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To protect genome integrity against invasive nucleic acids, most eukaryotic organisms have evolved gene silencing mechanisms, often involving small interfering RNAs (siRNAs). Endogenous banana streak virus (eBSV) integrants derived from three distinct species, present in Musa balbisiana (B) but not Musa acuminata (A) banana genomes are able to reconstitute functional episomal viruses in interspecific triploid AAB banana hybrids, causing banana streak disease. In contrast, the seedy diploid (BB) parent line, which harbours identical eBSV loci, does not develop disease. Illumina sequencing revealed that eBSV loci produce low-abundance transcripts covering most of the viral sequence and generate predominantly 24-nt siRNAs, in contrast to predominantly 21–22 nt viral siRNAs derived from the respective episomal viruses in M. acuminata or AAB hybrid plants. Remarkably, siRNA production is restricted to duplicated and inverted viral sequences present in eBSV loci. Using bisulfite sequencing we found that both siRNA-producing and non-producing sequences in the eBSV loci are methylated at 100 % symmetric CG and CHG sites and up to 50% in non-symmetric CHH sites unlike episomal virus DNA previously found to be largely unmethylated. Our data suggest that eBSVs are controlled at the epigenetic level in the BB diploids. This regulation prevents both their awakening and systemic infection of the plant, but probably also serves to confer the resistant phenotype observed in BB plants against mealybug-transmitted viral particles. These findings are thus of relevance to other plant resources hosting integrated viruses.

Banana bunchy top virus (BBTV) is a six-component ssDNA nanovirus transmitted by aphids, causing severe disease of monocot Musa plants, which originates from South-East Asia. Asian BBTV isolates are often associated with self-replicating alphasatellites. By Illumina sequencing analysis of banana aphids and leafs collected in Congo, we discovered an alphasatellite of previously unknown genus, phylogenetically related to alphasatellites of dicot nanoviruses. It was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads and altering relative abundance of its genomic components (formula). Consequently, the alphasatellite reduced virus transmission efficiency and delayed symptom appearance without impacting disease severity. BBTV and alphasatellite clones caused systemic infection in Nicotiana bentamiana, followed by recovery, and BBTV replication protein Rep (but not alphasatellite Rep) induced leaf chlorosis. Illumina sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands entire viral of the genome, allowed to map monodirectional Pol II transcription units for each viral mRNA and uncovered pervasive transcription of each component and alphasatellite in both polarities, likely generating double-stranded precursors of viral siRNAs. Consistent with latter hypothesis, viral DNA formulas resembled viral siRNA (but not mRNA) formulas. In summary, following African invasion, BBTV got associated with an alphasatellite, likely originating from a banana neighbour plant and having a negative impact on BBTV replication and transmission. Molecular analysis of BBTV- and alphasatelliteinfected plants revealed novel mechanistic aspects of viral DNA transcription by Pol II and siRNA production by the antiviral silencing machinery.

Banana bunchy top virus (BBTV) is the most important viral disease of banana and all the known varieties of banana are susceptible to the banana bunchy top disease (BBTD). BBTV is a multipartite virus with six circular single stranded DNA components (DNA-C, DNA-M, DNA-N, DNA-R, DNA-S and DNA-U3) of ~1 kb in size and are transmitted by banana aphids. Rolling circle amplification (RCA) was used to amplify the BBTV components from BBTV-infected banana plants and viruliferous aphids, RCA products sequenced with Illumina deep sequencing and sequencing reads were de novo assembled to reconstruct the consensus BBTV genome. A single dominant variant of DNA-C, DNA-N and DNA-R components, two variants of DNA-M (M1 and M2) and DNA-S (S1 and S2) and at least five variants of DNA-U3 (U3-1, U3-2, U3-3, U3-4 and U3-5) were obtained to denote the complexity of BBTD. A single dominant variant of a novel alphasatellite was also found to be frequently associated with BBTV and when present, it was way more abundant than the BBTV components.Infectious clones are a prerequisite for molecular characterization of any viral disease and till date there is no report of an infectious clone for BBTV. We have cloned all the BBTV genomic components, including the alphasatellite, constructed partial dimer clones and evaluated the infectivity of these constructs in the host banana plant and the model plant Nicotiana benthamiana by agroinoculation. Preliminary analysis with PCR and immuno-capture PCR showed that Nicotiana benthamiana plants can act as a good model system to study BBTV infection.All the plants employ RNA silencing as a primary mode of defense against viruses and viruses counteract this defense mechanism by expression of a silencing suppression protein(s). To identify the silencing suppressor protein(s) of BBTV, we expressed the open reading frames of all the six BBTV components, cell-cycle link protein, movement protein, nuclear shuttle protein, replication initiator protein, coat protein, a protein of unknown function and the alpha-replicase under a constitutive promoter and screened them for their ability to suppress RNA silencing in the GFP-transgenic Nicotiana benthamiana 16c system. Summary of the complexity in BBTV genome, infectivity of the cloned components, role of the alphasatellite in BBTD, RNA silencing-mediated host defense against BBTV and the ability of BBTV ORFs to suppress RNA silencing will be presented.

Plantain is a natural hybrid coming from interspecific crossing between two major banana species: Musa acuminata (genome A) and Musa balbisiana (genome B). All B genomes contain viral integrations of Banana streak virus (BSV), responsible for the banana streak disease, which affects plant growth and fruit production. The plantain genome (AAB) contains viral integrations of two BSV species (referred as endogenous BSV, or eBSV) and can develop eBSV-derived infections following biotic and abiotic stresses. However, no banana streak disease epidemic has been reported so far on plantain cultures. We suppose that plantain can regulate eBSV-derived infections, probably through RNA silencing acquired through the co-evolution history between B genome and BSV.During my PhD, I followed eBSV-derived infections kinetics in naturals (plantain) and artificial AAB hybrids, on whole mats and for a complete crop cycle. Virus distribution was heterogeneous across the mats, with some suckers showing severe infection while others remained un- or slightly infected. I observed viral titter fluctuations across time, with extreme cases of complete recovery in natural hybrids. Overall, average viral charge was higher in artificial hybrids compared to natural hybrids.Analysis of small virus-derived RNA profiles (from northern blots and deep sequencing) from contrasted samples taken at different stages of the infection, and from both artificial and natural hybrids are in progress. The results aiming to a better understanding of the defence mechanism involved in the infection control will be presented.

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.

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové – BSIMV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. Using deep sequencing of total siRNAs of PKW we underlined the presence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSIMV by blasting sequences against the 3 BSV species genomes. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. vsRNA are enriched in 24-nt class which represent about 75% of the total 21-24nt siRNA matching eBSV. We also demonstrated that eBSV are highly methylated in the three different sequence contexts (CG, CHH and CHG) throughout the whole sequence of eBSVs with no difference in methylation profile between siRNA producing and non producing areas. Interestingly, methylation patterns of all three eBSV are similar whereas they are located in different genomic context; eBSOLV being in a TE rich area whereas eBSIMV and eBSGFV are in genes rich region. It seems that eBSV are controlled mainly by epigenetic mechanisms similar to those described for transposable elements (TE). All together, our data indicate that eBSVs in PKW genome are likely silenced at the transcriptional level and this is probably responsible for the natural resistance of this genotype to the activation of such infectious eBSV as well as infection by external BSV particles.

Banana streak viruses (BSV), which infect bananas and plantains (Musa spp.) worldwide, became a couple of decade ago, the major constraint for banana breeding programmes and Musa germplasm exchange. Indeed, the movement of germplasm and propagation of virus-free planting material have been restricted due to the discovery, in the genome of Musa balbisiana (noted B genome), of infective BSV integrations (eBSV) which can spontaneously release functional BSV virions following stresses. Our objective is to both study the mechanisms underlying the regulation of eBSV and gain knowledge on the BSV biology. Unfortunately to date, BSV is known to infect banana only which is a difficult/complex plant to work with because the life cycle is long, mutant databases are not available, transient and stable transformations are difficult and laborious for instance. To overcome most of those difficulties, we tried to infect the model plant Arabidopsis thaliana (A.t.). We firstly stably transformed two different At ecotypes with a viral clone containing more than one length copy (1,5) of BSOLV DNA genome via agrobacterium. This clone, previously selected as infectious in banana plants, contains twice the promoter region because of the poly A signal is located in front of the promoter. Out of the thirty T2 transformants we analysed, none of them showed abnormal phenotype or BSV symptoms. Besides, RT-PCR experiments showed that viral transcription was undetectable suggesting that T-DNA expression was inhibited at the transcriptional level. Using methylation sensitive restriction enzymes we indeed evidenced that the T-DNA was methylated. To overcome this problem, we transformed several A.t. mutants affected at different stage of the epigenetic RNA silencing pathway. Screening and characterization of primary transformants is on the way but preliminary results on dcl234 triple mutant background indicate that BSV transcription is detectable. Immuno-capture PCR experiments and electron microscopy observations will be soon carried out to look for the presence of the virus. (Texte intégral)

Banana streak virus (BSV) belongs to the genus Badnavirus of plant pararetroviruses (family Caulimoviridae) that replicate by reverse transcription of pregenomic (pg)RNA and encapsidate circular non-covalently closed double stranded genomic DNA of ca. 7.4-8 kb to form episomal viruses. A complex of BSV species cause the streak disease transmitted by mealybugs in bananas and plantains worldwide. Gaining the knowledge on transcriptional regulation of episomal BSV is an important step forward in developing efficient strategies to fight against BSV. Schenk et al., 2001 have shown that the 1.3 and 2.1 kb fragments of the intergenic region of BSV Cavendish (BSCAV) and BSV Mysore (BSMYV) species, respectively, exhibited a promoter activity in a wide range of plant species including monocots, dicots, gymnosperm and fern. In 2005, Remans et al. have identified four elements in the intergenic region that are important for the activity of the BSCAV promoter. Here we mapped the pgRNA promoter of BSV Obino l'Ewai (BSOLV) species which is the most widespread BSV species on earth. Using transient expression in N. benthamiana of several deletion constructs of the intergenic region fused with the GFP reporter gene, we found that the BSOLV core promoter activity is regulated by mainly two elements. One element located downstream of a putative transcription start site (TSS) in the pgRNA leader region (5'UTR region with a strong secondary structure) has a negative effect decreasing the core promoter activity by 95%, whereas the second element located between positions - 450 and -180 upstream of the TSS enhances it by 90%. Interestingly, using Arabidopsis transgenic plants carriyng stably-integrated BSOLV promoter-reporter gene constructs, we observed the reporter gene expression in all types of the studied organs including roots, leaves, stem, reproductive organs, siliques and seeds, indicating that BSV is transcriptionally-active in any plant tissue. Besides, our work also suggests that infective BSV integrants characterised in the genome of Musa balbisiana subspecies (banana B genome), called endogenous (e)BSVs, can potentially give rise to BSV episomes in any plant tissue because their nucleic acid sequences (including promoter sequences) are over 99% identical to their counterpart episomal viruses (Chabannes et al., 2013). (Texte intégral)

BACKGROUND and OBJECTIVES Viruses are major constraints to the production and exchange of banana and plantain (Musa spp.) germplasm in the world. Several viruses are recognized to affect Musa spp: Banana bunchy top virus (BBTV), Banana streak GF virus (BSGFV), Banana streak OL virus (BSOLV), Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Cucumber mosaic virus (CMV), Banana bract mosaic virus, (BBrMV). To date, in Democratic Republic of Congo (DRC), except BBTV, others banana viruses are not yet reported. The main aim of this work was to elucidate the epidemiological situation, genetic variability and origin of Musa spp. viruses in DRC. MATERIALS and METHODS Epidemiological surveys were conducted from 2011-2013, in DRC, to search main viruses of Musa spp. Up to 666 samples collected from 122 locations; Samples were analysed using PCR based method. Detected viruses were sequenced and phylogenetic studies were performed. RESULTS BBTV is the most widespread virus (frequency=44,8%) in DRC. Molecular analyses support infrequent BBTV dispersal events between continents. This study reveals a low genetic variability of DRC BBTV isolates. They are close, gather in the South Pacific group and are linked with isolates of other sub-Saharan Africa (SSA) countries, but no with Egyptian isolates. Since the earliest BBTV report in SSA was in DRC in 1950, it is likely that BBTV spread from DRC to other SSA countries rather than from Egypt and that at least two BBTV introductions occurred in Africa. Haplotypes analysis based on the coding region m-Rep protein showed 38 haplotypes (haplotype diversity value of 0.944±0.013). In Bas Congo, symptoms of Banana streak disease were associated to the presence of BSV species (frequency=19,9%); the most widespread were BSGFV (frequency=67,2%) and (BSOLV) (frequency=24,2%). The sequenced BSOLV isolate from Musa acuminata, gather in the clade I recognized to gathering the BSV species with endogenous counterpart. CMV was also detected (frequency=9,3%). Genetic diversity analysis reveals the high similarity between DRC CMV isolates with a South African isolate). BBrMV was not detected in collected samples. CONCLUSION DRC is a place of high prevalence of banana viruses, indicating the need for an improved quality of planting material. DRC seems to be the primary center of BBTV spread in Africa. BSV species data revealed the activation of endogenous BSV and the transmission of BSV species from plantain (B genome) to banana (A genome), in natural conditions. While for CMV, data suggest the hypothesis of a unique introduction through infected planting material and the distribution of a single strain of CMV. The BBrMV still absent in DRC. (Texte intégral)

BACKGROUND and OBJECTIVES Banana bunchy top virus (BBTV), is the most devastating and widespread banana virus. Banana and plantain (Musa spp.) are major crops in terms of household income and food security in Democratic republic of Congo (DRC) (1). Despite the large territory under banana and plantain cultivation in the country, the genetic characterization of BBTV isolates has been undertaken for two provinces. The main objectives of this work were to understand the genetic variability of DRC BBTV isolates and to determine their origin. MATERIALS and METHODS In this study, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasai Oriental). 52 full sequences of DNAR and 30 full sequences of all BBTV components were analysed. RESULTS Full genome sequencing of DNA-R components was performed revealing a low genetic variation (98-100% nucleotide identity) amongst the BBTV isolates detected in seven of 11 DRC provinces. The phylogenetic tree showed that all DRC isolates gather a unique clade in the South Pacific group of BBTV. Based on the coding region for replication initiator protein, haplotype diversity was estimated to be 0.944±0.013, with 30 different haplotypes from 68 isolates in DRC. In addition, fives isolates were selected from each province for total genome sequencing, confirming low genetic variation among isolates from seven provinces (97-100% nucleotide identity). CONCLUSION This study strengthens the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country. Analysis of the geographical dispersion reveals specific haplotypes groups according to the different agro-environmental conditions.

Breeding new interspecific banana hybrid varieties with pests and disease resistance characters is instrumental for the development of pesticide-free agroecological banana farming systems. Such breeding relies on the combined use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of M. balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus (BSV) in interspecific hybrids harbouring both M. acuminata and M. balbisiana genomes. In this work, we show that M. balbisiana diploid genitors available for breeding host at least one infectious eBSV. We also show how we segregated infectious and non-infectious eBSV alleles in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. This work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.

Thirty-five unique partial sugarcane bacilliform virus (SCBV) sequences extending over 529 bp were identified in sugarcane samples from Guadeloupe diagnosed by Immunocapture-PCR (IC-PCR) using specific badnavirus primers. Phylogenetic analysis of these sequences along with the two known genome sequences of Sugarcane bacilliform Mor virus (SCBMV) and Sugarcane bacilliform IM virus (SCBIMV) revealed high molecular variability in the SCBV genome. Seven phylogenetic groups, named A to G, were characterized: virus isolates from groups A-B, C and D are proposed to be members of three additional SCBV species. The two (7446 and 7444 bp) and one (7317 bp) complete sequences of SCBV isolates from groups A and D, respectively, likely represented the genome of two new species. Phylogenetic analysis of the complete genome and RT/RNase H sequences confirmed the polyphyletic structure of SCBV isolates and the absence of a clear separation between SCBV and Banana streak virus (BSV) isolates within badnavirus group 1. These results showed that reconsideration of taxonomy and classification of SCBV and BSV are necessary.

The banana streak disease is due to a complex of distinct Banana streak viruses (BSVs) species showing a wide genetic diversity. Banana streak viruses (BSVs) are double stranded DNA pararetroviruses belonging to the family Caulimoviridae, genus Badnavirus. Outbreaks of BSVs causing banana streak disease have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. In addition, the banana genome (Musa sp.) is invaded by numerous badnavirus sequences. The majority of these viral integrants is mostly defective as a result of pseudogenisation driven by the host genome evolution. They are just called BEV (banana endogenous virus sequences) because episomal particles corresponding to the integrated counterparts have not been identified so far. Conversely, only few viral integrants named endogenous BSV (eBSVs) can release a functional BSV genome following stresses. All the badnaviral sequences described so far are spread among the three main clades of the badnavirus genus diversity. Our group established that BSVs are distributed among Clade I and Clade III. Clade I gathers BSV species distributed worldwide whereas Clade III is dedicated to BSV species only present in Uganda. eBSVs exclusively correspond to BSV species of Clade I and are endogenous to the Musa balbisiana (B) genomes only. We elucidated their sequence and organization for three BSV species (BSOLV, BSGFV and BSIMV) present in the seedy banana diploid PKW (BB). In addition, we established that all BEVs sequences belong to Clade II. All together our results allowed us to propose an evolutionary scheme of badnavirus and banana co-evolution, which is presented here.

Banana streak viruses (BSV) infect bananas and plantains (Musa spp.) worldwide. BSV became a major constraint for banana breeding programmes and germplasm exchanges since the discovery a couple of decade ago of the presence of infectious BSV integrations (eBSV) in the genome of Musa balbisiana (B genome) which can under certain stress conditions released spontaneously fonctional and infectious BSV. To date, BSV is known to only infect banana but for several reasons working with banana is very time consuming and not an easy task (space needed, long life cycle, no mutant database available, transient and stable transformations are difficult and laborious...). To overcome most of those problems and to both understand the mechanisms underlying the regulation of eBSV activation as well as eBSV non-activation and gain knowledge in the BSV biology, we tried to establish infection of Arabidopsis thaliana (At). We stably transformed two different ecotypes of At via agrobacterium using a more than a full length BSV viral genome clone containing twice the promoter region because of the poly A signal is in front of the promoter. T2 transformants did not show any abnormal phenotype neither BSV symptoms. RT-PCR experiments showed that viral transcription was very weak if not undetectable amongst more than 30 independent transformants. One hypothesis to explain this absence of transcription could be due to methylation of the viral genome. Using the cleavage activity of the McrBC methylation-dependent enzyme which recognizes 5-methylcytosines (a hallmark of plant DNA methylation) we demonstrated for all independent transformants tested (4 in total) than the viral construct was always methylated hampering potentially the BSV transcription. To validate and explore this hypothesis, we are crossing selected transformants with mutants affected at different stage of the epigenetic pathway.

The B genome of banana (Musa sp.) harbours integrations of Banana streak virus (eBSV) for at least three BSV species, whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses contexts. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated, more or less extensively rearranged containing at least one full length viral genome. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSIMV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. Using deep sequencing of total siRNAs of PKW we underlined the presence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSIMV by blasting sequences against the 3 BSV species genomes. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. vsRNA are enriched in 24-nt class which represent about 75% of the total 21-24nt siRNA matching eBSV. We also demonstrated that eBSV are highly methylated in the three different sequence contexts (CG, CHH and CHG) throughout the whole sequence of eBSVs with no difference in methylation profile between siRNA producing and non producing areas. Interestingly, methylation patterns of all three eBSV are similar whereas they are located in different genomic context; eBSOLV being in a TE rich area whereas eBSIMV and eBSGFV are in genes rich region. It seems that eBSV are controlled mainly by epigenetic mechanisms similar to those described for transposable elements (TE). All together, our data indicate that eBSVs in PKW genome are likely silenced at the transcriptional level and this is probably responsible for the natural resistance of this genotype to the activation of such infectious eBSV as well as infection by external BSV particles.

French West Indies Biological Resources Centre for Tropical Plants (CRB-PT) maintains several germplasm collections of tropical crops and wild relatives, including a collection of more than 450 yam accessions (Dioscorea spp). The purpose of this Centre is to conserve and distribute virus-free germplasm to end users. To this aim, virus populations infecting conserved accessions are characterized and appropriate detection tools are created or optimized, then implemented for the sanitation of infected germplasm. Several badnavirus species have been reported in yams. Recently, endogenous Dioscorea badnaviral sequences (eDBVs) were described in the genome of African yams of the D. cayenensis-rotundata complex [1, 2]. These sequences interfere with badnavirus PCR-based detection methods and are therefore a constraint for the accurate diagnostic of badnaviruses in yams. To address this problem, an analysis of the diversity of eDBVs was carried out in D. rotundata, using virus-free seedlings. It showed that sequences from at least four distinct Badnavirus species are integrated in yam genomes, and that these viral species belong to the groups 5, 8, 9 and 12 defined by Kenyon et al. [3]. This finding enabled the development and implementation of a reliable technique for the specific detection of badnavirus episomal forms in yams. Using this method, we showed that the CRB-PT yam collection hosts different strains of Dioscorea bacilliform AL virus (DBALV; group 8) and yet unreported episomal forms of one badnavirus species of group 9, for which only endogenous forms were known so far. The genome of this new yam badnavirus species was amplified from an infected Dioscorea trifida accession, cloned and sequenced. Its organization is similar to that of DBALV and Dioscorea bacilliform SN virus (DBSNV; group 4). However, phylogenetic analyses showed that it is a novel and distinct badnavirus species, for which the name Dioscorea bacilliform TR virus (DBTRV) is proposed. This first report of the occurrence of endogenous sequences (eDBV8 and eDBV9) from extant badnavirus species in yams should suggest that some eDBVs could be infectious. Conversely, molecular evidence supporting the role of these eDBVs in antiviral defense will also be presented.

International audience; Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed ‘endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

Infections of banana and plantain by banana streak viruses (BSV) can occur in the absence of vector-mediated transmission, through the activation of infectious endogenous BSV sequences (eBSVs). Such infectious eBSVs are present in the genome of Musa balbisiana spp, which are important progenitors for breeding improved banana varieties. Once activated by biotic or abiotic stresses, these viral integrants cause spontaneous infection in both natural and synthetic interspecific hybrids harbouring the M. balbisiana (B) genome. Therefore, the presence of infectious eBSVs within B genomes is the main constraint for breeding banana and plantain interspecific hybrids and for exchanging Musa germplasm. The sequence and organization of eBSVs in the diploid M. balbisiana genitor Pisang Klutuk Wulung (PKW) was elucidated for BSV species Obino l'EwaI (BSOLV), Goldfinger (BSGFV) and ImovO (BSIMV). This work showed that integration of BSGFV and BSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of BSImV is monoallelic. Allele-specific molecular markers were developed and used to genotype M. balbisiana germplasm, unveiling important differences between accessions and the presence of modified eBSV alleles in several accessions. Breeding improved M. balbisiana progenitors devoid of infectious eBSGFV and/or eBSOLV alleles was achieved through self-pollination and chromosome doubling of haploid lines. Both approaches lead to M. balbisiana cultivars devoid of infectious eBSOLV and/or eBSGFV resulting from the segregation of eBSOLV and eBSGFV alleles. These results pave the way to the safe use of M. balbisiana in breeding programs, and open new perspectives for breeding improved banana and plantain hybrid varieties. (Texte intégral)

National audience

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), a deep sequencing of total siRNAs of PKW was performed using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV by blasting sequences against the 3 BSV species genomes. vsRNA are enriched in 24-nt class thus eBSV in PKW genome are likely silenced at the transcriptional level. Interestingly, we show that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. All together, those data seem indicate these eBSV induce a natural resistance driven by gene silencing mechanisms based on their complex molecular re-arranged structure which could lead to dsRNA formation. (Texte intégral)

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double ? stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (Lockhart, 1990). In the mid-ninetys Ndowora et al. (1999), revealed the presence of multiple integrations of Banana streak virus in the genome of banana (Musa sp.) whereas this badnavirus does not require integration for its replication. Some endogenous BSV (eBSV) sequences in the Musa balbisiana genome are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. Seedy M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV whatever the viral origin. We postulated this resistance is mediated by gene silencing and performed deep sequencing of total small RNAs of PKW using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV by blasting small RNAs against the viral genome counterparts. VsRNAs are systematically enriched in 24-nt class. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences but are directly correlated with the structure of each integration. Finally, preliminary data using Southern blotting coupled with methylation-dependent restriction enzyme (McrBC) digestion indicated that each eBSV is heavily methylated. All together, our findings indicate that eBSVs in PKW are likely silenced at the transcriptional level. In parallel, we deep sequenced small RNAs from Cavendish banana plants (lacking eBSV integrations) infected independently by BSOLV, BSGFV, BSImV and 3 other distinct BSV species in order to evaluate silencing-ased regulation of episomal virus infection. Abundunt vsRNAs were found to be enriched in 21-nt class and most of the hot spots of siRNA production are located in the coding sequences. Our methylation analysis indicated that episomal BSV DNA is not methylated and therefore, is likely silenced only at the post-transcriptional level.

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double-stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (Lockhart, 1990). While the first two ORFs encode two small proteins of unknown function, the third ORF (~210 kD) encodes a polyprotein that can be cleaved to yield the viral coat protein and proteins with homology to aspartic protease, reverse transcriptase and RNaseH. Little information is available about antiviral defense response of the host plant on BSV or other members of Caulimoviridae. RNA silencing, also known as RNA interference (RNAi), is an ancient gene regulation and cell defense mechanism, which exists in most eukaryotes (Xie and Qi, 2008). Plants have adapted the RNA silencing machinery into an antiviral defense system. Interestingly, Arabidopsis plants infected with Cauliflower mosaic virus (CaMV), a type member of the genus Caulimovirus in the family Caulimoviridae, accumulate siRNAs of 21, 22 and 24 nt size classes, where the 24 nt species are the most predominant ones (Blevins et al., 2006; Moissiard and Voinnet, 2006). Further analysis showed that, the leader region (600 nt) of CaMV pregenomic RNA produces massive amounts of siRNAs with several hot and cold spots of siRNA generation (Blevins et al., 2011) to function as a decoy for the RNA silencing defense system of the plant. To determine whether the viral decoy strategy was universally used among viruses belonging to the family Caulimoviridae, we have performed a deep sequencing of total siRNAs of 6 Cavendish banana plants infected independently with one of the 6 BSV species we own in the laboratory. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from those 6 BSV species by blasting sequencing data against the 6 BSV species genomes. vsRNA are enriched in 21-nt class thus BSV are likely silenced at the post-transcriptional level. Besides, our data unequivocally show that the decoy strategy used by the CaMV is not employed by the BSV since most of the hot spots of siRNA production are located in ORF1 and 2. Information generated about siRNAs derived from BSV genome could help us to design silencing-based transgenic and non-transgenic (RNA vaccination) approaches to obtain BSV resistance in banana crop. (Texte intégral)

Banana streak viruses (BSV) infect bananas and plantains worldwide. They are naturally transmitted by mealybugs; however infections can also occur in the absence of vectormediated transmission, through the activation of infectious endogenous BSV sequences (eBSVs). Infectious eBSVs are present in the genome of Musa balbisiana spp, which are important progenitors for breeding improved banana varieties. Once activated by biotic or abiotic stresses, these viral sequences cause spontaneous infection in both natural and synthetic interspecific hybrids harbouring the M. balbisiana genome, denoted B [1]. Therefore, the presence of infectious eBSVs within B genomes is currently the main constraint for breeding banana and plantain interspecific hybrids and for exchanging Musa germplasm. The sequence and organization of eBSVs in the diploid M. balbisiana genitor Pisang Klutuk Wulung (PKW) was elucidated [2], showing that integration of infectious eBSGFV [3] and eBSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of infectious eBSImV is monoallelic [2]. Taking advantage of the development of allele-specific molecular markers [2; 4], eBSV signatures were established for all M. balbisiana genitors of the CIRAD Guadeloupe Musa collection. This work unveiled important differences between accessions. All combinations of infectious and non-infectious alleles were observed for the three BSV species, as well as complete and uncomplete integrants when compared to those described in PKW. Breeding improved M. balbisiana progenitors devoid of infectious eBSGFV and/or eBSOLV alleles was undertaken through self-pollination and chromosome doubling of haploid lines. Both approaches successfully lead to M. balbisiana cultivars devoid of infectious eBSOLV and/or eBSGFV resulting from the segregation of eBSOLV and eBSGFV alleles. Improved lines of one particular M. balbisiana cultivar, cv. Honduras, originally free of eBSImV, were shown to be free of infectious eBSV. These results pave the way to the safe use of M. balbisiana in breeding programs, and open new perspectives for breeding improved banana and plantain hybrid varieties. (Texte intégral)

The banana genome (Musa sp.) is invaded by numerous badnavirus sequences including those of banana streak viruses (BSVs). Badnaviruses are double stranded DNA pararetroviruses belonging to the family Caulimoviridae with any integration step into the host genome during their life cycle. The majority of viral integrants is mostly defective as a result of pseudogenisation driven by the banana host genome evolution. Conversely, some from BSVs only and named endogenous BSVs (eBSVs), can release a functional viral genome following stresses. All the badnavirus sequences described so far in banana [1] are spread among the three main groups structuring the badnavirus genus diversity [2]. We established that eBSVs are restricted to group 1, have an episomal counterpart, and are present in Musa balbisiana (B) genomes only [1;3;4] whereas the others are restricted to group 2 with any episomal counterpart described so far. We elucidated the sequence and the allelic organization for three BSV species (BSOLV, BSGFV and BSImV) present in the B genome of the seedy diploid PKW (BB), and developed several PCR and deCAPS markers for a specific eBSV genotyping [3-5]. Then, we investigated the early evolutionary stages of infectious eBSV through the study of their distribution, their polymorphism of insertion and their structure evolution when possible, among the banana B genomes diversity available through a sampling of BB seedy diploids extended to interspecific hybrids exhibiting different levels of ploidy for the B genome. We selected our sampling according to the two areas of sympatry between M. acuminata and M. balbisiana representing the centers of origin for the most largely cultivated AAB cultivars. One was in India and the other one in East Asia going from Philippines to New Guinea (Perrier et al, 2009). We used the PCR markers and Southern blots analysis to characterize PKW-related eBSV allelic polymorphism and we coded these results in order to create a common dissimilarity matrix with which we interpreted eBSV distribution. As a result, three dendrograms of PKW-related eBSV were constructed for each BSV species using the neighbor joining (NJ) method, as well as one dendogram resulting from NJ analysis of all three BSV species together. We found that the known phylogeny of banana accessions based on M. acuminata genomes can help to elucidate eBSV structural diversity, and that eBSV polymorphisms can shed light on the particularly unresolved question of M. balbisiana diversity. We propose for the first time a banana phylogeny driven by the M. balbisiana genome. A evolutionary scheme of BSV/eBSV banana evolution is finally proposed.

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double-stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (1). In the mid-ninetys Ndowora et al. (1999), revealed the presence of multiple integrations of Banana streak virus in the genome of banana (Musa sp.) whereas this badnavirus does not require integration for its replication. Some endogenous BSV (eBSV) sequences in the Musa balbisiana genome are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. Seedy M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger ?BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV whatever the viral origin. We postulated this resistance is mediated by gene silencing and performed deep sequencing of total small RNAs of PKW using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV species by blasting small RNAs against the viral genome counterparts. VsRNAs are systematically enriched in 24?nt class. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences but are directly correlated with the structure of each integration. Finally, preliminary data using PCR coupled with methylation-dependent restriction enzyme (McrBC) digestion indicated that eBSimV is heavily methylated. All together, our findings indicate that eBSVs in PKW are likely silenced at the transcriptional level.

The nuclear genome of banana plants is invaded by numerous viral sequences of banana streak viruses (BSV), a DNA virus belonging to the family Caulimoviridae. These integrations are mostly defective as a result of pseudogenisation driven by the host genome evolution. Conversely some named infectious, can release a functional viral genome following activating stresses. We characterized the infectious endogenous BSV (eBSV) for three BSV species (BSOLV, BSGFV and BSImV) present within the Musa balbisiana B genome of the seedy diploid Pisang Klutuk Wulung (PKW). Our aim is to study PKW-related BSV integrations among the diversity of the banana B genomes in order to retrace the evolutionary BSV and banana stories We extended on purpose the M. balbisiana diversity by the addition of interspecific hybrids with M. acuminata showing different levels of ploidy for the B genome (ABB, AAB, AB) of the banana sample in order to include unsampled or extinct M. balbisiana ressources. We also based the analysis referring to the two areas of sympatry between M. acuminata and M. balbisiana and the centers of origin for the most largely cultivated AAB cultivars. One was in India and the other one in East Asia going from Philippines to New Guinea (Perrier et al, 2009). We characterized the PKW-related eBSV allelic polymorphism using PCR markers (described in poster Galzi & Duroy et al.) and Southern blots on 77 accessions. We codified the results of Southern blot and PCR in order to calculate a common dissimilarity matrix and interpret the eBSV distribution. As a result, three dendrograms of PKW-related eBSV made with the Neighbor Joining (NJ) method on the 77 banana accessions, for each BSV species, are presented as well as one dendogram resulting of NJ analysis for the three BSV species all together . We show that the known phylogeny of banana accessions can enlighten the eBSV structure diversity and that eBSV polymorphisms can help to understand the particularly unresolved M. balbisiana diversity. An evolutionary scheme of BSV/eBSV banana evolution will be proposed. (Texte intégral)

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (BSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. We postulated these eBSV induced a natural resistance driven by gene silencing mechanisms based on their complex molecular re-arranged structure which could lead to dsRNA hairpins formation. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), a deep sequencing of total siRNAs of PKW was performed using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from BSOLV, BSGFV and BSImV by blasting sequences against the 3 BSV species genomes. vsRNA are enriched in 24-nt class thus eBSV in PKW genome are likely silenced at the transcriptional level. A repartition of the vsRNA population matching eBSV will be also presented in order to determine hot and cold spots of vsRNA generation. (Texte intégral)

Il est désormais acquis que des génomes viraux font partie intégrante du génome de nombreuses plantes. Ces intégrations virales sont majoritairement sous la forme de résidus de séquences dégradées. Elles sont principalement issues de virus appartenant à la famille Caulimoviridae, de génome ADN double brin circulaire, n'ayant pas d'obligation d'intégrer le génome de leur plante hôte au cours de leur cycle de multiplication. Notre étude vise à analyser le contexte d'intégration afin de comprendre les causes évolutives du maintien des séquences du Banana streak virus (eBSV) dans le génome du bananier de l'espèce Musa balbisiana, tant pour le virus que pour la plante (2, 3). En effet, l'originalité du pathosystème Banana streak virus (BSV) / Musa balbisiana est que certains eBSV sont conservés et à l'origine de particules virales infectieuses. Le BSV est actuellement la contrainte majeure des programmes d'amélioration génétique utilisant le génome B car porteur d'eBSV pouvant se révéler infectieux. Cinq évènements d'intégration différents pour quatre espèces de BSV ont été mis en évidence chez le bananier diploide naturel Musa balbisiana, le cv Pisang Klutuk Wulung (PKW) bananier modèle d'études. Ces intégrations, fragmentées et complexes sont supérieures à la taille d'un génome viral et se sont révélées être transmises de manière allélique dans la descendance pour deux des quatre espèces de BSV (1 et Baurens et al., non publié). Des marqueurs PCR de structure et d'intégration ont été développés pour chacun de ces eBSV constituant la signature d'intégration pour une espèce BSV donnée. L'ensemble des signatures constitue le passeport eBSV d'un individu. La répartition des eBSV au sein des Musa est en cours d'analyse afin de retracer l'histoire évolutive de ces intégrations. L'échantillonnage retenu est 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 (4). Les premiers résultats des passeports eBSV des différents bananiers obtenus après PCR et l'analyse des structures par southern blots seront présentés ainsi que les premières interprétations. (Texte intégral)

The genome of banana (Musa sp.) harbours multiple integrations of several species of Banana streak virus (BSV), certainly resulting from illegitimate recombination between host and viral DNA. Surprisingly, this badnavirus does not require integration for its replication. Some integrations, only existing in the Musa balbisiana genome (denoted B), are infectious by releasing a functional viral genome following stresses such as in vitro culture and interspecific crosses. To date, four widespread species of BSV (Goldfinger - BSGFV, Imové - BSImV, Mysore - BSMysV and Obino l'Ewai - BSOLV) have been reported as integrated into the B genome with three of them as infectious (eBSGFV, eBSImV and eBSOLV). In order to study BSV expression from such viral integrants, a characterisation of BSV integrants (eBSV) was undergone i) by studying a Musa BAC library obtained from the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) containing the four eBSV species described above and known as eBSV carrier, ii) by using the cytological FISH technique and iii) an interspecific genetic cross using PKW as virus-free female parent. Very low copies of integrations were recorded for each BSV species. The full characterisation of eBSGFV was recently performed in our lab (Gayral et al., 2008). eBSGFV results from a single event of integration corresponding to an allelic insertion of at least one full-length viral genome extensively rearranged with several viral regions duplicated. Although the four BSV species present important differences with each other, the organisation of eBSOLV and eBSImV looks like eBSGFV. Indeed, each of them is more or less extensively rearranged in PKW and is present as allelic insertions at the same locus. In contrary, the non infectious eBSMysV presents two independent insertions sites. Interestingly, M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbor infectious eBSVs in their genome but are nevertheless resistant to any multiplication of BSV. The mechanisms underlying such resistance are believed to be driven by epigenetic phenomena but no evidence has been obtained so far in banana plants. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), we have obtained, for the first time, experimental evidence of virus-derived small RNA in PKW. Deep sequencing of the population of siRNAs using the Illumina ultra-high-throughput technology is on the way. This would help us to detect RNA molecules of very low bundance to single-nucleotide resolution and create a complete map of eBSV siRNAs. This deep-sequencing analysis is done by FASTERIS AG, a Swiss member of our COST action. (Texte intégral)

Le génome de la banana (Musa sp.) possède des intégrations de plusieurs espèces du virus de la mosaïque en tiret du bananier (BSV), intégrations résultant probablement de recombinaison homologue entre le génome du bananier et celui du virus. De façon surprenante, ce pararétrovirus ne nécessite pas d'étape d'intégration pour effectuer son cycle de réplication. Certaines de ces intégrations, seulement présentes dans le génome Musa balbisiana (noté B) sont infectieuses c'est à dire qu'elles sont capables sous certaines conditions de stress de produire des particules virales circulaires fonctionnelles à l'origine de l'infection systémique de la plante. A ce jour, quatre espèces de BSV (Goldfinger -BSGFV, Imové - BSImV, Mysore - BSMysV and Obino l'Ewai - BSOLV) sont trouvées intégrées dans le génome B du bananier dont trois d'entre elles (BSGFV, BSImV et BSOLV) sont infectieuses (eBSV) (Gayral et al., 2008, Iskra-Caruana et al., 2010, Baurens et al., non publié). Afin d'étudier et de comprendre les mécanismes d'activation et d'expression de ces intégrations virales, nous avons entrepris leur caractérisation moléculaire via l'utilisation d'une banque BAC issue du bananier sauvage diploïde M. balbisiana cv. Pisang Klutuk Wulung (PKW) qui contient les quatre espèces de BSV décrites ci dessus. De plus, nous avons déterminé le nombre d'intégration de chacune de ces espèces et leur nature infectieuse grâce à l'utilisation d'une population F1 issue d'un croisement interspécifique utilisant PKW comme parent femelle. Nous avons ainsi pu démontrer récemment que les trois intégrations BSGFV, BSImV et BSOLV présentent une organisation assez similaire puisque chacune d'entre elles résultent d'un seul événement d'intégration avec deux allèles de structure très réarrangée mais dont l'un deux possèdent au moins une fois le génome viral en totalité. Contrairement aux trois autres espèces BSV, l'intégration BSMysV qui n'est pas infectieuse, présente deux intégrations différentes hemizygotes à deux loci indépendants. Dans un avenir proche, nous prévoyons de développer la technique de FISH (Fluorescence In Situ Hybridization) afin de pouvoir déterminer la localisation chromosomique de chacune des intégrations pour les différentes espèces de BSV. (Texte intégral)

Des séquences virales intégrées (EPRV) sont présentes dans le génome de nombreuses plantes, ces intégrations sont soit dégradées et fossiles d'évènement d'intégrations soit semblent récentes, stables, n'évoluant que peu et pouvant être infectieuses. Y a-t-il un bénéfice au maintien de ces séquences dans le génome de la plante, telle est la question que l'on peut se poser. Les séquences virales intégrées, assimilées à des transgènes, engendreraient la production de petits ARN qui pourraient jouer un rôle dans les mécanismes de défense virale de type RNA interference que ce soit au niveau transcriptionnel ou posttranscriptionnel. Une des hypothèses forte relative au maintien des EPRV dans le génome des plantes serait qu'ils participent aux mécanismes de résistance de la plante aux attaques virales. Nous allons donc préciser si les eBSV sont maintenus ou non dans le génome des bananiers, et étudier les conséquences évolutives observables de l'intégration de séquences virale dans le génome du bananier. Pour cela nous allons utilisés les eBSV infectieux d'une part et non infectieux d'autre part présent chez Pisang Klutuk Wulung (PKW) pour les 4 espèces BSV caractérisé. Nous formulerons deux hypothèses de travail. La première s'appuie sur le fait que les eBSV sont issus d'intégrations récentes et évoluent moins vite que les BSV libres. Ainsi ces informations tendront à montrer que les eBSV évoluent sous contraintes sélectives neutres et que les raisons de leur maintien dans le génome des bananiers s'expliquent par le fait que leur intégration est récente et située dans des zones du génome de la plante où la pression de sélection est faible. La deuxième hypothèse s'appuie sur les observations et études préliminaires qui montrent d'une manière générale que les eBSV sont faiblement transcrits, que des stress génomiques sont à l'origine de leur activation et que le mécanisme d'interférence ARN est fonctionnel. Ces informations tendent à indiquer que les eBSV sont associés à un avantage évolutif pour les bananiers sous la forme d'une résistance constitutive induite par le maintien des eBSV. Cet avantage conduirait à une sélection positive des séquences EPRV et donc à un maintien dans le génome de la plante.(Texte intégral)

Le génome de nombreuses plantes présente des invasions accidentelles de séquences pararétrovirales appartenant à la famille des Caulimoviridae. Aucun de ces virus à ADN double brin circulaire n'a d'étapes d'intégration au génome hôte dans son cycle viral. Ces intégrations peuvent servir les fonctions de l'hôte comme sembler neutres ou encore avoir une activité infectieuse. Le génome bananier présente un grand nombre d'intégrations correspondant à différentes espèces virales du Banana streak virus- BSV. Seules des intégrations du génome de l'espèce Musa balbisiana sont infectieuses et capables de restituer un génome viral fonctionnel. Les intégrations d'au moins deux espèces de BSV R espèce Golfinger BSGfV et espèce Imové BSImV - présentes dans un même génome hôte, le bananier sauvage diploïde Musa balbisiana Pisang Klutuk Wulung RPKW sont décrites comme infectieuses. Nous nous sommes intéressés à leur caractérisation après séquençage de clones BAC de PKW contenant le génome viral de chacune des deux espèces. L'analyse de leur ségrégation lors d'un croisement génétique utilisant PKW comme parent a montré une insertion allélique résultat d'un évènement unique d'intégration. Leur organisation montre la juxtaposition de plusieurs séquences virales partielles plus ou moins réarrangées représentant plus d'un génome viral en totalité. Bien que différentes les unes des autres, elles permettent la restitution d'un génome viral fonctionnel. Nous décrirons pour chacune leurs structures moléculaires et génétiques et aborderons leur capacité à être infectieuses via un mécanisme de recombinaison homologue. (Texte intégral)

Banana streak viruses are among the most widely distributed viruses of banana and are responsible for banana streak disease. Natural field spread occurs by either mealybugs or use of infected planting material, such as suckers. Banana streak viruses are pararetroviruses belonging to the genus Badnavirus, in the family Caulimoviridae. Like all members of the Badnavirus genus, they have bacilliform virions, 30 x 150 nm in size and a circular dsDNA genome of 7.4kbp. Fifteen years ago, an increasing number of outbreaks of banana streak disease were reported worldwide. Many occured in banana breeding lines and micropropagated inter-specific banana hybrids. The origin of infections in new hybrids and tissue-cultured plants was linked to the presence of viral DNA sequences integrated into the Musa balbisiana genome. Although integration is not an essential step in the replication cycle of pararetroviruses, it is assumed that under stress conditions some endogenous banana streak viruses could become infectious by reconstituting a complete replication-competent viral genome. Several serological and molecular tools have been developed to detect either virions or endogenous banana streak viruses. They specificity and potential to prevent and control outbreaks of banana streak disease is discussed.

Lors d'introduction aux Antilles de vitroplants de bananiers, les services de la protection des végétaux ont diagnostiqué au cours de contrôles routiniers utilisant la méthode ELISA, le BBrMV. Ce virus est responsable d'une grave maladie des bananiers restreinte à quelques pays d'Asie et du Pacifique La présence du virus a été confirmée par des approches moléculaires qui ont révélé la faiblesse des tests de détection moléculaires disponibles jusqu'alors. Un alignement des séquences disponibles sur genebank a permis de désigner des amorces spécifiques permettant l'amplification d'une partie conservée pour tous les isolats du gène de la capside. Une méthode performante utilisant des kits commerciaux a été développée au CIRAD. L'objectif était de proposer une détection adaptée aux exigences des quarantaines et des échanges de matériel végétal pour lesquels le taux de virus dans les échantillons testés est le plus souvent relativement faible. La méthode d'immunocapture (IC) one-step RT-PCR permet la détection du BBrMV dans des échantillons de bananiers testés de manière isolée jusqu'à une dilution de 1x10-10 et dans un mélange de 10 échantillons de plante. Nous avons proposé cette méthode comme la méthode standard internationale pour détecter le BBrMV afin de répondre à la demande pressante de la DGAL de disposer rapidement d'un outil performant pour contrôler cette maladie.