Copyright 1999 by the Genetics Society of America Transposition of the Autonomous Fot1 Element in the Filamentous Fungus Fusarium oxysporum Quirico Migheli,1 Richard LaugeÂ, Jean-Michel DavieÁre, Catherine Gerlinger, Fiona Kaper, Thierry Langin and Marie-JoseÂe Daboussi Institut de GeÂneÂtique et Microbiologie, Universite Paris-sud, BaÃtiment 400, F-91405, France Manuscript received July 10, 1998 Accepted for publication November 20, 1998 ABSTRACT Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia1 revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so- called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences ¯anking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an ef®cient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element. Y their ability to move from one location in the (Kinsey and Helber 1989; Oliver 1992). However, B genome to another, transposable elements act as our picture of fungal transposons has recently been insertional mutagens. The resulting mutated gene can enlarged as the result of work on other species such then be isolated by using the inserted transposon as a as plant pathogens, industrial and ®eld strains. Active tag. Transposons have been used successfully to clone retroelements or DNA transposons exist in such fungal genes from many organisms (for review see Berg and genomes (Oliver 1992; Dobinson and Hamer 1993; Howe 1989), including different plant species such as Daboussi 1996). We have thus initiated studies to de- maize and snapdragon (Balcells et al. 1991; Gierl and velop a gene tagging system with the Fot1 element of Saedler 1992; Walbot 1992), Arabidopsis (Aarts et the fungal plant pathogen Fusarium oxysporum, the ®rst al. 1993; Bancroft et al. 1993; Long et al. 1993), and active DNA transposon reported in ®lamentous fungi Petunia (Chuck et al. 1993); animals like nematodes (Daboussi et al. 1992). (Greenwald 1985; Moerman et al. 1986), insects Fot1 was discovered as an insertion within the nitrate (Cooley et al. 1988; Engels 1989), and yeast (Boeke reductase (nia) gene. It is 1928 bp long and has short 1989) or bacteria (Berg et al. 1989). (44 bp) inverted terminal repeats. Fot1 expresses one Despite the great importance of ®lamentous fungi in mRNA of 1.7 kb, which extends over most of the element ecology, animal and plant pathology, or in metabolic and encodes a putative transposase of 542 amino acids production, no transposon-based gene tagging system (Deschamps et al. 1999). Like Tc1-mariner elements has so far been developed for these microorganisms. (Robertson 1995), Fot1 duplicates 2 bp (TA) upon This is probably because laboratory strains of the two integration, but the transposase encoded by this ele- best-studied ascomycetes, Neurospora crassa and Aspergil- ment shares no signi®cant sequence similarity with the lus nidulans, appear to be devoid of active transposons Tc1-mariner transposases. However, the Fot1 transposase has recently been related to other transposases includ- ing the Tiggers element from humans and the pogo ele- ment from Drosophila (Smit and Riggs 1996). These Corresponding author: Marie-JoseÂe Daboussi, Institut de GeÂneÂtique similarities between the putative transposases and other et Microbiologie, Universite Paris-sud, BaÃtiment 400, 91405 Orsay Cedex, France. E-mail: [email protected] shared features suggest that Fot1 belongs to the Tc1- 1Present address: DI.VA.PRA., University of Torino, via Leonardo da mariner superfamily of DNA transposons. Vinci, I-10095 Grugliasco (To), Italy. The Fot1 element is widely distributed in the F. oxy- Genetics 151: 1005±1013 (March 1999) 1006 Q. Migheli et al. sporum species with a variable copy number, ranging trol of the A. nidulans trypC promoter, which was inserted into from 0 to .100 (Daboussi and Langin 1994; this the SnaBI site in the coding region of Fot1. A third plasmid, pEC62-fr, with a frameshift mutation in the Fot1 coding region, study). To determine whether cloned Fot1 elements can was constructed by digesting pEC62 with XmaCI followed by transpose autonomously, mobility was assessed in strains ®lling-in with Klenow and recircularizing the construct. These devoid of endogenous elements by use of a phenotypic three plasmids contain a EcoRI fragment consisting of the niaD assay for excision. We present evidence that Fot1 trans- gene interrupted by a defective Fot1-62 copy but differing in poses in different strains of F. oxysporum. The success of size according to the alterations in the transposase gene, e.g., 3.7 kb in pEC62DBamHI, 6.0 kb in pEC62-hph C, and 4.6 kb these assays suggests that Fot1 will provide a valuable in pEC62-fr. tool for tagging genes involved in pathogenicity in this Transformation experiments: Protoplast preparation and economically important fungus. In addition, we report polyethylene glycol-mediated transformation of the nitrate re- on the mechanism of Fot1 transposition. ductase-de®cient mutants were conducted according to Lan- gin et al. (1990). A total of 5 mg of pAN7-1 plus 5 mgof one of the plasmids pEC136 and pEC62 was used in each cotransformation. Hygromycin-B-resistant colonies were trans- MATERIALS AND METHODS ferred to minimal medium containing 200 mg/ml of hygro- Fungal strains and media: The following wild-type strains mycin B (Sigma, St. Louis) and 20 mm glutamine, and single of F. oxysporum (all obtained from C. Alabouvette, INRA, Dijon, spore isolates were puri®ed on the same medium. A mycelium- France) were used: FOM24, pathogenic on melon, whose ge- agar plug of each hygromycin-B-resistant transformant was nome contains .100 copies of the Fot1 element; two non- placed on a Petri dish containing 10 ml PDA covered by a pathogenic strains, FO5 with one Fot1 copy and FO47 that is Hybond N nylon membrane (Amersham, Arlington Heights, 8 free of Fot1. From these strains, nitrate reductase-de®cient IL). After 2 days of incubation at 26 membranes were treated 3 mutants were selected on the basis of resistance to chlorate as for 30 min in NaOH 0.5 m,5 SSC (Sambrook et al. 1989) 3 described in Daboussi et al. (1989). Mutants nia321 (FOM24), and for 30 min in Tris-HCl 0.5 m, pH 7.5, 10 SSC on a nia13 (FO5), and nia1 (FO47), with point mutations in the rotary shaker at room temperature. DNA cross-linking was nitrate reductase structural gene (nia), were used as recipient performed by exposing the membranes to UV at 254 nm for 1 min. Cotransformed colonies were identi®ed by hybridiza- strains in the transformation experiments. Interestingly, the 32 nia13 (FO5) mutant lost the unique Fot1 copy present in the tion to the P-labeled 2.7-kb EcoRI fragment of the niaD gene. wild-type FO5 strain (T. Langin, J. M. Daviere, D. Fernandez DNA preparation and Southern blot analysis: DNA for and M. J. Daboussi, unpublished results) and thus is consid- Southern blot analysis and for inverse polymerase chain reac- ered in transformation experiments as a strain free of Fot1. tion (IPCR) was obtained by a miniprep extraction method niaD37, niaD136, and niaD62 are the mutants containing a (Langin et al. 1990). For Southern blot analysis z10 mgof Fot1 insertion within the niaD gene of A. nidulans introduced DNA was digested in the presence of 100 units of the restriction by transformation into the nia321 FOM24 recipient strain enzymes EcoRI, XhoI, BglII, or SacII (Boehringer, Indianapolis) (Daboussi and Langin 1994; Deschamps et al. 1999). The and fractionated through a 0.6% agarose gel. Southern trans- minimal and complete media and culture conditions used fer was performed by standard methods (Sambrook et al. were described previously (Daboussi-Bareyre 1980). Strains 1989) onto Hybond N nylon membranes (Amersham) and were stored as sporulated mycelium-potato dextrose agar the DNA was ®xed to the membranes by UV cross-linking. (PDA) plugs under mineral oil at 128. DNA probes correspond to PCR products. Primers used for Vector constructions: Plasmid p11DNdeI was ®rst generated the ampli®cation of the niaD-speci®c probe were niaD-144 (59- from plasmid pMJT2 (Langin et al. 1990), which contains the GTTCATGCCGTGGTCGCTGCG-39) and niaD-145 (59-CCC complete niaD gene of A. nidulans on a 7.6-kb BamHI frag- GGCCAAAGCCTCGAATTCG-39). For the Fot1-speci®c probe, ment, by removing the NdeI fragment, leaving only the 2.7- a unique primer (Ft1) deduced from the inverted terminal kb EcoRI fragment containing most of the niaD gene in the repeat sequence was used (59-AGTCAAGCACCC ATGTAACC BamHI/NdeI fragment. pFox15 was then derived from p11- GACCCCCCC-39). An hph-speci®c probe was obtained using DNdeI by the addition of a 5.0-kb NdeI fragment chosen at hph1(59-CAGCGAGAGCCTGACCTATTGC-39) and hph2(59- random from F. oxysporum strain FOM24 to increase the fre- GCCATCGGTCCAGACGGCCGCGC-39).