J. Gener., Vol. 75, Number 3, December 1996, pp. 325-339. ((') Indian Academy of Sciences Fungal transposable elements: generators of diversity and genetic tools M. J. DABOUSSI Institut de G6n6tique et Microbiologie, Universit~ Paris-Sud, 91405 Orsay, France Abstract. Representatives of several classes of transposable elements (TEs) have been characterized in a broad range of fungal species. The studies indicate flint these elements are ancient and ubiquitous components of fungal genomes. Some of these elements have been shown to actively affect gene structure and function in several ways: inactivation of gene expression upon insertion, modification of the nucleotide sequence through excision, and probably by inducing extensive chromosomal rearrangements. The ability o['TEs to generate a high degree of genetic diversity may therefore be important in the evolution of the fungal genome. TEs also have many potential applications in genetic research, including inserfional mumgenesis and population fingerprinting, as well as gene transfer within and between species. All these genetic approaches are important as tools in studies &molecular biology and evolution of fungal species, many of which lack a functional sexual cycle. Keywords. Transposable elements; mutagenesis; filamentous fungi; genetic application. 1. Introduction Filamentous fungi have proved to be valuable in the development of many fundamen- tal ideas: these include the one gene-one enzyme concept (Neurospora crassa), models for recombination (Ascobolus immersus), and regulation of gene expression (Aspergillus nidulans). However, there is one area where fungi, particularly filamentous fungi, were long silent; that is the area of transposable elements (TEs). It is now more than four decades since Barbara McClintock developed the concept of transposable genetic elements through genetic studies in maize. Since then, such elements have been found in a wide range of organisms from bacteria to higher eukaryotes (see Berg and Howe 1989 for a review) and are presumed to be ubiquitous. However, with the notable exception of Ty elements in the yeast Saccharomyces cerevisiae (Boeke 1989), very little was known until a few years ago about the incidence and properties of TEs in other fungal species. This was probably due to the fact that the laboratory strains of the best-studied ascomycetes, A. nidulans and N. crassa, appear to be devoid of active transposons. In another ascomycete, A. immersus, genetic elements with properties of classical transposons associated wih genetic instabilities were reported but not characterized at the molecular level (Decaris et al. 1978). In the past few years our picture of fungal transposons has grown considerably as a result of work with species from different phylogenetic groups and ecological situations: plant pathogens, and industrial and field strains (see reviews by Oliver 1992 and Dobinson and Hamer 1993). Many different classes of TEs are now known in different species of ascomycetes (listed in this review), and TEs have recently been reported in phycomycetes (Avalos et al. 1996) and basidiornycetes (Gaskell et al. 1995), opening a door of opportunity for genetic research. The aim of this paper is to review progress made in the molecular-genetic analysis of these fungal transposons. Emphasis will be on what is known about their mechanisms of transposition and their consequences to 325 326 M. J. Daboussi the host genome, the spread of transposons within and between species, and finally the potential use of transposons for genetic research. 2. Structural features of fungal transposons Fungal transposons have been identified using a variety of strategies (figure 1) that exploit the two main characteristics of TEs: (i) they are present in multiple copies in the genome and thus can be discovered by analysing dispersed repetitive sequences, and (ii) they can move from one position to another in the genome, and therefore can be trapped through their insertion in a selected gene. Using the first strategy, different transposon- like sequences have been recognized by comparison with the known transposon sequences from other organisms, although we do not know if these transposon-like sequences are still active. The most satisfactory way to screen for active transposons is the transposon trapping approach, which is based on spontaneous inactivation of a cloned gene. This strategy is usually applied to genes whose mutant phenotypes can be positively screened. Examples of this approach are the am gene coding for glutamate dehyd- rogenase which allowed the discovery of the Tad element in N. crassa (Kinsey and Helber 1989), and mutations in the nia gene encoding nitrate reductase which can be selected through resistance to chlorate (Cove 1976). Trapping using the nia gene has been very efficient in Fusarium oxysporum (Daboussi and Langin 1994) and has been successfully applied to other fungal species, leading to identification of at least eight different mobile elements. Finally, some other elements have been found using heterologous hybridiz- ation or have been discovered fortuitously through DNA sequencing. The TEs or sets of related sequences identified in fungal genomes reflect the whole spectrum of eukaryotic transposable elements. Their general structural features are summarized in figure 1. Representatives of the two classes of transposable elements are found in fungi: those that transpose by reverse transcription of an RNA copy of the element (class I) and those that probably transpose directly through DNA copies (class II) (Finnegan 1989; McDonald 1993). Class I elements are LTR (long terminal repeats) retrotransposons that belong mostly to the gypsy group: Foret and skippy in F. oxysporum (Julien et al. 1992; Anaya and Roncero 1995), grh and MAGGYin Magnaporthe grisea (Dobinson et aI. 1993; Farman et aI. 1996a), CfT-1 in Cladosporium fulvum (McHale et al. 1989, 1992), Boty in Botrytis cinerea (Diolez et al. 1995), Afutl in Aspergillus fumigatus (Neuv6glise et al. 1996a) and Mars4 in Ascobolus immersus (Goyon et al. 1996). Only Mars2 of A. immersus is a copia-like element (Goyon et al. 1996). Several non-LTR retrotransposons with structural features of LINEs (long inter- spersed nuclear elements) have been characterized: Tad, an element of N. crassa, the first transposable element reported in fungi (Kinsey and Helber 1989), MGR583 in M. grisea (Hamer et. al. 1989), Palm in F. oxysporum (Mouyna et al. 1996), Mars1 in A. immersus (Goyon et al. 1996), and CgT-I in ColIetotrichum gloeosporioides (He et al. 1996). SINE (short interspersed nuclear element)-like elments have also been recog- nized: Nrsl in Nectria haematococca (Kim er aI. 1995), MGSR1 and Mg-SINE in M. grisea (Sone et al. 1993; Kachroo et aI. 1995), as well as Eg-RI and EGH in Erysiphe (Rasmussen et at. 1993; Wei et aI. 1996). Class II or DNA transposons of different types have also been isolated, most of them after transposition in the nia gene. Some of them are representatives of families Fungal transposable elements 327 Class h Retroelements LTR-RETROTRANSPOSON8 type Example 5' LTR ~Ta~ pOt ~ ? 3' LTR Feint t~) and skippy f3; iF. e~ysporumJ gypsy grh O) and MAGGYCt.;) (M. grisee) CtT-I r futvum); Boty ~r tin~ Afu110) (A. fumJg~tus); Mars4 (.4, immersus) 5' LTR gag pol 3' LTR copia Mars2 (~ (A. immersus) non LTFI-RETROTRANSPOSONS 5. gag? 3' Tad ~2)(N, erassa); PalmOJ iF. oxysporum) MGR583 (1)(M, grisea); Mars1 (~)(A. imrnersLts) CgT1 (IJ(C. gtoeospo[ioides) Nrs l O) (N. haematocc~eca) MGSRI {=)and Mg,BtNEII) (M. gfises) Eg-R1 ilt(E, graminis) and EGH (g(E. hordei) Class Ih DNA transposons Tc I/mariner impatel2) (F.oxysporumy Ant1 (~(A. niger) IR IR Fort {2~arid For2 (2~ (F.ox3,sporurn);Ta~ {2>(A.nigsO Pot2 (1~and MGR586 (~) (M. Grisea); Flipper {~J(B, cinerea); Puntr~ iN. crassa) A c-like Ascot ft) and Tasce I~) (A. imrnemus); restless (lilT. Inflaturn) Hop (a~ (F.oxysporctm) UnreLated Pc~ (P. chrysosporlum) Unclassified Guest (S)(N, cr~a) Figure. 1. Schematic representations of the structural features of fungal transposable ele- ments. For purposes of clarity, the overall size of the elements is not to scale. Arrows denote the short duplication (TSD) generated in the host genera~ during transposition. There are two types of class I elements. LTR-retrotransposons resemble retroviruses in having long terminal direct repeats (LTR) flanking an internal domain encoding proteins analogous to the gag and pol retroviral gone products. They are divided into two groups, the gypsy and the copia types, on the basis of the order of functional domains in the pot gone (PR, protease; RT, reverse transcriptase; RH, RNAase H; 1N, integrase) and the presence or not of a third ORF in a position similar to that of the viral env gone but with no structural or functional homology to it. The non-LTR-retrotransposons lack terminal repeats and carry a poly(A) taft at their 3' ends. They fall into two families similar to the long and short interspersed nuclear elements described in mammals and referred to as LINEs and SINEs respectively. LINE-like elements commonly possess two long ORFs, with similarities to ~]c~,]as well as RT and RH genes, and are often deleted in their 5' ends. SINE-like elements are short elements wl~ich contain an internal RNA polymerase Ill promoter with a bipartite structure represented by the A and B boxes and which rely on RT for mobilization but do not themselves encode the enzyme. Class II elements share the same basic design of short inverLed terminal repeats (IR) bordering one or more ORFs, encoding transposase, an enzyme required for their own transposition. Some very short elements with IR and potential to form a hairpin structure are difficult to classify at present. Numbers within brackets indicate the strategy used to identify the transposable element: (1) isolated by cloning band of repetitive DNA; (2) isolated after transposition within the nia gone, with the exception of Tad, which was transposed into the am gone, and Ascot, which preexisted as an insertion in the b2 gone; (3) isolated by differential screening with antibodies against RT; (4) isolated using a CfT-1 heterologous probe; (5) identified through gone sequcncing.