Matchmaker gal4 two hybrid system 3

Sequences encoding the TPR domains of Pex5a and Pex5b were inserted into pGBKT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech) between the EcoRI and BamHI restriction sites via Gibson assembly. The ORFs for GFP or GFP modified with C-terminal dodecamers of Um01966, Um10665, Um11001, and Um03158 including PTS1 motifs were cloned into pGADT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech) between the EcoRI and BamHI restriction sites. Either pGBKT7-Pex5aTPR or pGBKT7-Pex5bTPR were co-transformed with one of the pGADT7 plasmids into YTS398, a derivative AH109 of deleted for pex5 (Stehlik et al., 2020 (link)). Three independent transformants of each of the 10 combinations were grown in liquid synthetic defined (SD) medium lacking leucine and tryptophan to an OD₆₀₀ of approx. 1. Cells were washed once with sterile water and 4 µl of fivefold or fiftyfold dilutions (OD₆₀₀ = 0.2 or 0.02) were spotted on solid SD medium lacking leucine and tryptophan as growth control, and on SD medium lacking leucine, tryptophan and histidine, and containing 1.5 mM 3-amino-1,2,4-triazole to test for protein – protein interaction. Plates were incubated for 3 days at 30°C.

The sequence encoding the TPR domains of Pex5 was inserted into pGBKT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech). The ORFs for GFP, GFP-SKL, or GFP-PTS1_Ptc5 were cloned into pGADT7 (Matchmaker GAL4 Two-Hybrid System 3; Clontech). pGBKT7-Pex5_TPR and derivatives of pGADT7 were co-transformed into YTS398. The transformed strains were grown in liquid SD medium, lacking leucine and tryptophan, to an OD₆₀₀ of 1.0. Serial dilutions of each strain were spotted on solid SD lacking leucine and tryptophan as growth control, and on SD medium lacking leucine, tryptophan, histidine to test for protein–protein interaction.

A yeast two-hybrid screen was performed to determine physical interactions of Smo1 and to investigate its function as a RasGAP, using the Matchmaker GAL4 Two-Hybrid system 3 (Takara Clontech). SMO1, RAS2, and RAS1 cDNA were cloned into the bait vector pGBKT7 using primer combinations 5′-smoGB/3′-smoGB, 5′-ras2GB/3′ras2GB, and 5′-ras1GB/ras1GB. SMO1, RAS2, and GEF1 cDNA were cloned into the prey vector PGADT7 using primer combinations 5′-smoGA/3′-smoGA, 5′-ras2GA/3′ras2GA, and 5′-gef1GA/gef1GA. Cloning was performed using in-fusion cloning (Takara Clontech). Sequencing was performed to ensure that constructs were in-frame (MWG operon). Yeast two-hybrid analysis was then carried out using the Matchmaker GAL4 Two-Hybrid system 3 (Takara Clontech) according to the manufacturer’s instructions (Wilson et al. 2010 (link)). For in vivo co-immunoprecipitation studies, total protein was extracted from lyophilized M. oryzae mycelium of strains expressing Smo1:GFP and ToxA:GFP (control) after growth in liquid CM for 48 hr. Protein extracts were co-immunoprecipitated using the GFP-Trap protocol, according to the manufacturer’s protocol (ChromoTek). Protein extracts were prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) and separated by SDS-PAGE. Gels were cut into slices and LC-MS/MS analysis was performed at the University of Bristol Proteomics Facility.