Agrobacterium tumefaciens-mediated transformation of filamentous fungi consists of (i) induction of A. tumefaciens culture harbouring a binary vector, (ii) co-incubation of bacteria with fungal spores on a solid support, and (iii) selection of transformants. During the induction, vir genes on the helper component of the binary vector are activated, conditioning A. tumefaciens for transformation. During co-cultivation, T-DNA part of the binary vector system is transferred into fungal nucleus and inserted into the genome. Transformants are selected on a medium with appropriate antibiotic. In order to maximize the number of transformants, the ratio of A. tumefaciens cells to fungal spores and the duration of the co-cultivation need be optimized. The procedure takes two to three weeks for fast-growing fungi.
Kingdom Fungi consists of about two million species1, which include pathogens and symbionts of plants and animals and major biomass decomposers essential for all ecosystems. From the human perspective, fungal fruit bodies are consumed directly and many traditional food products are manufactured by fungal fermentation. Industrially produced fungal cultures serve as the source of enzymes, antibiotics, vitamins, amino acids and other substances2, 3.
The growing number of sequences of fungal genes and entire fungal genomes, together with transcriptomics and proteomics data accumulating on model species, have generated numerous of hypotheses about the biological function of fungal genes. Many of these hypotheses exist as sequence annotations and are based on indirect evidence such as sequence comparisons and data on the regulation of gene expression. Verifying the accuracy of this annotation often is the major limiting step in the analysis of fungal genomes. These hypotheses normally are tested by gene inactivation, which can be achieved either by gene disruption via homologous recombination or by RNAi-mediated gene silencing. A crucial prerequisite for both methods is an efficient genetic transformation protocol.
There are four techniques suitable for the genetic transformation of filamentous fungi
i. treatment of protoplasts with polyethylene glycol,
iii. biolistic methods
iv. Agrobacterium tumefaciens.
Before de Groot et al.'s introduction of A. tumefaciens into fungal transformation4, the most common method for the genetic transformation of filamentous fungi was treatment of a mixture of fungal protoplasts or spheroplasts and DNA with calcium salt and polyethylene glycol. The difficulty of protoplast preparation and regeneration, high batch-to-batch variation in the quality of enzyme cocktails used to digest fungal cell wall, and the low yields of transformants for some species were all important drawbacks. Electroporation and biolistic techniques were used rarely with relatively low yields5 and unstable transformants6 being common complaints. Since 1998, A. tumefaciens-mediated transformation of fungi has become a viable substitute for protoplast/polyethylene glycol method7.
A. tumefaciens is a soil-borne plant pathogenic bacterium that genetically manipulates its hosts by transferring a fragment of DNA (T-DNA) from its plasmid (Ti plasmid) into the plant genome8. T-DNA contains genes encoding enzymes of biosynthetic pathways for phytohormones and opines. Expression of these genes leads to the formation of tumors which serve the pathogen as an ecological niche and supplies it with nutrients. By replacing these genes with a cassette containing a gene to be transferred into the plant genome and a selectable marker, the A. tumefaciens-mediated transformation process can, and has been, used extensively for the past 20 years to genetically engineer plants. Protocols for A. tumefaciens-mediated transformation of dicotyledons such as Arabidopsis thaliana9 and soybean10 and monocotyledons such as maize11 and rice12 are well established. Since the discovery by de Groot et al.4 that chemical treatment of A. tumefaciens also enables this bacterium to genetically transform fungal mycelium, this technique has become available for the manipulation of fungal genomes. Similarly to the plant transformation process, a binary vector system consisting of a small plasmid with T-DNA and a second, large plasmid carrying the genes necessary for the process (vir genes) usually is used. The technique is suitable for both large-scale random insertional mutagenesis and targeted gene replacement. Although the majority of fungal species transformed using A. tumefaciens so far were Ascomycetes (58 species), the method has also been successfully applied to Basidiomycetes (15 species), Zygomycetes (4 species) and even fungus-like protists Oomycetes (3 species). A direct comparison of the protoplast method with A. tumefaciens-mediated transformation is not possible because the efficiencies are calculated in different ways: the number of transformants per µg DNA is used in the former and the number of transformants per plate or experiment in the latter method. In our hands, establishing A. tumefaciens-mediated transformation for a new species is faster and easier as compared to the protoplast method.
Fungal transformation mediated by A. tumefaciens consists of three steps: Induction of a bacterial culture harboring appropriate plasmids, co-incubation of the culture with fresh fungal spores on a solid support, and selection of transformants on a medium with a selection agents (figure 1). We optimized the method extensively and have used it routinely for the transformation of five species during the last six years.