The Almighty Fungi: The Revolutionary Neurospora crassa

“This brief paper, revolutionary in both its methods and its findings, changed the genetic landscape for all time”.

This is how Norman Horowitz started a historical account celebrating the 50^th anniversary of the landmark paper by Beadle and Tatum, published in 1941(Horowitz 1991). This work with the filamentous ascomycete fungus Neurospora crassa (Beadle and Tatum 1941), started off a series of important breakthroughs that brought the fields of biochemistry and genetics together and initiated a revolution: the explosive development of biochemical genetics and molecular biology. Undeniably, the one-to-one relationship between genes and enzymes (the “one gene, one enzyme” hypothesis), a concept derived from this and follow-up work, had a tremendous impact on biology.

These studies introduced a new experimental organism to geneticists, Neurospora crassa. Since then, Neurospora has been instrumental for our understanding of several basic and fundamental aspects of biology (see below). Studies with this fungi pioneered the use of microorganisms in genetics and in this brief overview, I will try to describe part of what has made N. crassa one of the most important model organisms in modern biology. I apologize to authors whose work I won’t discuss due to space constraints (in a blog… weird right?).

Edward L. Tatum (1909-1975). (Image from Nobelprize.org)

The first historical report of Neurospora crassa dates back to 1843 (nearly 100 years before the Beadle and Tatum paper), when it was reported as a contaminant of French bakeries. Several years later, in the mid-1920s, the modern history of this fungus started with the work of Bernard Dodge who worked out the basic genetics of the organism. He was fascinated with the benefits this fungus could provide to genetics, even to the point of suggesting T.H. Morgan, the father of Drosophila genetics, to use it in his lab. In fact, Carl Lindegren (which is regarded as the father of Neurospora genetics), who together with Dodge developed genetics in this organism during the 1930s, started working with Neurospora at Morgan’s Lab, in California.

As mentioned above, the work by Beadle and Tatum, and subsequent work, opened the path to a new field: biochemical genetics. The first biochemical mutants were described in Neurospora crassa in their 1941 paper and this work and particularly its methodology, led to the generation of these “biochemical mutations” in bacteria (allowing progress in this organism as suitable markers could now be used) and to the demonstration of recombination in E. coli: together these discoveries, derived directly from work in Neurospora, allowed for the development of bacterial genetics. These sorts of mutations were later introduced into yeast and other microorganisms. Notably also, the discovery of temperature-sensitive mutants, an extremely useful tool in the history of molecular biology, was first reported and studied in Neurospora as was the first suppressor of a biochemical mutation.

George W. Beadle (1903-1989). (Image from Nobelprize.org)

What made N. crassa such a good model for genetics? First, Neurospora was typically haploid, with only a short diploid stage prior to meiosis. Also, its fast growth rate, ease of culture and simple nutritional requirements, orthodox Mendelian genetics, the ability to determine the genotypes of all four products of individual meioses and susceptibility to mutagenesis, made it an ideal candidate for such a role. Further, it was non pathogenic.

Several groups became interested in Neurospora crassa and it quickly became the subject of intensive research leading to significant contributions, some even the first reported for any eukaryote. By using Neurospora mutants, Mary Mitchell reported the first example of gene conversion. Barbara McClintock, famous for her work in transposition, showed for the first time, that fungal chromosomes where typically eukaryotic. Further, several aspects of metabolism and cytogenetics where first studied. The coordinate control of unlinked genes involved in the same biosynthetic pathway was described and “cross-pathway” control of amino acid biosynthesis was shown. Also, the study of nitrogen, sulfur and phosphate metabolism became models for eukaryotes (Davis 2000).

The study of mitochondrial function and biogenesis has benefited deeply from research with Neurospora crassa. In 1953, the first maternally transmitted non-mendelian mutants were described, which were later shown to be due to mutations in mitochondrial DNA. Further, the first sequencing of a mitochondrial nucleic acid came from this fungus and self-splicing of a mitochondrial intron was demonstrated for the first time. Neurospora also made significant contributions to the study of the mechanisms underlying protein import into mitochondria. Moreover, mitochondrial plasmids were discovered and characterized.

Neurospora crassa has also been important, and in fact, continues to advance knowledge in several other areas of biological research, including recombination, DNA repair, differentiation, morphogenesis and cell biology, and notably, DNA methylation and silencing, findings applicable not only to the fungi, but to other eukaryotes as well.

Neurospora crassa can be described as a “paranoid organism” in that it tenaciously defends its genome using several protective mechanisms, including transcriptional and post-transcriptional gene silencing strategies. One of the latter, termed “quelling”, involves double stranded RNA and is mechanistically related to RNA interference (RNAi). Further, new classes of small RNAs and their functions are currently being studied (Lee et al. 2009). Moreover, a process called Repeat-induced point mutation (RIP), described so far only in fungi, represents a fascinating genome defense strategy with evolutionary consequences for Neurospora. Notably, this was the first described genome defense system in eukaryotes. During a sexual cross, and prior to karyogamy, the haploid genomes are scanned for duplicated sequences, and if found (the size and linkage of the duplicated sequences are relevant), both copies become riddled with polarized transition mutations (G:C to A:T). Notably, the sequences mutated by RIP are typically methylated at their remaining cytosines, which can lead to transcriptional silencing. This process can efficiently silence transposable elements (relics of such elements displaying the effects of RIP, can be found in the Neurospora genome), and it’s presumably the cause of the paucity of highly similar gene pairs in this fungus.

As mentioned above, work done in Neurospora crassa was essential for the development of bacterial genetics, and many prokaryotic organisms emerged as models, including their phages, for several biological processes. In the 1950s, E. coli emerged as the new model organism where most new findings of universal interest were made, particularly in the fields of biochemistry and microbial genetics. Nevertheless, Neurospora remained a wellspring of knowledge for areas like the ones mentioned above (meiotic recombination, mitochondrial biology, developmental biology, chromosome dynamics and others) and also, it soon became apparent than eukaryotic biology was indubitably different from E. coli’s, which made Neurospora the sole model eukaryote. This was until the rise of the well-known Saccharomyces cerevisiae, baker’s yeast, in the 1960s-1970s, which do to its ease of culturing (comparable to E. coli’s) and genetics, became a widely used organism in which cell biology, biochemistry and molecular biology would soon be greatly advanced.  What has to be kept in mind though, and is partly what still makes Neurospora a great model organism, is that this genetically tractable, easy culturable and fascinating organism is quite distinct from yeast, and developmentally more complex, which allows to perform studies and obtain information that cannot be obtained from yeast from complex biological processes, like development and differentiation, post-transcriptional gene silencing, photobiology and emergent properties like circadian rhythms (see below), which allow Neurospora to complement the knowledge derived from yeast, highlighting the use of fungi in the advancement of molecular biology (for a review on this subject, see (Brambl 2009). Further, Neurospora crassa remains a model for the filamentous fungi which account for nearly 70% of the approximately 250,000 species of fungi.

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Image from the Perkins Lab (http://www.stanford.edu/group.neurospora)

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Besides its undeniable historical contribution (reviewed extensively in (Davis 2000), it remains a useful organism in several areas of biology and a source of new discoveries, as mentioned before, not only applicable to the fungi. Due to a highly cooperative community, its genome sequence was published in 2003, representing the first reported from a filamentous fungi and a comprehensive functional genomics program got started (Dunlap et al. 2007). Molecular tools in this organism have greatly advanced in the last few years, a variety of selectable markers and regulatable promoters are available and targeted gene disruption is now trivial (which has allowed for the generation of over 8,000 KO strains for the nearly 10,000 genes present in Neurospora’s ~40Mb haploid genome, which are readily available for the community). At this point, it may not come as surprise that the famous Fungal Genetics Conference, which takes place every two years, was originally the Neurospora Information Conference.

The role of Neurospora crassa in circadian rhythms

I’d like to finish this post by briefly highlighting the importance of Neurospora crassa in our current understanding of the molecular basis of circadian rhythms, biological oscillations in biochemistry, physiology and behavior that display a period of ~ 24 hours. Studies using Neurospora (and Drosophila) have allowed us to position molecular feedback loops at the core of these rhythms (Dunlap 1999; Dunlap 2008). The first rhythm mutants were described concurrently in both of these species and the molecular cloning of “clock genes” (frq in N. crassa and per in Drosophila) occurred also around the same time in these organisms. Notably, the cloning of frq represented the first case of rescue of a behavioural mutation through DNA transformation. Work in Neurospora was the first to use genetic engineering to manipulate these clock genes. Also, the mechanisms for light-induced phase shifting and temperature entrainment were first studied in this fungus. Notably, many of these mechanisms work similarly in mammals and, interestingly, the general molecular design of the central oscillator is conserved in higher eukaryotes. Further, the first systematic genome-wide screen for “clock-controlled genes” (and the coining of that term) and the study of clock-regulated transcription, were done in Neurospora. Neurospora crassa continues to be a great model not only for circadian biology, but for several other important biological processes, for which fascinating discoveries are being made regularly. A lot still awaits to be unveiled.

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Alejandro is a PhD student at Pontificia Universidad Católica de Chile, where he studies the molecular basis of circadian rhythms using Neurospora crassa as a model (as you would imagine). He blogs at MolBio Research Highlights.

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Selected references

Beadle, G. W., and E. L. Tatum, 1941 Genetic Control of Biochemical Reactions in Neurospora. Proc Natl Acad Sci U S A 27: 499-506.

Brambl, R., 2009 Fungal physiology and the origins of molecular biology. Microbiology 155: 3799-3809.

Davis, R., 2000 Neurospora: Contributions of a Model Organism. Oxford University Press, New York.

Dunlap, J. C., 1999 Molecular bases for circadian clocks. Cell 96: 271-290.

Dunlap, J. C., 2008 Salad days in the rhythms trade. Genetics 178: 1-13.

Dunlap, J. C., K. A. Borkovich, M. R. Henn, G. E. Turner, M. S. Sachs et al., 2007 Enabling a community to dissect an organism: overview of the Neurospora functional genomics project. Adv Genet 57: 49-96.

Horowitz, N. H., 1991 Fifty years ago: the Neurospora revolution. Genetics 127: 631-635.

Lee, H. C., S. S. Chang, S. Choudhary, A. P. Aalto, M. Maiti et al., 2009 qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459: 274-277.

Leading Neurospora image, photo credit: Namboori B. Raju, Stanford University.

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See other articles in the series:

Drosophila melanogaster- The fruit fly.

Research’s Next Top Model (Zebrafish)

EEK! The Mouse.

Getting to Know Your Worms (C. elegans)

Saccharomyces cerevisiae a.k.a Budding/Baker’s/Brewer’s Yeast

We’re Gonna Need a Bigger Lab: Large Animal Models in Research

Tetrahymena: Little Creature, Big Discoveries

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