Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans

Summary Penicillium nalgiovense was transformed with the amdS gene from Aspergillus nidulans as a selectable marker. The vector apparently integrated at multiple sites into the chromosomes of the transformants, which were mitotically stable. A transformation efficiency of 12 transformants/g vector DNA was achieved when the expression phase was prolonged to 8 h.     

A transformation procedure for Botryotinia squamosa

Summary The onion leaf blight fungus, Botrytis squamosa, was transformed to hygromycin B resistance using a plasmid (pDH25) containing a bacterial gene for hygromycin phosphotransferase (hph) fused to promoter elements from Aspergillus. Southern hybridization of transformants indicated that single or multiple copies of the vector were integrated into heterologous regions of the B. squamosa genome. Free plasmid was found in undigested preparations of transformant DNA, but was not detected after 3–5 passages of selective transfer. Most transformants were mitotically stable in both selective and non-selective growth; however, both genetic rearrangements and loss of integrated DNA occurred during vegetative growth.     

Transformation of the mycoparasite Gliocladium

Summary Gliocladium roseum and G. virens are saprophytic fungi with biological control activity against various plant pathogens, including those causing seedling diseases in cotton. Genetic transformation systems were developed to provide the potential for incorporating additional traits to improve the biocontrol efficacy of Gliocladium. Gliocladium roseum protoplasts were transformed with G. virens genomic DNA. The 6.7 kb plasmid pH1S containing a bacterial hygromycin B resistance gene, hygB, was used to transform G. virens. Up to ten methionine-independent G. roseum transformants were recovered per microgram of G. virens DNA. Transformation frequencies as high as 150 hygromycin B-resistant transformants per microgram of circular palsmid DNA were observed with electroporation at a field strength of 500 V/cm. Total DNA was isolated from G. virens transformants and hybridized to purified hygB or pBR322 (the vector used in the pH1S construct) DNA. The hygB DNA was integrated into genomic DNA. Precise excision of the plasmid by two different restriction endonucleases provided evidence for the presence of multiple tandem copies in some transformants. The presence of multiple bands in digests of other transformants suggested multiple sites of integration.     

Characterization of inl + transformants of Neurospora crassa obtained with a recombinant cosmid-pool

Summary We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl ⁺ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl ⁺ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F₁ progeny of the transformants. We were able to recover eleven different plasmids from the F₁ progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl ⁺ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.     

High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V)

Summary A cosmid vector, suitable for library construction and DNA transformation in filamentous fungi, has been constructed and a reliable and highly efficient PEG-mediated DNA transformation system for F. solani f. sp. cucurbitae, based on resistance to hygromycin B, has been developed for use with this vector. This transformation system yielded 10⁴ transformants per g of DNA when using 10⁷ protoplasts. Factors important in achieving high efficiency included: the maintenance of an osmoticum in all transformation steps, PEG 4000 concentration, and the ratio of transforming vector DNA to protoplasts. Approximately 60% of transformants stably integrated vector DNA. Molecular analysis revealed multiple copies of the plasmid integrated into the genome at one or more sites. The frequency of transformation achieved will facilitate the isolation of genes from this fungus by complementation.     

Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per g of DNA. The phenotype of the stable Ura+transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.     

Transformation of the thermophilic fungus Humicola grisea var. thermoidea and overproduction of Humicola glucoamylase

Summary The thermophilic fungus Humicola grisea var. thermoidea was successfully transformed using a bleomycin-phleomycin resistance gene linked to regulatory sequences from Aspergillus nidulans. Transformation was achieved using the lithium acetate method with young mycelia, and transformants were obtained at a frequency of 0.5–2 per g of plasmid DNA. Vector DNA used in transformations was integrated in the genome of Humicola in varying patterns and copy number, and transformants were mitotically stable. Extra copies of an Humicola gla1 gene encoding glucoamylase (GAM) were introduced into the genome of several Humicola strains by transformation, with the result that some transformants produced almost 3-fold more GAM in comparison to the untransformed parental strains.     

Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata

An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA [romoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.     

Expression of the Escherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi

Summary The plant pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed using two positive selection systems, one based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the other on the Neurospora crassa -tubulin gene bml which encodes resistance to the methyl benzimidazole carbamate fungicides. Both selection systems gave a transformation frequency of 1–20 transformants g^–1 DNA. The vector DNA was integrated into the genome and the number and sites of integration varied among the transformants. The hph transformants were mitotically stable and the transformed gene was transmitted through spores. In contrast the bml transformants were less stable.     

Transformation of the mycoparasite Parasitella simplex to neomycin resistance

Summary The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.     

Cloning of the Trichoderma reesei pyrG gene and its use as a homologous marker for a high-frequency transformation system

Summary The Trichoderma reesei orotidine-5-phosphate decarboxylase gene was isolated by heterologous hybridization with the corresponding Neurospora gene as a probe. A 2.7 kb SalI fragment, which exclusively hybridized to the Neurospora gene, was subcloned in pGEM-5Zf(+). This subclone was termed pFG1 and was used to transform a Trichoderma reesei pyrG^- negative mutant to PYR⁺. The transformation frequency in this homologous system was up to 12000 transformants per g DNA. About one-fifth of the transformants tested were abortive. Perfect mitotic stability was found in half of the non-abortive transformants, correlating with vector integration at homologous and ectopic loci. In the unstable transformants the transforming DNA appears to be present in the form of extrachromosomal elements.     

NiaA,the structural nitrate reductase gene of Phytophthora infestans: isolation,characterization and expression analysis in Aspergillus nidulans

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia ^- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia ^- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli -glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.     

A homologous and self-replicating system for efficient transformation ofFusarium oxysporum

A highly efficient transformation system has been developed forFusarium oxysporum f. sp.lycopersici based on the complementation of a nitrate-reductase mutant with the homologousnitI gene and on the presence ofARS and telomeric sequences in the vector. Preliminary transformation experiments with theniaD gene fromAspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with theF. oxysporum nitl gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicativeF. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per g of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment fromF. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with theARS from different organisms has shown that this fragment contained the commonly identifiedARS consensus sequence, 5A/TTTTATA/GTTTA/T3 and, in addition to this core, ten copies of theARS-box, TNTA/GAA3. Adjacent to this presumedARS, the telomeric hexanucleotide sequence (TTAGGG)_n was present in six tandem copies followed by 18 copies of its complementary sequence.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Transformation of Trichoderma viride using the Neurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene from Aspergillus oryzae

Summary A pyrG mutant of Trichoderma viride, a very efficient cellulase producer, was isolated from among 5-fluoroorotic acid-resistant mutants. The mutation was complemented with the pyr4 gene of Neurospora crassa and used as a selection marker for the transformation of T. viride. A plasmid vector, pDJB1-Taa, carrying both the pyr4 gene and a gene encoding Taka-amylase A from Aspergillus oryzae, was constructed and introduced into protoplasts of T. viride pyrG^-. The transformation frequency was 1–10 transformants (3 on average) per g DNA. One transformant showed highly elevated -amylase production (about 17 times higher than the recipient level) and the integration of more than one copy of the Taka-amylase gene.     

Gibberella pulicaris transformants: state of transforming DNA during asexual and sexual growth

A genetically fertile, trichothecene-producing plant pathogen, Gibberella pulicaris (Fusarium sambucinum), was transformed with three different vectors: cosHyg1, pUCH1, and pDH25. All three vectors carry hph (encoding hygromycin B phosphotransferase) as the selectable marker. Transformation frequency was 0.03 transformants per mg of DNA for pDH25 and 0.5 for pUCH1 or cosHyg1. The vector DNA sequences integrated at different sites into the fungal genome. Transformants were classified into three types based upon distinctive integration patterns: type A contained a single, intact copy of the vector at one site per genome; type B contained multiple tandem copies or a combination of single and multiple tandem copies at one or more sites per genome; type C contained a partial vector copy at one site per genome. While the transformants with cosHyg1 and pUCH1 were type A or B, type C was unique to pDH25 transformants. Type A and C transformants were both meiotically and mitotically stable. However, type B multiple inserts were unstable in mitosis and meiosis since: (1) multiple tandem copies were deleted: (2) rearrangements occurred during premeiosis; and (3) inserts in one of the type B transformants became methylated during premeiosis. Differential expression of transforming sequences between spore germination and mycelial growth was also observed among type B transformants. The ability to transform G. pulicaris with the resulting varied features of integration patterns and the behavior of transforming DNA during mitosis and meiosis provides a means to isolate, manipulate, and study cloned genes in this mycotoxin-producing plant pathogen.Mention of companies or products by name does not imply the endorsement by the U.S. Department of Agriculture over others not cited     

Highly-efficient transformation of the homobasidiomycete Schizophyllum commune to phleomycin resistance

Regulatory sequences of the glyceraldehyde-3-phosphate-dehydrogenase (GPD) gene from the homobasidiomycete Schizophyllum commune were fused to the coding sequence of the ble gene from Streptoalloteichus hindustanus, which codes for a phleomycin-binding protein. The resulting construct transformed S. commune to phleomycin resistance at a high frequency (up to 10⁴ transformants/g DNA per 10⁷ protoplasts) when regeneration was done in 0.5 M MgSO₄. A similar construct with regulatory sequences from Aspergillus nidulans failed to give transformants, showing the importance of homologous regulatory sequences for the expression of genes in S. commune. The homologous GPD promoter could be deleted up to position -130 without any effect on the number of phleomycin-resistant transformants. This is the first effective stable transformation system in a homobasidiomycete employing antibiotic resistance.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.