A1 hd25

The expression of NPR1 tagged with either GFP (NPR1-GFP) or cyan fluorescent protein (CFP) (NPR1-CFP) was investigated in both intact leaves and protoplasts of stable NPR1-GFP transgenic plants. Confocal laser scanning microscopes (A1 HD25 from Nikon, Japan; FluoView 300 from Olympus, Tokyo, Japan; and STELLARIS 8 from Leica, Wetzlar, Germany) and a fluorescence microscope (THUNDER Imager from Leica, Wetzlar, Germany) equipped with a high-resolution CCD camera (A1 HD25 from Nikon, Tokyo, Japan) were used to visualize the GFP and CFP fluorescence in the cells. Excitation and emission were set at 488 and 520 nm, respectively, for GFP, and at 450 and 470 nm, respectively, for CFP. Red chlorophyll fluorescence was visualized by exciting at 458 nm and a detecting emission at 647–720 nm. The intensity of fluorescence was quantified using the Image J software (https://imagej.nih.gov/ij/, accessed on 15 March 2023). Thirty images were acquired per independent experiment, and each experiment was repeated more than three times (n = 30, N > 3).

The coding sequence of HvFRF9 gene (stop codon removed) was amplified using the intermediate vector, pEASY-Blunt-HvFRF9, and ligated into SUPER1300-35S-GFP and P131-35S-YFP vectors, respectively. The ligation products were used to transform competent E. coli Trans1-T1 cells, and positive clones were obtained and sequenced. SUPER1300-35S-HvFRF9-GFP and P131-35S-HvFRF9-YFP of subcellular localization vector were obtained from the plasmids of the correctly sequenced clones (to ensure that there was no frame shift and the complete fusion protein could be translated). SUPER1300-35S-HvFRF9-GFP, nuclear localization marker, arabidopsis protoplasts, and PEG4000 solution were mixed and placed in water bath for 15 min. Protoplasts were collected after washing with W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 5 mM glucose, and 2 mM MES). The fluorescence distribution was observed under a laser confocal microscope (A1 HD25, Nikon, Tokyo, Japan). P131-35S-HvFRF9-YFP plasmid was transferred into Agrobacterium tumefaciens EHA105, which was used to infect tobacco (Nicotiana benthamiana) at the 4–5 leaf stage. Between 36 and 72 h after infection, undamaged tobacco leaves were selected to make sample slides. The fluorescence distribution was observed under a laser confocal microscope (A1 HD25, Nikon, Tokyo, Japan). The culture conditions for tobacco were 24°C/26°C, 16 h/8 h, day/night.

For analysis of the chicken blastoderm, at least 10 blastoderms were pooled together and centrifuged at 2000 × g for 5 minutes. The resulting tissue masses were paraffin-embedded, and then 4-μm thick sections were cut and placed on slides. Tissue sections or cell slides were fixed in cold methanol and acetone (1:1 mixture) for 2 hours, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. This was followed by blocking using 10% donkey or goat FBS for 30 minutes at 37°C. The sections were incubated overnight at 4°C with primary antibodies against H3S10P (Cat: 3377T; CST; 1:200), PCNA (Cat: abs120180; Absin, Shanghai, China; 1:200), PKC (Cat: NB600-201; Novus, Littleton, CO, USA; 1:100) or P65 (Cat: NBP2-24541; Novus; 1:100). The slides were washed with PBS and then incubated with FITC-conjugated or PE-conjugated secondary antibodies (Abcam) for 2 hours at room temperature. The cell nuclei were counterstained with DAPI (Invitrogen) for 15 minutes at room temperature. The slides were then examined by laser scanning confocal microscopy (Nikon A1HD25; Nikon Corporation, Tokyo, Japan).