Neurospora Initiative

The Neurospora Fungal Genome Initiative

Steering Committee:

Jay C. Dunlap (Chair) Professor of Biochemistry, Dartmouth Medical School
Robert L. Metzenberg - Professor of Physiological Chemistry, University of Wisconsin, past -President, Genetics Society of America
Charles Yanofsky - American Cancer Society Professor of Molecular Biology Stanford University; Member, National Academy of Sciences
Jonathan Arnold , - Director, Fungal Genome Resource, University of Georgia
Rodolfo Aramayo - Stanford University
Donald Natvig - Associate Professor of Biology, Co-Director, Neurospora Genomics Center, University of New Mexico
J. Patrick Jordan, - Director, Southern Regional Research Center, USDA Agricultural Research Service
Bruce Roe - Director, DNA Sequencing Center, University of Oklahoma

SUMMARY

! The Significance: The Kingdom of the Fungi includes over 1.5 million 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 are major pathogens of animals (including people) and of plants, collectively costing in excess of ca. $30 billion annually on a worldwide basis. Some of the fundamental discoveries in biology have come from the study of fungi, such as what a gene does.

The Issues: We do not understand how fungi work.

We cannot engineer fungi to maximize production because we do not understand how fungi produce and secrete chemicals; therefore, 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 and improvement of strains is driven solely by empirical means.
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 is 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 towards this goal is to catalog all the genes that constitute the repertoire of fungal physiological capacity, and to associate these genes with biological function. Since 90 percent of all fungi are filamentous,

the most efficient and cost effective means of understanding fungi is to catalog the protein coding regions and then determine the sequence of DNA in the genome of the best understood filament! ous 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 1.5 million 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 (which form fruiting bodies or "mushrooms"). As a group, the fungi have an enormous impact on the United States and world economies: yeasts are used extensively in the brewing industry, filamentous fungi! are used both for the production of foodstuffs and indus! trial 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.

Cultivated mushrooms now constitutes an $800 million per annum industry in the US that has grown over 10 fold in size during the past two decades.
Industrial production of enzymes, largely by filamentous fungi, constitutes a $1.2 billion per annum industry.
Industrial production of chemicals by filamentous fungi constitutes a ca. $32 billion per annum industry. The US is a net importer of some chemicals such as citrate representing in excess of a $billion annually.
Pharmaceutical manufacture using fungi constitutes a $23 billion per year industry worldwide. Penicillin and similar º-lactams, all produced in fungi, are the world's second largest se! lling antibiotics; penicillin production alone accounts for $4.4 billion/yr worldwide, and some of the cephalosporins are derived from penicillin precursors.
Worldwide sales annually of leading antifungal drugs (Diflucan, Sporanox, Nizoral,and Lamisil) were approximately $2 billion per year.
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%.
Fungi are of major importance in basic research. They are genuine eukaryotes having cell and genome structures and metabolic organization similar to that of other eukaryotes like plants and animals. However, because they have smaller genomes and are uniquely experimentally tractable, fungi are universally used as model organisms for understanding all aspects of basic cell! ular regulation including cell cycle progression, gene reg! ulation, circadian timing, recombination, secretion, and development. The Nobel Prize has been awarded for research on one filamentous fungus, Neurospora crassa.

A Closer Look - Why Determine the DNA Sequence of a Fungal Genome?

The information encoded in the genomic DNA of an organism contains the entire repertoire of the organism's metabolic and developmental capacity - everything that it can do or become. It represents the pinnacle of an organism's evolutionary growth over eons of history, where variation induced by mutation has been refined in the crucible of evolution.

By determining the sequence of a genome, all of the proteins encoded by 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 informat! ion 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 several bacteria and for baker's yeast. 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 yeasts. For instance in the best described system, Neurospora crassa, greater than 50% of the protein coding regions sequenced in an ongoing pilot project do not correspond to genes found in yeast; they are novel. Since filamentous fungi constitute 90 percent of all fungi and are the basis of a multi-billion dollar US industry, these novel genes are of tremendous economic importance.
By extending genomic sequence information from yeast to filamentous fungi and eventually to Basidiomyc! etes, the range of growth, metabolic capacity, and developm! ental capacity at our fingertips can be greatly extended. Unlike yeast (which like bacteria is a unicellular organism), most fungi have a coenocytic growth habit and are genuinely multicellular at all stages of their life cycles.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.Fungi have substantially greater metabolic versatility than yeast. Fungi can grow at pH's from 2.5 to 11, at temperatures form 10íC to 48íC, at salt concentrations over 4 M, and on a staggering number of different and exotic carbon and nitrogen sources. The potential here for bioremediation is only beginning to be exploited.
As a group fungi are known for the exceptional ease with which they can be genetically and molecular! ly manipulated. Generally, once fungi can be cultivated they can be genetically transformed with exogenous DNA in a site-directed fashion, thus paving the way for genetic engineering of one fungus using developmental or metabolic capacities encoded by a different fungus.

The determination of complete genomic sequences of filamentous fungi is now an important and readily achievable goal. Initially the DNA sequence itself will be most informative in proportion to extent to which the genome sequenced is already understood genetically.

A Closer Look - Why Sequence Neurospora?

A recent 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 i! n any organism is now economically feasible, the true utility of the sequence cannot be fully realized until the string of A', T's, G, 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 organisms' genetic capacity is devoted to housekeeping functions that all organisms must be able to do - 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 for heterokaryons, genes that function in the cell cycle, in the biological clock, in sexual development - 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 800 different genes identified by function in Neurospora crassa, 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 ma! p of Neurospora is universally recognized as one of the mos! t painstakingly documented in existence. Over 800 genes have been mapped relative to one another, over 300 cloned genes and 250 RFLP markers allow precise links between the genetic and the physical map (which will soon completed).
The field of biochemical genetics was invented using Neurospora; there is over 50 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 first clear connection between genes and biochemical function.
Research on Neurospora is ongoing. Reflecting the presence of a large and vibrant research community, the largest devoted to a fungus except for yeast, nearly twice as many research papers are published annually on Neurospora than on any other filamentous fungus; within the past 30 years nearly 5000 papers have been published on Neurospora. Research on Neurospora is frequently seen in the mo! st prestigious scientific and lay science journals - Science, Nature, Cell - twice as cover articles in as many years.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, the role of methylation in gene expression, to name a few.
Information on Neurospora is readily applicable to other agriculturally important, industrially important, or pathogenic fungi. Although Neurospora is nonpathogenic, it is phylogenetically very closely allied with and genetically similar to several important plant pathogens including Cochliobolus (corn leaf blight which caused crop loss in excess of $ XX million in 19XX), Fusarium, and Magnaporthe grisea (the rice blast fungus).Neurospora is now being used for industrial manufacture on a small scale (Ne! ugenesis Inc.) and it is closely related to several indus! trially important organisms - WHAT ARE THESE??.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".
The infrastructure for exploiting Neurospora for fungal genomics exists now. There is an ongoing Neurospora Genome Project devoted to the sequencing of expressed genes. This work has established that over 50% of the genes expressed in Neurospora are not to be found in either yeast or in any other organisms with sequenced genes in the Genbank repository of all published DNA sequences
The physical map of Neurospora will be completed within a year through work being done at the Fungal Genome Resource at the University of Georgia under National Science Foundation and Georgia Research Alliance auspices.

There is an enormous wealth of genetic diversity awaiting discovery in the fungi, and the legacy of Neurospo! ra genetics is the key to elucidating the biological function of this genetic diversity.Today the 50 years of forefront research on this organism 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 prosecute the global sequencing effort. In this work our initial goals are twofold:

to establish the unique nature of fungal genes. This goal is largely complete as the result of the ongoing work at the Neurospora Genome Center in New Mexico (see above) where sequencing of ESTs has established that over 50% of expressed genes in Neurospora are not found in either yeast or in the GenBank repository that includes all sequenced genes in the literature.!
to complete the sequence of a fully functional ! chromosomal element from Neurospora. This will serve as a benchmark to establish our credibility in high through-put sequencing. This work will be completed chiefly at the Neurospora Genome Center in New Mexico and at the Fungal Genome Resource center at the University of Georgia, although we will encourage the development of four additional satellite centers .

We will then go for the funding to complete the entire sequence in Phase II, an overall effort that we expect will include the complete sequencing of Aspergillus nidulans and acquisition of partial sequences from several additional important fungal pathogens, industrial production strains, and cultured organisms.

Our Funding Base

Phase I - ESTs. Funding for ongoing work on sequencing of expressed genes in Phase I is coming from the National Science Foundation (ca. $500K over three years). - Chromosomal element. We must identify sources for the estimat! ed $400K of funding needed for the sequencing of the chromosomal element. We anticipate that partial funding will come from state governments and from private concerns. However, it is essential that we identify Private Sector Allies committed to the support of this Initiative.
Phase II Complete Genomic Sequences from Multiple Organisms. Careful estimates from several sources familiar with large scale genomic sequencing suggest that on the order to $30 million will be required over 5 years to complete the DNA sequencing of Neurospora crassa, Aspergillus nidulans, and parts of other genomes. Due to the magnitude of the effort anticipated in Phase II, we will require federal funding and, upon the completion of Phase I, will begin to organism a full scale public relations effort to educate members of Congress in important districts concerning the benefits of this Initiative. The Neurospora research community is large and well organized, and ! the 700+ member fungal genetics community, although less ! well organized, can be mobilized behind this effort. Several key Senators and Congressional Representatives come from states or Districts that will benefit greatly from this initiative, and we are confident that they will be receptive. However, it is clear that we will require the serious support of Private Sector Allies as we begin to contact Congress at the outset of Phase II.

To move forward we need your help.

Requested Action

We need your support in two ways.

First, for us to successfully educate members of Congress, it is critical that we be able to call upon your written support. An Initiative driven only by academic research interests is not likely to draw the necessary interest in Congress, whereas an Initiative supported by agricultural and industrial interests in the private sector is likely to draw attention.

Secondly, it is essential that we identify Private Sector Allies willing to financially suppo! rt the chromosomal sequencing aspect of Phase I. A commitment corresponding to one postdoctoral or technician equivalent for one year will allow us to prosecute the work immediately and to complete it is a timely manner, although partial but significant support at any level will allow the work to begin.

The Profits

Industry will profit from this Initiative through the development of an accessible database cataloging the total metabolic capacity of fungi. This can be used to design rational plans for genetic engineering of fungi.
Agriculture will benefit from this Initiative through the enhanced understanding of fungal metabolism that should identify aspects for fungicide development and that will promote understanding of the mechanisms of spore development and of mushroom fruiting.
Academia will profit from the development of closer links to the industrial and agricultural community, and from the! development of a database that will foster creative rese! arch.