3.2. Agrobacterium‑mediated transformation

Agrobacterium tumefaciens is a plant pathogenic bacterium which causes crown gall disease in plants [58]. This bacterium contains tumor-inducing plasmid (Ti-plasmid) which can enter the plant cell and insert of small transfer DNA (T-DNA) into the infected plant cell genome. Using this principle, A. tumefaciens is used as a vector for gene transfer in various filamentous fungi. AMT was first used for genetic transformation in species of Aspergillus in 1998 by de Groot et al. [59]. Until now, AMT has been used for the exogenous DNA introduction in more than 10 fungal species (Table 2). Among these studies, Michielse et al. [63] well described the detailed protocol in A. awamori.

Lists of AMT in species of Aspergillus.

The AMT procedure includes construction of binary factor, transformation of A. tumefaciens with binary factor, preparation of fungal conidia, Agrobacterium-fungal cocultivation, and transformation selection (Figure 1(B)). AMT is different from PMT and its advantage is that it does not required protoplasts for host starter cell types [49]. Some researchers have used protoplasts as starting materials, but in most studies, conidia or germinated conidia are mainly used for starting materials [59,63]. AMT also has several advantages such as high transformation frequency, high gene-replacement frequency, and single-copy T-DNA integration [63]. As a result of conducting several transformation methods such as AMT, PMT, EP, or BT, AMT exerts high transformation efficiency and stability in A. giganteus and A. fumigatus [51,62]. However, unlike PMT, AMT requires the processes of construction of a binary vector, which containing a fungal selective marker and the gene of interest, and co-culturing A. tumefaciens and fungi, so it takes a longer time compared to other transformation methods. To make AMT a success, therefore, it is important to construct the appropriate vectors and optimize the culture condition, such as the ratio of Agrobacterium:conidia concentration, cocultivation condition, and acetosyringone concentration, to induce the vir gene [63].

EP is a highly efficient method for introducing exogenous DNA into a cell by applying a high-voltage electric pulse [76]. EP was used for the first time in A. nidulans, and at this time, protoplasts are being used for recipient cells of exogenous DNA [77]. To increase efficiency and reduce time by removing the protoplast preparation process, several research groups used germinated conidia or conidia in Aspergillus spp. including A. oryzae, A. niger, and A. nidulans [78–80] (Table 3). In addition, several factors should be optimized, including electric field intensity, pulse condition, DNA concentration, and buffer composition [49]. Although EP is a simple and convenient method compared to other transformation methods, expensive instrumentation is required for EP (Figure 1(C)). Currently, EP is rarely used by researchers for fungal studies.

Lists of EP-mediated transformation in species of Aspergillus.