Differential gene expression analysis of MAT1-1 and MAT1-2 strains in Aspergillus oryzae

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 RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.