Marie-Josée Daboussi

Fot1, a new family of fungal transposable elements

SummaryWe report here the discovery of a family of transposable elements, which we refer to as Fotl elements, in the fungal plant pathogen Fusarium oxysporum. The first element was identified as an insertion in the gene encoding nitrate reductase. It is 1928 by long, has 44 by inverted terminal repeats, contains a large open reading frame and is flanked by a 2 by (TA) target site duplication. This element shares significant structural similarities with a class of transposons that includes Tc1 from Caenorhabditis elegans and therefore represents a new class of transposable elements in fungi.

The transposable element impala, a fungal member of the Tc1-mariner superfamily

A new transposable element has been isolated from an unstable niaD mutant of the fungus Fusarium oxysporum. This element, called impala, is 1280 nucleotides long and has inverted repeats of 27 bp. Impala inserts into a TA site and leaves behind a “footprint” when it excises. The inserted element, impala-160, is cis-active, but is probably trans-defective owing to several stop codons and frameshifts. Similarities exist between the inverted repeats of impala and those of transposons belonging to the widely dispersed mariner and Tc1 families. Moreover, translation of the open reading frame revealed three regions showing high similarities with Tc1 from Caenorhabditis elegans and with the mariner element of Drosophila mauritiana. The overall comparison shows that impala occupies an intermediate position between the mariner and Tcl-like elements, suggesting that all these elements belong to the same superfamily. The degree of relatedness observed between these elements, described in different kingdoms, raises the question of their origin and evolution.

Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans

A gene transfer system originally developed for Fusarium oxysporum has been applied to seven species of filamentous fungi of agricultural and industrial importance. This transformation system relies on the selection of mutants deficient in nitrate reductase by positive screening. Such mutants were recovered easily in all the fungi tested--without mutagenic treatments--through their resistance to chlorate. They were transformed by a plasmid vector (pAN301) carrying the Aspergillus nidulans wild-type gene (niaD). Transformation frequencies ranged from one to ten transformants/micrograms plasmid DNA. The general properties of the transformants were analyzed. Most of them are mitotically stable, and the integration of the vector into the host genome frequently occurred in a tandem fashion.

Genome organization in Fusarium oxysporum: clusters of class II transposons.

Abstract Several families of transposable elements (TEs) are present in the genome of the phytopathogenic fungus Fusarium oxysporum. They are present in copy numbers ranging from just a few elements to tens or hundreds per genome. Sequence analysis of contiguous stretches of genomic DNA surrounding insertion sites of one family revealed that they are packed with repeated sequences. We have carried out a detailed study of the composition and arrangement of these repeats in three chromosomal regions. We found that they are essentially mixtures of several types of TEs, most of them being DNA transposons, different from those previously characterized. Some repeats are frequently reiterated and many of them are inserted into other elements. Parts of these regions are also duplications. These regions appear prone to rearrangement and transposition and are subject to rapid reorganization.

Systematic Numbering of Vegetative Compatibility Groups in the Plant Pathogenic Fungus Fusarium oxysporum

First author: Plant Pathology Department, University of Florida, Gainesville 32611-0680; second author: Laboratoire de Recherches sur la Flore Pathogene du Sol, INRA, 17 rue Sully, B.V. 154

Heterologous transposition in Aspergillus nidulans

Aspergillus nidulans is one of the model ascomycete fungi. Transposition events have never been described in this organism. We have determined that this organism has at least 13 copies of a Fot1‐related element. These copies are transcribed, non‐methylated and polymorphic in various wild isolates. In spite of this, we have failed to isolate transposon insertions when the resident niaD gene is used as a transposon trap. This contrasts with the situation described previously in Fusarium oxysporum. We show that two elements of F. oxysporum, Fot1 and impala, transpose efficiently in A. nidulans. We have developed the impala system by tagging it with the yA gene. This permits the visual detection of the transposon by the colour of the conidiospores. We demonstrate that no endogenous transposase of A. nidulans is able to act in trans on a defective impala element, whereas its own transposase driven by two different promoters is able to mobilize this element. The frequency of excision of these modified elements is between 10−4 and 10−5. Loss of the transposable element occurs in about 10% of all excision events. In the remaining 90%, the transposon seems to be integrated at random positions in the genome. The availability of mitochondrially inherited mutations has allowed us to demonstrate that hybrid dysgenesis is apparently absent in A. nidulans. The development of this system opens the way to investigating the mechanism underlying the paucity of transposition events leading to visible phenotypes. It should allow us to develop efficient gene‐tagging tools, useful in this and other fungi.

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

Abstract The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.␣oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events.

Transposon impala, a novel tool for gene tagging in the rice blast fungus Magnaporthe grisea.

impala, a Tc1-mariner transposable element from Fusarium oxysporum, was introduced into the rice blast fungus Magnaporthe grisea to develop transposon-based insertional mutagenesis. A construct (pNIL160) containing an autonomous impala copy inserted in the promoter of niaD encoding Aspergillus nidulans nitrate reductase was introduced by transformation into a M. grisea nitrate reductase-deficient mutant. impala excision was monitored by restoration of prototrophy for nitrate. Southern analysis of niaD+ revertants revealed that impala was able to excise and reinsert at new loci in M. grisea. As observed for its host Fusarium oxysporum, impala inserted at a TA site left a typical excision footprint of 5 bp. We have shown that a defective impala copy was inactive in M. grisea, yet it can be activated by a functional impala transposase. A transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants. Mutants either altered for their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained. Complementation of Rev77 with a 3-kb genomic fragment from a wild-type locus was successful, demonstrating the tagging of a pathogenicity gene by impala. This gene, called ORP1, is essential for penetration of host leaves by M. grisea and has no sequence homology to known genes.

Novel Polyketide Synthase from Nectria haematococca

ABSTRACT 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: β-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.

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.