Another strategy for identification of pathogenicity genes has been to introduce transposons in fungal genomic DNA cloned into cosmid vectors (Hamer et al. 2001). The insertion site of the transposon is sequenced to assess what function has been disrupted. Each tagged insert is then transformed into the source fungus to create a disruption of the sequence in the genome by gene replacement. This method has been adapted to a high throughput scheme and the resulting mutants are then tested for phenotypic alterations using a battery of tests. The success of this scheme is not dependent on access to the full genome sequence of a fungus.
Agrobacterium tumefaciens T-DNA is also being used to directly mutagenize the genome of a number of fungi (De Groot et al. 1998; Mullins et al. 2001; Rho et al. 2001) as well as plants such as rice (An et al. 2001) and Arabidopsis (see the following: Krysan et al. 1999). M. grisea and F. oxysporum mutants altered in virulence have been identified (Mullins et al. 2001; Rho et al. 2001). The randomness of insertion in the target genome and the possibility that some mutants may be caused by secondary events needs to be thoroughly evaluated in fungi, nevertheless, this method provides a straightforward way of tagging genes in fungi and appears to be superior to restriction enzyme-mediated insertion (REMI) mutagenesis in which the transforming DNA containing a selectable marker is linearized and transformed with a restriction enzyme to promote insertion at restricted sites in the genome (Balhadere et al. 1999; Bolker et al. 1995; Lu et al. 1994; Sweigard et al. 1998). This method did not generate certain kinds of expected mutants presumably due to the lack of sites for this enzyme in the genes or the inaccessibility of the genes to enzyme because of their lack of expression under the conditions of growth and/or transformation. Mutants lacking a DNA insertion in the mutated gene have also been documented using this method. Thus reisolation of the mutant gene is not simple.
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