Tagging pathogenicity genes in Fusarium graminearum using the transposon system Mimp/Impala

The number of predicted genes present in the genome of Fusarium graminearum is estimated to be around 14,000. For many genes the function is yet unknown and consequently there is a need for a high-throughput method for functional analyses of genes. We applied a transposon mutagenesis strategy using a mite element (mimp1) activated by a transposase (impala). Previously we have shown that the double component system mimp1/impala transposase is fully functional in F. graminearum (Dufresne et al., 2006). Transposition characteristics and high reinsertion frequency were found to be the same as in the original host species, F. oxysporum, allowing the application of this double component system for the generation of large transposon mutant collections. We transformed the double component system into F. graminearum strain FG820, selected 100 revertants and determined the sequence flanking the mimp1 reinsertion sites using TAIL-PCR and the F. graminearum genome sequence. In 53% of the isolates mimp1 reinserted close to or in genes. Subsequently, a pilot collection of around 300 revertants from the same transformant (FG820-6-11) was screened for growth on a large set of media and for pathogenicity on wheat. Several revertants with altered phenotypes were identified and in one of them mimp1 reinserted into an ORF encoding a transcription factor (FG820-6-11-r112). The relationship between mimp1 insertion and the mutant phenotype is currently investigated by functional complementation. Our results indicate that this novel double component transposon system is a powerful mutagenesis tool for high-throughput analysis of F. graminearum and potentially other ascomycete fungi