“Fungi are Essential for the Survival of the Planet”
Fungi are key actors in biodiversity. We are gradually discovering the fundamental role they play in the proper operation of ecosystems. Specialist in this field, Francis Martin, a microbiologist at INRA, has discovered how these organisms dialogue with trees
Photo © Jean-Christophe Verhaegen / AFP Photo
For almost forty years, Francis Martin has devoted his life to studying the relationship between fungi and trees. More specifically, that of mycorrhiza, the beneficial symbiosis that exists between plants and fungi, and which facilitates the exchange of nutrients. More than just swapping sugars and minerals, a real dialogue exists between the fungus and its host. This dialogue relies on complex mechanisms, that Francis Martin and his team are attempting to decode. Already with some interesting results to their name, as they have revealed the existence of a dozen communication proteins essential in getting the symbiosis going. Fungi associated with trees form vast underground networks of filaments and in the autumn produce the remarkable fructifications that brighten up the forest floor. They are like a guild of architects essential for the construction of the forest. Delving into their study means discovering a fascinating world, of which the public is often unaware.
La Recherche • How many species of fungus can we find in our temperate forests?
Francis Martin • It’s highly variable. Year 2018 was an especially dry year, so there were few species of mushroom on the forest floor. The year before, on the other hand, was exceptional. Some days, walking through the Vosges forests, I photographed the fructifications (the reproductive organ of fungi) of more than one hundred species. These coloured structures, often called “mushrooms” were produced abundantly, following a wave of spring heat followed by heavy summer rain. For the first time in the Lorraine region, I even gathered specimens of Caesar’s mushroom, a heat-loving fungus that usually grows in the south of France. The fructifications however only reveal a fraction of the fungal diversity of forest floors. We realised this when we sequenced DNA extracted from the soil of deciduous or coniferous forests in the Vosges and Morvan regions, where we identified over 1,000 species of mycorrhizal and decayer fungi. The symbiosis between trees and fungi is called mycorrhiza (from the Greek myco “fungus” and rhiza “root”). There are two types in a forest: ectomycorrhiza (*) and endomycorrhiza or arbuscular mycorrhizal fungi.
How do you explain the difference between the number of observable fungi and what we detect when we analyse the soil?
This remains quite a mystery. By observation, we know the main factors that trigger the growth of fungal fructifications, like ceps and other mushrooms: summer rain and storms followed by lovely sunny days, but also a few colder nights. In the underground fungal networks, these conditions trigger poorly known phenomena which lead to the formation of a microscopic embryo; growing rapidly in just a few days, it leads to the characteristic mushroom with its foot and spore carrying cap. In mycorrhizal fungi, it may well be the plant that controls this complex process. The autumn fructification period coincides in many mycorrhizal fungi species (chanterelle, boletus, russula, etc.), storing amino acids and sugars that leave the aging leaves to gather in the trees’ stems and roots. This massive intake of sugar by the roots could feed the associated fungi and provide the additional energy and nutrients necessary to build the fructifications - one hectare of forest can host up to 400 kg of ceps. This fructification can take a variety of forms and periods. Some fungi fructify every year, others every ten years. Some very rare species only appear in forests that are centuries old. This is the case for the noble Polyporus, which only colonises very old conifers in the high-altitude forests of Oregon, in the United States.
Is fructification not essential for the survival of the fungus?
Fungi can survive for years underground in a vegetative (or clonal) state; their filaments divide and some form vast underground colonies several hundreds of square metres in size. The largest fungus in the world, which lives in the Malheur National Forest in east Oregon, is probably 2,500 years old. It is a single fungus that covers almost 9 km², a parasitic armillaria that is rampant underground and sends its blackish filaments from tree to tree to devour them. On the Swiss Jurassic plains and in the forest of Donon in the Vosges, I was lucky enough to study a few of these gigantic underground organisms. We encountered a yellow boletus that was over 150 years old. In fact, there are roughly two major groups of mycorrhizal fungi: those that prefer vegetative reproduction, such as the Suillus luteus, and those, like the Laccaria bicolor which undergo frenetic sexual activity and produce hundreds of fructifications over a few square metres and billions of spores.
What explains this difference in reproductive strategy?
There are several hypotheses. Those species which engage in sexual reproduction are often considered as pioneers or specialists in disturbed environments; intense mixing of their genetic heritage through sexual reproduction could promote mutations and the appearance of new combinations of genes, enabling them to adapt to new environments. On the contrary, species that prefer vegetative reproduction are supposed to be characteristic of ancient, very stable, mature forests. However, in the surveys we conducted in several mature, undisturbed beech and pine forests, we identified thousands of ephemeral Laccaria and a few, venerable hundred-year old boletes that coexisted. The mystery remains!
Mycorrhizae are at the heart of your research. Where does this interest stem from?
That’s a long story! In the 1980s, Professor Pierre Gadal, a specialist in plant physiology at Nancy university, met François Le Tacon, a microbiologist at the INRA forest research centre. With a series of experiments, the latter showed that the Austrian black pine tolerates limey soils in natural conditions only in the presence of soil fungi (1). Otherwise, its leaves become discoloured by chlorosis linked to disturbed nitrogen nutrition. Inoculating young trees with ectomycorrhizal fungi restores the normal nitrogen metabolism and tolerance to lime. To understand the physiological mechanisms at play, my two mentors suggested that I write my thesis on the assimilation of mineral nitrogen by the black pine, mycorrhized and non-mycorrhized. After I was recruited by the INRA, I continued on this issue. I drew up metabolic maps of several species of ectomycorrhizal fungi using techniques such as in vivo nuclear magnetic resonance imaging. Then, thanks to the development of molecular biology techniques at the end of the 1990s, we started an ambitious research programme to identify and characterise the signals and the genes in the tree and the fungus that were essential for the developments of mycorrhizal symbiosis - the symbiotic toolbox so to speak.
How are you studying symbiosis today?
We are attempting to explain the ecological characteristics of trees and their associated fungi by decoding their genomes and analysing the expression of their genes. The publication in 2006 in Science of the first tree genome (poplar), was quite a revolution! Jerry Tuskan, this project’s coordinator at the Joint Genome Institute in the United States, immediately asked me if I knew of any fungi associated with poplars that would be interesting to sequence. The aim was to study the microbiome of this tree even before the term was invented. As a priority, I suggested sequencing an ectomycorrhizal fungus, Laccaria bicolor, one endomycorrhizal fungus, Glomus intraradices and a pathogenic fungus, poplar rust. In 2008 in Nature, we published the genome of the first symbiotic fungus. For a dozen years, we have had the complete poplar genome (over 45,000 genes) and a complete Laccaria bicolor genome (about 20,000 genes). We can accurately follow the evolution in the expression of various sets of genes during the development of mycorrhizal symbiosis using transcriptomics tools (**), working on this poplar-Laccaria bicolor pair.
What did the study on this pair teach you about how mycorrhizae function?
Often, mycorrhization is reduced to an exchange of nutrients - sugars for minerals - between the fungus and the tree. But it’s not that simple. Whatever fungi or host trees are involved in the symbiotic interaction, we observe the same phenomena, i.e. induction and repression of several hundreds of genes in both partners. Of these “symbiotic” genes, dozens are indeed involved in exchanging minerals and sugars, membrane transporters or assimilating enzymes. The interaction also causes the induction of dozens of genes involved in cell development, hormonal metabolism and signalling pathways. We are studying genes that are only expressed during the interaction as a priority. We have discovered that they are involved in the molecular dialogue that controls the start-up and operation of the symbiosis, and that some of these communication proteins (that we call “effectors”) are able to modify the functioning of the host plant. These little proteins (less than 250 amino acids) are secreted at the symbiotic interface by hyphal threads that colonise the root. This type of effector is known in bacteria and parasitic fungi. Some of these proteins then rapidly penetrate the cells of the host plant, migrate to their nucleus and interfere with the regulatory proteins that command the expression of defence genes. In other words, symbiotic fungi, like parasitic fungi, take control of the main communication systems and send their “missiles” to interfere with the host’s system and manipulate it. Laccaria bicolor for example, is able to fool the poplar’s immune system to invade the root and take up residence (see inset).
Is the poplar easily fooled?
Not at all! This is one of most exciting research projects. With our American and Australian colleagues, we wondered if the plant was not also using effectors to control the mycorrhizal fungus. We scanned the poplar genome looking for genes coding for little secreted proteins, whose expression was strongly stimulated during the symbiotic interaction. More than a dozen proteins met the criteria. By synthesizing them and monitoring their progression using a fluorescent marker, we tested whether these plant proteins entered the cells of Laccaria. Currently, we are focusing on one of them, PtSSP1, which migrates rapidly to the fungal nucleus and interacts there with a regulatory protein. Unravelling this complex molecular dialogue between tree and fungus will probably take us a few years.
At the same time, you are continuing to study the evolution of forest fungi using comparative genomics, winning the La Recherche prize in 2016. Have you confirmed the scenario put forward at the time?
The article that won suggested an evolutionary scenario for ectomycorrhizal symbiotic fungi. We showed that this type of fungus was descended from fungi that rot wood and forest-litter (leaves and organic waste on the forest floor) and had gradually lost its ability to break down wood lignocellulose to establish symbiosis with the trees. At the same time, we discovered that the communication proteins were present in the genomes of all mycorrhizal fungi. We proposed this scenario by studying a dozen symbiotic fungi genomes – quite a small sample when you know that there are thousands of mycorrhizal fungi. Thanks to huge progress in high-speed sequencing, we now have genomes for 1,500 fungi. As part of the project called “1,000 fungal genomes”, we have sequenced over one hundred mycorrhizal fungi: we can now compare them to over 300 wood and litter-rotting fungi that share the same ecosystems – temperate and boreal forests. This analysis required a considerable collective effort and months of computer calculations, but we did confirm our hypothesis: forest rotting fungi are indeed the ancestors of ectomycorrhizal symbiotic fungi. As the latter have lost their ability to decompose organic matter in the litter and lignocellulose, they are totally dependent on their host plant to provide them with sugars. Even more surprisingly, this evolutionary process repeated one hundred times over in most higher fungi families. Remarkable evolutionary convergence.
What work remains to be done in his field?
Studies on the evolution of forest fungi, decayers and symbiotic, rely on several hundreds of genomes. There are over 20,000 species of mycorrhizal fungi; we would like to verify this evolutionary scenario on a larger sample of species from more varied ecosystems. Large groups of mycorrhizal fungi, such as Cortinarius and Chanterelles, are abundant in our forests, where they play a major ecological role. The problem: they are very difficult to cultivate, or the genome DNA essential for sequencing is very difficult to obtain. In the absence of cultures, we are trying to extract DNA from fructifications collected in the forest in the autumn. In 2017, we launched an international participative science programme, in cooperation with French, American and Australian amateur mycological associations. We hope that they will be able to provide us with samples of these rare and recalcitrant species that interest us.
What would forests be like without fungi?
Our readers need to realise that fungi are essential to the survival of our planet. Often invisible, they play a fundamental role in the proper functioning of land and possibly marine ecosystems. It is crucial to better understand the multiple ecological services they provide and study the impact of human activity and climate change on them. It is a question of the survival of the human species. Humans enable plants to flourish – even though some are serious predators of cultivated plants. Decaying fungi for example, have been recycling carbon for almost 300 million years. Without these cleaners of the forests and fields, which transform gigatonnes of dead wood and leaves reinjected into the ecosystem, we would be submerged. Symbiotic fungi are just as essential, as they help plants to feed and protect them from attack by pathogens. Without fungi, our forests would look nothing like they do today.
(1) A. J. Clément et al., Oecolog. Plantar., 12, 111, 1977. (*) Ectomycorrhiza is a type of symbiosis that appeared 180 million years ago with the ancestors of current pine trees. It only concerns about 10% of plants, mainly tree species in temperate forests, but they cover more than one third of the planet. The most common mycorrhiza, endomycorrhiza or arbuscular mycorrhiza, concern 80% of plants. (**) Transcriptomics is the study of all the RNA messengers that are produced when a genome is transcribed.
THE LITTLE PROTEIN THAT FOOLS THE PLANT DEFENCE SYSTEM
How does Laccaria bicolor manage to fool the tree’s defence system? Using a small protein called MISSP7 (Mycorrhiza-induced Small Secreted Protein 7). It is released into the symbiotic interface, absorbed by the plant and penetrates its nuclei in a matter of minutes. It then interacts with a regulating protein, which is the co-receptor for the plant’s alert system, methyl jasmonate. When a foreign body enters the cells of a plant leaf or root, it destroys some of the cells, which contain lipid compounds in their membranes which produce methyl jasmonate. This volatile compound binds to a receptor protein and serves to protect the whole plant from attack. In the absence of foreign microbes, this receptor acts as an interrupter, blocking the coordindated expression of around one hundred genes coding for defence proteins. In the presence of methyl jasmonate, the regulator protein is rapidly decayed and the expression of defence genes is triggered. When the mycorrhized root is colonised by Laccaria, the proteins MISSP7 binds to the methyl jasmonate coreceptor and prevents it from decaying. This interaction prevents the defence mechanisms from starting up, enabling the symbiotic fungus to colonise the roots.
> INTERVIEWEE
Francis Martin
Microbiologist at INRA, he has discovered how mushrooms, key players in biodiversity, interact with trees.
1981 • Francis Martin joined the forestry centre at the Institut National de la Recherche Agronomique (INRA), in Nancy. 1987 • Became the youngest director of research at INRA. 2001-2009 • Director of the Tree/microorganism interactions research unit. 2012 • Winner of the INRA Lauriers d'excellence for his work on tree/microorganism interactions and fungal genomics. SINCE 2012 • He is director of the Tree Laboratory on the biology of trees and forest ecosystems.
