ABSTRACT
impala, a Tc1-mariner transposable element from Fusarium oxysporum, was introduced into the rice blast fungus Magnaporthe grisea to develop transposon-based insertional mutagenesis. A construct (pNIL160) containing an autonomous impala copy inserted in the promoter of niaD encoding Aspergillus nidulans nitrate reductase was introduced by transformation into a M. grisea nitrate reductase-deficient mutant. impala excision was monitored by restoration of prototrophy for nitrate. Southern analysis of niaD+ revertants revealed that impala was able to excise and reinsert at new loci in M. grisea. As observed for its host Fusarium oxysporum, impala inserted at a TA site left a typical excision footprint of 5 bp. We have shown that a defective impala copy was inactive in M. grisea, yet it can be activated by a functional impala transposase. A transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants. Mutants either altered for their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained. Complementation of Rev77 with a 3-kb genomic fragment from a wild-type locus was successful, demonstrating the tagging of a pathogenicity gene by impala. This gene, called ORP1, is essential for penetration of host leaves by M. grisea and has no sequence homology to known genes.
Subject(s)
DNA Transposable Elements , Fungal Proteins/genetics , Magnaporthe/genetics , Oryza/microbiology , Schizosaccharomyces pombe Proteins , Amino Acid Sequence , Blotting, Southern , Cell Cycle Proteins/genetics , Cell Cycle Proteins/isolation & purification , Cell Cycle Proteins/metabolism , DNA, Fungal/analysis , DNA-Binding Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Fusarium/genetics , Magnaporthe/pathogenicity , Molecular Sequence Data , Mutagenesis, Insertional , Nitrate Reductase , Nitrate Reductases/genetics , Nitrate Reductases/metabolism , Open Reading Frames , Sequence Alignment , Sequence Analysis, DNA , Transformation, Genetic , Transposases/geneticsABSTRACT
The fungal wheat pathogen Mycosphaerella graminicola was transformed to carbendazim and hygromycin B resistance. A beta-tubulin gene from a M. graminicola strain resistant to the fungicide carbendazim was cloned and used to transform a sensitive strain to carbendazim resistance. Hygromycin B-resistant transformants (up to 8 per microgram of transforming DNA) arose at a higher rate than beta-tubulin transformants (up to 0.6 per microgram of DNA). Transformants were able to infect wheat and were morphologically similar to recipient strains.
