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To establish compatible interactions with their hosts, plant pathogenic fungi secrete an array of molecules to modulate host physiology and overcome host immunity. These molecules, known as effectors, include small secreted proteins but also secondary metabolites (SMs) and small interfering RNAs (siRNAs).

The major goals of our team are to understand the mode of action and plant targets of fungal effectors and the mechanisms of communication between fungi and plants. For this work we are using two fungal models that have contrasting trophic behaviors and host ranges, namely Botrytis cinerea, a necrotrophic and polyphagous plant pathogen, and Colletotrichum higginsianum, a hemibiotrophic pathogen of Arabidopsis thaliana and other brassicaceae. Rich genomic/transcriptomic data and genetic tools are available for both fungi, as well as abundant mutants and transgenic lines of A. thaliana, a host that is shared by both pathogens. 

We are currently interested in the following scientific questions:

• What is the role of fungal secondary metabolites (SMs) as effectors in the infection process ? What are their modes of action and plant targets ?
• What are the genomic determinants of host specialization? Could small RNAs be involved ?
• What are the mechanisms of trans-kingdom communication between fungi and plants ? In particular, what is the role of fungal extracellular vesicles (EVs) in the infection process ?
• What are the plant signals that reprogram fungal effector gene expression during infection ? 

Botrytis cinerea

 

The ascomycete Botrytis cinerea is responsible for grey mould disease on grapevine (Adrian et al. 2024) and other crops important for French agriculture including tomatoes and strawberries. As a necrotrophic fungus, B. cinerea induces rapid death of the host cells after penetration of the plant tissues (reviewed in Hahn, Viaud & van Kan, 2014). During this process, the fungus secretes several toxic compounds, including the secondary metabolites botrydial and botcinic acid, that play a role in killing host cells (Dalmais et al., 2011).

Our functional studies are mainly conducted with the international reference strain B05.10 whose genome has been fully sequenced and assembled (Amselem et al., 2011 ; van Kan et al., 2017). In order to study host adaptation, we have additionally selected two field strains, isolated from tomato (Sl3) and grapevine (Vv3) and have obtained their full genomes (Simon et al., 2022; see below). These genomes and annotated genes are available at NCBI and on our bioinfobioger web portal. Annotation of B. cinerea transposable elements can be accessed from Data INRAE. Reverse genetics (targeted mutagenesis) and classical genetics (sexual crosses, see picture above) are used to investigate the molecular bases of the fungal-plant interactions. Targeted K.O. mutants generated in our team or published by the Botrytis community can be assessed on a dedicated page of bioinfobioger.

Colletotrichum genus

 

The genus Colletotrichum comprises over 200 recognized species, including many that provoke devastating anthracnose diseases on monocot and dicot crops world-wide (Crouch et al 2014), as well as some endophytic species (Hiruma et al 2016, Hacquard et al 2016). C. higginsianum causes major economic losses on many cultivated Brassicaceae but also infects Arabidopsis thaliana, providing a model pathosystem in which both partners can be genetically manipulated. Like most members of the genus, C. higginsianum is a hemibiotroph, with an initial symptomless biotrophic phase when the fungus grows inside living plant cells, followed by a destructive necrotrophic phase when it kills host cells ahead of infection. Spores germinate on the plant surface to produce a darkly pigmented cell called an appressorium, which breaks through the plant cuticle and cell wall using a combination of mechanical force and enzymes. Bulbous biotrophic hyphae then invade living epidermal cells surrounded by the host plasma membrane.

Re-sequencing the genome of C. higginsianum reference strain IMI 349063 using single molecule real-time sequencing produced a gapless assembly of all twelve chromosomes (Dallery et al., 2017). The genome assembly and gene annotations are available at NCBI and on our bioinfobioger web portal. Annotation of C. higginsianum transposable elements can be accessed from URGI. Likewise, we recently sequenced the complete genome of C. destructivum reference strain LARS 709, a sister species to C. higginsianum, the causal agent of Medicago anthracnose (Lapalu et al. 2024, NCBI).

Fungal secondary metabolites

Beyond the small number of SMs isolated from each ascomycete fungal species, genome sequencing has revealed that they harbour a large number and diversity of Biosynthetic Gene Clusters (BGCs). About 40 BGCs could be identified from the genome of B. cinerea (Collado and Viaud, 2016), while 78 BGCs were found in C. higginsianum genome (Dallery et al. 2017). In collaboration with the I2BC (Université Paris-Saclay), a computational tool was developed to efficiently identify the genes encoding different families of fungal SM key enzymes (Oliveira et al. 2023).

Reverse genetics approaches have allowed us to link several BGCs to the corresponding SMs. In B. cinerea, we characterized the BGCs responsible for synthesis of two phytotoxins, namely the sesquiterpene botrydial (Porquier et al., 2016) and the polyketide botcinic acid that both act as virulence factors in B. cinerea (Dalmais et al., 2011, Porquier et al. 2019). These two toxins are not exclusively produced during the infection of the plant and are likely also involved in the interactions with other microorganisms (Vignati et al. 2020). Other SMs with antibacterial activities are investigated through a collaboration with the I2BC (OI Microbes-GS LSH project ‘Filantrope’ 2023-2024).

Which fungal biosynthetic gene clusters are activated during infection? Are they contributing to the success of infection?

Molecular plant–fungal interaction studies have mainly focused on small secreted protein effectors. However, accumulating evidence shows that numerous fungal SMs are produced at all stages of plant colonization (Kelloniemi et al. 2015, Dallery et al. 2017). While some of them act as phytotoxins (like botcinic acid and botrydial mentioned above), some others are sensu stricto effectors that manipulate plant immunity (See the blog post « Effecteurs : subterfuge et manipulation”). Previous work revealed that a subset of 14 BGCs is specifically expressed by C. higginsianum during the early biotrophic phase of plant infection, i.e. when host cells are still alive, suggesting the chemical products are not primarily phytotoxins but may have effector-like activities.

The roles of several BGCs that are upregulated during the infection process of B. cinerea and C. higginsianum are being investigated (Aude Geistodt-Kiener’s PhD 2019-2023, Axelle Deroubaix’s PhD 2023-2026). For this purpose, molecular genetics tools were developed for the deletion of these large, multi-gene loci together with subsequent elimination of the marker genes. This tool enabled the reuse of marker genes to target other BGCs and generate strains with multiple BGCs deleted and allowed us to show that loss of several biotrophy-specific BGCs drastically reduced pathogenicity (Young Scientist ANR project ‘ShySM’ 2024-2028). However, these metabolites that are specifically produced in planta have yet to be characterized and their plant molecular targets are completely unknown.

What are the fungal secondary metabolites induced by the host(s) plant(s)?

Though some BGCs were shown to be required for full infection, the nature of the SMs produced in planta remains unknown. A first approach builds on the knowledge of the regulatory networks of secondary metabolism (ANR Projects ‘FunApp’ 2013-2018, ‘HerbiFun’ 2017-2021). For example, botrydial and botcinic acid biosynthesis are regulated by specific transcription factors (Porquier et al., 2016, 2019) and global regulators including the Velvet complex (Schumacher et al. 2013; 2015). In addition, we investigated the role of nucleosome positioning (Clairet et al., 2021) and those of histone modifiers in the expression of BGCs. Our results indicated that histone methylation plays a role in the regulation of SM production in both C. higginsianum (Dallery et al., 2019a, 2019b) and B. cinerea. Nevertheless, genetic manipulation of chromatin modifiers activated in axenic cultures the biosynthesis of very few of the infection-specific SMs. As an alternative, we developed a more targeted approach by transferring the BGCs of interest (i.e. ) into a heterologous host, namely Saccharomyces cerevisiae. As a proof of concept, we designed a set of polycistronic backbone plasmids with auto-inducible promoters and applied it to the putative Colletochlorin BGC. This tool allowed to assign for the first time the Colletochlorins to their BGC (Geistodt-Kiener et al. 2023, SPE project ‘UNCHAIN’). This approach is now used to identify other biotrophy related SMs (ANR project ‘ShySM’ 2024-2028) and a new class a fungal SMs that remains unexplored, the dikaritins (Laure Bardiot’s PhD and ANR project ‘FRiPPon’ 2024-2028 in collaboration with the Museum National d’Histoire Naturelle and the Centre Mondial de l’Innovation-Roullier).

What are their functions and plant targets?

The purification of several families of SMs allowed us to investigate their bioactivities. Using chemical genetics screens, we found that Higginsianin B was a potent inhibitor of jasmonate-mediated plant defences. With a consortium of 5 European labs including BIOGER, ICSN (CNRS, Gif-sur-Yvette), Leibniz IPB (Halle, Germany), MPIPZ (Cologne, Germany) and the University of Athens (Greece), we were able to show Higginsianin B inhibits two of the three proteolytic activities of the 26S proteasome (Dallery et al., 2020). In the frame of the ‘PROLIFIC’ project funded by the LabEx/Graduate School of Research ‘Saclay Plant Science’ and the Plant Health and Environment division of INRAE, we are now implementing untargeted methods for the identification of plant molecular targets of fungal SMs in collaboration with IJPB (Versailles) and PAPPSO (Le Moulon).

The bioactivities of purified SMs (either from fungi or plants) are also investigated using a range of assays including antifungal (nephelometry) and antibacterial activities developed in the frame of the SPS project ‘MetSpe’.

Genomic determinants of host specialization in the polyphagous pathogen Botrytis cinerea

While B. cinerea is notorious for having a broad host range and is considered a generalist, genomic population studies at BIOGER revealed that this species actually corresponds to multiple co-existing populations with some of them showing a certain level of host specialization on tomato or grape (Mercier et al. 2019). These two pathosystems provide the opportunity to investigate the molecular determinants of host specialization. By characterizing single-nucleotide polymorphisms in 32 representative isolates, genes with footprints of positive selection and/or divergent selection were identified. In particular, the data suggested that cellulases, pectinases, and enzymes involved in oxidative stress responses may contribute to specialization on tomato (Mercier et al. 2021). To access additional genomic determinants such as Transposable Elements (TEs) and TE-derived small RNAs, the full genome assemblies of the strains Sl3 and Vv3 representing populations specialized to tomato and grapevine, respectively was obtained through PacBio sequencing. This revealed that the main population specialized on grapevine harbors a specific set of accessory chromosomes, TEs and TE-derived small RNAs (Simon et al., 2022; See repertoires of TEs in the figure). We are now investigating the role of these genomic elements in the interaction between B. cinerea and grapevine by functional approaches in collaboration with the Agroecology unit in Dijon (ANR Project ‘VITAE’).

The role of extracellular vesicles in fungal-plant communication

Extracellular vesicles (EVs) are small, membranous particles that are released by cells of all three domains of life and which vary in their size, cargo, biogenesis pathways and functions. Many recent studies have highlighted the importance of EVs in intercellular communication, including cross-kingdom communication between fungal pathogens and their plant hosts. In the ERA-CAPS project ‘Exosomes’ (2018-2022, ANR-17-CAPS-0004) we studied the role of fungal EVs in the Colletotrichum-Arabidopsis pathosystem, in collaboration with scientists at Indiana University (USA), the Danforth Plant Science Center (USA) and Copenhagen University (Denmark). We found that Colletotrichum produces EVs at different infection stages, including at the interface between biotrophic hyphae and living plant cells (Figure, a). Working with Brian Rutter and Roger Innes (Indiana University), EVs were isolated from cultured C. higginsianum mycelia using density-gradient ultracentrifugation. Proteomic analysis of these EVs identified several hundred proteins, including some involved in vesicle trafficking, fungal cell wall remodeling and secondary metabolite biosynthesis. Transgenic fungal strains expressing fluorescent-tagged EV marker proteins were generated and used to localize the proteins during infection by confocal microscopy (See Figure, b). Part of this work was published in the Journal of Extracellular Vesicles (Rutter, Chu et al. 2022).

Many questions remain about fungal EVs and their role in infection: How are they produced? What molecules do they transport during plant infection? Does this cargo change at different stages of infection? How do EVs cross the fungal cell wall and how are they taken up by plant cells? The recent recruitment of Julien Pernier (2024) as a permanent researcher in our team will allow us to address some of these questions to obtain a better understanding of the role EVs in fungal pathogenicity.

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