The Neurospora - Fungal Genome Initiative
Mary Anne Nelson - Chair, Neurospora Genome Policy Committee, Associate Professor, University of New Mexico
Robert L. Metzenberg - Member, National Academy of Sciences, Professor, Stanford University; Past -President, Genetics Society of America
Jay Dunlap - Professor and Chair of Genetics, Dartmouth Medical School
Katherine Borkovich - Associate Professor, University of Texas-Houston Medical School
Matthew Sachs - Associate Professor, Oregon Graduate Institute
Ulrich Schulte - Director, German Neurospora Genome Initiative, Institute of Biochemistry, Heinrich-Heine-University, Duesseldorf, Germany
Eric Selker - Professor, Institute of Molecular Biology, University of Oregon
The Significance: The Kingdom of the Fungi includes over 250,000 different species and contains members central to every ecosystem on our planet. Fungi are universally consumed as food and are used for the industrial manufacture of chemicals and enzymes, collectively representing industries that contribute ca. $35 billion to the US economy each year. Additionally, fungi damage wood, fabric, and optical equipment, and are major pathogens both of animals (including people) and of plants, collectively costing in excess of ca. $30 billion annually on a worldwide basis.
The Issues: Although we do know a great deal about fungal culture and metabolism, we do not understand how fungi work.
We understand very little about the means through which animal or plant pathogens recognize or colonize hosts, so treatment of infections and defense against pathogens are largely determined by trial and error.
The Goal: To move fungal research from the era of empiricism to the era of rational design. To achieve this we must understand how fungi work and what makes them work. The critical first step toward this goal is to catalog all the genes that constitute the repertoire of fungal physiological capacity, and associate these genes with biological function. Hence,
We cannot engineer fungi to maximize production because we do not understand how fungi produce and secrete chemicals; hence nearly all strain improvement is driven empirically, by trial and error.
We understand very little about what makes fungi fruit, so that cultivation of mushrooms is limited to just a few of many possible species; most strain improvement is driven empirically.
the most efficient and cost effective means of understanding filamentous fungi is to determine the nucleotide sequence of DNA in the genome and then catalog the protein coding regions of the best understood filamentous fungus, Neurospora crassa.
Once we have determined the road map for this best understood organism, we will be in a position to acquire and interpret genome information from the more economically important but vastly unexplored fungi, including industrially important fungi, cultivated strains, and plant and animal pathogens - organisms that comprise the bulk of the known species.
A Closer Look - Why Fungi?
Fungi, plants, and animals represent the three phylogenetic kingdoms within the eukaryotes (non bacteria). Within the approximately 250,000 different species of fungi, about 75% belong to the Ascomycetes (approximately 90% of which are filamentous fungi, the remainder being yeasts) and 25% are Basidiomycetes (whose fruiting bodies are commonly known as "mushrooms"). As a group, the fungi have an enormous impact on the United States and world economies: yeast is used extensively in the brewing industry, filamentous fungi are used both for the production of foodstuffs and industrial production of enzymes and chemicals, and Basidiomycetes are consumed as food all over the world. Fungi are known to infect nearly all food crops and represent the most universal and costly pathogens. Filamentous fungi are widely used as organisms for basic research. Mycorrhizal fungi (those that grow interdependently with the roots of plants) are critical in providing plants with nutrients on which agriculture depends.
Filamentous fungi rank with bacteria as the most serious human and animal pathogens. Because they are eukaryotes, treatment of opportunistic infections by fungi poses special risks and challenges not encountered with bacterial infections.
Pharmaceutical manufacture using fungi constitutes a multi-billion dollar per year industry. Penicillin and similar beta-lactams, all produced in fungi, are the world's largest selling antibiotics.
Estimates are that 10% of the world's food supply is lost each year due to fungal contamination. Within the US alone, fungicide sales grew by 13.7% last year to nearly $600 million. Annual losses in peanut production in the US due to fungal infection can run as high as 30%.
Cultivated mushrooms now constitute an $800 million per year industry in the US that has grown over 10 fold during the past two decades.
Industrial production of chemicals by filamentous fungi constitutes a ca. $32 billion per year industry. The US is a net importer of some of these chemicals such as citrate, representing in excess of $1 billion annually. Industrial production of enzymes, largely by filamentous fungi, constitutes a $1.2 billion per year industry.
Fungi are of major importance in basic and biomedical research. They are genuine eukaryotes having cell and genome structures and metabolic organization similar to that of other eukaryotes including plants and animals. However, because they have smaller genomes and are eminently experimentally tractable, fungi are universally used as model organisms for understanding all aspects of basic cellular regulation. These regulatory networks include cell cycle progression, gene regulation, circadian timing, recombination, secretion, and multicellular development. The Nobel Prize has been awarded for research using one filamentous fungus, Neurospora crassa.
A Closer Look - Why Determine the DNA Sequence of a Filamentous Fungal Genome?
The information encoded in the genomic DNA of an organism describes the entire repertoire of the organism's metabolic and developmental capacity - everything that it can do or become. It represents the repository of an organism's evolutionary knowledge acquired through adaptation over eons of history, where variation induced by mutation has been refined in the crucible of natural selection.
By determining the sequence of a genome, all of the proteins present in the organism can be identified and all of the developmental and metabolic potential of an organism becomes available for manipulation both in that organism and, potentially, for re-engineering in any other organism.
The initial key to this treasury of information is the DNA sequence.
Fungal genomes, although containing a metabolic potential of staggering diversity, are small and well within the reach of current DNA sequencing technologies. The entire genomic DNA sequence has now been determined for many bacteria and a few eukaryotes. Filamentous fungal genomes range in size from two to five times that of yeast.
Reflecting their increased genetic complexity, filamentous fungi contain more genes than do yeasts. For instance in the best described system, Neurospora, greater than 50% of the protein coding regions sequenced in an ongoing pilot project do not correspond to genes found in yeast or identified as yet in any organism; they are novel.
By extending genomic sequence information from yeast to filamentous fungi and eventually to Basidiomycetes, the ability to understand and manipulate many aspects of growth, metabolism, and development will be at our fingertips.
Unlike yeast (which like bacteria are unicellular), most fungi have a coenocytic growth habit (having many nuclei in large cytoplasmic compartments), are truly multicellular during nearly all stages of their life cycles, and differentiate into a variety of cell types.
As a group, fungi are known for the exceptional ease with which they can be genetically and molecularly manipulated. Generally, if a fungus can be cultivated it can be genetically transformed with exogenous DNA, thus paving the way for genetic engineering of any fungus using developmental or metabolic capacities encoded by any other fungus.
Filamentous fungi produce an extraordinary number of secondary metabolites, including known mycotoxins such as aflatoxin and commercially important antibiotics such as penicillin.
Unlike yeast, most fungi form distinct asexual spores that constitute the chief means of dispersal of the organism, and therefore the chief means of infection for pathogens.
Filamentous fungi have substantially greater metabolic versatility than yeast. They can grow at pH ranging from 2.5 to 11, at salt concentrations over 4 M, at temperatures from 5 C to 60 C, and on a staggering number of different and exotic carbon and nitrogen sources. The potential for bioremediation has never been seriously exploited; for example, Neurospora could play a critical role in hemicellulose degradation.
Circadian rhythms occur in Neurospora and mammals but not in yeast, and Neurospora is one of the model organisms for the study of biological clocks. But why does Neurospora need a circadian rhythm? The Neurospora genome sequence would contribute to understanding of this complicated phenomenon, one of many we would like to address using a biocomplexity approach. A readily testable hypothesis considers the response to light as a possible adaptation to temperature changes.
Determination of complete genomic sequences of filamentous fungi is now an important and readily achievable goal.
The DNA sequence itself will be most informative in proportion to the extent to which the genome sequenced is already understood genetically.
A Closer Look - Why Sequence Neurospora?
A cover article in The Scientist (Nov. 25, 1996) hailed "Functional Genomics - unraveling how genes work" as the currently hottest area for biotechnology investment, and they found (not surprisingly) that a lot of the smart money was going into start-up companies working on model eukaryotic organisms and not on people or plants. The reason for this is that, although the acquisition of DNA sequences in any organism is now economically feasible, the true utility of the sequence cannot be fully realized until the string of A's, T's, G's, and C's comprising the sequence can be interpreted as a series of genes encoding the proteins that confer metabolic and developmental potential on the organism - until this connection between gene and function is made, the sequence is mute.
It has been estimated that roughly 1/3 of any organism's genetic capacity is devoted to housekeeping functions that all organisms must be able to carry out - metabolizing simple sugars, making proteins, replicating DNA, etc. The DNA sequences of these genes will be similar enough in all organisms that the presence of these genes in a raw DNA sequence is identifiable and interpretable. However, genes that are not highly conserved in all organisms - the truly interesting genes in terms of development of fungal spores, elaboration of mushroom fruiting bodies, synthesis and secretion of secondary metabolites such as penicillin, capacity for pathogenesis in plants and animals and ability of hyphae to fuse and produce heterokaryons, genes that function in the cell cycle, control of the biological clock, and sexual development - these genes cannot be recognized just from the DNA sequence because no other genes like them have ever been identified.
Herein lies the reason that venture capitalists are investing in functional genomics companies that can exploit the knowledge base of model organisms - and the reason why, in the context of filamentous fungi, Neurospora is ideal for launching the genome initiative. Neurospora has been a dominant force in filamentous fungal research for the past half century.
Most importantly, there are over 1000 different genes identified by function in Neurospora, on the order of twice as many as identified in any other filamentous fungus and more than 10 fold more than any Basidiomycete.
The genetic map of Neurospora is universally recognized as one of the most painstakingly documented in existence. Over 1000 genes have been mapped relative to one another, and over 500 cloned and studied genes, 350 strains bearing chromosomal translocations and 250 RFLP markers allow precise links connecting the genetic map (showing the relative positions of genes on chromosomes) with the physical map (the relative positions of cloned DNA pieces on chromosomes). These data pave the way for determining and interpreting the genomic DNA sequence. EST sequencing has identified nearly 5000 genes.
The field of biochemical genetics was invented using Neurospora; there is over 60 years of research in connecting genes with biochemical function. Nobel Prize winning work on Neurospora resulted in the development of the one gene-one enzyme hypothesis, the landmark connection between genes and biochemical function.
Research on Neurospora is ongoing, reflecting the presence of a large and vibrant research community having the greatest number of scientists devoted to any fungus except for yeast. Nearly twice as many research papers are published annually on Neurospora as on any other filamentous fungus, well over 5000 papers within the past 30 years.
Research on Neurospora is frequently seen in the most prestigious scientific and lay science journals - Science, Nature, Cell - twice as cover articles in as many years.
Information on Neurospora is readily applicable to other agriculturally important, industrially important, or pathogenic fungi.
Research on Neurospora is at the cutting edge of many fields of fungal genetics, development, and filamentous fungal cell biology - heterothallism, sexual development, temporal regulation, cytoplasmic movement, signal transduction, meiotic drive, transvection, and the role of methylation in gene expression, to name a few.
Although Neurospora is nonpathogenic, it is phylogenetically very closely allied with and genetically similar to several important plant pathogens including Cochliobolus (Southern corn leaf blight, which resulted in crop losses in excess of $1.5 billion in 1970 alone), Fusarium, and Magnaporthe grisea (the rice blast fungus).
The infrastructure for exploiting Neurospora for fungal genomics exists now.
Neurospora is now being used for industrial manufacture on a small scale (Neugenesis Inc.) and it is closely related to several industrially important organisms such as Trichoderma reesii.
Neurospora sp. is widely consumed as a foodstuff in parts of Southeast Asia as the microbial agent in a cultured pressed peanut or soybean cake called "oncham".
There is an ongoing Neurospora Genome Project devoted to the sequencing of expressed genes. This effort has sequenced about one third of the expressed genes, and it has established that over 50% of the genes expressed in Neurospora are not to be found in either yeast or in the Genbank repository of all sequenced genes in all organisms.
The physical map of Neurospora will be completed within a year through work being done at the Fungal Genome Resource Center at the University of Georgia.
There is an enormous wealth of genetic diversity awaiting discovery in the fungi, and the legacy of Neurospora genetics is an invaluable key to elucidating the biological function of this genetic diversity.
Today the 60 years of cutting edge research on Neurospora constitutes a priceless legacy for interpreting what comes out of the DNA sequence - a Rosetta Stone really - and one not available in an organism chosen for its temporary, topical interest.
The Neurospora - Fungal Genome Initiative
Our Plan of Attack
We are now engaged in Phase I of an overall plan to execute the global sequencing effort. In this work our initial goals were three-fold:
to establish the unique nature of fungal genes. This goal is largely complete as the result of the ongoing work at the University of New Mexico (see above), and more recently in a collaboration between Dartmouth and the University of Oklahoma, that has established that over 50% of expressed genes in Neurospora are not found in yeast or in the Genbank repository that includes all sequenced genes.
The Whitehead Institute under Eric Lander has committed its impressive resources to completing this project at cost in Phase II, so long as funding can be secured. They can guarantee accuracy of l error in between 10,000 and 100,000 bases, better than the available yeast sequence, and can complete the project within months from when funding is secure. After an NSF Science and Technology Center devoted to Neurospora and Fungal Genomics failed in the Blue Ribbon panel by a narrow margin at the 11th hour, NSF (Dr. Maryanna Henkart) agreed to give serious consideration to a proposal from the Neurospora community to complete the sequence at the Whitehead. However, there were reasonable concerns about the magnitude of the request, and some amount of cost sharing with NIH would be viewed as an extremely positive sign.
to complete the physical map of Neurospora. This will serve as the basis for subsequent functional genomics work. Being completed at the University of Georgia, it is now well over half done with a firm target date of 2000 for completion.
to establish a funding base sufficient to complete the project. To date we have contributed to successful efforts to raise over $5 million devoted exclusively to Neurospora genomics through efforts chiefly in the US and in Germany. This funding has yielded the present EST databases, cosmid and BAC libraries, the physical map, and the entire sequence of two of the seven chromosomes, Linkage Groups II and V, about 20% of the genome. We now seek an additional $5-6 million to complete the sequence of the entire genome.
The fastest and surest method to find out how filamentous fungi work is to correlate genomic sequence with metabolic and developmental phenotype. This is best done in the organism where the most genes have been characterized based on mutant phenotypes, Neurospora.
The best way to guarantee rapid future progress in the genomics and genetics of all fungi is to provide informational resources to the largest and most active intellectual community within the filamentous fungi - the community whose efforts have trained many of the scientists working on all filamentous fungi today - Neurospora.
For more information contact:
Prof. Mary Anne Nelson, Department of Biology, University of New Mexico, Albuquerque, NM 87131. Phone: (505)277-2629; FAX (505)277-0304; email: email@example.com
Prof. Jay C. Dunlap, Department of Genetics, Dartmouth Medical School, Hanover, NH 03755.
Phone: (603)650-1108; FAX (603)650-1128; email: firstname.lastname@example.org