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 phytopathogenic fungus Septoria nodorum to hygromycin B resistance

Summary The phytopathogenic fungus Septoria nodorum has been transformed using a plasmid (pAN7-1) containing the Escherichia coli hygromycin phosphotransferase gene (hph). Large, stable hygromycin-resistant transformant colonies appeared at frequencies between 2 and 25 per g DNA when wheat-adapted and barley-adapted wild type strains were used as recipients. These transformants grew at hygromycin concentrations up to ten times that which inhibits the wild types. A second type of colony also developed on transformation plates. These appeared at higher frequencies, grew less vigorously and could not be subcultured in the presence of hygromycin. They are believed to be abortive transformants. Southern hybridization analyses indicated that transformation takes place via the integration of plasmid DNA into the fungal chromosomal DNA. Multiple integrations occur producing tandemly iterated arrays of plasmid molecules. Some transformants arose as heterokaryons. These could be resolved by propagation through a single spore and transformants purified in this way remained mitotically stable. All of 1,025 transformants tested were unchanged in pathogenicity. Reisolates from leaves retained their hygromycin-resistance, indicating that transformants remain stable during growth in plant tissue. Cotransformation of an unselected plasmid (p3SR2) carrying the Aspergillus nidulans amdS gene occurred at a high frequency.     

Transformation of Botrytis cinerea with the hygromycin B resistance gene,hph

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), confering hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2–10 transformants per g of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.     

Biolistic transformation of conidia of Botryotinia fuckeliana

Botryotinia fuckeliana, the causal agent of grey mould, was biolistically transformed to hygromycin B resistance using a plasmid (pOHT) containing a bacterial hygromycin phosphotransferase gene fused to regulatory sequences from Aspergillus nidulans. Multiple copies of the plasmid, precipitated onto tungsten particles, were delivered into the conidia by a helium-driven gene gun. Southern analysis showed that the plasmid was integrated into the fungal genome at one single locus. After five subsequent transfers on selective medium, all transformants were mitotically stable. When propagated on non-selective medium, four out of eight transformants retained their resistance to hygromycin B. Southern analysis of the fifth generation of transformants showed that no genetic rearrangements occurred during vegetative growth of stable transformants.     

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     

Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals

Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E. coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pkil (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively. Transformation frequencies of over 1800–2500 transformants/g DNA were obtained, which is a 15–20-fold increase over that with pAN7-1, which contains hph between A. nidulans expression signals. Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pkil promoter and the cbh2 terminator integrated into the genome. Evidence for preferentially ectopic integration is given.     

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.     

Genetic transformation of the fungal plant wilt pathogen,Fusarium oxysporum

Summary A system for transformation of the fungal plant pathogen Fusarium oxysporum has been developed. The system employs plasmids which contain a bacterial hygromycin B phosphotransferase gene (hph) linked to Aspergillus regulatory sequences and which confer hygromycin B resistance in Fusarium. Transformation resulted from integration of the vectors into heterologous regions of the Fusarium genome and occurred at a frequency of approximately one transformant per µg DNA. No evidence was found for autonomous replication of the vector in the fungus. The transformed, drug resistant phenotype was mitotically stable with or without selection. However, modification of integrated DNA could occur during vegetative growth.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the antitumor clavaric acid-producing basidiomycete <Emphasis Type="Italic">Hypholoma sublateritium</Emphasis>

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.     

Transformation of the oomycete pathogen Phytophthora megasperma f. sp. glycinea occurs by DNA integration into single or multiple chromosomes

A procedure for stable transformation was developed for Phytophthora megasperma f. sp. glycinea, an oomycete pathogen of soybean. Transformants were obtained using a bacterial hygromycin resistance gene fused to a promoter and terminator from the ham34 gene of another oomycete, Bremia lactucae. Vector DNA, alone or complexed to cationic liposomes, was introduced into protoplasts using polyethylene glycol and CaCl₂. DNA and RNA hybridization, and phosphotransferase assays, confirmed the presence and expression of vector DNA in the transformants. Hybridization to electrophoretically separated chromosomes of P. m. glycinea showed that vector DNA had integrated into only one chromosome in four transformants, and into multiple chromosomes in one transformant.     

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.     

Transformation of Acremonium coenophialum,a protective fungal symbiont of the grass Festuca arundinacea

Summary Acremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue). An electroporative transformation system was developed for A. coenophialum. Segments of DNA 5 to the -tubulin gene (tub2) of the closely related ascomycete Epichloë typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30–200% depending on the plasmid construct. Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.     

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant -tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10–20 transformants per g of DNA per 4-8×10⁷ viable protoplasts. Intergration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems is the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.     

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.     

Transformation of Trichoderma species with dominant selectable markers

Summary We have developed a transformation system for Trichoderma hamatum and Trichoderma harzianum Rifai, using dominant markers for selection based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the -tubulin gene (bml) from Neurospora crassa, respectively. Transformation frequencies and protoplast regeneration were low in both species. All the T. hamatum hygromycin-resistant transformants analysed were mitotically stable, in contrast to those of T. harzianum derived by benomyl resistance, in which only 50% of the transformants analysed were stable. Molecular analysis of transformants showed the integration of the transforming DNA into the genome and indicated that the number and sites of integration varied among the transformants.     

Development of transformation system for <Emphasis Type="Italic">Trichophyton rubrum</Emphasis> by electroporation of germinated conidia

Dermatophytes are the fungi that can cause infections of skin, hair, and nails due to their ability to utilize keratin. The genetic transformation systems of dermatophytes were successfully applied to Trichophyton mentagrophytes and Microsporum canis. Here we describe the procedure for genetic transformation of Trichophyton rubrum by electroporation of their germinated conidia. A linearized transformation vector (pCHSH75-Pch/GFP/TtrpC) containing bacterial hygromycin B phosphotransferase gene (hph) and green fluorescent protein gene (egfp) was introduced into the germinated conidia of T. rubrum by electroporation. PCR reaction analysis showed that egfp gene was integrated randomly and Southern blotting analysis demonstrated a single integration of hph gene into the chromosomal DNA of randomly selected transformant. In this work we report the efficient transformation and selection of the stable T. rubrum transformants.     

Development of a transformation system for the filamentous,ML-236B (compactin) — producing fungus Penicillium citrinum

Summary We present here the first report of a transformation system developed for the filamentous, ML-236B (compactin)-producing fungus Penicillium citrinum. Hygromycin B-resistant colonies were obtained after treatment of protoplasts with a vector containing an Escherichia coli hygromycin B phosphotransferase gene fused to a 3-phosphoglycerate kinase promoter from Aspergillus nidulans. The transformation rate was 194 transformants per g circular DNA per 4x10⁵ viable protoplasts under optimized transformation conditions. Transformation took place via the integration of plasmid DNA into the fungal chromosomal DNA. Most of the integration events appeared to produce tandemly iterated arrays of plasmid molecules at different sites in the chromosome. The transformed, drug-resistant, phenotype and the integrated plasmids were mitotically stable with or without selection in a majority of cases. The demonstration of such a transformation system is an essential first step in the application of recombinant DNA technology to strain improvement and for the production of novel ML-236B derivatives.     

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15–20 g of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pANsCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.Dedicated to Professor Dr. K. Esser on the occasion of his 70th birthday