• Abstract and Figures
• Public Full-text

Idnurm et al. Fungal Biol Biotechnol (2017) 4:6 DOI 10.1186/s40694-017-0035-0 Fungal Biology and Biotechnology REVIEW Open Access A silver bullet in a golden age of functional genomics: the impact of Agrobacterium‑mediated transformation of fungi Alexander Idnurm1* , Andy M. Bailey2, Timothy C. Cairns3, Candace E. Elliott1, Gary D. Foster2, Giuseppe Ianiri4 and Junhyun Jeon5 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 genet- ics, 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. Addition- ally, in several cases AtMT has paved the way for the development of new species to act as models for specifc 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 Background additional metaphor; DNA is shot into a fungal genome In 1998, Dunn-Coleman and Wang published a commen- to cause damage to a key gene, and thereby that can pro- tary on a newly described system for the transformation vide information on the strengths and weaknesses of the of foreign DNA into flamentous fungi using Agrobacte‑ fungus. Tis remarkable use of a plant pathogenic bacte- rium tumefaciens: the catchy term in their article’s title rium, A. tumefaciens, to transform fungi had frst been was that this method was potentially a “silver bullet” [1]. demonstrated in the model yeast Saccharomyces cerevi‑ We think of a “silver bullet” as a missile to combat were- siae just a few years earlier [2, 3], and then extended in wolves or other fantasy monsters, yet here there is an 1998 to seven species of flamentous fungi in both the Ascomycota and Basidiomycota lineages [4]. Within a decade from its frst reported use in S. cerevisiae, by 2005 *Correspondence: [email protected] over 50 fungal species had been transformed with A. 1 School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia tumefaciens [5]. In the decade since then, the use of A. Full list of author information is available at the end of the article tumefaciens mediated transformation (AtMT) continued © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Idnurm et al. Fungal Biol Biotechnol (2017) 4:6 Page 2 of 28 to expand to become a standard experimental technique more permanently into the genome [9]. Te Agrobacte‑ within the tool-box for gene manipulation in many fungal rium genus is within the family Rhizobiaceae and as such species. For some species it became the easiest or even it is closely related to the genus Rhizobium, members the only method by which to introduce foreign DNA. In of which also form intimate associations with plants to other species, it emerged as a powerful technique for for- fx atmospheric nitrogen. A. tumefaciens was renamed ward genetics, for use in the creation of large collections Rhizobium radiobacter [10], although the community of strains carrying random T-DNA insertions and their using this species as a transformation technology for analysis, for reverse genetics to create specifc targeted fungi continues to use the name A. tumefaciens. gene replacements, or for manipulation of gene expres- Before the development of genome sequencing pro- sion for biotechnological benefts. jects, the only known example of horizontal gene trans- In this review we describe the rise and infuence of fer from bacteria to eukaryotes was the trans-conjugation AtMT on the understanding of fundamental aspects of mediated by A. tumefaciens [11]. Agrobacterium naturally fungi. We describe species or groups of fungi in which exists in an environment where it encounters numer- AtMT has had greatest impact, some of the limitations ous hosts, including fungi that are likely to be present at that have subsequently emerged in applications, and the plant wounds, which induce T-DNA transfer. Knight areas of research or fungal species in which this trans- et al. [12] demonstrated that it is entirely feasible that formation technology did not have as great an impact. such transformation events happen in a natural environ- Understanding how this technology was implemented ment. Tey co-cultivated the plant pathogenic fungus can guide or anticipate the benefts of future technologies Verticillium albo-atrum on plant material alongside an for advancing research on fungi. Agrobacterium strain containing a plasmid that could It is not possible to include specifc details from all the potentially transform fungi, and observed transforma- publications reporting the use of AtMT on fungi, even if tion of the fungus under these in planta conditions [12]. covering those since the review by Michielse et al. [5]. A Of course, in the wild such an event is unlikely to deliver PubMed search of “Agrobacterium and fungus” returns any benefcial DNA sequence into the fungus, so may more than 900 papers, and as an example in the Crypto‑ well not confer any selectable advantage, but it is inter- coccus neoformans species complex alone AtMT has been esting to speculate on the frequency of such events over used in more than 30 studies. Other comprehensive and an evolutionary timescale: indeed, in addition to plants insightful reviews address specifc aspects of this tech- [9], genome sequencing projects have identifed Agrobac‑ nique, e.g. diferent vectors that are available [6], or the terium-like DNA in the genomes of some fungi such as proteins encoded by A. tumefaciens that are required Aspergillus oryzae [13]. to transform organisms [7, 8], which this review aims Plant molecular biologists altered wild strains of A. to complement. Finally, “impact” is relative in that what tumefaciens to their own advantage. Te bacterial strains may appear important to one set of researchers may not and genetic material were modifed to prevent gall for- to another set, while individual people may have per- mation, and to establish systems in which DNA for sonal favorite experiments or discoveries made using the transformation into a plant can be placed between two technique. direct repeats of 25 bp (the left and right borders of the transfer or T-DNA) (Fig. 1). From the perspective of Agrobacterium tumefaciens and how it transforms bacterial genetics, rather than transformation it is more species accurate to describe the movement of the T-DNA from Agrobacterium tumefaciens is a plant pathogen in the the bacterium into the eukaryotic host as a trans-conju- class α-Proteobacterium that is best known as one of gation method of gene transfer, in this case a conjugation nature’s natural agents in creating genetically modifed mechanism that is capable of occurring across diferent organisms. In this process, the bacterium inserts a piece species. Te promiscuous nature of A. tumefaciens, to of a plasmid into the nucleus of the plant host cell, and in target a wide diversity of hosts, enabled it to be applied to the wild that bacterial DNA encodes proteins that mod- numerous other eukaryotic species, with members of the ify the plant growth in favor of the bacterium. In most fungi being the best examples beyond the model plant circumstances, this results in the formation of a non- Arabidopsis thaliana [11]. proliferative gall or tumor-like growth on a plant, with One of the initial downsides of transformation of fungi alterations in the recipient genome that are not normally with Agrobacterium was the perceived infexibility with carried on into subsequent plant generations. However, the vector systems. Some of the Agrobacterium vectors analysis of the genome of sweet potato indicates that in were rather unwieldy, with limited restriction sites for rare cases these transformation events can be integrated conventional cloning and, having been developed for Idnurm et al. Fungal Biol Biotechnol (2017) 4:6 Page 3 of 28 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.

Load more

  28

Copy Link