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* Abstract The past 10 years have been productive in the characterization of fungal transposable elements (TEs). All eukaryotic TEs described are found including an extraordinary prevalence of active members of the pogo family. The role of TEs in mutation and genome organization is well documented, leading to significant advances in our perception of the mechanisms underlying genetic changes in these organisms. TE-mediated changes, associated with transposition and recombination, provide a broad range of genetic variation, which is useful for natural populations in their adaptation to environmental constraints, especially for those lacking the sexual stage. Interestingly, some fungal species have evolved distinct silencing mechanisms that are regarded as host defense systems against TEs. The examination of forces acting on the evolutionary dynamics of TEs should provide important insights into the interactions between TEs and the fungal genome. Another issue of major significance is the practical applications of TEs in gene tagging and population analysis, which will undoubtedly facilitate research in systematic biology and functional genomics.
Key Words mobile elements, mutagenic effects, genome evolution, epigenetic regulation, genetic tools
CONTENTS INTRODUCTION OVERVIEW OF FUNGAL TRANSPOSABLE ELEMENTS Retroelements DNA Transposons MUTAGENIC EFFECTS OF TRANSPOSABLE ELEMENTS Variation Mediated by Transposition Transposon-Mediated Rearrangements Transposable Elements as Tools for Gene Tagging CONTROL OF TRANSPOSABLE ELEMENTS Stress Activation Epigenetic Silencing Mechanisms DYNAMICS OF TRANSPOSABLE ELEMENTS IN HOST GENOMES Loss and Gain of Transposable Elements Evidence for Horizontal Transmission Tools for the Analysis of Population Structure and Epidemiology CONCLUDING REMARKS
Transposable elements (TEs) are present in the genome of all species of the three domains of life: eubacteria, archaeabacteria, and eukaryotes. Although TEs were known to exist in bacteria, plant, animals, and yeast since the 1970s and the beginning of the 1980s (6,21), they were more recently described in filamentous fungi. Evidence of the presence and activity of TEs in hyphal fungi came from studies on diverse and poorly characterized species, most of which lack the sexual stage. Curiously, the analysis of thousands of spontaneous and induced mutants in ascomycetes used as models for fungal genetics, e.g., Aspergillus nidulans and Neurospora crassa, had no evidence for TE activity, with the exception of Ascobolus immersus (29). Advances in the molecular analysis of these genomes revealed that they contain silent TEs. This lack of activity might be the consequence of continuous selection for phenotypic stability, but this might also be due to various gene silencing mechanisms that inactivate repeated sequences, including TEs (17, 38, 133).
An enormous increase in our understanding of the biology of fungal TEs has occurred in the past decade because of the diversity of fungal research in organisms playing an important role in agriculture, medicine, and biotechnology. These efforts led to the discovery of many types of TEs, covering the entire spectrum of eukaryotic TEs (23, 74). Surprisingly, they include active members of families, such as pogo and Mutator, that are generally found in a restricted host range. Several types of retroelements and DNA transposons remain active and are demonstrated to induce a variety of genomic modifications. These mutagenic properties have also been exploited in the development of a powerful strategy for gene isolation, "transposon tagging." Given their abundance, estimated in some species at about 14% (50), TEs are viewed as central agents in the evolutionary restructuration of fungal genomes. Their dynamics include different mechanisms, such as transposition (normal or aberrant), ectopic recombination, horizontal transmission, amplification bursts, degradation, and epigenetic inactivation. Moreover, the examination of TE distribution in natural populations provided valuable information concerning ecological and epidemiological considerations.
We review the current status of research on fungal TEs, with an emphasis on how these elements might be a useful source of diversity in the evolution of the fungal genome. Following a short description of the different classes of TEs found in fungi, a synthesis of the present knowledge concerning their mechanisms of transposition, their impact on the host genome, their spread within and between species, and finally their potential use for genetic research is presented.
OVERVIEW OF FUNGAL TRANSPOSABLE ELEMENTS
Eukaryotic TEs are divided into two main classes by their mode of transposition and structural organization (43). Class I elements, or retroelements, transpose by a "copy-and-paste" mechanism by the reverse transcription of an RNA intermediate. This class can be subdivided into LTR retrotransposons, which are flanked by long terminal repeats (LTRs) sharing an overall organization similar to retrovimses, and non-LTR retroelements, which have structural features of long and short interspersed nuclear elements (LINEs and SINEs, respectively). Class II TEs, also called DNA transposons, are flanked by two terminal inverted repeats (TIRs) and transpose directly through a DNA form by a "cut-and-paste" mechanism. Both classes are subdivided into distinct superfamilies on the basis of structural features, internal organization, the size of the target site duplication (TSD) generated upon insertion, and sequence similarities at the DNA and protein levels (Figure 1). Most but not all TE families include both autonomous and nonautonomous elements. Proteins supplied by autonomous elements, generally belonging to die same family, can transactivate nonautonomous elements.
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Fungal TEs have been identified by a variety of strategies, mainly by the characterization of dispersed repetitive sequence or by trapping them in a target gene. The nitrate reductase gene has been particularly useful for this purpose because chlorate resistance can select loss-of-function mutants (23). Other elements were found by heterologous hybridization or polymerase chain reaction (PCR) amplification with degenerated primers deduced from conserved domains (23). Finally, as genome segments began to be cloned and sequenced, the discovery of new TEs accelerated (10, 59). The TEs presented in Figure 1 are found in three orders of fungi, Ascomycota, Basidiomycota, and Zygomycota. However, most were identified in Ascomycota species. This bias is probably due to the number of researchers working on ascomycetes, using A. nidulans, N. crassa, A. immersus, and Podospora anserina as models, or their involvement in plant interactions and biotechnology processes. The 60 TE sequences have been assigned to the major groups previously described in the different kingdoms (13).
Approximately 30 elements corresponding to LTR retrotransposons [Ty3/gypsy (Metaviridae) and Ty1/copia (Pseudoviridae) (64)] and non-LTR retrotransposons (LINEs and SINEs) were recognized. The majority corresponds to the Ty3/gypsy group and is dispersed among the three fungal orders. A phylogenetic analysis of these elements revealed that they are closely related (22, 92). Several elements, such as CfT-1, skippy, MAGGY, and grh, are characterized by the presence of a chromodomain at the C-terminal end of the integrase (100) and are included in a new family of LTR retrotransposons that uses self-priming to initiate reverse transcription (92). The transpositional activity of some elements has been documented either on the basis of heterologous transposition, as for MAGGY (113), or due to the absence of polymorphism between LTRs, the presence of virus-like particles associated with reverse transcriptase activity, an increase in copy number, and the polymorphism of the genomic position of different copies for CfT-1 and skippy (2, 106). Other elements, such as Foret (67), DAB1 (7), Afutl (115), and Yeti (55), appeared to be remnants because numerous stop codons and frameshift mutations were found in the reverse transcriptase domain. Few Ty1/copia-like elements have been characterized; they are inactive, as indicated by the occurrence of multiple deletions and mutations in conserved coding regions. Their low abundance relative to the Ty3/gypsy-like elements could reflect a lesser distribution in filamentous fungi or a location in genomic regions not represented in the libraries. For instance, the Tcen element is exclusively found in centromeric regions of the N. crassa genome (10).
Several non-LTR retrotransposons have also been characterized. Tad, the first TE reported in fungi, was discovered in a particular strain of N. crassa (83) and demonstrated to transpose through internuclear transfer between genetically marked nuclei in forced heterokaryons (81). Extensive analysis of other Neurospora strains (4) has revealed the presence of multiple copies of Tad-related sequences in most strains, but all contain numerous mutations resulting from the repeat-induced point mutation (RIP) process (see below). A family of sequences with the hallmarks of SINE-like elements, e.g., small size (70-500 bp), presence of an internal binding site for RNA polymerase II, and adenine-rich 3' ends, has also been identified in several species. One element, Foxy, was demonstrated to be active by new insertions following gamma irradiation (107).
Fungal transposons can be assigned to four distinct superfamilies, namely, Tc1/ mariner, hAT, Mutator, and MITEs. The Tc1/mariner superfamily is probably the most widespread in nature (126). It can be divided into different families, Tc1, mariner, Ant1, and pogo (130). The elements are approximately 1.3-2.4 kb in length, with TIRs of variable size and a TA target site. The transposase contains a signature of three acidic amino acids (DDE or DDD), which forms the …