Figure S1. Amino acid sequence alignment of the B-type ARRs. B-type response regulators receiver domain is highlighted in green. In ARR18, the aspartate in position 70 (D70) is conserved in all B-type ARRs and predicted to be targeted for phosphorylation. The closest aspartate in proximity is also highly conserved and in position 75 (D75). The output domain includes the nuclear localization signal (NLS, highlighted blue) and GARP DNA-binding domain (highlighted yellow). Amino acid residues in red are conserved throughout the whole family of B-type ARRs.

Figure S2. Expression analysis of FRET-FLIMcontrol constructs. Confocal images ofN.  benthamiana epidermal leaf cells expressingthe indicated GFP and mCherry fusion proteins. The emissionchannels for the GFP, RFP fluorescence and the overlay with thebright field image are indicated above. The bars represent10 μm.

Figure S3. ARR18 DNA-binding activity appearsnot to be affected by the Asp70 mutation. Reporter gene activationassay where Arabidopsis protoplasts were co-transfected with eitherthe PARR5:uidA reporter gene construct and theP35S:NAN transformation efficiency standard alone(control) or in combination with the different effector plasmidsindicated: wild type ARR18 (18), gain-of-functionARR18D70E (18D70E) or loss-of-functionARR18D70N (18D70N) proteins. DNA bindingcompetition was performed by keeping the wild type ARR18 (18) at aconstant level and increasing the ratio of ARR18D70E(18D70E) or ARR18D70N (18D70N) asindicated (1:3 and 1:6). Mean values and standard deviations ofGUS/NAN activity were calculated from four independent transfectionexperiments. Keeping the wild type ARR18 protein amount constantand increasing those of the gain-of-function ARR18D70Eversion, resulted in a reporter gene activity similar to thatobserved for the ARR18D70E single transformation. Incontrast, an increased ratio of the ARR18D70N proteinrevealed an inhibition of the reporter gene activity when comparedto the wild type ARR18 single transformation. This suggests thatboth ARR18D70E and ARR18D70N compete withwild type ARR18 for activation of the5′-(A/G)GAT(T/C)-3′ promoter region in vivo.

Figure S4. Characterization of the Arabidopsis ARR18 T-DNAinsertion mutant and ectopic-ARR18-GFP overexpressing lines. (a)Confocal images of wild type (Ws-2) and transgenic Arabidopsis rootcells overexpressing ARR18-GFP (ARR18-GFPOX1 andARR18-GFPOX2). The emission channel for the GFPfluorescence, the overlay with the bright field image and anenlargement are indicated above. The bars represent 10 μm.(b) Western blot analysis of protein extracts from the ARR18ectopic-overexpressors (ARR18-GFPOX1,ARR18-GFPOX2) described above. Immuno detection of theARR18-GFP fusion proteins was carried out with an antibody againstthe GFP-tag and wild type Arabidopsis (Ws-2) was used as a negativecontrol. (c) Semi-Quantitative RT-PCR analysis of the steady-stateexpression level of ARR18 transcript performed withARR18-specific primers and with CAB3-specific primers aspositive control. Three different cycle numbers were used (20, 25and 30). To exclude any cross contamination, the RT-PCR wasperformed also in the absence of cDNA (control).

Figure S5. ARR18 interaction with AHP proteins. (a) Yeast clonesexpressing BD-ARR18RD and the indicated AD-AHP cloneswere cultivated for 4 days at 28°C on either vector selectivemedia (CSM-L-, W-) or interaction selective media (CSM-L-, W-,Ade-). The pGADT7 vector expressing only the AD domain was used asa negative control. The specific β-galactosidase activity wasmeasured in the extracts of three independent yeast clonesexpressing BD-ARR18RD and the indicated AD fusionproteins. In the western blot, the detection of theBD-ARR18RD was carried out with an antibody directedagainst the c-myc-tag (α-myc) and that of the AD fusionproteins with an antibody against the HA-tag (α-HA). CLSMimages of tobacco leaf cells expressing the indicated YFP-C fusionsof full-length ARR18 (b) or ARR18RD (c) and YFP-Nfusions of AHP1 to AHP6. The upper panel shows the fluorescenceimages and the bottom panel, the overlays with the bright fieldimages. The bars represent 10 μm. (c) Immunodetection ofthe AHPs-YFP-N fusion proteins was carried out with an antibodyagainst the c-myc-tag (α-myc). Detection of the ARR18- andARR18RD-YFP-C fusion protein was carried out with anantibody against the HA-tag (α-HA). N, negative controlconsisting of a protein extract from a non-transformed tobaccoleaf.

Table S1. Constructs and primers list used in the different cloning strategies. Table S1 shows all the entry clones used in this study and the cloning method (Topo cloning, BP cloning and Site directed mutagenesis cloning). The forward and reverse primers used to amplify the desired genes are also listed.

Methods S1. Protein–protein interactionstudies in yeast.

Methods S2. BiFC interaction studies.

Methods S3. Construction of transgenicArabidopsis lines and characterization of the ArabidopsisARR18 T-DNA insertion mutant.

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