High-Throughput Production of Gene Replacement Mutants in Neurospora crassa

Park, Gyungsoon; Colot, Hildur V.; Collopy, Patrick D.; Kryštofová, Svetlana; Crew, Christopher M.; Ringelberg, Carol S.; Litvinkova, Liubov; Altamirano, Lorena; Li, Liande; Curilla, Susan; Wang, Wei; Gorrochótegui-Escalante, Norma; Dunlap, Jay C.; Borkovich, Katherine A.

doi:10.1007/978-1-61779-040-9_13

Cited by 54 publications

(45 citation statements)

References 17 publications

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“…A majority of the viable S/T protein kinase mutants exhibit growth or developmental phenotypes. We analyzed vegetative hyphal growth, asexual development, and sexual development in the 77 viable knockout mutants (13,67). Neurospora grows vegetatively by apical extension, branching, and fusion of basal hyphae.…”

Section: Resultsmentioning

confidence: 99%

“…Spot testing was used to identify progeny that lacked the mus-51 or mus-52 mutation (both marked with bar, conferring resistance to phosphinothricin [2]) and to determine mating type. Homokaryotic mutant strains were confirmed by Southern blot analysis as described previously (13,67).…”

Section: Methodsmentioning

confidence: 99%

“…Transformants were selected on FGS solid medium containing phosphinothricin (66). Homokaryotic strains were isolated by serial plating of macroconidia as described above and then confirmed by Southern blot analysis (67).…”

Section: Methodsmentioning

confidence: 99%

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Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa

Park

Servin

Turner³

et al. 2011

Eukaryot Cell

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110

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Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa

Park

Servin

Turner³

et al. 2011

Eukaryot Cell

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110

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“…The availability of an almost complete knockout library for N. crassa has accelerated research with this model fungus (99). Similar efforts are underway for Magnaporthe oryzae (20).…”

Section: High-throughput Transcriptomes and Functional Analysismentioning

confidence: 99%

Trichoderma Research in the Genome Era

Mukherjee

Horwitz

Herrera-Estrella³

et al. 2013

Annu. Rev. Phytopathol.

379

231

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Trichoderma species are widely used in agriculture and industry as biopesticides and sources of enzymes, respectively. These fungi reproduce asexually by production of conidia and chlamydospores and in wild habitats by ascospores. Trichoderma species are efficient mycoparasites and prolific producers of secondary metabolites, some of which have clinical importance. However, the ecological or biological significance of this metabolite diversity is sorely lagging behind the chemical significance. Many strains produce elicitors and induce resistance in plants through colonization of roots. Seven species have now been sequenced. Comparison of a primarily saprophytic species with two mycoparasitic species has provided striking contrasts and has established that mycoparasitism is an ancestral trait of this genus. Among the interesting outcomes of genome comparison is the discovery of a vast repertoire of secondary metabolism pathways and of numerous small cysteine-rich secreted proteins. Genomics has also facilitated investigation of sexual crossing in Trichoderma reesei, suggesting the possibility of strain improvement through hybridization.

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“…Construction of a T. reesei strain defective in the NHEJ pathway represents a milestone toward high-throughput gene deletion (Guangtao et al 2009), and later such a strain became available also for T. virens (Catalano et al 2011). The strongly increased efficiency of homologous integration paved the way to construction of a knockout library as already available for N. crassa (Park et al 2011). This approach can be further refined by adding recombination-mediated marker reuse systems (Hartl and Seiboth 2005;Steiger et al 2011).…”

Section: Tools For Genome-wide Investigation and Beyondmentioning

confidence: 98%