Agrobacterium-mediated transformation as a tool for functional genomics in fungi.
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.
Michielse
Caroline B
CB
Institute of Biology, Clusius Laboratory, Fungal Genetics Research Group, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
Hooykaas
Paul J J
PJ
van den Hondel
Cees A M J J
CA
Ram
Arthur F J
AF
D001426:Bacterial Proteins; D001483:Base Sequence; D004271:DNA, Fungal; D016681:Genome, Fungal; D023281:Genomics / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001426:Bacterial Proteins; D004271:DNA, Fungal