Fig. 5
Flg22 induces RGS1 phosphorylation at Ser431. a Flg22 induces phosphorylation of RGS1 C-terminus (RGS1CT). Col-0 protoplasts expressing RGS1CT-HA were treated with 1 μM flg22 for 5 min before protein extraction. Protein samples were treated with (+) or without (−) λ protein phosphatase (PPase) before immunoblot analysis. b Phospho-peptides identified from mass spectrometric analyses. RGS1CT-FLAG was transiently expressed in protoplasts, affinity purified following flg22 treatment and subjected to LC-MS/MS analysis. c Alignment of the regulatory tail of RGS proteins from different plant species. At Arabidopsis thaliana, Al Arabidopsis lyrata, Cs Camelina sativa, Gm Glycine max, Mt Medicago truncatula, Sl Solanum lycopersicum, Vv Vitis vinifera. Position of Ser431 is indicated by an asterisk. d RGS1 Ser431 is phosphorylated upon flg22, elf18, chitin, and Pep2 treatment and substitution of Ser431 with Ala in RGS1CT abolishes pattern-induced phosphorylation. The indicated constructs were transiently expressed in WT protoplasts, which were then treated with indicated flg22, elf18, chitin, or Pep2, and RGS1CT protein mobility was determined by immunoblot analyses. e AvrAC blocks flg22- and chitin-induced RGS1 phosphorylation at Ser431. RGS1CT-FLAG were transiently expressed in WT protoplasts along with AvrAC-HA, and RGS1CT phosphorylation was detected by immunoblot. f Chitin-induced RGS1CT phosphorylation is compromised in rlck vii-4 mutant. RGS1CT-FLAG was expressed in protoplasts prepared from WT and rlck vii-4 mutant plants. After flg22 or chitin treatment of protoplasts, the protein mobility was detected by immunoblot. g Pep2-induced RGS1CT phosphorylation is compromised in rlck vii-8 mutants. RGS1CT-FLAG protein mobility was examined in WT and rlck vii-8 protoplasts. h BIK1 phosphorylates RGS1CT-FLAG in vitro. RGS1CT-FLAG was expressed and purified from protoplasts, incubated with HIS-tagged BIK1 and BIK1K105E recombinant proteins, and RGS1CT-FLAG protein mobility was detected with immunoblot analyses. Numbers on top of the gel blots indicate ratio of phosphorylated (upper band) to non-phosphorylated (lower band) RGS1CT-FLAG. The experiments were performed two (f) or three (a, d, e, g, h) times with similar results
