The plasmids pg5′CAoatg1, pg3′downAoatg1, the Entry Clone plasmid containing the A. oryzae adeA gene as a selective marker (constructed in our laboratory),

and the destination vector pDEST™R4-R3 (Invitrogen) were then subjected to the Gateway LR reaction using the Gateway LR Clonase Reaction Mix (Invitrogen) to generate plasmid pgA1EG. Using plasmid pgΔAoatg1 as a template, the sequence containing the deletion cassette, which consisted of the C-terminal region of Aoatg1 (0.8 kb), egfp and adeA genes (2.9 kb), and 1.5-kb downstream region of Aoatg1, was amplified by PCR with the primers pg5′aoatg1locusF and pg3′aoatg1-locusdownR, and then transformed into A. oryzae

NSRku70-1-1. selleck products The recombination of the Aoatg1 and egfp genes was confirmed by Southern blotting using a 2.0-kb fragment of the region downstream of Aoatg1 MAPK inhibitor as a probe, which was generated by PCR with the primers downAoatg1-F and downAoatg1-R. The plasmid pgaA1, which harbored the amyB promoter, Aoatg1 gene, and selection marker niaD, was constructed to overexpress AoAtg1 under control of the amyB promoter using the Multisite Gateway cloning system. The pgaA1 plasmid was transformed into A. oryzae niaD300. We first identified an A. oryzae ATG1 homolog, Aoatg1, in the A. oryzae genome database (http://www.bio.nite.go.jp/dogan/project/view/AO) using the BLAST algorithm. 5′-and 3′-RACE analyses revealed that Aoatg1 contained one intron and two exons, and encoded a predicted polypeptide of 986 amino acids with a calculated molecular mass of 107 kDa. AoAtg1 displayed 25% identity to Atg1 of S. cerevisiae and, as determined from the Pfam database, had an Atg1 kinase domain identified in the Pfam database (http://pfam.sanger.ac.uk/) (Supporting Information, Fig. S1). To determine the localization of AoAtg1, we constructed strain A1EG, which expressed the fusion protein AoAtg1–EGFP under control of the native promoter. After culturing A1EG for 24 h at 30 °C in CD + m medium to promote growth, the

strain was transferred (-)-p-Bromotetramisole Oxalate to nitrogen-deprived medium (CD − N) and further cultured for 4 h to induce autophagy. In CD + m medium, AoAtg1–EGFP localized to PAS-like structures and in the cytoplasm (Fig. 1, left). After starvation in CD − N medium, the number of punctate fluorescent spots had clearly increased (Fig. 1, right). These results were consistent with the reported localization of Atg1–GFP in S. cerevisiae, in which the number of the PAS increased after the induction of autophagy (Cheong et al., 2008). To investigate the function of AoAtg1, we disrupted Aoatg1 by the replacement with the selective marker adeA and confirmed the mutation by Southern blot analysis (Fig. S2).