Gateway reaction

To generate the lentiviral vector pLTiTSA-Smad4-Flag plasmid, the Smad4-Flag cDNA was excised from a pRK5-SMAD4-Flag plasmid [71 (link)] using EcoRI/SalI digestion, gel purified and ligated into a pENTR SfiI Shuttle previously linearized with EcoRI/XhoI. Subsequently, the SMAD4-Flag insert was transferred into a Gateway modified pLTiTSA GW (TREtight-GW-IRES-tomato, SV40-rtTA) [72 (link)] via Gateway reaction (Invitrogen).

Candidate genes were first identified by a search for best similarity (blast) with known oxidosqualene cyclases from GenBank, using the Plant & Food Research EST database (Newcomb et al., 2006) and the apple genome (Velasco et al., 2010). Full‐length coding sequences for MdOSC4 and MdOSC5 were obtained by PCR amplification from a cDNA library made from RNA extracted from the fruit skin of ‘Merton Russet’ and ‘Royal Gala’, respectively. The resulting products were cloned into the plant transformation vector pHEX2 using Gateway reactions (Invitrogen, Mulgrave, Victoria, Australia) and transformed into Agrobacterium tumefaciens strain GV3101 (Hellens et al., 2005).
CYP716A from apple was identified by a blast search in the Plant & Food Research EST database and the genome, using the sequence of the characterized triterpene hydroxylase from Medicago truncatula (CYP716A12) (Fukushima et al., 2011). This EST was then full‐length sequenced and cloned into pSAK277S and transformed into A. tumefaciens strain GV3101 as described previously (Hellens et al., 2005).
GenBank accession numbers are as follows: CYP716A175, EB148173; MdOSC1, FJ032006; MdOSC3, FJ032008; MdOSC4, KT383435; MdOSC5, KT383436.

The transactivation assays were performed in N. benthamiana leaves as described previously^{35 (link)}. About 2-kb TaJAZ1 promoter was fused with the luciferase reporter gene LUC by Gateway reactions (Invitrogen) into the plant binary vector pGWB35 to generate the reporter construct TaJAZ1_pro:LUC^{42 (link)}. The full-length TaJAZ1 and TaMYC4 were separately cloned into the plant binary vector pGWB5 and pEarlyGate 101 to generate the effector constructs 35S:TaJAZ1-GFP and 35S:TaMYC4-YFP. Different combinations of reporters and effectors were coinfiltrated into N. benthamiana, and the LUC signals were observed and analyzed 48 h after infiltration by using Night SHADE LB 985 system (Berthold, Germany).

The transcriptional activity assays were performed in N. benthamiana leaves as previously described [23 (link)]. The 2-kb CO promoter sequence was amplified from Col-0 genome DNA, and fused with the luciferase reporter gene LUC through Gateway reactions (Invitrogen) into the plant binary vector pGWB35 [34 (link)] to generate the reporter construct CO_pro:LUC. For the construction of the effectors, the full-length coding sequences of indicated genes were amplified and cloned into the plant binary vector pGWB17 [34 (link)]. The reporter and effector constructs were separately introduced into Agrobacterium strain GV3101 (pMP90), to carry out the co-infiltration in N. benthamiana leaves. LUC activities were observed and quantified 48 h after infiltration using NightSHADE LB 985 (Berthold). In each experiment, 10 independent N. benthamiana leaves were infiltrated and analyzed, and totally three biological replications were performed with quantification.

AtMiro2 cDNA (LIC6 vector containing the coding sequence for AT3G63150) was purchased from the Arabidopsis Biological Resource Centre. The full AtMiro2 coding sequence was cloned using Gateway reactions (Invitrogen) into the binary destination vector PB7WGF2 creating an N-terminal GFP fusion. Using this as template, the Quickchange site-directed mutagenesis kit (Agilent Technologies) was used according to the manufactures’ instructions to sequentially introduce point mutations into both GTP-binding domains, to create KKVV (K23>V and K434>V) and SSNN (S28>N and S439>N) double mutants.