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: Findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.
Abstract: Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity
1 citations
TL;DR: In this paper, the authors characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in Fusarium graminearum.
Abstract: Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.
1 citations
01 Jan 2017
TL;DR: The VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1.
Abstract: Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.
1 citations
Cites methods from "Efficient four fragment cloning for..."
...higginsianum by amplifying 1,5 kb sequences (Supplemental table 1) flanking the coding sequence of FoAve1 and ChAve1, respectively, and cloned as previously described (Frandsen et al. 2008) into vector pRF-HU2 containing a nourseothricin cassette for selection....
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...…were generated in F. oxysporum and C. higginsianum by amplifying 1,5 kb sequences (Supplemental table 1) flanking the coding sequence of FoAve1 and ChAve1, respectively, and cloned as previously described (Frandsen et al. 2008) into vector pRF-HU2 containing a nourseothricin cassette for selection....
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01 Oct 2008
TL;DR: It is demonstrated that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual conidiospores, and it is furthermore shown that VdSGE1 deletion strains have lost pathogenicity on tomato.
Abstract: Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The ascomycete fungus Verticillium dahliae causes vascular wilt diseases in hundreds of dicotyledonous plant species, including important crops such as eggplant, lettuce, olive, spinach and tomato. The resting structures, microsclerotia, are triggered by root exudates to germinate and penetrate the roots after which the fungus grows into the xylem vessels. The fungus colonizes these vessels and interferes with the transportation of water and nutrients, resulting in the development of symptoms such as stunting, wilting, chlorosis and vascular browning. Verticillium wilt diseases are difficult to control due to the longevity of the microsclerotia, the broad host range of the pathogen, the inability of fungicides to kill the fungus once it has colonized the xylem vessels and the lack of natural resistance in many plant species. Chapter 1 is the introduction to this thesis that describes the identified pathogenicity and virulence factors of V. dahliae and strategies to identify these components. In spite of the economic importance of V. dahliae, relatively few pathogenicity genes have been identified in this species. With the availability of whole genome sequences and the development of functional genomics tools such as random mutagenesis, targeted mutagenesis, transcriptomics, RNA interference (RNAi) and comparative genomics, more strategies have become available to identify novel pathogenicity and virulence genes. Chapter 2 focuses on the identification of virulence and pathogenicity genes of V. dahliae by screening of a library of random T-DNA insertion mutants. Using Agrobacterium tumefaciens-mediated transformation, 900 T-DNA transformants with random insertions were generated and screened for altered virulence on susceptible tomato plants. This screening, followed by inverse PCR on selected transformants, resulted in the identification of 55 potential pathogenicity and virulence genes. One of the potential pathogenicity genes, VdNRS/ER, is a homolog of a nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER), which is presumably involved in the biosynthesis of UDP-rhamnose. Using targeted mutagenesis, VdNRS/ER was deleted from wild-type V. dahliae and the resulting deletion mutants were characterized. VdNRS/ER deletion mutants exhibit unaltered vegetative growth and sporulation, but the deletion mutants were no longer pathogenic on tomato and N. benthamiana and showed impaired root attachment on tomato seedlings. These data suggest that UDPrhamnose is required for pathogenesis of V. dahliae. Chapter 3 describes the role of the V. dahliae homolog of Sge1, a transcriptional regulator that was shown to play a role in pathogenicity and regulate effector gene expression in Fusarium oxysporum. In this chapter it is demonstrated that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual conidiospores. It is furthermore shown that VdSge1 deletion strains have lost pathogenicity on tomato. Since the VdSge1 deletion mutants are not able to infect and colonize tomato plants, a tomato cell suspension culture was used to the study the expression of Ave1, as well as nine other genes of which the expression is highly induced in planta. This assay revealed that VdSge1 is not required for the induction of the Ave1 effector that activates resistance mediated by Ve1 in tomato. Furthermore, the expression of one other putative effector gene was not affected by VdSge1 deletion. However, VdSge1 was shown to be required for the expression of six putative effector genes, whereas expression of the remaining two putative effectors genes was negatively regulated. Thus, the data show that VdSge1 is required for V. dahliae pathogenicity and differentially regulates effector gene expression. Chapter 4 describes the functional characterization of the gene family encoding necrosis- and ethylene-inducing-like proteins (NLPs) of V. dahliae. The cytotoxic activity of NLP family members was determined using agroinfiltration into tobacco leaves. This resulted in the identification of two out of the seven NLPs, VdNLP1 and VdNLP2, that induced plant cell death. The genes encoding these cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato. Targeted deletion of VdNLP1 and VdNLP2 significantly reduced the virulence of V. dahliae on tomato and Arabidopsis plants. In contrast, only deletion of VdNLP1 affected virulence on N. benthamiana whereas deletion of NLP2 did not. However, subsequent transcriptional analysis revealed that VdNLP2 was not expressed in V. dahliae during colonization of N. benthamiana. Moreover, VdNLP2 also affects vegetative growth and conidiospore production. In conclusion, the expanded V. dahliae NLP family shows differential cytotoxic activity between family members and in planta induction of the cytotoxic NLP genes varies between plant hosts. In addition, VdNLP2 plays a role in vegetative growth and conidiospore production in addition to its contribution to virulence. Thus, evidence is provided for functional diversification within the V. dahliae NLP family. Finally in Chapter 5, the major findings of this thesis are discussed and placed in a broader perspective.
1 citations
Cites methods from "Efficient four fragment cloning for..."
...The resulting amplicons were cloned into pRF-HU2 as described previously (Frandsen et al 2008)....
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01 Jan 2010
TL;DR: The USER™ technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction.
Abstract: Background: Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very timeefficient method for engineering of big and complex plasmids. Results: By application of the USER™ system, we engineered a collection of binary vectors, termed UCE (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient expression. Conclusions: The USER™ technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction.
1 citations
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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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