Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
TL;DR: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.
Abstract: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.
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TL;DR: To the authors' knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring As pergillus rot on grape berries.
36 citations
Cites methods from "Efficient four fragment cloning for..."
...The amplified fragment was cloned into the plasmid vector pRF-HUE 131 (Frandsen et al., 2008), a binary vector designed to be used with the USER 132 friendly cloning technique (New England Biolabs)....
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TL;DR: The Destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts.
Abstract: Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-l-valine and N-methyl-l-alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.
35 citations
Cites methods from "Efficient four fragment cloning for..."
...PCR amplifications for USER-based cloning were conducted as described (Nour-Eldin et al. 2006; Geu-Flores et al. 2007; Frandsen et al. 2008)....
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TL;DR: This is the first report describing the transformation of these two important postharvest pathogens with GFP and the use of transformed strains to study compatible and non-host pathogen interactions on oranges and apples.
32 citations
Cites methods from "Efficient four fragment cloning for..."
...The egfp gene included in this plasmid was obtained from plasmid pEGFPC3 and was cloned in plasmid pRF-HUE (Frandsen et al., 2008) under the control of the Aspergillus nidulans gpdA promotor....
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TL;DR: Comparison of the NRPSs from genome-sequenced producers of chrysogine identified a candidate NRPS cluster comprising five additional genes named chry2-6, indicating that the gene cluster is responsible for chrysogsine biosynthesis.
Abstract: Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSs from genome-sequenced producers of chrysogine we identified a candidate NRPS cluster comprising five additional genes named chry2–6. Deletion of the two-module NRPS (NRPS14 = chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14 enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.
32 citations
TL;DR: A USER-ATMT dual-selection (DS) binary vector is generated, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker.
Abstract: The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.
32 citations
References
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TL;DR: These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA.
Abstract: Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.
2,471 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Several laboratories have solved this problem by dividing the replacement constructs into two, a technique known as bipartite gene-targeting or split-marker recombination [6- 8]. In this technique, the two HRS's are fused with two thirds of either the 3' or 5'end of the selection marker gene, by fusion-PCR [ 9 ]....
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TL;DR: Questions are addressed, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.
Abstract: Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.
1,488 citations
"Efficient four fragment cloning for..." refers background in this paper
...The use of Proofreading DNA polymerase is essential when making targeted genome modifications in fungi, due to the close spacing of fungal genes [26], which often means that the HRS extends into neighbouring genes or their regulatory sequences....
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TL;DR: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Abstract: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
1,185 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Examples are the Xi-cloning, InFusion cloning, Ligase independent cloning (LIC-PCR), Recombinational cloning and USER Friendly cloning techniques [16-20]....
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TL;DR: This study describes a method for rapidly creating knockout mutants in which it makes use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics.
Abstract: The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
1,074 citations
"Efficient four fragment cloning for..." refers background or methods in this paper
...crassa, Colot and coworks [1], also allows for efficient four fragment cloning....
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...Contrary to Saccharomyces cerevisiae, where 30 bp is sufficient, many filamentous fungi require longer HRS [1], eg Fusarium graminearum needs 400 bp [2] 1500 bp is reported for Aspergillus niger [3] and around 1000 bp for Neurospora crassa [4]....
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...Vector construction for targeted replacement of genes is reduced to design of two primer pairs, which will permit automation of the experimental design as required for high-throughput knockout projects [1]....
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...Recombinational cloning of the two required HRS with a selection marker gene and a vector backbone is carried out in yeast, followed by PCR amplification of the two HRS and selection marker gene [1]....
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TL;DR: It is reported that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungi.
Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.
893 citations
"Efficient four fragment cloning for..." refers background in this paper
...The Agrobacterium tumefaciens mediated transformation (ATMT) technology [10] has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types [11]....
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