Understanding Fungi through Their Genomes

In the 1800s, fungi led the way to modern microbiology and biochemistry. Peering through a microscope, Pasteur was the first person to observe tiny living creatures. Uncertain about what he was seeing, he classified their activities into “organized ferments” and, when changes occurred without any observable microorganisms, “unorganized ferments.” When it became known that the unorganized ferments were the metabolic products of organized ferments, he later suggested the term “enzyme” (en = in, zyme = yeast).

One of the best studied of these enzymes, diastase, is a product of germinating barley and was used in the malting step of beer production. In 1894 Jokichi Takamine began inoculating thin layers of rice and barley with spores from Aspergillus oryzae to optimize production of the enzyme, which converts starch to malt sugar. Much like contemporary scientists, Takamine showed a strong entrepreneurial streak and patented his process, later marketing diastase as a malting enzyme and a digestive aid to treat dyspepsia.

In 1928 medicine vaulted firmly into the modern age with Alexander Fleming's discovery of penicillin, the first “wonder drug.” This spurred all sorts of studies into fungal physiology, fermentation technology, and industrial strain development. In 1941 George Beadle and Edward Tatum found the first connection between genes and biochemical function. The one gene–one enzyme hypothesis could not have been formed without the help of Neurospora crassa.

When genetics progressed to genomics, Saccharomyces cerevisiae became the second species and the first eukaryote to be sequenced. But since this genome sequence became available in 1996, research tools in yeast have bounded forward in huge leaps, while studies of filamental fungi have lagged behind. The gap will soon close.

Over the last two years, a large group of scientists from across the spectrum of mycology and genomics has worked to sequence a broad array of fungal species. In June, the National Human Genome Research Institute (NHGRI), a component of the National Institutes of Health, agreed to fund the costs of the first 7 of 15 proposed species of the Fungal Genome Research Initiative. Researchers in the fungal genomics field anticipate an explosion of information that not only will influence the fields of medicine, industry, and phylogeny but will make mycology one of the hottest fields in science.

“When a young scientist who might not have ever given fungi a serious thought sees the depth and breadth of genomic information that we are going to have in this field,” says University of California–Berkeley systematist John Taylor, “this person is going to have to give mycology a serious second look.”

What is available

Within mycology, only yeast researchers have powerful genomic technologies at their fingertips. The S. cerevisiae sequence is well annotated, and the organism can be manipulated with a number of genetic tools such as expression profiling with microarrays, serial analysis of gene expression, protein tagging, two-hybrid interactions, and transposon insertions.

Filamental genetics, however, is much less developed and bits of sequence are flung all across the Internet. A search of 379 genome-project Web sites finds 16 with filamental genetic information representing only 12 species. and some of these Web sites are owned by industry and thus contain proprietary information.

After mining the sequence for information, Cereon/Monsanto released their proprietary, low-resolution sequence of Aspergillus nidulans. Access, of course, comes with strings. Researchers using the database have to acknowledge Monsanto in their papers and give the corporation first crack at licensing any agricultural applications stemming from the research. Still, the restrictions have done little to slow the obvious zeal in accessing the genome.

In the last year, however, things have started to improve with the public release of the genome sequences of three other fungi. But for some academics, it was too little, too late. “If you want to be serious about genetic research, then you need the genomic information,” says Olen Yoder, who left a tenured faculty position at Cornell University to join Syngenta at their La Jolla, California, research lab. An agricultural research group, Syngenta boasts that it has high-resolution sequences of five fungal genomes. “That just wasn't available to me in academia, so I left. and I know a number of people who did the same thing.”

The squeaky wheel gets the oil

“As a field, mycologists work well together,” Taylor says, a statement echoed by many others. This collegial atmosphere was a definite plus in securing funds for what some refer to as mycology's Manhattan Project. According to Taylor, a core filamental fungi group began coalescing only within the last decade, springing out of the Neurospora Information Conference, a small biannual meeting that Taylor admits was something of a scientific backwater.

“When I first went to the meeting there were fewer than 100 people,” he says. “Still, it was bigger than the previous year's and there was a great deal of excitement.” In addition to Neurospora researchers, the meeting managed to attract scientists working on other filamental fungi, and things really took off when a charismatic leader, Bill Timberlake, figured out how to introduce genetic material into Aspergillus. As more scientists began attending, the conference morphed into the Fungal Genetics Conference. It still convenes every two years at Asilomar, in Pacific Grove, California, and has grown so popular that people have to be turned away.

Another important reason for greater interest in mycology was the release of the S. cerevisiae genome sequence in 1996. Combine all the filamental scientists in the world, and you still have fewer people than the number who study this single species of yeast. After the sequence was published, some yeast scientists probably began looking for ways to validate their findings in other organisms, or maybe they saw something more fundamental about their field.

“Suddenly you've got about 10,000 people chasing only 6000 genes,” says Rytas Vilgalys, a mycologist at Duke University. “That's been good for mycology, because the yeast people are smart and organized and they're looking for something to do.”

In a series of meetings and symposia, various members of the fungal genomics community began working together on a comprehensive initiative for fungal genomics. They chose an initial 15 species with importance in biomedicine, agriculture, industry, eukaryotic biology, and evolution. The Whitehead Institute for Biomedical Research, which has already sequenced Magnaporthe grisea and N. crassa, was involved from the beginning, and when NHGRI released some funds, seven species were chosen for the initial sequencing. Of course, not everyone got his or her pick.

Researchers complain that the initial seven are heavily weighted toward human pathogens, but because NHGRI is under the National Institutes of Health, this comes as no surprise. They also point out that fungi have become fatal pathogens only in recent years. In the first part of the 20th century, the most important fungal pathogens were both nonfatal: ringworm and thrush. The change from morbidity to mortality is a recent consequence of therapies for patients with AIDS, transplants, or cancer. Although drugs allow people to survive longer with these conditions, fungal infections can quickly undo those gains.

Less than 6 percent of the complete microbial genomes are plant pathogens, and James Sweigard, a senior assistant biologist at DuPont, sees cause for criticism. “The trouble with plant pathogens is that they kill more people than human pathogens, but they do not do this in a very dramatic way,” he says. “Famine isn't a problem in the developed world.”

Others feel that the heavy focus on human pathogens leaves out species with less obvious importance, such as chytrids, which have been discovered recently as pathogens of amphibians. Joyce Longcore of the University of Maine says that though chytrids are a little-studied group, they have great importance when it comes to phylogeny. “I do wish that a chytrid that was more at the base of the fungal clade had made the list.”

Still, despite the moderate criticism of the list, every researcher acknowledges that scientific compromises made to secure funding will matter little over time. “Maybe I didn't get what I wanted, but any sequence that we get is going to inform my research and the research of all of us in this field,” Sweigard says.

How to sequence

Genetically, fungi have slightly larger and more complicated genomes than bacteria. Like us, they have introns, but they are only about 1/300th the size of introns in Homo sapiens. To tackle the sequencing, the Whitehead Institute plans to do a deep-shotgun sequence. Specifically, they intend to produce a fine-resolution map by sequencing fragments at “10× coverage,” which involves randomly shearing the DNA of multiple clones and then putting the pieces into 4-kilobase (kb) plasmids (90 percent) and much larger 40 kb fosmids (10 percent). For 10× coverage, 10 times the amount of the complete genome is sequenced in a redundant manner. The resulting pieces are strung together by matching overlapping sequences, like copies of the same puzzle jigsawed different ways, producing a high-quality map of the genome.

A deep-shotgun approach leaves some gaps, but the process is quick and cheap. Moreover, the resulting sequence and physical map (that is, where sequences are found on the chromosome) are useful for just about every purpose. A much lower-resolution 3× map of A. nidulans proved extremely valuable for years to Monsanto, and since it was published on a Web site only a year ago, the site has received almost 3 million hits.

If a researcher later runs into a gap and needs to fill it in, copies of the various vectors will be available at the Fungal Genetics Stock Center. The FGSC has been around for almost 30 years and maintains a large number of fungal clones, including over 8000 strains of Neurospora. “At a 10× coverage, you get several thousand gaps,” says Ralph Dean, director of the Fungal Genomics Lab at North Carolina State University. “In 90 percent of these cases, we already have the DNA; we just have to sequence it. But in 10 percent of the cases, we don't have the DNA, so you really end up with a few hundred real gaps.”

This filling in of gaps, or closure, can be as time consuming and difficult as the actual sequencing. You can reach a point of diminishing returns, where getting every base pair correct is not very cost-effective. There is also the issue of annotating the genome, that is, locating the individual genes in the sequence. Both closure and annotation can be done piecemeal or systematically in one quick shot. Dean hopes it will be the latter, but that depends on the availability of resources. “What we're hoping for is that we can prove to funding agencies that this is just as important as the sequencing and that funding will be made available,” he says.

A fungus among us

As lower eukaryotes, fungi are more closely related to humans than are other microorganisms, such as bacteria and viruses. With what little fungal sequence is available, homologues for 30 percent of human proteins can be found, almost twice what is known from S. cerevisiae alone. The fungus A. nidulans has been the source of much of our knowledge about the genetics of tubulin and microtubules and is also an important model for studying mitosis. Oddly enough, it is this very resemblance to humans that makes fungi a particularly nasty human pathogen, because most therapies designed to kill fungi also harm humans.

“We're building a drug company, and one of our tasks is discovering antifungals,” says Peter Hecht, president of Microbia, a three-year-old company. “To do this you have to find targets of intervention where you can cripple the pathogen. This can be quite difficult, but what you'd like to find are targets critical to the pathogen, but which are not found in humans.”

Also, fungal metabolites have been harnessed for other important pharmaceuticals. The statins were first discovered during antifungal screens in Japan. In high doses these drugs are quite toxic. However, at lower doses they are sold as cholesterol-lowering drugs, a $12 billion worldwide market. Because of the big payoff, pharmaceutical companies have turned all sorts of fungi into minibiofactories, manufacturing statins, antibiotics, fungicides, and other drugs. “At Microbia, we're not just out to find new or safer pharmaceuticals,” Hecht says. “We want to manipulate the regulatory controls inside fungal cells to help manufacturers goose up their microfactories.”

When fungi attack

Although fungi are not important pathogens in humans, they make up 80 to 90 percent of the disease microbes preying on plants. Rice blast caused by Magnaporthe grisea leads to losses of crops capable of feeding 60 million people annually. and like another crop pathogen, Ustilago maydis (corn smut), it is also an important model for studying host–pathogen interactions.

The genome for M. grisea is now complete, as is the genome for rice, and Ralph Dean sees only vast possibilities. “We've now got two genomes, a host and a pathogen,” he says. “So now we're going to be studying host and pathogen interactions not just at the molecular level, but also genetically. This really hasn't been done before—understanding how two species interact at the genomic level.” He also points out that as other fungal species become available, they will start cross-referencing the M. grisea pathways with other fungal pathogens.

“These genomes will certainly impact us,” Sweigard says. DuPont has now moved away from the pathogen side to the host side of disease, attempting to modify species such as corn to make them more resistant to pathogens. “We do directive genetics, asking, ‘Does the pathogen need this gene to be a pathogen?’ If it does need this gene, then we counter the metabolic process by modifying the plant.”

“We're interested in why certain fungi are pathogens and how their genomes differ from others, such as saprophytes,” says Gillian Turgeon, who collaborates with Olen Yoder at Syngenta. “The ultimate goal is to control the pathogen in the field, and with the species' blueprint, it just shifts the kind of questions you can ask.”

During a mutagenesis screen several years ago, Turgeon and colleagues discovered a mutant with reduced pathogenicity. After sequencing the mutated gene, they realized they had discovered a novel gene, and by searching their proprietary genome databases, they discovered the gene had been highly conserved across many different pathogen species.

“There are general pathogen factors, and then there are specific factors for each host,” Turgeon explains. “General factors are more interesting, because this allows you to control more fungi.” After mutagenizing the gene in other pathogens, she and her colleagues found it reduced fungal disease by around 60 percent in many different plant species. and the gene can also be found in human pathogens. “What we'd like to do, and we have several ongoing collaborations, is to test the gene's virulence in some of the human pathogens. We do not have the data yet, but we are hot on the trail.”

The ongoing challenge

With every technological advance, from light microscopy to electron microscopy to genetics, the systematics of fungi gets reinvented. Systematic groupings once thought solid are found to be artificial, and species that were thought to be close are discovered to be distant relatives. “They keep changing the names all the time!” says one medical researcher. “I guess this keeps people busy and employed.”

Rytas Vilgalys says the confusion is due to the peculiar nature of fungi. “Morphology is misleading, and for fungi we don't really have a fossil record,” he says. “Genetics is really the only way to understand these evolutionary relationships.”

Take gilled mushrooms, for example. For decades, species with gills were grouped together, even though gills disappear and reappear randomly throughout evolution. There are many examples in which a gilled mushroom made the transition to a puffball-like derivative, and some gilled species can produce both gills and pores. Trying to understand how organisms fit into groups and how these groups are related becomes an act of futility. “It's not [like the situation] in other groups, like birds,” Vilgalys says. “There's no way to resolve morphology with phylogeny. You'd go nuts!”

Ecologically, it also becomes useless to understand which morphology leads to greater fitness. “It may not even be useful to think which came first, gills or pores, because species might go back and forth,” Vilgalys adds. “There may be some directionality, but some recent rigorous analysis with evolutionary models of how often species have gone from gills to pores or gills to puffballs has found that there is no discernable pattern.”

“What genetics did is to take some of the opinion out of systematics,” John Taylor comments. “You could go to a meeting of taxonomists and someone would say ‘I think this is related to this because I say so.’ End of story. It just devolved into ‘I've studied these things for 50 years and know more about them than you do.’ Now people have to put their data into a matrix and see how it looks.”

He does point out, however, that molecular markers are not any better than other characters; it's just that the huge quantity of them evens out opinion and allows statistical analysis. and with genomics, you just get a huge number of data points.

“I'm not really sure what we'll find,” he says. “It might be that things are just more complicated than we expected.”

Comparisons of fungal divergence from Archaea and bacteria, for instance, were thought to be simple when the only character chosen was a single RNA subunit. “When people started looking at other genes, it became fuzzier. Now the branching order depends on which gene you pick. The hope is that by choosing more genes you get better resolution, but the reality is that you sometimes get confusion.”

Magic mushrooms

What will the future bring for mycology? Taylor is not quite sure. “I'll be perfectly honest,” he says, “I really don't know, but I think it's going be more than we can imagine.”

Like Ralph Dean, Peter Hecht sees only vast possibilities. At Microbia researchers are examining genomes with the goal of making fungi the engineers of complex biosynthetic pathways leading directly to finished drug products. This saves all the time and energy of modifying a metabolite through traditional chemistry. As Hecht sees it, chemistry becomes irrelevant in the future as pharmaceutical companies move to direct bioprocessing. “The really fun part starts when the information is made available,” he says.

and for Yoder, genomic information is the chance at a lifelong dream. “Maybe I wasn't visionary enough, but back in grad school I never imagined we would have this sort of information available,” he says. “I remember a group of us sitting around and having these late-night discussions about what caused pathogenicity. Was it one gene? Was it many genes? and if so, how many? We didn't have a clue, because there weren't any data. It was just talk. Now I'm going to have a chance at possibly figuring this out, or at least a small part of it.”

He pauses for a moment. “It's not philosophy anymore. This is real science.”

The 15 species of fungi to be sequenced as part of the Fungal Genome Initiative are listed on the right in colors corresponding to their positions on the fungal phylogenetic tree. Geologic time is shown on the horizontal axis. The fungi were selected so that genomic comparisons could be made between species spanning a wide range of geologic time. Figure provided by J. W. Taylor.

Chytrids are the earliest-diverging lineage of fungi and have been reported around the globe as a cause for the decline of amphibians. Micrograph shows epidermal cells of a northern leopard frog that have been invaded with the thalli of Batrachochytrium dendrobatidis. The species reproduces by asexual zoospores, and, like other chytrids, its cells retain centrioles. Arrow indicates zoospores within a sporangium and arrowheads indicate empty sporangia. Micrograph: Joyce Longcore, University of Maine.

When environmental conditions are just right, certain fungi send out reproductive fruiting bodies to spread spores. The sampling above represents just a small number of the mushrooms found in North Carolina, and their elegant shapes and splashy colors exemplify the term “butterflies of the soil.” Photographs: Rytas Vilgalys, Duke University.

Industry is keeping a close eye on fungal genome work, because the combined annual sales of fungal pharmaceuticals amount to more than $5 billion annually. Photograph: Etchell Cordero, Microbia.

Ranging from microscopic yeast and molds to oversized mushrooms, fungi have been a part of human civilization since before recorded history. As agents of food spoilage, they have long been the bane of societies attempting to store provisions, but fungi can also be vital to the diets of people around the planet. Without them, we would not have mushrooms on our pizza, mold-ripened cheeses, soy sauce, or sliced bread. and let's not forget about cold beer.