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

Feel++ : A Versatile High Performance Finite Element Embedded Library into C++

International audienceA Domain Specific Language for PDEs embedded in C++ providing a syntax very close to the mathematical language in order to describe the variational formulation: Supports generalized arbitrary order Galerkin methods (cG, dG) in 1D, 2D, 3D Supports simplex, hypercube and high order meshes Supports finite elements: Lagrange, Hermite, Nedelec, Raviart-Thomas Supports seamless parallel computing Supports seamless interpolation between grids/function spaces Supports symbolic computation thanks to GiNaC Supports large scale parallel linear and non-linear solvers (PETSc/SLEPc) Supports hybrid computing: MPI, multi-thread, GPU (HARTS) Supports in-situ visualization with ParaView</ul

Expression of an Arabidopsis aspartic protease in Pelargonium : [Abstract L36]

Arabidopsis thaliana transgenic plants with constitutive over-expression of the aspartic protease gene At2g28010 (named CDS10) showed a bushy, multi-branching dwarf phenotype. In order to obtain compact plants of ornamental interest with an analogous phenotype in Pelargonium zonale, a tall cultivar (Boda Gitana Salmon) was transformed to over express the A. thaliana CDS 10 gene under the 35S promoter. Twenty seven transgenic lines were obtained with different levels of expression after gold particle bombardment and regeneration. Some of them showed indeed a bushy phenotype with a higher number of branches and a dwarf phenotype. However, an increase in the number of branches correlated with a decrease in the number of petals in the flowers. So the plants that were of interest from the compact habit point of view, had lost the double flower trait, and exhibited only 5 petals/flower which were also smaller than those from double flowers from the non transformed plants. Intermediate phenotypes with semidouble flowers and higher number of branches but without a compact phenotype were also observed. In order to determine if it was genotype related two other cultivars were transformed, Mirada Violet and Mirada Simple Pink double and single flower cultivars respectively. Transgenic plants showed indeed a higher number of branches and single flowers. Even if the busy phenotype was of interest in order to get a higher number of cuttings/plant and a compact phenotype, the pleiotropic effects of the over-expression of the A. thaliana CDS 10 gene on the flowers are too strong meaning it is only of interest in single flowered cultivars which are a small share of the market. (Résumé d'auteur

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

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

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

The chromosome segregation in a created Musa interspecific tetraploid (AAAB) hybrid can lead to eBSV elimination. [P.51]

Banana cultivars (including plantain) are derived from natural hybridization between wild diploid Musa species and subspecies. The main species involved are Musa acuminata (A genome) that is encountered in all banana cultivars, and M. balbisiana (B genome) in many of them. The usual strategy to produce improved AAB plantain like hybrids exploits residual fertility of existing triploids cultivars and relies on two successive crosses. The first cross involves a plantain (AAB) and a highly fertile diploid (AA) banana to produce offsprings from which a tetraploid is selected. In a second step, this selected tetraploid is crossed with another diploid accession to produce triploid offspring from which improved hybrids may be selected. However, plantain breeding was hampered since the mid-1990s by the discovery in the B genome of infectious endogenous sequences of banana streak viruses (eBSVs), which in interspecific genome context, are able to produce BSV particles causing banana streak disease (BSD). Understanding chromosomes segregation and recombination during meiosis, in inter-specific hybrids, is essential for breeding because it determines the possibility of combining the agronomical attributes of both species and the possibility to eliminate by crossing over undesirable traits such as eBSV. Here we monitored the distribution of infectious eBSV sequences among a F1 triploid AAB population produced by inter-specific cross between CRBP39 (AAAB) as female parent and the diploid M. acuminata (AA) Pahang as male. CRBP39 is a new created tetraploid carrying both infectious alleles of eBSV Goldfinger (eBSGFV) and of eBSV Obino l'ewai (eBSOLV) while Pahang do not possess any eBSV for these two BSV species. Results showed the distribution of these eBSVs was skewed toward an excess in the progeny but demonstrated that interspecific chromosome recombination occur frequently between M. balbisiana and M. acuminata genomes. Interestingly, we showed that both eBSVs were absent from 24 offsprings harboring nevertheless segment of B genome, which represent promising material for breeding. The results obtained in this study, that are based on plant material used in conventional (4x/2x) breeding strategy, open undoubtedly new positive perspectives for the safe exploitation of M. balbisiana characteristics in breeding programs. (Résumé d'auteur

Hemodynamic simulations in the cerebral venous network: A study on the influence of different modeling assumptions

International audienceBlood flow computations in complex geometries are of major interest in various cardio-vascular applications. However, deriving an appropriate computational model is still an open issue and a central question is how to incorporate and quantify uncertainties due to different modeling assumptions. The present work is intended as a first step in this direction, in the particular case of blood flow in the cerebral venous system. After a careful evaluation of the influence of the computational methodology, the study investigates the impact on the velocity field and the wall shear stress of three inflow boundary conditions, two strategies for treating the outflow boundary condition and two different viscosity models. The results demonstrate that the effect of setting the inflow boundary condition on the forces created by blood flow, is likely greater than for other modeling assumptions, the other important factor being the blood viscosity model, especially in wall shear stress computations. They suggest that improvements on the one hand on the mathematical and computational approach, and on the other hand on available data for their treatment are needed