How single cells can shed light on ‘fungal dark matter’

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Researchers have developed a way to generate genomes from single cells of uncultivated fungi.

Fungi can be found on forest floors, in swamps, and in houses, ranging in size from smaller than the period on your smartphone’s keyboard to stretching over several city blocks.

Scientists estimate more than a million species live on this planet, but most of that diversity remains unknown because the fungi have avoided detection and scientists have not cultured them for study in laboratories.

Now, scientists are using single-cell genomics to expand the fungal tree of life. They tested the approach on several uncultivated fungal species representing early diverging fungi, the earliest evolutionary branches in the fungal genealogy that provide a repertoire of important and valuable gene products. The findings appear in Nature Microbiology.

Tracing the fungal family tree

“Most of the phylogenetic diversity represents early diverging fungi. We know from environmental DNA surveys that they’re common in many habitats, but they’re presumably microscopic so you really have to look for them,” says co-senior author of the study Tim James, an associate professor in the ecology and evolutionary biology department at the University of Michigan.

“We don’t know what they look like and we know we can’t culture them, since what you can culture is not representative of what you see in environmental DNA,” James says. “We would love to be able to look at a given sample and identify what the cells might look like, but we also want to look at the genomes of the organisms and infer what they’re like. That’s where single-cell genomics comes in.”

“This work was a proof-of-principle that the single-cell genomics approach can reconstruct near-complete fungal genomes.”

Through projects such as the US Department of Energy’s Joint Genome Institute’s 1,000 Fungal Genomes, researchers aim to expand the known fraction of fungal diversity with representative genome sequences for various lineages. Even with such efforts though, the majority of available genomes belong to just two major lineages, Ascomycota and Basidiomycota. The early-diverging lineages that are closer to the base of the Fungal Tree of Life have few representative genomes.

“Conceptually, this is a pilot project,” says Joint Genome Institute data scientist and first author Steven Ahrendt. “This is a similar idea to the approach JGI has taken with microbial dark matter—that the species are out there, but they don’t show up in plate-based culturing.”

‘Fungal dark matter’

The researchers applied the single-cell genomics approach to eight fungi, seven of which belong to the early diverging lineages Cryptomycota, Chytridiomycota, and Zoopagomycota. In addition, six of the seven fungi are mycoparasites, or fungi that attack other fungi. As such, they need to be able to infest the hosts without harming themselves. These species were grown in co-culture with their hosts, and then researchers isolated the spores of the parasites for sampling.

“That mycoparasitic lifestyle might be a factor in why these species are unculturable,” Ahrendt says.

Looking at the genomes of the six mycoparasites, the team found that essential metabolism genes for pathways involving thiamine, urea, and sulfate, among others, were missing, which could make culturing them difficult.

James notes that the fungi researchers used in the study came from a wide range of mycoparasitic strategies and represent a large amount of evolutionary time, which made it difficult for the team to identify novel sets of genes that could shed light on the mycoparasitic lifestyle. What the study really highlights, he adds, is that the single-cell approach is feasible for what he calls “fungal dark matter.”

The fungal single cells yielded anywhere from 6 percent of the genome to 88 percent, but combining the single cells yielded genome co-assemblies ranging from 73 percent complete up to 99 percent complete.

Tweaking the pipeline

There are around 2,000 described species of early diverging fungi, and about 120,000 described species of the Ascomycota and Basidiomycota, James says.

“We’ve described maybe 5 percent of the fungal diversity, and we’re in an era where we can start to get at that missing piece of the diversity,” he says.

The single-cell genomics approach will be applied to a JGI Community Science Program proposal that James is leading and that involves 50 unknown early diverging fungi from aquatic environments.

“What I’d really like to see is people take up this approach and tweak the pipeline to fit different organismal groups,” he says. “This pilot just started the exploration by looking at unicellular aquatic organisms, and yet we have organisms in soil, in plants, and so on.”

“This work was a proof-of-principle that the single-cell genomics approach can reconstruct near-complete fungal genomes and provide insights into phylogenetic position and metabolic capacities of diverse unculturable species from environmental samples,” says JGI Fungal Program head and co-senior author Igor Grigoriev.

“Several genomes in this study represent the first references for fungal phyla containing mostly species that have not been or cannot be cultured. Having genome sequences and metabolic reconstructions of a broad diversity of uncultured fungal species enable us to better understand fungal evolution and expand the catalogs of gene, enzymes, and pathways,” Grigoriev says.

The genomes of the species referenced in the study are available in JGI’s fungal portal, MycoCosm, as well as on GenBank.

Additional researchers who contributed to this work are from University of California, Berkeley; Ottawa Hospital Research Institute, Canada; Aix-Marseille University, France; Institut National de la Recherche Agronomique, France; King Abdulaziz University, Saudi Arabia; and University of Florida.

Source: University of Michigan