Tcs sp8 confocal laser scanning platform

By using ClonExpressII One Step Cloning Kits (Vazyme, Piscataway, NJ, United States), full-length LlNAC2 was inserted into vector pBI121-GFP at XhoI and SalI sites. The pBI121-LlNAC2-GFP plasmid and the pBI121-GFP empty vector were transformed into Agrobacterium tumefaciens GV3101 and infiltrated separately into N. benthamiana leaves. After infiltration, the plants were grown in a growth room under controlled conditions (22/16 °C, 16 h light/8 h dark, 65% relative humidity, and 1000 lx light intensity) for 32 h. GFP fluorescence signals were excited at 488 nm and detected under Leica TCS SP8 Confocal Laser Scanning Platform (Leica SP8, Leica, America) using a 500–530 nm emission filter.

Full-length RcNAC72 was cloned into the pSPYNE173 vector, while RcDREB2A and RcABF4 were cloned into the pSPYCE (M) vector. As described in the above subcellular localization test method, pSPYNE173-RcNAC72 and pSPYCE-RcDREB2A were co-injected into tobacco leaves, along with pSPYNE173-RcNAC72 and pSPYCE-RcABF4. The GFP signals were observed through Leica TCS SP8 Confocal Laser Scanning Platform (Leica SP8, Leica, USA). The primers used above are listed in Table S1.

PmDAMs were cloned into pCambia1300-YFP-N and pCambia1300-YFP-C vectors. Co-expression was executed on N. benthamiana leaves as described in subcellular localization assessments. Chimeric fluorescence from expressed fusion proteins was checked 2 days after infiltration. Images were generated through Leica TCS SP8 Confocal Laser Scanning Platform. YFPs were excited at 514 nm. Specific primers for BiFC analysis were used (Supplementary Table 6).

The sequences coding for PmCBFs were cloned into supper1300-GFP plasmid using specific primers (Supplementary Table S4) and In-Fusion HD Cloning Kit System (Clonetech, Mountain View, CA, USA) to obtain 35S::PmCBF::GFP fusion. The GFP construct (supper1300-PmCBF), checked after sequencing, was transferred to Nicotiana benthamiana following agroinfiltration. These plasmids were transformed into GV3101, Agrobacterium tumefaciens strains, followed by culturing on LB medium bearing rifampicin (50 μg/mL), kanamycin (50 μg/mL) and gentamicin (50 μg/mL) at 28 °C. Agrobacteria were harvested and suspended twice in an infiltration buffer with 150 µM acetosyringone, 10 mM MES and 10 mM MgCl₂. Bacterial suspension culture concentrations were measured using spectroscopy at an optical density of 600 nm and the concentrations were adjusted at 0.5–0.8. The mixture was maintained at room temperature for 2–3 h in the dark. Using syringes, the bacterial culture was infiltrated on the abaxial surfaces of the leaves and sampling was performed after two days for location assessment. Nuclear position was precisely assessed by incubating N. benthamiana leaf tissues with the nucleus marker DAPI. Finally, infiltrated leaves were observed under Leica TCS SP8 Confocal Laser Scanning Platform (excitation/emission settings: 405 nm for DAPI and 488 nm for GFP).

Pair-wise cloning of the full-length cDNA sequences of PmCBFs into pCambia1300-YFP-C and pCambia1300-YFP-N was performed to obtain BiFC constructs. Co-expression was executed on N. benthamiana leaves, as described in subcellular localization assessments. Chimeric fluorescence from expressed fusion proteins was observed 2–3 days post-infiltration using Leica TCS SP8 Confocal Laser Scanning Platform (YFPs excited at 514 nm). Specific primers were used for BiFC assays (Supplementary Table S6).

Full-length LlNAC2 and zinc finger homeodomain protein LlZFHD4 (sequence shown in Supplementary Figure S2) were cloned into the pSPYNE173 vector, LlDREB1 and LlZFHD4 were cloned into the pSPYCE(M) vector [56 (link)], respectively. Co-expression was executed on N. benthamiana leaves as described in subcellular localization assessments. After infiltration, the plants were grown under 24 h dark and then 16 h light/8 h dark for 32 h. YFPs were excited at 514 nm. Images were generated through Leica TCS SP8 Confocal Laser Scanning Platform using a 500–530 nm emission filter.