Based on the construction of two binary vectors containing hygromycin B, the use of Agrobacterium tumefaciens-mediated transformation as a successful method for insertional mutagenesis in rice blast fungus Magnaporthe grisea was reported. A library including more than 4000 transformants with a high transformation efficiency of over 300 hygromycin B resistant transformants per 1×106 conidia of M. grisea was generated. 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. Through testing pathogenicity of mutational transformants by a rapid barley leaf assay method, 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 was identified. Phenotypic analysis showed that the mutant A1-412 was significantly reduced in conidiation only accounting for 7% conidia of the wild type strain, and was unable to form appressorium on hydrophobic surfaces and its germination was slightly delayed. Southern blot analysis showed that T-DNA inserted into the A1-412 genome was single copy. These data suggest that an important biological process blocked in A1-412 was likely to be due to the insertion of T-DNA and the subsequent disruption of gene function.
LIU Peng-juan ,WANG Zheng-yi ,WANG Qiu-hua et al. Agrobacterium tumefaciens-Mediated Transformation of Magnaporthe grisea and Identification of Pathogenicity Defective Mutant[J]. , 2006, 20(3): 231-237 .