Fungi: shining a light on a hidden kingdom and its fascinating powers

Ceps, chanterelles, boletes, horns of plenty... When we think of fungi, we spontaneously imagine all kinds of forest species, capped and standing in the shade of a tree. Yet this familiar image captures only the tiniest part of the fungal world and a misleading one at that. It shows merely part of the anatomy of certain fungi, the bulk of which is hidden underground. "What we commonly call a fungus is in fact only a fleeting fruiting body," confirms Fabienne Malagnac, a lecturer at the Institute for Integrative Cell Biology (I2BC- Univ. Paris-Saclay/National Centre for Scientific Research, CNRS/French Alternative Energies and Atomic Energy Commission, CEA). "It's like only ever thinking about a plant's fruit while the rest remains buried."

First appearing on Earth over 900 million years ago, fungi have not always occupied their current position on the phylogenetic tree. For centuries, they were wrongly placed alongside plants, largely because of their immobility. "It took a very long time for fungi to be recognised as their own kingdom, clearly distinct from plants," explains the geneticist. The fungal kingdom was only officially established in 1969. Since then, it has become clear that these organisms are more closely related to animals than to plants. They even display some striking differences to plants, such as the way they generate energy. Fungi do not photosynthesise and produce no chlorophyll. "They use an extracorporeal digestion process, releasing enzymes to break down substrates present in their environment." Another difference lies in their cell walls, which do not contain cellulose as in plants, but chitin, the same molecule found in the shells of certain animals such as crustaceans.

Some 200,000 species of fungi have been described to date, including over 30,000 in France. Globally, however, the fungal kingdom may exceed 1.5 million species, according to the most widely cited estimates. It has to be said that this enormous diversity makes life difficult for the scientists who study them. Among fungi are the fly agaric with its iconic redcap and white spots, the moulds that thrive in bathroom grouting and the yeasts essential for making bread and beer. Based on their studies, scientists have successfully imposed some order on the fungal kingdom. "90% of so-called 'true fungi' species belong to a large group called dikarya, which includes basidiomycetes and ascomycetes, the two most-represented divisions," explains Pierre Grognet, also a lecturer at I2BC. Still, this classification does not completely solve the conundrum of the extreme diversity of organisms. "In fungi, there may be species in the same group that are as different from one another as humans are from fish, for example."

Although fungi long remained an underexplored field, it is now clear that they play vital ecological roles. Many species have the ability to form symbiotic associations with plants and trees. By attaching to plant roots, fungi draw water and nutrients from the soil and share them with their host. In return, the plant provides some of its sugar resources to the fungus. "This is called a mycorrhiza," notes the microbiologist. "In a temperate European forest, an estimated 80% of plants live in association with a fungus." These mycorrhizae are far more than just a system for sharing food, they create underground communication networks that are invaluable for plant survival. Fungi are also essential for recycling dead organic matter. "When a leaf or tree trunk falls in the forest, fungi are the only organisms that can produce the enzymes needed to begin breaking down the plant matter," emphasises Fabienne Malagnac. Without this fungal action, it would be impossible for insects and bacteria to attack the material. It would also be impossible to form humus, the forest layer produced by decomposing plant matter.

A saprophyte that has fascinated geneticists for eighty years

Fungi that feed on dead organic matter are known as saprophytes. One of these is the focus of research by Fabienne Malagnac and Pierre Grognet: Podospora anserina, a species that lives on the droppings of herbivores. "A horse's digestive efficiency is very poor," explains the scientist. "So it's organisms like Podospora anserina that finish the job." Like all filamentous species, P. anserina is composed of countless hyphae, fine filaments that form an interwoven network known as the mycelium. Through this structure, the fungus explores its environment, secretes enzymes and absorbs nutrients. This is also how it spreads, by producing spores. The P. anserina species is very familiar to scientists. 

Since the 1940s, it has served as a model organism, and not only because it's easy to cultivate. This ascomycete has a determined lifespan, unlike the majority of fungi which are capable of growing indefinitely. "There comes a time when it stops growing and the fungus dies. And this is genetically determined," confirms the lecturer. By modifying or inactivating certain genes in P. anserina, it is possible to create mutants that live either longer or shorter lives. This distinctive feature has led to countless studies that seek to understand the process of senescence - the decline in cellular functions - associated with ageing. 

Although it may not live long, P. anserina has been captivating geneticists for eighty years. In their I2BC laboratory, Fabienne Malagnac, Pierre Grognet and their teamare also generating fungal mutants, but with the aim of understanding a different mechanism. "We're studying a genome defence mechanism called RIP, short for repeat-induced point mutation, which targets repetitive DNA sequences," explains the scientist. Within every genome, there are DNA sequences that are capable of moving and duplicating themselves in several places. Studies have shown that these mobile elements, which are a major source of genomic instability, are abundant in animals and plants, but far less so in fungi such as P. anserina. "These organisms have found highly effective ways to inactivate them," highlights Pierre Grognet. "RIP is one such system: as soon as an element occurs more than once in the genome, RIP inactivates it by introducing mutations into its sequence." If genes are located within repetitive sequences, RIP irreversibly mutates them, silencing their expression. In addition to this action, the system triggers so-called epigenetic changes. These do not modify the DNA sequence - the succession of nucleotides - itself, but regulate its expression through the addition of biochemical marks.

RIP is interesting to geneticists because of its uniqueness: "it's the only known case where an organism deliberately modifies its genome and transmits the introduced mutations to its descendants." So, how does RIP work? That is the question the I2BC team is trying to answer. "We still don't know how the system recognises repetitive sequences," explains Fabienne Malagnac. However, laboratory studies show that one of the key factors behind the emergence of this RIP also controls the reproduction of the fungus. In fact, a mutant strain unable to perform RIP also turns out to be sterile. "Transcriptomic analyses reveal that this mutant contains many deregulated genes, and these genes all play vital roles in reproduction," notes Pierre Grognet. "With the team's collection of 400 P. anserina mutants, one of our strategies is to select promising candidates and test whether they are involved in the mechanism." 

Mechanisms such as RIP are essential for understanding fungal biology. This is because these organisms are not only capable of forming symbiotic associations with certain plants, but many are also sources of disease, especially for crops. Fungi demonstrate a remarkable ability to adapt and evolve to overcome obstacles to infection, as illustrated by the RIP system. "It plays a major role in genome modification which fungi use to develop strategies for bypassing challenges," concludes the lecturer.

Understanding the adaptive mechanisms of fungi

For over twenty years, Tatiana Giraud, research director at the Ecology, Society and Evolution laboratory (ESE - UnivParis-Saclay/French National Centre for Scientific Research, CNRS/AgroParisTech), has also been fascinated by fungi. "They are important ecologically, agronomically and economically," she explains. "They are also remarkable models for the sciences of ecology and evolution because they have specific life cycles that can be used to test hypotheses in the laboratory." In addition to working with the I2BC team to unlock the secrets of P. anserina, the scientist studies various plant diseases caused by pathogens. One of these pathogens is Microbotryum, a genus of basidiomycetes that infects the Caryophyllaceae family, which includes carnations and campions. Unlike many pathogens, Microbotryum does not kill its host. On the contrary, it even prolongs the plant's life to ensure the production of its own spores. To achieve this, the fungus aborts the plant's ovules and replaces its pollen with its fungal spores. "It completely sterilises the plant, which can no longer produce pollen or seeds," says the researcher. The infection is visible in the anthers, the part of the flower that normally contains pollen, which are covered with a black powder comprised of fungal spores. "The advantage is that the disease is very easy to spot. That's why it's called anther smut."

Although the disease has long been observed, advances in genetics are providing unprecedented insights into it. "When I first started working on this disease, we thought it was just a single species of Microbotryum infecting all Caryophyllaceae. But genetic studies revealed that each species within this genus is highly specialised, infecting only a single plant species," explains Tatiana Giraud. Even more intriguingly, the different species show very different genetic profiles. This means that, as it has evolved, the genus Microbotryum has adapted specifically to hundreds of different plants. This illustrates the very particular relationship fungi maintain with the plants they infect. "There really is a coevolution between fungi and plants," says the researcher. Plants don't simply give in to the invaders, they recognise certain molecules produced by fungal species and trigger defence strategies to block infection. All the fungus needs to do is to evolve so that its target does not recognise it. "It's often compared to the Red Queen in Alice in Wonderland who has to keep running just to stay in the same place because the landscape keeps moving. It's the same here: the fungus must evolve in response to its host, and the host evolves in return."

Using genome assemblies from different Microbotryum species, Tatiana Giraud and her team are investigating the pathogen's adaptation mechanisms. Their observations show that this basidiomycete employs a particular strategy to bypass plant defences. Instead of losing the genes that produce molecules that are recognised by the host and activate its defences, as many pathogens do, these genes evolve extremely rapidly. "We discovered that small proteins involved in attacking the plant evolve much faster than other regions of the genome." This mode of operation appears to be used by all Microbotryum species, regardless of the target species. For now, the role of all the molecules associated with these genes remains unclear, but several are likely to play key roles in the interaction with the host plant. These results are important for understanding the evolution of pathogenic fungi, the emergence of new species and their ability to adapt to new hosts. 

Tatiana Giraud and her team are also studying the evolution of Microbotryum sex chromosomes. In fungi, there are no males or females, instead individuals display mating types, the number of which varies by species and which determine whether individuals are compatible for reproduction. These mating types are controlled by specific chromosomes which, in Microbotryum, display an absence of recombination. In other words, no further genetic exchange occurs in the relevant regions between the two sex chromosomes. This phenomenon is similar to what happens with human X and Y chromosomes, leading to their divergence. "For a long time, it was thought that this divergence was linked to differences between males and females. But since that doesn't exist in fungi, it means other evolutionary explanations must be at play, and that completely changes the direction in which we need to look."

Secondary metabolites, key figures in invasions

While Microbotryum attacks plants of limited economic importance, other species are more destructive. According to estimates, 10% to 20% of the world's crops are lost every year to fungi. The common fungicides used to combat them represent one of agriculture's largest expenses, with dramatic consequences for human health and biodiversity loss. The fight against these invasions and the pathogens responsible for them is central to the research at the laboratory of Biology of fungal plant pathogens: from genomes to agro-ecosystems (BIOGER - Univ.  Paris-Saclay/French National Research Institute for Agriculture, Food and  Environment, INRAE). "We have several teams working on different aspects of phytopathology," explains Jean-Félix Dallery, a researcher at the laboratory. "In my team, we focus on the molecular level and, in particular, the roles of the molecules involved in infection from a mechanistic perspective. The aim is to understand what molecule X does and why it's important for infection." To infect plants, fungi produce a whole arsenal of molecules, also known as secondary metabolites, that help suppress plant defences, disperse the pathogen or divert nutrients from the host. 

For their research, Jean-Félix Dallery and his team also have their own model organism: Colletotrichum higginsianum.This ascomycete infects members of the Brassicaceae family, which includes thale cress (Arabidopsis thaliana) as well as cultivated species such as cabbage and radish. However, the pathogen has a rather unusual life cycle; it is hemibiotrophic. It has a first biotrophic life stage during which the plant remains alive, followed by a second necrotrophic stage in which it feeds on the dead tissue of its host, causing black spot disease or anthracnose. "Collelotrichum higginsianum is not the only hemibiotroph, but it is one of the few fungi whose biotrophy is limited to the first infected cell," notes the researcher. Necrotrophy therefore begins even before the pathogen has spread to the plant's adjacent cells. Together with his colleagues at BIOGER, Jean-Félix Dallery has studied the first phase of the cycle, gaining a greater understanding of it through laboratory experiments on A. thaliana plants.

Once on the surface of a leaf, the fungal spore germinates and produces a short hypha. Very quickly, this filament develops a structure called the appressorium. This exerts immense pressure until it breaks through the plant's cell walls and penetrates the plant. However, the fungus does not rely on mechanical action alone. At this stage, it has already begun producing the infamous secondary metabolites. It is now known that these molecules are produced in several stages by enzymes. For each metabolite, the enzymes are encoded by different genes, which are grouped together in a single region of the genome known as a biosynthetic gene cluster (BGC). "A transcriptomics study revealed that not all the genes are expressed at the same time or under the same conditions. Fourteen BGCs were identified that are specifically induced in the presence of the plant during the biotrophic stage." The aim now is to understand their role. By deleting two of these clusters in C. higginsianum, the scientists observed a very pronounced phenotype, either a complete failure to penetrate the plant or a strong delay in establishing infection.

The explanation lies in the plant's defences. When the plant detects a hostile presence, it mounts several responses, one of which is to form a shield around the appressorium to prevent penetration. After removing certain genes in the fungus, Jean-Félix Dallery and his team found that the plant produced many more shields. "This suggests that the gene is potentially useful in a process that prevents the plant from generating these defences." Conversely, other mutants appear quite capable of penetrating the plant despite the presence of more shields. To investigate further, the team has launched a project, the first step of which is to delete all fourteen BGCs and observe the effects on both pathogen and host. At the same time, they are continuing their work on the two identified BGCs and the associated metabolites, which they are attempting to produce, in order to determine their structure and role in infection. "It's important to understand the role of these genes, because it opens the way to developing control methods that could stop this fungus from penetrating plants altogether," emphasises Jean-Félix Dallery.

However, developing control methods is not the only goal. Beyond their natural bioactivity, fungal secondary metabolites are also a promising avenue for discovering molecules that could be potentially useful for society. "Penicillin is a secondary metabolite synthesised by moulds and is anantibiotic that has saved millions of lives," reflects the researcher. All the more reason then, to continue shining a light on the remarkable, yet still too often overlooked, capacities of fungi.

References :

• Fabienne Malagnac et Philippe Silar, Les champignons redécouverts, Belin, 2013.
• Carlier et al., Loss of EZH2-like or SU(VAR)3–9-like proteins causes simultaneous perturbations in H3K27and H3K9 tri-methylation and associated developmentaldefects in the fungus P. anserina, Epigenetics & Chromatin, 2021.
• Lucotte et al., Repeated loss of function at HD mating-type genes and of recombination in anther-smutfungi, Nature communications, 2025.
• Geistodt-Kiener et al., Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites, Metabolic Engineering, 2023.