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Team Leader : Armelle Marais (armelle.marais-colombel@inrae.fr)

Team description and research objectives :

Plant viruses are ubiquitous and highly diverse obligate pathogens. They are difficult to control and can severely affect yield of several important crops. They are also an underestimated component of natural ecosystems and account for half of all emerging plant pathogens. The multidisciplinary Plant Viruses (Viro) team brings together expertise in plant virology, genetics, molecular and cell biology, biochemistry, metabolomics, metagenomics and bioinformatics. Using a translational approach, we seek to understand the virus diversity, the plant-virus interactions, and the genetic basis of plant responses to virus infections in a multi-stress environment. With these approaches we aim to meet the challenges of agro-ecological transition. Viro team focuses on three major axes: (i) developing and using tools to determine and analyze viral diversity, (ii) identifying the mechanisms and factors controlling plant-virus interactions, and (iii) exploring plant genetic diversity to investigate the genetic basis of their response to viruses under multiple stress conditions. Each of these areas has both basic and applied aspects. Applications mainly concern viral diagnosis and etiology (axis 1) and the development and use of resistant and tolerant plants (axes 2 and 3) as part of global agro-ecological solutions. To this end, the team works closely with major players in the plant industry (Arvalis, Limagrain, Syngenta, etc.). The team also offers the services of two associated technology platforms: "Viral indexing by sequencing" and "Viral vectors". The three axes are interconnected, as knowledge of viral diversity is often a useful entry point for studies of plant-virus interactions and genetic bases.

Research topics :

1 - Analysis of viral diversity at different scales and translational research applications: etiology, diagnostics, taxonomy

Contacts :
Thierry Candresse (thierry.candresse@inrae.fr) - Armelle Marais-Colombel (armelle.marais-colombel@inrae.fr)

Viruses are undoubtedly the most diverse biological agents. Individually, they are also highly variable and their error-prone replication endows them with very high evolutionary potential. These characteristics have a number of practical implications for control efforts, from difficulties encountered identifying and detecting disease agents to host resistance instability. Our team is actively engaged in the diagnostics and viral characterization field, with particular emphasis on the use of high-throughput sequencing (HTS). We develop these approaches (i) to solve etiological problems and their practical application to virological diagnosis, (ii) to answer viral ecology questions on the functioning of viral populations.

In recent years, we have made a significant contribution to the characterization of the grapevine virome in the context of collaborative projects with grower organizations. This effort has been extended by two PNDV (Plan National du Dépérissement du Vignoble) projects addressing the mycovirome (fungi virus) associated with grapevines. A PhD, as part of the Marie Curie InextVir European training network, sought to characterize the virome of fruit trees of the genus Prunus. This work led to the characterization of six new viruses for which molecular detection tests have been developed. With the same objective, the viral indexing sequencing platform operated by the team is open to all stakeholders in the phytosanitary field and supports collaborative projects on a large number of crops. These actions lead to the characterization of viral populations, the identification and characterization of the agents responsible for diseases of unknown aetiology and the development of efficient diagnostic tools. Over the years, these virus characterization activities have generated a collection of reference viral isolates housed in our high-level containment greenhouse. This collection has now been integrated into the EU-funded EVA-Global (European Virus Archive) infrastructure project, which aims to increase the number and quality standards of viral isolates and facilitate their distribution. Our virus characterization know-how has recently been mobilized to contribute to the PNRI (Plan National de Recherche et d'Innovation) on beet yellowing viral diseases, with the aim of reducing the dependence of crops on insecticides used to control aphid vectors of yellows viruses.

While they can be applied to isolated plants for diagnostic purposes, HTS approaches can also be applied to plant populations, with the goal to identify all plant viruses in a given environment and gain an understanding of viral ecology. Our efforts in viral metagenomics have two objectives. The first is methodological and has resulted, for example, in the development of the VirAnnot pipeline, allowing for the first time to reproducibly estimate the specific richness of viromes. The second concerns the description and comparison of phytoviromes in different environments and, in viral ecology approaches, the understanding of the factors structuring phytoviral populations in time and space or the identification of viral fluxes between wild and cultivated compartments of agroecosystems. These studies have highlighted the complexity and diversity of the viromes associated with plants, and demonstrated their highly dynamic nature. A recent PhD project, part of the Marie Curie InextVir European training network, has led to a better understanding of the contrasting viromes associated with cultivated carrot and wild carrot populations and provides a basis for further research into the biological and ecological barriers limiting virus movement at the agro-ecological interface. This research theme is also developed within the framework of the ANR Phytovirus project (coordination P. Roumagnac CIRAD Montpellier) which seeks to address fundamental questions such as the relationship linking hosts diversity and viral diversity or the contribution of “viral enemy release” to the success of invasive plant species.

The PPR Deep Impact project (Programme Prioritaire de Recherche, coordination C. Mougel INRAE Rennes) aims to design a new generation of agro-ecological solutions to increase plant resistance to biotic stresses, while respecting the environment. By combining various approaches, the aim of the project is to explore the contribution of the rapeseed and wheat microbiomes to the health of these crops, with a view to reducing dependence on pesticides. In this context, our team is in charge of characterizing the virome component of the microbiome of these two crops. Our researches into viral metagenomics aimed at better understanding the diversity and functioning of phytovirus populations, will intensify in the coming years, in particular as a consequence of the resurgence of aphid-transmitted viruses in field crops resulting from the ban of neonicotinoid insecticides, and the positive impact of climate change on insect vector populations. Our aim is to answer questions about virus circulation between crops and reservoirs, virus dynamics in vector populations, the ability of viruses to evolve towards deployed plant resistances, or the unintended effects on virus populations of new agricultural practices.

2 - Molecular bases of plant-virus interactions and viral adaptation

To invade plants, plant viruses reroute host cellular functions for their own benefits. The completion of the viral cycle results from a complex interplay between virus- and host-encoded factors, also called susceptibility factors. In this scheme, absence or non-adequacy of a single susceptibility factor may lead to full or partial resistance to viruses, with potential practical implications.

• Functional studies to identify susceptibility factors involved in plant virus propagation

Contact : Sylvie German-Retana (sylvie.german-retana@inrae.fr) - Nathalie Arvy (nathalie.arvy@inrae.fr)

Plasmodesmata (PD) are symplasmic tunnels between cells that are the gateway for plant virus movement. In the frame of the ANR-PotyMove project (ANR-16 CE20-0008-01, 2016-2023) we identified and validated two new plant factors involved in potyvirus propagation: the Remorin protein, associated with particular membrane domains called lipid rafts and plasmodesmata (collaboration with S. Mongrand, CNRS, Laboratoire de Biogenèse membranaire, LBM-Bordeaux) and AtHVA22a (Arabidopsis homolog of Hordeum vulgare abscisic acid responsive gene 22) which is partially associated with plasmodesmata and appears to be part of a wider network of proteins capable of modifying specific structures in the plant cell's endoplasmic reticulum. The transfer of these results to tomato in a translational research approach is still in progress (Collaboration with Jean-Luc Gallois, INRAE, GAFL-Avignon).

The ANR-LiDroVir project, (ANR-23-CE20-0026-01, 2024-2028), aims to study the role of lipid droplets (LDs) in the potyvirus cycle, in collaboration with the teams of Claire Bréhélin (CNRS, LBM-Bordeaux,) and Jean-Luc Gallois (INRAE, GAFL-Avignon). As well as being involved in cellular lipid metabolism and the response to environmental stresses, LDs are hijacked by RNA (+) animal viruses to enable/favor their replication and/or encapsidation in viral replication compartments (VRCs). This raises the question of the role of these LDs in the cycle of plant viruses. Indeed, our recent unpublished results show that i) a RNA (+) phytovirus induces a significant accumulation of neutral lipids and a considerable increase in the number of LDs, ii) these LDs are relocated to VRCs in infected leaves, and iii) phytovirus propagation is altered in plants mutated for genes involved in LD synthesis. On the basis of this demonstration, the LidroVir project aims to identify potential new players in the potyvirus cycle and seek out other original resistance targets. Interestingly, in animals, the homologs of AtHVA22 have been shown to regulate lipid metabolism by interacting with seipin (a structural protein of LDs). The LiDroVir project might allow to replace other candidates identified in Potymove (besides AtHVA22a) in a network of plant proteins involved in LDs and crucial for potyvirus replication and movement.

 

• Modulation of the structure-function of viral proteins: Exploring adaptation mechanisms

Contacts : Guillaume Lafforgue (guillaume.lafforgue@inrae.fr) - Thierry Michon (thierry.michon@inrae.fr)

 

The high mutation frequency of viruses boosts their evolutionary potential and compromises the durability of host resistance. Certain viral proteins have so-called intrinsically disordered regions (IDR) which do not have a stable structure, but which nevertheless fulfill important functions in the infectious cycle of the virus. These structurally unconstrained IDRs have a mutational robustness which could facilitate the adaptation of the virus to its host. By combining experimental evolutionary approaches in plants and biochemical methods, we evaluated the contribution of IDRs to bypassing host resistance. Our experimental data, obtained in vivo, allow for the first time, to support the hypothesis that IDRs can directly modulate the adaptability of viruses. As part of the ANR ID4VISA21 project, we are currently exploring the contribution of potyvirus proteome IDRs to host adaptation and the possible correlations between IDRs and host range size using in silico approaches.

 

 

• Luteovirus-plant molecular interactions

Contacts: Syed S. Zaidi (syed.zaidi@inrae.fr) - Sylvaine Boissinot (sylvaine.boissinot@inrae.fr)

Luteoviruses are responsible for the yellow dwarf disease in cereals, and are among the most economically important viral pathogens in the world. They infect major food crops including wheat, causes substantial yield losses that can reach 80%. Phloem-feeding aphids act as luteovirus vectors. In the absence of natural resistance to these viruses, the primary method for disease management is to control the insect vector population, mainly through pesticides. However, these problematic pesticides accumulate in the environment, cause the evolution of pesticide-resistant insects, harm beneficial insects, and pose potential health risks to humans and wildlife. Consequently, these neonicotinoid-based pesticides are being banned in the EU, creating an urgent need for the alternative solutions for the farmers.

Our team uses high throughput plant-virus interactomics to generate topologically-resolved interaction maps. Focusing on the viruses barley yellow dwarf virus (BYDV)-PAV and BYDV-PAS in cereal crops, our team is exploring the formation of viral replication compartments and the role of lipids in BYDV infection. The ultimate goal is to develop eco-friendly resistance strategies against BYDV, significantly reducing the reliance on pesticides and contributing to sustainable agricultural practices. The team combines advanced techniques like proximity labeling, correlative microscopy, proteomics, and genome editing, promising to deliver seminal insights and innovations in plant biology.

 

Aim: Phloem is a deeply buried plant tissue that acts as a highway for nutrient transport. BYDV is a specialized phloem-restricted pathogen which has evolved to exploit this nutrient-rich and protected environment. Little is known about the host-pathogen molecular interactions leading to phloem-restriction of the virus, organelle membrane reorganization, and selective movement through different types of plasmodesmata. Understanding phloem-restricted BYDV pathogenesis is urgent for devising eco-friendly resistance strategies. Addressing the knowledge gap in understanding phloem-restricted pathogenesis and identifying plant proteins involved in this mechanism are the central aim of our group. Genes encoding some of these plant proteins are expected to be susceptibility factors, and therefore potential sources of resistance.

Facilities: Our team leverages state-of-the-art equipment including Confocal Laser Scanning Microscopy for advanced cell imaging and Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) for high-throughput proteomics. We also utilize virus-induced gene silencing (VIGS) and specialized aphid-mediated virus inoculation techniques for targeted research in monocots.

Scientific Partnership: Given the interdisciplinary nature of our project, we engage in robust collaborations with leading national and international laboratories. These partnerships enhance our research capabilities and broaden the impact of our work in plant virology.

Industry Collaboration: Acknowledging the significant socioeconomic implications of our research, we maintain active collaborations with industry partners. Our contributions are particularly notable in the areas of resistance gene discovery and in the quantification of virus responses in genetic materials, offering valuable insights and services to the agricultural sector

 

A) Wheat protoplast transformation, and confirmation of gene expression: Note that pZmUBI-GFP expression is higher compared to p35S-GFP.
B) Subcellular localization of Barley yellow dwarf virus (BYDV) movement protein (MP): BYDV-MP fused with yellow fluorescent protein (YFP) was observed with plasmodesmata marker aniline blue. The overlay indicates the plasmodesmata localization of BYDV-MP (white arrows).
C) Wheat varieties maintained at the insectarium for BYDV phenotyping
D) Leaf yellowing – characteristic BYDV symptom on wheat leaves
E) Insectarium where BYDV-viruliferous and healthy aphids (insect vector of BYDV) are maintained. The viruliferous Rhopalosiphum padi is used for BYDV inoculation in different hosts including wheat, barley, and oats.
F) An aphid (R. padi) colony maintained on barley.

 

 

 

 

3 - Genetic basis of plant tolerance to viruses under multi-stress conditions

Contact : Valérie Schurdi-Levraud (valerie.schurdi-levraud@inrae.fr; valerie.schurdi-levraud@u-bordeaux.fr)

Virus infection is one of the most alarming biotic threats in agrosystems, due to the impact of climate change on the spatial and temporal distribution of vectors and viruses. Global changes, and in particular the increased frequency of periods of high temperature and drought, are likely to favor the redistribution of many plant pathogens and the emergence of new pathogens/strains. In addition, little is known about the effect of heat and/or drought on the robustness of resistance, but also on the defense responses and signaling elements produced by the plant during viral infection.

In this context, understanding plant responses to viral infection under abiotic constraints such as rising temperatures and/or drought is of great importance in finding sustainable agricultural solutions. Until now, breeding virus-resistant varieties has been considered the best response to the viral threat. But this solution has the disadvantage of generating strong selection pressures on pathogen populations, contributing to pathogen evolution and resistance breakdown. It is also often associated with a yield penalty linked to the trade-off between resistance and growth.

With a view to sustainable agriculture and the agroecological transition, we need to reconsider the genetic basis of the response to viruses in the context of multiple stresses, and evaluate this response in terms of tolerance, i.e. the trade-off between stress response and growth maintenance. The ultimate aim is to be able to offer farmers tolerant varieties as part of a global agroecological solution.

 

 

Arabidopsis in the field !
Evaluation of the response of a collection of Arabidopsis thaliana genotypes to TuMV (turnip mosaic virus) under multiple stress conditions in the field. (photo B. Rubio) and search for genetic basis of response using GWAs (Manhattan plot of viral load)

Our team focuses on exploring genetic diversity in Arabidopsis thaliana and Solanum pimpenillifolium. Using multiple phenotyping approaches (disease score, viral load, dry biomass, flowering date, fitness, targeted and non-targeted metabolism, ecophysiological parameters, etc.), quantitative genetics and pan-genomic analyses (GWAs), we are looking for the genetic and functional bases of the "tolerance" response. Firstly, on worldwide and local collections of A. thaliana genotypes TuMV (turnip mosaic virus) outdoor infection (collaborations F. Roux INRAE LIPME Toulouse, J. Bergelson U. Chicago/ New York University, USA), this study revealed a more complex genetic architecture than under controlled conditions. We were able to identify new loci involved in the interaction. In collaboration with the META team of our UMR, we have also shown that certain metabolites can be used as markers and predictors of plant response.

This pioneering study highlights the value of analyzing plant-virus interactions in the complex multi-stress context plants face in eco and agrosystems (Thesis B. Rubio, 2017) to decipher the genetic basis of the tolerance.

This same question is now being asked within the framework of the Bordeaux Plant Science (BPS) Grand Project de Recherche funded by the University of Bordeaux, France and the ANR GreenTolerance. We are studying S. pimpenillifolium in collaboration with LIPME, Toulouse, France and the company Syngenta France, and A. thaliana facing CMV (cucumber mosaic virus) under heatwave conditions or TMV (tobacco mosaic virus). The focus is on the contribution of metabolites as markers and predictors of response.