Extended Data Fig. 2: Features of effector proteins identified in pooled screens.

(a) Outline of the pooled ORFeome screens for protein stability regulators. (b) 5 days after completion of the selection, cells infected with the ORFeome-PYL1 library were treated with 100 µM abscisic acid (ABA) for 48 h and top 10% and bottom 10% of cells with EGFP/BFP ratio were sorted. About 1.5 million cells were sorted in duplicate for both subpopulations to reach a coverage of 100x. Biological replicates correlations were similar between unsorted (R² = 0.74) and top 10% (R² = 0.77) subpopulations, and a bit lower for the bottom 10% subpopulation (R² = 0.60). (c) Similar FACS gates were used for the sorting of the ORFeome-vhhGFP infected cells 5 days after completion of the selection. 1.8 million cells were sorted in triplicate, with similar replicate correlations between unsorted (R² = 0.75 to 0.80), top 10% (R² = 0.88 to 0.91), and bottom 10% (R² = 0.86 to 0.95) subpopulations. (d) Enrichment of ORFs in the low EGFP pool infected with the ORFeome-vhhGFP compared to unsorted cells. Significantly enriched ORFs are shown in blue. (e) Enrichment of ORFs in the high EGFP pool infected with the ORFeome-vhhGFP compared to unsorted cells. Significantly enriched ORFs are shown in red. (f-g) Enrichment of Gene Ontology (GO) categories in the ORFeome-PYL1 screen hits (f) and ORFeome-vhhGFP screen hits (g). (h) Enriched GO categories in the overlapping hits between ORFeome-vhhGFP and ORFeome-PYL1 stabilization screens. CC, cellular component; MF, molecular function; BP, biological process. (i) Enrichment of PFAM and Interpro domains in the degradation screen hits. (j) Enrichment of PFAM and Interpro domains in the stabilizer hits. (k-l) Comparison of protein half-lifes²¹ (k) and protein steady state levels²² (l) between hits and all tested proteins. Statistical significance was calculated with a two-tailed t-test. ns, non-significant; *, p≤0.0181; **, p≤0.0064; ****, p≤0.0001.

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