Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes
Fig 3
flg22-induces changes in AtRGS1/ RLKs complex dynamics.
(A) flg22 rapidly causes AtRGS1 to move away from BIR1 and toward BAK1. FRET analysis (acceptor photobleaching) of N. benthamiana cells expressing BAK1-CFP, BIR1-CFP and AtRGS1-YFP in the presence of flg22 (1μM) at the indicated time points is shown. Error bars represent standard error of the mean (SEM) of regions of interests (ROIs) n = 3 to 16. (B) Top: The dynamics of the BIR1-AtRGS1 is modulated by the phosphorylated C-terminal domain. FRET efficiency in N. benthamiana cells expressing BIR1-CFP AtRGS1-YFP or AtRGS1ΔCt-YFP in the presence of a low concentration of flg22 (100 nM) is shown over time. Error bars represent SEM of ROIs (n = 4 to 23). Bottom: BAK1 interaction requires the carboxy-terminal domain of AtRGS1. FRET efficiency in N. benthamiana cells expressing BAK1-CFP and AtRGS1-YFP or AtRGS1ΔCt-YFP in the presence of a low concentration of flg22 (100 nM) is shown over time. Error bars represent SEM of ROIs (n = 3 to 17). All experiments were repeated at least two times. It is important to note that panel A shows results of flg22 at a moderate concentration (1 μM) and panel B show the results of flg22 at a low concentration (100 nM), hence the different time courses. Student's t tests were conducted to compare the FRET Efficiency % of flg22 treated leaves at the indicated time points to 0 min. ***, Student's t test significant at P value < 0.0001; **, Student's t test significant at P value < 0.01; *, Student's t test significant at p < 0.05. AtRGS1-YFP vs AtRGS1ΔCt-YFP interactions in B are shown by a letter: aaa, aa or a.