Agrobacterium Tumefaciens-mediated Transformation Of Magnaporthe Grisea

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 Magnaporthe grisea. We generated a library of more than1114 transformants with a high transformation efficiency of over 300 hygromycin B resistant transformants per 1×10⁶ conidia of M. grisea. All of the hygromycin B resistant transformants tested were mitotically stable after several subcultures onto complete medium without hygromycin.A rapid barley leaf assay method was used to screen pathogenicity-reduced mutants from these transformants. We identified four interesting mutants, A1-134,A1-452,A2-1-8 and A1-3-6, which were non- or reduced in pathogenicity to both barley and rice. Further phenotypic analysis showed that the A1-134 was significantly reduced in conidiation with only 7.8% conidia of the wild type strain, while A1-452 was increased in conidiation. A2-1-8 and A1-3-6 mutants with long shape conidium were unable to form appressoria and to infect hosts. With TAIL-PCR, we obtained the left genomic sequence flanking T-DNA from A1-134 mutant. By BLAST analyzing,we found the T-DNA insertional site located at 228bp downstream the stop codon of MGG₀6868.5, which was on the chromosome II . MGG₀6868.5 gene was made of 2475bp, including 5 extrons and 4 introns. The gene encodes acetolactate-synthase (ALS), a protein with 683 aa. Amino acid sequence of ALS was over 70% homologous to other filamentous fungi. The gene complemention vector has been constructed. Functional confirmation of ALS gene and identification of the tagging genes in other mutants were carrying on in the lab.