Subcellular Protein Localization

Stable homozygous lines expressing mVenusYFP-fusion proteins were used for all live imaging of CRK2. Transient expression of pFLS2::FLS2-GFP in N. benthamiana was used for FLS2 internalization controls. Transient transformation of Arabidopsis seedlings was used for CRK2 localization in the pldα1 mutant background and for colocalization with PDLP5. Seven-day-old seedlings were transferred to 12-well plates and treatments were applied as described in Table 2. Samples were mounted in the treatment solution and imaged immediately. Fluorescent images were obtained with a Leica TCS SP5 II HCS confocal microscope using standard YFP settings (CRK2-YFP) of 514 nm excitation and a detection range of 525–590 nm, standard GFP settings (FLS2-GFP) of 488 nm excitation and a detection range of 500–600 nm, or standard RFP settings (PDLP5-RFP) of 561 nm excitation and a detection range of 560–600 nm. Quantification of CRK2 colocalization with callose deposits or PDLP5 was achieved using the following equation: % colocalization = (number of colocalized spots/total number of CRK2 spots). Quantification of CRK2-YFP relocalization was achieved by calculating the percent enrichment at the relocalization domains (with Image J) using the following equation: % enrichment = (fluorescence intensity “spot”/fluorescence intensity general plasma membrane) × 100. Statistical significance was determined by one-way ANOVA with pooled t test using JMP Pro 13. Replicates are as indicated in the figure legends.