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.     

Transformation of Penicillium chrysogenum with a dominant selectable marker

Summary We have cloned a mutant oligomycin resistance allele of the mitochondrial ATP synthase subunit 9 gene from the filamentous fungus Penicillium chrysogenum. The gene was isolated using the equivalent gene from Aspergillus nidulans as a hybridisation probe. Using the cloned gene it is possible to select for oligomycin resistance in P. chrysogenum transformation experiments. This transformation system was used to introduce further copies of the P. chrysogenum isopenicillin N synthetase gene, which were stably maintained without selection. An assessment of the frequency with which homologous integration occurs was also made. With this system, it should prove possible to transform any strain of P. chrysogenum.     

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.     

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.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

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.     

Hygromycin B resistance as dominant selectable marker in yeast

Summary Saccharomyces cerevisiae is normally sensitive to the drug hygromycin B; a hygromycin B concentration of 200 µg/ml in agar plates is sufficient to completely inhibit growth. We constructed yeast-E. coli bifunctional plasmids which confer hygromycin B resistance to Saccharomyces cerevisiae. Promoters and amino terminal coding regions of a heat shock gene, a heat shock cognate gene, and the phosphoglycerate kinase gene from yeast were fused to a bacterial hygromycin B resistance gene. In all three cases, yeast cells containing plasmids with the hybrid hygromycin B resistance gene were resistant to high levels of the drug. Yeast cells containing these plasmids can also be directly selected after transformation by using hygromycin B. The intact bacterial hygromycin B resistance gene and the kanamycin resistance gene from Tn903 were also tested in yeast for their ability to confer resistance to hygromycin B and G418. The intact bacterial genes were not effective in conferring drug resistance to yeast cells.     

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 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 the rice blast fungus Magnaporthe grisea to hygromycin B resistance

Summary Low frequency, integrative transformation of three fertile hermaphroditic strains of Magnaporthe grisea has been achieved using plasmid pAN7-1 and cosmid pAN7-2, which contain an Escherichia coli hygromycin B phosphotransferase gene linked to Aspergillus nidulans regulatory sequences.     

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.     

Presence,transcription and translation of cellobiohydrolase genes in several Trichoderma species

Eight different species of Trichoderma (T. virgatum, T. longibrachiatum, T. harzianum, T. pseudokoningii, T. polysporum, T. koningii, T. todica, T. saturnisporum), and three strains of T. reesei [QM 6a (wildtype), QM 9123 and QM 9414 (derived mutants)] were found to contain single copies of the cellobiohydrolase genes cbh1 and cbh2 in their genome. This was demonstrated by hybridization of the respective chromosomal DNAs with the corresponding gene fragments of T. reesei QM 9414. According to the relative position of cbh1 and cbh2 in Southern blots, T. harzianum, T. virgatum and T. saturnisporum were clearly distinguishable as unique species. Despite the presence of both cbh genes, these species did not form detectable cellobiohydrolase (CBH) I or II, or exhibit any cellulase activity. All other taxa were identical with respect to the genomic position of cbh1, formed two groups with respect to the position of cbh2, and produced varying amounts of CBH I and II. In all cases CBH I and II production correlated with the relative amount of cbh1- and cbh2-mRNA found. This was particularly true for the three strains of T. reesei, which secreted different amounts of CBH I and II, their efficiency to transcribe cbh1 and cbh2 having been increased as a result of mutation for higher cellulase production.Communicated by J.R. Schweyen     

Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants

We have constructed a dominant selectable marker, PHT1, for transformation of the basidiomycete Coprinus cinereus. PHT1 consists of a bacterial hygromycin B resistance gene fused to the promoter and terminator regions of the C. cinereusβ-tubulin gene. We found in transformation experiments that PHT1 confers hygromycin B resistance to all strains of C. cinereus tested, that it integrates without apparent bias into the genome, and that it is stable through meiotic crosses. We used a plasmid containing this marker, pPHT1, for restriction enzyme-mediated integration (REMI) and found that this technique could increase transformation efficiencies more than seven-fold. In REMI experiments using KpnI, the integrated DNA was flanked by intact KpnI sites in 53% of the cases examined, single-copy insertions represented 60% of the integration events, and most multicopy insertions were oriented head-to-tail. A screen of REMI-generated transformants yielded sporulation-defective mutants at a frequency of 1.2%. Genetic analysis showed that in six of nine mutants examined, the defect in spore formation is most likely a direct result of the pPHT1 insertion, and in three of these mutants a single pPHT1 locus was shown to cosegregate with the sporulation defect. We used semi-random PCR to isolate the genomic DNA adjacent to one pPHT1 insertion in a sporulation-defective mutant and found that we had disrupted the C. cinereusspo11 gene. Thus, REMI, in combination with pPHT1, is a powerful tool for the dissection of the meiotic process in C. cinereus. Received: 26 February / 13 July 1999     

The use of DNA-fingerprint analysis in the classification of some species of the Trichoderma aggregate

Summary We have analyzed nine different species of the filamentous fungus Trichoderma and three strains of T. reesei for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides [(CT)₈, (GTG)₅, and (GACA)₄]. On the basis of the DNA-fingerprints obtained, the Trichoderma aggregate is re-classified into five groups: I (T. reesei, T. todica), II (T. polysporum, T. longibrachiatum, T. koningii, and T. pseudokoningii), III (T. virgatum), IV (T. saturnisporum) and V (T. harzianum). These results contradict the claim that T. reesei is a subspecies of T. longibrachiatum. Furthermore, hybridization with (CA)₈ allowed a subdivision of group II, wherein T. pseudokoningii formed a subgroup, IIb, which is highly homologous with, but distinct from subgroup IIa. The results show that RFLP analysis may be used to re-classify the Trichoderma aggregate.     

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 Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker

Summary The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.     

Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca

Summary A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts. Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate. The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis. No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.     

Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.     

The oliC3 gene of Aspergillus niger: isolation,sequence and use as a selectable marker for transformation

Summary The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycinresistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5 untranslated sequences with those from other fungi.