A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi

Abstract

The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

“…The development of the T-DNA binary vector system allows for convenient introduction of the gene of interest into the bacterium T-DNA. 28 The Agrobacterium genus contains strains that can transform a wide range of host cells, including plants, fungi, and mammalian cells. 29 The schematic overview of ATMT system is provided in Fig.…”

“…31,32 It is more likely to integrate a single copy of gene than restriction enzyme mediated transformation (REMI), allowing precise genetic manipulations sequentially in the same locus. 28 In addition, ATMT is applicable to many fungal species, removing the restriction on using model species for molecular biology experiments. 28 Thus, ATMT is widely used in the production of transgenic crops and in the plant and fungal genetic studies.…”

21st century miniguide to fungal biotechnology

“…Indeed, a major attraction of AMT is the variety of starting materials that can be used: protoplasts, spores, mycelium, and pieces of fruit body tissues have all produced successful transformation. The approach seems to be applicable to the full range of fungi (zygomycetes, Ascomycota and Basidiomycota) and shows great potential for fungal biotechnology and medicine (Michielse et al, 2005;Sugui et al, 2005;Idnurm et al, 2017).…”

GFP transformation sheds more light on a widespread mycoparasitic interaction

“…To develop Ampelomyces strains constitutively expressing GFP we utilized ATMT, 'a silver bullet in a golden age of functional genomics' 54 . The transformation ratios were similar to those achieved in other studies 55 .…”