For in vitro analysis, GST-MoRgs7, GST-MoRgs75A, and His-MoSep1 were expressed in E. coli DE3 cells and purified [41 (link)]. We used the Pro-Q Diamond Phosphorylation gel stain (Thermo Fisher Scientific), a phosphor-protein gel-staining fluorescence dye in this assay. A kinase reaction buffer (100 mM phosphate-buffered saline, pH 7.5, 10 mM MgCl2, 1 mM ascorbic acid) was mixed with MoRgs7 and His-MoSep1, MoRgs75A, and His-MoSep1, respectively. The subsequent experiments were performed according to the previously described protocol [21 (link)].
For in vivo analysis, conidia were prepared from various transformants as described above and were filtered through three layers of lens paper before resuspending in sterile water (2 ×105 spores mL-1) [42 (link)]. For appressorium protein extraction, droplets (5 mL) of spore suspensions were placed on strips of onion epidermis, incubated under humid conditions at room temperature for 6 h, and onion epidermis grounded for protein extraction [43 (link)]. Protein extraction was the same as described above and phosphorylation analysis was performed as according to the protocol, phosphatase inhibitors (P0044, sigma) and alkaline phosphatase (P6774, sigma) [21 (link)].
For in vivo analysis, conidia were prepared from various transformants as described above and were filtered through three layers of lens paper before resuspending in sterile water (2 ×105 spores mL-1) [42 (link)]. For appressorium protein extraction, droplets (5 mL) of spore suspensions were placed on strips of onion epidermis, incubated under humid conditions at room temperature for 6 h, and onion epidermis grounded for protein extraction [43 (link)]. Protein extraction was the same as described above and phosphorylation analysis was performed as according to the protocol, phosphatase inhibitors (P0044, sigma) and alkaline phosphatase (P6774, sigma) [21 (link)].