Plant viruses and the vectors, especially hemipterans like aphids, that they use for plant-to-plant transmission, cause tremendous damage in agriculture. Conventional control strategies rely on insecticides to eliminate the aphids, but appearance of resistances, ecological issues and new legislation demand alternative control strategies that are more respectful of the environment and compatible with sustainable agricultural practices. Evidence emerges that viruses manipulate plant traits, vector behavior and vector performance to optimize their transmission. Thus, research targeting plant viruses, vectors and hosts, and exploring the tritrophic interactions between them may show ways to more sustainable pest control strategies. This is the objective of our project. We want to identify the molecular mechanisms driving these interactions on all three levels, with an emphasis on their role in transmission. For this, we will study two viruses sharing the same hosts and aphid vectors, and representing the two most common transmission modes used by plant viruses, non-circulative (NC) and circulative (C) transmission. NC viruses are transmitted by binding to and being released from a specific site on the external mouthparts of vectors, whereas C viruses must traverse the intestine of their aphid vectors, cycle through the hemocoel and invade the salivary glands, before they can be inoculated with saliva when aphids feed on a new host. In a comparative study, we want to elaborate the similarities and differences between NC and C transmission and to determine whether they can be host-specific. For this, we use NC Cauliflower mosaic virus (CaMV) and C Turnip mosaic virus (TuYV) as viruses, the model plant Arabidopsis thaliana and the biofuel plant Camelina sativa as hosts for the two viruses, and the economically important pest, green peach aphid (Myzus persicae L.), as a vector transmitting both viruses. Our first objective is to confirm, by aphid performance analysis (individual growth rate, fecundity, starvation survival, arrestment, behavior), previous studies showing that these viruses do modify aphid behavior and traits in a way favorable for transmission, and analyze these traits in detail. Then we will identify and characterize virus-induced sRNAs and viral proteins involved in aphid modifications. This will be achieved by analyzing aphid performance on plants expressing the candidates stably or transiently. It is then our second objective to create, by RNA profiling, a comprehensive catalog of aphid and plant genes whose transcription is altered by viral infection and/or aphid infestation. Our third objective is to validate and characterize, by functional analysis, the host and aphid genes and pathways identified by RNA profiling and that are primarily involved in modifying plant and aphid traits in a way conducive for transmission. Taken together, this project will show which host and vector traits are modified by the viral infection for a better transmission, which viral factors provoke these changes, and which host and vector genes and pathways are targeted by the two viruses.

Genetic exchanges are one of the main mechanisms generating within-population diversity that selection can act on. Even though some extremely successful genotypes may be thus produced, many deleterious genotypes are also produced; the net balance that could result from systematic experimental gene exchange between two distinct genotypes is unknown, and this is true for any organisms. This project aims at filling this gap by using as model system multipartite viruses, whose genome is divided in several segments packaged independently, each viral particle carrying only one segment and each segment encoding only one gene. Multipartite viruses can exchange entire genomic segments, an additional mechanism of genomic shuffling termed reassortment. Using the octopartite nanovirus faba bean necrotic stunt virus (FBNSV), a parasite of legume plants, we will measure several viral fitness components for intra- and inter-specific single segment (and thus single gene) reassortants. This will allow us to estimate whether reassorting is on average beneficial/detrimental and whether specific viral genes/functions are more likely to produce beneficial reassortants. Competition experiments between reassorting and parental-genotypes will show whether mechanisms favoring species/genotype genomic integrity are at play, and experimental evolution will show whether deleterious effects of reassortment may be alleviated during coevolution of reassorting segments with the rest of the genome. Finally, transmission experiments will investigate whether reassorting may facilitate the transmission of multipartite viruses by allowing for the non-concomitant transmission of different genomic segments. This project may thus provide (i) the first systematic characterization of the fitness and biological trait effects of reassortment; (ii) a potential mechanism through which the between-host transmission cost of multipartitism may be alleviated.

In recent years, the number of identified non-proteinogenic amino acid-containing secondary metabolites and their biosynthetic gene clusters has greatly expanded in bacteria. A new family of stand-alone adenylation (A) domains involved in the incorporation of ß-amino acids has been previously described. Based on the protein structural analyses of three members of this new family, new ß-amino acid specificity-conferring codes have been proposed. A common specific feature of these stand-alone A domains is that they are co-encoded with a stand-alone acyl carrier protein domain. Based on these specific features, the French partner (BGPI) has identified two new stand-alone A domains expected to be involved in the incorporation of a ß-amino acid. These stand-alone A domains are present in two loci belonging to important pathogenic bacteria. According to their annotation, these loci encode two different new unknown molecules, respectively. Interestingly, by a genetic approach, these loci have both been shown to be required for the bacterial virulence. However, the chemical structure of the secondary metabolites synthesized by these loci remains unknown and the presence of a ß-amino acid has never been yet suspected and explored. This project aims at elucidating the chemical structure of the molecules encoded by these two loci, deciphering their biosynthesis pathways, and refining the functional assignment of their biosynthesis genes. This project will contribute to a better understanding of the incorporation of ß-amino acids by stand-alone A domains in bacteria. Since the targeted microorganisms are important pathogens, this project will essentially contribute to a deeper understanding of their pathogenicity. Consequently, the fight against these pathogens should therefore be facilitated by new data arising from the project. Since genome mining is an important approach to discover new loci encoding new natural products with potentially novel biological activities, this project proposes to mine bacterial genomic sequences available in Genbank for the presence of new loci encoding a stand-alone A domain specific of a ß amino acid. Depending on the biological activity of the characterized ß-amino acid-containing secondary metabolites, this project potentially could lead to industrial applications as antibiotics or plant protection agents. Patents obtained would potentially also contribute to the visibility of the project. The French and German partners BGPI and TU Berlin have developed a long-standing collaboration since 2005, working together on several projects including the structural characterization of the potent antibiotic albicidin.

Emerging plant viruses are a threat worldwide. Studying their life cycles in details is a prerequisite to reveal alternative control strategies. We will target the family Nanoviridae for both practical and fundamental reasons: i) it represents a huge threat for Musaceae crop (genus Babuvirus) and for legumes (genus Nanovirus), and ii) it has adopted the enigmatic “multipartite” organization, with its genome composed of several nucleic acid segments each encapsidated individually. Nanoviruses being multipartite viruses with the highest number of genome segments described thus far, they are perfect models to investigate processes that might be specific to this viral genomic architecture most frequently adopted by plant viruses. In particular, how such viruses can efficiently infect a high proportion of cells/hosts with at least one copy of each of their numerous genome segments remains elusive. It is deemed impossible in the literature that actually questions the conceptual frame with which we try to comprehend the multipartite viral systems. Consistently, we recently showed that a nanovirus do not function in a way that fits the current concepts in virology. The virus spreads distinct genome segments in distinct individual cells of the host. These segments functionally complement across cells and thereby define a pluricellular way of life. This unprecedented discovery in virology now requires in depth investigation to decipher the mechanisms allowing such a surprising viral lifestyle. More specifically the Krenz group analyzes the viral proteins role during virus-host interactions, while the Blanc group studies the within-host viral population dynamics and the virus-vector interactions during plant-to-plant transmission. Through this project, we will join forces to study the full lifecycle. We propose to decipher the biochemical and biological properties of various nanoviral gene products interacting with host plants and aphid vectors. We aim to understand how distinct viral genome segments initially expressed in distinct plant cells actually function, how they can communicate and complement at a distance and at a supra-cellular scale. We will analyze the properties of the viral gene products with a focus on those with yet unknown function, and on properties that could be involved in trafficking among cells for complementation. Likewise, we will strive to understand how virus particles successfully travel through the body of their aphid vectors, ensuring that all segments are acquired, transported across aphid’s cell barriers, and inoculated together. Thus, beyond the urgent need to better understand the biology of nanoviruses, an emerging threat worldwide, another ambitious goal of this project is to decipher the means by which a multipartite virus can sustain a pluricellular lifestyle, and thereby definitely coin this discovery as a new research horizon in plant virology and beyond.

CONTEXT, POSITIONING AND OBJECTIVES: Our aim is to increase our understanding of the role of pathogenicity factors of plant-pathogenic fungi, called effectors, in the infection process and in changes in virulence after host-shifts. Our central hypothesis is that the first step of host-shifts (the fixation of immune-escape mutations enabling to overcome non-host resistance) is followed by a second step of fine-tuning during which effector variants that efficiently interact with their target proteins in the novel host species are selected. Attempts to probe into the evolutionary, molecular and functional drivers of effector diversification have been hindered by the lack of large effector families identified in fungi, and thus the lack of good criteria to prioritize effectors for functional analysis. In this project, we overcome the methodological and conceptual barrier imposed by effector hyperdiversity by building on our recent discovery of an important, structurally conserved, but sequence-diverse family of effectors called MAX (for Magnaporthe Avrs and ToxB) in the model organism Magnaporthe oryzae, a fungal pathogen causing blast disease on rice and other cereals. In the highly multi-disciplinary MagMAX project, we will generate and integrate knowledge on protein structure, mode of action and genetic diversity to address four specific objectives: (1) Infer the diversification history of effector repertoires after host-shifts, (2) Understand the features determining the protein structure of effectors, (3) Decipher the virulence functions of fungal effectors by identifying the host proteins and processes they target, (4) Understand the molecular and eco-evolutionary factors driving fungal effector diversification. PROJECT ORGANISATION AND MEANS IMPLEMENTED: MagMAX will be coordinated by evolutionary microbiologist Dr Pierre Gladieux (INRA). MagMAX involves BGPI, which is among the best-qualified labs in functional and evolutionary studies of M. oryzae, and Centre de Biochimie Structurale, which is recognized for its expertise in studying protein biochemistry and structure biology. The project is organized in four work packages (WP) matching our four specific objectives. WP1 will infer the diversification history of the MAX effector repertoire, based on the production of high-quality re-sequencing data for a large sample set representative of the diversity of M. oryzae. WP2 will decipher the features governing the MAX effector fold, which is key to understand the effector’s mode of action, by modeling and experimental determination of their tertiary structure. WP3 will massively identify the host target proteins of MAX effectors using analyses of protein-protein interactions. WP4 will assess the hypothesis of molecular adaptation of MAX effectors to their targets combining information from other WPs about surface residues, surface of interactions and surface polymorphism. IMPACT: The world agricultural system needs to produce twice the amount of food before 2050. The emergence of new fungal plant pathogens poses a threat to global food security, and host-shifts are a major route for their emergence. Consequently, there is tremendous interest in identifying the factors driving the emergence of new fungal diseases. By combining diversity-, function- and structure-informed analysis of fungal effectors, our study will be pioneering, owing to its large-scale, multidisciplinarity and integrated nature, and as such should have a significant impact. Improved understanding of the biological features and molecular mechanisms underlying exploitation of new hosts will allow informed selection of which components of plant immunity to engineer to durably reduce the disease burden of cereals caused by fungi.