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.

Keywords: Functional genomics; Mycota; Pathogenicity genes; Rhizobium radiobacter; Transfer DNA.

Fig. 1

A summary of the transformation process of fungi by Agrobacterium tumefaciens. At step a, acetosyringone present in the transformation media is recognized by the bacterium, triggering b the expression of genes within the chromosomal DNA and from the helper plasmid. At stage c, the T-DNA is excised from the T-plasmid. The linear DNA is bound by VirD2, which is recognised by the type IV secretion system for export from the cell, along with other Vir proteins (d). At step e, the DNA enters the fungal nucleus guided by nuclear localization signals on the bacterial-encoded proteins. In the final event f, the T-DNA inserts into the chromosomal DNA of the fungus. The T-DNA should encode a selectable marker such that the integration events can be selected

Fig. 2

Phylogeny of major lineages within the fungi. The relationships are based on references [204, 243]; note that some nodes remain poorly defined. For hierarchy: phylum -mycota, subphylum -mycotina, class -mycetes. The Pezizomycotina subphylum is split into seven classes in the phylogeny. Groups in which species have not yet been transformed successfully by Agrobacterium are in bold

Fig. 3

Databases of T-DNA insertion sites, mutant phenotypes, and strain availability have been developed for some fungal species. A screen shot of the front page of the ATMT Database (http://atmt.snu.ac.kr) that features information on T-DNA mutants of P. oryzae, primarily based on information from [38]. Limited funding for maintaining such databases and strain resources after projects end may undermine the potential impact of large-scale mutant screens

Fig. 4

Two examples of T-DNA insertion bias into the 5′ non-coding (or flanking) regions of the genes in Cryptococcus neoformans (a basidiomycete yeast and human pathogen) and Pyricularia oryzae (a filamentous ascomycete and plant pathogen). a Example of a single gene in C. neoformans targeted by T-DNA insertion on five occasions [157, 159, 160] causing loss of pigmentation. A schematic drawing of the LAC1 gene represented by an arrow shows coding regions as brown boxes, introns as white boxes and upstream and downstream non-coding sequences as a grey line. T-DNA insertions from five independent transformation events each causing loss of pigmentation all lie within the upstream region, with none to date in the coding region. R and L refer to the left and right border and the relative positions of these when the T-DNA inserted; note that the same site is targeted by two independent insertion events in opposite orientation. The plate shows C. neoformans wild type and one of the T-DNA insertional mutants growing on medium containing the substrate for laccase, l-DOPA. b Example of insertional bias of T-DNA into the genome of P. oryzae transformants. Results and figure are modified from Ref. [40]. An analysis of the distribution of 799 insertion sites mapped into 50 bp windows illustrates a twofold higher insertion frequency in promoter or untranslated regions compared to coding regions. This is even more striking in that such regions have a higher proportion of AT nucleotides while in P. oryzae T-DNA insertions are preferentially into higher GC content DNA