Transposition of the miniature inverted-repeat transposable element mimp1 in the wheat pathogen Fusarium culmorum

Abstract

High-throughput methods are needed for functional genomics analysis in Fusarium culmorum, the cause of crown and foot rot on wheat and a type B trichothecene producer. Our aim was to develop and test the efficacy of a double-component system based on the ability of the impala transposase to transactivate the miniature inverted-repeat transposable element mimp1 of Fusarium oxysporum. We report, for the first time, the application of a tagging system based on a heterologous transposon and of splinkerette-polymerase chain reaction to identify mimp1 flanking regions in the filamentous fungus F. culmorum. Similar to previous observations in Fusarium graminearum, mimp1 transposes in F. culmorum by a cut-and-paste mechanism into TA dinucleotides, which are duplicated on insertion. mimp1 was reinserted in open reading frames in 16.4% (i.e. 10 of 61) of the strains analysed, probably spanning throughout the entire genome of F. culmorum. The effectiveness of the mimp1/impala double-component system for gene tagging in F. culmorum was confirmed phenotypically for a putative aurofusarin gene. This system also allowed the identification of two genes putatively involved in oxidative stress-coping capabilities in F. culmorum, as well as a sequence specific to this fungus, thus suggesting the valuable exploratory role of this tool.

Figure 1

Southern blot analysis of revertants mimp1/impala. Genomic DNA of co‐transformant M7 and derived revertants was digested by XbaI and membranes were successively probed with: (A) a 419‐bp‐long niaD probe obtained by polymerase chain reaction (PCR) on the pAN301 plasmid using primers niaCG1 and niaCG2; and (B) a 120‐bp‐long mimp1‐specific probe amplified from the pNm1H18 plasmid using the primers mi1 and SacF. The revertants showed a single niaD band of 1.7 kb, i.e. 224 bp shorter than the corresponding band of the co‐transformant (approximately 1.9 kb), thereby demonstrating the excision of mimp1 from the nitrate reductase gene (A). The asterisks indicate reinsertion events of the mimp1 element. In lane 5 (black rectangle), the excision of mimp1 was not followed by a reinsertion event (B).

Figure 2

Putative localization of the distribution of mimp1 reinsertion sites in the closest genome available to F. culmorum: Fusarium graminearum (Gibberella zeae). The transposon reinserted evenly in all four chromosomes of G. zeae and was located in 23% of the analysed flanking regions within an open reading frame (ORF) or an intron.