Fig. 1: Identification of PRAF/RLD proteins as BASL physical partners. | Nature Communications

Fig. 1: Identification of PRAF/RLD proteins as BASL physical partners.

From: Connected function of PRAF/RLD and GNOM in membrane trafficking controls intrinsic cell polarity in plants

Fig. 1

a Localization of BASL (green) in Arabidopsis stomatal asymmetric cell division (ACD). PrC protodermal cell, MMC meristemoid mother cell, M meristemoid, SLGC stomatal lineage ground cell. Bottom schematic shows BASL polarization requires MAPK-mediate protein phosphorylation and the plasma membrane-associated BRX proteins. Unknown regulators (orange) are anticipated to mediate BASL polarization. PM, plasma membrane; NE, nuclear envelope. b Results of GFP-BASL co-immunoprecipitation (IP) coupled with mass spectrometry. The ubiquitous 35S promoter was used to drive the expression of GFP-BASL in wild-type plants and proteins associated with GFP-BASL were isolated by GFP-trap agarose beads. GFP alone driven by the BASL promoter was used as control. For details, see Supplementary Data 1. c Subdomains of PRAF/RLD and BASL, respectively. “N”, “I”, and “C” stand for N-terminal, Internal and C-terminal domain of the protein, respectively. “NLS”, nuclear localization signal. Dashed red boxes highlight identified interacting domain/motif. d Phylogenetic tree for the PRAF/RLD family, generated by Clustal W and based on the full-length proteins. Bold indicates proteins further characterized in this study. The AGI numbers can be found in “Methods”. e–g Pairwise yeast two-hybrid assays. Bait, designated proteins fused with Gal4 DNA-binding domain (BD). Prey: “-“ indicates Gal4 activation domain (AD) only, and the others are AD fused with designated protein or protein domain. 3F > 3K (BASL FxxFxF mutated to KxxKxK). “Test” indicates interaction assays on synthetic dropout media (-LeuTrpHis, -LTH); “Control” indicates yeast growth in rich media (-LeuTrp, -LT). When needed, specific concentrations of 3-Amino-1,2,4-triazole (3-AT) were supplied to suppress bait auto-activation. h Bimolecular fluorescence complementation (BiFC) assays in N. benthamiana leaf epidermal cells. Positive YFP signals indicate positive protein-protein interactions (BASL with PRAF4). Deletion of the BRX domain (PRAF4ΔBRX) or mutating the FxxFxF motif (BASL_3F > 3K) abolished YFP signals. White arrows show the interaction occurs in a polarized manner. Data represent the results of three independent experiments. Scale bars = 25 μm. (z), z-stacked confocal image.

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