Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
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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
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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
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Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen
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Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus
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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.
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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.
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