Origine de la biodiversité des séquences BSV détectées en Afrique de l'Est

Le Banana streak virus (BSV), du genre Badnavirus présente une structuration polyphylétique en trois clades bien distincts. Cette structuration complexe demande à être élucidée sachant que le BSV peut exister à la fois sous forme libre et intégrée au génome de son hôte. Des échantillons ont été prélevés en Ouganda, seul pays d'épidémie ancienne où la variabilité moléculaire du BSV est particulièrement importante puisque les trois clades y ont été détectés, contrairement à d'autres régions du monde. Afin d'étudier la variabilité moléculaire du BSV dans cette zone en lien avec son origine intégrée ou pas, le virus a été recherché par immuno-captures PCR avec des amorces spécifiques des espèces BSV du clade I, puis par PCR sur les extraits d'ADN totaux avec des amorces spécifiques des espèces BSV du clade II et III. Par ailleurs, la nature libre ou intégrée des séquences BSV dans le génome de bananier a été étudiée par hybridation en Southern Blot. (Texte intégral

Sequential integrations of badnaviruses into the M. acuminata and M. balbisiana genomes

Banana streak virus (BSV) is a double stranded DNA pararetrovirus belonging to the genus Badnavirus and triggering necrotic mosaic lesions on banana plants. BSV exhibits a higher biodiversity than other badnaviruses as a consequence of two phenomena: the epidemic process of the disease through contamination of plant to plant by vector and the release of infectious viral genomes by the banana genome harboring endogenous sequences. BSV sequences described so far are spread among the three groups of the badnavirus diversity. BSV-like sequences belonging to group 2 seem to be all integrated into the banana genome since no infectious episomal particles have been identified so far. We describe here the characterization of such integrations in thirty-four plant samples belonging to different families of the Zingiberale order with a banana sampling representative of the Musa diversity. We have firstly looked for group 2 sequences using PCR with 7 sets of primers which are specific of the 7 BSV-like species identified so far in this group. We then used Southern blot approach with viral probes corresponding to each BSV-like species. Sequenced PCR products of the RT-RNase H region of the viral genome have been used for the construction of a phylogenetic tree. We found all the 7 BSV-like species within the A genomes whereas only 4 species were within B genomes. Interestingly, two new species have been discovered in M. balbisiana genomes only. Additionally, four BSV-like species seem to be integrated in M. schizocarpa, M. basjoo, M. ornata and M. itinerans but these results have to be confirmed. The integration patterns observed by Southern blot analysis show multiple and different integrations of BSV-like species into M. acuminata plants conversely to M. balbisiana plants where patterns are highly conserved. The data suggest that BSV-like integrations likely occurred for some of them before the speciation M. acuminata/M. balbisiana whereas others occurred afterwards. (Texte intégral

Evolution of hazardous integrations of Banana streak virus in the genome of the wild banana (Musa balbisiana)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus responsible for banana streak disease. Even though integration is not an essential step in the replication cycle of BSV, the nuclear genome of banana and plantain (genus Musa) contains viral integrations called BSV Endogenous Pararetrovirus (BSV EPRV). Triggered by stresses, EPRV found in Musa balbisiana reconstitute an infectious viral genome. We showed the wild diploid M. balbisiana Pisang Klutuk Wulung (PKW) harbours pathogenic BSV-EPRV and is furthermore resistant to the virus. In these conditions, how to explain the presence of such viral integrants fixed in the host genome in terms of cost and benefits for both plant and virus? In order to highlight this question, we retraced the evolutionary history of infectious EPRV of BSV Golfinger species (BSGfV) integrated at a single locus in the genome of M. balbisiana PKW. This integrant was characterized in PKW by sequencing BAC clones containing BSGfV sequences. This BSGfV EPRV is composed of back-to-back viral sequences representing more than a whole genome. We developed molecular markers to explore the polymorphism of BSGfV integration patterns among M. balbisiana genotypes and other Musa species representing the genetic diversity of the genus. We observed a strong conserved pattern of BSGfV EPRV in all M. balbisiana genotypes. Among relative species, we found BSGfV EPRV only in M. boman showing a modified pattern. These results suggest that BSGfV integrated its host recently. Phylogenetic analysis of sequence data from both virus and EPRV confirmed this result. The consequences of deleterious viral sequences rapidly fixed in Musa genomes will be discussed. (Texte intégral

Integrations of Banana streak virus sequences in the genome of the banana Musa balbisiana : Endogenous viruses of host genes?

Banana streak virus or BSV (Badnavirus) is a double stranded DNA pararetrovirus causing leaf streak mosaic disease. Recently, numerous outbreaks of the disease occurred in all banana producing areas in interspecific Musa hybrids (M. acuminata x M. balbisiana) originating from virus-free parents. These infections correlated with BSV DNA sequences integrated in the M. balbisiana genome only, called endogenous pararetroviruses (EPRVs). Although integration is not needed for the replication cycle, some BSV EPRVs could become infectious under stress conditions by reconstituting a replication-competent genome after homologous recombination events. Surprisingly, even though the wild M. balbisiana Pisang Klutug Wulung (PKW) harbours pathogenic BSV EPRV, it is resistant to the virus. In these conditions, how to explain such viral integrants fixed in the host genome in terms of cost and benefits for both plant and virus? In order to highlight this question, we retraced the evolutionary history of infectious EPRVs of Golfinger species (BSGfV) integrated at a single locus in the genome of PKW. The structure of this EPRV was characterized in PKW by sequencing BAC clones containing BSGfV EPRVs. The integrant is composed of back-to-back viral sequences representing more than a whole genome. We developed molecular markers to explore the polymorphism of BSGfV integration patterns among M. balbisiana genotypes and other Musa species representing the genetic diversity of the genus. BSGfV EPRVs showed the same integration pattern as PKW in all M. balbisiana and a modified pattern in the relative species M. boman. Any BSGfV EPRV was observed in the other Musa species. We assume that BSGfV integrated its host recently. Phylogenetic analysis combining sequence data from both virus and EPRV confirmed this result. The consequences for Musa harbouring potentially deleterious sequences which have rapidly been fixed will be discussed. (Texte intégral

Molecular characterization of a pathogenic integration of the Golfinger Gf species of banana streak virus in the genome of Musa balbisiana

As several other plants, the genome of banana and plantain suffered integrations of pararetroviral sequences named EPRV (Endogenous pararetrovirus) even tough these integrations are not a part of the replication cycle of this virus. An original situation concerns the genome of Musa balbisiana which harbours banana streak virus (BSV) EPRV able to reconstitute pathogenic viral genomes under specific conditions. In order to assess the risk of spreading BSV through the diffusion of micro propagated banana plants and the creation of new lines habouring EPRVs as well as to understand the evolutionary forces that explain the presence and preservation of EPRVs in Musa genome, we proposed to characterise pathogenic BSV EPRVs of the Goldfinger species. The analysing of BAC library of Musa balbisiana cv Pisang Klutuk Wulung (PKW) permitted to characterise the integration pattern of BSGfV EPRVs as two similar integrants. Each integrant is composed of back-to-back viral sequences representing more than a whole genome. We developed molecular markers (PCR, PCR-RFLP) to distinguish each others. Then, we analysed the BSGfV EPRV segregation in the triploids hybrid progeny (AAB) resulting from crosses between virus free parents PKW (BB) and IDN110T (AAAA). There are found to be allelic, located as the same locus. Afterwards, we checked for BSGfV expression by searching the presence of virions by IC-PCR in the same AAB progeny in order to precise their allelic origin. Our results confirm that both allelic EPRV could be involved in the restitution of virions. We proposed a model of viral genome releasing based on the number of recombination events necessary to explain the genotype frequency observed between the two alleles. (Texte intégral

Distribution des intégrations de BSV au sein de la diversité Musa

Il est désormais acquis que des séquences virales font partie intégrante du génome de nombreuses plantes. Il est à noter que ce sont les virus de la famille des Caulimoviridae qui sont les plus représentés, 5 des 6 genres constituant cette famille ont été décrits dans le génome des plantes bien qu'aucun d'eux ne nécessite une intégration dans leur cycle viral. Mon travail porte sur l'étude des intégrations de BSV (Banana streak virus) dans le génome de l'espèce Musa balbisiana pour 4 espèces de BSV. Ces intégrations, fragmentées et complexes sont supérieures à la taille d'un génome viral et se sont révélées fonctionnelles car capable de restituer un génome viral infectieux produisant des particules virales. L'objectif de mon étude est d'analyser le contexte d'insertion et d'étudier l'intérêt du maintien de ces séquences tant pour le virus que pour la plante. Pour cela mon étude porte sur un échantillonnage représentatif de la diversité des bananiers tenant compte des deux centres géographiques (Inde et Philippines) d'origine de la diversité interspécifique Musa acuminata et Musa balbisiana. (Résumé d'auteur

Integrated virus and infection risk: does the banana streak virus (bsv) threat the banana culture? [Poster-P242]

Cultivated bananas mainly stem from intra- and interspecific crosses between two species: Musa acuminata (A genome) and Musa balbisiana (B genome). The B genome harbors viral sequences of Banana streak virus (BSV), inducing the banana streak disease and affecting growth and fruits production. The plantain is a natural hybrid (AAB) containing these integrations. BSV integrations can release functional virus following stresses, leading to spontaneous infections. However, no epidemic has been reported so far. We suspect the plantain to control this viral infection using a RNA silencing mechanism, resulting from the co-evolution between viral integrations and B genome. My PhD work aims to investigate BSV infections in plantain, from eBSV activation to complete recovery (qPCR, immunolabelling); as well as the defense mechanism set up by the host (Northern blot, NGS, in situ hybridization). I aim to propose a BSV infection evolution profile in plantain, to further test the virus transmission to a healthy plant. Indeed, other banana trees among which Cavendish cultivar (AAA, producing “dessert bananas”), without B genome, are extremely susceptible to BSV. The plantain culture intensification, associated with global changes, could threat the dessert bananas culture. My project will help answering this arising question of BSV epidemic risk

Distribution and conservation of Banana Streak Virus (BSV) within banana Musa balbisiana genome: what impact on host and virus evolution ?

The nuclear genome of several plants isinvaded by numerous viral sequences. These integrations correspond to accidental events mainly resulting from illegitimate recombination of DNA viruses belonging to the family Caulimoviridae with plant DNA whereas integration into the host genome is not required for viral replication. These integrations are for the most part defective as a result of pseudogenisation driven by the host genome evolution. Conversely some, named infectious, could release a functional viral genome following activating stresses. Our aim is to study the evolving integration context of such infectious integrants for Banana streak virus among the diversity of the banana B genome in order to retrace the evolutionary BSV story and understand their impact on host and virus evolution in terms of cost/benefit. To answer this question, we propose to characterize the infectious integrants (eBSV) among a representative sampling of Musa balbisiana diversity and to hypothesize a contribution of eBSV towards plant virus resistance through an RNA interfering mechanism versus virus maintain through interspecific crosses. (Texte intégral