Identification of Pathogenicity Mutants of the Rice Blast Fungus Magnaporthe grisea by Insertional Mutagenesis

Abstract

Restriction enzyme-mediated DNA integration (REMI) mutagenesis was used to identify mutants of Magnaporthe grisea impaired in pathogenicity. Three REMI protocols were evaluated and the frequency of REMIs determined. An REMI library of 3,527 M. grisea transformants was generated in three genetic backgrounds, and 1,150 transformants were screened for defects in pathogenicity with a barley cut leaf assay. Five mutants were identified and characterized. Two mutants (2029 and 2050) were impaired in appressorium function. Two other mutants, 125 and 130, were altered in conidial morphology, conidiogenesis, and appressorium function. Mutant 130 was also a methionine auxotroph and methionine auxotrophy co-segregated with the reduction in pathogenicity. An additional mutant, 80, showed reduced pathogenicity on blast-susceptible rice cultivars but was fully pathogenic on barley. The reduction of pathogenicity in mutant 80 was associated with a delay in conidial germination and appressorium development. Genetic analysis suggested single-gene segregation for each mutant, but only two of the mutations co-segregated with the hygromycin resistance marker. The genetic loci in mutants 2029, 2050, 125, 130, and 80 were termed PDE1, PDE2, IGD1, MET1, and GDE1, respectively. pde1 and pde2 were non-allelic to cpkA, a mutation in the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A with a very similar phenotype. The results indicate the utility of REMI for studying fungal pathogenicity, but also highlight the requirement for rigorous genetic and phenotypic analysis.