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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12 Jun 2018
TL;DR: In this paper, the authors present methods, systems, and compositions for seamless nucleic acid assembly using an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids.
Abstract: Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.
6 citations
TL;DR: The recently obtained genome sequence and transcriptome analysis was exploited to inform the design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi and provides for the first time the ability to stably introduce transgenes and over-express target M.lychnidis-dioicae genes.
Abstract: Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.
6 citations
Cites methods from "Efficient four fragment cloning for..."
...Techniques for targeted gene replacement have been developed in Fusarium (Frandsen et al. 2012, 2008), and this is definitely worth investigating in the future for M. lychnidis-dioicae....
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TL;DR: VdAve1L2 is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota, which opens up strategies for more targeted biocontrol against microbial plant pathogens.
Abstract: Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health, and may thus be targeted by pathogen effectors as such. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in absence of the corresponding immune receptor Ve1. We now identified a multiallelic V. dahliae gene displaying ~65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L). Interestingly, VdAve1L shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve1 locus, does not recognize VdAve1L. Furthermore, we show that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum. Altogether, VdAve1L2 is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens.
5 citations
TL;DR: A single repetitive element is strongly associated with centromeric regions in some but not all Verticillium species, indicating a role of repetitive elements in the function, organization and rapid evolution of centromeres in a set of closely related fungal species.
Abstract: Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the ten species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (~150 kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen V. dahliae. Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single repetitive element is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, yet only a minority could be linked to centromeres, suggesting that centromeres played a minor role in chromosomal evolution. Nevertheless, the size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus Verticillium.
4 citations
Cites methods from "Efficient four fragment cloning for..."
...dahliae, a recombinant 543 DNA fragment was constructed into the binary vector PRF-HU2 (79) or PRF-GU2 for 544 homologous recombination....
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...To construct the N-terminally FLAG-tagged CenH3 strain of V. dahliae, a recombinant DNA fragment was constructed into the binary vector PRF-HU2 (79) or PRF-GU2 for homologous recombination....
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TL;DR: FgCWM1 encodes a CWM protein that plays an important role in the cell wall integrity and pathogenicity of F. graminearum, characterized and confirmed by its expression in Saccharomyces cerevisiae.
Abstract: Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease of wheat. Salicylic acid (SA) is involved in the resistance of wheat to F. graminearum. Cell wall mannoprotein (CWM) is known to trigger defense responses in plants, but its role in the pathogenicity of F. graminearum remains unclear. Here, we characterized FgCWM1 (FG05_11315), encoding a CWM in F. graminearum. FgCWM1 was highly expressed in wheat spikes by 24 h after initial inoculation and was upregulated by SA. Disruption of FgCWM1 (ΔFgCWM1) reduced mannose and protein accumulation in the fungal cell wall, especially under SA treatment, and resulted in defective fungal cell walls, leading to increased fungal sensitivity to SA. The positive role of FgCWM1 in mannose and protein accumulation was confirmed by its expression in Saccharomyces cerevisiae. Compared with wild type (WT), ΔFgCWM1 exhibited reduced pathogenicity toward wheat, but it produced the same amount of deoxynivalenol both in culture and in spikes. Complementation of ΔFgCWM1 with FgCWM1 restored the WT phenotype. Localization analyses revealed that FgCWM1 was distributed on the cell wall, consistent with its structural role. Thus, FgCWM1 encodes a CWM protein that plays an important role in the cell wall integrity and pathogenicity of F. graminearum.
4 citations
Additional excerpts
...tumefaciens-mediated transformation [44,45]....
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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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