Transposon impala, a novel tool for gene tagging in the rice blast fungus Magnaporthe grisea

impala, a Tcl-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 tan 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.