Abstract
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 μg/mL of G418 or 50 μg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.
References
Belden WJ, Larrondo LF, Froehlich AC et al (2007) The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output. Genes Dev 21:1494–1505. https://doi.org/10.1101/gad.1551707
Borkovich KA, Alex LA, Yarden O et al (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108. https://doi.org/10.1128/mmbr.68.1.1-108.2004
Cao X, Liu X, Li H et al (2018) Transcription factor CBF-1 is critical for circadian gene expression by modulating WHITE COLLAR complex recruitment to the frq locus. PLoS Genet 14:e1007570. https://doi.org/10.1371/journal.pgen.1007570
Colbere-Garapin F, Horodniceanu F, Kourilsky P et al (1981) A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol 150:1–14. https://doi.org/10.1016/0022-2836(81)90321-1
Colot HV, Park G, Turner GE et al (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. PNAS 103:10352–10357. https://doi.org/10.1073/pnas.0601456103
Davis R (2000) Neurospora contributions of a model organism. Oxford Univ. Press, New York 0-19-512236-4
Davis RH, Perkins DD (2002) Timeline: Neurospora: a model of model microbes. Nat Rev Genet 3:397–403. https://doi.org/10.1038/nrg797
Dunlap JC, Borkovich KA, Henn MR et al (2007) Enabling a community to dissect an organism: Overview of the Neurospora functional genomics project. Adv Genet 57:49–96. https://doi.org/10.1016/S0065-2660(06)57002-6
Galagan JE, Selker EU (2004) RIP: the evolutionary cost of genome defense. Trends Genet 20:417–423. https://doi.org/10.1016/j.tig.2004.07.007
Garceau NY, Liu Y, Loros JJ et al (1997) Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89:469–476. https://doi.org/10.1016/S0092-8674(00)80227-5
Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553. https://doi.org/10.1002/(SICI)1097-0061(199910)15:14%3C1541:AID-YEA476%3E3.0.CO;2-K
He Q, Cha J, He Q et al (2006) CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes Dev 20:2552–2565. https://doi.org/10.1101/gad.1463506
He Q, Cheng P, He Q et al (2005) The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev 19:1518–1531. https://doi.org/10.1101/gad.1322205
Hentges P, Van Driessche B, Tafforeau L et al (2005) Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22:1013–1019. https://doi.org/10.1002/yea.1291
Jiang H, Liu GL, Chi Z et al (2018) Genetics of trehalose biosynthesis in desert-derived Aureobasidium melanogenum and role of trehalose in the adaptation of the yeast to extreme environments. Curr Genet 64:479–491. https://doi.org/10.1007/s00294-017-0762-z
Jimenez A, Davies J (1980) Expression of a transposable antibiotic resistance element in Saccharomyces. Nature 287:869–871. https://doi.org/10.1038/287869a0
Kim MS, Kim SY, Yoon JK et al (2009) An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem Biophys Res Commun 390:983–988. https://doi.org/10.1016/j.bbrc.2009.10.089
Kluge J, Kuck U (2018) AcAxl2 and AcMst1 regulate arthrospore development and stress resistance in the cephalosporin C producer Acremonium chrysogenum. Curr Genet 64:713–727. https://doi.org/10.1007/s00294-017-0790-8
Krügel H, Fiedler G, Smith C et al (1993) Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127–131. https://doi.org/10.1016/0378-1119(93)90627-f
Lakin-Thomas P (2019) Circadian rhythms, metabolic oscillators, and the target of rapamycin (TOR) pathway: the Neurospora connection. Curr Genet 65:339–349. https://doi.org/10.1007/s00294-018-0897-6
Maerz S, Dettmann A, Ziv C et al (2009) Two NDR kinase-MOB complexes function as distinct modules during septum formation and tip extension in Neurospora crassa. Mol Microbiol 74:707–723. https://doi.org/10.1111/j.1365-2958.2009.06896.x
Maerz S, Ziv C, Vogt N et al (2008) The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa. Genetics 179:1313–1325. https://doi.org/10.1534/genetics.108.089425
Margolin B, Freitag M, Selker E (1997) Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Genetics Reports 44:34–36. https://doi.org/10.4148/1941-4765.1281
Millerioux Y, Clastre M, Simkin AJ et al (2011) Drug-resistant cassettes for the efficient transformation of Candida guilliermondii wild-type strains. FEMS Yeast Res 11:457–463. https://doi.org/10.1111/j.1567-1364.2011.00731.x
Pall ML (1993) The use of Ignite (Basta;glufosinate;phosphinothricin) to select transformants of bar-containing plasmids in Neurospora crassa. Fungal Genet Rep 40:58. https://doi.org/10.4148/1941-4765.1412
Reuss O, Vik A, Kolter R et al (2004) The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127. https://doi.org/10.1016/j.gene.2004.06.021
Sato M, Dhut S, Toda T (2005) New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe. Yeast 22:583–591. https://doi.org/10.1002/yea.1233
Seiler S, Vogt N, Ziv C et al (2006) The STE20/germinal center kinase POD6 interacts with the NDR kinase COT1 and is involved in polar tip extension in Neurospora crassa. Mol Biol Cell 17:4080–4092. https://doi.org/10.1091/mbc.e06-01-0072
Selitrennikoff C, Nelson R (1984) Putative transformation of Neurospora os-1 protoplasts by a plasmid containing a G-481 phosphotransferase gene. Fungal Genet Rep 31:43. https://doi.org/10.4148/1941-4765.1613
Shen J, Guo W, Köhler JR (2005) CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species. Infect Immun 73:1239–1242. https://doi.org/10.1128/IAI.73.2.1239-1242.2005
Staben C, Jensen B, Singer M et al (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet Rep 36:79. https://doi.org/10.4148/1941-4765.1519
Temporini ED, Alvarez ME, Mautino MR et al (2004) The Neurospora crassa cfp promoter drives a carbon source-dependent expression of transgenes in filamentous fungi. J Appl Microbiol 96:1256–1264. https://doi.org/10.1111/j.1365-2672.2004.02249.x
Tiwari A, Ngiilmei SD, Tamuli R (2018) The NcZrg-17 gene of Neurospora crassa encodes a cation diffusion facilitator transporter required for vegetative development, tolerance to endoplasmic reticulum stress and cellulose degradation under low zinc conditions. Curr Genet 64:811–819. https://doi.org/10.1007/s00294-017-0794-4
Virgilio S, Bertolini MC (2018) Functional diversity in the pH signaling pathway: an overview of the pathway regulation in Neurospora crassa. Curr Genet 64:529–534. https://doi.org/10.1007/s00294-017-0772-x
Wang K, Zhang Z, Chen X et al (2015) Transcription factor ADS-4 regulates adaptive responses and resistance to antifungal azole stress. Antimicrob Agents Chemother 59:5396–5404. https://doi.org/10.1128/AAC.00542-15
Xue W, Yin Y, Ismail F et al (2019) Transcription factor CCG-8 plays a pivotal role in azole adaptive responses of Neurospora crassa by regulating intracellular azole accumulation. Curr Genet 65:735–745. https://doi.org/10.1007/s00294-018-0924-7
Yang Y, Cheng P, Liu Y (2002) Regulation of the Neurospora circadian clock by casein kinase II. Genes Dev 16:994–1006. https://doi.org/10.1101/gad.965102
Zhao Y, Shen Y, Yang S et al (2010) Ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation in Neurospora crassa. J Biol Chem 285:4355–4365. https://doi.org/10.1074/jbc.M109.034710
Acknowledgements
We are very grateful to Prof. John McCusker for providing pA25 containing nat gene and Prof. Sen Wu for pPB-U6-SV40-Neo plasmid and to Yubo He and Yasmin Niamat for the crucial revision of this manuscript. This work is supported by the grants from the National Natural Science Foundation of China (31771383) to Q.H.
Author information
Authors and Affiliations
Contributions
Conceptualization, HL, and QH; methodology, LH, WG, JL, YM, HL, and QH; investigation, LH, WG, HL, and QH; data analysis, LH, WG, HL, and QH; writing, original draft, HL, WG, and QH; writing, review & editing, LH, HL, and QH; funding acquisition, QH; resources, HL, YW, and QH; supervision, QH.
Corresponding author
Ethics declarations
Conflicts of interest
None declared.
Additional information
Communicated by M. Kupiec.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
He, L., Guo, W., Li, J. et al. Two dominant selectable markers for genetic manipulation in Neurospora crassa. Curr Genet 66, 835–847 (2020). https://doi.org/10.1007/s00294-020-01063-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-020-01063-1