Gateway bp clonase enzyme mix

The coding region of CsUGT76F1 was amplified by PCR with forward and reverse primers (Supplementary Table S1). The PCR product was introduced into the vector pDONR207 using the Gateway BP Clonase Enzyme mix (Invitrogen, United States). Subsequently, CsUGT76F1 was transferred into the expression vector pCB2004 using the Gateway LR Clonase system (Invitrogen, United States). The recombinant pCB2004-CsUGT76F1 plasmid was transferred into the Agrobacterium tumefaciens EHA105-competent cells through electroporation. The positive cells were screened on the agar-solidified medium consisting 50 mg/L spectinomycin and 50 mg/L kanamycin, and genetic transformation was implemented via the leaf disk transformation method (Maiti et al., 1988 (link)). The transgenic plants were screened on Murashige and Skoog medium containing 25 mg/L phosphinothricin, and then identified using both genomic PCR and RT-PCR. Three lines with high transcript levels were used in the flavonoid profiling analyses.

The effector construct was constructed by recombining the CDS of PpERF3 with the flanking attB sites into the attP site of pDONR201 using GATEWAY™ BP Clonase™ Enzyme Mix (Invitrogen), followed by moving the PpERF3 fragment from pDONR201 to the pK2GW7 destination vector containing the attR sites by mixing both plasmids using GATEWAY™ LR Clonase™ Enzyme Mix (Invitrogen). The pK2GW7-PpERF3 fusion was driven by the 35S promoter. The reporter constructs were the PpNCED2/3 pro::GUS constructs described in the promoter activity assay. The reporter and effector constructs were transferred into Agrobacterium tumefaciens strain GV3101 and co-infiltrated into tobacco leaves. Each reporter-effector combination was infiltrated at least three times. Three days after infiltration, the leaves were used for GUS activity analyses. A fluorimeter (SpectraMax^® i3x Platform, USA) was used to measure fluorescence after the proteins were extracted from the infected tobacco leaves^{38 (link)}.

To generate TaARF15-A1 overexpression plants, the primers 15A1-F and 15A1-R (Supplementary Table S2) were used to amplify the coding sequence of the wheat cDNA, after which the sequence under the control of the CaMV 35S promoter was inserted into a pBI121 binary vector using Gateway BP Clonase enzyme mix (Invitrogen, United States). The construct was then transformed into Arabidopsis (Col-0) by Agrobacterium tumefaciens strain GV3101 (Herrera-Estrella et al., 2005 (link)) via the floral dip method. The transformed lines were first selected on half-strength Murashige and Skoog medium that contained 50 mg L^-1 kanamycin (Ahmed et al., 2012 (link)) and then screened by PCR. The resistant seedlings were subsequently transferred to a mixture of soil and vermiculite (1:1) at 22°C under a 16/8-h light/dark cycle with 70% relative humidity, after which homozygous lines were generated by self-fertilization. Plants from the F₃ generation and wild-type Arabidopsis were used for morphological comparison. qRT-PCR was then used to detect the expression of TaARF15-A.1 in the transgenic plants. The total RNA was isolated from leaves of 20-day-old TaARF15-A1 overexpression plants and wild-type plants at 0, 0.5, and 2 h after 10 μM auxin treatment, and ACTIN2 served as an endogenous control. qRT-PCR for each line was based on three independent biological replicates.