Clonexpressii one step cloning kits

According to the instructions of ClonExpressII One Step Cloning Kits (Vazyme, Nanjing, China), the full length, N-terminal (1–526 bp) and C-terminal (527–1059 bp) of the RcNAC72 gene were inserted into the EcoRI and BamHI of the pGBKT7 vector. The recombinant plasmids and pGADT7 plasmid were transferred into Y2HGold yeast cells (Huayueyang, Beijing, China), referring to the Quick Easy Yeast Transformation Mix kit instructions (Clontech, San Jose, CA, USA). The transformed yeast cells were diluted 10-fold with sterile water and 10 μL of the diluted solution was spotted on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal media, respectively. These were cultured upside down at 30 °C for 3 days, and the yeast growth was observed.
The full length of RcDREB2A was inserted into pGBKT7 vector as a prey, and the full length of RcNAC72 was inserted into pGADT7 as a bait. As mentioned above, pGBKT7- RcDREB2A and pGADT7- RcNAC72 plasmids were jointly transferred into Y2H yeast. These yeast cells were observed on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal selective media. The primers used above are listed in Table S1.

The full length of the RcNAC72 gene with the terminator removed was inserted between the XhoI and SalI sites of the pBI121-GFP vector, using ClonExpress II One Step Cloning Kits (Vazyme, Nanjing, China). The specific operations were in accordance with the instructions. The constructed vector pBI121-RcDREB2A-GFP and pBI121-GFP plasmids were transformed into Agrobacterium tumefaciens GV3101 and the infection solutions were prepared respectively and injected into the tobacco leaves. The injected tobacco leaves were cut into approximately 1 cm × 1 cm sizes, placed on a glass slide with 100μL ddH₂O dripped in advance and covered with a cover glass. These leaves were imaged using a Leica TCS SP8 Confocal Laser Scanning Platform (Leica SP8, Leica, Buffalo Grove, IL, USA) under 488 mm laser excitation and 500–530 nm filter to observe the GFP positioning. The primers used above are listed in Table S1.

For the subcellular localization analysis of TaWRKY31, we designed primers with appropriate double restriction enzyme sites, XbaI and KpnI, based on the full-length TaWRKY31 and p35s-1301-GFP vector. The target gene sequence was amplified using these primers. The complete TaWRKY31 ORF without the termination codon was then inserted into the p35s-1301-GFP vector using the ClonExpressII One Step Cloning Kits (Vazyme, Nanjing, China). The p35s-1301-TaWRKY31-GFP plasmid and p35s-1301-GFP empty vector were transformed into Agrobacterium GV3101 and subsequently infiltrated into the abaxial surface of Nicotiana benthamiana leaves using 1-mL needleless syringes along with NLS-mCherry and P19 as controls [53 (link)]. The transformed tobacco plants were initially grown in a dark environment at 22℃ for 24 h and then transferred to controlled conditions with a temperature of 22℃, a photoperiod of 16 h/8 h, and an illumination intensity of 180 µmol·m^− 2·s^− 1 for 2 d. Green fluorescent protein (GFP) signals were observed using a laser confocal microscope (Andor, Belfast, UK) at an excitation wavelength of 488 nm. The specific primers used for this analysis are listed in Supplementary Table S1.