Agrobacterium-mediated transformation as a tool for functional genomics in fungi

Abstract

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.

Approaches to functional genomics in filamentous fungi

“…In recent years, efficient transformation systems have been developed for a wide range of filamentous fungi (reviewed in [1,4,5]). Successful transformation of fungi has been achieved by: CaCl 2 /polyethylene glycol [6][7][8], electroporation [9-15], particle bombardment [2,6,10,16,17], and Agrobacterium tumefaciens-mediated transformation (AMT) [1].…”

“…Tailoring the right strategy for a particular project is a complicated task with many variables to consider. While Agrobacterium-mediated transformation (AMT) has been developed as a powerful tool for both random and targeted gene disruption and is increasingly being seen as the system of choice for many fungi (reviewed in [1]), there are other methods that offer advantages in specific situations. Also, some successful transformation systems may not be suitable for particular uses.…”

“…Some of these reviews are restricted to quite specific areas of this topic. AMT and its application to functional genomics in fungi has been comprehensively reviewed recently [1]. A review of RNA silencing in fungi, still in press at the time of writing, by Hitoshi Nakayashiki has just been completed [3].…”

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

“…Agrobacterium tumefaciens-mediated transformation of filamentous fungi has become an essential tool for functional genomics (Michielse et al 2005). The homo-basidiomycete ectomycorrhizal fungi Paxillus involutus, Suillus bovinus, Hebeloma cylindrosporum, Pisolithus spp., and Laccaria bicolor, and the ascomycete Tuber borchii (Grimaldi et al 2005) besides several other non-mycorrhizal fungi (Michielse et al 2005), have successfully been transformed with Agrobacterium.…”

“…The homo-basidiomycete ectomycorrhizal fungi Paxillus involutus, Suillus bovinus, Hebeloma cylindrosporum, Pisolithus spp., and Laccaria bicolor, and the ascomycete Tuber borchii (Grimaldi et al 2005) besides several other non-mycorrhizal fungi (Michielse et al 2005), have successfully been transformed with Agrobacterium. Transformation of H. cylindrosporum was carried out using the binary vector pBGgHg, as it had already been shown to be efficient for T-DNA transfer and integration into the Hebeloma genome (Combier et al 2003).…”