Fig. 3: Single AA changes within the predicted ligand-binding interfaces affect the functionality of RLP32.

a AF3 predicts a high ipTM for RLP32 in complex with IF1 and the co-receptor BAK1. The AF3 guidelines state that ipTM values higher than 0.8 represent confident high-quality predictions. ipTM values between 0.6 and 0.8 are within a gray zone where predictions could be correct or incorrect. The AF3 cutoffs are depicted with a red and black dotted line. pLDDT scores can be found in Supplementary Fig. 2c, f. The ID is highlighted in black, other LRR-RP domains in dark gray. The ligand is depicted in yellow and BAK1 in blue. Residues highlighted in light gray were mutagenized from the receptor side in this study. b–d Diverse responses in N. benthamiana post heterologous expression of RLP32 and RLP32 variants and subsequent treatment with H₂O (white) or 1 μM IF1 (gray). Each biological replicate is represented by at least three technical replicates. Box plots indicate the median and the interquartile range (IQR), the whiskers extend to the most extreme data points within 1.5 times the IQR. Significance was tested by performing non-parametric two-sided Wilcoxon-Mann-Whitney tests between both mock- and ligand-treated RLP32 (variants) (depicted in green), as well as the ligand-treated RLP32 vs specific RLP32 variants, without adjustments for multiple comparisons. The asterisks indicate a significant difference of p < 0.05. b ROS production (4-60 min) in cumulative RLUs post treatment. Eight independent biological replicates (n = 8 plants) were performed. c Cytosolic Ca²⁺ concentrations (3–30 min) in cumulative RLUs post treatment. Seven independent biological replicates (n = 7 plants) were performed. d Ethylene accumulation after 4 h treatment. Data points are indicated as gray dots from four independent experiments. The Pdb file can be found in Supplementary Data 1 and the predicted local distance difference test (pLDDT) scores can be found in Supplementary Fig. 2c, f, i.