Molecular Mechanism Of Resistance To Isoprothiolane In Magnaporthe Oryzae

Rice blast is caused by the ascomycete fungus Magnaporthe oryzae,and is one of the most devastating diseases of rice worldwide.Isoprothiolane(IPT),a systemic fungicide,has been applied to control rice blast since 1970s,and it inhibits M.oryzae mycelial growth and infection peg formation.It was considered to be associated with trans-methylation in the biosynthesis of phosphatidylcholine similar with iprobenfos(IBP).However,its mode of action and target protein still have not been elucidated.In this study,whole-genome sequencing combined with Genome Analysis Toolkit analysis was applied to detect polymorphisms in lab-generated resistant mutants and their parental wild type isolates.In two genome-sequenced resistant mutants,single point mutations were detected in a gene encoding a putative Zn₂Cys₆ fungi specific transcription factor-like protein.Point mutations and insertion were verified in the Fungl?TF?MHR domain from three resistant mutants of the Zn₂Cys₆ transcription factor-like protein.Based on genetic transformation,the gene was confirmed to be associated with IPT resistance,and was designated as Mo IRR(Magnaporthe oryzae isoprothiolane resistance related).Resistant mutant and Mo IRR knock out transformants did not show any resistance to other fungicide except for IPT and IBP.No significant differences for mycelial growth,conidial production and conidial germination among wild type isolates,mutants and transformants were observed.Furthermore,all resistant mutants and transformants showed similar pathogenicity as the wild type isolates.These results indicated that Mo IRR plays an important role in resistance to IPT in M.oryzae.Interestingly,it was found that expression levels of type I PKS cluster biosynthetic genes were reduced in resistant mutants and in Mo IRR knock-out transformants,suggesting that Mo IRR is involved in polyketide biosynthesis.Transcriptome analysis of resistance mutant 1a?mut,knock out transformant?Mo IRR-1 and parental wild type H08-1a revealed that Mo IRR contributed to the biosynthesis of secondary metabolites and metabolic pathways,suggesting that biosynthesis of secondary metabolites and metabolic pathways may involve in resistance to IPT in M.oryzae.Two genes in high osmolarity pathway were significantly up regulated in?Mo IRR-1 and 1a?mut may be related with potential target of IPT.