Neurospora crassa

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1. Classification

a. Higher order taxa [1]

Domain: Eukaryota

Phylum: Fungi

Class: Sordariomycetes

Order: Sordariales

Family: Sordariaceae

Genus: Neurospora

Species: Neurospora crassa

2. Description and significance

Neurospora crassa (common name: red bread mold [2] is a rapidly growing filamentous fungus [3] known since its contamination of French bakeries in 1843 [4]. It has been used in scientific studies since 1927 [4] and is a model organism [5]. N. crassa has been predominantly used for genetic research [4], including the study of the one gene-one enzyme hypothesis in 1941 [6] and, in the past two decades, various researchers exploring DNA silencing mechanisms present in N. crassa.

3. Genome structure

The genome of N. crassa consists of 40 Mb [7]. It has a 50% G/C content, and approximately 44% of the genome is protein-coding [7]. This results in 10,082 protein-coding genes, with 9,200 genes coding for proteins that consist of over 100 amino acids [7]. Some genes code for light-sensing proteins unique to N. crassa for circadian rhythm regulation [7], and others code for ten G-protein-coupled receptors, three not previously identified in any fungus [7]. The genes also code for proteins used in the highly conserved mitogen-activated protein kinase pathways that regulate N. crassa’s growth [7].

On average, N. crassa has 1.7 introns per gene, with each intron consisting of approximately 134 nucleotides [7]. Additionally, only 10% of the genome consists of repeat sequences due to the mechanism of repeat-induced point mutations (RIP) in N. crassa [7].

Uniquely, N. crassa contains only a few genes in multigene families [7] and only a few highly similar gene pairs compared to other sequenced eukaryotes [7]. Despite this, N. crassa is a model organism for higher eukaryotes as it possesses complex features found in higher eukaryotes that other fungi lack [3]. These features include DNA and H3K27 methylation and silencing mechanisms, including RIP and RNAi [2]. Additionally, N. crassa’s tolerance to genetic manipulation and ease in complementation tests and mapping further make N. crassa a model organism [3].

4. Cell structure

Being a eukaryote [1], N. crassa contains all the necessary components of a eukaryotic cell: a nucleus containing its genetic material, an endoplasmic reticulum, a Golgi body, a cytoskeleton, mitochondria, vacuoles, ribosomes, and a cytoplasm enclosed by a plasma membrane [8]. The plasma membrane is surrounded by a cell wall primarily composed of chitin [8].

Throughout N. crassa’s life cycle, it consists of different cell types [8] due to varying environmental and internal factors [9]. While growing, N. crassa contains hyphae (singular: hypha) [10], large, multicellular, rapidly growing, tube-like branches that allow N. crassa to extend over considerable distances to absorb nutrients [11]. N. crassa also produces ascospores, football-shaped cells with thick black walls, for reproduction [8]. The thick walls allow the ascospores to resist environmental changes when released [8]. Heat from forest fires [9] and certain chemicals will cause the ascospore to germinate and produce a hypha [8], similar to a flower sprouting.

5. Metabolic processes

N. crassa is an obligate aerobe [12], a decomposer [13], and a heterotroph [13] that grows best at 32°C [3] in a synthetic media containing an organic carbon source, an inorganic nitrogen source, and the vitamin biotin [14]. It can use various organic carbon sources depending on the environmental conditions, but N. crassa best grows on dextrose at a concentration of 2% [15]. N. crassa can also use various inorganic nitrogen sources, including nitrate, nitrite, purines, amino acids, nucleic acids, and proteins [16]. N. crassa has no known preference for a specific nitrogen source [16]. N. crassa cannot synthesize biotin and requires supplementation in media to grow [14].

N. crassa is not known to possess a secondary metabolism outside of carotenoid and melanin pigment synthesis [7].

6. Ecology and Pathology

N. crassa grows on burned vegetation and trees after forest fires [9] in tropical and subtropical regions [17]. However, N. crassa has been found growing on coniferous trees in Montana and Alaska [9]. N. crassa also grows on carbohydrate-rich foods and residues from sugar cane processing [9] and in lumber yards and plywood factories [18].

N. crassa predominantly grows on the Scots pine (Pinus sylvestris) [9] and can have multiple ecological relationships with its host depending on host and environmental factors [9]. The relationship can switch between mutualistic, saprotrophic, or rarely pathogenic [9], but the mechanisms for doing so are unknown.

N. crassa is not known to be a pathogen of humans, animals, and plants [18], excluding rare ecological relationships with the Scots pine [9]. Instead, N. crassa is used to ferment koji in Japan and cassava in indigenous Brazil and produce Roquefort cheese in France [18].

N. crassa is a primary colonizer as dictated by its ability to colonize post-forest fire environments [19]. It capitalizes on the lack of competitors and the influx of nutrients and moisture from the burned trees to grow and become established [19]. This occurred in Tokyo in 1923, the Florida Everglades in 1999, and along the Rio Grande in New Mexico in 2000 [19].

7. Role in One Gene-One Enzyme Hypothesis

In 1941, George Beadle and Edward Tatum created mutants of N. crassa, discovering that they were all unable to synthesize a functional vitamin B, with some mutants only able to synthesize a portion and others unable to synthesize the entire vitamin [6]. From these results, Beadle and Tatum concluded that all the mutants had various mutations in one gene, resulting in the inability to produce a functional vitamin B [6]. They further applied this concept to all genes, concluding one gene must result in one enzyme [6], now called the one gene-one enzyme hypothesis [2]. The one gene-one enzyme hypothesis drastically altered how we view gene regulation [2, 5, 20], leading to extensive experimentation that informed our current understanding of genetics and molecular biology within eukaryotes.

8. Current Research: Silencing Mechanisms

a. Repeat-Induced Point Mutations

Repeat-induced point mutations (RIP), initially discovered in N. crassa [21], is a rapid process [22] that changes some of the pairings of the DNA bases from paired guanine and cytosine to paired adenine and thymine [22] in sequences with greater than 80% nucleotide similarity [7], ultimately resulting in the inactivation of these sequences [23]. Using N. crassa, it was discovered RIP works alongside RNAi to ensure rapid silencing [24]. N. crassa predominantly uses RIP to control repeated sequences and the movement of transposons by post-transcriptionally targeting the degradation of their RNA transcripts [24].

b. Quelling

N. crassa utilizes quelling to rapidly inactivate transgenes greater than 132 nucleotides [25, 26], indicating quelling is essential to maintain potentially harmful bacterial transposons in N. crassa’s natural environment [26, 27]. Because of its importance, quelling is dominant [26, 27] and works alongside RIP in N. crassa [21]. Although quelling can be reversed [25], the mechanism and purpose of reversal are unknown [26].

c. Meiotic Silencing by Unpaired DNA

N. crassa utilizes meiotic silencing by unpaired DNA (MSUD) during meiosis to temporarily silence unpaired genes [25, 28]. This results in MSUD predominately silencing transposons [28] and spore killers [21], protecting N. crassa from their harmful effects [[[#9. References |[28]]]. MSUD works alongside RIP [21] and RNAi [21, 25] in N. crassa.

d. Implications in Healthcare

The efficient detection mechanisms of N. crassa’s silencing mechanisms [22, 25] are proposed for use in healthcare settings [25]. N. crassa’s silencing mechanisms are rapid [22, 25] and can work alongside RNAi silencing mechanisms [25, 27, 29] already present in vivo. Due to this, N. crassa’s silencing mechanisms could replace CRISPR/Cas9 as the primary DNA homology recognition system [29].

N. crassa is also implicated in host-viral research as its RNAi-based silencing mechanisms result in no known viral infections [29]. Research for this is still in its early stages.

9. References

[1] Schoch CL, et al. NCBI Taxonomy: a comprehensive update on curation, resources, and tools. Database (Oxford). 2020: baaa062. PubMed: 32761142PMC: PMC7408187.

[2] Horowitz, N. H. (1985). Roots: the origins of molecular genetics: one gene, one enzyme. BioEssays, 3(1), 37-39. https://doi.org/10.1002/bies.950030110

[3] Aramayo, R., & Selker, E. U. (2013). Neurospora crassa, a model system for epigenetics research. Cold Spring Harbor Perspectives in Biology, 5(10), a017921. https://doi.org/10.1101/cshperspect.a017921

[4] Davis, R. H., & Perkins, D. D. (2002). Neurospora: a model of model microbes. Nature Reviews Genetics, 3(5), 397-403. https://doi.org/10.1038/nrg797

[5] Braun, E. L., Natvig, D. O., Werner-Washburne, M., & Nelson, M. A. (2004). Genomics in Neurospora crassa: From one-gene-one-enzyme to 10,000 genes. Applied Mycology and Biotechnology, 4, 295-313. https://doi.org/10.1016/S1874-5334(04)80015-7

[6] Beadle, G. W., & Tatum, E. L. (1941). Genetic control of biochemical reactions in Neurospora. Proceedings of the National Academy of Sciences, 27(11), 499-506. https://doi.org/10.1073/pnas.27.11.499

[7] Galagan, J., Calvo, S., Borkovich, K. et al. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868 (2003). https://doi.org/10.1038/nature01554

[8] Neurospora crassa: Basic Cell Structure. (2004, April 28). Fungal Genetic Stock Center; Kansas State University. https://www.fgsc.net//Neurospora/sectionB3.htm

[9] Kuo, HC., Hui, S., Choi, J. et al. Secret lifestyles of Neurospora crassa. Sci Rep 4, 5135 (2014). https://doi.org/10.1038/srep05135

[10] Verdín, J., Sánchez-León, E., Rico-Ramírez, A. M., Martínez-Núñez, L., Fajardo-Somera, R. A., & Riquelme, M. (2019). Off the wall: the rhyme and reason of Neurospora crassa hyphal morphogenesis. The Cell Surface, 5, 100020. https://doi.org/10.1016/j.tcsw.2019.100020

[11] Steinberg, G., Peñalva, M. A., Riquelme, M., Wösten, H. A., & Harris, S. D. (2017). Cell biology of hyphal growth. Microbiology Spectrum, 5(2), 10-1128. https://doi.org/10.1128/microbiolspec.funk-0034-2016

[12] Howell, N., Zuiches, C. A., & Munkres, K. D. (1971). Mitochondrial Biogenesis in Neurospora crassa: I. An Ultrastructural and Biochemical Investigation of the Effects of Anaerobiosis and Chloramphenicol Inhibition. The Journal of Cell Biology, 50(3), 721-736. https://doi.org/10.1083/jcb.50.3.721

[13] Gopaliya, D., Kumar, V., & Khare, S. K. (2021). Recent advances in itaconic acid production from microbial cell factories. Biocatalysis and Agricultural Biotechnology, 36, 102130. https://doi.org/10.1016/j.bcab.2021.102130

[14] Ryan, F. J., Beadle, G. W., & Tatum, E. L. (1943). The Tube Method of Measuring the Growth Rate of Neurospora. American Journal of Botany, 30(10), 784–799. https://doi.org/10.2307/2437554

[15] Florio, V. J. (2011), Proteomic Analysis of Neurospora crassa Using the Non-Preferred Carbon Source Acetic Acid [Master’s Thesis, Youngstown State University]. Digital.Maag Repository

[16] DeBusk, R. M., & Ogilvie, S. U. S. A. N. (1984). Nitrogen regulation of amino acid utilization by Neurospora crassa. Journal of Bacteriology, 160(2), 493-498. https://doi.org/10.1128/jb.160.2.493-498.1984

[17] Perkins, D. D., & Turner, B. C. (1987). Neurospora from natural populations: toward the population biology of a haploid eukaryote. Experimental Mycology, 12(2), 91-131. https://doi.org/10.1016/0147-5975(88)90001-1

[18] Perkins, D. D., & Davis, R. H. (2000). Evidence for safety of Neurospora species for academic and commercial uses. Applied and Environmental Microbiology, 66(12), 5107-5109. https://doi.org/10.1128/AEM.66.12.5107-5109.2000

[19] Jacobson, D. J., Powell, A. J., Dettman, J. R., Saenz, G. S., Barton, M. M., Hiltz, M. D., ... & Natvig, D. O. (2004). Neurospora in temperate forests of western North America. Mycologia, 96(1), 66-74. https://doi.org/10.1080/15572536.2005.11832998

[20] Hausmann, R., & Hausmann, R. (2002). Molecular clones. To Grasp the Essence of Life: A History of Molecular Biology, 227-235.

[21] Gladyshev, E. (2017). Repeat‐induced point mutation and other genome defense mechanisms in fungi. The Fungal Kingdom, 687-699. https://doi.org/10.1128%2Fmicrobiolspec.FUNK-0042-2017

[22] Singer, M. J., Marcotte, B. A., & Selker, E. U. (1995). DNA methylation associated with repeat-induced point mutation in Neurospora crassa. Molecular and Cellular Biology. 15(10), 5586–5597. https://doi.org/10.1128/MCB.15.10.5586

[23] Lewis, Z. A., Honda, S., Khlafallah, T. K., Jeffress, J. K., Freitag, M., Mohn, F., ... & Selker, E. U. (2009). Relics of repeat-induced point mutation direct heterochromatin formation in Neurospora crassa. Genome Research, 19(3), 427-437. https://doi.org/10.1101/gr.086231.108

[24] Chicas, A., Cogoni, C., & Macino, G. (2004). RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa. Nucleic Acids Research, 32(14), 4237-4243. https://doi.org/10.1093/nar/gkh764

[25] Rhoades, N., Nguyen, T. S., Witz, G., Cecere, G., Hammond, T., Mazur, A. K., & Gladyshev, E. (2021). Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa. Proceedings of the National Academy of Sciences, 118(33), e2108664118. https://doi.org/10.1073/pnas.2108664118

[26] Cogoni, C., Irelan, J. T., Schumacher, M., Schmidhauser, T. J., Selker, E. U., & Macino, G. (1996). Transgene silencing of the al‐1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA‐DNA interactions or DNA methylation. The EMBO Journal, 15(12), 3153-3163. https://doi.org/10.1002/j.1460-2075.1996.tb00678.x

[27] Fulci, V., & Macino, G. (2007). Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa. Current Opinion in Microbiology, 10(2), 199-203. https://doi.org/10.1016/j.mib.2007.03.016

[28] Shiu, P. K., Raju, N. B., Zickler, D., & Metzenberg, R. L. (2001). Meiotic silencing by unpaired DNA. Cell, 107(7), 905-916. https://doi.org/10.1016/S0092-8674(01)00609-2

[29] Honda, S., Eusebio-Cope, A., Miyashita, S., Yokoyama, A., Aulia, A., Shahi, S., ... & Suzuki, N. (2020). Establishment of Neurospora crassa as a model organism for fungal virology.Nature Communications, 11(1), 5627. https://doi.org/10.1038/s41467-020-19355-y


Edited by Elizabeth Bagshaw, student of Jennifer Bhatnagar for BI311: General Microbiology, 2023, Boston University.