Réassortiments chez un phytovirus octopartite ˸ l'étude du faba bean necrotic stunt virus (FBNSV) du genre Nanovirus

The viral multipartite genomic architecture and packaging strategy, in which the genome is divided in multiple segments separately encapsidated, stands as an evolutionary conundrum associated with theoretical high fitness costs associated to the maintenance of genome integrity. Earlier studies within the research team showed the capacity of the faba bean necrotic stunt virus (FBNSV; Nanovirus genus, genome composed of 8 segments) to accumulate each segment in distinct host cells and to complement the system at the supra-cellular level by exchanging gene products among interconnected cells, thereby greatly reducing the theorized intra-host necessary MOI. The aim of this doctoral research was to delve into the mechanisms through which nanoviruses manage to maintain their genome integrity, with a specific focus on reassortments and on their effects on the fitness of resulting hybrid genotypes. A reassortment corresponds to the replacement of one or more segments by homologous ones from a distinct parental genotype. The primary objective was to conduct a systematic comprehensive phenotypic characterization of all 16 possible single-segment reassortants involving two distinct FBNSV isolates.The objectives were addressed through the utilization of experimental techniques such as agro-inoculation, aphid inoculation, qPCR, phenotyping of infected plants, and confocal microscopy. Our findings revealed the FBNSV capacity to transmit its distinct segments from host-to-host non-concomitantly and to eventually reconstitute a complete genome, thereby significantly diminishing the costs related to maintenance of the genomic integrity.These findings, along with the capacity of nanovirus genomic segments to effectively complement each other at the supra-cellular level, significantly expand the spatial scale at which reassortment can occur. A thorough literature review on reassortments in single-stranded DNA multipartite viruses underscored that the replication of reassorted segments appears as the primary constraint for the success of the new generated genotypesThis doctoral research has led to the production of two new infectious clones, [AZ;15] and [AZ;10_12b], each representing distinct FBNSV isolates. Together with the isolate [JKI-2000], which was already available at the start of the PhD, we now possess three infectious clones representing isolates from the three main FBNSV phylogenetic clades. These clones will facilitate additional investigations of genetic exchanges, mixed infections, and ecological dynamics.The phenotypic characterization of isolates was conducted across three host plants (faba beans, lentils, and vetches) and one aphid vector (Aphis craccivora), who appeared to be common to all three viral isolates holding significant implications on ecological dynamics of nanovirus natural populations. This analysis revealed phenotypic diversity among isolates, notably regarding their genome formula, across all host plants and aphid vectors. Finally, the systematic phenotypic characterization of single-segment reassortants between [AZ;15] and [AZ;10_12b] isolates demonstrated the viability of all 16 possible combinations across three host plants. While these findings hold potential ecological and evolutionary significance for nanoviruses, it is important to emphasize that they were obtained under controlled environment with no competition from other genotypes.The results from this doctoral research significantly expand the genetic resources and opportunities available for exploring nanovirus biology, reassortment dynamics, and the strategies employed by ssDNA multipartite viruses in general, at least nanoviruses, to mitigate the costs associated with the maintenance of genome integrity.L'architecture génomique virale multipartite, pour laquelle le génome est divisé en plusieurs segments encapsidés séparément, est une énigme évolutive dû à des coûts théoriques importants associés au maintien de l'intégrité génomioque. Des études antérieures au sein de l'équipe ont démontré la capacité du faba bean necrotic stunt virus (FBNSV ; genre Nanovirus, génome composé de 8 segments) à accumuler ses segments dans des cellules hôtes distinctes et à complémenter le génome au niveau supra-cellulaire réduisant ainsi considérablement le coût intra-hôte théorisée.L'objectif de cette thèse était d'explorer les mécanismes par lesquels les nanovirus parviennent à maintenir l'intégrité de leur génome, en se focalisant sur les réassortiments et leurs effets sur la fitness des génotypes hybrides résultants. Un réassortiment correspond au remplacement d'un ou plusieurs segments par des segments homologues provenant d'un génotype parental distinct. L'objectif principal était de mener une caractérisation phénotypique systématique des 16 réassortiments possibles impliquant un seul segment entre deux isolats distincts du FBNSV.Les objectifs ont été abordés grâce à l'utilisation de techniques expérimentales telles que l'agro-inoculation, l'inoculation par pucerons, la qPCR, le phénotypage des plantes infectées et la microscopie confocale. Nos résultats ont révélé la capacité du FBNSV à transmettre ses segments distincts d'hôte à hôte de manière non concomitante et à reconstituer finalement un génome complet, réduisant ainsi significativement les coûts liés au maintien de l'intégrité génomique inter-hôte.Ces découvertes, associées à la capacité des segments génomiques des nanovirus à se complémenter efficacement au niveau supra-cellulaire, élargissent considérablement l'échelle spatiale à laquelle les réassortiments peuvent se produire. Une revue approfondie de la littérature sur les réassortiments dans les virus multipartites à ADN simple brin a mis en évidence que la réplication des segments réassortis semble être la principale contrainte pour la viabilité des nouveaux génotypes produits.Cette recherche doctorale a conduit à la création de deux nouveaux clones infectieux, [AZ;15] et [AZ;10_12b], représentant chacun des isolats distincts de FBNSV. En combinant l'isolat [JKI-2000], déjà disponible au début du doctorat, nous disposons désormais de trois clones infectieux représentant des isolats des trois principaux clades phylogénétiques du FBNSV. Ces clones faciliteront de nouvelles investigations sur les échanges génétiques, les infections mixtes et la dynamique écologique.La caractérisation phénotypique des isolats a été réalisée sur trois plantes hôtes (fèves, lentilles et vesces) et un puceron vecteur (Aphis craccivora). Les trois isolats sont capables d'infecter ces hôtes et vecteur ce qui a des implications significatives sur la dynamique écologique des populations naturelles de ces nanovirus. Cette analyse a révélé une diversité phénotypique entre les isolats, notamment en ce qui concerne leur formule génomique, sur toutes les plantes hôtes et au sein du puceron vecteur.Enfin, la caractérisation phénotypique systématique des réassortiments impliquant un seul segment entre les isolats [AZ;15] et [AZ;10_12b] a démontré la viabilité de toutes les 16 combinaisons possibles sur trois plantes hôtes. Bien que ces découvertes aient un potentiel écologique et évolutif significatif pour les nanovirus, il est important de souligner qu'elles ont été obtenues dans un environnement contrôlé sans concurrence d'autres génotypes.Les résultats de cette recherche doctorale élargissent considérablement les ressources génétiques et les opportunités disponibles pour explorer la biologie des nanovirus, la dynamique des réassortiments et les stratégies employées par les virus à ADN monocaténaire multipartite, du moins les nanovirus, pour atténuer les coûts associés au maintien de leur intégrité génomique

Reassortments in single-stranded DNA multipartite viruses: Confronting expectations based on molecular constraints with field observations

International audienceSingle-stranded DNA multipartite viruses, which mostly consist of members of the genus Begomovirus, family Geminiviridae, and all members of the family Nanoviridae, partly resolve the cost of genomic integrity maintenance through two remarkable capacities. They are able to systemically infect a host even when their genomic segments are not together in the same host cell, and these segments can be separately transmitted by insect vectors from host to host. These capacities potentially allow such viruses to reassort at a much larger spatial scale, since reassortants could arise from parental genotypes that do not co-infect the same cell or even the same host. To assess the limitations affecting reassortment and their implications in genome integrity maintenance, the objective of this review is to identify putative molecular constraints influencing reassorted segments throughout the infection cycle and to confront expectations based on these constraints with empirical observations. Trans-replication of the reassorted segments emerges as the major constraint, while encapsidation, viral movement, and transmission compatibilities appear more permissive. Confronting the available molecular data and the resulting predictions on reassortments to field population surveys reveals notable discrepancies, particularly a surprising rarity of interspecific natural reassortments within the Nanoviridae family. These apparent discrepancies unveil important knowledge gaps in the biology of ssDNA multipartite viruses and call for further investigation on the role of reassortment in their biology

Nonconcomitant host-to-host transmission of multipartite virus genome segments may lead to complete genome reconstitution

International audienceBecause multipartite viruses package their genome segments in different viral particles, they face a potentially huge cost if the entire genomic information, i.e., all genome segments, needs to be present concomitantly for the infection to function. Previous work with the octapartite faba bean necrotic stunt virus (FBNSV; family Nanoviridae , genus Nanovirus ) showed that this issue can be resolved at the within-host level through a supracellular functioning; all viral segments do not need to be present within the same host cell but may complement each other through intercellular trafficking of their products (protein or messenger RNA [mRNA]). Here, we report on whether FBNSV can as well decrease the genomic integrity cost during between-host transmission. Using viable infections lacking nonessential virus segments, we show that full-genome infections can be reconstituted and function through separate acquisition and/or inoculation of complementary sets of genome segments in recipient hosts. This separate acquisition/inoculation can occur either through the transmission of different segment sets by different individual aphid vectors or by the sequential acquisition by the same aphid of complementary sets of segments from different hosts. The possibility of a separate between-host transmission of different genome segments thus offers a way to at least partially resolve the genomic maintenance problem faced by multipartite viruses

Genome‐wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection

Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages