Agrobacterium Tumefaciens-mediated Transformation Of Magnaporthe Grisea And Identification Of A Pathogenicity Defective Mutant

Magnaporthe grisea is a causal agent of rice blast and diseases of many other different grasses. Understanding of the molecular basis of this disease is not only benificial for rice blast control, but also can serve as a model for revealing of other fungal pathogen-plant interactions. Obviously, identification of genes required for pathogenicity is a key step toward achieving this goal. DNA insertional mutagenesis is one of the most effective ways to identify pathogenicity genes of phytopathogenic fungi. Agrobacterium tumefaciens-mediated transformation is a new technique for insertional mutagenesis of filamentous fungi, and it has many advantages such as high efficiency, low cost, easy operation and good repetition.In this paper, based on the construction of two binary vectors, we report the use of Agrobacterium tumefaciens-mediated transformation as a successful method for insertional mutagenesis in the rice blast fungus M. grisea. We generated a library of more than 4000 transformants with a high transformation efficiency of over 300 hygromycin B resistant transformants per 1 × 10~6 conidia of M. grisea. All of the hygromycin B-resistant transformants tested were mitotically stable after several subcultures onto complete medium without hygromycin. Genomic Southern blot analysis showed that about two thirds of the transformants were single T-DNA insertional events. A rapid barley leaf assay method was used to screen mutational transformants for reduced pathogenicity. We identified an interesting mutant, A1-412, which was completely nonpathogenic to both barley and rice and also lost the ability to undergo infectious growth through abraded leaves. Further phenotypic analysis showed that the Al-412 was significantly reduced in conidiation with only 7% conidia of the wild type strain, was unable to form appressorium on hydrophobic surfaces and was...