Leted and non-deleted versions of an OsGRF4 cDNA had been amplified from NJ6. The resultant amplicons had been inserted into the pSY-735-35S-cYFP-HA or pSY-736-35S-nYFP-EE vectors37 to create fusion constructs. Co-transfection of constructs (e.g., those encoding nYFP-OsGRF4 and cYFP-SLR1) into tobacco leaf epidermal cells by Agrobacterium-mediated infiltration enabled testing for protein-protein interaction. Following 48h incubation in the dark, the YFP signal was examined and photographed making use of a confocal microscope (Zeiss LSM710). Every BiFC assay was repeated at the least three instances. Relevant primer sequences are given in Supplementary Data Table 6.Co-immunoprecipitation (Co-IP) and western blotting Full-length OsGRF4, OsGIF1 and SLR1 cDNAs had been amplified, after which inserted into either the pUC-35S-HA-RBS or the pUC-35S-Flag-RBS vector as previously described38. A. thaliana protoplasts have been transfected with one hundred g of plasmid and after that incubated overnight in low light intensity conditions. Total protein was then extracted from harvested protoplasts by treating with 50 mM HEPES (pH7.5), 150 mM KCl, 1 mM EDTA (pH8), 0.3 Trition-X 100, 1 mM DTT with added proteinase inhibitor cocktail (Roche LifeScience). Lysates have been incubated with magnetic beads conjugated with an antiDDDDK-tag antibody (MBL, M185-11) at four for no less than four hours. The magnetic beads were then rinsed six times with the extraction buffer and eluted with 3 lag peptide (SigmaAldrich, F4709). Immunoprecipitates were electrophoretically separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare). FD&C Green No. 3 custom synthesis proteins had been detected by immunoblot using the antibodies anti-Flag (Sigma, F1804) and anti-HA (MBL, M180-7). InNature. Author manuscript; readily available in PMC 2019 February 15.Li et al.Pageaddition, the OsGRF4, SLR1, OsLhca1, OsLhca3, OsLhca4, OsLhcb2, OsPsaD and OsPsaE proteins had been detected by probing the membrane with anti-OsGRF4 antibodies (Abmart), anti-SLR1 antibodies (ABclonal Technology), anti-OsLhca1 antibodies (Agrisera, AS01005), anti-OsLhca3 antibodies (Agrisera, AS01007), anti-OsLhca4 antibodies (Agrisera, AS01008), anti-OsLhcb2 antibodies (Agrisera, AS01003), anti-OsPsaD antibodies (Agrisera, AS09461) and anti-OsPsaE antibodies (Agrisera, AS08324A), respectively. Uncropped blots were shown in Supplementary Details Figure. 1. Relevant primer sequences are given in Supplementary Information Table 6. EMSA assays EMSA was performed as previously described with minor modifications39. Full-length OsGIF1 and SLR1 cDNAs had been amplified and cloned into the pCold-TF vector (Takara). His-OsGIF1 and His-SLR1 recombinant proteins were purified using Ni-NTA agarose (QIAGEN, 30210), following the manufacturer’s guidelines. GST (Glutathione Stransferase) and GST-OsGRF4 recombinant protein were expressed inside the Escherichia coli BL21 (DE3) strain then purified utilizing Glutathione Sepharose 4B beads (GE Activated T Cell Inhibitors Reagents Healthcare, 17-0756-01). 42 bp DNA probes have been artificially amplified and labelled utilizing a biotin label kit (Biosune). DNA gel shift assays have been performed applying the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, 20148). Relevant primer sequences are offered in Supplementary Info Table 8. RNA-seq analysis Total RNAs had been extracted from 3-week-old rice plants grown below higher N conditions (1.25 mM NH4NO3) working with the QIAGEN RNeasy plant mini kit (QIAGEN, 74904) following the manufacturer’s guidelines. 3 replicate RNA-seq libraries have been prepared fr.