The sequences of FgRab1 cDNA, FgGyp1 cDNA, TBC domain and R357K point mutant of FgGyp1 were cloned into the MBP (Maltose-binding protein) vector pMAL-c2X, using their respective primers listed in Supplementary Table 2 , and expressed. The protein products were isolated and purified for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer’s protocols. GTP hydrolysis was assayed based on previous reports (Xiong et al., 2012 (link); Zheng et al., 2020 (link)). Briefly, the recombinant proteins MBP–FgGyp1, MBP–FgGyp1TBC (TBC), MBP–FgGyp1R357K (R357), and MBP–FgRab1 were expressed in BL21 Escherichia coli strain and isolated by Amylose resin (Sangon Biotech, NO.C500096) as per the instructions of the manufacturer. A colorimetry-based kit was used (Sigma-Aldrich, Catalog Number MAK113) to assay for the GTPase activity of Gyp1, and FgRab1 was incubated with FgGyp1, FgGyp1TBC (TBC), FgGyp1R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.
Amylose resin
Manufactured by Sangon
Amylose resin is a chromatographic resin used for the purification of proteins and other biomolecules. It consists of amylose, a linear polysaccharide derived from starch, which is covalently bound to a solid support matrix. The amylose chains interact with target molecules through hydrogen bonding and other non-covalent interactions, allowing for the selective capture and separation of these molecules from complex mixtures.
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Lab products found in correlation
4 protocols using amylose resin
1
GTPase Activity Assay for FgRab1 and FgGyp1
2
Cloning and Purification of SmMYB1
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cDNA from SmMYB1 was amplified by PCR and cloned into a pETMALC-H vector (Supplementary Fig. S3 ), which was then transformed into Escherichia coli BL21 cells. All the primers used are listed in Supplementary Table S1 . SmMYB1 proteins were expressed in E. coli via induction with 0.5 mM isopropyl b-d -1-thiogalactopyranoside (IPTG), followed by incubation at 37 °C for 3 h and purification using amylose resin (Shanghai Sangon Biotech Co., Ltd.).
3
Purification and Characterization of SlPHR Transcription Factors
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The coding sequences of SlPHR1, SlPHR4, SlPHR10, SlPHR11, and SlPHR12 were amplified from the corresponding LacZi-SlPHR recombinant using specific primers (SlPHRs-MBP-F/R) and inserted into the pMAL-c5X vector using T4 DNA Polymerase. SlPHR1-, SlPHR4-, SlPHR10-, SlPHR11-, and SlPHR12-MBP proteins were induced in E. coli BL21 (DE3) cells harboring the corresponding vectors and purified using Amylose Resin (Sangon Biotech, C500096). Biotin probes for P1BS and P1BS mutation (P1BSm) synthesized commercially. Probes were incubated with streptavidin agarose for 12 h at 4°C before pull down. The purified proteins were incubated with beads at 4°C, and beads were washed with PBS 5 times. Then, supernatants were detected by immunoblot (Wu, 2006; Yang et al., 2018) .
4
Biochemical Analysis of Fungal Rab1 GTPase Regulation
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The sequences of FgRab1 cDNA, FgGyp1 cDNA, TBC domain and R357K point mutant of FgGyp1 were cloned into the MBP (Maltose-binding protein) vector pMAL-c2X, using their respective primers listed in Supplementary Table 2, and expressed. The protein products were isolated and purified for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer's protocols. GTP hydrolysis was assayed based on previous reports (Xiong et al., 2012; (link)Zheng et al., 2020) (link). Briefly, the recombinant proteins MBP-FgGyp1, MBP-FgGyp1 TBC (TBC), MBP-FgGyp1 R357K (R357), and MBP-FgRab1 were expressed in BL21 Escherichia coli strain and isolated by Amylose resin (Sangon Biotech, NO.C500096) as per the instructions of the manufacturer. A colorimetry-based kit was used (Sigma-Aldrich, Catalog Number MAK113) to assay for the GTPase activity of Gyp1, and FgRab1 was incubated with FgGyp1, FgGyp1 TBC (TBC), FgGyp1 R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.
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