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 int roduced into the rice blast fungus Magnaporthe grisea to develop transposon -based insertional mutagenesis, A construct (pNIL160) containing an autonom ous impala copy inserted in the promoter of niaD encoding Aspergillus nidul ans nitrate reductase was introduced by transformation into a M. grisea nit rate reductase-deficient mutant. impala excision was monitored by restorati on of prototrophy for nitrate. Southern analysis of niaD(+) revertants reve aled 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 l eft a typical excision footprint of 5 bp, We have shown that a defective im pala copy was inactive in M. grisea, yet it tan be activated by a functiona l impala transposase. A. transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants, Mutants either altered fo r their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained, Comp lementation of Rev77 with a 3-kb genomic fragment from a wild-type locus wa s successful, demonstrating the tagging of a pathogenicity gene by impala. This gene, called ORP1, is essential for penetration of host leaves by M, g risea and has no sequence homology to known genes.