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

DNA insertional mutagenesis is one of the most effective ways to identify pathogenicity genes of phytopathogenic fungi. In this paper, the binary vector, pATMT1, was transformed into Agrobacterium tumefaciens strain AGL-1 using electric transformation approsch. A library of more than 1500 hygromycin resistant transformants with a high transformation efficiency of over 250 transformants per 1×10⁶ conidia of Magnaporthe grisea was successfully generated by Agrobacterium tumefaciens-mediated transformation (ATMT). All tested hygromycin resistant transformants were mitotically stable after several subcultures onto complete medium without hygromycin. A rapid barley leaf assay method was used to screen mutational transformants for reduced pathogenicity. We obtained two pathogenicity defective mutants, A1-192 and A1-139. In addition, five mycelium growth reduced and eight conidiation redued transformants were identified.A1-192 strain was unable to infect the leaves of susceptible barley and rice. Similarly, A1-139 mutant lost the ability to cause disease on both unwounded and wounded leaves of barley and rice, indicating penetration and infectious growth were blocked in A1-139 mutant. Further phenotypic analysis showed that A1-139 was significantly reduced in conidiation with only 0.7% conidia of the wild type strain and lost the ability to form appressoria on hydrophobic surfaces.Southern blot analysis showed that single copy T-DNA was inserted into the A1-192 genome, whereas two copy T-DNA insertional events were occurred in A1-139. These data suggested that an important biological process blocked in A1-192 was likely to be due to the insertion of T-DNA and the subsequent disruption of gene function. However, the phenotypic alterations in A1-139 might be caused by one or two loci of T-DNA insertion.T-DNA flanking sequences of A1-192 and A1-139 mutants were amplified by an improved nested inverse-PCR and HiTAIL-PCR protocols, The PCR amplicons were cloned and sequenced. The results of BLAST search and alignment analysis showed that the T-DNA inserted site in A1-192 mutant was located at the position of 456bp upstream from the start codon of a MAPK gene-MPS1(MGG₀4943.5), and the two T-DNA inserted sites in A1-139 were located in the codon DNA sequence (CDS) of two genes (MGG₀1057.5 and MGG₁0568.5).