Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis

Abstract

Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. 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 A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens 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 A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, 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 A. aculeatus.

Figure 1

Distribution of the regions with microhomology between the host genome and the left-border (A) and right-border (B) sequences. The open bars show the distributions of T-DNA possessing each microhomologous region, and the solid lines show the expected length of microhomology.

Figure 2

A frequency distribution for different size classes of recipient genome deletions among 13 T-DNA integration sites for which the sequences of both junctions were determined.

Figure 3

Complementation of albino mutants. (A) Diagram of the transformation vector for the A. aculeatus alb1 and alb2 mutants. (B) Pigmented colonies formed in transformants of alb1 with pAUR-pksP (left), but transformants with pAUR325 remained albino (right).