Mushrooms get sick too, but how does their immune system work?

Just like animals and plants, mushrooms can get sick due to infections caused by viruses, bacteria, or other fungi. And when disease strikes a mushroom farm, the economic impact can be significant. Yet little is known about how mushrooms defend themselves against pathogens, and research in this area remains limited. Erik Beijen explored the immune system of mushrooms for his PhD project. Today, he defends his dissertation.

Fungi do not have the best reputation, as people might primarily associate them with skin infections or rotting fruit. But mushrooms, the fruiting bodies of certain fungi, and their names often spark the imagination. Think of witches' butter, the dark-scaled knight, and the iconic fly agaric, red with white spots. Nevertheless, when it comes to scientific research, mushrooms tend to be somewhat overlooked.

According to Beijen, this may be partly because mushrooms have less economic value than plants. Yet mushrooms are also consumed by humans, and diseases can be costly for mushroom growers. For instance, the pathogenic fungus Lecanicillium fungicola alone causes around 300 million euros in damage annually in Europe. Beijen points out that gaining a better understanding of the mushroom immune system could help control diseases in economically important species in the future.

Schizophyllum commune

Beijen conducted his research on Schizophyllum commune, a mushroom that primarily lives on dead wood and can be found almost worldwide. This species grows quickly and can produce mushrooms in the lab within just 10 days. In addition, there are well-developed tools for genetic analysis and modification of this fungus. Because of these advantages, the fungus is used relatively often in research. It is also a common model organism in Robin Ohm’s Fungal Genomics group, where Beijen carried out his doctoral work.

Since so little is known, I focused on the low-hanging fruit.

Gene expression

To better understand the immune system of Schizophyllum commune, Beijen followed a series of steps. He began by growing the fungus in petri dishes, either on its own or alongside a pathogenic bacterium or fungus. He then collected samples from both the isolated fungus and the ones exposed to pathogens. Next, he examined gene expression in these samples: which genes are ‘switched on’ and being transcribed into RNA, the molecules that are ultimately translated into proteins. Because many genes are not ‘on’ all the time, and only get activated when the proteins they encode are needed.

Beijen: “We first looked at which genes were ‘on’ in Schizophyllum when it came into contact with pathogens, but ‘off’ when there was no contact. That led us to identify more than ten transcription factors, proteins that can switch a whole range of other genes on or off. We saw that the genes encoding these transcription factors were more actively transcribed into RNA when the fungus encountered pathogens. From these, we selected three genes that we knew we would be able to switch off.”

Switching off genes

Because that was exactly Beijen’s plan: to switch off the genes that code for the transcription factors and see which other genes were affected as a result. He used CRISPR-Cas9, a kind of molecular “DNA scissors” that can cut DNA at precise locations, to deactivate these specific genes.

“Using CRISPR-Cas9, we completely removed the genes from Schizophyllum's DNA. When we grew the genetically modified fungi alongside the same pathogens, we saw they were less capable of defending themselves,” Beijen indicates. “That shows that these transcription factors do indeed play a role in the mushroom's defense system.”

Offense and defense

Next, Beijen examined which genes were activated in unmodified Schizophyllum when it came into contact with pathogens, but not in the genetically modified version. This allowed him to identify genes that are likely switched on by the transcription factors.

It has been great to challenge myself with four years of fundamental research like this. But I now feel ready to work on a product or application

“These include genes that makes Schizophyllum secrete certain substances, allowing it to defend itself or even actively attack the invading pathogen. We also found genes known to pump toxic substances, like those produced by pathogens, out of the cells. So it makes sense that these particular genes are activated when the fungus comes into contact with pathogens.”

White button mushroom

Beijen says these are just the first steps in unraveling how the mushroom immune system works. “Since so little is known, I focused on the low-hanging fruit. Also, while Schizophyllum is sometimes eaten in Asia, it does not hold much economic value here. It would be great if we could apply this research to mushrooms like the white button mushroom.”

More applied

Before studying biology, Beijen was not particularly interested in mushrooms. But when he took the course Eukaryotic Microbiology during his studies, Professor Han Wösten was able to spark his interest in fungi.

Beijen will now spend another year as a postdoctoral researcher in Utrecht, hoping to find time to make a start with studying the white button mushroom. After that, he would like to continue in research, but with a more applied focus. “It has been great to challenge myself with four years of fundamental research like this. But I have a strong societal drive and now feel ready to work on a product or application.”