Agrobacterium tumefaciens-Mediated Transformation of the Lichen Fungus,Umbilicaria muehlenbergii

Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway.     

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.     

Strong preference for the integration of transforming DNA via homologous recombination in Trichoderma atroviride

Trichoderma species play important roles in nature as plant growth promotors and antagonists of phytopathogenic fungi, and are used as models to study photomorphogenesis. Molecular tools have been implemented to manipulate and improve these fungi. However, instability of transformants or very low frequency of homologous recombination has been reported. Here, we report the fate of transforming DNA, demonstrating that it can follow two different fates. When a vector contains sequences also present in the Trichoderma atroviride genome, it mainly integrates by homologous recombination generating stable recombinant strains. In contrast, vectors with no sequence homology to the T. atroviride genome generate unstable transformants, losing the transforming DNA in the first generation of conidia produced without selection where, surprisingly, the vector behaves as autoreplicative. Integration by homologous recombination was demonstrated when transformants were generated with a truncated version of the blr2 gene, resulting in insertional mutants with phenotypes identical to those of knockout mutants. Our results indicate that T. atroviride is highly efficient in integrating DNA by homologous recombination and that plasmid vectors with no sequence homology to the genome are maintained for several generations in T. atroviride if kept under selective pressure even though they lacked fungal autonomous replication sequences.     

Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system

Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2^loxP/loxP mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Microprojectile bombardment as a reliable method for transformation of the mucoralean fungus Absidia glauca

Genetic analysis of all Mucor-like fungi is severely impaired by the low efficiency of transformation systems and the genetic instability of the introduced plasmid constructs. The transformation efficiency of one of the model systems among mucoralean fungi, Absidia glauca, was improved considerably by microprojectile bombardment. For this purpose, a plasmid was constructed conferring (i) neomycin resistance as a selective marker and (ii) fluorescence due to expression of the gfp gene from the jellyfish Aequorea victoria. Compared with previous techniques, this method offers increased efficiency, with considerably easier handling than procedures based on protoplasts and, therefore, improved reliability. The uninucleate sporangiospores of A. glauca can be transformed early during the germination process. At this stage the number of nuclei ranges between 1 and 2. Thus, the abundance of transgenic nuclei in the coenocytic mycelia is high, and fewer problems are encountered with detecting low expression levels of the genes used for selection and monitoring of transformants.     

An efficient gene disruption method using a positive–negative split-selection marker and <Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation for <Emphasis Type="Italic">Nomuraea rileyi</Emphasis>

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0–4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ～3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A.?nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

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. Received: 2 September 1996 / Accepted: 7 April 1997     

A set of Hansenula polymorpha integrative vectors to construct lacZ fusions

A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around 100 transformants per microgram of plasmid DNA; in all transformants checked by Southern blotting the plasmid was integrated into the genome locus corresponding to the gene plasmid marker. PCR showed that about 50% of the transformants contained more than one plasmid copy per genome. Experiments carried out using the developed plasmids to determine the strength of the gene promoters involved in nitrate assimilation in H. polymorpha revealed that, in the presence of nitrate, the nitrate reductase gene promoter (YNR1) was the strongest, followed by nitrite reductase (YNI1) and nitrate transporter (YNT1). Received: 5 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Cloning of the Ribosomal Protein L41 Gene of Phaffia rhodozyma and Its Use as a Drug Resistance Marker for Transformation

The ribosomal protein L41 gene of Phaffia rhodozyma was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of P. rhodozyma. Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/μg of DNA with an integrated copy number of about seven per haploid genome.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5′ and 3′ untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Expression of liver fluke antigens by mouse cells

Fasciola hepatica cDNA carried on bacterial plasmids was used in conjunction with marker plasmid DNA to co-transform mouse tissue culture cells using the calcium phosphate procedure. Two systems were used: mouse L cells lacking thymidine kinase activity (Ltk⁻) in conjunction with plasmids pFH4 or pFH1 (carrying parasite DNA) and pHSV-106 (carrying the thymidine kinase gene from herpes simplex virus); and C127 mouse cells with the plasmids pFH4 or pFH1 and the plasmid pBPV-MMTneo(342-12) which carries the bovine papilloma virus genome and, as a selective marker, a gene conferring resistance to the antibiotic geneticin. Both procedures gave rise to transformants which expressed liver fluke antigens: these were detected by a fluorescent antibody test (incorporating flow cytometry) using fluke-infected sheep serum as first antibody. Stability of antigen expression characterised C 127 derived transformants. Ltk⁻ transformants ceased expression within a few weeks.     

丝状真菌遗传筛选系统的研究及其应用

随着基因组时代的发展,主要丝状真菌基因组测序基本完成而被广泛应用于工业、农业、医药等领域。然而丝状真菌的遗传转化效率极低,为了保证在大量非转化子背景下能筛选到目标转化子,恰当的筛选标记显得尤为重要。目前,在丝状真菌的遗传转化过程中常用的筛选标记可分为两类:药物抗性筛选标记和营养缺陷型筛选标记。但两者均具有一定的局限性,为此科研人员利用最新研究的基因组编辑技术对筛选标记加以修饰改造,以更好地用于遗传筛选。本文综述了目前常用的遗传筛选系统的分子机制及运用基因组编辑技术改造的新型筛选标记理论,为丝状真菌在各领域更广泛的应用奠定基础。     

pSH-CUP重组质粒的构建及面包酵母酸性海藻糖 酶基因的敲除

设计含有与面包酵母(Saccharomyces cerevisiae BY-6)编码酸性海藻糖酶ATH基因内部部分序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除单元,转化面包酵母获得G418阳性克隆.将铜抗性基因(cuP1-MT1)导入Cre重组酶表达质粒pSH47,得到重组质粒pSH-CUZ,并转化阳性克隆,以铜抗性筛选面包酵母转化子.半乳糖诱导表达Cre酶切除Kanr基因.重组质粒pSH-CUP的构建,不仅解决了酵母转化子筛选标记问题和非酵母基因的引入,而且使LoxP-kanMX-loxP基因敲除体系在进行真核生物基因敲除时更加方便可行.     

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

The bacterial GUS (β-glucuronidase) gene has been used as a reporter gene in plants and bacteria and was recently expressed in filamentous fungi. Here, we report the application of GUS for the establishment of transient and stable gene expression systems in the phytopathogenic fungus Cochliobolus heterostrophus. The utility of the transient expression system is demonstrated in applications involving promoter analysis and in tests of various parameters of a transformation system, for comparing the rates of stable and transient transformation events using GUS as sole screening marker and for comparing different transformation systems using either GUS or a dominant selection marker. For these purposes two plasmids were constructed harbouring the GUS gene and the hph gene of Escherichia coli which confers resistance to the antibiotic hygromycin B (HygB), ligated either to the P1 or GPD1 (glyceraldehyde 3 phosphate dehydrogenase) promoter of C. heterostrophus. In transient expression studies the first appearance of GUS activity was observed within 2 h after transformation and maximal values were obtained after 7 or 10 h, depending on the promoter fused to the GUS gene. At peak activity, the GPD1 promoter was revealed to be five fold stronger than the P1 promoter. The same difference in promoter strenght was observed when the vectors were stably integrated in the fungal genome. Using the GUS gene as a colour selection marker in plate assays, it was possible to detect transformants and monitor the process of transient gene expression visually. Blue transformants obtained by screening for the GUS phenotype were mitotically unstable. Transformants obtained by selecting for HygB resistance were mitotically stable and expressed the β-glucuronidase gene constitutively. GUS activity in fungal colonies was detected fluorometrically in a nondestructive plate assay. The pathogenicity of these strains was unaltered compared with wild type. The GUS phenotype allowed selective blue staining of the colonizing mycelia on maize leaves.     

Polyethylene glycol-mediated transformation of fused egfp-hph gene under the control of gpd promoter in Pleurotus eryngii

Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90–210 transformants were produced per μg plasmid DNA per 10⁷ viable protoplasts.     

A selectable and excisable marker system for the rapid creation of recombinant poxviruses

Background

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.

Methodology/Principal Findings

Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.

Conclusion/Significance

The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.