The Obtaining Of A Nonpathogenic Mutant Ly-130 Of Magnaporthe Grisea And A Primarily Functional Study On The Tagged Gene MgPRG1

The rice blast disease caused by the fungal pathogen Magnaporthe grisea is one of the most destructive diseases of rice.To identify the fungal pathogenicity genes and to understand their molecular mechanisms for pathogenicity are important for the control of rice blast disease.By Agrobacterium tumefaciens-mediated transformation (ATMT)technique,we obtained a nonpathogenic mutant Ly-130 and identified a T-DNA tagged gene.Furthermore,we primarily demonstrated MgPRG1 function by complementation analysis and detected its expression and subeellular localization by GFP(green fluorescent protein)fusion expression.Obtaining of a nonpathogenie mutant Ly-130 DNA insertional mutagenesis is one of the most effective methods to identify pathogenicity genes in fungal pathogen.By ATMT technique,we isolated a Ly-130 mutant of M.grisea,width was completely nonpathogenic to both susceptible barley and rice.Phenotypic analysis showed that the mutant was significantly reduced in vegetative growth,conidiation and unable to form appressoria on hydrophobic surfaces.The colony color of the mutant was lighter than that of the wild type strain Guy11.No perithecia were observed when the mutant was crossed with a standard tester strain TH3.Identification of MgPRG1 gene To identify the disrupted gene in Ly-130,the genomic DNA flanking T-DNA was amplified using hiTAIL-PCR.The tertiary 852bp PCR product for the right border was purified and cloned into pGEM-T easy vector. The sequencing and BLAST results showed that T-DNA inserted in a gene of MGG₁4008(chromosome V)with 1617bp containing 4 exons and 3 introns.The T-DNA insert site was located at 1111bp downstream the start codon in the third exon. This gene encodes a predicted protein of 467 amino acids.After NCBI BLAST search, we did not find any high homogenous sequences to the predicted protein.It has respective similarity of 48.25%,43.85%and 33.25%to the sequences of XP₉63944.1 from Neurospora crassa,XP₀01912592.1 from Podospora anserine and XP₀01220992.1 from Chaetomium globosum.Moreover,no functional analysis has been done on these sequences.We thus named the corresponding gene MgPRG1 (for Magnaporthe grisea pathogenicity-related gene 1).Complementation of the mutant Ly-130 To determine whether the phenotypes of Ly-130 were caused by a T-DNA insertion of MgPRG1,we constructed a complementation vector pPRG1-GFP containing the MgPRG1 and GFP fusion.We transformed pPRG1-GFP into the Ly-130 mutant and obtained 58 transformants. Complementation analysis showed that the selected transformant Ly-130-R recovered all the wide type phenotypes in vegetative growth,conidiation,appressorium formatiom,mating and pathogenicity.Furthermore,confocal microscopy on PRG1-GFP expression showed that MgPRG1 expressed in the cytoplasm of fungal mycelia,conidia and appressoria and that MgPRG1 expression in appressoria increased during appressorial development.