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Research

Botrytis cinerea is responsible for grey mould disease on grapevine and other crops important for French agriculture including tomatoes and strawberries. B. cinerea is a necrotroph and penetration is usually accompanied by rapid death of the host cells (reviewed in Hahn, Viaud & van Kan, 2014). During this process, B. cinerea secretes several toxic compounds, including the secondary metabolites botrydial and botcinic acid, that play a role in killing host cells (Dalmais et al., 2011). The genomes of the reference strains B05.10 and T4 have been fully sequenced. Up-to-date gene annotations and list of inactivated genes are available at botportal and botmutant interfaces. Annotations of transposable elements in the B05.10 genome are available under the DOI 10.15454/TFYH9N.

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The genus Colletotrichum comprises over 190 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 Genbank. Annotation of C. higginsianum transposable elements can be accessed from URGI.

 

Which fungal secondary metabolites (SMs) are produced during infection ?

What are their functions and plant targets ?

How are they regulated during the infection process ?

In recent years, reverse genetics approaches have allowed us to link several secondary metabolism Biosynthetic Gene Clusters (BGCs) to specific SMs. In B. cinerea, we characterized the BCGs 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). These two toxins are not exclusively produced during the infection of the plant and are likely also involved in the interactions with other microorganisms (Collaboration with F. Pieckenstain, University of Chascomus , Argentina). Strikingly, B. cinerea and a few other phytopathogenic fungi are able to produce the well-known plant hormone Abscisic Acid (ABA). While in plants ABA is known to be derived from carotenoids, fungi produce ABA by a very different pathway through the cyclisation of farnesyl diphosphate followed by several oxidation steps. In collaboration with the group of I.G. Collado (University of Cadiz, Spain), we identified the gene encoding the sesquiterpene cyclase (STC) which acts as the key enzyme for ABA production in fungi (Izquierdo-Bueno et al. 2018) but the enigmatic role of ABA in fungal/plant interactions remains to be elucidated.

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This work was scooped by Life Sciences UPSaclay.

Sequencing the genomes of ascomycete fungi has revealed that they harbour a large number and diversity of BGCs. About 40 BGCs could be identified from the genome of the B. cinerea (Collado and Viaud, 2016), while 77 BGCs were found in the C. higginsianum genome (Dallery et al. 2017). Transcriptomic studies showed that a significant proportion of B. cinerea BGCs are induced during the development of grey mould on grape berries (Kelloniemi et al. 2015), and to a lesser extent during the development of noble rot (Collaboration with S. Delrot, INRA, Bordeaux). Likewise in C. higginsianum, 14 out of 77 BGCs are specifically induced during host penetration and the biotrophic phase of the infection (Dallery et al 2017), suggesting the fungus produces an array of metabolites very early when host cells are still alive. These fungal molecules are therefore unlikely to be cytotoxic and may instead function similar to effector proteins for host manipulation. However, these metabolites have yet to be characterized and their plant targets are completely unknown.

One of the main aims of the ECPP group is to exploit our knowledge of fungal genomes and their regulatory networks to produce infection-specific fungal SMs during growth in vitro to then study the mode of action of the purified molecules and identify their plant targets. This approach builds on our recent identification of pathway-specific regulators and global regulators of secondary metabolism in both Botrytis and Colletotrichum. For example, botrydial synthesis is regulated by a specific transcription factor (Porquier et al., 2016) and global regulators including the Velvet complex (Collaboration with J. Schumacher, BAM, Berlin, Germany; Schumacher et al. 2013; 2015). In addition, we investigated the role of nucleosome positioning (Collaboration with I. Fudal, BIOGER and N. Ponts, MycSA, INRA, Bordeaux; SPE Project Nucleosomes) and those of histone modifiers in the expression of BCGs. Our recent results indicate that histone methylation plays a role in the regulation of SM production in both C. higginsianum and B. cinerea.

Within the frame of the ANR-funded Project ‘HerbiFun’, we are searching for phytotoxic fungal metabolites that could be developed as novel herbicides. This project involves collaboration with the groups of J. Ouazzani (ICSN, CNRS, Gif), O. Lespinet (I2BC, CNRS, Gif), and O. André (De Sangosse biopesticide company) as well as with MH Lebrun (EGIP, BIOGER). SMs produced in vitro by selected mutants are currently being extracted and purified by our collaborators at ICSN and screened for phytotoxic activity on model plants, crops and weeds by De Sangosse. Bioguided fractionation will then be used to identify the active SMs, which will be further studied to determine their chemical structure (ICSN).

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-  Contacts: muriel.viaud@inra.fr for Botrytis, richard.oconnell@inra.fr for Colletotrichum

 

Functional analysis of secreted protein effectors

The C. higginsianum genome encodes ~350 putative effector proteins (ChECs) secreted during infection. By tagging 61 ChECs with GFP for transient expression in Nicotiana benthamiana, we found 16 were targeted to specific subcellular locations (Robin et al. 2018). Half of these were imported into plant nuclei, while the rest were addressed to plant compartments not previously reported for effectors of any other filamentous pathogens, namely microtubules (3), Golgi (1) and peroxisomes (3). The fact that multiple ChECs converge on peroxisomes and microtubules suggests that fungal manipulation of plant functions associated with these structures could be critical for Colletotrichum pathogenesis and work is on-going to elucidate their functions and host targets.

Two other effectors containing chitin-binding LysM domains, ChELP1 and ChELP2, accumulate at the interface between biotrophic hyphae and the plant plasma membrane (Takahara et al. 2016). Both proteins bind fungal cell wall chitin with high affinity and specificity and both suppress the chitin-triggered activation of defense-related MAP kinases in Arabidopsis. By gene silencing, we showed these effectors are required for fungal virulence and play dual roles in both appressorium function and host defense suppression.

 

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-  Contact: richard.oconnell@inra.fr

 

Extracellular vesicles: what are their roles in bi-directional plant-fungal communication ?

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The ERA-CAPS-funded project ‘Exosomes’ (2018-2021) involves collaboration with Roger Innes (Coordinator, Indiana University, USA), Blake Meyers (Danforth Plant Science Center, USA) and Hans Thordal-Christensen (University of Copenhagen, Denmark). Exosomes are small extracellular vesicles (EVs) secreted from cells by the fusion of multivesicular bodies with the plasma membrane. Although they are implicated in the intercellular transport of sRNAs and proteins in animals, little is known about EV functions in plants or fungi. Using the C. higginsianum-A. thaliana interaction as a model, the project aims to determine whether plant and fungal EVs carry sRNAs that target each other’s genes, how plant and fungal EVs are produced, and how plants and fungi exchange EVs. We will analyse the cargoes (proteins and sRNAs) of C. higginsianum EVs purified from cultures and infected plants. To study the cell biology of EV biogenesis in Colletotrichum, we will use reverse genetics approaches and tagged protein markers for confocal and transmission electron microscopy.

 

-  Contact: richard.oconnell@inra.fr

 

How are the host signals that regulate fungal gene expression sensed and transduced ?

The INRA SPE project ‘IGgI PHOP’ (R. Laugé, 2018-2020) aims to identify fungal receptors involved in the perception of host signal(s), primarily in B. cinerea. The work is based on two complementary approaches. (i) An unbiased forward genetics screen that allows the selection of spontaneous fungal perception mutants on culture media mimicking in planta conditions. And (ii) a reverse genetics approach wherein candidate fungal receptor genes will be mutated. These will be selected from lower and higher Eukaryotes genes known to be involved in signal perception (non-self, environmental, host and pathogen perception). The putative function of genes in signal perception / transduction will then be validated by targeted gene replacement, i.e. verification of the loss of activation of in planta-induced genes, and phenotyping on host plants.

 

-  Contact: richard.lauge@inra.fr

 

What are the genetic determinants of partial host specialisation in B. cinerea ?

B. cinerea is considered a generalist pathogen attacking >500 plant genera but recent data have indicated that the population structure is linked to the host plant (Walker et al. 2015). Together with cross-inoculation data, this suggests the occurrence of partial host specialisation (PhD work of A. Mercier). In the frame of a common project with A.S. Walker (AMAR), P. Gladieux (BGPI, INRA, Montpellier), T. Giraud (ESE, CNRS, Orsay), and B. Poinssot (AgroEcologie, INRA Dijon), we use population genomic approaches to identify the underlying genomic determinants of partial host specialisation (BASC project Daphné and SPE project Paris-Match 2016-2018).

 

-  Contacts: muriel.viaud@inra.fr, anne-sophie.walker@inra.fr

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