Evolution of the Fot1 Transposons in the Genus Fusarium: Discontinuous Distribution and Epigenetic Inactivation

Marie-Josée Daboussi, Jean-Michel Davière1, Stéphane Graziani and Thierry Langin2

Institut de Génétique et Microbiologie, Université Paris-Sud


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
To understand the evolution of Fot1, a member of the pogo family widely dispersed in ascomycetes, we have performed a phylogenetic survey across the genus Fusarium divided into six sections. The taxonomic distribution of Fot1 is not homogeneous but patchy; it is prevalent in the Fusarium oxysporum complex, absent in closely related sections, and found in five species from the most distant section Martiella. Multiple copies of Fot1 were sequenced from each strain in which the element occurs. In three species, the Fot1 nucleotide sequence is 98% identical to that from F. oxysporum (Fox), whereas nucleotide divergence for host genes is markedly higher: 11% for partial nuclear 28S rDNA and up to 30% for the gene encoding nitrate reductase (nia). In two species, sequence divergence of Fot1-related elements relative to Fox ranged from 7% to 23% (16% average). Most of the sequence differences (82%) were C-to-T and G-to-A transitions. These mutations are distributed throughout the Fot1 sequences, although they tend to be concentrated in the middle portion of the elements. Analysis of the local sequence context of transitions revealed a hierarchy of site preferences. These characteristics are typical of the repeat-induced point mutation process, first discovered in Neurospora crassa. The spotty distribution of Fot1 elements among species together with the high degree of similarity between Fot1 sequences present in distant species strongly suggests a case of horizontal transfer.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Members of the Fusarium oxysporum complex (hereafter referred to as the FO complex) are common soilborne pathogens that cause diseases in a wide range of economically important crops. This complex provides an interesting example of interspecific variation, and effort has been focused on understanding the molecular mechanisms generating population variability. In the absence of a sexual stage, the origin and maintenance of variability probably require mechanisms ensuring high mutation rates. In this respect, the activity of transposable elements has been postulated to explain part of the genetic variability in this complex. Indeed, 12 families of transposable elements have been identified, including representatives of both retroelements and DNA transposons (for a review, see Daboussi and Langin 1994Citation ; Hua-Van et al. 2000Citation ).

Among these, Fot1 is the most abundant in a variety of strains in terms of copy number and the best characterized for its effect on target gene expression (Daboussi and Langin 1994Citation ; Deschamps et al. 1999Citation ). Fot1 is related to pogo transposons, which are, together with Tc1 and mariner elements, grouped in a large superfamily of transposable elements, the Tc1-mariner superfamily (Smit and Riggs 1996Citation ; Plasterk, Izsvak, and Ivics 1999). Fot1 is 1,928 bp in length and contains a single gene encoding a transposase enzyme that is flanked by terminal inverted repeats. Like all Tc1-mariner elements, Fot1 transposes by an excision-reinsertion mechanism and specifically inserts into a TA site (Daboussi and Langin 1994Citation ).

Fot1 is widely distributed in the FO complex. It is present essentially as full-length copies, with a variable copy number ranging from zero to more than one hundred (Daboussi and Langin 1994Citation ). This family is very homogeneous in size and exhibits a very low level (<2%) of polymorphism within the FO complex (Langin et al. 2002Citation ). The presence of Fot1 in most of the 12 formae speciales analyzed suggests that it is an ancient component of the FO complex, which has been vertically transmitted during the evolution of host specialization. Analysis of the pattern of Fot1 in a large sample of strains suggests that its distribution is the result of a combination of different evolutionary processes: vertical inactivation, stochastic loss, burst of amplification, and horizontal transmission (Langin et al. 2002Citation ).

The recent identification of Fot1-like elements in different ascomycetes (for a review, see Daboussi 1996Citation ; Kempken and Kuck 1998Citation ) suggests that they are ancient components of this phylum. At present, information on the origin of these elements is lacking, and questions dealing with the mechanisms by which they spread through different organisms need to be addressed. Are they ancient components of the genus Fusarium? In this case, they might be distributed throughout the genus. Alternatively, are they restricted to the FO complex, with the possibility of transferring laterally to other species? In order to evaluate the evolutionary forces that determine the distribution of Fot1, we carried out hybridization experiments to detect their presence in representative species of Fusarium.

Fusarium is a large genus of hyphomycetes whose classification has been controversial and often confusing (Nelson 1991Citation ; O'Donnell, Cigelnik, and Nirenberg 1998Citation ). Taxa recognized within this genus include sections, species, and varieties. This classification is essentially based on the microanatomy of the anamorph. Teleomorphs described for fusaria in 9 of 11 sections (Windels 1991Citation ) all belong to the Hypocreales either in Gibberella, Nectria, or Neocosmospora (O'Donnell 2000Citation ). Within some sections, the teleomorph has been found for some, but not all, species. In the past 10 years, the use of molecular tools has brought a degree of uniformity to the systematics of the fusaria, and the evolutionary relationships of some species have been investigated through rDNA sequence analysis (Guadet et al. 1989Citation ; Bruns, White, and Taylor 1991Citation ; O'Donnell 1992Citation ) and sequence analysis of portions of single-copy nuclear genes (Donaldson et al. 1995Citation ; O'Donnell, Cigelnik, and Nirenberg 1998Citation ; O'Donnell et al. 2000Citation ).

In this paper, we report on the distribution of sequences homologous to Fot1 in Fusarium and on the divergence of the Fot1 elements with that of the host gene. In the course of this investigation, we found degenerate Fot1 elements characterized by numerous CG-to-TA transitions. Comparison with the RIP (repeat-induced point mutation) process described in Neurospora crassa (Selker et al. 1987Citation ), in which duplicated sequences are inactivated, is presented.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Strains
Strains examined for the presence of sequences homologous to Fot1 and their sources are listed in table 1 . They were kindly provided by different laboratories cited in the legend to the table.


View this table:
[in this window]
[in a new window]
 
Table 1 Distribution of Fot1-Related Sequences Within Fusariuma

 
DNA Preparation and Southern Blot Analysis
Preparation of genomic DNA and Southern blotting were carried out as described in Daboussi, Langin, and Brygoo (1992)Citation . Hybridization experiments were performed at high stringency (hybridizations at 65°C, washing at 65°C in 2x SSC/0.1% SDS for 1 h, and then in 0.2x SSC/0.1% SDS for 30 min). Probes were labeled by random primer labeling (Feinberg and Vogelstein 1984Citation ). The Fot1 probe was derived from the PCR product obtained from pIN37 (Daboussi, Langin, and Brygoo 1992Citation ) using a primer deduced from the inverted terminal repeats (ITRs). The nia probe corresponds to the 4-kb HindIII genomic fragment present in pNH24 (Diolez et al. 1993Citation ).

Polymerase Chain Reaction
PCR experiments were conducted in a Biomed 60 Thermal Cycler (Braun). Three hundred nanograms of genomic DNA (see table 1 ) was used as a template. A primer deduced from the ITRs (positions 1–29) was used to amplify putative Fot1 sequences. Amplification of the D1 and D2 domains located at the 5'-end of the 28S rDNA was conducted by using two primers: PN4 (5'-CCTTGGTCCGTGTTTCAAGACGGG-3') and PN9 (5'-CTTAAGCATATCAATAAGCGGAGG-3'). Cycling parameters were 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 30 cycles. Amplification of a portion of the nia gene was performed using two degenerate primers designed to amplify the region of the intron—primer 541 (positions 1408–1427 as numbered in Diolez et al. 1993Citation ): 5'-ATGATGAATCAATCCCITGGTT-3' and primer 680 (positions 1866–1884): 5'-TTTCIGCAGTTTTCGCTGTCT-3'. The following cycling parameters were used: three cycles of denaturation (1 min at 94°C), annealing of primers (1 min at 42°C), and extension (1 min at 72°C), followed by 30 cycles of denaturation (1 min at 94°C), annealing of primers (1 min at 50°C), and extension (1 min at 72°C). The Taq DNA polymerase (Appligene) was used according to the supplier's specifications. Amplified products were separated on a 0.8% agarose gel, purified by using the Jetsorb kit (Bioprobe), and subcloned into the pGEM-T vector (Promega).

Cloning
Cloning of the Neo2 copy in Neocosmospora sp. Southern blotting of genomic DNA from Neocosmospora sp. was carried out using different restriction enzymes, and hybridization with Fot1 as a probe was performed at low stringency (50°C) (data not shown). A SacI digest was conducted to obtain a second copy of Fot1. Genomic DNA was separated on a 0.8% agarose gel, and the region of the gel containing the 1.4-kb Fot1 fragment was excised and cloned in pUC19.

Sequence Alignments and Phylogenetic Analysis
Sequences were determined using commercially available primers and Taq cycle sequencing with fluorescent-labeled dideoxynucleotide terminators. All sequencing reactions were run on a 373 ABI sequencer. Nucleotide sequence data reported in this paper are deposited in the EMBL/GenBank databases under the following accession numbers: AF403139, nitrate reductase Nectria haematococca; AF403143, nitrate reductase Fusarium solani f. sp. pisi; AF403144, nitrate reductase F. coeruleum; AF403140, nitrate reductase Gibberella fujikuroi; AF403141, nitrate reductase F. solani var. minus; AF403142, nitrate reductase Neocosmospora; AF434909, Fot1, Neocosmospora, Neo1 copy; AF434910, Fot1, Neocosmospora, Neo2 copy; AF443562, Fot1, F. solani var. minus; AF443563, Fot1, F. solani sp.

Sequences were analyzed using the DNA strider package. Sequence alignments were obtained using the Pileup program (GCG package). Maximum parsimony phylogenetic analyses were performed with PAUP, version 3.1.1 (Swofford 1991Citation ). Analysis of nucleotidic environment of RIP was performed on a Microsoft Excel 2000 worksheet, developed by S. Graziani, containing generic formulas for sequence comparison.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Distribution of Homologous Fot1 Sequences Within Fusarium
To test for the presence of Fot1 sequences, we carried out hybridization experiments on a total of 45 strains representing 16 species distributed among the different sections of Fusarium recognized by Gerlach (1981)Citation . We also employed a more specific PCR assay using either a primer corresponding to the inverted repeats of Fot1-37 or a set of internal primers. To obtain a more precise description of the phylogenetic distribution of homologous Fot1 sequences within the genus, we used as a frame, the phylogenetic tree established by Guadet et al. (1989)Citation by direct sequencing of two portions of the rRNA.

The points to be made from the data presented in table 1 and figure 1A are as follows:



View larger version (73K):
[in this window]
[in a new window]
 
Fig. 1.—Southern blot hybridization of species of fusaria. See also table 1 . Genomic DNA was digested with EcoRI, and the filter was probed with a full-length Fot1 PCR product (A) and a nia genomic fragment (B).

 
  1. Homologous Fot1 sequences are not limited to F. oxysporum. Of the 16 species surveyed, 5 showed evidence of Fot1 hybridization. All these species belong to section Martiella, namely, F. solani var. minus, F. javanicum var. radicicola, F. caucasicum, Neocosmospora sp., and F. solani sp. These strains showed between 2 and 12 hybridizing bands with differences in the intensity of the signals (fig. 1A ). In other species, particularly those more closely related to F. oxysporum, the presence of Fot1-related elements was not detected even under hybridization conditions of moderate stringency or by PCR amplification using Fot1 primers. These results indicate that some species may be free of Fot1 elements or may contain very divergent elements.
  2. The Fot1-hybridization signals in the five Martiella strains are strong; however, no hybridization was observed when the filter was probed with the F. oxysporum nia gene (fig. 1B ). The nia probe gave strong signals in strains belonging to section Liseola (G. fujikuroi complex; anamorph F. moniliforme, syn. F. verticillioides), as expected from the close phylogenetic relationship between these species and F. oxysporum (Guadet et al. 1989Citation ; O'Donnell, Cigelnik, and Nirenberg 1998Citation ), but no hybridization was detected with the Fot1 probe. Another single-copy sequence from F. oxysporum with unknown function gave the same pattern of hybridization as the nia probe (data not shown).

Nucleotidic Polymorphism of Fot1 Elements
To examine the Fot1 elements further, we amplified Fot1 by using a primer deduced from the ITRs. PCR products of the size expected for full-length elements (1.9 kb) were cloned, and three clones from three strains were sequenced. The aligned Fot1 sequences from F. solani var. minus (Min1), Neocosmospora spp. (Neo1), and F. solani (Fus1) compared with the Fot1-37 sequence from F. oxysporum (Fox) are presented in figure 2A .



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2.—A, Aligned DNA sequences of Fot1 from F. solani var. minus (Min1), Neocosmospora sp. (Neo1), and F. solani (Fus1) compared with Fot1-37 sequence (Fox) from F. oxysporum (Daboussi, Langin, and Brygoo 1992Citation ). Dots denote bases identical to those in Fox, and dashes denote deletions. C-to-T and G-to-A transitions are in bold. The ORF is indicated by brackets. B, Alignment of four Fot1 sequences. Only sites differing among the sequences are shown. The number at the top indicates the position of the variable sites in the Fot1 sequence

 
For F. solani var. minus, one copy, designated as Min1, was completely sequenced, whereas another, Min2, was sequenced in two regions spanning nucleotide positions 1–460 and 1550–1900, which includes the 5' and 3' noncoding regions and approximately 500 nucleotides of the open reading frame (ORF). The Min1 and Min2 sequences differ in the common region (780 bp) by eight nucleotides (six substitutions in the ORF). This divergence of 1% is in the same range as that observed between Fot1 copies within the FO complex (Langin et al. 2002Citation ). A comparison of Min1 with Fox gave an average divergence of about 2% (40 nucleotides that differ out of 1,856 bp). This high level of nucleotide-sequence identity accounts for the strong hybridization signal observed in Southern blot analysis. The modifications observed are evenly distributed along the sequence. One consistent difference between the Fox and Min1 sequences is a single nucleotide deletion at position 1720, also present in Min2, which determines a shift in the reading frame relative to Fox. The coding potential of Min1 has been determined. This copy possesses an uninterrupted ORF encoding a polypeptide of 535 residues. When the putative protein encoded by this element was compared with the Fox transposase (data not shown), the overall amino acid identity was 97% (16 changes out of 522 amino acids) over the coding region up to the deletion. However, the 13 amino acids 3' to the deletion are completely different.

For F. caucasicum (Cau) and F. javanicum var. radicicola (Rad), partial sequencing of one Fot1 product from each strain (900–1440) revealed a low level of nucleotide-sequence divergence relative to Fox, in the same range (1%–3%) as that reported for Min. The differences presented in figure 2B showed that the Fot1 product from the three distant species shared the same substitutions at different sites (1004, 1227, 1344, and 1353). This finding suggests a common evolutionary origin.

For Neocosmospora sp. and F. solani, in which only two Fot1-hybridizing bands were observed, sequence divergence of Fot1 copies (Neo1) and (Fus1) relative to Fox ranged from 7.5% to 23% (15% average), depending on the region examined (figs. 2A and 3A ). There are no gaps in the alignment, but there was a remarkable frequency of C-to-T and G-to-A transitions. In Neo1, 259 out of the 313 modifications (83%) observed among the 1,856 nucleotides correspond to CG-to-TA transitions. Without considering these, nucleotide divergence is around 3%. Partial sequencing (1465–1900) of a second Fot1 copy (Neo2) revealed 98% nucleotide identity with Neo1 (9 differences out of 435 nucleotides; see fig. 3B ). The high rate of transitions results in the inactivation of the transposase because no ORF can be detected in the three frames (fig. 3C ). In Fus1, CG-to-TA transitions also represent 82% of the modifications, with a dense distribution in the middle of Fot1 similar to that observed in Neo1. We also observed that Neo1 and Fus1 elements share many substitutions with Fox, as observed for Min1, Rad, and Cau elements, which supports their close relationships.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3.—A, Distribution of CG-to-TA mutations in Neo1. Of the 313 differences between Fox and Neo1, 136 are C-to-T transitions (lower vertical bars), and 123 are G-to-A transitions (upper vertical bars). B, Alignment of Neo sequences. Only sites differing in Neo1 and Neo2 sequences are shown. The numbers at the top indicate the positions of the variable sites. Identities with Fox are indicated by a period and deletions by dashes. Gray boxes denote CG-to-TA transitions. C, Consequences of transitions on the function of Neo1: no large ORFs can be found in the three frames

 
Comparison of nia and Fot1 Phylogenies
The remarkably close similarity between the Fox and Min elements is suggestive of horizontal transfer, but it is desirable to compare the Fot1 elements with nontransposable sequences from the same distant species. The gene for nitrate reductase is highly conserved in evolution (Kinghorn and Campbell 1989Citation ). This gene has been cloned in different fungi, including Aspergillus nidulans (Johnstone et al. 1990Citation ), N. crassa (Okamoto, Fu, and Marzluf 1991Citation ), F. oxysporum (Diolez et al. 1993Citation ), and Leptosphaeria maculans (Williams, Davis, and Howlett 1994Citation ). The aligned sequences allowed us to design degenerate primers from two conserved domains located on either side of the unique intron in the Fusarium gene, yielding an amplicon of 500 bp. The nia nucleotide sequences were compared among Fusarium species representative of different lineages. The coding regions, consisting of part of the two exons, could be aligned easily; however, the introns were too variable in length and too polymorphic to align (data not shown).

Comparisons based on 340 bp of exon 1 (table 2 ) show that the nia regions of G. fujikuroi and F. oxysporum are 95% and 98% identical, respectively, at the DNA and amino acid level. In contrast, the nia regions of F. solani and Neocosmospora are only 73% and 69% identical, respectively, at the DNA and amino acid level, with that of F. oxysporum. These results confirm that F. solani and Neocosmospora are not closely related to F. oxysporum because the divergence between these species is roughly in the same range as that observed with N. crassa, a species from a different genus. Maximum parsimony analysis of the aligned nia DNA sequences yielded a tree (fig. 4) concordant with partial 28S rDNA sequences, but the evolution rate of these two genes appeared different. Examination of nucleotide substitutions along different branches of the tree revealed that most of them are in the third codon position. (F. solani and Neocosmospora differ by 30 nucleotide changes, but there are differences only in 2 amino acids; F. oxysporum and G. fujikuroi differ by 22 nucleotide changes, and there are differences in only 3 amino acids.)


View this table:
[in this window]
[in a new window]
 
Table 2 Pairwise Identity Among the nia Sequences

 
Is the Fot1 Element from Neocosmospora a Product of RIP?
The high frequency of transition mutations CG-to-TA observed in the Neo1 sequence relative to Fox (fig. 2A ) is reminiscent of a process known as RIP, as described by Selker et al. (1987)Citation , Selker and Garrett (1988)Citation , Cambareri et al. (1989)Citation , and Selker (1990)Citation in N. crassa.

In order to determine if the polarized transition mutations in Neo1 and Fus1 have the characteristics of RIP, the pattern and distribution of mutations were examined (table 3 ). In Neo1, 136 out of 476 cytosine residues (28%) and 123 out of 474 guanine residues (26%) were replaced by thymine or adenine residues, respectively. In Fus1, the frequency of transitions appears lower (18%) because of the fact that 700 bp of the central region in which the density of transitions is higher (see fig. 3A ) has not been sequenced. Among the 259 CG-to-TA transitions in Neo1, there are 136 G-to-A changes and 123 C-to-T changes. We observed the same proportion in Fus1. The distribution of these changes along the sequence is indicated, respectively, as bars below and above the central horizontal line (fig. 3A ). Mutations are concentrated in the middle of the Fot1 element. For example, 27 CG-to-TA transitions, corresponding to 56% of the changes, occurred in the first 300 nucleotides, 14 (63% of the changes) in the 300 last nucleotides, whereas 87 (97% of the changes) were observed within the 300 bp internal region (between positions 700 and 1000).


View this table:
[in this window]
[in a new window]
 
Table 3 Site Specificity of CG-to TA Mutations in Neo1 from Neocosmospora sp. and Fus1 from F. solani

 
Analysis of the local sequence context of CG-to-TA mutations in Neo1 and Fus1 revealed that these mutations do not occur randomly. Considering the transitions C-to-T, they occurred primarily at sites with an adenine or a guanine 3' to the changed cytosine (85%–95%) and rarely where there was a cytosine 3' to the substituted cytosine (table 3 ). For example, 52% of the CpA dinucleotides changed, whereas 36%, 8%, and 4%, respectively, of CpG, CpT, and CpC dinucleotides changed. We observed the same bias for complementary nucleotides 5' to altered G nucleotides, and therefore we summed the totals (table 3 ). The hierarchy of site preferences in Fusarium is CpA/TpG > CpG/GpC > CpT/ApG = CpC/GpG. We also observed that C-to-T mutations occurred most frequently when an adenine or a guanine is downstream a CpA (82%) and to a lesser extent, a CpG (61%). The data also reveal that the identity of the bases immediately 5' to the altered cytosine did not appear to deviate from a random distribution (table 3 ). Interestingly, the mutations at the CpG dinucleotide are generally (66%) double mutations, with the C converted to T and the G to A. These changes can result from at least two runs of RIP, one run leading to the change of the C to T on one strand, leading to a CpA dinucleotide on the other strand. The fact that this dinucleotide has been shown to correspond to a preferential site for RIP increases the probability of obtaining a TA dinucleotide.

Because RIP affects both copies of a duplication in N. crassa, we determined the pattern of transitions in the other copy, Neo2. This copy was cloned by screening a Neocosmospora genomic library and identified as different from Neo1 by analyzing the flanking regions. Partial sequencing of Neo2 revealed that most CG-to-TA mutations were at the same position as in Neo1. However, nine differences were detected with a different pattern of RIP at four positions (fig. 3C ).

Sequences mutated by RIP are also typically methylated at most of the remaining cytosine residues (Selker et al. 1987Citation ; Cambareri et al. 1989Citation ; Grayburn and Selker 1989Citation ). In order to analyze the methylation status of Neo1 in genomic DNA, we carried out Southern hybridization using two pairs of isoschizomers that show differential sensitivity to cytosine methylation—HpaII and MspI or Sau3AI and NdeII (data not shown). No difference was detected in hybridization patterns between the isoschizomers, indicating that cytosine residues were not selectively methylated.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Based largely on Southern blot hybridization, we presented the detection of several Fot1 elements in strains of the Martiella section that are distantly related to F. oxysporum, as deduced from rDNA phylogenies. In contrast, Fot1-related elements were not found in species belonging to section Liseola, which is a putative sister group of the FO complex (Guadet et al. 1989Citation ). Even on using a more sensitive PCR assay based on internal primers, which are able to detect truncated copies, or on the ITR primer, which is able to amplify full-length elements, this assay was unable to detect Fot1 elements within a large sample of strains from section Liseola.

To account for the discontinuous distribution of Fot1 within Fusarium, there are two possible explanations, which are not mutually exclusive (fig. 5 ). The first postulates that these elements are ancient transposons present in the common ancestor of most of the species and retained in some lineages but eliminated by stochastic loss or RIP in others (fig. 5A ). This hypothesis requires several independent stochastic losses and suggests a low rate of Fot1 evolution relative to cellular genes. The alternative is to consider that Fot1 elements have been transferred horizontally from one species into others in the recent past (fig. 5B ). Several results support the recent invasion hypothesis: (1) mainly, the remarkably high level of identity between Fot1 sequences present in distant species illustrated by F. oxysporum and F. solani (98% identity) compared with those of nontransposable sequences from the same genomes, 75% for the nia gene and 81% for the rDNA; (2) the fact that four out of the five strains containing Fot1 are representative of the same lineage supports the recent introduction of Fot1 from F. oxysporum into the most recent common ancestor of this lineage; and (3) the comparison of Fot1 sequences present in strains from the section Martiella revealed many shared nucleotide changes and a shared deletion, indicating that these elements have a common origin. If horizontal transmission of Fot1 elements appears to be the most likely explanation for our data, screening of a larger number of strains in the different lineages using PCR assays with degenerate primers could help reinforce the horizontal transfer hypothesis. In addition, it would be interesting to eliminate the hypothesis that Fot1 has a restricted host range in Fusarium because of the host factors required for transposition. The fact that Fot1 elements are able to function in A. nidulans (Li Destri Nicosia et al. 2001) and Magnaporthe grisea (Villalba et al. 2001Citation ) suggests that transposition of this element is not highly dependent on host factors. However, confirmation could be obtained by determining the ability of an autonomous element (Migheli et al. 1999Citation ) to transpose in species of Fusarium in which Fot1was absent.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5.—Alternative hypotheses to explain the discontinuous distribution of Fot1 in Fusarium and inconsistencies between the phylogenies of the species based on nontransposable sequences. A, Existence of Fot1 elements in the most recent common ancestor distributed in several lineages (arrows). This element is lost in some lineages (lozenges) and maintained in others (strains in black and gray boxes). Black boxes represent strains in which an RIP-like process has been identified. B, Potential horizontal transfer of Fot1, symbolized by arrows, from F. oxysporum and two distant lineages

 
Accepting the hypothesis of horizontal transfer in some lineages still leaves questions about the behavior of the transferred copies, which range in number from 2 to 12. We have shown that in all the species, the Fot1 copy we cloned and sequenced contains a deletion at position 1720, which determines a shift in the ORF. The last 13 amino acids from the deletion are completely different, suggesting that this copy should be inactive. In this case, it is surprising to observe that the divergence is not greater than that observed for active elements in F. oxysporum. One explanation is that there are actually some selective constraints, perhaps because inactive elements have regulatory effects. This hypothesis has been advanced to explain the average nucleotide identity between inactive and active mariner elements in natural populations of Drosophila simulans (Capy et al. 1992Citation ). Another explanation is that the deletion does not affect the activity of the transposase. Indeed, the 3' part of the transposase might be less important, as reported for the ant1 transposable element identified in Aspergillus niger which seems to be active, although the terminal part of the transposase appeared to be different (Glayzer et al. 1995Citation ). Whether copies sharing the deletion are able to catalyze transposition remains to be determined. This will be tested in F. oxysporum by the ability of these copies to mobilize a defective Fot1 element through an excision test (Daboussi and Langin 1994Citation ).

In two strains collected from nature, a homothallic Neocosmospora sp. and an asexual or heterothallic F. solani, Fot1 elements were present in two copies, which are heavily mutated with multiple CG-to-TA transitions. The consequence of these mutations, which represent 82% of the changes, is the inactivation of the transposase. In fungi, mechanisms that inactivate transposable elements or duplicated sequences have been studied intensively in two species. RIP in N. crassa (Selker et al. 1987Citation ) and for methylation-induced premeiotically (MIP) in Ascobolus immersus (Goyon and Faugeron 1989Citation ) are closely related processes: both occur during the sexual cycle during the time between fertilization and karyogamy, both involve homologous DNA segments larger than a few hundred base pairs, and both lead to the simultaneous alteration of the two duplicated elements. One significant difference between the two processes resides in the nature of the alteration: only C-methylation occurs in MIP, whereas point mutations and C-methylation take place in RIP. Mutations by RIP are essentially C-to-T and G-to-A transitions. The most probable explanation for transition mutations is that deamination of 5-methylcytosines generate thymine, subsequently leading to a base change from guanine to adenine in the complementary strand during replication. Mutations by RIP do not occur randomly; C-to-T mutations occur principally (75%) at CpA/TpA sites (Cambareri et al. 1989Citation ; Selker 1990Citation ). These processes have been envisioned as mechanisms ensuring genome stability and serving as genome-defense mechanisms that guard against the deleterious effects of multicopy transposable elements and aberrant gene duplications. Firstly, they might control the mobility of transposons by inactivating them and hence limiting the number of repeats. Secondly, the rapid divergence of DNA repeats, in the case of RIP, or methylation, in the case of MIP, prevents homologous recombination, hence preventing chromosomal rearrangements by ectopic recombination (Kricker Drake, and Radman 1992Citation ; Rossignol and Faugeron 1994Citation ; Selker 1997Citation ; Maloisel and Rossignol 1998Citation ; Colot and Rossignol 1999Citation ). Consistent with this interpretation, different types of transposable elements found in N. crassa (Tad, DAB1, and Punt) show hallmarks of RIP (Kinsey et al. 1994Citation ; Bibbins, Cummings, and Connerton 1Citation 998; Margolin et al. 1998Citation ), and in A. immersus, the only mobile element identified, Ascot, escapes MIP most likely because of its short size (Colot et al. 1995Citation ). Studies on transposable elements in other fungi such as M. grisea (Nakayashiki et al. 1999Citation ), Aspergillus fumigatus (Neuvéglise et al. 1996Citation ), F. oxysporum (Julien, Poirier-Hamon, and Brygoo 1992Citation ; Hua-Van et al. 1998Citation ; Hua-Van, Langin, and Daboussi 2001Citation ), and Podospora anserina (Hamann, Feller, and Osiewacz 2000a, 2000b;Citation Graïa et al. 2001Citation ) revealed degenerate elements characterized by numerous CG-to-TA transitions. Their presence in fungal species in which sexual reproduction has not been found may reflect the operation of an RIP-like process at a time when they had a sexual cycle or the existence of an RIP-like process in vegetative cells.

In our study on Neocosmospora sp. and F. solani, several features indicate that Fot1 elements have been subjected to an RIP-like process: (1) the numerous CG-to-TA transitions; (2) the density of mutations which appear lighter at the borders than in the middle portion of Fot1; (3) the alterations in both Fot1 copies (Neo1 and Neo2); and (4) a preference for CpA/TpG dinucleotides (52%). However, some differences relative to RIP in N. crassa can be observed: (1) the second most common target of RIP is CpG in Fusarium (36%) and CpT in N. crassa (18%); (2) no methylation in sequences mutated by RIP has been detected in Fusarium; and (3) sequences altered by an RIP-like process have been found in a homothallic strain. These variations reflect the diversity of situations found in fungal species relative to the ability to detect methylation associated with RIP, the preferred target sites for transitions (CpA or TpA or CpG), and the status of strains in which RIP has been detected (asexual, homothallic, and heterothallic).

The situation described in our study addressed different questions. Is the RIP-like process still active in Neocosmospora sp.? MIP and RIP are active in heterothallic strains. It has been suggested that premeiotic inactivation may reflect a regulatory function related to the expression of the two nuclei of opposite mating type and that this function is required in heterothallic species such as N. crassa and A. immersus but not in a homothallic species such as Sordaria macrospora (Le Chevanton, Leblon, and Lebilcot 1989Citation ). Indeed, although sequences altered by RIP have been identified in homothallic strains of N. crassa, there is no demonstration that this process can operate in such strains. One way to demonstrate this consists in the introduction of autonomous Fot1 copies in Neocosmospora sp. and in the analysis of nucleotide sequence variations after different rounds of meiosis. This could also be tested using the nia gene, whose inactivation can be detected through the resistance to chlorate. Does an RIP-like process operate in other lineages? The discovery of an RIP-like process in strains belonging to two lineages addresses the question of the occurrence of such a mechanism within Fusarium. The apparent discontinuous distribution of Fot1 within Fusarium could be because of the presence of degenerate elements, reflecting the activity of an RIP-like process in other lineages. As observed in Neurospora, most or all the species showed the presence of Tad-related elements, which have been mutated extensively by RIP. These results suggest that a common ancestor hosted Tad and that RIP has inactivated almost all the copies (Kinsey et al. 1994Citation ). Finding relics of Fot1 in different lineages would have significant implications of an RIP-like process throughout Fusarium.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.—Phylogenetic relationships of Fusarium species based on nia sequences, using sequences of A. nidulans to root the tree. Sequences from the PCR-amplified exon 1 region were aligned using Clustal V. Phylogenetic trees were inferred with maximum parsimony (PAUP, version 3.1.1; Swofford 1991Citation ). Bootstrap values (100 replications) are given for each node; only values >50% are indicated

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Kerry O'Donnell for helpful comments on the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique (Génome action) and the Ministère de l'Education nationale (Biotechnology program). J.-M.D. was supported by a fellowship from MENERSR and S.G. by a fellowship from CNRS.


    Footnotes
 
Pierre Capy, Reviewing Editor

;thPresent address: Department of Agricultural Sciences, University of Bristol, U.K Back

;thPresent address: Institut de Biotechnologie des plantes, Université Paris-Sud, Orsay, France Back

Keywords: Fusarium transposable elements Fot1 horizontal transmission evolution rate RIP-like process Back

Address for correspondence and reprints: Marie-Josée Daboussi, Institut de Génétique et Microbiologie, Bât 400, Université Paris-Sud, 91405 Orsay, France. daboussi{at}igmors.u-psud.fr Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Bibbins M., N. J. Cummings, I. F. Connerton, 1998 DAB1: a degenerate retrotransposon-like element from Neurospora crassa Mol. Gen. Genet 258:431-436[ISI][Medline]

    Bruns T. D., T. J. White, J. W. Taylor, 1991 Fungal molecular systematics Annu. Rev. Ecol. Syst 22:525-564[ISI]

    Cambareri E. B., B. C. Jensen, E. Schabtach, E. U. Selker, 1989 Repeat-induced G-C to A-T mutations in Neurospora Science 244:1571-1575[ISI][Medline]

    Capy P., A. Koga, J. R. David, D. L. Hartl, 1992 Sequence analysis of active elements in natural populations of Drosophila simulans Genetics 130:499-506[Abstract/Free Full Text]

    Colot V., C. Goyon, G. Faugeron, J. L. Rossignol, 1995 Methylation of repeated DNA sequences and genome stability in Ascobolus immersus Can. J. Bot 73:s221-s225[ISI]

    Colot V., J. L. Rossignol, 1999 Eukaryotic DNA methylation as an evolutionay device Bioessays 21:402-411[ISI][Medline]

    Daboussi M. J., 1996 Fungal transposable elements: generators of diversity and genetic tools J. Genet 75:325-329[ISI]

    Daboussi M. J., T. Langin, 1994 Transposable elements in the fungal plant pathogen Fusarium oxysporum Genetica 93:49-59[ISI]

    Daboussi M. J., T. Langin, Y. Brygoo, 1992 Fot1, a new family of fungal transposable elements Mol. Gen. Genet 232:12-16[ISI][Medline]

    Deschamps F., T. Langin, P. Maurer, C. Gerlinger, B. Felenbok, M. J. Daboussi, 1999 Specific expression of the Fusarium transposon Fot1 and effects on target gene transcription Mol. Microbiol 31:1373-1383[ISI][Medline]

    Donaldson G. C., L. A. Ball, P. E. Axelrood, N. L. Glass, 1995 Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating Fusarium species associated to conifers Appl. Environ. Microbiol 61:1331-1340[Abstract]

    Diolez A., T. Langin, C. Gerlinger, Y. Brygoo, M. J. Daboussi, 1993 The nia gene of Fusarium oxysporum: isolation, sequence and development of a homologous transformation system Gene 131:61-67[ISI][Medline]

    Feinberg A. P., B. Vogelstein, 1984 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Anal. Biochem 137:266-267[ISI][Medline]

    Gerlach W., 1981 Present concept of Fusarium classification Pp. 413–426 in P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. Fusarium: diseases, biology and taxonomy. The Pennsylvania State University Press, Pa

    Glayzer D. C., I. N. Roberts, D. B. Archer, R. P. Oliver, 1995 The isolation of Ant1, a transposable element from Aspergillus niger Mol. Gen. Genet 249:432-438[ISI][Medline]

    Goyon C., G. Faugeron, 1989 Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences Mol. Cell. Biol 9:2818-2827[ISI][Medline]

    Gra;auIa F., O. Lespinet, B. Rimbault, M. Dequard-Chablat, E. Coppin, M. Picard, 2001 Genome quality control: RIP (repeat-induced point mutation) comes to Podospora Mol. Microbiol 40:586-595.[ISI][Medline]

    Grayburn W. S., E. U. Selker, 1989 A natural case of RIP: degeneration of the DNA sequence in an ancestral duplication Mol. Cell. Biol 9:4416-4421[ISI][Medline]

    Guadet J., J. Julien, J. F. Lafay, Y. Brygoo, 1989 Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison Mol. Biol. Evol 6:227-242[Abstract]

    Hamann A., F. Feller, H. D. Osiewacz, 2000a The degenerate DNA transposon Pat and repeat-induced point mutation (RIP) in Podospora anserina Mol. Gen. Genet 263:1061-1069[ISI][Medline]

    ———. 2000b Yeti—a degenerate gypsy-like LTR retrotransposon in the filamentous ascomycete Podospora anserina Curr. Genet 38:132-140[ISI][Medline]

    Hua-Van A., J. M. Davière, F. Kaper, T. Langin, M. J. Daboussi, 2000 Genome organization in Fusarium oxysporum: clusters of class II transposons Curr. Genet 37:339-347[ISI][Medline]

    Hua-Van A., F. Hericourt, P. Capy, M. J. Daboussi, T. Langin, 1998 Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum Mol. Gen. Genet 259:354-362[ISI][Medline]

    Hua-Van A., T. Langin, M. J. Daboussi, 2001 Evolutionary history of the impala element in Fusarium oxysporum Mol. Biol. Evol. 18:1959–1969

    Johnstone I. L., P. C. McCabe, P. Greaves, S. J. Gurr, G. E. Cole, M. A. D. Brow, S. E. Unkles, A. J. Clutterbuck, J. R. Kinghorn, M. A. Innis, 1990 Isolation and characterization of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans Gene 90:181-192[ISI][Medline]

    Julien J., S. Poirier-Hamon, Y. Brygoo, 1992 Foret1, a reverse transcriptase-like sequence in the filamentous fungus Fusarium oxysporum Nucleic Acids Res 20:3933-3937[Abstract]

    Kempken F., U. Kuck, 1998 Transposons in filamentous fungi—facts and perspectives Bioessays 20:652-659[ISI][Medline]

    Kinghorn J. R., E. I. Campbell, 1989 Amino acid sequence relationships between bacterial, fungal and plant nitrate reductase and nitrite reductases proteins Pp. 385–403 in J. L. Wray and J. R. Kinghorn, eds. Molecular and genetic aspects of nitrate assimilation. Oxford Science Publications, Oxford

    Kinsey J. A., P. W. Garrett-Engele, E. B. Cambareri, E. U. Selker, 1994 The Neurospora transposon Tad is sensitive to repeat-induced point mutation (RIP) Genetics 138:657-664.[Abstract/Free Full Text]

    Kricker M. C., J. W. Drake, M. Radman, 1992 Duplication targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes Proc. Natl. Acad. Sci. USA 89:1075-1779.[Abstract]

    Langin T., J. M. Davière, D. Fernandez, M. P. Dubois, M. J. Daboussi, 2002 Widespread occurrence of the Fot1 transposon within the Fusarium oxysporum species Gene (in press)

    Le Chevanton L., G. Leblon, S. Lebilcot, 1989 Duplications created by transformation in Sordaria macrospora are not inactivated during meiosis Mol. Gen. Genet 218:390-396[ISI][Medline]

    Li Destri Nicosia M. G., C. Masson, S. Demais, A. Hua-Van, M. J. Daboussi, C. Scazzocchio, 2001 Heterologous transposition in Aspergillus nidulans Mol. Microbiol. 39:1330–1344

    Maloisel L., J. L. Rossignol, 1998 Suppression of crossing-over by DNA methylation in Ascobolus Genes Dev 12:1381-1389[Abstract/Free Full Text]

    Margolin B. S., P. W. Garett-Engele, J. N. Stevens, D. Y. Fritz, C. Garett-Engele, R. L. Metzenberg, E. U. Selker, 1998 A methylated Neurospora 5S rRNA contains a transposable element inactivated by repeat-induced point mutation Genetics 149:1787-1797[Abstract/Free Full Text]

    Migheli Q., R. Laugé, J. M. Davière, C. Gerlinger, F. Kaper, T. Langin, M. J. Daboussi, 1999 Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum Genetics 151:1005-1013[Abstract/Free Full Text]

    Nakayashiki H., N. Nishimoto, K. Ikeda, Y. Tosa, S. Mayama, 1999 Degenerate MAGGY elements in a subgroup of Pyricularia grisea: a possible example of successful capture of a genetic invader by a fungal genome Mol. Gen. Genet 261:958-966[ISI][Medline]

    Nelson P. E., 1991 History of Fusarium systematics Phytopathology 81:1045-1051[ISI]

    Neuvéglise C., J. Sarfati, J. P. Latgé, S. Paris, 1996 Afut1, a retrotransposon-like element from Aspergillus fumigatus Nucleic Acids Res 24:1428-1434[Abstract/Free Full Text]

    O'Donnell K., 1992 Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris) Curr. Genet 22:213-220[ISI][Medline]

    ———. 2000 Molecular phylogeny of the Nectria haematococca-Fusarium solani species complex Mycologia 92:919–938

    O'Donnell K., E. Cigelnik, H. I. Nirenberg, 1998 Molecular systematics and phylogeography of the Gibberella fujikuroi species complex Mycologia 90:465-493[ISI]

    O'Donnell K., H. C. Kistler, B. K. Tacke, H. H. Casper, 2000 Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab Proc. Natl. Acad. Sci. USA 97:7905-7910.[Abstract/Free Full Text]

    Okamoto P. M., Y. H. Fu, G. A. Marzluf, 1991 Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover Mol. Gen. Genet 227:213-223[ISI][Medline]

    Plasterk R. H. A., S. Izsvak, Z. Ivics, 1999 Resident aliens: the Tc1-mariner superfamily of transposable elements Trends Genet 15:326-332[ISI][Medline]

    Rossignol J. L., G. Faugeron, 1994 Gene inactivation triggered by recognition between DNA repeats Experientia 50:307-317[ISI][Medline]

    Selker E. U., 1990 Premeiotic instability of repeated sequences in Neurospora crassa Annu. Rev. Genet 24:579-613[ISI][Medline]

    ———. 1997 Epigenetic phenomena in filamentous fungi: useful paradigms or repeat-induced confusion? Trends Genet 13:296-301[ISI][Medline]

    Selker E. U., E. B. Cambareri, B. C. Jensen, K. R. Haack, 1987 Rearrangements of duplicated DNA in specialized cells of Neurospora crassa Cell 51:741-752[ISI][Medline]

    Selker E. U., P. W. Garrett, 1988 DNA sequence duplications trigger gene inactivation in Neurospora crassa Proc. Natl. Acad. Sci. USA 85:6870-6874[Abstract]

    Smit A. F., A. D. Riggs, 1996 Tiggers and DNA transposon fossils in the human genome Proc. Natl. Acad. Sci. USA 93:1443-1448[Abstract/Free Full Text]

    Swofford D. L., 1991 PAUP: phylogenetic analysis using parsimony. Version 3.0 Illinois Natural History Survey, Champaign

    Villalba F., M. H. Lebrun, A. Hua-Van, M. J. Daboussi, M. C. Grosjean-Cournoyer, 2001 Transposon tagging in the rice blast fungus Magnaporthe grisea using impala, a Tc1-mariner element from Fusarium oxysporum Mol. Plant-Microbe Interact. 14:308–315

    Williams R. S. B., M. A. Davis, B. J. Howlett, 1994 Nitrate reductase of the ascomycetous fungus, Leptosphaeria maculans: gene sequence and chromosomal location Mol. Gen. Genet 244:1-8[ISI][Medline]

    Windels C. E., 1991 Current status of Fusarium taxonomy Phytopathology 81:1048-1051[ISI]

Accepted for publication December 4, 2001.