Fig. 5: The rice cell wall-derived specific oligosaccharides are perceived by OsCERK1.
a ROS burst induced by rice cell wall-derived specific oligosaccharides is abolished in oscerk1 mutant. The WT and oscerk1 suspension cells were elicited with sterile water (Mock), filtered supernatants (1/100, v/v) of MoCel12A-hydrolyzed rice cell walls (CW), 10 μM BGTRIB, 10 μM BGTETC, and 5 μg/ml chitin in ROS burst assays, respectively. flg22 (100 nM) served as a positive control. Values are means ± SD (n = 8 biological replicates). RLU, relative light units. b ROS generation induced by rice cell wall-derived specific oligosaccharides in oscebip mutant. Others are as in (a). c ROS generation induced by rice cell wall-derived specific oligosaccharides in OsRac1-RNAi plants. Others are as in (a). d Microscale thermophoresis (MST) assays show the binding of OsCERK1-ECD with the specific oligosaccharides. The solid curve is the fit of the data points to the standard Kd-fit function. Kd, dissociation constant. Bars represent ± SD (n = 3 biological replicates). e MST assays show that OsCEBiP-ECD does not bind the oligosaccharides. Others are as in (d). f BGTRIB and BGTETC triggered heterodimerization of OsCERK1 and OsCEBiP in rice. OsCERK1-FLAG and OsCEBiP-HA were co-expressed in WT rice protoplasts. GUS-FLAG served as a negative control. The protoplasts were treated with 5 ug/ml chitin, filtered supernatants (1/100, v/v) of MoCel12A-hydrolyzed rice cell walls (CW), 10 μM BGTRIB and 10 μM BGTETC for 15 min. Co-immunoprecipitation was performed using anti-FLAG antibody and subjected to immunoblot analysis with anti-FLAG or anti-HA antibody as indicated. The experiment was repeated three times with similar results. g BGTRIB and BGTETC triggered OsCERK1 dimerization in rice. FLAG- and HA-tagged OsCERK1 were co-expressed in WT rice protoplasts. Others are as in (f).
