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.
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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.
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The high-affinity phosphodiesterase BcPde2 has impact on growth, differentiation and virulence of the phytopathogenic ascomycete Botrytis cinerea.
TL;DR: The functional characterization of the two PDEs in the ascomycete B. cinerea showed that BcPde2 accumulates at the plasma membrane, but is also localized in the cytoplasm, but also accumulates in so far unknown mobile vesicles.
acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant.
Da-Woon Chung,Charles Greenwald,Srijana Upadhyay,Shengli Ding,Heather H. Wilkinson,Daniel J. Ebbole,Brian D. Shaw +6 more
TL;DR: It is concluded that the biochemical activity of the medA orthologs is conserved for conidiation, and therefore the evolutionary origins of asexual development in distinct fungal lineages can be tested.
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Hyphal ontogeny in Neurospora crassa : a model organism for all seasons
TL;DR: Some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa are summarized and the emerging questions that should be addressed are summarized.
A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling
TL;DR: Using promoter::RFP and promoter::GFP constructs, it is demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided forConidia- specific gene expression or for a significant increase in their expression during conidiation.
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Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea
TL;DR: It is shown that the transcription factor Bap1 and the response regulator B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B.cinerea and proves that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.
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TL;DR: A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome), finding that 17 percent were essential for viability in rich medium.
Molecular Bases for Circadian Clocks
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The genome sequence of the filamentous fungus Neurospora crassa
James E. Galagan,Sarah E. Calvo,Katherine A. Borkovich,Eric U. Selker,Nick O. Read,David B. Jaffe,William Fitzhugh,Li-Jun Ma,Serge Smirnov,Seth Purcell,Bushra Rehman,Timothy Elkins,Reinhard Engels,Shunguang Wang,Cydney B. Nielsen,Jonathan Butler,Matthew G. Endrizzi,Dayong Qui,Peter Ianakiev,Deborah Bell-Pedersen,Mary Anne Nelson,Margaret Werner-Washburne,Claude P. Selitrennikoff,John A. Kinsey,Edward L. Braun,Alex Zelter,Alex Zelter,Ulrich Schulte,Gregory O. Kothe,Gregory Jedd,Werner Mewes,Chuck Staben,Edward M. Marcotte,David Greenberg,Alice Roy,Karen Foley,Jerome Naylor,Nicole Stange-Thomann,Robert Barrett,Sante Gnerre,Michael Kamal,Manolis Kamvysselis,Evan Mauceli,Cord Bielke,Stephen Rudd,Dmitrij Frishman,Svetlana Krystofova,Carolyn G. Rasmussen,Robert L. Metzenberg,David D. Perkins,Scott Kroken,Carlo Cogoni,Giuseppe Macino,David E. A. Catcheside,Weixi Li,Robert J. Pratt,Stephen A. Osmani,Colin P.C. DeSouza,Louise Glass,Marc J. Orbach,J. Andrew Berglund,Rodger B. Voelker,Oded Yarden,Michael Plamann,Stephan Seiler,Jay C. Dunlap,Alan Radford,Rodolfo Aramayo,Donald O. Natvig,Lisa A. Alex,Gertrud Mannhaupt,Daniel J. Ebbole,Michael Freitag,Ian T. Paulsen,Matthew S. Sachs,Eric S. Lander,Chad Nusbaum,Bruce W. Birren +77 more
TL;DR: A high-quality draft sequence of the N. crassa genome is reported, suggesting that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.
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