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.     

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.     

Transformation of Trichoderma harzianum by high-voltage electric pulse

Summary We have developed conditions for an efficient method of genetic transformation in Trichoderma harzianum, using high-voltage electroporation. Transformation was obtained with a plasmid carrying the Escherichia coli, hygromycin B phosphotransferase gene as a dominant selectable marker, and the gpd promoter and trpC terminator from Aspergillus nidulans. The transformation frequency is up to 400 transformants per g of plasmid DNA. The transformants were phenotypically 100% stable; they were also mitotically stable. Hybridization experiments suggest that the transforming DNA might be integrated at the same position in the T. harzianum genome. This report opens possibilities for improving transformation systems that have already been described for fungi, or else for transforming filamentous fungi where the use of polyethylene glycol is not efficient.     

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 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.     

Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

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.     

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 Podospora anserina with a dominant resistance gene

Summary The Podospora anserina immortal mutant incoloris, vivax was transformed to benomyl resistance with a -tubuline gene from a resistant Neurospora crassa strain. The transforming plasmid was integrated into the genome of the transformants, and subsequent Southern analysis and retransformation experiments provided no evidence for autonomous replication. Non-homologous integration was demonstrated in some of the transformants. Resistance to benomyl varied widely among the transformants and was conserved after the transformants were grown on non-selective medium.     

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 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.     

Meiotic and mitotic stability of transforming DNA in the phytopathogenic fungus Magnaporthe grisea

Summary Magnaporthe grisea was transformed with cosmid pAN7-2 encoding hygromycin B resistance but containing no homology with the M. grisea genome. Rearrangement of the integrated DNA was detected in several hygromycin B-resistant progeny from cross Guy11-T10-1 (a single-copy integration site transformant)x2539 (sensitive wild-type parent), but not in hygromycin B-resistant progeny from four other crosses. Transformants produced typical lesions when inoculated onto host plants. Southern hybridization revealed rearrangements of integrated DNA in single conidial isolates of highcopy transformant 2539-T1-1 re-isolated from host plants, characterized by excision of one or more copies of the transforming plasmid. Plasmid loss and rearrangement were also observed within single conidial isolates derived from transformant 2539-T1-1 following ten asexual generations on non-selective agar medium. These examples of instability of integrated DNA in M. grisea transformants suggest that caution should be exercised in the use of transformation for assessing the phenotypic effects of specific introduced genes.     

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.     

Transformation ofGaeumannomyces graminis to benomyl resistance

Summary Gaeumannomyces graminis var.graminis andtritici were transformed to benomyl resistance using pBT3, a plasmid encoding fungicide-resistant -tubulin. Either circular or linear plasmid DNA producedG. graminis var.graminis transformants in which plasmid DNA was integrated into the fungal genome. There was no evidence for autonomous plasmid replication in any of the transformants examined. 4/11 linear DNA transformants had a single plasmid copy, whereas 8/9 circular DNA transformants had multiple copies of the plasmid. Integration of transforming DNA occurred by nonhomologous recombination in all (20/20) of these transformants.     

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 Fulvia fulva,a fungal pathogen of tomato,to hygromycin B resistance

Summary A transformation system for the tomato pathogen Fulvia fulva has been developed. Hygromycin B resistant colonies were obtained after treatment of protoplasts with a plasmid containing an E. coli hygromycin B phosphotransferase gene fused to an Aspergillus nidulans promoter. The DNA was stably integrated into the genome. The number and sites of integrations varied among transformants. The demonstration of transformation opens the way for the molecular genetic analysis of the interaction of Fulvia with tomato.     

The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain

Summary Six uridine auxotroph mutants of Trichoderma reesei QM 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as OMP-decarboxylase negative (pyr ^-) by a radiometric enzyme assay. Transformation to uridine prototrophy was achieved with the pyr4 gene of Neurospora crassa (up to 1500 transformants/g) and with pyrA of Aspergillus niger (700–800 transformants/g). In many transformants the PYR⁺ function seems to be present as extrachromosomal DNA. There is evidence for a correlation between the stability of transformants and integration of the vector in the genome whereas unstable transformants are obtained when autonomous replication of the plasmid occurs.     

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.     

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 Aspergillus giganteus to hygromycin B resistance

Summary A wild strain of A. giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A. nidulans sequences. Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome. Between 6 and 30 resistant colonies were obtained per g DNA per 3×10³ viable protoplasts. Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants.