Extended Data Fig. 10: Chitin-induced EDS1 phosphorylation by PBL19 promotes antifungal immunity.
a, PBL19-mediated EDS1 phosphorylation sites in vitro identified by mass spectrometry. Identified phosphosites with peptide ion scores over 20 are considered reliable. Mass error in parts per million (ppm) = [1-(experimentally determined mass)/(predicted mass)]×106. b, Phosphodeficient 5A mutations of EDS1 cannot fully abolish PBL19-mediated phosphorylation in vitro. GST was used as a control. c, Four in vitro phosphorylation sites (orange) are located on the protein surface of EDS1. Note that another phosphorylation site (Thr623) corresponds to the last residue of EDS1 and is located in a disordered tail missing in the deciphered structure. d,e, Phos-tag electrophoresis reveals that chitin-induced EDS1 phosphorylation depends on the kinase activity of PBL19 (d) and is dramatically reduced in phosphodeficient EDS15A (e). Indicated proteins were transiently expressed in pbl19 null protoplasts. f,g, Phosphomimetic EDS15D tends to be enriched in the cytoplasm relative to phosphodeficient EDS15A in pbl19 null plants (f) or protoplasts (g). In (f) and (g), the abundances of EDS1 or derivatives were quantified by Image J based on the densitometric ratios against tubulin (Input fraction), histone H3 (Nucleic-enriched fraction), and tubulin (Nucleic-depleted fraction), respectively. h, Phosphomimetic EDS15D can suppress the susceptibility of pbl19 null plants to V. dahliae infection. i, Complementary expression of EDS15D enhances plant resistance to V. dahliae relative to wild-type plants, whereas that of EDS15A impairs plant resistance. The experiments in (b) and (g) were repeated twice and those in (d), (e) and (f) were repeated three times with similar results.
