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

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.

Genome-wide comparative analysis of pogo-like transposable elements in different Fusarium species.

The recent availability of genome sequences of four different Fusarium species offers the opportunity to perform extensive comparative analyses, in particular of repeated sequences. In a recent work, the overall content of such sequences in the genomes of three phylogenetically related Fusarium species, F. graminearum, F. verticillioides, and F. oxysporum f. sp. lycopersici has been estimated. In this study, we present an exhaustive characterization of pogo-like elements, named Fots, in four Fusarium genomes. Overall 10 Fot and two Fot-related miniature inverted-repeat transposable element families were identified, revealing a diversification of multiple lineages of pogo-like elements, some of which accompanied by a gain of introns. This analysis also showed that such elements are present in an unusual high proportion in the genomes of F. oxysporum f. sp. lycopersici and Nectria haematococca (anamorph F. solani f. sp. pisi) in contrast with most other fungal genomes in which retroelements are the most represented. Interestingly, our analysis showed that the most numerous Fot families all contain potentially active or mobilisable copies, thus conferring a mutagenic potential of these transposable elements and consequently a role in strain adaptation and genome evolution. This role is strongly reinforced when examining their genomic distribution which is clearly biased with a high proportion (more than 80%) located on strain- or species-specific regions enriched in genes involved in pathogenicity and/or adaptation. Finally, the different reproductive characteristics of the four Fusarium species allowed us to investigate the impact of the process of repeat-induced point mutations on the expansion and diversification of Fot elements.

Development of impala-based transposon systems for gene tagging in filamentous fungi.

Genome sequences of many filamentous fungi are now available and additional genomes are currently being sequenced. One of the next strategic goals is to generate collections of tagged genes in order to establish a link between the several thousands of predicted genes and their function. Transposable elements have been invaluable for the identification and isolation of genes of interest as insertion of a transposon both disrupts and tags a gene. In an effort to exploit active transposons identified in the genome of Fusarium oxysporum as insertional mutagens, a binary system including the tagging element, MITE, and the transposase of a Tc1 element has been established as an efficient tool for gene-tagging in Fusarium graminearum. In this chapter, we provide an overview of the techniques used and highlight some of the critical steps for the application of this tool to other fungal species.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large-scale transposon tagging.

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia(+) revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016, displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans. The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.

Genome-wide analysis of the Fusarium oxysporum mimp family of MITEs and mobilization of both native and de novo created mimps.

We have performed a genome-wide analysis of the mimp family of miniature inverted-repeat transposable elements, taking advantage of the recent release of the F. oxysporum genome sequence. Using different approaches, we detected 103 mimp elements, corresponding to 75 nonredundant copies, half of which are located on a single small chromosome. Phylogenetic analysis identified at least six subfamilies, all remarkably homogeneous in size and sequence. Based on high sequence identity in the terminal inverted repeats (TIRs), mimp elements were connected to different impala members. To gain insights into the mechanisms at the origin and amplification of mimps, we studied the potential of impala to cross-mobilize different mimps, native but also created de novo by inserting a short DNA segment between two TIRs. Our results show that TIR sequences are the main requirement for mobilization but that additional parameters in the internal region are likely to influence transposition efficiency. Finally, we show that integration site preference of native versus newly transposed mimps greatly varies in the host genomes used in this study.

Transposon-tagging identifies novel pathogenicity genes in Fusarium graminearum.

With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.

Transposition of a fungal miniature inverted-repeat transposable element through the action of a Tc1-like transposase.

The mimp1 element previously identified in the ascomycete fungus Fusarium oxysporum has hallmarks of miniature inverted-repeat transposable elements (MITEs): short size, terminal inverted repeats (TIRs), structural homogeneity, and a stable secondary structure. Since mimp1 has no coding capacity, its mobilization requires a transposase-encoding element. On the basis of the similarity of TIRs and target-site preference with the autonomous Tc1-like element impala, together with a correlated distribution of both elements among the Fusarium genus, we investigated the ability of mimp1 to jump upon expression of the impala transposase provided in trans. Under these conditions, we present evidence that mimp1 transposes by a cut-and-paste mechanism into TA dinucleotides, which are duplicated upon insertion. Our results also show that mimp1 reinserts very frequently in genic regions for at least one-third of the cases. We also show that the mimp1/impala double-component system is fully functional in the heterologous species F. graminearum, allowing the development of a highly efficient tool for gene tagging in filamentous fungi.

Bistability and hysteresis of the 'Secteur' differentiation are controlled by a two-gene locus in Nectria haematococca.

BACKGROUND: Bistability and hysteresis are increasingly recognized as major properties of regulatory networks governing numerous biological phenomena, such as differentiation and cell cycle progression. The full scope of the underlying molecular mechanisms leading to bistability and hysteresis remains elusive. Nectria haemaotcocca, a saprophytic or pathogenic fungus with sexual reproduction, exhibits a bistable morphological modification characterized by a reduced growth rate and an intense pigmentation. Bistability is triggered by the presence or absence of sigma, a cytoplasmic determinant. This determinant spreads in an infectious manner in the hyphae of the growing margin, insuring hysteresis of the differentiation. RESULTS: Seven mutants specifically affected in the generation of sigma were selected through two different screening strategies. The s1 and s2 mutations completely abolish the generation of sigma and of its morphological expression, the Secteur. The remaining five mutations promote its constitutive generation, which determines an intense pigmentation but not growth alteration. The seven mutations map at the same locus, Ses (for 'Secteur-specific'). The s2 mutant was obtained by an insertional mutagenesis strategy, which permitted the cloning of the Ses locus. Sequence and transcription analysis reveals that Ses is composed of two closely linked genes, SesA, mutated in the s1 and s2 mutant strains, and SesB, mutated in the s* mutant strains. SesB shares sequence similarity with animal and fungal putative proteins, with potential esterase/lipase/thioesterase activity, whereas SesA is similar to proteins of unknown function present only in the filamentous fungi Fusarium graminearum and Podospora anserina. CONCLUSIONS: The cloning of Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Atypical regulatory relations between the two proteins may account for the hysteresis of Secteur differentiation.

Novel polyketide synthase from Nectria haematococca.

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: beta-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.

Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation.

Penicillium griseoroseum has been studied because of its efficient pectinases production. In this work, the Penicillium griseoroseum nitrate reductase gene was characterized, transcriptionally analyzed in different nitrogen sources, and used to create a phylogenetic tree and to develop a homologous transformation system. The regulatory region contained consensus signals involved in nitrogen metabolism and the structural region was possibly interrupted by 6 introns coding for a deduced protein with 864 amino acids. RT-PCR analysis revealed high amounts of niaD transcript in the presence of nitrate. Transcription was repressed by ammonium, urea, and glutamine showing an efficient turnover of the niaD mRNA. Phylogenetics analysis showed distinct groups clearly separated in accordance with the classical taxonomy. A mutant with a 122-bp deletion was used in homologous transformation experiments and showed a transformation frequency of 14 transformants/microg DNA. All analyzed transformants showed that both single- and double-crossover recombination occurred at the niaD locus. The establishment of this homologous transformation system is an essential step for the improvement of pectinase production in Penicillium griseoroseum.

Transposable elements in filamentous fungi.

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.

Hop, an active Mutator-like element in the genome of the fungus Fusarium oxysporum.

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.

Impala, a transposon from Fusarium oxysporum, is active in the genome of Penicillium griseoroseum.

An autonomous impala transposon trapped in Fusarium oxysporum by insertion within the niaD gene encoding nitrate reductase was introduced in the genome of the fungus Penicillium griseoroseum, a producer of pectinase enzymes. Through a phenotypic assay, we demonstrate that this element is able to excise from the niaD gene and to reinsert at new genomic positions. As in the original host, impala inserts into a TA site and footprints left by impala excisions are generally 5 bp. The fact that impala is able to transpose in P. griseoroseum offers the opportunity to develop a gene-tagging system based on this element with the objective to detect and clone genes related in pectinase production.

Evolution of the Fot1 transposons in the genus Fusarium: discontinuous distribution and epigenetic inactivation.

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.