Evolution of Plant - Microbe Interactions

The AM symbiosis evolved with first land plants 450 million years ago. Most land plants, vascular and non-vascular, are able to form this symbiosis with AM fungi. However, a few lineages - such as the Brassicaceae or the mosses - have lost this ability. 

Evolution of the symbiotic gene regulatory networks and metabolism

Among these genes, many transcription factors have been characterized, including GRAS (NSP1, NSP2, RAM1, RAD1), AP2 (WRI5s, CBX1) and CYCLOPS/IPD3. Altogether these TFs are thought to regulate the spatial and temporal expression of downstream target genes. Interactions between these TFs have been documented suggesting the occurrence of hetero-complexes. 

The symbiotic TFs regulate the expression of a large number of enzymes have been identified. Other genes coding for enzymes putatively involved in the production of secondary metabolites are also induced in these cells. However, the role played by these enzymes and the metabolites they generate remain unknown. 

In this context, we are addressing the following questions:

                What is the ancestral function of the symbiotic TFs?

                How did they get recruited into symbiotic gene regulatory networks?  

                Does an arbuscule-specific secondary metabolism exist?

                What is the function of these metabolites?

Characterizing novel "fungal" symbiotic mechanisms 

Studying the genomes and secretomes of AM fungi we have discovered multiple secreted peptides and small molecules. Some of them seems to be accumulated during AMS with multiple host species, suggesting a generalized function. 

We are now trying to understand the function of these small peptides and molecules during AMS by addressing the following questions:

                Do host plants perceive fungal-derived molecules and peptides?

                Does the presence of these molecules affect AMS ?                     

The nitrogen-fixing Root-Nodule (RN) symbiosis is formed between nitrogen-fixing bacteria and species belonging to four closely related orders of angiosperms (Fabales, Fagales, Cucurbitales and Rosales – the NFN clade). 

In this association, the bacterial symbiont colonizes either intra or intercellularly the plant root, a colonization mediated by the host plant. In parallel, division of specific plant cell tissues lead to the formation of a new organ, the root nodule. Nitrogen fixation takes place within the colonized nodule.

Loss of the RN symbiosis

Within the four orders of the NFN clade, only few genera restricted to ten out of thirty families contains RN-forming species. For decades, the favored hypothesis to explain this scattered distribution was that RN symbiosis evolved independently in a convergent manner multiple times (up to sixteen). Because of its restriction to the NFN-clade it was proposed that a “predisposition” to evolve RN symbiosis occurred before the radiation of the four orders (Soltis et al. 1995). 

Recently we, and others, have demonstrated that the RN symbiosis was lost multiple times in the four orders of the NFN-clade (Griesmann et al. 2018; van Velzen et al. 2018 – Figure 2). These losses left “genomic evidence” with the convergent losses of at least two genes (NIN and RPG) known to be essential for intracellular infection by nitrogen-fixing bacteria. This finding suggests that, rather than a predisposition and multiple gains, the most likely hypothesis to explain the current distribution of the RN symbiosis is a single gain at the base of the NFN-clade followed by multiple losses. 

To test this hypothesis in depth, analizing additional non-RN-forming species within the NFN-clade is required. In that context, we partcipate in the 10KP initiative that aims at sequencing the genomes of 10 000 plant species (https://db.cngb.org/10kp/). By sequencing genomes covering the entire diveristy of the NFN-clade we should be able to support or reject the "single gain - multiple losses" hypothesis. 

Origin of the RN symbiosis

If the RN symbiosis evolved only once, it can be assumed that genetic modifications that occurred at the base of the NFN-clade were essential for this process. As a partner of the “Enabling Nutrient Symbiosis in Agriculture” project (www.ensa.ac.uk) funded by Bill & Melinda Gates Agricultural Innovations, we are aiming at discovering these modification. 

Following this initial symbiosis, multiple other intracellular symbioses have evolved in plants as diverse as orchids, Ericaceae such as cranberry, legumes or the Jungermanniales, a group of bryophytes. These symbioses provide numerous benefits, improving plant nutrient acquisition and fitness. 

Despite their absolute importance in terrestrial ecosystems, the molecular mechanisms underlying the origin and subsequent evolution of intracellular symbioses in plants remain poorly understood. As a first insight, we have found that a single genetic pathway may regulate all these diverse intracellular symbioses (Radharkishnan et al. Nature Plants 2020) using comparative phylogenomics.

In a first objective, we will use CRISPR/Cas9 in the bryophyte Marchantia paleacea to test the conservation across land plants of symbiotic mechanisms known in angiosperms. Then, we will decipher how these mechanisms evolved by comparing land plants with their closest algal relatives. In a second objective, we will conduct transcriptomics coupled with genetic manipulations of most known intracellular symbioses in plants. This will allow determining how the ability to host intracellularly microbial symbionts recruited in the environment evolved repeatedly in land plants and how functional specificity evolved in these different symbioses. Lastly, we will investigate why the evolution of intracellular symbioses is constrained to a unique genetic pathway.

Lichens are formed by photobionts (green algae or cyanobacteria) and mycobionts (mostly ascomycete fungi) in associations where the fungus surrounds the alga. Multiple algal species from the chlorophytes are able to form this association, however the molecular mechanisms that have allowed this symbiosis to evolve remain unknown (Puginier et al. 2022). We aim at identifying these mechanisms to understand how lichenization evolved. 

Marchantia has been found colonized by a large number of endophytes in natural ecosystems (Nelson et al. 2018) and has been shown to be suceptible host for angiosperm pathogens (Carella et al. 2018, Gimenez-Ibanez et al. 2019). A number of mechanisms involved in Marchantia immunity has been described based on direct comparisons with our current knowledge in angiosperms. By contrast, Marchantia-specific resistance mechanisms have been completely overlooked and forward approaches completely ignored.

Model species: Marchantia polymorpha

Approaches: Population & Quantitative Genetics - Metabolomics - Reverse genetics 

Developping pathosystems in Marchantia

As a first step to identify novel resistance mechanisms in Marchantia we are developping new pathosystems. For this purpose, we are testing fungi, oomycetes and bacteria known to infect angiosperms or isolated from Marchantia. 

We are currently determining whether compatible interactions are governed by the same infection-mechanisms on the side of the microbial partner. 

Identification of novel resistance mechanisms by GWAS

We have gathered a unique collection of hundred Marchantia polymorpha accessions and sequenced them as part of the 10KP initiative. With this resource we are now addressing the following question:

                    What are the loci linked to resistance to pathogens in Marchantia?

Interactions between organisms affect gene and ecosystem diversity. Given that most species are constantly challenged by both mutualistic and parasitic microorganisms, we propose that mutualism and parasitism could influence each other. Here, using plants as a model we will combine our complementary expertise to test the hypothesis that parasitism and mutualism impact the evolution of each other. To do this, we first propose to identify genes involved in parasitism or mutualism using genetic approaches (GWAS, selection scans) and to compare their evolution within species of two deeply divergent clades of land plants, angiosperms and liverworts, including in species which have lost mutualism (e.g. Arabidopsis thaliana and Marchantia polymorpha). Then, through phylogenomic approaches across the entire embryophyte phylogeny, we will finely describe the evolution of these genes and infer selective processes to understand how selection can manage the interplay between mutualism and parasitism.