Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis

<jats:title>Abstract</jats:title> <jats:p> <jats:italic>Agrobacterium tumefaciens</jats:italic>-mediated transformation (AMT) was applied to <jats:italic>Aspergillus aculeatus</jats:italic>. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of <jats:italic>A. tumefaciens</jats:italic> and <jats:italic>A. aculeatus</jats:italic> cells, the duration of co-cultivation, and the status of <jats:italic>A. aculeatus</jats:italic> cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 10<jats:sup>4</jats:sup> conidia or 217 transformants per 10<jats:sup>7</jats:sup> conidia were obtained under the optimized conditions when <jats:italic>A. tumefaciens</jats:italic> co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino <jats:italic>A. aculeatus</jats:italic> mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene <jats:italic>AapksP</jats:italic>, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in <jats:italic>A. aculeatus</jats:italic>.</jats:p>