Poster image credit Sonja Inske.

Microbiology doesn’t often go down well at parties. You don’t score points for explaining the microbial diversity in a bowl of peanuts as your guests slowly shuffle away. However, we have a handful of show-stopping facts and anecdotes that are impressive even to the most cynical among us. One of my favourites is when you ask people what is the largest organism on the planet? Is it an elephant? Is it a blue whale? No. Of course, it’s the Humongous Fungus. Not only can fungi be humongous, but they can also form fairy rings which have inspired mythical stories for centuries. But there’s more to the fungi than just being mythical and humongous. A recent publication suggests that they also have far fewer mutations than you would expect from a typical eukaryotic cell.

Fairy rings in the wild. Photo credit Markus Hiltunen. 

The inception of this work began 20 years ago when during her PhD. Hanna Johannesson dreamed of a study to sample fairy rings, but not to look at mutations. She was interested in the parasexual life cycle of these fungi by which the organisms exchange genetic information without meiosis or any specific sexual structures. But without funding, the study was put on ice until Hanna was a professor, sitting across from a masters student in her office at Uppsala University. 

Markus Hiltunen was a student who had a deep interest in the fungus Marasmius oreades. The problem was, Hanna didn’t have funding for the study that her and Markus wanted to do, so during their discussions, she proffered many alternative projects, while Markus sat characteristically silent. But he refused to give up on his dream PhD project and Hanna realised that despite the lack of resources, if anyone could make this work it was Markus.

“I have been interested in mushrooms most of my life.” explained Markus. “I spent a lot of my childhood running around in the forest where I came across many different kinds of mushrooms. As a biologist, my main interest is in genetics, as genetics can explain a lot of what we see in nature. When I met Hanna she was mostly working on Ascomycete fungi, which is not my main group of fungi, but the kind of questions her group was working on still really appealed to me. When she gave me the opportunity to study similar questions in mushroom-forming fungi I was hooked.”

The challenge was not finding the fairy rings, the challenge was finding complete ones. Normally the fruiting bodies only form a partial ring, but for this study Johannesson and her team wanted a full ring to get the broadest possible set of samples. “Fairy rings are like a whole evolutionary experiment in one organism,'' explained Johannesson. That’s because they start from a single cell at the centre of the fairy ring and grow outwards. As the mycelia extend outward over decades and produce fruiting bodies, the centre of the ring slowly dies. By sequencing the DNA from the fruiting bodies around the circumference of the fairy ring, you can measuring decades worth of evolution from a single origin. 

So they set out to look for indicators of the parasexual life cycle of Marasmius oreades by sampling the fruiting bodies of the fairy rings and sequencing them to look for patterns of variation in the DNA. They went in search of the fairy rings in Sweden, where the team is based. “They’re not difficult to find,” says Johannesson, “they’re everywhere.” And in Sweden, they are everywhere, you can find them in almost any forest or grassland if you look out for the characteristic discolouration of the grass. Markus found one example of a complete fairy ring in a graveyard close to the University and more examples were quickly spotted, around Uppsala and in Vadstena which is a town on a lake to the south of Uppsala. 

They hypothesised that comparing the genomes from fruiting bodies around the ring would show exactly how the parasexual life cycle was shaping the  genetic information of the ring. However, their sequencing turned up empty, at least they didn’t see any evidence of the life cycle in the DNA sequences, but then they started to look at mutation rates and the results surprised them.

Now Hanna, Markus and a team of researchers have published findings that suggest that these fungi have a way of protecting their genome from mutations. When screening for mutations they found almost none, which was surprising. After calculating the mutation rates, they saw that they were much lower than expected when compared to many other eukaryotic cell types. Despite living for hundreds of years, these fungi simply didn’t really accumulate errors in their genetic code.  

This finding is truly unique. While other studies have shown lower mutation rates in fungi and plants, the mechanism has often been to produce special structures such as the meristem or rhizomorphs. These structures sequester and protect genetic material specifically for transmission so that errors cannot accumulate. However, M. oreades doesn’t have any of these structures, meaning that each cell in the mycelium is capable of preventing mutations from forming. The implications are huge, but the real question is how do they do this?

Whatever the mechanism, it has to be able to function in every cell in the mycelium. One hypothesis is that M. oreades has more faithful proofreading in the DNA polymerases it used to copy the DNA. Higher fidelity polymerases will introduce less errors in the genetic code leading to less mutations which would fit perfectly with the experimental results. 

However, Duur Aanen, professor of evolutionary biology from Wageningen University says that, evolutionarily speaking, this is a very costly way to reduce mutations. “It’s hard to imagine that there would be sufficient selective pressure for such an elaborate mechanism,” explains Aanen. Johannesson and her team also saw no evidence for this in their genetic data. 

Another hypothesis comes from looking at ciliate as Aanen explains in a recent dispatch article. One way that some ciliates improve the fidelity of their DNA polymerase is by silencing transcription. They have two nuclei, one large used to transcribe mRNA and allow the cell to function, and one small, termed a micronucleus. The micronucleus is transcriptionally silent, meaning that there’s not much going on, the only time the DNA is touched, is to be replicated and passed on when a cell divides. Apparently, under these conditions, the DNA polymerase can be very high fidelity. The problem here is that there is no evidence of transcription being silenced in M. oreades. 

A theory that is gaining more and more empirical support is the ‘immortal strand hypothesis’. It started in the 70s when John Cairns published several theories in Nature on how stem cells can protect against mutations leading to cancer. In DNA replication, one DNA strand acts as a template for the DNA polymerase. This template strand, or immortal strand is unchanged and never incorporates errors. If cells can protect and pass on this strand preferentially over the replicated strand then mutations will be kept very low.

The immortal strand hypothesis has been controversial in the past as there wasn’t much empirical evidence to support it. However, that has started to change. With the improvements and increasing use of next generation sequencing, mining genetic data for mutation rates have slowly built up a pile of evidence that points squarely at the immortal strand hypothesis as the protector of genome integrity, at least in some cases.

Although evidence is now stacking up in favour of the immortal strand hypothesis, we’re still waiting for the irrefutable experimental evidence. While possible, it is difficult for a variety of reasons. Firstly, the time scales needed to do these experiments are not conducive to many laboratory set ups. And in some of the model systems used to propose the immortal strand hypothesis such as stem cells, it’s hard to grow cells to high enough densities to see the results. However, both Johannesson and Aanen are confident that this experiment will soon be done in fungi. 

It appears that fungi go to a lot of effort to avoid mutation, does this mean that they’ve given up on variation and adaptation? Maybe it’s not necessary in their particular environmental niche? Actually not at all. “When you look at the lifespan of a fungus like M. oreades, it has a similar mutation rate to many other eukaryotes. However, because the fairy rings live for so much longer, they have to reduce their mutation rate to avoid too many deleterious effects,” says Johannesson.

This raises an obvious if not far fetched questions, can studying fungi help humans to become immortal, or at least prevent mutations that cause diseases like cancer? “We’ve been using studies on stem cells and cancer to inform our work on fungi, so actually, it’s the other way around,” explains Duur Aanen. That being said, the recent work on fungi suggests that fundamental principles to reduce copy errors during cell division are shared between stem cells and fungal cells. Fungi could be a useful experimental model to study cancer, not only for single cancer cells, but also for how cancer tissues behave. 

References

Hiltunen, M., Grudzinska-Sterno, M., Wallerman, O., Ryberg, M. and Johannesson, H., 2019. Maintenance of High Genome Integrity over Vegetative Growth in the Fairy-Ring Mushroom Marasmius oreades. Current Biology.

Aanen, D.K., Germline Evolution: Sequestered Cells or Immortal Strands? 2019. Current Biology. p. R799-R801

Cairns, J., 1975. Mutation selection and the natural history of cancer. Nature, 255(5505), p.197.

Aanen, D.K., 2014. How a long-lived fungus keeps mutations in check. Science, 346(6212), pp.922-923.

Aanen, D.K. and Debets, A.J., 2019. Mutation-rate plasticity and the germline of unicellular organisms. Proceedings of the Royal Society B, 286(1902), p.20190128.

Werner, B. and Sottoriva, A., 2018. Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates. PLoS computational biology, 14(6), p.e1006233.

Anderson, J.B. and Catona, S., 2014. Genomewide mutation dynamic within a long-lived individual of Armillaria gallica. Mycologia, 106(4), pp.642-648.

Anderson, J.B., Bruhn, J.N., Kasimer, D., Wang, H., Rodrigue, N. and Smith, M.L., 2018. Clonal evolution and genome stability in a 2500-year-old fungal individual. Proceedings of the Royal Society B, 285(1893), p.20182233.