Extended Data Fig. 5: Structure-function analysis of RLP42 required for pg9(At) recognition.
From: Distinct immune sensor systems for fungal endopolygalacturonases in closely related Brassicaceae
a, Immunoblot analysis using anti-GFP antibody of protein extracts from N. benthamiana leaves alone or transiently expressing GFP or RLP42 chimeric and mutant proteins. The relative band intensities of GFP to loading (Ponceau S staining) were calculated and shown at the bottom of the blot. b, Total ROS production (relative light units, RLU) in N. benthamiana leaf discs transiently transformed with chimeric constructs or mutant constructs as indicated and treated with water (mock), 1 µM flg22, or 1 µM pg9(At). Data points are indicated as grey dots from three independent experiments (n with exact numbers are indicated on top of the box plots) and plotted as box plots (centre line: median, bounds of box: the first and third quartiles, whiskers: 1.5x the interquartile range, error bar: minima and maxima). Statistically significant differences to the response observed in RLP42-expressing plant are indicated (two-sided Student’s t-test). c, Summary of RLP42 chimeric and mutant proteins used in this study. d, Ethylene accumulation after treatment with serial dilutions of pg9(At) in N. benthamiana leaves transiently transformed with RLP42, and RLP42 D153V, H321S, or E696K mutant constructs. Bars represent means ± standard deviation of three replicates. Assays were performed in triplicate with similar results. e, BAK1 recruitment to RLP42 receptor mutant proteins. Proteins extracted from N. benthamiana leaves co-expressing RLP42/mutant-GFP and BAK1-Myc treated with water (-) or 1 µM pg9(At) (+) for 5 min were used for co-immunoprecipitation with GFP-trap beads, and immunoblotting with tag-specific antibodies. Assays were performed in triplicate with similar results.
