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 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 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 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.     

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 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.     

The transformation of protoplasts of Leptosphaeria maculans to hygromycin B resistance

Summary Conditions are described for the efficient isolation and regeneration of protoplasts of a fungal pathogen of brassicas, Leptosphaeria maculans. Treatment of the protoplasts with DNA of the plasmid pAN7-1 (containing an E. coli hygromycin phosphotransferase gene with Aspergillus nidulans expression signals) and plating under selective conditions resulted in the formation of hygromycin 13-resistant colonies. Southern blot analysis of resistant colonies indicated that single copies of the plasmid had integrated into different sites in the genome. In twelve of the transformants analysed so far, the integration is stable through mitosis. The demonstration of efficient transformation is an essential first step in the molecular analysis of pathogenicity of this commercially important pathogen.     

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.     

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.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

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.     

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     

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.     

<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.     

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.     

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 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.     

Mitotic stability of transforming DNA is determined by its chromosomal configuration in the fungus Cochliobolus heterostrophus

Summary Cochliobolus heterostrophus was transformed with a plasmid (pH1S) containing a bacterial gene (hygB), which confers resistance to the antibiotic hygromycin B when under control of an 838-bp fragment of promoter 1 from C. heterostrophus. The plasmid integrated at either homologous (52% single copy, 33% tandemly repeated copies) or ectopic (4% single copy, 11% tandemly repeated copies) sites on different chromosomes, resulting in four distinct configurations of integrated DNA. All four configurations were highly stable during mitotic growth; virtually no loss of integrated DNA was detected after five subcultures on nonselective medium or after seven cycles of pathogenesis on maize, the normal host of this fungus. However, deletion of integrated DNA was detected after eight or more disease cycles. The frequency of deletion depended on the configuration of the recombinant chromosome. A single copy of pH1S integrated at an homologous site was flanked by direct repeats of the target sequence and was least stable; up to 50% of the population lacked integrated DNA after 12 disease cycles. A single copy integrated at an ectopic site had no repeated DNA directly associated with it and was the most stable; no deletions were detected after 12 disease cycles. Tandemly repeated copies of pH1S integrated at either homologous or ectopic sites appeared to have intermediate stability; 2–18% of each population lost at least one copy after 12 disease cycles, although in no case were all copies deleted. Cytosine residues of integrated DNA were methylated during mitotic growth, but this had no apparent effect on the expression of hygB.     

Transformation of Penicillium roqueforti to phleomycin- and to hygromycin B-resistance

Summary A strain of Penicillium roqueforti was transformed to hygromycin B and phleomycin resistance using resistance genes under the control of A. nidulans sequences. The transformation efficiency ranged from 0.15 to 1 transformant per g DNA per 10⁶ viable protoplasts when transformants were selected on medium containing a high antibiotic concentration (7–10 times the minimum inhibitory concentration). Transformation resulted from either single copy or tandem integration of the phleomycin vector while the hygromycin vector was modified during integration. The transformed antibiotic-resistant phenotypes were mitotically stable with or without selective pressure.     

Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum

Summary The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per g of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycinfree medium. All tranformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.