The PP2C and AC domain-coding sequences of BAC and BACS1407P were isolated from the wild-type and mutant strains and inserted into pET28a+ (Supplementary Figure S4 ) to produce the BAC-His6 PP2C-AC and BACS1407P-His6 PP2C-AC vectors, respectively. These vectors were transformed into Escherichia coli BL21 cells by a heat-shock method. Target proteins were produced by the addition of 1.5 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) at 30°C. The BAC PP2C-AC domain and BACS1407P PP2C-AC domain were individually linked to six histamines (His6) and expressed as fusion proteins; eventually, the BAC-His6 PP2C-AC domain and BACS1407P-His6 PP2C-AC domain fusion proteins were produced and then purified using the Ni-NTA 6FF Sefinose (TM) Resin Kit (Shenggong, Shanghai, China).
For the gel retardation assay, the purified protein was run in SDS-PAGE or mixed with Phos-tag (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to examine the phosphorylation levels of different proteins following the reported method (Gou et al., 2015 (link)). In this assay, the phosphate group on the protein binds the Phos-tag with the manganese ion in the gel. Eventually, the relative mass of the protein becomes larger, and therefore, the mobility in the gel becomes slower. The variance of the position of the protein is representative of the phosphorylation levels between different proteins. The fusion proteins were specified by binding with anti-His6 and secondary antibody goat anti-mouse IgG HRP (AB-M-M100, GOOD HERE, Hangzhou, China) according to the manufacturer’s instructions. This binding was detected by the Western Blot test reagent dye solution (34,580, Thermo Scientific™, Shanghai, China), where the second antibody was bound to HRP, and the substrate of ECL produced chemiluminescence after being catalyzed by HRP.
For the total protein phosphorylation level test, the mycelia growth on cellophane-covered CM medium for 3 days under light or dark conditions, and the protein extraction buffer (Yang et al., 2013 (link)) was used to extract mycelia protein. The anti-phosphoserine antibody (ab9332, Abcam, United Kingdom) was used to analyze the total phosphorylation level.
For the gel retardation assay, the purified protein was run in SDS-PAGE or mixed with Phos-tag (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to examine the phosphorylation levels of different proteins following the reported method (Gou et al., 2015 (link)). In this assay, the phosphate group on the protein binds the Phos-tag with the manganese ion in the gel. Eventually, the relative mass of the protein becomes larger, and therefore, the mobility in the gel becomes slower. The variance of the position of the protein is representative of the phosphorylation levels between different proteins. The fusion proteins were specified by binding with anti-His6 and secondary antibody goat anti-mouse IgG HRP (AB-M-M100, GOOD HERE, Hangzhou, China) according to the manufacturer’s instructions. This binding was detected by the Western Blot test reagent dye solution (34,580, Thermo Scientific™, Shanghai, China), where the second antibody was bound to HRP, and the substrate of ECL produced chemiluminescence after being catalyzed by HRP.
For the total protein phosphorylation level test, the mycelia growth on cellophane-covered CM medium for 3 days under light or dark conditions, and the protein extraction buffer (Yang et al., 2013 (link)) was used to extract mycelia protein. The anti-phosphoserine antibody (ab9332, Abcam, United Kingdom) was used to analyze the total phosphorylation level.
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