Split-ubiquitin yeast two-hybrid assay.

The A. fumigatus gprM, gpaA, gpaB, and gpaC open reading frames were synthesized and cloned into the pTMBV4 and pDL2XN plasmids, respectively (41). The genes of interest were expressed in Saccharomyces cerevisiae strain NMY32. The NMY32 strain was initially transformed with the gprM-bearing plasmid and later with one of the gpaA-, gpaB-, or gpaC-bearing plasmids. Then, we used these transformants for the split-ubiquitin membrane-based yeast two-hybrid assay. First, these strains were inoculated onto SD-Leu-Trp selective culture medium and grown overnight. The absorbance of the inoculum was measured through spectrophotometry, and later, all samples were standardized to an optical density at 600 nm (OD₆₀₀) of 1. Tenfold serial dilutions were made, and these dilutions were spotted on agar plates with different selective culture media, including SD-Leu-Trp plus X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), SD-Leu-Trp-Ade, SD-Leu-Trp-His, SD-Leu-Trp-His plus 3-AT (3-amino-1,2,4-triazole), or SD-Leu-Trp-Ade-His medium. The agar plates were incubated for 3 days at 30°C.