Gateway cloning

The wild-type copy of xopX and its 14-3-3 protein binding motif mutants were cloned by Gateway cloning (Invitrogen, California) from the pENTR clones to the BiFC vector pDEST-VYCE(R)GW carrying the C-terminal region of the Venus Fluorescent Protein (VFP) (Gehl et al., 2009) by Gateway® cloning (Invitrogen, California) from the pENTR clones to yield the constructs as listed in Supplementary table S2. The eight rice 14-3-3 genes cloned in the BiFC vector pDEST-VYNE(R)GW carrying the N-terminal region of VFP were used from a previous study (Deb et al., 2019) . To check for XopQ-XopX interaction, xopQ was cloned in pDEST-VYNE(R)GW. These binary vectors obtained were then electroporated into the A.
tumefaciens strain AGL1 (Supplementary table S2). A suspension of two strains expressing the gene-nVFP/cVFP fusions were grown to 0.8 O.D. 600 , resuspended in infiltration buffer (10mM MES, 10mM MgCl 2 , 100µM acetosyringone, pH 5.6) and used for transient expression in N. benthamiana. VFP signals were examined 48h after infiltration under a LSM880 confocal microscope (Carl Zeiss, Germany) using 20x objectives and He-Ne laser at 488nm excitation. Images were analyzed using the ZEN software. Each set was repeated three times.

To overexpress bzr1-1D and bes1-D, the full-length bzr1-1D and bes1-D coding sequences plus 10 bp upstream of the start codon ATG were first amplified from cDNA reverse transcribed from mRNA isolated from bzr1-1D and bes1-D plants, respectively. The coding sequences were subsequently inserted downstream of the 35S promoter in pB2GW7 (Karimi et al., 2005) by Gateway cloning (Invitrogen). To construct FLAG:BIM1, the full-length BIM1 coding sequence minus the start codon was fused in frame with FLAG (at the 5 0 end) in pENTR3 (Invitrogen), and subsequently the fusion sequence was inserted downstream of the 35S promoter in pB2GW7 (Karimi et al., 2005) by Gateway cloning. For ELF6:FLAG construction, the full-length ELF6 coding sequence minus the stop codon was fused in frame with FLAG in pPZPY112 at BamHI and KpnI (Hou et al., 2010) . The oligos used for cloning are described in Supplemental Table 2.
To create FLC mE-box and FLC mBRRE , an 8.2-kb FLC genomic fragment consisting of the 1.8-kb promoter, 5.6-kb genomic coding region, and 0.8-kb 3 0 region, bearing a mutated E-box (CATATG to TCTAAA; Yin et al., 2005) or a mutated BRRE (CGTGTG to AAAAAA; He et al., 2005) in the first intron (mutated by overlapping PCR with oligos specified in Supplemental Table 2), were cloned into the binary vector pPZP212 at SalI and SacI (Hajdukiewicz et al., 1994) .