Clonexpress 2

The DcaWOX2, 3a, 5, 9, 11b, and 13a coding sequences were amplified by PCR using specific primers and Phanta^® Max SuperFidelity DNA Polymerase (Vazyme Biotech Co., Ltd.; Nanjing, China). The amplified sequences were inserted into the pRI101-GFP vector using ClonExpress^® II (Vazyme Biotech Co., Ltd.; Nanjing, China). The 35S: GFP-DcaWOX constructs were inserted into Agrobacterium tumefaciens EHA105 cells via electroporation for the subsequent injection into Nicotiana benthamiana leaves. A laser confocal microscope was used to examine the tobacco leaves after a 3-day incubation in a greenhouse, as previously described [35 (link)].

The open reading frames of selected IbbHLH-5/-25/-69 L/-106/-123/-43 L/-148/-154 L/-181 L/-206/-212 L/-215 were cloned and then fused into pGBKT7 or pGADT7 vectors by ClonExpress II (Vazyme, Nanjing, China) recombination reaction. The empty pGBKT7, each recombinant pGBKT7-IbbHLH, and recombinant pGBKT7-IbbHLH and pGADT7-IbbHLH plasmids were then subjected to Y2HGold yeast transformation [63 (link), 64 (link)]. Yeast dilutions were then dropped on SD/-Trp, SD/-Trp-His-Ade medium with or without AureobasidinA (AbA) for transactivation activity analysis. Yeast dilutions were dropped on SD/-Trp-Leu, SD/-Trp-Leu-His-Ade medium with or without AbA for protein interaction detection. All the plate was incubated upside down at 28 °C for three days to detect the growth phenotype of yeasts. The related primers are found in Additional file 16.

The DNA sequence encoding full-length IniA (residues 1–602) from M. smegmatis mc²155 was PCR-amplified using Phanta Max DNA polymerase (Vazyme) and inserted into the NcoI-HindIII sites of pET-28a vector (GE Healthcare) using ClonExpress^®II (One Step Cloning Kit, Vazyme). This construct includes a C-terminal 6 × His-tag. For expression in M. smegmatis, the encoding sequence of IniA-GFP fusion protein was inserted into the SspI site of the engineered plasmid pMV261 and the mutant construct (residues L480-K492 was mutated into GGGGSGGGGS liker) was generated by recombinant circle PCR. Site-directed mutagenesis was performed using the TaKaRa MutanBEST Kit. The mutants were introduced by the PCR method using the IniA expression plasmid as a template, with pairs of primers encoding the mutations at the sites of substitution. DNA sequencing of the constructs was performed to validate that the mutagenesis experiments were successful. All primer sequences used in this study are shown in the Supplementary Table 1.
E. coli BL21 (DE3) ATCC strain (Supplementary Table 2) was used to express IniA for purification. The M. smegmatis mc²155 strain was used to observe cellular localization of IniA.

The ClonExpress II or ClonExpress MultiS kit (Vazyme, China) was used to construct the following plasmids by recombination cloning following the manufacturer’s instructions. All oligonucleotide primers used are listed in Table S1.
pBI121-6myc-DCL3 was generated by cloning the coding sequence (NM_001161190.2, 4743 bp) of DCL3 from Col-0 plants into the BamHI-linearized vector pBI121-Myc.
To generate pBI121-6myc-DCL3_mRIII and pBI121-6myc-DCL3_ΔRBD constructs, corresponding sequences were amplified from pBI121-6myc-DCL3, and the PCR products were cloned into the BamHI-linearized vector pBI121-Myc. For the plocex-DCL3-EGFP, plocex-DCL3_mRIII-EGFP and plocex-DCL3_ΔRBD constructs, the DCL3, DCL3_mRIII and DCL3_ΔRBD sequences were cloned into the SpeI/BamHI-linear vector plocex-EGFP.
For generating translational fusion with glutathione S-transferase (GST) protein, the fragments (F1, F2, F3, F4 and F5) of DCL3 were amplified from pBI121-6myc-DCL3 and cloned into the BamHI/XhoI-linearized vector pGEX-4T-2.

The mitochondrial location vector pDsRED2-Mito (pDRM) was purchased from Waryong Biotechnology (Beijing, China). Homologous recombination was performed to clone PCK2 coding sequences into pDRM, according to the instructions of the one-step cloning kit ClonExpress II (Vazyme, Nanjing, China). Briefly, pDRM was linearized by restriction enzyme BamH I and Not I (NEB, Ipswich, MA, USA). PCK2 mRNA sequence was cloned using primers containing BamH I and Not I restriction sites and homologous sequences with the terminal sequences of linearized vector at the 5′-end (Suppl. Table S1). Then the linearized vector and the PCK2 coding sequences were ligated using ligase Exnase II supplied by the kit, and the ligation product was transfected into DH.5α. The monoclonal was picked, and pDRM-PCK2 vectors were purified using GeneJET Plasmid Midiprep Kit (Thermo Fisher, Waltham, MA, USA) and verified by sequencing.

For yeast one-hybrid experiments, the full-length coding sequences of AabHLH2 and AabHLH3 were amplified with primers P7 F/R and P8 F/R and then cloned into the pB42AD (EcoRI/XhoI) by using ClonExpress II (Vazyme, China) to generate the effector vectors. The artificial synthesized triplicate cis-element segments contain possible bHLH binding elements from the promoters of ADS, CYP71AV1, DBR2, and ALDH1, which were inserted into the pLacZ (EcoRI/XhoI) to create the reporter vectors. The sequences of artificially synthesized triplicate cis-elements were listed in Supplementary Table 1. The combinations of pB42AD-AabHLH2 and pB42AD-AabHLH3 with different cis-element motifs were co-transformed into the yeast strain EGY48 by using the LiAc method. The transformant yeasts were cultivated on synthetic-defined SD/-Trp/-Ura dropout (Clontech, Dalian) selected plates at 30°C and positive clones were transferred to and grown on SD/-Trp/-Ura plates with X-gal to test the interaction.