Fig. 1: Establishment of TurboID-mediated proximity labeling in Arabidopsis for identification of proximal proteins of RRS1/RPS4 complex.

A Analysis of ability of RRS1-R-TurboID or RPS4-TurboID construct to induce cell death in response to AvrRps4 or PopP2. Each tobacco leaf section was coinfiltrated to transiently express the indicated constructs together with either mCherry, AvrRps4 or PopP2. Photograph was taken at 4 days post infiltration (dpi). B Diagram of the expression cassettes used for the expression of TurboID. GFP containing a NLS was fused to the N-terminus while a FLAG tag was added to the C-terminus of TurboID. Expression was under the control of Arabidopsis SSR16 (Small Subunit Ribosomal Protein 1 promoter, named as pAt2 in TSL ‘moderate promoter’ database for moderate expression in Arabidopsis) and nopaline synthase terminator. C RRS1-R-TurboID fusion protein complements loss of resistance to Pst DC3000 (AvrRps4) observed in the rrs1arrs1b mutant. Bacterial growth was measured 3 days post infiltration (dpi). Data are shown as means ± SD (n = 4 and 6 biological replicates, respectively). The corresponding p values can be found in the Source Data. D TurboID-based analysis of RPS4 biotinylation by RRS1-R-TurboID in Nb. Nb leaves were agroinfiltrated with the indicated constructs and biotin was infiltrated into the previously agroinfiltrated leaves at 36 h post-agroinfiltration (hpi). mCherry-TurboID served as a negative control. IP was carried out using samples collected 3 h after biotin treatment with anti-HA beads. The FLAG-tagged proteins were detected using anti-FLAG antibody and biotinylated proteins were detected using Streptavidin-HRP antibody, respectively. E Biotinylation of RRS1-R and its vicinal proteins in RRS1-R-TurboID transgenic plants. Total protein extracts from seedlings with or without biotin treatment were immunoblotted with Streptavidin-HRP and anti-FLAG antibody for detection of biotinylated proteins (top panel) and expression of TurboID (lower panel), respectively. Non-treated seedlings were used as controls to visualize the background activity of TurboID with endogenous biotin. Col-0 seedlings served as a control. The asterisks indicate the positions of naturally biotinylated proteins. F Streptavidin pull-down analysis of biotinylated proteins by TurboID-tagged RRS1-R. GFP-fused TurboID served as a control. Immunoblotting analysis of the Streptavidin pull-down products were probed with Streptavidin-HRP (top panel) and anti-FLAG antibody (lower panel).