Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
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
Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing
Ronnie de Jonge,H. Peter van Esse,Karunakaran Maruthachalam,Melvin D. Bolton,Parthasarathy Santhanam,Mojtaba Keykha Saber,Zhao Zhang,Toshiyuki Usami,Bart Lievens,Krishna V. Subbarao,Bart P. H. J. Thomma +10 more
TL;DR: It is demonstrated that Ave1 activates Ve1-mediated resistance and markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis, and that Verticillium acquired Ave1 from plants through horizontal gene transfer.
Journal ArticleDOI
Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen
Ronnie de Jonge,Melvin D. Bolton,Anja Kombrink,Grardy C. M. van den Berg,Koste A. Yadeta,Bart P. H. J. Thomma +5 more
TL;DR: It is shown that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific genomic regions that act as a source for genetic variation to mediate aggressiveness.
Journal ArticleDOI
Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus
Marina Marcet-Houben,Ana-Rosa Ballester,Beatriz de la Fuente,Eleonora Harries,Jose F. Marcos,Luis González-Candelas,Toni Gabaldón +6 more
TL;DR: The complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.
Journal ArticleDOI
Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity
Ana-Rosa Ballester,Marina Marcet-Houben,Elena Levin,Noa Sela,Cristina Selma-Lázaro,Lourdes Carmona,Michael Wisniewski,Samir Droby,Luis González-Candelas,Toni Gabaldón +9 more
TL;DR: Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection and it was demonstrated that neither patulin nor citrinin are required by P. expandum to successfully infect apples.
Book ChapterDOI
USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.
TL;DR: This chapter presents a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
References
More filters
Journal ArticleDOI
Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker.
TL;DR: A polyketide synthase gene (pks12) from Fusarium graminearum is cloned and the disruption of the pks12 gene is used as a visible marker for transformation-mediated homologous recombination and the transformation procedure is optimized to achieve a high rate of homologously recombination.
Journal ArticleDOI
A simple protocol for isolation of fungal DNA
TL;DR: Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s, good enough for PCR analysis and Dot blot hybridization.
Journal ArticleDOI
Easy and rapid method of zygosity determination in transgenic mice by SYBR® Green real-time quantitative PCR with a simple data analysis
TL;DR: It is shown that the real-time quantitative PCR using SYBR® Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene.
Patent
Rapid and enzymeless cloning of nucleic acid fragments
TL;DR: In this paper, a method for cloning a nucleic acid fragment into a vector by flanking the fragment with first and second adapter sequences, and contacting it with the vector having sequences homologous to the first two adapter sequences under conditions such that the nucleic amino acid fragment is incorporated into the vector by homologic recombination in vivo in a host cell.