ERA-CAPS - Appel Europe-USA pour renforcer la recherche transnationale en biologie moléculaire végétale

Role of extracellular vesicles in plant-microbe interactions – Exosomes

Role of extracellular vesicles in plant-microbe interactions

Exosomes are small extracellular vesicles (EVs) secreted from cells by the fusion of multivesicular bodies (endosomes) with the plasma membrane. Although they are implicated in intercellular transport of sRNAs and proteins in animals and plants, very little is known about EV composition or functions in filamentous fungi.

Our aim is to understand the role EVs in plant-microbe interactions, specifically what do plant and fungal EVs contain, how are they produced, and what is their role in host-pathogen communication?

The ERA-CAPS project ‘Exosomes’ involves collaboration with Roger Innes (Coordinator, Indiana University, USA), Blake Meyers (Danforth Plant Science Center, USA) and Hans Thordal-Christensen (University of Copenhagen, Denmark). As experimental systems, we are using Arabidopsis thaliana infected by Colletotrichum higginsianum and Medicago truncatula infected by C. destructivum. Our collaborators focus on plant EVs, in particular: (a) The impact of pathogens on the protein and sRNA content of plant EVs; (b) The localization of plant sRNAs at infection sites; (c) The identification of plant genes involved in EV biogenesis. At BIOGER, we focus on fungal EVs, in particular: (a) Developing procedures to isolate EVs from Colletotrichum growing in vitro, and analysing the protein and sRNA composition of these EVs (with the Innes and Meyers Labs). (b) The cell biology and dynamics of fungal EV formation, using fluorescent protein-tagged markers for confocal microscopy. (c) Analysis of EV biogenesis and release at plant-fungal interfaces using transmission electron microscopy. (d) Characterizing the phenotype of plant mutants affected in vesicle trafficking / secretion upon infection by adapted and non-adapted fungal pathogens (with the Thordal-Christensen and Innes Labs).

Combinations of ultrafiltration, ultracentrifugation and size exclusion chromatography are to isolate EVs from a range of fungal cell types. Details of the methods will be presented in the final report, after publication. The quality of EV preparations is assessed using nanoparticle tracking analysis and TEM negative staining. The composition of the isolated vesicles is then determined using proteomics and sRNA sequencing. A variety of candidate EV marker proteins are tagged with fluorescent proteins for confocal and super-resolution microscopy. High-pressure freezing and freeze-substitution are used to prepare cells and tissues for transmission electron microscopy of EV biogenesis and release at plant-fungal interfaces.

Results will be presented in the final report, after publication

Future perspectives of the project will be presented in the final report.

Scientific production and patents will be presented in the final report.

The goal of this research is to determine the role of extracellular vesicles (EVs) in plant-microbe interactions. EVs are known to mediate intercellular transfer of small RNAs (sRNAs) in mammalian and insect systems, but very little is known about the function of EVs in plants or fungi. Our team has developed methods to purify plant EVs and has demonstrated that EVs are enriched in stress response proteins, and carry sRNAs. We hypothesize that plant EVs also mediate intercellular signaling, including trans-kingdom transfer of sRNAs. The experiments detailed in this proposal will examine whether plant and fungal EVs carry sRNAs that target each other’s genes, how plant EVs are produced, and how plants and fungi exchange EVs.
These questions will be addressed using soybean and Arabidopsis as host plants, and Colletotrichum spp., Pseudomonas syringae, and Bradyrhizobium japonicum as microbes. We will isolate EVs from plants infected with these microbes and will quantitatively assess their protein and sRNA contents. These analyses will provide insight into how proteins and sRNAs are targeted to EVs, and how EVs differ between types of interactions. These analyses will also reveal, for the first time, the contents of EVs from a fungal plant pathogen. Most significantly, these analyses will enable us to establish a network of trans-kingdom gene regulation between plants and fungi.
EV-mediated transfer of sRNAs between host cells and fungal cells will be imaged using superresolution microscopy and RNA-PAINT. In addition, genes required for EV biosynthesis will be identified using reverse genetics. Mutants with defects in EV biogenesis will be further analyzed using 3D electron microscopy, allowing us to image plant-fungal interfaces at unprecedented resolution, capturing all stages of EV production, release, and uptake.
The above objectives will be accomplished by a team of four laboratories with highly complementary expertise. The Innes lab is pioneering the study of EVs, and brings over 25 years of experience comparing soybean and Arabidopsis immune systems. The Meyers lab is an international leader in the sRNA field, and has performed extensive characterization of soybean sRNAs. The O’Connell lab is an international leader in Colletotrichum pathogenesis, and has pioneered EM methods for imaging fungal infection sites. The Thordal-Christensen lab has amassed a collection of Arabidopsis endomembrane trafficking mutants, which will be assessed for defects in EV production. With this collection of expertise and resources, our group will be able to make rapid progress toward understanding the roles of EVs in plant-microbe interactions.
This research will enable development of durably disease resistant crops via improving novel disease resistance mechanisms such as host-induced gene silencing. Development of crops with durable disease resistance is a key to the creation of a sustainable and secure food supply, the ultimate goal of ERA-CAPS.

Project coordination

Richard O'Connell (UMR Biologie et Gestion des Risques en Agriculture, INRA)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

BIOGER UMR Biologie et Gestion des Risques en Agriculture, INRA

Help of the ANR 199,508 euros
Beginning and duration of the scientific project: April 2018 - 36 Months

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