Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa

Tabilo-Agurto, Cyndi; Rio-Pinilla, Veronica Del; Eltit-Villarroel, Valeria; Goity, Alejandra; Muñoz-Guzmán, Felipe; Larrondo, Luis F.

doi:10.1128/mbio.03291-22

Cited by 2 publications

(1 citation statement)

References 98 publications

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“…The orange bread mold Neurospora crassa has a vast and rich history as a model system ( 1 , 2 ), and is perhaps the best understood filamentous fungus. Because of ease of culture, facile genetics, rapid growth rate, and an available genome sequence [encoding about 10,000 protein-coding genes ( 3 )], together with an expanding molecular toolkit ( 4 – 6 ), Neurospora has contributed to key findings in several research fields and continues to represent a powerful model, particularly for animal and plant pathogens, and for agriculturally and industrially relevant species ( 7 , 8 ). As such, and building on decades of research, a functional genomics characterization of N. crassa has the potential to greatly contribute to our understanding of eukaryotic biology.…”

Section: Introductionmentioning

confidence: 99%

Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype

Montenegro-Montero,

Goity,

Canessa

et al. 2023

Microbiol Spectr

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Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa , and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δ ada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so / ham-1 , and show that it is the absence of this gene—and not of ada-3 —that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations—of a largely unclear nature—that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures.

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Section: Introductionmentioning

confidence: 99%

Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype

Montenegro-Montero,

Goity,

Canessa

et al. 2023

Microbiol Spectr

Self Cite

View full text Add to dashboard Cite

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Transcriptional rewiring of an evolutionarily conserved circadian clock

Goity,

Dovzhenok,

Lim

et al. 2024

EMBO J

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Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a “lights-on timer” logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.

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Developing a Temperature-Inducible Transcriptional Rheostat in Neurospora crassa

Abstract: A timely and dynamic response to strong temperature fluctuations is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors.

Cited by 2 publications

References 98 publications

Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype

Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype

Transcriptional rewiring of an evolutionarily conserved circadian clock

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