Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae

Abstract

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.

Fig 1

Mating type genes of Aspergillus oryzae. (A) Schematic arrangements of the MAT loci in A. oryzae strains RIB40 and AO6. Arrows represent putative ORFs in regions adjoining the MAT loci. (B) Amino acid sequence alignment of MAT1-2 proteins from A. oryzae, A. flavus, A. fumigatus, and A. nidulans. The conserved HMG domain is boxed. Arrowheads indicate the locations of conserved introns.

Fig 2

Existence of both the MAT1-1 and MAT1-2 mating types in industrial A. oryzae strains from the RIB and IAM culture collections. (A) Construct for Southern blot analysis. (B) Mating type identification by Southern blot analysis. DNA fragments from the MAT1-1 and MAT1-2 genes were used as probes. MAT1-1 strains were RIB40, AO1 (RIB128), RIB177, RIB430, RIB609, and IAM2609. MAT1-2 strains were AO6, IAM2735, IAM2959, IAM2960, and RIB647.

Fig 3

Replacement of the MAT1-1 gene. (A) Strategy for replacement of the MAT1-1 gene with MAT1-2 by pyrG marker recycling. pyrG was excised by homologous recombination with direct repeats of the 0.3-kb upstream region of the MAT1-2 gene. (B) Verification of MAT gene replacement by Southern blot analysis. DNA fragments from MAT1-1 and MAT1-2 were used as probes. (C) Southern blot analysis of the strain from which pyrG had been excised. A DNA fragment from the MAT1-2 gene was used as a probe.

Fig 4

Analysis of gene expression in the MAT1-1, MAT1-2, and ΔMAT strains by qRT-PCR. qRT-PCR was performed for the AoppgA gene, putatively involved in the mating process, and for genes upregulated in the MAT1-2 strain (AO080505000070 and AoEST5207) to confirm the changes in gene expression detected by DNA microarray experiments. The actin transcript levels were used as internal standards, and the transcript levels for MAT1-1 were set at 100%. The data shown are means ± standard errors from three independent experiments.