Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
Reads0
Chats0
TLDR
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.read more
Citations
More filters
Journal ArticleDOI
The FTF gene family regulates virulence and expression of SIX effectors in Fusarium oxysporum.
Jonathan Niño-Sánchez,Virginia Casado-Del Castillo,Vega Tello,José J. de Vega-Bartol,Brisa Ramos,Serenella A. Sukno,Jose Maria Diaz Minguez +6 more
TL;DR: The role of the F TF gene expansion, mostly the FTF1 paralogues, as a regulator of virulence in F. oxysporum is demonstrated and the control of effector expression is suggested to be the mechanism involved.
Journal ArticleDOI
LaeA and VeA are involved in growth morphology, asexual development, and mycotoxin production in Alternaria alternata.
TL;DR: The role of LaeA and VeA, two regulatory proteins belonging to the velvet family, which have been described to be involved in several functions in many fungi including secondary metabolism, are examined and it is found that deletion of lae and veA genes greatly reduced sporulation and strongly compromised mycotoxin production.
Patent
De novo synthesized gene libraries
TL;DR: De novo synthesized large libraries of nucleic acids are provided in this article with low error rates. And devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described here.
Journal ArticleDOI
Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis
Simon Hartung Jørgensen,Rasmus John Normand Frandsen,Kristian Fog Nielsen,Erik Lysøe,Teis Esben Sondergaard,Reinhard Wimmer,Henriette Giese,Jens Laurids Sørensen +7 more
TL;DR: This work focused on PKS14, which has only been shown to be expressed during plant infections or when cultivated on rice or corn meal (RM) based media, and introduced a constitutive promoter in front of P KS14 and cultivated two of the resulting mutants on RM medium, which led to the production of two compounds.
Book ChapterDOI
Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation
TL;DR: Two efficient single-step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT) compatible binary vectors are described.
References
More filters
Journal ArticleDOI
A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions
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.
Journal ArticleDOI
Fungal secondary metabolism — from biochemistry to genomics
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.
Journal ArticleDOI
Ligation-independent cloning of PCR products (LIC-PCR)
C Aslanidis,P.J. de Jong +1 more
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.
Journal ArticleDOI
A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.
Hildur V. Colot,Gyungsoon Park,Gloria E. Turner,Carol S. Ringelberg,Christopher M. Crew,Liubov Litvinkova,Richard L. Weiss,Katherine A. Borkovich,Jay C. Dunlap +8 more
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.
Journal ArticleDOI
Agrobacterium tumefaciens-mediated transformation of filamentous fungi.
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.