Poly(I:C) causes BIK1 phosphorylation and PD callose deposition in a SERK1-dependent manner. A to C) Analysis of BIK1 phosphorylation in poly(I:C)-treated A. thaliana Col-0 protoplasts. A) Poly(I:C) treatment induces BIK1 phosphorylation as shown by a protein mobility shift detected by immunoblot analysis. Protoplasts expressing BIK1-HA were nontreated (mock) or treated with 1 µM flg22 or 0.5 µg/µL poly(I:C), lysed after 20 min and treated or nontreated with CIP for 1 h before immunoblot analysis using HA-HRP antibody. BIK1 band intensities were quantified using Image Lab (Bio-Rad). Quantification of BIK1 phosphorylation (upper panel) calculated as ratio of intensity of the upper band (phosphorylated BIK1, pBIK1) to the sum intensities of shifted and nonshifted bands (pBIK1 + BIK1) (no treatment set to 0.0). CCB, Coomassie Brilliant Blue staining of Rubisco as gel loading control (lower panel). B) Poly(I:C)-induced BIK1 phosphorylation is blocked by 1 µM of the kinase inhibitor K-252a added 1 h before poly(I:C) treatment. Rubisco detection by CBB staining is shown as gel loading control (lower panel). Experimental conditions and quantification of BIK1 phosphorylation as in A). C) SERK1 enhances poly(I:C)-induced BIK1 phosphorylation. Protoplasts from WT Col-0 or serk1-1 mutants were transfected with BIK1-HA together with or without SERK1-FLAG and followed by treatment with or without 0.5 µg/µL poly(I:C). Phosphorylated BIK1 band intensities were quantified as in A). The middle panel shows SERK1-FLAG expression. Rubisco detection by CBB staining is shown as gel loading control (lower panel). D and E) Efficient poly(I:C)-induced PD callose deposition depends on SERK1. D) Callose fluorescence at PD seen upon aniline blue staining of epidermal cells of WT Col-0 plants and serk1-1 mutants treated with water (control) or 0.5 µg/µL poly(I:C). Inlays show enlargements of the areas within the dashed boxes. Scale bar, 10 µm. E) Mean callose content in PD determined in 9 leaf disks taken from 3 plants per treatment (dots). The total number of evaluated PD is given at the base of each column. Error bars show the Sem. One way ANOVA of the mean values (P = <0.0001) followed by Sidak's test for pairwise comparisons. ****, P = < 0.0001; ns = nonsignificant. The mean callose intensity levels indicate a drastic inhibition of the poly(I:C)-induced PD callose deposition response in serk1 as compared to the WT.