the full-length coding sequences (CDSs) of PuMYB21 and PuMYB54 without terminate codons were inserted into the pRI101-eGFP vector using NdeI and KpnI restriction enzyme sites to form the fusion expression vectors PuMYB21-eGFP and PuMYB54-eGFP under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. The fusion expression vectors were transferred into A. tumefaciens strain GV3101 and used to transform onion epidermal cells as previously described⁵⁹ . Laser scanning confocal microscopy (TCS SP8-SE; Leica, Wetzlar, Germany) was used to detect fluorescence signals.

Subcellular localization was performed using the Dong et al. (2021) (link). Primers (FveARF2-GFP-F and FveARF2-GFP-R) were designed to amplify the FveARF2 CDS region without a termination codon (Table S1). The LA Taq polymerase (Takara, Dalian, China) was used for PCR, which was performed according to the following procedure: (1) 95 C for 5 min, (2) 95°C for 30 s, 55°C for 30 s, and 72°C for 2 min 30 s for 35 cycles, and (3) 72°C for 10 min. XbaI and BamHI were used to digest the pRI101-GFP vector and 35S::FveARF2-GFP (FveARF2-GFP) was constructed using T₄ DNA ligase (Takara, Dalian, China). The pRI101-GFP vector containing 35S::GFP (GFP) was used as a control. FveARF2-GFP and pRI101-GFP were transformed into the Agrobacterium strain GV3101, and the bacterial liquid culture was used to inoculate N. benthamiana leaves. A laser confocal fluorescence microscope (TCS SP8-SE; Leica, Wechsler, Germany) was used to examine GFP fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 505–530 nm.