Heterologous transposition in Aspergillus nidulans

Nicosia, Maria Giulia Li Destri; Brocard-Masson, Corinne; Demais, Stéphane; Van, Aurelie Hua; Daboussi, Marie‐Josée; Scazzocchio, Claudio

doi:10.1046/j.1365-2958.2001.02323.x

Cited by 16 publications

(45 citation statements)

References 25 publications

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“…Furthermore, comparison of the integration sites to the A. fumigatus database revealed that in two of the transformants analyzed, a T-DNA insertion close to or into an open reading frame was found. Although the transformants displayed no aberrant phenotype, this shows that T-DNA can be used effectively as a gene tag in filamentous fungi, in addition to transposon mutagenesis, which is developed for various fungi (14,18,39). The sequence data of the T-DNA rescues suggest that the T-DNA had integrated at random and via nonhomologous recombination, since no extensive homologies were found between the T-DNA and the integration site or among the integration sites themselves.…”

Section: Discussionmentioning

confidence: 95%

Agrobacterium-Mediated Transformation ofAspergillus awamoriin the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

et al. 2004

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Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.Agrobacterium tumefaciens is capable of transferring a part of its tumor-inducing (Ti) plasmid (the transferred DNA, or T-DNA) to eukaryotic cells, where it stably integrates into the host genome. A. tumefaciens is now widely used for the transformation of plants, and more recently also for transformation of yeasts and fungi (3, 10). Proteins encoded by the virulence region of the Ti plasmid mediate T-DNA processing and transfer (13, 45). The VirD2 protein, in the presence of VirD1, recognizes the ends of the T-DNA, which is delimited by two T-DNA border repeats, and nicks the bottom strand of these 24-bp border repeats between the second and third bases. These nicks are then used as initiation and termination sites for the displacement of a linear single-stranded copy of the bottom strand of the T-DNA region, the T strand. The VirD2 protein stays covalently attached to the T strand via its tyrosine residue (Tyr29) (40). The right border is usually the initiation point for T-strand production due to the presence of a socalled overdrive located at that border. VirC1, one of the products of the virC locus, can bind this 25-bp overdrive sequence and thereby enhance T-strand production (32). The other product of the virC locus, the VirC2 protein, is also thought to play a role in efficient T-DNA transfer (43); however, its precise role in T-DNA formation is unknown. The T-strand-VirD2 complex is transported to the host via a piluslike structure consisting of VirD4 and 11 different VirB proteins. Once in the host, the T strand is coated with the singlestranded DNA binding protein VirE2, which is transported to the host independently of the T strand (36). Binding of VirE2 to the T strand protects it against nucleolytic degradation and turns the T strand into a transportable conformation, which facilitates the nuclear import of the T strand. The T complex, composed of the T strand, VirD2, and VirE2, is imported into the nucleus, where the T strand integrates stably into the host genome. Besides nicking the T-DNA borders, the VirD2 protein also plays a role in tar...

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Section: Discussionmentioning

confidence: 95%

Agrobacterium-Mediated Transformation ofAspergillus awamoriin the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

et al. 2004

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“…The entire cassette was extracted from plasmid pEO62 (Li Destri Nicosia et al 2001) as an EcoRI/HindIII fragment and cloned into plasmid pBC1004 (Carroll et al 1994), which permits hygromycin selection. pNm1H18 was constructed by introduction of a mimp1 element into the first intron of the niaD gene (Figure 3).…”

Section: Methodsmentioning

confidence: 99%

Transposition of a Fungal Miniature Inverted-Repeat Transposable Element Through the Action of a Tc1-Like Transposase

et al. 2007

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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. M INIATURE inverted-repeat transposable elements (MITEs) are a particular group of transposable elements (TEs), first described in plants (Bureau and

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“…Transposons are molecular tools widely used in vitro and/or in vivo for bacteria (30) and yeasts (31,47), but only very recently have they been applied in the filamentous fungal kingdom (9,24). In particular, impala160, a class II transposable element of the Tc1-mariner family (45), has been identified by transposon trapping in the phytopathogenic fungus Fusarium oxysporum (32) and has been shown to transpose efficiently in Fusarium species (28) as well as in Aspergillus nidulans (35) and Magnaporthe grisea (52). The results presented here show that impala160 is also functional in A. fumigatus and can be used to generate a collection of random heterozygous diploids.…”

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confidence: 99%

Identification of Essential Genes in the Human Fungal Pathogen Aspergillus fumigatus by Transposon Mutagenesis

et al. 2003

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The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.

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Heterologous transposition in Aspergillus nidulans

Cited by 16 publications

References 25 publications

Agrobacterium-Mediated Transformation ofAspergillus awamoriin the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Agrobacterium-Mediated Transformation ofAspergillus awamoriin the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Transposition of a Fungal Miniature Inverted-Repeat Transposable Element Through the Action of a Tc1-Like Transposase

Identification of Essential Genes in the Human Fungal Pathogen Aspergillus fumigatus by Transposon Mutagenesis

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