Extended Data Fig. 6: Effect of pectic fragments and NO on LURE1 properties.
From: FERONIA controls pectin- and nitric oxide-mediated male–female interaction
a–c, Wide-field images of control, GSNO-, SNP- and PGA-treated LURE1–GFP-expressing ovules from unpollinated pistils. Data are average ± s.d. n, number of pistils. Representative of three independent experiments. P values were obtained by two-tailed t-tests; numbers in plots denote the number of ovules examined. d, SDS–PAGE of purified E.-coli-produced MBP, MBP–LURE1 (arrowheads) and the extracellular domain of its receptor PRK6 (His6–HA–PRK6(ecd)) (arrow). M, molecular mass markers. e, MBP–LURE1 attracts pollen tubes. Top, purified MBP–LURE1 was used in pollen tube attraction assays1,37 in semi-pollen tube growth cultures. Arrowheads, tips of pollen tubes at the time of bead application. Bottom, histogram shows MBP–LURE1 dose-dependent pollen-tube-attraction activity. Attraction efficiency was similar to that in a previous study1. Mock treatment (0) used MBP in gelatin beads. Scale bars, 100 μm. Data are representative of multiple independent protein preparations using a similar range of MBP–LURE1 concentrations. f–h, GSNO treatment of MBP–LURE1. f, MBP–LURE1 was mock-treated or incubated with GSNO before application for SDS–PAGE in the absence of β-mercaptoethanol, followed by Coomassie-blue staining. g, An immunoblot by anti-MBP antibody of an experiment similar to that in f. Arrow, double arrowheads and bracket indicate monomeric MBP–LURE1, dimer-sized and higher-molecular-weight forms, respectively. h, Comparison of GSNO treatment of MBP and MBP–LURE1 shows no notable effect on the molecular weight of MBP. Collectively comparable observations were made in at least six independent experiments. i, j, Dot blot assay for LURE1 interaction with its receptor PRK626,27. i, Equal volumes (1.5 μl) of increasing concentrations of HA–PRK6(ecd) were applied to filter in triplicate rows for interaction with MBP–LURE1, then processed for immunodetection of bound MBP–LURE1 by anti-MBP antibody. Ponceau-S-stained filter illustrates quantitative spotting of HA–PRK(ecd) to the membrane. Data plot (right) shows signal intensity from the bound MBP–LURE1. Data are averaged from the triplicate binding samples ± s.d., showing a Kd of about 1.21 ± 0.28 μM, approximating the previously reported affinity26. j, Dot blot assay for PRK6(ecd) interaction with control (0) and MBP–LURE1 treated with increasing concentration of GSNO. PRK6(ecd) on membrane was incubated with MBP–LURE1, and the interaction was detected by anti-MBP. Ponceau-S-stained filters confirmed quantitative spotting of PRK6(ecd). The result from the 12-μg bait blots (top row) is shown in Fig. 4c. k, Effect of DTT on GSNO-induced inhibition of MBP–LURE1 binding to HA–PRK6(ecd). MBP–LURE1 was preincubated with combinations of DTT and GSNO as shown, before application to an HA–PRK6(ecd) filter for binding. Ponceau-S-stained filters illustrate comparable HA–PRK6(ecd) applied to all filters. Results indicate reducing conditions mitigated GSNO inhibition. Collectively, similar observations were made in three independent experiments (i, j) or two for k; the triplicated and duplicated dots in i, k served as technical replicates. Box plots: centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points.
