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