Transposon impala, a Novel Tool for Gene Tagging in the Rice Blast Fungus Magnaporthe grisea

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.

Heterologous transposition in Aspergillus nidulans

“…We show that heterologous transposition is possible at high frequency in A. nidulans. Both elements have been shown to be autonomous in F. oxysporum, and impala has been shown to be active in the closely related Fusarium moniliforme (Hua- Van et al, 2000). Heterologous transposition across different genera has not been shown before in fungi for DNA transposons, but it has been shown recently for retrotransposons (Nakayashiki et al, 1999).…”

Approaches to functional genomics in filamentous fungi

“…Of that library, 1.2% had a copy of Impala integrated in an essential gene. Impala has also been used successfully to tag genes in A. nidulans and Magnaporthe grisea [94,95].…”

The biology and potential for genetic research of transposable elements in filamentous fungi

“…The Tcl/mariner superfamily is the most abundant, the most studied elements of this superfamily being the fot1 and impala transposons of Fusarium oxysporum (Daboussi et al, 1992;Daboussi and Langin, 1994;Langin et al, 1995;Fernandez et al, 1998;Hua-van et al, 1998;Deschamps et al, 1999;Migheli et al, 1999;Chiocchetti et al, 1999;Hua-van et al, 2000;Rosevitch and Kistler, 2000;Hua-van et al, 2001ab;Villalba et al, 2001;Daviére et al, 2001;Hua-van et al, 2002;Daboussi et al, 2002;Daboussi and Capy, 2003). Members of this superfamily have inverted terminal repetitions (TIRs) of variable size and a Thymine/adenine (TA) target site.…”

“…The fot1 and impala elements have also been used to asses the genetic diversity of F. oxysporum isolates from different French soils (Edel et al, 2001). Villalba et al (2001) introduced the impala element into M. grisea where transposition of the element was revealed by excision of the niaD gene promoter and molecular analysis of the revertents using hybridization and sequencing. One mycelial growth mutant and a non- 806 Transposable elements in filamentous fungi Julien et al, 1992Anaya and Roncero, 1995Dobinson et al, 1993Farman et al, 1996bNakayashiki et al, 2001Kang, 2001McHale et al, 1992Zhu and Oudemans, 2000Diolez et al, 1995Kaneko et al, 2000Nielsen et al, 2001Neuvéglise et al, 1996Goyon et al, 1996Bibbins et al, 1998Hamann et al, 2000bMurata and Yamada, 2000Ruiz-Pérez et al, 1996 LTR retrotransposon Pseudoviridae (copia) mars 2, 3 tcen nht2…”

“…The fot1 transposition was demonstrated in A. nidulans (Li Destri et al, 2001) while the impala element is capable of transposition in several ascomycetes species, for example F. moniliforme (Hua-van et al, 2001b), M. grisea (Villalba et al, 2001), A. nidulans (Li Destri et al, 2001), A. fumigatus (Firon et al, 2003), C. gloeosporioides (Li Destri et al, 2002), and P. griseoroseum (De Queiroz and Daboussi, 2003). The isolation of genes of interest, such as a gene involved in M. grisea pathogenicity (Villalba et al, 2001), genes involved in A. nidulans development and metabolism (Brocard-Masson, 2001) and different genes essential for the growth of A. fumigatus (Firon et al, 2003) support the development of insertional mutagenesis tools in filamentous fungi. Other elements have also shown activity in heterologous species, e.g.…”