Is Banana streak virus/Arabidopsis thaliana a viable pathosystem? [P.33]

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. (Résumé d'auteur

How eBSV polymorphism could enlighten BSV and banana evolution story?

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

Endogenous banana streak virus sequences (eBSV) are likely transcriptionally silenced in the resistant seedy diploid Musa balbisiana Pisang Klutuk Wulung (PKW). [P.50]

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. (Résumé d'auteur

Banana plants use post-transcriptional gene silencing to control banana streak virus infection

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

Natural resistance of banana genotypes to banana streak virus is probably driven by transcriptional gene silencing

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

Molecular characterisation of integrated sequences of Banana streak virus in the banana plant genome

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 pararetrovirus does not require integration for its replication. Some integrations, only existing in the Musa balbisiana genome (denoted B), are infectious by reconstituting a functional viral genome. 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 and as infectious, under stress conditions, resulting in viral infection of the banana plant. In order to study BSV expression from such viral integrants, a characterisation of infectious integrants (eBSV) was undergone by studying both a Musa BAC library obtained from the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) containing the four BSV species described above and one interspecific genetic cross using carrier PKW. The organization of eBSGfV was fully characterized recently in our lab (Gayral et al., 2008). eBSGfV results from a single event of integration corresponding to an allelic insertion extensively rearranged, containing at least one full-length viral genome. Although the four BSV species present important differences with each other, the organisation of the three other eBSVs looks like eBSGFV. Indeed, preliminary data indicate that each of them is extensively rearranged in PKW and present as two insertions at the samelocus. This suggests an allelic insertion resulting from a single even of integration. Experimental evidences to demonstrate BSV expression and to validate the infectious nature of every eBSV are on the way. (Résumé d'auteur

A scenario of co-evolution between badnaviruses and Musa sp.. [P.22]

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. (Résumé d'auteur

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

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

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