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.

PDE1 Encodes a P-Type ATPase Involved in Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe grisea

“…This, in turn, triggers multiple mitogen-activated protein kinase cascades, resulting in differentiation of the appressorium, transport of lipid and carbohydrate reserves to the infection cell, and generation of appressorial turgor (Mitchell and Dean, 1995; Xu and Hamer, 1996;Choi and Dean, 1997;Liu and Dean, 1997; Xu et al, 1997 Xu et al, , 1998Adachi and Hamer, 1998;Dixon et al, 1999; Thines et al, 2000). How appressorium development culminates in plant infection, however, is less clear because the genetic components required for penetration peg formation have not yet been identified.Our aim in this study was to identify and characterize the PDE1 gene, which had been defined previously by identification of an insertional mutant impaired in plant infection (Balhadère et al, 1999). When we analyzed the plant infection process in pde1 mutants at the ultrastructural level, we found that cuticle penetration was reduced dramatically, and the only penetration structures observed were nonpolarized hyphae that were limited to the initial epidermal cell.…”

“…Among the mutants we identified was a penetration-defective mutant called pde1 . The pde1 mutant exhibited reduced appressorium-mediated penetration and produced very few disease symptoms when inoculated onto a susceptible plant host (Balhadère et al, 1999). In this article, we report that PDE1 encodes a P-type ATPase belonging to a family of aminophospholipid translocases.…”

Approaches to functional genomics in filamentous fungi

“…Although the enzyme type and concentration have an important impact on the number of transformants and the frequency of single-copy integrations, there are no clear rules for selecting either [56,64,66], and the npg concentration of RE that produces the maximum number of transformants is enzyme and host dependent [66][67][68]. REMI can give rise to a significant number of different integration events including single insertion with deletion of flanking RE sites, ectopic integration in the absence of an appropriate RE site, tandem insertion and large genome deletions or inversions [50,56,64,65,68,69]. The high frequency with which the mutant phenotype is not linked to the integrated DNA greatly complicates the use of this system for gene discovery.…”

Promoter trapping in Magnaporthe grisea

“…Pathogenicity of some transformants was assessed with a rice CO-39 cut leaf assay (Balhadère et al, 1999). Leaf segments were excised from the second leaf of a 14-d-old rice seedling.…”

“…In recent years, many techniques have been developed to identify functional genes in M. grisea (Kamakura et al, 1999;Rauyaree et al, 2001;Takano et al, 2003;Irie et al, 2003;Lu et al, 2005). Of these techniques, insertional mutation method including REMI (restriction enzyme-mediated insertional mutagenesis) (Balhadère et al, 1999) and ATMT (Agrobacterium tumefaciens-mediated transformation) (Rho et al, 2001) is a powerful way for functional genomic analysis. This technique has been successfully used to transform diverse filamentous fungi and isolate important genes (Mullins and Kang, 2001) relying on screening a large number of transformants.…”