Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

Transformation of Curvularia lunata IM 2901 with pAN7-1 influences selected physiological properties of the fungus

Genetic analysis of Curvularia lunata IM 2901 transformants, previously obtained by electroporation with plasmid pAN7-1, was carried out. Isolates displayed several differences in hygromycin B resistance and their physiology. It was shown that plasmid pAN7-1 was integrated in different copy numbers and at different positions in the genome of the strains studied. Both the wild type and pAN7-1 isolates, when growing in liquid media, produced an extracellular emulsifying agent. The transformants differed in their growth kinetics, intensity of surfactant production and in the efficiency of cortexolone 11beta-hydroxylation, in comparison with the wild type. The micro-organisms varied in susceptibility to the lytic enzyme complex (Novozyme 234), which indicated the presence of differences in their cell wall composition and/or in architecture caused by an integrated plasmid pAN7-1.     

粟长蠕孢菌的原生质体转化

本文报道了利用具有潮霉素抗性标记的质粒(pAN7-1)对粟长蠕孢菌原生质体进行转化的结果。经pAN7-1质粒DNA转化处理的粟长蠕孢菌原生质体在含潮霉素(200μg/ml)的选择性培养基上出现两类转化子。一类是正常转化子,其转化率为2个转化子/μg DNA;另一类是流产转化子,其产生频率为500—600个转化子/μg DNA。DNA杂交分析结果表明,在正常转化子中质粒DNA以首尾相接、重复排列的形式整合入受体菌染色体DNA。初筛获得的转化子多数以异核状态存在,经单孢分离纯化后可通过有丝分裂稳定传代。     

Genetic transformation of Trichosporon cutaneum with a plasmid, pAN 7–1, from filamentous fungi

Genetic transformation of a biological oxygen demand (BOD)-sensing yeast, Trichosporon cutaneum IFO 10466 was carried out using a plasmid shuttle vector, pAN 7–1, between Escherichia coli and Aspergillus niger and Aspergillus nidulans. The hygromycin B-resistant transformants were obtained with the plasmid by using the spheroplasts. The transformation frequency was up to 70 colonies per g of plasmid DNA. Southern-blot analysis and transformation test of E. coli using DNA extracts from hygromycin B-resistant transformants of IFO 10466 indicated that pAN 7–1 existed independently in the cells without integration into the chromosome. These results suggest that pAN 7–1 has potential availability for introduction and expression of the external gene such as a bioluminescence gene, lux or luc, to create a luminous yeast for BOD sensing. © Rapid Science Ltd. 1998     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

赤霉菌原生质体DNA转化*

Methods for the protoplast preparation and DNA transformation of Gibberella fujikuroi were established. The protoplasts were transformed with plasmid pAN7-1, which carries hygromycin B resistant gene (hPh), and the transformation frequency was about 10. Some transformants grew vigorously on the selectivemedium containing 400μg / ml of hygromycin B, while the minimum inhibitory concentration (MIC) of the antibiotic to the recipient strain was oniy 20μg / ml. Southern hybridization showed that the hph gene ha…     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Genetic transformation of the pathogenic fungus Wangiella dermatitidis

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10–203 HmB-resistant colonies/g pAN7-1 on medium containing 100 g HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20–40 g/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17–50 transformants/g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.     

Transformation of the medicinal basidiomycete Trametes versicolor to hygromycin B resistance by restriction enzyme mediated integration

Trametes versicolor, a white-rot basidiomycete, degrades cellulose and lignin as well as many recalcitrant chemicals. There have been many reports about the cloning of laccase and peroxidase genes of T. versicolor which are involved in lignin degradation. In order to analyze a gene function and introduce foreign genes into an organism, genetic transformation is required. Here we have successfully transformed T. versicolor to hygromycin B resistance using pAN 7-1 plasmid by restriction enzyme mediated integration and have obtained many mutants in peroxidase activity and growing patterns. The transformation frequency was 25-50 transformants (microg plasmid DNA)(-1). The transformants were quite stable after 10 consecutive transfers in non-selectable medium.     

Heterologous transformation ofClaviceps purpurea

Summary A transformation system based on dominant selection markers was established for an industrialClaviceps purpurea strain. Transformants could be obtained by using plasmid pAN 7-1 carrying a bacterial gene for hygromycin (hph) resistance fused to a fungal promoter or by plasmid p3SR2 which carries the acetamidase gene (amdS) fromAspergillus nidulans.     

Heterologous transformation of Cochliobolus lunatus

A DNA mediated transformation system has been developed for the filamentous fungus Cochliobolus lunatus. Transformants were obtained by using plasmid pAN 7-1 carrying the Escherichia coli hygromycin B phosphotransferase gene (hph) fused to an Aspergillus nidulans promoter. The integration of plasmid pAN 7-1 into the fungal genome altered the ability of this microorganism to transform progesterone.     

Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes

Summary Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15–21 transformants/ plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the -tubuline gene of a benomyl-resistant Neurospora crassa mutant, the cotransformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractination of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus. Offprint requests to: K. Esser     

Rapid detection of bacterial hygromycin B phosphotransferase in Aspergillus niger transformants

Summary A rapid method is presented for determining in vitro the activity of bacterial hygromycin B phosphotransferase expressed in Aspergillus niger transformants. Even low amounts of this enzyme can be detected with this assay. In addition, there is evidence for a correlation between phosphotransferase activity and the level of resistance to high concentrations of the antibiotic hygromycin B in A. niger transformants.Dedicated to Prof. Dr. Dr. h. c. Karl Esser on the occasion of his 65th birthday     

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.     

Fate of transformed Trichoderma harzianum in the phylloplane of tomato plants

A propiconazole-resistant Trichoderma harzianum strain with high phylloplane survival capability was transformed with the E. coli hygromycin B phosphotransferase gene (hph), coding for hygromycin B resistance. Four transformants were analysed for survival ability on the phylloplane of tomato plants grown under glasshouse conditions in comparison with their prototype and a yellow, hygromycin B-sensitive mutant. Over 2 weeks, the four transformants showed higher survival rates in comparison with the wild-type strain. The yellow mutant TF3/973 did not significantly differ in survival from the transformants. Both hygromycin B resistance and mitotic stability of transformants were evaluated during growth in vitro and after reisolation from tomato phylloplane. Hybridization patterns with the complete plasmid indicated that all four transformants were mitotically stable after several rounds of vegetative growth without selective pressure and during 2 weeks on tomato plants. None of the transformants had lost the ability to grow in the presence of both propiconazole and hygromycin B after growth under the same conditions. The results are discussed in relation to risk assessment of the release of transgenic fungi.     

Genetic introduction of foreign genes to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> by restriction enzyme-mediated integration

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 10⁶ mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.     

Transformation of the fungusPyrenopeziza brassicae,cause of light leaf spot of brassicas,and complementation of mutants using a genomic library

An efficient gene transfer system is a prerequisite for the molecular genetic analysis of pathogenicity and other genes of plant pathogens. A transformation procedure for the fungusPyrenopeziza brassicae was therefore devised. Three plasmids, encoding hygromycin resistance (pAN7-1, pAN7-2) or phleomycin resistance (pAN8-1), were used to transform conidial protoplasts ofP. brassicae in the presence of calcium chloride and polyethylene glycol. Transformation arose due to integration of transforming DNA, apparently at random sites, and multiple integration events were common. The frequency of transformation was variable but similar to that reported for other phytopathogenic fungi (up to 20 μg⁻¹ DNA) and was increased when homologous DNA was included in the vector. The pathogenicity of the transformants was unchanged by transformation and, when reisolated from inoculated host tissue, the transformants were found to have retained their antibiotic resistance. The transformation technique was used to complement adeninerequiring and extracellular enzyme-deficient, UV-induced mutants to prototrophy and extracellular protease production, respectively, with cosmids from a genomic library of the fungus.     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants

Summary A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hyg^r strains.Dedicated to Dr. T.A. Trautner on the occasion of his 60^th birthday