Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Notes High-efficiency transformation system for the biocontrol agents, Trichoderma spp.

We have developed an efficient transformation system based on the use of polyethylene glycol and CaCl2 for the biocontrol agents, Trichoderma spp. Transformation was obtained with the plasmid pAN7-1, carrying a bacterial hygromycin-resistance gene as a selectable marker, under the control of Aspergillus nidulans heterologous expression signals. The system described here yielded 200-800 transformants per microgram of DNA. Transformants contained several copies of the plasmid integrated into their genome, apparently at the same site in the different transformants analysed. Stability of the transformants was achieved by inserting a 2.4kb homologous DNA fragment into pAN7-1. Southern blot analysis indicated that integration in the stable transformants occurs through non-homologous recombination.     

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

Cloning an Aspergillus nidulans developmental gene by transformation.

We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A. nidulans developmental gene brlA by visual selection. The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved. The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

Summary When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Efficient Transformation of the Astaxanthin-Producing Yeast Phaffia Rhodozyma

An efficient transformation system for the astaxanthin-producing yeast Phaffia rhodozyma was developed based on electroporation that routinely yields approximately 1000 transformants per g of plasmid DNA. The high transformation efficiency depends on vector integration in the ribosomal DNA (rDNA) and the presence of the homologous glycolytic glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and terminator to drive the expression of the transposon Tn5 encoded kanamycin resistance gene (Km^R) as a selective marker. Using this system stable transformants were obtained, carrying multiple plasmid copies. Plasmid copy number could be markedly increased by deletion of the gpd terminator from the transforming plasmid.     

红曲霉不同转化方法比较

为了研究红曲霉聚酮体途径,考察和比较了4种不同的转化方法以建立有效的红曲霉遗传转化系统。以潮霉素作为抗性筛选标记,pBC-Hygro作为转化载体,用基于原生质体的传统转化和电击转化、基于萌发孢子的电击转化以及REMI技术转化红曲霉。发现基于萌发孢子的电击转化由于转化率极低而不适于红曲霉转化。基于原生质体的传统转化和电击转化尽管每微克DNA分别能获得135个转化子和125个转化子,但因转化子稳定性差也适合红曲霉转化的转化。应用REMI技术,转化率提高约20倍,每微克DNA 2500个转化子,70%～75%的转化子的稳定,非常适合于红曲霉的转化。      

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.     

Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.     

A versatile and efficient gene-targeting system for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.     

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker.     

Stable transformation of the cyanobacterium Synechocystis sp. PCC 6803 induced by UV irradiation.

Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 X 10(8) cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmid is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV-inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 X 10(-4) and 1.5 X 10(-5), respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.     

Efficient Agrobacterium-mediated transformation of <Emphasis Type="Italic">Lotus japonicus</Emphasis> with reliable antibiotic selection

We have developed a new procedure for transforming a model legume, Lotus japonicus, that yields transformed plants from transverse cotyledon segments without contamination from the presence of non-transformants that survived the antibiotic selection. L. japonicus was transformed with the HPT gene as a selectable marker and the GUS reporter gene, both of which were driven by cauliflower mosaic virus 35S promoter. The efficacy of selection with hygromycin was tested using the assay of GUS activity in putative transformants. The integration of the GUS gene was also confirmed by polymerase chain reaction analysis of the genomic DNA. The integrated T-DNA was stable and inherited as a dominant trait. This procedure may have potential effectiveness and application in large-scale transformation for insertional mutagenesis or gene tagging.