Figure 4.

SSPbP53 interacts with xylem PLCPs. A. Yeast-two-hybrid (Y2H) assays using the clubroot pathogen SSPbP53 as the bait and Arabidopsis PLCP-cys domains representing different subfamilies as the prey. Growth of yeast cells on SD-3 selective media represents protein–protein interaction, while growth of the same cells on SD-2 media confirms yeast transformation. Yeast transformed with the empty vectors served as negative controls. B. In vitro pull-down assay using GST-AtPLCP-cys to immunoprecipitate His-SSPbP53 protein. Input and output (immunoprecipitated proteins) were detected by western blotting (WB) using anti-GST and anti-His antibodies. E. coli transformed with empty GST-vector was used as negative control. Immunoprecipitated fraction was visualized through Ponceau stain (PS). C. Pre-incubation of GST-XCP1-cys with E-64 inhibits the interaction with His-SSPbP53. Input and output (immunoprecipitated proteins) were visualized by western blotting (WB) using anti-GST and anti-His antibodies. Immunoprecipitated fraction was visualized through Ponceau stain (PS). D. Schematic representation of SSPbP53 and SSPbP53^ΔL1 interaction with XCP1. E. Proteolytic activity of His-XCP1-cys, measured by digestion of a fluorescent casein substrate, is inhibited by purified His-SSPbP53 protein, but not by SSPbP53^ΔL1. Fluorescence was measured at 485/538 nm excitation/emission. Mean ± standard deviation (n = 3). Statistically significant differences based on the two-tailed Student’s t-test are indicated by asterisks (*) where p < 0.001 is represented by (***)