The resultant plasmids were named pg5′DAA and pg3′DAA, respectively.

The pg5′DAA, pgEaA, and pg3′DAA plasmids were used by LR recombination, and the DNA cassette from the resultant plasmid digested with PstI was used for A. oryzae transformation. For A. oryzae transformation, the DNA fragments or plasmids were introduced into each host strain using a standard method. Confirmation of aipA disruptants and transformants that contain a single plasmid with the niaD Epacadostat selective marker by Southern blot analysis was performed as described previously (Higuchi et al., 2009b). For YTH screening, we used the Matchmaker™ Library Construction and Screening Kit (Clontech) according to the manufacturer’s instructions. We constructed an A. oryzae cDNA library expressed in yeast as follows: total RNA (1 μg) from A. oryzae strain RIB40 [wild type (WT)] cultured in Czapek-Dox (CD) medium (0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, Small Molecule Compound Library and 2% glucose, pH 5.5) for 24 h was prepared using an RNeasy Plant Mini Kit (Qiagen). For the generation of first-strand cDNA, a random primer (CDS III/6 primer) was utilized. Double-strand cDNA and pGADT7-Rec were transformed into S. cerevisiae strain AH109, and the resulting transformants were used as the cDNA library for YTH screening.

For the generation of a strain as bait in YTH screening, the Aoabp1 coding sequence was introduced into the SmaI site of pGBKT7 and the resultant plasmid selleck chemicals was transformed to the S. cerevisiae strain Y187. For the generation of bait- and prey-expressing strains, pGBKT7 and circularized pGADT7-Rec, respectively, were used as vector plasmids. As negative controls, strains transformed with empty vectors were

used. Mated strains with both bait and prey proteins were grown on SD/−Leu/−Trp, SD/−Leu/−Trp/−His, SD/−Leu/−Trp/−His with 2 mM 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of S. cerevisiae His3p, or SD/−Leu/−Trp/−His/−Ade plates at 30 °C for 2 or 3 days. GST-fused bait proteins were prepared as follows: the coding sequences of two SH3 domains of AoAbp1 or AipA were introduced into pGEX-6P-1. The resultant plasmids, including empty pGEX-6P-1 as a negative control, were transformed to the Escherichia coli strain BL21 (DE3) pLysS. Glutathione Sepharose 4B (GE Healthcare) was utilized for the GST pull-down assay. Twenty microliters of glutathione sepharose beads (1 : 1 slurry) were washed mildly five times with 500 μL cold phosphate-buffered saline (PBS). Five hundred microliters of lysate with GST or GST-bait was added to the bead solution, which was then mixed with constant rotation at 4 °C for 2 h. After centrifugation at 500 g for 30 s at 4 °C, the beads were gently washed five times with 500 μL cold PBS containing 1% Triton X-100.