Restriction Enzyme-mediated Integration Research Articles

Functional analysis of the homoserine O-acetyltransferase gene and its identification as a selectable marker in Gibberella zeae.

We used restriction enzyme-mediated integration (REMI) to identify a methionine auxotrophic mutant of Gibberella zeae, an important cereal pathogen. In addition to its methionine requirement, the G. zeae REMI mutant designated Z43R3912 showed pleiotropic phenotypes, including reduced virulence on host plants and lack of sexual development. Outcrossing of Z43R3912 with a mat1-1 deletion strain confirmed that the mutation of Z43R3912 was tagged with the hygromycin B resistance marker. The vector insertion site in Z43R3912 was identified within the ORF designated GzmetE, encoding a putative homoserine O-acetyltrasferase (HOA). Gene disruption analyses confirmed that GzmetE was responsible for the pleiotropic phenotypes of Z43R3912. Genetic complementation of the G. zeae methionine auxotroph with an intact copy of the Aspergillus nidulans metE and GzmetE genes suggests that the HOA gene can be used as a selectable marker for transformation of G. zeae.

Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton mentagrophytes.

Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.

Development of a Robust Screening Method for Pathogenicity of Colletotrichum spp. on Strawberry Seedlings Enabling Forward Genetic Studies.

Generation and screening for nonpathogenic mutants is a popular tool for identifying pathogenicity-related genes. Successful application of this technique for plant fungal pathosystems requires reliable and rapid screening procedures. This study reports on the development of a rapid in vitro bioassay enabling large-scale screening and isolation of nonpathogenic mutants of Colletotrichum gloeosporioides and C. acutatum on strawberry seedlings. Inoculation was carried out on strawberry seedlings at two different developmental stages: 12-week-old (young) and 15-week-old (older) seedlings. A comparison was made between two inoculation techniques, (i) foliar dip and (ii) root soak, at two incubation temperatures (19 and 25°C). Mortality of young seedlings was observed 4 days after inoculation with both species, reaching 50% within 10 days, using both techniques at 25°C. However, mortality of older seedlings was delayed by 4 days compared with that in the young seedlings when using the root-soak method. Disease development decreased in young and older seedlings at the lower temperature. This method also was reliable in determining pathogenicity of the cucurbit-specific C. magna that did not cause disease symptoms on strawberry by either inoculation method. The proposed method enabled screening of more than 980 restriction enzyme-mediated integration mutants resulting in a selection of five reduced-virulence isolates. Initial characterization of some of these mutants revealed large differences in germination and appressorial formation compared with pathogenic isolates.

Insertional mutagenesis and characterization of a polyketide synthase gene ( PKS1) required for melanin biosynthesis in Bipolaris oryzae

A polyketide synthase gene named PKS1, involved in the melanin biosynthesis pathway of the phytopathogenic fungus Bipolaris oryzae, was isolated using restriction enzyme-mediated integration. Sequence analysis showed that the PKS1 encodes a putative protein that has 2155 amino acids and significant similarity to other fungal polyketide synthases. Targeted disruption of the PKS1 gene showed that it is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that PKS1 transcripts were specifically enhanced by near-ultraviolet radiation (300–400 nm) and that its temporal transcriptional patterns were similar to those of THR1 and SCD1 genes involved in the melanin biosynthesis pathway of B. oryzae.

The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain

We identified and characterized elongationless3 ( eln3-1), a restriction enzyme-mediated integration (REMI) mutation affecting fruiting body morphogenesis in Coprinus cinereus. The mutant produces an aberrant fruiting body in which the stipe hardly elongates during fruiting body maturation. In the wild type, cylindrical stipe cells, elongation growth of which is responsible for stipe elongation, make side-by-side contact with one another and run parallel to the stipe axis, whereas in the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells. This disorganization of the stipe tissue, together with reduced elongation of the stipe cells, causes the mutant stipe short and bulgy. After a plasmid rescue, the eln3 gene was identified as a DNA fragment that complements the eln3-1 mutation. The eln3 ORF is predicted to encode a protein of 927 amino acids with a general glycosyltransferase domain and to be located in the plasma membrane. Transcription of the eln3 gene is specifically activated in rapidly elongating stipes. Possible involvement of the putative Eln3 enzyme in cell-to-cell connection is discussed. Index Descriptors: Homobasidiomycete; Coprinus cinereus; Fruiting body morphogenesis; REMI mutant; Stipe elongation; Cell-to-cell connection; Membrane protein; Glycosyltransferase

The eln3 gene involved in fruiting body morphogenesis of Coprinus cinereus encodes a putative membrane protein with a general glycosyltransferase domain

We identified and characterized elongationless3 (eln3-1), a restriction enzyme-mediated integration (REMI) mutation affecting fruiting body morphogenesis in Coprinus cinereus. The mutant produces an aberrant fruiting body in which the stipe hardly elongates during fruiting body maturation. In the wild type, cylindrical stipe cells, elongation growth of which is responsible for stipe elongation, make side-by-side contact with one another and run parallel to the stipe axis, whereas in the mutant, the organization of the stipe tissue is disturbed and much space is produced between stipe cells. This disorganization of the stipe tissue, together with reduced elongation of the stipe cells, causes the mutant stipe short and bulgy. After a plasmid rescue, the eln3 gene was identified as a DNA fragment that complements the eln3-1 mutation. The eln3 ORF is predicted to encode a protein of 927 amino acids with a general glycosyltransferase domain and to be located in the plasma membrane. Transcription of the eln3 gene is specifically activated in rapidly elongating stipes. Possible involvement of the putative Eln3 enzyme in cell-to-cell connection is discussed. Index Descriptors: Homobasidiomycete; Coprinus cinereus; Fruiting body morphogenesis; REMI mutant; Stipe elongation; Cell-to-cell connection; Membrane protein; Glycosyltransferase

Genetic transformation and mutant isolation inGanoderma lucidumby restriction enzyme-mediated integration

Genetic transformation and mutant isolation in<i>Ganoderma lucidum</i>by restriction enzyme-mediated integration

Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration

A white-rot basidiomycete Ganoderma lucidum has long been used as a medicinal mushroom in Asia, and it has an array of enzymes important for wood degrading activity. There have been many reports about the ingredients which show health aiding effects. In order to analyze gene functions and introduce foreign genes into this fungus, genetic transformation is required. We have successfully transformed G. lucidum to geneticin resistance using pJS205-1 which has the antibiotic resistance genes against geneticin and phosphinothricin. Many different mutants have been generated during the transformation by restriction enzyme mediated integration, and the transformation yield was 4–17 transformants (μg plasmid DNA) −1. The plasmid was integrated stably into the recipient chromosome, which was confirmed by PCR with the plasmid-specific primers.

Cloning of the pathogenicity-related gene FPD1 in Fusarium oxysporum f. sp. lycopersici

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.

Three decades of fungal transformation: novel technologies.

Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research. In addition, they are excellent models for molecular and genetic studies. An important key in the advancement of genetics and molecular biology of a given organism is the development of genetic transformation systems. This technology makes possible the analysis and manipulation of the genome of the organism of interest. Thirty years from the first report of transformation of a fungus, transformation of many other fungi has been achieved. However, the development of gene tagging systems generally applicable to a wide range of filamentous fungi has remained elusive. A widely used gene tagging strategy for filamentous fungi is restriction enzyme mediated integration (REMI). In recent years numerous reports have been published describing the effective application of REMI. However, REMI shows certain disadvantages for some fungi. Recently a very promising alternative strategy has been reported based on the use of the soil bacterium Agrobacterium tumefaciens. Using this system a well-defined DNA segment (T-DNA) is transferred, which integrates by illegitimate recombination and is 100-1000 times more efficient than conventional methods. The T-DNA can be used as an efficient tool to generate recombinant strains where DNA is integrated as a single copy, allowing the generation of collections of gene-tagged mutants of the fungus of interest.

Gdt2 regulates the transition of Dictyostelium cells from growth to differentiation.

BackgroundDictyostelium life cycle consists of two distinct phases – growth and development. The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known.ResultsWe have isolated a REMI (restriction enzyme-mediated integration) mutant, which prematurely initiates multicellular development. When grown on a bacterial lawn, these cells aggregate before the bacteria are completely cleared. In bacterial suspension, mutant cells express the developmental marker discoidin Iγ even at low cell densities and high concentrations of bacteria. In the absence of nutrients, mutant cells aggregate more rapidly than wild type, but the rest of development is unaffected and normal fruiting bodies are formed. The disrupted gene shows substantial homology to the recently described gdt1 gene, and therefore was named gdt2. While GDT1 and GDT2 are similar in many ways, there are intriguing differences. GDT2 contains a well conserved protein kinase domain, unlike GDT1, whose kinase domain is probably non-functional. The gdt2 and gdt1 mRNAs are regulated differently, with gdt2 but not gdt1 expressed throughout development. The phenotypes of gdt2- and gdt1- mutants are related but not identical. While both initiate development early, gdt2- cells grow at a normal rate, unlike gdt1- mutants. Protein kinase A levels and activity are essentially normal in growing gdt2- mutants, implying that GDT2 regulates a pathway that acts separately from PKA. Gdt1 and gdt2 are the first identified members of a family containing at least eight closely related genes.ConclusionsWe have isolated and characterised a new gene, gdt2, which acts to restrain development until conditions are appropriate. We also described a family of related genes in the Dictyostelium genome. We hypothesise that different family members might control similar cellular processes, but respond to different environmental cues.

Isolation and characterization of mutations that affect nuclear migration for dikaryosis in Coprinus cinereus.

We identified 14 Coprinus cinereus variants defective in nuclear migration for dikaryosis after restriction enzyme-mediated integration mutagenesis. All the variants were able to donate nuclei but failed to accept nuclei in compatible matings. Of the 14 variants, six were due to single-gene mutations. We characterized num1, a gene responsible for one of the mutations. The num1 ORF interrupted by three introns is predicted to encode a protein of 217 amino acids. The Num1 protein has two leucine-zipper motifs, one at the N-terminal region and the other at the C-terminal region. Num1 is also predicted to form a coiled-coil structure in its C-terminal half. The num1-1 mutant allele is predicted to produce a truncated peptide with only one leucine-zipper motif at the N-terminal region. A plasmid carrying a sequence for the truncated peptide could inhibit nuclear migration when introduced into a wild-type strain, showing that the num1-1 mutant allele is dominant negative. The transcription of num1 is down-regulated when the pathway regulated by B mating-type genes is activated.

Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis.

To identify plant-induced genes in the maize pathogenic fungus Ustilago maydis we have developed a genetic screen that combines REMI (restriction enzyme mediated integration) mutagenesis with enhancer trapping using the gene for Green Fluorescent Protein (GFP) as vital reporter. Of 2,350 insertion mutants isolated, three were shown to express GFP only after the fungus had come into contact with the host maize plant. One of the genes tagged was mfa1, which encodes the pheromone precursor, while the second gene, pig2, codes for a product that showed similarity to protein disulfide isomerase. The third integration event had occurred in a locus which we designated the p -locus. This locus contains 11 genes in a 24-kb stretch. Of these, pig3, 4, 5, 6 and 7 show a plant-regulated expression pattern, while the other genes found at the locus (designated npi) do not. Of the plant-regulated genes only two were found to be similar to database entries: the pig4 product is related to membrane transporters of the major facilitator family, while the pig6 protein shows similarity to multidrug transporters. Detailed expression studies revealed that the five plant-regulated genes at the p -locus differ in their expression profiles. Mutants deleted for each of them showed no apparent phenotype, while the npi1 gene appeared to be essential. A viable deletion encompassing the entire p -locus could be generated when npi1 function was provided ectopically. This deletion mutant also showed no obvious alteration in virulence.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Tagging and localization of a phospholipase D gene in Coprinellus congregatus by restriction enzyme-mediated integration and pulsed-field gel electrophoresis

We have identified a phospholipase D gene ( pld) fragment from a transformant generated by restriction enzyme-mediated integration in Coprinellus congregatus, which is a mushroom-forming basidiomycete. A fragment of pld of this fungus has been cloned from a transformant by gene tagging. The transformation vector has been inserted into pld and this has resulted in a decreased enzyme activity of the transformant compared with the wild strain. C. congregatus has seven chromosomes, the range of its genome size is 1.6–4.7 Mb and pld is located at chromosome 4 where the transformation vector has been inserted.

A novel gene trap method using terminator-REMI and 3′ rapid amplification of cDNA ends (RACE) in Dictyostelium

We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase ( bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3′ proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3′ rapid amplification of cDNA ends (3′-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3′ ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3′ sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.

The Efficient Transformation of Pleurotus ostreatus using REMI Method

Restriction enzyme-mediated integration (REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 μg of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 μg of linearized pTRura3-2 DNA was added into 1×107 protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

PHOMOPSIS VITICOLA IS EASILY TRANSFORMED WITH hph AND Bmlr GENES

Phomopsis viticola (Sacc.) Sacc. is the phytopathogenic fungus causing a severe disease of grapevine known as Phomopsis cane and leaf spot. Protoplasts from mycelium of P. viticola were successfully transformed either with plasmids carrying the bacterial hph gene, conferring resistance to hygromycin B (pAN7-1, pOHT, pOHT-AMA1), or the Bmlr gene from Neurospora crassa, causing resistance to benzimidazole fungicides (pBT6). Up to more than 300 transformants per mg of plasmid DNA were obtained with the hph marker gene. The highest effectiveness was obtained with pOHT, whereas pBT6 yielded around 25 transformants per mg of plasmid DNA. Southern blot analysis showed the occurrence of multiple integration events in the fungal genome of all tested plasmids. Experiments of co-transformation with pOHT and pBT6 were successful and about 70% of transformants were resistant to both hygromycin B and benomyl. The “Instant Gene Bank” technique and mutagenesis through Restriction Enzyme Mediated Integration (REMI) were attempted. As reported for other phytopathogenic fungi, the REMI technique proved to be a powerful method for obtaining mutant strains with variation in phenotypic traits.

Construction of a homologous selectable marker gene for Lentinula edodes transformation.

We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 microg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.